Victor J Hruby

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• Giri, A. K., & Hruby, V. J. (2014). Investigational peptide and peptidomimetic μ and δ opioid receptor agonists in the relief of pain. Expert Opinion on Investigational Drugs, 23(2), 227-241.
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  Abstract: Introduction: Current methods for treating prolonged and neuropathic pain are inadequate and lead to toxicities that greatly diminish quality of life. Therefore, new approaches to the treatment of pain states are needed to address these problems. Areas covered: The review primarily reviews approaches that have been taken in the peer-reviewed literature of multivalent ligands that interact with both μ and δ opioid receptors as agonists, and in some cases, also with pharmacophores for antagonist ligands that interact with other receptors as antagonists to block pain. Expert opinion: Although there are a number of drugs currently on the market for the treatment of pain; none of them are 100% successful. In the authors' opinion, it is clear that new directions and modalities are needed to better address the treatment of prolonged and neuropathic pain; one drug or class clearly is not the answer for all pain therapy. Undoubtedly, there are many different phenotypes of prolonged and neuropathic pain and this should be one avenue to further develop appropriate therapies. © Informa UK, Ltd.
• Vardanyan, R. S., & Hruby, V. J. (2014). Fentanyl-related compounds and derivatives: Current status and future prospects for pharmaceutical applications. Future Medicinal Chemistry, 6(4), 385-412.
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  Abstract: Fentanyl and its analogs have been mainstays for the treatment of severe to moderate pain for many years. In this review, we outline the structural and corresponding synthetic strategies that have been used to understand the structure-biological activity relationship in fentanyl-related compounds and derivatives and their biological activity profiles. We discuss how changes in the scaffold structure can change biological and pharmacological activities. Finally, recent efforts to design and synthesize novel multivalent ligands that act as mu and delta opioid receptors and NK-1 receptors are discussed. © 2014 Future Science Ltd.
• Alleti, R., Vagner, J., Dehigaspitiya, D. C., Moberg, V. E., Elshan, N. G., Tafreshi, N. K., Brabez, N., Weber, C. S., Lynch, R. M., Hruby, V. J., Gillies, R. J., Morse, D. L., & Mash, E. A. (2013). Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors. Bioorganic and Medicinal Chemistry, 21(17), 5029-5038.
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  PMID: 23890524;PMCID: PMC3773999;Abstract: Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited K d values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported. © 2013 Elsevier Ltd. All rights reserved.
• Barkey, N. M., Preihs, C., Cornnell, H. H., Martinez, G., Carie, A., Vagner, J., Liping, X. u., Lloyd, M. C., Lynch, V. M., Hruby, V. J., Sessler, J. L., Sill, K. N., Gillies, R. J., & Morse, D. L. (2013). Development and in vivo quantitative magnetic resonance imaging of polymer micelles targeted to the melanocortin 1 receptor. Journal of Medicinal Chemistry, 56(16), 6330-6338.
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  PMID: 23863078;PMCID: PMC3807968;Abstract: Recent emphasis has focused on the development of rationally designed polymer-based micelle carriers for drug delivery. The current work tests the hypothesis that target specificity can be enhanced by micelles with cancer-specific ligands. In particular, we describe the synthesis and characterization of a new gadolinium texaphyrin (Gd-Tx) complex encapsulated in an IVECT micellar system, stabilized through Fe(III) cross-linking and targeted with multiple copies of a specific ligand for the melanocortin 1 receptor (MC1R), which has been evaluated as a cell-surface marker for melanoma. On the basis of comparative MRI experiments, we have been able to demonstrate that these Gd-Tx micelles are able to target MC1R-expressing xenograft tumors in vitro and in vivo more effectively than various control systems, including untargeted or un-cross-linked Gd-Tx micelles. Taken in concert, the findings reported herein support the conclusion that appropriately designed micelles are able to deliver contrast agent payloads to tumors expressing the MC1R. © 2013 American Chemical Society.
• Brabez, N., Nguyen, K. L., Saunders, K., Lacy, R., Liping, X. u., Gillies, R. J., Lynch, R. M., Chassaing, G., Lavielle, S., & Hruby, V. J. (2013). Synthesis and evaluation of cholecystokinin trimers: A multivalent approach to pancreatic cancer detection and treatment. Bioorganic and Medicinal Chemistry Letters, 23(8), 2422-2425.
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  PMID: 23489620;Abstract: In the quest for novel tools for early detection and treatment of cancer, we propose the use of multimers targeting overexpressed receptors at the cancer cell surface. Indeed, multimers are prone to create multivalent interactions, more potent and specific than their corresponding monovalent versions, thus enabling the potential for early detection. There is a lack of tools for early detection of pancreatic cancer, one of the deadliest forms of cancer, but CCK2-R overexpression on pancreatic cancer cells makes CCK based multimers potential markers for these cells. In this Letter, we describe the synthesis and evaluation of CCK trimers targeting overexpressed CCK2-R. © 2013 Elsevier Ltd. All rights reserved.
• Brabez, N., Saunders, K., Nguyen, K. L., Jayasundera, T., Weber, C., Lynch, R. M., Chassaing, G., Lavielle, S., & Hruby, V. J. (2013). Multivalent interactions: Synthesis and evaluation of melanotropin multimers - Tools for melanoma targeting. ACS Medicinal Chemistry Letters, 4(1), 98-102.
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  Abstract: To develop agents for early detection and selective treatment of melanomas, high affinity and high specificity molecular tools are required. Enhanced specificity may be obtained by simultaneously binding to multiple cell surface targets via the use of multimeric analogues of naturally occurring ligands. Trimers targeting overexpressed melanocortin receptors have been found to be potential candidates for this purpose. In the present letter, we describe the synthesis and study of multimers based on a dendrimer-like scaffold. The binding affinity and activity results revealed that dendrimers promote multivalent interactions via statistical and/or cooperative effects on binding. Moreover, viability studies showed no significant toxicity at micromolar concentrations, which will allow these molecular complexes to be used in vivo. Finally, imaging studies showed effective internalization for all of the molecules, confirming their potential as delivery agents. © 2012 American Chemical Society.
• Cai, M., Stankova, M., Muthu, D., Mayorov, A., Yang, Z., Trivedi, D., Cabello, C., & Hruby, V. J. (2013). An unusual conformation of γ-melanocyte-stimulating hormone analogues leads to a selective human melanocortin 1 receptor antagonist for targeting melanoma cells. Biochemistry, 52(4), 752-764.
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  PMID: 23276279;PMCID: PMC3641192;Abstract: γ-MSH (γ-melanocyte-stimulating hormone, H-Tyr-Val-Met-Gly-His- Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide is like most peptides susceptible to proteolysis in vivo, which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable ligand with specific receptor binding, we have engineered peptides by cyclizing γ-MSH using a thioether bridge. A number of novel cyclic truncated γ-MSH analogues were designed and synthesized, in which a thioether bridge was incorporated between a cysteine side chain and an N-terminal bromoacyl group. One of these peptides, cyclo-[(CH2) 3CO-Gly1-His2-d-Phe3-Arg 4-d-Trp5-Cys(S-)6]-Asp7-Arg 8-Phe9-Gly10-NH2, demonstrated potent antagonist activity and receptor selectivity for the human melanocortin 1 receptor (hMC1R) (IC50 = 17 nM). This novel peptide is the most selective antagonist for the hMC1R to date. Further pharmacological studies have shown that this peptide can specifically target melanoma cells. The nuclear magnetic resonance analysis of this peptide in a membrane-like environment revealed a new turn structure, specific to the hMC1R antagonist, at the C-terminus, where the side chain and backbone conformation of d-Trp5 and Phe9 of the peptide contribute to hMC1R selectivity. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine. © 2012 American Chemical Society.
• Hruby, V. J., & Cai, M. (2013). Design of peptide and peptidomimetic ligands with novel pharmacological activity profiles. Annual Review of Pharmacology and Toxicology, 53, 557-580.
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  PMID: 23294313;Abstract: Peptide hormones and neurotransmitters are of central importance in most aspects of intercellular communication and are involved in virtually all degenerative diseases. In this review, we discuss physicochemical approaches to the design of novel peptide and peptidomimetic agonists, antagonists, inverse agonists, and related compounds that have unique biological activity profiles, reduced toxic side effects, and, if desired, the ability to cross the blood-brain barrier. Designing ligands for specific biological and medical needs is emphasized, as is the close collaboration of chemists and biologists to maximize the chances for success. Special emphasis is placed on the use of conformational (φ-ψ space) and topographical (χ space) considerations in design. © 2013 by Annual Reviews. All rights reserved.
• Largent-Milnes, T., Brookshire, S. W., Skinner Jr., D. P., Hanlon, K. E., Giuvelis, D., Yamamoto, T., Davis, P., Campos, C. R., Nair, P., Deekonda, S., Bilsky, E. J., Porreca, F., Hruby, V. J., & Vanderah, T. W. (2013). Building a better analgesic: Multifunctional compounds that address injury-induced pathology to enhance analgesic efficacy while eliminating unwanted side effects. Journal of Pharmacology and Experimental Therapeutics, 347(1), 7-19.
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  PMID: 23860305;PMCID: PMC3781412;Abstract: The most highly abused prescription drugs are opioids used for the treatment of pain. Physician-reported drug-seeking behavior has resulted in a significant health concern among doctors trying to adequately treat pain while limiting the misuse or diversionof pain medications. In addition to abuse liability, opioid use is associated with unwanted side effects that complicate pain management, including opioid-induced emesis and constipation. This has resulted in restricting long-term doses of opioids and inadequate treatment of both acute and chronic debilitating pain, demonstrating a compelling need for novel analgesics. Recent reports indicate that adaptations in endogenous substance P/neurokinin-1 receptor (NK1) are induced by chronic pain and sustained opioid exposure, and these changes may contribute to processes responsible for opioid abuse liability, emesis, and analgesic tolerance. Here, we describe a multifunctional mu-/delta-opioid agonist/NK1 antagonist compound [Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(CF3)2 (TY027)] that has a preclinical profile of excellent antinociceptive efficacy, low abuse liability, and no opioid-related emesis or constipation. In rodent models of acute and neuropathic pain, TY027 demonstrates analgesic efficacy following central or systemic administration with a plasma half-life of more than 4 hours and central nervous system penetration. These data demonstrate that an innovative opioid designed to contest the pathology created by chronic pain and sustained opioids results in antinociceptive efficacy in rodent models, with significantly fewer side effects than morphine. Such rationally designed, multitargeted compounds are a promising therapeutic approach in treating patients who suffer from acute and chronic pain. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
• Lee, Y. S., Hongchang, Q. u., Davis, P., Ma, S., Vardanyan, R., Lai, J., Porreca, F., & Hruby, V. J. (2013). Chiral effect of a Phe residue in position 3 of the Dmt¹-l (or d)-Tic² analogues on opioid functional activities. ACS Medicinal Chemistry Letters, 4(7), 656-659.
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  Abstract: In this letter, we describe a structure-activity relationships study, specifically related to the chirality of third amino acid residue in our H-Dmt-l(or d)-Tic analogues, of which C-terminus is attached to a piperidinyl moiety. Observed selectivities and functional activities of these analogues demonstrated that the chiralities of the second and third position residues are crucial for determining whether these ligands act as antagonists or agonists at the δ opioid receptor, but not at the μ opioid receptor. © 2013 American Chemical Society.
• Mollica, A., Pinnen, F., Costante, R., Locatelli, M., Stefanucci, A., Pieretti, S., Davis, P., Lai, J., Rankin, D., Porreca, F., & Hruby, V. J. (2013). Biological active analogues of the opioid peptide biphalin: Mixed α/β³-peptides. Journal of Medicinal Chemistry, 56(8), 3419-3423.
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  PMID: 23547584;PMCID: PMC3942533;Abstract: Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-β3 amino acids. The derivative 1 containing hβ3 Phe in place of Phe showed good μ- and δ-receptor affinities (Kiδ = 0.72 nM; K iμ = 1.1 nM) and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma. © 2013 American Chemical Society.
• Munawar, M. A., Hall, G. B., Roberts, S. A., & Hruby, V. J. (2013). Tert-Butyl 4-(3,4-dichloroanilino)piperidine-1-carboxylate. Acta Crystallographica Section E: Structure Reports Online, 69(2), o205.
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  PMID: 23424488;PMCID: PMC3569265;Abstract: In the title compound, C16H22Cl2N2O 2, the substituted piperidine ring adopts a chair conformation with both substituents in equatorial positions. In the crystal, N - H⋯O and C - H⋯O hydrogen bonds connect molecules into ribbons along the a-axis direction.
• Nair, P., Yamamoto, T., Largent-Milnes, T. M., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2013). Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic and Medicinal Chemistry Letters, 23(17), 4975-4978.
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  PMID: 23899615;PMCID: PMC3810412;Abstract: The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr1-DAla2-Gly3-Phe 4-Gly5-Trp6-O-[3′,5′-Bzl(CF 3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo. © 2013 Elsevier Ltd. All rights reserved.
• Petrov, R. R., Lee, Y. S., Vardanyan, R. S., Liu, L., Ma, S., Davis, P., Lai, J., Porreca, F., Vanderah, T. W., & Hruby, V. J. (2013). Effect of anchoring 4-anilidopiperidines to opioid peptides. Bioorganic and Medicinal Chemistry Letters, 23(11), 3434-3437.
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  PMID: 23623418;PMCID: PMC3942532;Abstract: We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides. © 2013 Elsevier Ltd. All rights reserved.
• Podolsky, A. T., Sandweiss, A., Jackie, H. u., Bilsky, E. J., Cain, J. P., Kumirov, V. K., Lee, Y. S., Hruby, V. J., Vardanyan, R. S., & Vanderah, T. W. (2013). Novel fentanyl-based dual μ/δ-opioid agonists for the treatment of acute and chronic pain. Life Sciences, 93(25-26), 1010-1016.
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  PMID: 24084045;Abstract: Approximately one third of the adult U.S. population suffers from some type of on-going, chronic pain annually, and many more will have some type of acute pain associated with trauma or surgery. First-line therapies for moderate to severe pain include prescriptions for common mu opioid receptor agonists such as morphine and its various derivatives. The epidemic use, misuse and diversion of prescription opioids have highlighted just one of the adverse effects of mu opioid analgesics. Alternative approaches include novel opioids that target delta or kappa opioid receptors, or compounds that interact with two or more of the opioid receptors. Aims: Here we report the pharmacology of a newly synthesized bifunctional opioid agonist (RV-Jim-C3) derived from combined structures of fentanyl and enkephalin in rodents. RV-Jim-C3 has high affinity binding to both mu and delta opioid receptors. Main methods: Mice and rats were used to test RV-Jim-C3 in a tailflick test with and without opioid selective antagonist for antinociception. RV-Jim-C3 was tested for anti-inflammatory and antihypersensitivity effects in a model of formalin-induced flinching and spinal nerve ligation. To rule out motor impairment, rotarod was tested in rats. Key findings: RV-Jim-C3 demonstrates potent-efficacious activity in several in vivo pain models including inflammatory pain, antihyperalgesia and antiallodynic with no significant motor impairment. Significance This is the first report of a fentanyl-based structure with delta and mu opioid receptor activity that exhibits outstanding antinociceptive efficacy in neuropathic pain, reducing the propensity of unwanted side effects driven by current therapies that are unifunctional mu opioid agonists. © 2013 Elsevier Inc. All rights reserved.
• Rinne, P., Nordlund, W., Heinonen, I., Penttinen, A., Saraste, A., Ruohonen, S. T., Mäkelä, S., Vähätalo, L., Kaipio, K., Cai, M., Hruby, V. J., Ruohonen, S., & Savontaus, E. (2013). α-Melanocyte-stimulating hormone regulates vascular NO availability and protects against endothelial dysfunction. Cardiovascular Research, 97(2), 360-368.
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  PMID: 23131503;PMCID: PMC3543993;Abstract: Aimsα-Melanocyte-stimulating hormone (α-MSH), derived from the precursor molecule pro-opiomelanocortin, exerts potent anti-inflammatory actions in the vasculature, but its role in circulatory regulation remains unclear. Therefore, we sought to investigate whether α-MSH could regulate the local control of blood vessel tone.Methods and resultsUsing in vivo and ex vivo methods to assess vascular reactivity, we found that α-MSH improved endothelium-dependent vasodilatation in the mouse aorta and coronary circulation without directly contracting or relaxing blood vessels. α-MSH promoted vasodilatation by enhancing endothelial nitric oxide (NO) formation and by improving sensitivity to endothelium-independent blood vessel relaxation. Using cultured human endothelial cells to elucidate the involved molecular mechanisms, we show that α-MSH increased the expression and phosphorylation of endothelial NO synthase in these cells. The observed effects were regulated by melanocortin 1 (MC1) receptors expressed in the endothelium. In keeping with the vascular protective role of α-MSH, in vivo treatment with stable analogues of α-MSH ameliorated endothelial dysfunction associated with aging and diet-induced obesity in mice.ConclusionThe present study identifies α-MSH and endothelial MC1 receptors as a new signalling pathway contributing to the regulation of NO availability and vascular function. These findings suggest applicability of α-MSH analogues for therapeutic use in pathological conditions that are characterized by vascular dysfunction. © 2013 The Author.
• Liping, X. u., Josan, J. S., Vagner, J., Caplan, M. R., Hruby, V. J., Mash, E. A., Lynch, R. M., Morse, D. L., & Gillies, R. J. (2012). Heterobivalent ligands target cell-surface receptor combinations in vivo. Proceedings of the National Academy of Sciences of the United States of America, 109(52), 21295-21300.
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  PMID: 23236171;PMCID: PMC3535626;Abstract: A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and speci fi city. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantifi ed by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.
• Liu, J., Ying, J., T., V., Hruby, V. J., & Satyanarayanajois, S. D. (2012). Erratum: Structure-activity studies of peptides from the "hot-spot" region of human CD2 protein: Development of peptides for immunomodulation (Journal of Medicinal Chemistry (2005) 48 (6236-6249) DOI:10.1021/jm0503547). Journal of Medicinal Chemistry, 55(16), 7295-.
• Liu, Z., Mehta, S. J., & Hruby, V. J. (2012). Strategies for asymmetric synthesis of amino acids withγ,δ -Unsaturation. Organic Preparations and Procedures International, 44(3), 222-255.
• Liu, Z., Mehta, S. J., Lee, K., Grossman, B., Hongchang, Q. u., Xuyuan, G. u., Nichol, G. S., & Hruby, V. J. (2012). Thio-Claisen rearrangement used in preparing anti-β-functionalized γ,δ-unsaturated amino acids: Scope and limitations. Journal of Organic Chemistry, 77(3), 1289-1300.
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  PMID: 22283513;Abstract: Multifunctionalized amino acids, especially amino acids with unsaturation, are important, demanding building blocks in peptide chemistry. Here we present a summary of our most recent study using the thio-Claisen rearrangement for the synthesis of anti-β-functionalized γ,δ-unsaturated amino acids. Investigations on scope, limitations, chemoselectivities and stereoselectivities regarding an FeBr 3-catalyzed allylation strategy and a thio-enolate dianion formation strategy for asymmetric thio-Claisen rearrangement are documented. An explanation of the chirality crossover observed between the Eschenmoser-Claisen rearrangement and the thio-Claisen rearrangement is proposed. Novel optically active N α-protected amino acids with biologically interesting functional groups were prepared for the first time. © 2011 American Chemical Society.
• Mollica, A., Pinnen, F., Stefanucci, A., Feliciani, F., Campestre, C., Mannina, L., Sobolev, A. P., Lucente, G., Davis, P., Lai, J., Ma, S., Porreca, F., & Hruby, V. J. (2012). The cis-4-amino-l-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues. Journal of Medicinal Chemistry, 55(7), 3027-3035.
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  PMID: 22394120;Abstract: Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH 2) is an endogenous tetrapeptide that combines potency and efficacy with high affinity and selectivity toward the μ opioid receptor, the most responsible for analgesic effects in the central nervous system. The presence of the Pro 2 represents a crucial factor for the ligand structural and conformational properties. Proline is in fact an efficient stereochemical spacer, capable of inducing favorable spatial orientation of aromatic rings, a key factor for ligand recognition and interaction with receptors. Here the Pro 2 has been replaced by 4(S)-NH 2-2(S)-proline (cAmp), a proline/GABA cis-chimera residue. This bivalent amino acid maintains the capacity to influenc the tetrapeptide conformation and offers the opportunity to generate new linear models and unusually constrained cyclic analogues characterized by an N-terminal Tyr bearing a free α-amino group. The results indicate that the new analogues do not show affinity for both δ and κ opioid receptors and bind only poorly to the μ receptors (for cyclopeptide 9: K iμ = 660 nM; GPI (IC 50) = 1.4% at 1 μM; for linear tetrapeptide acid 13: K iμ = 2000 nM; GPI (IC 50) = 0% at 1 μM; for linear tetrapeptide amide 15: K iμ = 310 nM; GPI (IC 50) = 894 nM). © 2012 American Chemical Society.
• Mollica, A., Pinnen, F., Stefanucci, A., Mannina, L., Sobolev, A. P., Lucente, G., Davis, P., Lai, J., Ma, S., Porreca, F., & Hruby, V. J. (2012). Cis-4-amino-l-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues: Part 2. Journal of Medicinal Chemistry, 55(19), 8477-8482.
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  PMID: 22966938;Abstract: Recently, we reported synthesis and activity of a constrained cyclic analogue of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH2) and related linear models containing the cis-4-amino-l-proline (cAmp) in place of native Pro2. In the present article, the adopted rationale is the possible modulation of the receptor affinity of the cAmp containing EM-2 analogues by assigning a different stereochemistry to the Phe3 and Phe4 residues present in the ring. Thus, eight more analogues with different absolute configuration at the chiral center of the aromatic residues in positions 3 and 4 have been synthesized and their opioid activity examined. The stereochemical change at the α-carbon atoms leads to a meaningful enhancement of the affinity and activity toward μ opioid receptors with respect to the prototype compound 9: e.g., 9a, Kiμ = 63 nM, GPI (IC50) = 480 nM; 9b, Kiμ = 38 nM, GPI (IC50) = 330 nM. © 2012 American Chemical Society.
• Tumati, S., Largent-Milnes, T. M., Keresztes, A. I., Yamamoto, T., Vanderah, T. W., Roeske, W. R., Hruby, V. J., & Varga, E. V. (2012). Tachykinin NK ₁ receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation. European Journal of Pharmacology, 684(1-3), 64-70.
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  PMID: 22724132;PMCID: PMC3565540;Abstract: Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK 1 (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK 1 receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK 1 receptor antagonist (N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection)) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK 1 antagonist pharmacophore yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK 1 receptor antagonists may provide a novel paradigm for long-term pain management. © 2012 Elsevier B.V. All rights reserved.
• Barkey, N. M., Tafreshi, N. K., Josan, J. S., R., C., Sill, K. N., Hruby, V. J., Gillies, R. J., Morse, D. L., & Vagner, J. (2011). Development of melanoma-targeted polymer micelles by conjugation of a melanocortin 1 receptor (MC1R) specific ligand. Journal of Medicinal Chemistry, 54(23), 8078-8084.
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  PMID: 22011200;PMCID: PMC3302579;Abstract: The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH 2) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity. © 2011 American Chemical Society.
• Brabez, N., Lynch, R. M., Liping, X. u., Gillies, R. J., Chassaing, G., Lavielle, S., & Hruby, V. J. (2011). Design, synthesis, and biological studies of efficient multivalent melanotropin ligands: Tools toward melanoma diagnosis and treatment. Journal of Medicinal Chemistry, 54(20), 7375-7384.
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  PMID: 21928837;PMCID: PMC3521580;Abstract: To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms. © 2011 American Chemical Society.
• Cai, M., Liu, Z., Hongchang, Q. u., Fan, H., Zheng, Z., & Hruby, V. J. (2011). Utilize conjugated melanotropins for the earlier diagnosis and treatment of melanoma. European Journal of Pharmacology, 660(1), 188-193.
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  PMID: 21315067;PMCID: PMC3095766;Abstract: Peptides serve as effective drugs and contrast agents in the clinic today. However the inherent drawbacks of peptide structures can limit their efficacy as drugs. To overcome this we have been developing new methods to create 'tailor-made' peptides and peptide mimetics with improved pharmacological and physical properties. In this work we introduce novel peptide and small molecule conjugated molecules for earlier diagnosis and treatment of melanoma.
• Grieco, P., Brancaccio, D., Novellino, E., Hruby, V. J., & Carotenuto, A. (2011). Conformational study on cyclic melanocortin ligands and new insight into their binding mode at the MC4 receptor. European Journal of Medicinal Chemistry, 46(9), 3721-3733.
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  PMID: 21652123;Abstract: The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. Understanding the basis of the ligand-receptor interactions is crucial for the design of potent and selective ligands for these receptors. The conformational preferences of the cyclic melanocortin ligands MTII (Ac-Nle 4-c[Asp 5-His 6-DPhe 7-Arg 8-Trp 9-Lys 10]-NH 2) and SHU9119 (Ac-Nle 4-c[Asp 5-His 6-DNal(2′) 7-Arg 8-Trp 9-Lys 10]-NH 2), which show agonist and antagonist activity at the h-MC4R, respectively, were comprehensively investigated by solution NMR spectroscopy in different environments. In particular, water and water/DMSO (8:2) solutions were used as isotropic solutions and an aqueous solution of DPC (dodecylphosphocholine) micelles was used as a membrane mimetic environment. NMR-derived conformations of these two ligands were docked within h-MC4R models. NMR and docking studies revealed intriguing differences which can help explain the different activities of these two ligands. © 2011 Elsevier Masson SAS. All rights reserved.
• Hanlon, K. E., Herman, D. S., Agnes, R. S., Largent-Milnes, T. M., Kumarasinghe, I. R., Ma, S. W., Guo, W., Lee, Y., Ossipov, M. H., Hruby, V. J., Lai, J., Porreca, F., & Vanderah, T. W. (2011). Novel peptide ligands with dual acting pharmacophores designed for the pathophysiology of neuropathic pain. Brain Research, 1395, 1-11.
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  PMID: 21550594;PMCID: PMC3105124;Abstract: The conventional design of high affinity drugs targeted to a single molecule has not resulted in clinically useful therapies for pain relief. Recent reviews have suggested that newly designed analgesic drugs should incorporate multiple targets. The distributions of cholecystokinin (CCK) and CCK receptors in the central nervous system (CNS) overlap significantly with endogenous opioid systems and can be dually targeted. CCK has been shown to act as an endogenous "anti-analgesic" peptide and neuropathic pain conditions promote endogenous CCK release in CNS regions of pain modulation. Administration of CCK into nuclei of the rostral ventromedial medulla induces pronociceptive behaviors in rats. RSA 504 and RSA 601 are novel bifunctional compounds developed to target neuropathic pain by simultaneously acting as agonists at two distinct opioid receptors and antagonizing CCK receptors in the CNS. RSA 504 and RSA 601 demonstrate agonist activity in vitro and antihypersensitivity to mechanical and thermal stimuli in vivo using the spinal nerve ligation model of neuropathic pain. Intrathecal administration of RSA 504 and RSA 601 did not demonstrate antinociceptive tolerance over 7 days of administration and did not display motor impairment or sedation using a rotarod. These are the first behavioral studies that demonstrate how multi-targeted molecule design can address the pathology of neuropathic pain. These compounds with δ and μ opioid agonist activity and CCK antagonist activity within one molecule offer a novel approach with efficacy for neuropathic pain while lacking the side effects typically caused by conventional opioid therapies. © 2011 Elsevier B.V. All rights reserved.
• Hruby, V. J. (2011). Collaborations Between Chemists and Biologists. Collaborative Computational Technologies for Biomedical Research, 113-120.
• Hruby, V. J., Cai, M., Cain, J., Nyberg, J., & Trivedi, D. (2011). Design of novel melanocortin receptor ligands: Multiple receptors, complex pharmacology, the challenge. European Journal of Pharmacology, 660(1), 88-93.
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  PMID: 21208601;PMCID: PMC3138524;Abstract: The major pharmacophore for the melanocortin 1, 3, 4 and 5 receptors is the sequence -His-Phe-Arg-Trp-. There is a need for potent, biologically stable, receptor selective ligands, both agonists and antagonists, for these receptors. In this report we briefly examine the structural and biophysical approaches we have taken to develop selective agonist and antagonist ligands that can cross (or not) the blood brain barrier. Remaining questions and unmet needs are also discussed.
• Hruby, V. J., Cai, M., Nyberg, J., & Muthu, D. (2011). Approaches to the rational design of selective melanocortin receptor antagonists. Expert Opinion on Drug Discovery, 6(5), 543-557.
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  PMID: 22646078;Abstract: Introduction: When establishing the physiological roles of specific receptors in normal and disease states, it is critical to have selective antagonist ligands for each receptor in a receptor system with several subtypes. The melanocortin receptors have five subtypes referred to as the melanocortin 1 receptor, melanocortin 2 receptor, melanocortin 3 receptor, melanocortin 4 receptor and melanocortin 5 receptor, and they are of critical importance for many aspects of human health and disease. Areas covered: This article reviews the current efforts to design selective antagonistic ligands for the five human melanocortin receptors summarizing the currently published orthosteric and allosteric antagonists for each of these receptors. Expert opinion: Though there has been progress, there are still few drugs available that address the many significant biological activities and diseases that are associated with these receptors, which is possibly due to the lack of receptor selectivity that these designed ligands are currently showing. The authors believe that further studies into the antagonists' 3D conformational and topographical properties in addition to future mutagenesis studies will provide greater insight into these ligands which could play a role in the treatment of various diseases in the future. © 2011 Informa UK, Ltd.
• Josan, J. S., Handl, H. L., Sankaranarayanan, R., Liping, X. u., Lynch, R. M., Vagner, J., Mash, E. A., Hruby, V. J., & Gillies, R. J. (2011). Cell-specific targeting by heterobivalent ligands. Bioconjugate Chemistry, 22(7), 1270-1278.
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  PMID: 21639139;PMCID: PMC3381984;Abstract: Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach - to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle 4, dPhe 7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20 - 50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH 2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. © 2011 American Chemical Society.
• Lee, Y. S., Kulkarani, V., Cowell, S. M., Ma, S., Davis, P., Hanlon, K. E., Vanderah, T. W., Lai, J., Porreca, F., Vardanyan, R., & Hruby, V. J. (2011). Development of potent μ and δ opioid agonists with high lipophilicity. Journal of Medicinal Chemistry, 54(1), 382-386.
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  PMID: 21128594;PMCID: PMC3136578;Abstract: An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-ylpropionamide moiety at the C-terminal was performed and has resulted in highly potent ligands at μ and δ opioid receptors. In general, ligands with the substitution of d-Nle2 and halogenation of the aromatic ring of Phe4 showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay. © 2010 American Chemical Society.
• Mayorov, A. V., Cai, M., Palmer, E. S., Tanaka, D. K., Cain, J. P., Dedek, M. M., Tan, B., Trivedi, D., & Hruby, V. J. (2011). Cyclic lactam hybrid α-MSH/Agouti-related protein (AGRP) analogues with nanomolar range binding affinities at the human melanocortin receptors. Bioorganic and Medicinal Chemistry Letters, 21(10), 3099-3102.
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  PMID: 21486697;PMCID: PMC3216836;Abstract: A novel hybrid melanocortin pharmacophore was designed based on the topographical similarities between the pharmacophores of Agouti related protein (AGRP) an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. When employed in two different 23-membered macrocyclic lactam peptide templates, the designed hybrid AGRP/MSH pharmacophore yielded non-competitive ligands with nanomolar range binding affinities. The topography-based pharmacophore hybridization strategy will prove useful in development of unique non-competitive melanocortin receptor modulators. © 2011 Elsevier Ltd. All rights reserved.
• Mollica, A., Pinnen, F., Feliciani, F., Stefanucci, A., Lucente, G., Davis, P., Porreca, F., Ma, S., Lai, J., & Hruby, V. J. (2011). New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers.. Amino acids, 40(5), 1503-1511.
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  PMID: 20924622;Abstract: We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4' positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-L-phenylalanine residues at 4 and 4' positions. Binding values are: Kμ(i)=0.51 nM and Kδ(i)=12.8 nM for compound 9, Kμ(i)=0.09 nM and Kδ(i)=0.11 nM for analogue 10.
• Vardanyan, R. S., Danagulyan, G. G., Mkrtchyan, A. D., & Hruby, V. J. (2011). Recyclization reactions of 1-alkylpyrimidinium salts. Heterocyclic Communications, 17(3-4), 129-133.
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  Abstract: The reaction of 4-amino-2-benzyl-1-methyl-5-ethoxycarbonylpyrimidinium iodide ( 3 ) with alcoholic methylamine resulted in the formation of the methylimine of 2-amino-4- hydroxy-6-methylamino-5-phenylpyridine-3-carbaldehyde ( 5 ). Heating of the same pyrimidinium salt in benzylamine gave a mixture of products of two C - C recyclizations: 2-benzyl-4-benzylamino-5- carbamoylpyrimidine ( 7 ) and the benzylimine of 4-amino-2-benzyl-6- benzylaminopyrimidine- 5-carbaldehyde ( 8 ). The reaction of 2-amino-1,4-dimethyl-5- ethoxycarbonylpyrimidinium iodide ( 10 ) with KOH ethanolic solution gave a single product of C - C-recyclization: 2-amino- 5-acetyl-4-hydroxypyrimidine ( 11 ). © by Walter de Gruyter.
• Vardanyan, R., Kumirov, V. K., & Hruby, V. J. (2011). Improved synthesis of d,l-fluorocitric acid. Journal of Fluorine Chemistry, 132(11), 920-924.
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  Abstract: We propose a decagram synthesis of commercially unavailable fluorocitric acid. The synthesis begins with benzyl fluoroacetate, which is converted to dibenzyl 2-fluoro-3-oxosuccinate, followed by condensation with malonic acid or its monobenzyl ester and subsequent hydrogenolysis. © 2011 Published by Elsevier B.V. All rights reserved.
• Vardanyan, R., Kumirov, V. K., Nichol, G. S., Davis, P., Liktor-Busa, E., Rankin, D., Varga, E., Vanderah, T., Porreca, F., Lai, J., & Hruby, V. J. (2011). Synthesis and biological evaluation of new opioid agonist and neurokinin-1 antagonist bivalent ligands. Bioorganic and Medicinal Chemistry, 19(20), 6135-6142.
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  PMID: 21925887;Abstract: Newly designed bivalent ligands - opioid agonist/NK1-antagonists have been synthesized. The synthesis of new starting materials - carboxy-derivatives of Fentanyl (1a-1c) was developed. These products have been transformed to 'isoimidium perchlorates' (2a-c). The new isoimidium perchlorates have been successfully implemented in nucleophilic addition reactions, with l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester to give the target compounds - amides (3a-c). Perchlorates (2a-c) successfully undergo reactions with other nucleophiles such as alcohols, amines or hydrazines. The obtained compound 3b exhibited μ-opioid agonist activity and NK1-antagonist activity and may serve as a useful lead compound for the further design of a new series of opioid agonist/NK1-antagonist compounds. © 2011 Elsevier Ltd. All rights reserved.
• Yamamoto, T., Nair, P., Largent-Milnes, T. M., Jacobsen, N. E., Davis, P., Ma, S., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2011). Discovery of a potent and efficacious peptide derivative for δ/μ opioid agonist/neurokinin 1 antagonist activity with a 2′,6′- Dimethyl- l -tyrosine: In vitro, in vivo, and NMR-based structural studies. Journal of Medicinal Chemistry, 54(7), 2029-2038.
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  PMID: 21366266;PMCID: PMC3090346;Abstract: Multivalent ligands with δ/μ opioid agonist and NK1 antagonist activities have shown promising analgesic potency without detectable sign of toxicities, including motor skill impairment and opioid-induced tolerance. To improve their biological activities and metabolic stability, structural optimization was performed on our peptide-derived lead compounds by introducing 2′,6′-dimethyl-l-tyrosine (Dmt) instead of Tyr at the first position. The compound 7 (Dmt-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3′, 5′-(CF3)2-Bzl]) showed improved multivalent bioactivities compared to those of the lead compounds, had more than 6 h half-life in rat plasma, and had significant antinociceptive efficacy in vivo. The NMR structural analysis suggested that Dmt1 incorporation in compound 7 induces the structured conformation in the opioid pharmacophore (N-terminus) and simultaneously shifts the orientation of the NK1 pharmacophore (C-terminus), consistent with its affinities and activities at both opioid and NK1 receptors. These results indicate that compound 7 is a valuable research tool to seek a novel analgesic drug. © 2011 American Chemical Society.
• Zhijun, W. u., & Hruby, V. J. (2011). Backbone alignment modeling of the structure-activity relationships of opioid ligands. Journal of Chemical Information and Modeling, 51(5), 1151-1164.
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  PMID: 21488692;Abstract: Opioid studies are an important area of modern medicinal chemistry research. In this study we have provided innovative considerations to some long-standing problems in opioid studies, specifically the opioid pharmacophore and the potential binding modes of opioid ligands. Based on a new peptide backbone-alignment concept that we have developed along with this study, we discuss a wide variety of opioid ligands with respect to their structure - activity relationships. (Figure presented). © 2011 American Chemical Society.
• Doedens, L., Opperer, F., Cai, M., Beck, J. G., Dedek, M., Palmer, E., Hruby, V. J., & Kessler, H. (2010). Multiple N -methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: Pharmacological and conformational studies. Journal of the American Chemical Society, 132(23), 8115-8128.
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  PMID: 20496895;PMCID: PMC2895553;Abstract: Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation that resembles other α-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains. © 2010 American Chemical Society.
• Giordano, C., Sansone, A., Masi, A., Lucente, G., Punzi, P., Mollica, A., Pinnen, F., Feliciani, F., Cacciatore, I., Davis, P., Lai, J., Ma, S., Porreca, F., & Hruby, V. (2010). Synthesis and activity of endomorphin-2 and morphiceptin analogues with proline surrogates in position 2. European Journal of Medicinal Chemistry, 45(10), 4594-4600.
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  PMID: 20692738;Abstract: The opioid agonists endomorphins (Tyr-Pro-Trp-Phe-NH2; EM1 and Tyr-Pro-Phe-Phe-NH2; EM2) and morphiceptin (Tyr-Pro-Phe-Pro-NH 2) exhibit an extremely high selectivity for μ-opioid receptor. Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2 the S and R residues of β-homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs) and of 3- pyrrolidinesulphonic acid (βPrs) have been synthesized and their binding affinity and functional activity have been investigated. The highest μ-receptor affinity is shown by [(S)βPrs2]EM2 analogue (6e) which represents the first example of a β-sulphonamido analogue in the field of opioid peptides. © 2010 Elsevier Masson SAS. All rights reserved.
• Hruby, V. J., Alves, I., Cowell, S., Salamon, Z., & Tollin, G. (2010). Use of plasmon waveguide resonance (PWR) spectroscopy for examining binding, signaling and lipid domain partitioning of membrane proteins. Life Sciences, 86(15-16), 569-574.
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  PMID: 19281827;Abstract: Aims: Due to their anisotropic properties and other factors, it has been difficult to determine the conformational and dynamic properties of integral membrane proteins such as G-protein coupled receptors (GPCRs), growth factor receptors, ion channels, etc. in response to ligands and subsequent signaling. Herein a novel methodology is presented that allows such studies to be performed while maintaining the receptors in a membrane environment. Main method: Plasmon waveguide resonance (PWR) spectroscopy is a relatively new biophysical method which allows one to directly observe structural and dynamic changes which occur on interaction of GPCRs (and other integral membrane proteins) with ligands and signaling molecules. The delta opioid receptor (DOR) and its ligands serve as an excellent model system to illustrate the new insights into GPCR signaling that can be obtained by this method. Key findings: Among our key findings are: 1) it is possible to obtain the following information directly and without any need for labels (radioactive, fluorescent, etc.): binding affinities, and the ability to distinguish between agonists, antagonists, inverse agonist, and partial agonists without a need for second messenger analysis; 2) it is possible to determine directly, again without a need for labels, G-protein binding to variously occupied or unoccupied DORs, and to determine which α-subtype is involved in allowing structurally different agonist ligands to have differential effects; 3) GTPγS binding can be examined directly; and 4) binding of the DOR with different ligands leads to differential segregation of the ligand-receptor complex into lipid rafts. Significance: The implications of these discoveries suggest a need to modify our current views of GPCR-ligand interactions and signaling. © 2009 Elsevier Inc.
• Juni, A., Cai, M., Stankova, M., Waxman, A. R., Arout, C., Klein, G., Dahan, A., Hruby, V. J., Mogil, J. S., & Kest, B. (2010). Sex-specific mediation of opioid-induced hyperalgesia by the melanocortin-1 receptor. Anesthesiology, 112(1), 181-188.
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  PMID: 19996949;Abstract: Background: N-Methyl-d-aspartate receptor antagonists reverse hyperalgesia during morphine infusion in male mice only. Because the melanocortin-1 receptor can act as a female-specific counterpart to N-methyl-d-aspartate receptors in κ-opioid analgesic mechanisms, the authors assessed the contribution of melanocortin-1 receptors to the sex-specific mechanisms underlying morphine hyperalgesia. Methods: The tail-withdrawal test was used to compare the nociceptive responses of male and female C57BL/6J (B6) mice with those of C57BL/6J-Mc1r (e/e) mice, spontaneous mutants of the B6 Background lacking functional melanocortin-1 receptors, during continuous morphine infusion (1.6 and 40.0 mgkg • 24 h). Separate groups of hyperalgesic B6 and outbred CD-1 mice were injected with MK-801 or MSG606, selective N-methyl-d-aspartate and melanocortin-1 receptor antagonists, respectively. Results: Morphine infusion (40.0 mg • kg • 24 h) reduced baseline withdrawal latencies by 45-55% in B6 mice of both sexes, indicating hyperalgesia; this increased nociception was manifest in male e/e mice only. Although MK-801 reversed hyperalgesia in male mice only, increasing latencies by 72%, MSG606 increased latencies by approximately 60% exclusively in females. A lower morphine infusion dose (1.6 mg • kg • 24 h) reduced baseline withdrawal latencies by 45-52% in B6 and e/e mice of both sexes, which was reversed by MK-801, but not MSG606, in both male and female B6 mice. Conclusions: The data indicate the sex-specific mediation of high-dose morphine-induced hyperalgesia by N-methyl-d-aspartate and melanocortin-1 receptors in male and female mice, respectively, suggesting a broader relevance of this known sexual dimorphism. The data further indicate that the neural substrates contributing to hyperalgesia are morphine dose-dependent. © 2010 American Society of Anesthesiologists, Inc.
• Largent-Milnes, T., Yamamoto, T., Nair, P., Moulton, J. W., Hruby, V. J., Lai, J., Porreca, F., & Vanderah, T. W. (2010). Spinal or systemic TY005, a peptidic opioid agonist/neurokinin 1 antagonist, attenuates pain with reduced tolerance. British Journal of Pharmacology, 161(5), 986-1001.
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  PMID: 20977451;PMCID: PMC2998681;Abstract: BACKGROUND AND PURPOSE The use of opioids in treating pain is limited due to significant side effects including somnolence, constipation, analgesic tolerance, addiction and respiratory depression. Pre-clinical studies have shown that neurokinin 1 (NK 1) receptor antagonists block opioid-induced antinociceptive tolerance and may inhibit opioid-induced rewarding behaviours. Here, we have characterized a bifunctional peptide with both opioid agonist and NK 1 antagonist pharmacophores in a rodent model of neuropathic pain. EXPERIMENTAL APPROACH Rats were evaluated for behavioural responses to both tactile and thermal stimuli in either an uninjured, sham- or nerve-injured state. TY005 (Tyr-DAla-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bn(CF 3) 2) was delivered spinally or systemically to assess the antinociceptive effects after acute exposure. Motor skills were evaluated using the rotarod test to determine potential sedative effects. Spinal TY005 was given chronically to sham- or nerve-injured animals to determine the development of tolerance. KEY RESULTS Bolus injections of TY005 produced dose-dependent antinociception in non-injured animals and alleviated nerve injury-induced thermal and tactile hypersensitivities (i.e. antihyperalgesia) more effectively than morphine. Sedative effects were not evident from the rotarod test at doses that were antihyperalgesic, nor at doses threefold higher. Repeated administration of TY005 did not lead to the development of antihyperalgesic tolerance or alter sensory thresholds. CONCLUSIONS AND IMPLICATIONS Collectively, the data suggest that opioid agonist/NK 1 antagonist bifunctional peptides represent a promising novel approach to the management of chronic pain without the development of tolerance, reducing the need for escalation of doses and unwanted side effects associated with opiates alone. © 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.
• Lee, Y. S., Fernandes, S., Kulkarani, V., Mayorov, A., Davis, P., Ma, S., Brown, K., Gillies, R. J., Lai, J., Porreca, F., & Hruby, V. J. (2010). Design and synthesis of trivalent ligands targeting opioid, cholecystokinin, and melanocortin receptors for the treatment of pain. Bioorganic and Medicinal Chemistry Letters, 20(14), 4080-4084.
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  PMID: 20547453;PMCID: PMC2917332;Abstract: It has been known that co-administration of morphine with either cholecystokinin (CCK) receptor or melanocortin (MC) receptor antagonists enhance morphine's analgesic efficacy by reducing serious side effects such as tolerance and addiction. 1-4 Considering these synergistic effects, we have designed trivalent ligands in which all three different pharmacophores for opioid, CCK, and MC receptors are combined in such a way as to conserve their own topographical pharmacophore structures. These ligands, excluding the cyclic compound, were synthesized by solid phase synthesis using Rink-amide resin under microwave assistance in very high yields. These trivalent ligands bind to their respective receptors well demonstrating that the topographical pharmacophore structures for the three receptors were retained for receptor binding. Ligand 10 was a lead compound to show the best biological activities at all three receptors. © 2010 Elsevier Ltd. All rights reserved.
• Liping, X. u., Vagner, J., Alleti, R., Rao, V., Jagadish, B., Morse, D. L., Hruby, V. J., Gillies, R. J., & Mash, E. A. (2010). Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors. Bioorganic and Medicinal Chemistry Letters, 20(8), 2489-2492.
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  PMID: 20304640;PMCID: PMC2857964;Abstract: A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low μM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH2, exhibited a Kd for hMC4R of 9.1 ± 1.4 μM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-α-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects. © 2010 Elsevier Ltd. All rights reserved.
• Liu, Z., Hongchang, Q. u., Xuyuan, G. u., Lee, K., Grossman, B., Kumirov, V. K., & Hruby, V. J. (2010). Novel anti-β-functionalized γ,δ-unsaturated amino acids via a thio-Claisen rearrangement. Tetrahedron Letters, 51(27), 3518-3520.
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  Abstract: A significantly improved thio-Claisen rearrangement method was developed for preparing anti-β-functionalized γ,δ-unsaturated amino acids, which are extremely useful nonproteinogenic amino acids used in chemistry and biology research. The mild reaction condition successfully introduced base labile functional groups into the amino acids with excellent anti/syn selectivities. © 2010 Elsevier Ltd. All rights reserved.
• Mayorov, A. V., Cai, M., Palmer, E. S., Liu, Z., Cain, J. P., Vagner, J., Trivedi, D., & Hruby, V. J. (2010). Solid-phase peptide head-to-side chain cyclodimerization: Discovery of C₂-symmetric cyclic lactam hybrid α-melanocyte-stimulating hormone (MSH)/agouti-signaling protein (ASIP) analogues with potent activities at the human melanocortin receptors. Peptides, 31(10), 1894-1905.
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  PMID: 20688117;PMCID: PMC3041174;Abstract: A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the agouti-signaling protein (ASIP), an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide lactam cyclodimerization yielded nanomolar range (25-120 nM) hMC1R-selective full and partial agonists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the development of novel GPCR peptide ligands. © 2010 Elsevier Inc. All rights reserved.
• Morse, D. L., Balagurunathan, Y., Hostetter, G., Trissal, M., Tafreshi, N. K., Burke, N., Lloyd, M., Enkemann, S., Coppola, D., Hruby, V. J., Gillies, R. J., & Han, H. (2010). Identification of novel pancreatic adenocarcinoma cell-surface targets by gene expression profiling and tissue microarray. Biochemical Pharmacology, 80(5), 748-754.
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  PMID: 20510208;PMCID: PMC2914681;Abstract: Pancreatic cancer has a high mortality rate, which is generally related to the initial diagnosis coming at late stage disease combined with a lack of effective treatment options. Novel agents that selectively detect pancreatic cancer have potential for use in the molecular imaging of cancer, allowing for non-invasive determination of tumor therapeutic response and molecular characterization of the disease. Such agents may also be used for the targeted delivery of therapy to tumor cells while decreasing systemic effects. Using complementary assays of mRNA expression profiling to determine elevated expression in pancreatic cancer tissues relative to normal pancreas tissues, and validation of protein expression by immunohistochemistry on tissue microarray, we have identified cell-surface targets with potential for imaging and therapeutic agent development. Expression profiles of 2177 cell-surface genes for 28 pancreatic tumor specimens and 4 normal pancreas tissue samples were evaluated. Expression in normal tissues was evaluated using array data from 103 samples representing 28 organ sites as well as mining published data. One-hundred seventy unique targets were highly expressed in 2 or more of the pancreatic tumor specimens and were not expressed in the normal pancreas samples. Two targets (TLR2 and ABCC3) were further validated for protein expression by tissue microarray (TMA) based immunohistochemistry. These validated targets have potential for the development of diagnostic imaging and therapeutic agents for pancreatic cancer. © 2010 Elsevier Inc.
• Nichol, G. S., Kumirov, V. K., Vardanyan, R., & Hruby, V. J. (2010). Proton sharing and transfer in some zwitterionic compounds based on 4-oxo-4-((1-phenethylpiperidin-4-yl)(phenyl)amino)alcanoic acids. CrystEngComm, 12(11), 3651-3657.
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  Abstract: Three compounds, each derived from Fentanyl and differing essentially only in the length of a carboxylic acid chain, were synthesized and yielded four crystal structures three of which share several structural similarities, including the length of the chain, while the fourth, with a shorter chain, is quite different. The chain length has a significant influence on the crystal structures formed. The 'three atom' chain compounds are all solvated zwitterions which feature a hydrogen-bonded 'dimer' between adjacent zwitterions. The formation of this large dimer leaves available a second carboxylate O atom to take part in hydrogen bonding interactions with solvent molecules. The shorter 'two atom' chain compound was difficult to crystallize and required the use of synchrotron radiation to measure X-ray diffraction data. It does not form the same dimer motif observed in the 'three atom' chain compounds and has not formally formed a zwitterion; although there is evidence of proton sharing or disorder X-ray data are insufficient to create a disordered model, and the compound was modeled as formally neutral based on O-H and N-H distances. Room temperature analyses showed the proton transfer behavior to be independent of crystal temperature, and nuclear magnetic resonance studies show proton transfer behavior in solution. The formation of a zwitterionic hydrogen-bonded dimer is implicated in providing some stability during crystal growth of the easily crystallized 'three atom' chain compounds. © 2010 The Royal Society of Chemistry.
• Nichol, G. S., Vardanyan, R., & Hruby, V. J. (2010). Synthesis and crystallographic study of N'-(1-benzylpiperidin-4-yl) acetohydrazide. Journal of Chemical Crystallography, 40(11), 961-964.
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  Abstract: As part of a study into new Fentanyl-derived opioid compounds with potent analgesic activity and reduced side effects the starting material title compound, C14H21N3O (1), was synthesized and characterized by NMR spectroscopy and single-crystal X-ray diffraction. The crystal structure is monoclinic Cc with unit cell parameters a = 14.1480(3) Å, b = 14.1720(4) Å, c = 27.6701(7) Å, β = 96.956(1)°, α = γ = 90°. The compound has crystallized with four crystallographically unique molecules in the asymmetric unit; each molecule has a very similar conformation and an analysis of the structure shows that although all four unique molecules overlay very well there is no evidence of pseudo-symmetry which would relate the molecules in the higher symmetry space group C2/c. The crystal packing consists of two separate hydrogen bonded chains which are linked together to form a thick 2D structure in the ab plane. Index Abstract: The compound N'-(1-benzylpiperidin-4-yl)acetohydrazide crystallizes with four crystallographically unique molecules in the asymmetric unit; each molecule has a very similar conformation but there is no evidence of pseudo-symmetry.[Figure not available: see fulltext.] © 2010 Springer Science+Business Media, LLC.
• R., C., Vagner, J., Lynch, R., Gillies, R. J., & Hruby, V. J. (2010). Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions. Analytical Biochemistry, 398(1), 15-23.
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  PMID: 19852924;PMCID: PMC2830854;Abstract: Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0 M) prior to the luminescent enhancement step. [Nle4,d-Phe7]-α-melanocyte-stimulating hormone (NDP-α-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. © 2009 Elsevier Inc. All rights reserved.
• Torino, D., Mollica, A., Pinnen, F., Feliciani, F., Lucente, G., Fabrizi, G., Portalone, G., Davis, P., Lai, J., Ma, S., Porreca, F., & Hruby, V. J. (2010). Synthesis and evaluation of new endomorphin-2 analogues containing (Z)-α,β-didehydrophenylalanine (δ^ZPhe) residues. Journal of Medicinal Chemistry, 53(11), 4550-4554.
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  PMID: 20476738;PMCID: PMC2918392;Abstract: New endomorphin-2 (EM-2) analogues incorporating (Z)-α,β- didehydrophenylalanine (δZPhe) in place of the native phenylalanine in EM-2 are reported. Tyr-Pro-δZPhe-Phe-NH 2 {[δZPhe3]EM-2} (1), Tyr-Pro-Phe-δZPhe-NH2 {[δZPhe 4]EM-2} (2), and Tyr-Pro-δZPhe- δZPhe-NH2 {[δZPhe 3,4]EM-2}(3) have been synthesized, their opioid receptor binding affinities and tissue bioassay activities were determined, and their conformational properties were examined. Compound 2 shows high μ opioid receptor selectivity and μ agonist activity comparable to those of the native peptide. The conformation adopted in solution and in the crystal by N-Boc-Tyr-Pro-δZPhe-Phe-NH2 (8) is reported. © 2010 American Chemical Society.
• Yamamoto, T., Nair, P., Jacobsen, N. E., Kulkarni, V., Davis, P., Ma, S., Navratilova, E., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2010). Biological and conformational evaluation of bifunctional compounds for opioid receptor agonists and neurokinin 1 receptor antagonists possessing two penicillamines. Journal of Medicinal Chemistry, 53(15), 5491-5501.
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  PMID: 20617791;PMCID: PMC2943425;Abstract: Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with ?-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3?,5?-(CF 3)2-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold ?-selectivity over the ?-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant ?-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug. © 2010 American Chemical Society.
• Cai, M., Nyberg, J., & Hruby, V. J. (2009). Melanotropins as drugs for the treatment of obesity and other feeding disorders: Potential and problems. Current Topics in Medicinal Chemistry, 9(6), 554-563.
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  PMID: 19689365;Abstract: Current biological and pharmacological evidence suggests that the melanocortin 4 and melanocortin 3 receptors which are seven transmembrane G-protein coupled receptors (GPCRs) are involved in various aspects of energy balance and feeding behaviors in animals including humans. The natural endogenous ligands for these receptors are products of the gene pro-opiomelanocortin (POMC), and include α-melanocyte stimulating hormone, γ-melanocyte stimulating hormone and perhaps other modified products of POMC. Thus well designed agonists and antagonists of these ligands might serve as drugs for the treatment of feeding disorders. However, these melanotropin peptides also can have other biological activities that involve the MC3R and MC4R, and these other biological properties will need to be modulated in ligands that are likely to be useful drugs for feeding disorders. Current progress in these areas with special emphasis on the MC3R will be discussed along with possible new directions that might be fruitful in these important aspects of contemporary biology and medicine. © 2009 Bentham Science Publishers Ltd.
• Chen, M., Cai, M., McPherson, D., Hruby, V., Harmon, C. M., & Yang, Y. (2009). Contribution of the transmembrane domain 6 of melanocortin-4 receptor to peptide [Pro⁵, dNal (2′)⁸]-γ-MSH selectivity. Biochemical Pharmacology, 77(1), 114-124.
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  PMID: 18930713;PMCID: PMC2701352;Abstract: The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with dNal (2′) in γ-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro5, dNal (2′)8]-γ-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro5, dNal (2′)8]-γ-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro5, dNal (2′)8]-γ-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro5, dNal (2′)8]-γ-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, dNal (2′)-γ-MSH selective activity at MC4R. © 2008 Elsevier Inc. All rights reserved.
• Fernandes, S. M., Sankaranarayanan, R., Liping, X. u., Josan, J., Vagner, J., Gillies, R., & Hruby, V. J. (2009). Synthesis of bivalent MSH ligands and evaluation of their binding to hMC4R using MSH lanthaligand.. Advances in experimental medicine and biology, 611, 433-434.
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  PMID: 19400253;
• Hongchang, Q. u., Cai, M., Mayorov, A. V., Grieco, P., Zingsheim, M., Trivedi, D., & Hruby, V. J. (2009). Substitution of arginine with proline and proline derivatives in melanocyte-stimulating hormones leads to selectivity for human melanocortin 4 receptor. Journal of Medicinal Chemistry, 52(12), 3627-3635.
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  PMID: 19473029;PMCID: PMC2775485;Abstract: A new series of melanotropin analogues with His or Arg residues in the core pharmacophores of MTII, SHU9119, and Ac-NDP-γ-MSH-NH2 replaced by Pro or trans-/cis-4-guanidinyl-Pro derivatives were designed and synthesized to introduce selectivity toward the human melanocortin 4 receptor (hMC4R). Analogues 1, 2, 3, 6, 7, 8 were found to be hMC4R selective. Second messenger studies have demonstrated that analogues 1 and 2 are insurmountable inhibitors of MTII agonist activity at the hMC4R. Molecular modeling studies suggest that the hMC4R selectivity is due to a β-turn shift induced by the Pro ring that makes the global minimum structures of these analogues resemble the NMR solution structure of the hASIP melanocortin receptor binding motif. Substitution of His in MTII also provided functional selectivity for the hMC3R or the hMC4R. These findings are important for a better understanding of the selectivity mechanism at the hMC3R/hMC4R and the development of therapeutic ligands selectively targeting the hMC4R. © 2009 American Chemical Society.
• Hruby, V. J. (2009). Organic chemistry and biology: Chemical biology through the eyes of collaboration. Journal of Organic Chemistry, 74(24), 9245-9264.
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  PMID: 20000552;PMCID: PMC3041175;Abstract: (Figure Presented) From a scientific perspective, efforts to understand biology including what constitutes health and disease has become a chemical problem. However, chemists and biologists "see" the problems of understanding biology from different perspectives, and this has retarded progress in solving the problems especially as they relate to health and disease. This suggests that close collaboration between chemists and biologists is not only necessary but essential for progress in both the biology and chemistry that will provide solutions to the global questions of biology. This perspective has directed my scientific efforts for the past 45 years, and in this overview I provide my perspective of how the applications of synthetic chemistry, structural design, and numerous other chemical principles have intersected in my collaborations with biologists to provide new tools, new science, and new insights that were only made possible and fruitful by these collaborations. © 2009 American Chemical Society.
• Hruby, V. J., Cai, M., Dedek, M., Hongchang, Q. u., Palmer, E., Mayorov, A., Trivedi, D., Tsaprailis, G., & Yang, Y. (2009). Peptide and non-peptide mimetics utilize different pathways for signal transduction.. Advances in experimental medicine and biology, 611, 305-307.
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  PMID: 19400204;
• Josan, J. S., Morse, D. L., Liping, X. u., Trissal, M., Baggett, B., Davis, P., Vagner, J., Gillies, R. J., & Hruby, V. J. (2009). Solid-phase synthetic strategy and bioevaluation of a labeled δ-opioid receptor ligand Dmt-Tic-Lys for In Vivo imaging. Organic Letters, 11(12), 2479-2482.
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  PMID: 19445485;PMCID: PMC2756606;Abstract: A general solid-phase synthetic strategy is developed to prepare fluorescent and/or lanthanide-labeled derivatives of the δ-opioid receptor (δOR) ligand H-Dmt-Tic-Lys(R)-OH. The high δ-OR affinity {K i = 3 nM) and desirable in vivo characteristics of the Cy5 derivative 1 suggest its usefulness for structure - function studies and receptor localization and as a high-contrast noninvasive molecular marker for live imaging ex vivo or in vivo. © 2009 American Chemical Society.
• Josan, J. S., Sankaranarayanan, R., Handl, H. L., Fernandes, S., Liping, X. u., Vagner, J., Gillies, R. J., & Hruby, V. J. (2009). Heterobivalent ligands crosslink multiple cell-surface receptors--a step towards personal medicine.. Advances in experimental medicine and biology, 611, 413-414.
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  PMID: 19400245;
• Kessler, H., Chatterjee, J., Doedens, L., Operrer, F., Biron, E., Hoyer, D., Schmid, H., Gilon, C., Hruby, V. J., & Mierke, D. F. (2009). New perspective in peptide chemistry by n-alkylation. Advances in Experimental Medicine and Biology, 611, 229-231.
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  PMID: 19400173;
• Kulkarni, V. V., Lee, Y. S., Salibay, C., Davis, P., Ma, S., Porreca, F., & Hruby, V. J. (2009). Novel analogues of bifunctional ligands for opioid and melanocortin 4 receptor. Advances in Experimental Medicine and Biology, 611, 195-196.
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  PMID: 19400157;
• Kumarasinghe, I. R., Hruby, V. J., & Nichol, G. S. (2009). 1-(4-Methoxy-phen-yl)-3-phenyl-1H-pyrazol-5-amine. Acta Crystallographica Section E: Structure Reports Online, 65(5), o1170.
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  PMID: 21583972;PMCID: PMC2977835;Abstract: The synthesis of the title compound, C16H15N3O, is regiospecific and single-crystal X-ray diffraction provides the only means of unambiguous structural analysis, with the benzene ring bonded to the imine C atom. The phenyl ring and the essentially planar (r.m.s. deviation 0.0354 Å) methoxy-benzene group are rotated by 29.41 (5) and 37.01 (5)°, respectively, from the central pyrazole ring. An inter-molecular N - H⋯N hydrogen bond links symmetry-related mol-ecules into a C(5) chain, which runs parallel to the b axis.
• Kumarasinghe, I. R., Hruby, V. J., & Nichol, G. S. (2009). 3-[1-(4-Sulfamoylphenyl)-5-p-tolyl-1H-pyrazol-3-yl]propanoic acid and 3-[5-(4-bromophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-3-yl]propanoic acid-dichloromethane-diethyl etherwater (2/0.72/1/1). Acta Crystallographica Section C: Crystal Structure Communications, 65(6), o296-o299.
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  PMID: 19498242;PMCID: PMC2724996;Abstract: The structures of two related analogues 3-[1-(4-Sulfamoylphenyl)-5-p-tolyl- 1H-pyrazol-3-yl]propanoic acids and 3-[5-(4-bromophenyl)-1-(4-sulfamoylphenyl)- 1H-pyrazol-3-yl]propanoic acid-dichloromathane-diethyl ether-water has been reported. The compound was synthesized by a two step procedure using NaHMDS in place of LiHMDS. Nonsteroidal anti-inflammatory drugs are divided into three different categories. These structures makes use of hydrogen bonding and while compound forms a straight forward unsolvated Z equaling to structure compound crystallizes as an unusual mixed solvate with two crystallographically unique molecules of the pyrazole derivative present in the asymmetric unit. The molecules of dichloromethane are also found between the sheets and although there are no significant interactions between dichloromethane and adjacent molecules.
• Kumarasinghe, I. R., Hruby, V. J., & Nichol, G. S. (2009). 3-[5-(4-Chloro-phen-yl)-1-(4-methoxy-phen-yl)-1H-pyrazol-3-yl]propionic acid and the corresponding methyl ester. Acta Crystallographica Section C: Crystal Structure Communications, 65(4), o163-o166.
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  PMID: 19346614;PMCID: PMC2724994;Abstract: The synthesis of 3-[5-(4-chloro-phen-yl)-1-(4-methoxy-phen-yl)-1H-pyrazol- 3-yl]propionic acid, C19H17ClN2O3, (I), and its corresponding methyl ester, methyl 3-[5-(4-chloro-phen-yl)-1-(4-methoxy-phen-yl)-1H-pyrazol-3-yl]propionate, C20H19ClN2O3, (II), is regiospecific. However, correct identification of the regioisomer formed by spectroscopic techniques is not trivial and single-crystal X-ray analysis provided the only means of unambiguous structure determination. Compound (I) crystallizes with Z′ = 2. The propionic acid groups of the two crystallographically unique mol-ecules form a hydrogen-bonded dimer, as is typical of carboxylic acid groups in the solid state. Conformational differences between the meth-oxy-benzene and pyrazole rings give rise to two unique mol-ecules. The structure of (II) features just one mol-ecule in the asymmetric unit and the crystal packing makes greater use than (I) of weak C - H⋯A inter-actions, despite the lack of any functional groups for classical hydrogen bonding. © 2009 International Union of Crystallography.
• Lee, Y., Petrov, R., Kulkarni, V., Min, B. J., Ma, S., Davis, P., Oyarzo, J., Vanderah, T., Lai, J., Porreca, F., Vardanyan, R., & Hruby, V. J. (2009). Development of mu/delta opioid ligands: enkephalin analogues containing 4-anilidopiperidine moiety.. Advances in experimental medicine and biology, 611, 517-518.
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  PMID: 19400292;
• Liping, X. u., Vagner, J., Josan, J., Lynch, R. M., Morse, D. L., Baggett, B., Han, H., Mash, E. A., Hruby, V. J., & Gillies, R. J. (2009). Enhanced targeting with heterobivalent ligands. Molecular Cancer Therapeutics, 8(8), 2356-2365.
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  PMID: 19671749;PMCID: PMC3662534;Abstract: A novel approach to specifically target tumor cells for detection and treatment is the proposed use of hetero-multivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligandswith two ormore different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the humanmelanocortin 4 receptor (MC4R) expressed in combination with either the human δ-opioid receptor (δOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptorswere characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-α-MSH, and Eu-CCK8 for the δOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a δOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, comparedwith cellswith single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers. Copyright © 2009 American Association for Cancer Research.
• Liu, Z., Hongchang, Q. u., Xuyuan, G. u., Min, B. J., Nyberg, J., & Hruby, V. J. (2009). Enantioselective synthesis of anti-β-substituted γ,δ- unsaturated amino acids: A highly selective asymmetric Thio-Claisen rearrangement (Organic Letters (2008) 10). Organic Letters, 11(2), 497-.
• Min, B. J., Mayorov, A. V., Cai, M., Palmer, E., & Hruby, V. J. (2009). Design and parallel synthesis of new bicyclic small molecules for targeting the melanocortin receptors. Advances in Experimental Medicine and Biology, 611, 187-188.
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  PMID: 19400153;
• Nair, P., Yamamoto, T., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Largent, T., Ma, S., Yamamura, H. I., Vanderah, T., Lai, J., Porreca, F., & Hruby, V. J. (2009). Novel bifunctional peptides as opioid agonists and NK-1 antagonists.. Advances in experimental medicine and biology, 611, 537-538.
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  PMID: 19400302;
• Salamon, Z., Tollin, G., Alves, I., & Hruby, V. (2009). Chapter 6 Plasmon Resonance Methods in Membrane Protein Biology. Applications to GPCR Signaling. Methods in Enzymology, 461(B), 123-146.
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  PMID: 19480917;Abstract: Plasmon waveguide resonance (PWR) spectroscopy, a variant of surface plasmon resonance (SPR) spectrometry, allows one to examine changes in conformation of anisotropic structures such as membranes and membrane-associated proteins such as G-protein-coupled receptors (GPCRs). The binding and resulting structural changes that accompany interactions of membrane protein with ligands (agonists, antagonists, inverse agonist, etc.), G-proteins, and other effectors and modulators of signaling can be directly examined with this technique. In this chapter we outline the instrumentation used for these studies, the experimental methods that allow determination of the structural changes, and thermodynamic and kinetic parameters that can be obtained from these studies. © 2009 Elsevier Inc. All rights reserved.
• Torino, D., Mollica, A., Pinnen, F., Lucente, G., Feliciani, F., Davis, P., Lai, J., Ma, S., Porreca, F., & Hruby, V. J. (2009). Synthesis and evaluation of new endomorphin analogues modified at the Pro² residue. Bioorganic and Medicinal Chemistry Letters, 19(15), 4115-4118.
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  PMID: 19560919;PMCID: PMC2737817;Abstract: Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Δ3Pro), azetidine-3-carboxylic acid (3Aze) and dehydro-alanine (ΔAla) have been used to prepare [Δ3Pro2]EM-2 (1), [Aze2]EM-1 (2), [Aze2]EM-2 (3), [3Aze2]EM-1 (4), [3Aze2]EM-2 (5) and [ΔAla2]EM-2 (6). Binding assays and functional bioactivities for μ- and δ-receptors are reported. The highest affinity, bioactivity and selectivity are shown by peptides 2 and 3 containing the Aze residue. © 2009 Elsevier Ltd. All rights reserved.
• Vardanyan, R., Vijay, G., Nichol, G. S., Liu, L., Kumarasinghe, I., Davis, P., Vanderah, T., Porreca, F., Lai, J., & Hruby, V. J. (2009). Synthesis and investigations of double-pharmacophore ligands for treatment of chronic and neuropathic pain. Bioorganic and Medicinal Chemistry, 17(14), 5044-5053.
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  PMID: 19540763;PMCID: PMC2759397;Abstract: Acids 9a-f as possible bivalent ligands designed as a structural combination of opioid μ-agonist (Fentanyl) and NSAID (Indomethacin) activities and produced compounds which were tested as analgesics. The obtained series of compounds exhibits low affinity and activity both at opioid receptors and as cyclooxygenase (COX) inhibitors. One explanation of the weak opioid activity could be stereochemical peculiarities of these bivalent compounds which differ significantly from the fentanyl skeleton. The absence of significant COX inhibitory properties could be explained by the required substitution of an acyl fragment in the indomethacin structure for 4-piperidyl.
• Yamamoto, T., Nair, P., Jacobsen, N. E., Vagner, J., Kulkarni, V., Davis, P., Ma, S., Navratilova, E., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2009). Improving metabolic stability by glycosylation: Bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of Medicinal Chemistry, 52(16), 5164-5175.
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  PMID: 20560643;PMCID: PMC3521585;Abstract: In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3′,5′-Bzl(CF3) 2) which was a previously reported bifunctional compound with δ/μ opioid agonist and neurokinin-1 receptor antagonist activities and with a half-life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3′,5′-Bzl(CF 3)2) was found to have effective bifunctional activities with a well-defined conformation with two β-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 ± 9% of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and δ selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug. © 2009 American Chemical Society.
• Yamamoto, T., Nair, P., Ma, S., Davis, P., Yamamura, H. I., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2009). The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety. Bioorganic and Medicinal Chemistry, 17(20), 7337-7343.
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  PMID: 19762245;PMCID: PMC2775479;Abstract: In order to improve metabolic stability, a ring structure with a cystine moiety was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3′,5′-(CF3)2Bzl]), which is a lead compound of our developing bifunctional peptide possessing opioid agonist and NK1 antagonist activities. TY038 (Tyr-cyclo[d-Cys-Gly-Phe-Met-Pro-d-Cys]-Trp-NH-[3′,5′-(CF3)2Bzl]) was found as a highly selective δ opioid agonist over μ receptor in conventional tissue-based assays, together with an effective NK1 antagonist activity and good metabolic stability with more than 24 h half life in rat plasma. © 2009 Elsevier Ltd. All rights reserved.
• Yang, Y., Cai, M., Chen, M., Hongchang, Q. u., McPherson, D., Hruby, V., & Harmon, C. M. (2009). Key amino acid residues in the melanocortin-4 receptor for nonpeptide THIQ specific binding and signaling. Regulatory Peptides, 155(1-3), 46-54.
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  PMID: 19303903;PMCID: PMC3216638;Abstract: Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and glucose homeostasis. Synthetic nonpeptide compound N- (3R)-1 4-tetrahydroisoquinolinium-3-ylcarbonyl-(1R)-1-(4-chlorobenzyl)-2-4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl-2-oxoethylamine (THIQ) is a potent agonist at MC4R but not at hMC2R. In this study, we utilized two approaches (chimeric receptor and site-directed mutagenesis) to narrow down the key amino acid residues of MC4R responsible for THIQ binding and signaling. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TMs) of the human MC4R (hMC4R) with the homologous regions of hMC2R were constructed. Our results indicate that the cassette substitutions of these TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter THIQ binding affinity and potency except the substitution of the hMC4R TM3, suggesting that the conserved amino acid residues in these TMs of the hMC4R are main potential candidates for THIQ binding and signaling while non conserved residues in TM3 of MC4R may also be involved. Nineteen MC4R mutants were then created, including 13 conserved amino acid residues and 6 non-conserved amino acid residues. Our results indicate that seven conserved residue [E100 (TM2), D122 (TM3), D126 (TM3), F254 (TM6), W258 (TM6), F261 (TM6), H264 (TM6)] are important for THIQ binding and three non-conserved residues [N123 (TM3), I129 (TM3) and S131 (TM3)] are involved in THIQ selectivity. In conclusion, our results suggest that THIQ utilize both conserved and non-conserved amino acid residues for binding and signaling at hMC4R and non conserved residues may be responsible for MC4R selectivity. © 2009 Elsevier B.V. All rights reserved.
• Yang, Y., Hruby, V. J., Chen, M., Crasto, C., Cai, M., & Harmon, C. M. (2009). Novel binding motif of ACTH analogues at the melanocortin receptors. Biochemistry, 48(41), 9775-9784.
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  PMID: 19743876;PMCID: PMC2775456;Abstract: The melanocortin receptor (MCR) subtype family is a member of theGPCRsuperfamily, and each of them has a different pharmacological profile with regard to the relative potency of the endogenous and synthetic melanocortin peptides. α-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R, and MC5R. In this study, we examined the role of Phe7 in ACTH on human (h) MC1R, MC3R, and MC4R binding and signaling. Our results indicate that substitution of Phe7 with D-Nal(2′)7 in ACTH1-24 yields a pharmacological profile different from that for substitution of Phe7 with D-Nal(2′)7 in MSH in hMC1R, hMC3R, and hMC4R. N-D-Nal(2′)7-ACTH1-24 is an agonist at hMC3R and hMC4R which did not change the peptide from an agonist to an antagonist at hMC3R and hMC4R. Further experiments indicate that N-D-Nal(2′) 7-ACTH1-17 is the minimal peptide required for hMC3Rand hMC4R activation. Single-amino acid substitution studies of D-Nal(2′) 7-ACTH1-17 indicate that amino acid residues 15-17 in N-D-Nal(2′)7-ACTH1-17 are crucial for hMC3R and hMC4R activation. Substitutions of these amino acid residues reduced or abolished agonist activity at hMC3R and hMC4R. Conformational studies revealed a new β-turn (Arg8-Trp9-Gly10-Lys11) in N-D-Nal(2′)7-ACTH1-17, compared to the β-turn-like structure at NDP-α-MSH (His6-D-Phe7-Arg 8-Trp9). Our results suggest that NDP-α-MSH and N-D-Nal(2′)7-ACTH1-17 do not share the same binding site; the highly basic C-terminal fragment (Lys15-Lys16-Arg 17) of N-D-Nal(2′)7-ACTH1-17 induced a new β-turn, and this shift contributed the selective agonist activity at hMC3R and hMC4R. ©2009 American Chemical Society.
• Zhang, W., Gardell, S., Zhang, D., Xie, J. Y., Agnes, R. S., Badghisi, H., Hruby, V. J., Rance, N., Ossipov, M. H., Vanderah, T. W., Porreca, F., & Lai, J. (2009). Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors. Brain, 132(3), 778-787.
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  PMID: 19050032;PMCID: PMC2724921;Abstract: Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male SpragueDawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80 of these cells co-express both receptors. Agonist-directed cell lesion in the RVM with the cytotoxin, saporin, using either CCK-saporin to target CCK receptor expressing cells, or dermorphin-saporin to target MOR expressing cells, resulted in concomitant loss of CCK2 and MOR expressing cells, did not alter the basal sensory thresholds but abolished the hyperalgesia induced by microinjection of CCK into the RVM. The findings suggest that these CCK2-MOR co-expressing RVM neurons facilitate pain and can be directly activated by CCK input to the RVM. Furthermore, lesion of these RVM neurons did not affect the initial development of neuropathic pain in the hind paw upon injury to the sciatic nerve, but the abnormal pain states were short lived such that by about day 9 the sensory thresholds had reverted to pre-injury baselines despite the existing neuropathy. These data support our hypothesis and identify CCK2-MOR co-expressing neurons in the RVM as potential therapeutic targets for neuropathic pain.
• Agnes, R. S., Ying, J., Kövér, K. E., Lee, Y. S., Davis, P., Ma, S., Badghisi, H., Porreca, F., Lai, J., & Hruby, V. J. (2008). Structure-activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors. Peptides, 29(8), 1413-1423.
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  PMID: 18502541;PMCID: PMC2601673;Abstract: Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors. Compound 1 (Tyr-c[d-Cys-Gly-Trp-Cys]-Asp-Phe-NH2) showed potent binding and agonist activities at δ and μ opioid receptors but weak binding to CCK receptors. The NMR structure of the lead compound displayed similar conformational features of opioid and CCK ligands. © 2008 Elsevier Inc. All rights reserved.
• Balagurunathan, Y., Morse, D. L., Hostetter, G., Shanmugam, V., Stafford, P., Shack, S., Pearson, J., Trissal, M., Demeure, M. J., D., D., Hruby, V. J., Gillies, R. J., & Han, H. (2008). Gene expression profiling-based identification of cell-surface targets for developing multimeric ligands in pancreatic cancer. Molecular Cancer Therapeutics, 7(9), 3071-3080.
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  PMID: 18765825;PMCID: PMC2567866;Abstract: Multimeric ligands are ligands that contain multiple binding domains that simultaneously target multiple cell-surface proteins. Due to cooperative binding, multimeric ligands can have high avidity for cells (tumor) expressing all targeting proteins and only show minimal binding to cells (normal tissues) expressing none or only some of the targets. Identifying combinations of targets that concurrently express in tumor cells but not in normal cells is a challenging task. Here, we describe a novel approach for identifying such combinations using genome-wide gene expression profiling followed by immunohistochemistry. We first generated a database of mRNA gene expression profiles for 28 pancreatic cancer specimens and 103 normal tissue samples representing 28 unique tissue/cell types using DNA microarrays. The expression data for genes that encode proteins with cell-surface epitopes were then extracted from the database and analyzed using a novel multivariate rule-based computational approach to identify gene combinations that are expressed at an efficient binding level in tumors but not in normal tissues. These combinations were further ranked according to the proportion of tumor samples that expressed the sets at efficient levels. Protein expression of the genes contained in the topranked combinations was confirmed using immunohistochemistry on a pancreatic tumor tissue and normal tissue microarrays. Coexpression of targets was further validated by their combined expression in pancreatic cancer cell lines using immunocytochemistry. These validated gene combinations thus encompass a list of cell-surface targets that can be used to develop multimeric ligands for the imaging and treatment of pancreatic cancer. Copyright © 2008 American Association for Cancer Research.
• Georgieva, T., Devanathan, S., Stropova, D., Park, C. K., Salamon, Z., Tollin, G., Hruby, V. J., Roeske, W. R., Yamamura, H. I., & Varga, E. (2008). Unique agonist-bound cannabinoid CB₁ receptor conformations indicate agonist specificity in signaling. European Journal of Pharmacology, 581(1-2), 19-29.
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  PMID: 18162180;PMCID: PMC2279194;Abstract: Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (KD = 0.2 ± 0.03 nM and 2 ± 0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB1 receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940-and WIN 55,212-bound hCB1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, Gi1 (KD = 27 ± 8 nM and KD = 10.7 ± 4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type. © 2007 Elsevier B.V. All rights reserved.
• Grieco, P., Cai, M., Liu, L., Mayorov, A., Chandler, K., Trivedi, D., Lin, G., Campiglia, P., Novellino, E., & Hruby, V. J. (2008). Design and microwave-assisted synthesis of novel macrocyclic peptides active at melanocortin receptors: Discovery of potent and selective hMC5R receptor antagonists. Journal of Medicinal Chemistry, 51(9), 2701-2707.
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  PMID: 18412316;PMCID: PMC2756086;Abstract: Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9 (PG-20N) is a selective and competitive hMC5R antagonist, with IC50 of 130 ± 11 nM, and a pA2 value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC50 values of 38 ± 3 nM and 58 ± 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals. © 2008 American Chemical Society.
• Josan, J. S., Vagner, J., Handl, H. L., Sankaranarayanan, R., Gillies, R. J., & Hruby, V. J. (2008). Solid-phase synthesis of heterobivalent ligands targeted to melanocortin and cholecystokinin receptors. International Journal of Peptide Research and Therapeutics, 14(4), 293-300.
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  Abstract: Heteromultivalency provides a route to increase binding avidity and to high specificity when compared to monovalent ligands. The enhanced specificity can potentially serve as a unique platform to develop diagnostics and therapeutics. To develop new imaging agents based upon multivalency, we employed heterobivalent constructs of optimized ligands. In this report, we describe synthetic methods we have developed for the preparation of heterobivalent constructs consisting of ligands targeted simultaneously to the melanocortin receptor, hMC4R, and the cholecystokinin receptors, CCK-2R. Modeling data suggest that a linker distance span of 20-50 Å is needed to crosslink these two G-protein coupled receptors (GPCRs). The two ligands were tethered with linkers of varying rigidity and length, and flexible polyethylene glycol based PEGO chain or semi-rigid [poly(Pro-Gly)] linkers were employed for this purpose. The described synthetic strategy provides a modular way to assemble ligands and linkers on solid-phase supports. Examples of heterobivalent ligands are provided to illustrate the increased binding avidity to cells that express the complementary receptors. © 2008 Springer Science+Business Media, LLC.
• Lee, M., Kim, A., Conwell, I. M., Hruby, V., Mayorov, A., Cai, M., & Wardlaw, S. L. (2008). Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression. Peptides, 29(3), 440-447.
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  PMID: 18155809;PMCID: PMC2278043;Abstract: Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [d-Trp8]-γ-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake. © 2007 Elsevier Inc. All rights reserved.
• Lee, Y. S., Lee, Y. S., Agnes, R. S., Agnes, R. S., Cain, J. P., Cain, J. P., Kulkarni, V., Kulkarni, V., Cai, M., Cai, M., Salibay, C., Salibay, C., Ciano, K., Ciano, K., Petrov, R., Petrov, R., Mayorov, A., Mayorov, A., Vagner, J., , Vagner, J., et al. (2008). Opioid and melanocortin receptors: Do they have overlapping pharmacophores?. Biopolymers - Peptide Science Section, 90(3), 433-438.
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  PMID: 17657709;PMCID: PMC2693099;Abstract: We have identified compound 1 as a novel ligand for opiod and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the δ opiod receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe (p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the δ opioid receptor (Ki = 0.38 nM in binding assay, EC50 = 0.48 nM in GTP-γ-S binding assay, IC50 = 74 nM in MVD as an agonist instead of an antagonist and showed selective binding affinity (IC50 = 2.3 μM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opiod receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors. ©2007 Wiley Periodicals, Inc.
• Liu, Z., Hongchang, Q. u., Xuyuan, G. u., Min, B. J., Nyberg, J., & Hruby, V. J. (2008). Enantioselective synthesis of anti-β-substituted γ,δ- unsaturated amino acids: A highly selective asymmetric thio-Claisen rearrangement. Organic Letters, 10(18), 4105-4108.
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  PMID: 18702500;PMCID: PMC2654228;Abstract: (Chemical Equation Presented) A novel synthesis of optically active anti-β-substituted γ,δ-unsaturated amino acids via a thio-Claisen rearrangement has been achieved. A 2,5-diphenylpyrrolidine was used as a (C2-symmetric chiral auxiliary to control the stereochemistry, giving good yields and excellent diastereoselectivities and enantioselectivities. © 2008 American Chemical Society.
• Mayorov, A. V., Cai, M., Palmer, E. S., Dedek, M. M., Cain, J. P., R., A., Tan, B., Vagner, J., Trivedi, D., & Hruby, V. J. (2008). Structure-activity relationships of cyclic lactam analogues of α-melanocyte-stimulating hormone (α-MSH) targeting the human melanocortin-3 receptor. Journal of Medicinal Chemistry, 51(2), 187-195.
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  PMID: 18088090;PMCID: PMC2587288;Abstract: A variety of dicarboxylic acid linkers introduced between the α-amino group of Pro6 and the ε-amino group of Lys10 of the cyclic lactam α-melanocyte-stimulating hormone (α-MSH)-derived Pro6-D-Phe7/D-Nal(2′)7-Arg 8-Trp9-Lys10-NH2 pentapeptide template lead to nanomolar range and selective hMC3R agonists and antagonists. Replacement of the Pro6 residue and the dicarboxylic acid linker with 2,3-pyrazine-dicarboxylic acid furnished a highly selective nanomolar range hMC3R partial agonist (analogue 12, c[CO-2,3-pyrazine-CO-D-Phe-Arg-Trp-Lys]- NH2, EC50 = 27 nM, 70% max cAMP) and an hMC3R antagonist (analogue 13, c[CO-2,3-pyrazine-CO-D-Nal(2′)-Arg-Trp-Lys]-NH2, IC50 = 23 nM). Modeling experiments suggest that 2,3- pyrazinedicarboxylic acid stabilizes a β-turn-like structure with the D-Phe/D-Nal(2′) residues, which explains the high potency of the corresponding peptides. Placement of a Nle residue in position 6 produced a hMC3R/hMC5R antagonist (analogue 15, c[CO-(CH2)2-CO-Nle-D- Nal(2′)-Arg-Trp-Lys]-NH2, IC50 = 12 and 17 nM, respectively), similarly to the previously described cyclic γ-melanocyte- stimulating hormone (γ-MSH)-derived hMC3R/hMC5R antagonists. These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-3 receptor. © 2008 American Chemical Society.
• Min, B. J., Xuyuan, G. u., Yamamoto, T., Petrov, R. R., Hongchang, Q. u., Lee, Y. S., & Hruby, V. J. (2008). Synthesis of a novel benzyl-octahydropyrazino[1,2-a]pyrimidin-6-one derivative as a convenient internal bicyclic peptidomimetic. Tetrahedron Letters, 49(14), 2316-2319.
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  Abstract: A substituted hydropyrazino[1,2-a]pyrimidin-6-one derivative was synthesized stereoselectively via the intramolecular N-acyliminium ion cyclization between an amide nitrogen and an Nα-acetal derived from Cbz-protected aminopropyl-phenylalaninamide in very good yields. The formation of a single diastereomer is due to the low energy chairlike conformation of its bicyclic structure. This methodology provides a convenient tool to build internal bicyclic peptidomimetics. © 2008 Elsevier Ltd. All rights reserved.
• Sutton, G. M., Babin, M. J., Xuyuan, G. u., Hruby, V. J., & Butler, A. A. (2008). A derivative of the melanocortin receptor antagonist SHU9119 (PG932) increases food intake when administered peripherally. Peptides, 29(1), 104-111.
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  PMID: 18054119;PMCID: PMC2234266;Abstract: Melanocortin receptors are considered promising candidates for the treatment of behavioral and metabolic disorders ranging from obesity to anorexia and cachexia. These experiments examined the response of mice to peripheral injections of two compounds. PG932 is a derivative of SHU9119 which is non-selective antagonist of melanocortin-3 and melanocortin-4 receptors (Mc3r and Mc4r). PG946 is a derivative of a hybrid of α- and β-MSH, and is a moderately selective Mc3r antagonist. SHU9119 increases food intake when administered intracerebroventricularly but is without effect when injected into the periphery. In contrast, PG932 was found to be highly effective at stimulating food intake when administered peripherally by intraperitoneal injection. The orexigenic effect of PG932 required functional Mc4r, suggesting that inhibition of this receptor is involved in the stimulation of food intake. PG946 did not significantly affect on feeding behavior. PG932 is thus a useful new compound for studies examining the regulation of appetite and energy balance, and may also prove useful for the treatment of cachectic conditions. © 2007 Elsevier Inc. All rights reserved.
• Vagner, J., Hongchang, Q. u., & Hruby, V. J. (2008). Peptidomimetics, a synthetic tool of drug discovery. Current Opinion in Chemical Biology, 12(3), 292-296.
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  PMID: 18423417;PMCID: PMC2515564;Abstract: The demand for modified peptides with improved stability profiles and pharmacokinetic properties is driving extensive research effort in this field. Many structural modifications of peptides guided by rational design and molecular modeling have been established to develop novel synthetic approaches. Recent advances in the synthesis of conformationally restricted building blocks and peptide bond isosteres are discussed. © 2008 Elsevier Ltd. All rights reserved.
• Vagner, J., Liping, X. u., Handl, H. L., Josan, J. S., Morse, D. L., Mash, E. A., Gillies, R. J., & Hruby, V. J. (2008). Heterobivalent ligands crosslink multiple cell-surface receptors: The human melanocortin-4 and δ-opioid receptors. Angewandte Chemie - International Edition, 47(9), 1685-1688.
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  PMID: 18205159;PMCID: PMC2716288;Abstract: (Figure Presented) Together we bind: A series of synthetic heterobivalent ligands containing a fragment of melanocyte stimulating hormone analogue MSH(7) and the δ-opioid ligand deltorphin-II has been prepared. These ligands bind with higher affinity and with apparent cooperativity to cells expressing both hMC4R and d-opioid receptors. Binding affinities were evaluated using a lanthanide-based in-cyto time-resolved fluorescence binding assay. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.
• Yamamoto, T., Nair, P., Jacobsen, N. E., Davis, P., Ma, S., Navratilova, E., Moye, S., Lai, J., Yamamura, H. I., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2008). The importance of micelle-bound states for the bioactivities of bifunctional peptide derivatives for δ/μ opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of Medicinal Chemistry, 51(20), 6334-6347.
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  PMID: 18821747;PMCID: PMC2675940;Abstract: To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifunctional peptide derivatives of δ-preferring opioid agonists, and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe- Met-Pro-Leu-Trp-O-3,5-Bzl(CF3)2; 2: Tyr-D-Ala-Gly-Phe-Met- Pro-Leu-Trp-NH-3,5-Bzl(CF3)2; 3: Tyr-D-Ala-Gly-Phe-Met- Pro-Leu-Trp-NH-Bzl) were determined based on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological activities showed different trends in direct relation to the compound-specific conformations in DPC micelles. The important result is that not only were the NK1 antagonist activities different (the pharmacophore located at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was induced due to relatively small and limited structural modifications. © 2008 American Chemical Society.
• Yamamoto, T., Nair, P., Vagner, J., Largent-Milnes, T., Davis, P., Ma, S., Navratilova, E., Moye, S., Tumati, S., Lai, J., Yamamura, H. I., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2008). A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are δ/μ opioid receptor agonists and neurokinin 1 receptor antagonists. Journal of Medicinal Chemistry, 51(5), 1369-1376.
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  PMID: 18266313;PMCID: PMC2737825;Abstract: A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr(tBu)-D-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl (3) demonstrated high binding affinities at both δ and μ receptors (Ki = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation (Ke = 26 nM) and good affinity for the hNK1 receptor (Ki = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities. © 2008 American Chemical Society.
• Ballet, S., Mayorov, A. V., Cai, M., Tymecka, D., Chandler, K. B., Palmer, E. S., Rompaey, K. V., Misicka, A., Tourwé, D., & Hruby, V. J. (2007). Novel selective human melanocortin-3 receptor ligands: Use of the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold. Bioorganic and Medicinal Chemistry Letters, 17(9), 2492-2498.
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  PMID: 17314042;PMCID: PMC2442462;Abstract: In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-d-Nal(2′)-Arg-Trp-Lys]-NH2) by replacing the His-d-Phe and His-d-Nal(2′) fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx = d-Phe/pCl-d-Phe/d-Nal(2′)). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists. © 2007 Elsevier Ltd. All rights reserved.
• Bowen, M. E., Monguchi, Y., Sankaranarayanan, R., Vagner, J., Begay, L. J., Liping, X. u., Jagadish, B., Hruby, V. J., Gillies, R. J., & Mash, E. A. (2007). Design, synthesis, and validation of a branched flexible linker for bioactive peptides. Journal of Organic Chemistry, 72(5), 1675-1680.
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  PMID: 17279799;PMCID: PMC3538035;Abstract: A branched flexible linker that incorporates a fluorescent dansyl moiety was synthesized and used to connect two high affinity NDP-α-MSH ligands or two low affinity MSH(4) ligands. The linker was incorporated into the conjugate by solid-phase synthesis. In vitro biological evaluations showed that potency of binding to the human melanocortin 4 receptor was not diminished for linker-ligand combinations relative to the corresponding ligand alone. © 2007 American Chemical Society.
• Bowen, M. E., Monguchi, Y., Sankaranarayanan, R., Vagner, J., Begay, L. J., Liping, X. u., Jagadish, B., Hruby, V. J., Gillies, R. J., & Mash, E. A. (2007). Erratum: Design, synthesis, and validation of a branched flexible linker for bioactive peptides (Journal of Inorganic Chemistry (2007) 72 (1679-1680)). Journal of Organic Chemistry, 72(9), 3608-.
• Chen, M., Cai, M., Aprahamian, C. J., Georgeson, K. E., Hruby, V., Harmon, C. M., & Yang, Y. (2007). Contribution of the conserved amino acids of the melanocortin-4 receptor in D-[Nle⁴,Phe⁷]-α-melanocyte-stimulating hormone binding and signaling. Journal of Biological Chemistry, 282(30), 21712-21719.
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  PMID: 17545153;PMCID: PMC2704061;Abstract: Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for α-melanocortin-stimulating hormone (α-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for α-MSH binding and signaling. α-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and α-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter α-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for α-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu100 in TM2, Asp122, Asp126 in TM3 and Trp258, Phe 261, His264 in TM6 are involved in α-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for α-MSH binding and signaling. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
• Chen, M., Cai, M., Aprahamian, C. J., Georgeson, K. E., Hruby, V., Harmon, C. M., & Yang, Y. (2007). Erratum: Contribution of the conserved amino acids of the melanocortin-4 receptor in [Nle⁴,D-Phe⁷]-α-melanocyte-stimulating hormone binding and signaling (Journal of Biological Chemistry (2007) 282 (21712-21719)). Journal of Biological Chemistry, 282(46), 33896-.
• Gao, F., Handl, H., Vagner, J., Hruby, V., & Gillies, R. (2007). Convenient and efficient synthesis of a lanthanide ³⁺ - Coordinated, diethylene triamine pentaacetic acid labeled biopolymer as an assay for the cholecystokinin B receptor. Journal of Applied Polymer Science, 106(4), 2683-2688.
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  Abstract: To develop an assay for the cholecystokinin B receptor with an Eu 3+-labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide 3+-coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based highthroughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study. © 2007 Wiley Periodicals, Inc.
• Grieco, P., Cai, M., Han, G., Trivedi, D., Campiglia, P., Novellino, E., & Hruby, V. J. (2007). Further structure-activity studies of lactam derivatives of MT-II and SHU-9119: Their activity and selectivity at human melanocortin receptors 3, 4, and 5. Peptides, 28(6), 1191-1196.
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  PMID: 17482720;PMCID: PMC1955225;Abstract: Recently we have demonstrated that replacing His6 by constrained amino acids22Amino acid symbols denote L-configuration unless indicated otherwise. in the well-known antagonist SHU-9119 resulted in potent and selective antagonist ligands especially at the hMC3R and hMC5 receptors. With the aim to further explore position 6 in the sequence of SHU-9119 and MT-II, we have designed, synthesized, and pharmacologically characterized a series of peptide analogues of MT-II and SHU-9119 at the human melanocortin receptors subtypes MC3R, MC4R and MC5R. All these peptides were modified at position 6 with constrained amino acids which are commercially available. In this study, we have identified new selective ligands for the hMC4R, and an antagonist for the hMC3/hMC4 receptors. Additionally, we have discovered an interesting new selective antagonist at the hMC3R, Ac-Nle-c[Asp-βAla-DNal(2′)-Arg-Trp-Lys]-NH2 (2, PG-106) which represents an important tool in further biological investigations of the hMC3R. PG-106 will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R. © 2007 Elsevier Inc. All rights reserved.
• Handl, H. L., Sankaranarayanan, R., Josan, J. S., Vagner, J., Mash, E. A., Gillies, R. J., & Hruby, V. J. (2007). Synthesis and evaluation of bivalent NDP-α-MSH(7) peptide ligands for binding to the human melanocortin receptor 4 (hMC4R). Bioconjugate Chemistry, 18(4), 1101-1109.
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  PMID: 17591746;PMCID: PMC3538036;Abstract: We demonstrate the potential utility of multivalent ligands as targeting agents for cancer imaging or therapy by determining the binding of homobivalent ligands to their corresponding receptors. This manuscript details the synthesis and evaluation of a series of bivalent ligands containing two copies of the truncated heptapeptide version of [Nle4-D-Phe7]-α- melanocyte stimulating hormone (NDP-α-MSH), referred to as MSH(7). These were connected with various semirigid linkers containing Pro-Gly repeats, with or without flexible poly(ethylene glycol) (PEGO) moieties at their termini. Modeling data suggest a distance of 20-50 Å between the ligand binding sites of two adjacent G-protein coupled receptors, GPCRs. These bivalent ligands were observed to bind with higher affinity compared to their monovalent counterparts. Data suggest these ligands may be capable of cross-linking adjacent receptors. An optimal linker length of 25 ± 10 Å, inferred from these ligands, correlated well with the interreceptor distance estimated through modeling. Although there was no difference in maximal binding affinities between the ligands constructed with the Pro-Gly repeats versus those constructed with the PEGO inserts, the PEGO-containing ligands bound with high affinities over a greater range of linker lengths. © 2007 American Chemical Society.
• Hongchang, Q. u., Xuyuan, G. u., Liu, Z., Min, B. J., & Hruby, V. J. (2007). Asymmetric eschenmoser-claisen rearrangement for anti-β-substituted γ,δ-unsaturated amino acids. Organic Letters, 9(20), 3997-4000.
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  PMID: 17760455;PMCID: PMC2587277;Abstract: Optically active anti-β-substituted γ,δ-unsaturated amino acids are important synthetic building blocks in organic synthesis and for peptidomimetics. A novel asymmetric Eschenmoser-Claisen rearrangement with use of a C 2-symmetric chiral auxiliary was developed to generate this type of amino acid. Excellent diastereoselectivities and high enantioselectivities (87-93% ee) were obtained after the chiral auxiliary was removed via iodolactonization/zinc reduction. © 2007 American Chemical Society.
• Hruby, V. J. (2007). Professor Mac E. Hadley: creative scientist, superb teacher, dynamic collaborator and wonderful friend.. General and comparative endocrinology, 151(3), 358-360.
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  PMID: 17590901;
• Hruby, V. J., & Tollin, G. (2007). Plasmon-waveguide resonance (PWR) spectroscopy for directly viewing rates of GPCR/G-protein interactions and quantifying affinities. Current Opinion in Pharmacology, 7(5), 507-514.
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  PMID: 17869585;PMCID: PMC2151673;Abstract: Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that has been developed in our laboratories and applied to the study of membrane-associated proteins, especially GPCRs. It has high sensitivity and requires no labeling of materials, and it can monitor changes in proteolipid mass density and conformation in real time using plasmon excitation by light polarized both perpendicular and parallel to the resonator surface. Direct measurements will be described of the association of ligands and G-proteins to GPCRs incorporated into a self-assembled lipid bilayer deposited on the silica surface of a PWR resonator. These studies have provided new insights into the functioning of this important class of signaling proteins. © 2007 Elsevier Ltd. All rights reserved.
• Hruby, V. J., Cai, M., Cain, J. P., Mayorov, A. V., Dedek, M. M., & Trivedi, D. (2007). Design, synthesis and biological evaluation of ligands selective for the melanocortin-3 receptor. Current Topics in Medicinal Chemistry, 7(11), 1085-1097.
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  Abstract: The processed products of the proopiomelanocortin gene (ACTH, α-MSH, β-MSH, γ-MSH, etc.) interact with five melanocortin receptors, the MC1R, MC2R, MC3R, MC4R, and MC5R to modulate and control many important biological functions crucial for good health both peripherally (as hormones) and centrally (as neurotransmitters). Pivotal biological functions include pigmentation, adrenal function, response to stress, fear/flight, energy homeostasis, feeding behavior, sexual function and motivation, pain, immune response, and many others, and are believed to be involved in many disease states including pigmentary disorders, adrenal disorders, obesity, anorexia, prolonged and neuropathic pain, inflammatory response, etc. The melanocortin-3 receptor (MC3R) is found primarily in the brain and spinal cord and also in the periphery, and its biological functions are still not well understood. Here we review some of the biological functions attributed to the MC3R, and then examine in more detail efforts to design and synthesize ligands that are potent and selective for the MC3R, which might help resolve the many questions still remaining about its function. Though some progress has been made, there is still much to be done in this critical area. © 2007 Bentham Science Publishers Ltd.
• Hruby, V. J., Cai, M., Cain, J. P., Mayorov, A. V., Dedek, M. M., & Trivedi, D. (2007). Design, synthesis and biological evaluation of ligands selective for the melanocortin-3 receptor.. Current topics in medicinal chemistry, 7(11), 1107-1119.
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  PMID: 17584128;PMCID: PMC2274922;Abstract: The processed products of the proopiomelanocortin gene (ACTH, alpha-MSH, beta-MSH, gamma-MSH, etc.) interact with five melanocortin receptors, the MC1R, MC2R, MC3R, MC4R, and MC5R to modulate and control many important biological functions crucial for good health both peripherally (as hormones) and centrally (as neurotransmitters). Pivotal biological functions include pigmentation, adrenal function, response to stress, fear/flight, energy homeostasis, feeding behavior, sexual function and motivation, pain, immune response, and many others, and are believed to be involved in many disease states including pigmentary disorders, adrenal disorders, obesity, anorexia, prolonged and neuropathic pain, inflammatory response, etc. The melanocortin-3 receptor (MC3R) is found primarily in the brain and spinal cord and also in the periphery, and its biological functions are still not well understood. Here we review some of the biological functions attributed to the MC3R, and then examine in more detail efforts to design and synthesize ligands that are potent and selective for the MC3R, which might help resolve the many questions still remaining about its function. Though some progress has been made, there is still much to be done in this critical area.
• Jagadish, B., Sankaranarayanan, R., Liping, X. u., Richards, R., Vagner, J., Hruby, V. J., Gillies, R. J., & Mash, E. A. (2007). Squalene-derived flexible linkers for bioactive peptides. Bioorganic and Medicinal Chemistry Letters, 17(12), 3310-3313.
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  PMID: 17448660;PMCID: PMC2033335;Abstract: A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-α-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3 + 2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-α-MSH constructs, presumably due to slow off rates for this high affinity ligand. © 2007 Elsevier Ltd. All rights reserved.
• King, S. H., Mayorov, A. V., Balse-Srinivasan, P., Hruby, V. J., Vanderah, T. W., & Wessells, H. (2007). Melanocortin receptors, melanotropic peptides and penile erection. Current Topics in Medicinal Chemistry, 7(11), 1111-1119.
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  Abstract: Penile erection is a complex physiologic event resulting from the interactions of the nervous system on a highly specialized vascular organ. Activation of central nervous system melanocortinergic (MC) receptors with either endogenous or synthetic melanotropic ligands may initiate and/or facilitate spontaneous penile erection. While the CNS contains principally the MC3 and MC4 receptor subtypes, there is conflicting data as to which receptor mediates erection. Although the MC4R is emerging as the principle effector of MC induced erection, the role of the MC3R is poorly understood. Manipulation of each receptor subtype with newly synthesized receptor specific agonists and antagonists, as well as knockout mice, has elucidated their individual contributions. Novel data from our laboratories suggests that antagonism of forebrain MC3R may enhance melanocortin-induced erections. Furthermore, melanocortin agents may interact with better-studied systems such as oxytocinergic pathways at the hypothalamic, brainstem or spinal level. Current therapies for erectile dysfunction target end organ vascular tissue. Manipulation of MC receptors may provide an alternative, centrally mediated therapeutic approach for erectile and other sexual dysfunctions. The non-specific "superpotent" MC agonist, PT-141, which is the carboxylate derivative of MT-II, has reached phase II human trials. Through their centrally mediated activity, melanocortin agonists have potential to treat erectile dysfunction as well as possible applications to the unmet medical needs of decreased sexual motivation and loss of libido. © 2007 Bentham Science Publishers Ltd.
• King, S. H., Mayorov, A. V., Balse-Srinivasan, P., Hruby, V. J., Vanderah, T. W., & Wessells, H. (2007). Melanocortin receptors, melanotropic peptides and penile erection.. Current topics in medicinal chemistry, 7(11), 1098-1106.
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  PMID: 17584130;PMCID: PMC2694735;Abstract: Penile erection is a complex physiologic event resulting from the interactions of the nervous system on a highly specialized vascular organ. Activation of central nervous system melanocortinergic (MC) receptors with either endogenous or synthetic melanotropic ligands may initiate and/or facilitate spontaneous penile erection. While the CNS contains principally the MC3 and MC4 receptor subtypes, there is conflicting data as to which receptor mediates erection. Although the MC4R is emerging as the principle effector of MC induced erection, the role of the MC3R is poorly understood. Manipulation of each receptor subtype with newly synthesized receptor specific agonists and antagonists, as well as knockout mice, has elucidated their individual contributions. Novel data from our laboratories suggests that antagonism of forebrain MC3R may enhance melanocortin-induced erections. Furthermore, melanocortin agents may interact with better-studied systems such as oxytocinergic pathways at the hypothalamic, brainstem or spinal level. Current therapies for erectile dysfunction target end organ vascular tissue. Manipulation of MC receptors may provide an alternative, centrally mediated therapeutic approach for erectile and other sexual dysfunctions. The non-specific "superpotent" MC agonist, PT-141, which is the carboxylate derivative of MT-II, has reached phase II human trials. Through their centrally mediated activity, melanocortin agonists have potential to treat erectile dysfunction as well as possible applications to the unmet medical needs of decreased sexual motivation and loss of libido.
• Lee, Y. S., Nyberg, J., Moye, S., Agnes, R. S., Davis, P., Ma, S., Lai, J., Porreca, F., Vardanyan, R., & Hruby, V. J. (2007). Understanding the structural requirements of 4-anilidopiperidine analogues for biological activities at μ and δ opioid receptors. Bioorganic and Medicinal Chemistry Letters, 17(8), 2161-2165.
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  PMID: 17329100;PMCID: PMC2274923;Abstract: New 4-anilidopiperidine analogues in which the phenethyl group of fentanyl was replaced by several aromatic ring-contained amino acids (or acids) were synthesized to study the biological effect of the substituents on μ and δ opioid receptor interactions. These analogues showed broad (47 nM-76 μM) but selective (up to 17-fold) binding affinities at the μ opioid receptor over the δ opioid receptor, as predicted from the message-address concept. © 2007 Elsevier Ltd. All rights reserved.
• Mollica, A., Guardiani, G., Davis, P., Ma, S., Porreca, F., Lai, J., Mannina, L., Sobolev, A. P., & Hruby, V. J. (2007). Synthesis of stable and potent δ/μ opioid peptides: Analogues of H-Tyr-c[D-Cys-Gly-Phe-D-Cys]-OH by ring-closing metathesis. Journal of Medicinal Chemistry, 50(13), 3138-3142.
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  PMID: 17539621;PMCID: PMC2274921;Abstract: Ring-closing metathesis has emerged as a powerful tool in organic synthesis for generating cyclic structures via C-C double bond formation. Recently, it has been successfully used in peptide chemistry for obtaining cyclic molecules bridged through an olefin unit in place of the usual disulfide bond. Here, we describe this approach for obtaining cyclic olefin bridged analogues of H-Tyr-c[D-Cys-Gly-Phe-Cys]-OH. The synthesis of the new ligands was performed using the second generation Grubbs' catalyst. The resulting cis-8 (cDADAE) and trans-9 (tDADAE) were fully characterized and tested at δ, μ, and κ opioid receptors. Also the linear precursor 13 (lDADAE) and the hydrogenated derivative 11 (rDADAE) also were tested. All the cyclic products containing a olefmic bond are slightly selective but highly active and potent for the δ and μ opioid receptors. Activity toward the κ opioid receptors was absent or very low. © 2007 American Chemical Society.
• Navratilova, E., Waite, S., Stropova, D., Eaton, M. C., Alves, I. D., Hruby, V. J., Roeske, W. R., Yamamura, H. I., & Varga, E. V. (2007). Quantitative evaluation of human δ opioid receptor desensitization using the operational model of drug action. Molecular Pharmacology, 71(5), 1416-1426.
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  PMID: 17322005;PMCID: PMC2694736;Abstract: Agonist-mediated desensitization of the opioid receptors is thought to function as a protective mechanism against sustained opioid signaling and therefore may prevent the development of opioid tolerance. However, the exact molecular mechanism of opioid receptor desensitization remains unresolved because of difficulties in measuring and interpreting receptor desensitization. In the present study, we investigated deltorphin II-mediated rapid desensitization of the human δ opioid receptors (hDOR) by measuring guanosine 5′-O-(3-[35S]thio)-triphosphate binding and inhibition of cAMP accumulation. We developed a mathematical analysis based on the operational model of agonist action (Black et al., 1985) to calculate the proportion of desensitized receptors. This approach permits a correct analysis of the complex process of functional desensitization by taking into account receptor-effector coupling and the time dependence of agonist pretreatment. Finally, we compared hDOR desensitization with receptor phosphorylation at Ser363, the translocation of β-arrestin2, and hDOR internalization. We found that in Chinese hamster ovary cells expressing the hDOR, deltorphin II treatment leads to phosphorylation of Ser363, translocation of β-arrestin2 to the plasma membrane, receptor internalization, and uncoupling from G proteins. It is noteworthy that mutation of the primary phosphorylation site Ser363 to alanine had virtually no effect on agonist-induced β-arrestin2 translocation and receptor internalization yet significantly attenuated receptor desensitization. These results strongly indicate that phosphorylation of Ser363 is the primary mechanism of hDOR desensitization. Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics.
• Shinoda, K., Hruby, V. J., & Porreca, F. (2007). Antihyperalgesic effects of loperamide in a model of rat neuropathic pain are mediated by peripheral δ-opioid receptors. Neuroscience Letters, 411(2), 143-146.
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  PMID: 17110034;PMCID: PMC1876755;Abstract: The possible antihyperalgesic and antiallodynic activity of loperamide, an opioid agonist which does not readily penetrate the blood-brain barrier, were examined in the spinal nerve ligation model of experimental neuropathic pain. Intraperitoneal (i.p.) injection of loperamide effectively reversed thermal hyperalgesia. In contrast, loperamide had minimal effects on cold allodynia and no effects on mechanical allodynia. The antihyperalgesic action of loperamide against noxious heat was antagonized by naltrindole, a δ-opioid receptor selective antagonist, but not by pretreatment with β-funaltrexamine, a μ-opioid receptor selective antagonist, or administration of nor-binaltorphimine, a κ-opioid receptor selective antagonist. Furthermore, i.p. injection of [d-Ala2, Glu4]-deltorphin II, a δ-opioid receptor selective peptide agonist, also reversed thermal hyperalgesia. The present results suggest that thermal hyperalgesia in experimental neuropathic pain can be reduced through activation of peripheral δ-opioid receptors. The data suggest the possible application of peripherally restricted and δ-opioid receptor selective agonists in the treatment of some aspects of neuropathic pain without many of the side effects associated with centrally acting opioids and without the peripheral side effects of opioid agonists acting at μ-receptors. © 2006.
• Yamamoto, T., Nair, P., Davis, P., Ma, S., Navratilova, E., Moye, S., Tumati, S., Lai, J., Vanderah, T. W., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2007). Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for δ/μ opioid receptor agonists and neurokinin-1 receptor antagonists. Journal of Medicinal Chemistry, 50(12), 2779-2786.
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  PMID: 17516639;PMCID: PMC2365895;Abstract: A series of bifunctional peptides that act as agonists for δ and μ opioid receptors with δ selectivity and as antagonist for neurokinin-1 (NK1) receptors were designed and synthesized for potential application as analgesics in various pain states. The peptides were characterized using radioligand binding assays and functional assays using cell membrane and animal tissue. Optimization was performed on the fifth residue which serves as an address moiety for both receptor recognitions. It had critical effects on both activities at δ/μ opioid receptors and NK1 receptors. Among the synthesized peptides, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2 (5) and H-Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Trp-O-3,5- Bzl(CF3)2 (7) had excellent agonist activity for both δ opioid and μ opioid receptors and excellent antagonist activity for NK1 receptors. These results indicate that the rational design of multifunctional ligands with opioid agonist and neurokinin-1 antagonist activities can be accomplished and may provide a new tool for treatment of chronic and several pain states. © 2007 American Chemical Society.
• Yeon, S. L., Agnes, R. S., Davis, P., Ma, S., Badghisi, H., Lai, J., Porreca, F., & Hruby, V. J. (2007). Partial retro-inverso, retro, and inverso modifications of hydrazide linked bifunctional peptides for opioid and cholecystokinin (CCK) receptors. Journal of Medicinal Chemistry, 50(1), 165-168.
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  PMID: 17201419;PMCID: PMC2365893;Abstract: Partially modified retro-inverso, retro, and inverso isomers of hydrazide linked bifunctional peptides were designed, synthesized, and evaluated for bioactivities at δ/μ opioid receptors and CCK-1/CCK-2 receptors. All modifications of the CCK pharmacophore moiety affected bioactivities for the CCK-1 and CCK-2 receptors (up to 180-fold increase in the binding affinity with higher selectivity) and for the δ and μ opioid receptors. The results indicate that the opioid and CCK pharmacophores in one molecule interact with each other to induce topographical changes for both pharmacophores. © 2007 American Chemical Society.
• Yeon, S. L., Petrov, R., Park, C. K., Ma, S., Davis, P., Lai, J., Porreca, F., Vardanyan, R., & Hruby, V. J. (2007). Development of novel enkephalin analogues that have enhanced opioid activities at both μ and δ opioid receptors. Journal of Medicinal Chemistry, 50(22), 5528-5532.
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  PMID: 17927164;PMCID: PMC2678914;Abstract: Enkephalin analogues with a 4-anilidopiperidine scaffold have been designed and synthesized to achieve therapeutic benefit for the treatment of pain due to mixed μ and δ opioid agonist activities. Ligand 16, in which a Dmt-substituted enkephalin-like structure was linked to the N-phenyl-N- piperidin-4-yl propionamide moiety, showed very high binding affinities (0.4 nM) at μ and δ receptors with an increased hydrophobicity (aLogP = 2.96). This novel lead compound was found to have very potent agonist activities in MVD (1.8 nM) and GPI (8.5 nM) assays. © 2007 American Chemical Society.
• Zhou, M., Nakatani, E., Gronenberg, L. S., Tokimoto, T., Wirth, M. J., Hruby, V. J., Roberts, A., Lynch, R. M., & Ghosh, I. (2007). Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules. Bioconjugate Chemistry, 18(2), 323-332.
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  PMID: 17373766;Abstract: We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight (∼1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of α-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human δ-opioid receptor (hDOR). © 2007 American Chemical Society.
• Agnes, R. S., Lee, Y. S., Davis, P., Ma, S., Badghisi, H., Porreca, F., Lai, J., & Hruby, V. J. (2006). Structure-activity relationships of bifunctional peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors. Journal of Medicinal Chemistry, 49(10), 2868-2875.
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  PMID: 16686530;PMCID: PMC1484468;Abstract: Cholecystokinin (CCK) has been identified as a pronociceptive endogenous peptide which also possesses antiopioid actions. CCK may be upregulated in conditions of chronic pain or during sustained morphine administration resulting in attenuation of opioid-mediated pain relief. These complex interactions between opioids and endogenous CCK receptor systems have suggested the need for a new paradigm in drug design for some states of chronic pain. In these circumstances the rational design of potential drugs for the treatment of these conditions must be based on one ligand for multiple targets. We have designed a single peptide which can interact with δ and μ opioid receptors as agonists and with CCK receptors as antagonists. The ligands were designed based on a model of overlapping pharmacophores of opioid and CCK peptide ligands, which incorporates opioid pharmacophores at the N-terminal and CCK tetrapeptide pharmacophores at the C-terminal of the designed ligands. We measured binding and activities of our bifunctional peptides at opioid and CCK receptors. Compound 11 (Tyr-D-Ala-Gly-D-Trp-NMeNle-Asp-Phe-NH2) demonstrated opioid agonist properties at δ and μ receptors (IC50 = 63 ± 27 nM and 150 ± 65 nM, respectively in MVD and GPI tissue assays) and high binding affinity at CCK-1 and CCK-2 receptors (Ki = 320 and 1.5 nM, respectively). Compound 9 (Tyr-D-Nle-Gly-Trp-Nle-Asp-Phe-NH 2) displayed potent agonist activity at δ and μ receptors (IC50 = 23 ± 10 nM and 210 ± 52 nM, respectively in MVD and GPI tissue assays), with a balanced binding affinity for CCK-1 and CCK-2 receptors (Ki = 9.6 and 15 nM, respectively). These results provide evidence supporting the concept that opioid and CCK receptors have overlapping pharmacophores required for binding affinity and biological activity and that designing overlapping pharmacophores of two peptides into a single peptide is a valid drug design approach. © 2006 American Chemical Society.
• Alves, I. D., Cowell, S. M., Salamon, Z., Devanathan, S., Tollin, G., & Hruby, V. J. (2006). Erratum: Different structural states of the proteolipid membrane are produced by ligand binding to the human δ-opioid receptor as shown by plasmon-waveguide resonance spectroscopy (Molecular Pharmacology (2004) 65 (1248-1257)). Molecular Pharmacology, 69(1), 406-.
• Alves, I. D., Delaroche, D., Mouillac, B., Salamon, Z., Tollin, G., Hruby, V. J., Lavielle, S., & Sagan, S. (2006). The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy. Biochemistry, 45(16), 5309-5318.
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  PMID: 16618119;PMCID: PMC1865500;Abstract: Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O2)11]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (Kd1 = 0.14 ± 0.02 nM and Kd2 = 1.4 ± 0.18 nM, and Kd1 = 5.5 ± 0.7 nM and Kd2 = 620 ± 117 nM, respectively), whereas only one binding affinity (K d = 5.5 ± 0.4 nM) could be observed for propionyl[Met(O 2)11]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h). © 2006 American Chemical Society.
• Alves, I. D., Salamon, Z., Hruby, V. J., & Tollin, G. (2006). Erratum: Ligand modulation of lateral segregation of a G-protein-coupled receptor into lipid microdomains in sphingomyelin/phosphatidylcholine solid-supported bilayers (Biochemistry (June 28, 2005) 44, 25 (9168-9178)). Biochemistry, 45(12), 4044-.
• Alves, I. D., Salgado, G. F., Salamon, Z., Brown, M. F., Tollin, G., & Hruby, V. J. (2006). Erratum: Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy (Biophysical Journal (2005) 88, (198-210)). Biophysical Journal, 90(2), 709-.
• Cai, M., Varga, E. V., Stankova, M., Mayorov, A., Perry, J. W., Yamamura, H. I., Trivedi, D., & Hruby, V. J. (2006). Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: Differentiation of agonists and antagonists. Chemical Biology and Drug Design, 68(4), 183-193.
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  PMID: 17105482;PMCID: PMC2547351;Abstract: Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen. © 2006 The Authors.
• Cain, J. P., Mayorov, A. V., Cai, M., Wang, H., Tan, B., Chandler, K., Lee, Y., Petrov, R. R., Trivedi, D., & Hruby, V. J. (2006). Design, synthesis, and biological evaluation of a new class of small molecule peptide mimetics targeting the melanocortin receptors. Bioorganic and Medicinal Chemistry Letters, 16(20), 5462-5467.
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  PMID: 16931008;PMCID: PMC1810397;Abstract: A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported. © 2006 Elsevier Ltd. All rights reserved.
• Carducci, M. D., Xuyuan, G. u., Cole, J. R., & Hruby, V. J. (2006). [N-({(R)-2-[(N-Benzylprolyl)amino]-phenyl}phenylmethylene)-2(S) -(pent-4-enyl)glycinato]nickel(II). Acta Crystallographica Section E: Structure Reports Online, 62(6), m1219-m1220.
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  Abstract: The title compound, [Ni(C32H33N3O 3)], crystallized as a minor product during the purification of its 2(R)-pent-4-enyl diastereomer. Mixtures of the title compound and its enantiomer self-resolve. © 2006 International Union of Crystallography. All rights reserved.
• Grieco, P., Cai, M., Mayorov, A. V., Trivedi, D., & Hruby, V. J. (2006). Structure-activity studies of new melanocortin peptides containing an aromatic amino acid at the N-terminal position. Peptides, 27(2), 472-481.
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  PMID: 16303211;PMCID: PMC1483901;Abstract: Cyclic melanotropin peptides, designed with an aromatic amino acid substitution at the N-terminal position of the MT-II-type scaffold, were prepared by solid-phase peptide synthesis and evaluated for their ability to bind to and activate human melanocortin-1, -3, -4, and -5 receptors. The structure-activity studies of these MT-II analogues have identified a selective antagonist at the hMC4R (H-Phe-c[Asp-Pro-d-Nal(2′)-Arg-Trp-Gly-Lys]- NH2, pA2 = 8.7), a selective partial agonist at the hMC4R (H-d-Nal(2′)-c[Asp-Pro-d-Phe-Arg-Trp-Gly-Lys]-NH2, IC 50 = 11 nM, EC50 = 56 nM), and a selective partial agonist at the hMC3R (H-d-Phe-c[Asp-Pro-d-Phe-Arg-Trp-Lys]-NH2, IC 50 = 3.7 nM, EC50 = 4.9 nM). Aromatic amino acid substitution at the N-terminus in conjuction with the expansion of the 23-membered cyclic lactam MT-II scaffold to a 26-membered scaffold by addition of a Gly residue in position 10 leads to melanotropin peptides with enhanced receptor selectivity. © 2005 Elsevier Inc. All rights reserved.
• Hen, G., Yosefi, S., Simchaev, V., Shinder, D., Hruby, V. J., & Friedman-Einat, M. (2006). The melanocortin circuit in obese and lean strains of chicks. Journal of Endocrinology, 190(2), 527-535.
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  PMID: 16899585;PMCID: PMC2730167;Abstract: Agonists of membranal melanocortin 3 and 4 receptors (MC3/4Rs) are known to take part in the complex control mechanism of energy balance. In this study, we compared the physiological response to an exogenous MC3/4R agonist and the hypothalamic expression of proopic melanocortin (POMC) gene, encoding few MC3/4R ligands, between broiler and layer chicken strains. These strains, representing the two most prominent commercial strains of chickens grown for meat (broilers) and egg production (layers), differ in their food intake, fat accumulation, and reproductive performance and, therefore, form a good model of obese and lean phenotypes, respectively. A single i.v. injection of the synthetic peptide melanotan-II (MT-II; 1 mg/kg body weight) into the wing vein of feed-restricted birds led to attenuation of food intake upon exposure to feeding ad libitum in both broiler and layer chickens. A study of the POMC mRNA encoding the two prominent natural MC3/4R agonists, α-MSH and ACTH, also revealed a general similarity between the strains. Under feeding conditions ad libitum, POMC mRNA levels were highly similar in chicks of both strains and this level was significantly reduced upon feed restriction. However, POMC mRNA down-regulation upon feed restriction was more pronounced in layers than in broilers. These results suggest: (i) a role for MC3/4R, agonists in the control of appetite; (ii) that the physiological differences between broilers and layers are not related to unresponsiveness of broiler chickens to the satiety signal of MC3/4R ligands. Therefore, these findings suggest that artificial activation of this circuit in broiler chicks could help to accommodate with their agricultural shortcomings of overeating, fattening, and impaired reproduction. © 2006 Society for Endocrinology.
• Hongchang, Q. u., Carducci, M. D., Nichol, G. S., & Hruby, V. J. (2006). (1R,2S)-Benzyl N-[3-(diisopropylamino-carbonyl)-2-phenylprop-3-enyl] carbamate. Acta Crystallographica Section E: Structure Reports Online, 62(9), o3921-o3922.
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  Abstract: The title compound, C25H32N2O3, was synthesized as part of a series of related compounds using a modified Eschenmoser-Claisen rearrangement reaction. The compound is racemic and the structure features a centrosymmetric hydrogen-bonded dimerization along with some aromatic stacking stabilization. © 2006 International Union of Crystallography. All rights reserved.
• Hongchang, Q. u., Xuyuan, G. u., Min, B. J., Liu, Z., & Hruby, V. J. (2006). Synthesis of anti-β-substituted γ,δ-unsaturated amino acids via Eschenmoser-Claisen rearrangement. Organic Letters, 8(19), 4215-4218.
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  PMID: 16956190;PMCID: PMC2366803;Abstract: (Chemical Equation Presented) Anti-β-substituted γ,δ- unsaturated amino acids have been synthesized via a novel design of the Eschenmoser-Claisen rearrangement. The rearrangement gives good isolated yields and excellent diastereoselectivity due to (Z)-N,O-ketene acetal formation and the pseudochairlike conformations of the reaction intermediates. Upon reductive hydrolysis, important novel amino acids and amino alcohols were synthesized for the first time via this methodology. © 2006 American Chemical Society.
• Hruby, V. J., & Vagner, J. (2006). High throughput synthesis of peptides and peptidomimetics. Chimica Oggi, 24(4), 18-21.
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  Abstract: Peptide synthesis has been developed into one of the most efficient synthetic procedures in organic chemistry. The problems of orthogonal functional group protection and amide bond formation without racemization have been developed in a number of ingenious strategies. Optimization, in particular, has been achieved in stepwise solid phase synthesis. This in turn made possible the development of combinatorial synthesis allowing the synthesis of millions of peptide compounds of high purity in a few days. A variety of methodologies and strategies have been developed and continue to be developed to determine structures and to evaluate peptides and peptidomimetics. The development of methods for solid phase synthesis of a variety of organic and inorganic structures using similar strategies as in peptide synthesis are being vigorously pursued. However, existing instrumentation and technology is not sufficient to cover current demands for peptides, and thus new approaches and technologies for cost-effective synthesis of peptide arrays are needed.
• Hruby, V. J., Porreca, F., Yamamura, H. I., Tollin, G., Agnes, R. S., Lee, Y. S., Cai, M., Alves, I., Cowell, S., Varga, E., Davis, P., Salamon, Z., Roeske, W., Vanderah, T., & Lai, J. (2006). New paradigms and tools in drug design for pain and addiction. AAPS Journal, 8(3), E450-E460.
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  PMID: 17025262;PMCID: PMC1764851;Abstract: New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
• Lee, Y. S., Agnes, R. S., Badghisi, H., Davis, P., Ma, S., Lai, J., Porreca, F., & Hruby, V. J. (2006). Design and synthesis of novel hydrazide-linked bifunctional peptides as δ/μ opioid receptor agonists and CCK-1/CCK-2 receptor antagonists. Journal of Medicinal Chemistry, 49(5), 1773-1780.
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  PMID: 16509592;PMCID: PMC1614704;Abstract: A series of hydrazide-linked bifunctional peptides designed to act as agonists for δ/μ opioid receptors and antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors and in functional assays. SAR studies in the CCK region examined the structural requirements for the side chain groups at positions 1′, 2′, and 4′ and for the N-terminal protecting group, which are related to interactions not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities (0.1 - 10 nM) for both δ and μ opioid receptors generally showed lower binding affinities (micromolar range) at CCK-1 and CCK-2 receptors, but were potent CCK receptor antagonists in the GPI/LMMP assay (up to Ke = 6.5 nM). The results indicate that it is reasonable to design chimeric bifunctional peptide ligands for different G-protein coupled receptors in a single molecule. © 2006 American Chemical Society.
• Marks, D. L., Hruby, V., Brookhart, G., & Cone, R. D. (2006). The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R). Peptides, 27(2), 259-264.
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  PMID: 16274853;PMCID: PMC1679957;Abstract: High levels of binding sites for melanocortin peptides exist within the arcuate nucleus, and a functional response to melanocortin peptides has been demonstrated in arcuate POMC neurons. Because the MC3R is thought to function as an inhibitory autoreceptor on POMC neurons, we reasoned that peripheral injections of MC3R-specific agonists would act within the arcuate nucleus to inhibit POMC neurons and thereby stimulate feeding. We demonstrate that the peptidergic MC3R agonist, d-Trp8-γ-MSH, stimulates feeding via the MC3R when injected peripherally. These data provide the first evidence that feeding can be stimulated by peripheral injection of MC3R-specific agonists. © 2005 Elsevier Inc. All rights reserved.
• Mayorov, A. V., Cai, M., Chandler, K. B., Petrov, R. R., R., A., Zerui, Y. u., Tanaka, D. K., Trivedi, D., & Hruby, V. J. (2006). Development of cyclic γ-MSH analogues with selective hHMC3R agonist and hMC3R/hMC5R antagonist activities. Journal of Medicinal Chemistry, 49(6), 1946-1952.
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  PMID: 16539382;PMCID: PMC1484467;Abstract: A series of cyclic lactam analogues of γ-MSH (H-Tyr 1-Val2-Met3-Gly4-His 5-Phe6-Arg7-Trp8-Asp 9-Arg10-Phe11-Gly12-OH) with a bulky hydrophobic residue in the direct proximity to the pharmacophore (Xaa-D-Phe/D-Nal(2′)-Arg-Trp) were designed and synthesized by solid-phase methods. A variety of amino acids with a broad range of hydrophobic/hydrophilic properties was introduced in position 5 to further explore their complementary role in receptor selectivity. Biological evaluation of these peptides revealed several analogues with potent hMC3R agonist and hMC3R/hMC5R antagonist activities, and good receptor selectivity. Analogue 4, c[Nle-Arg-D-Phe-Arg-Trp- Glu]-NH2, was found to be a very potent and selective hMC3R agonist (EC50 = 1.2 nM, 112% act). In addition, analogue 13, c[Nle-Val-D-Nal(2′)-Arg-Trp-Glu]-NH2, was identified as an hMC3R/hMC5R antagonist with the best selectivity against the hMC4R in this series (pA2(hMC3R) = 8.4; pA2(hMC5R) = 8.7). These results indicate the significance of steric factors in melanocortin receptor selectivity and suggest that introduction of bulky residues in the direct proximity to the melanocortin pharmacophore is an effective approach to design of novel hMC3R and hMC5R selective ligands. © 2006 American Chemical Society.
• Mayorov, A. V., Han, S., Cai, M., Hammer, M. R., Trivedi, D., & Hruby, V. J. (2006). Effects of macrocycle size and rigidity on melanocortin receptor-1 and -5 selectivity in cyclic lactam α-melanocyte-stimulating hormone analogs. Chemical Biology and Drug Design, 67(5), 329-335.
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  PMID: 16784457;PMCID: PMC1851940;Abstract: The effects of the linker arm rigidity and size on melanocortin receptor selectivity were explored in a series of compounds using cyclic lactam α-melanocyte-stimulating hormone template. A variety of dicarboxylic acid linkers introduced between the α-amino group of His6 and the ε-amino group of Lys10 lead to high-affinity, selective human melanocortin receptor-1 and -5 (hMC1R and hMC5R) antagonists. The incorporation of hydrophilic functions into the linker arm was found to be unfavorable for both binding potency and receptor selectivity. Analogs 8 and 9 containing highly conformationally constrained hydrophobic linkers (m- and p-phthalic acids) were found to be selective nanomolar range hMC1R antagonists (IC50 = 7 and 4 nm, respectively), whereas the employment of a small conformationally constrained linker (maleic acid) resulted in a high-affinity (IC50 = 19 nm) and selective hMC5R antagonist (analog 12). These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-1 and -5 receptors. © 2006 The Authors.
• Mollica, A., Davis, P., Ma, S., Porreca, F., Lai, J., & Hruby, V. J. (2006). Synthesis and biological activity of the first cyclic biphalin analogues. Bioorganic and Medicinal Chemistry Letters, 16(2), 367-372.
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  PMID: 16275086;Abstract: Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH ← Phe ← Gly ← D-Ala ← Tyr). In this study we present the design, synthesis, and biological evaluation of the first cyclic biphalin analogues. d-Alanine residues in positions 2, 2′ of the parent peptide were replaced by d- and l-cysteine and an intramolecular disulfide bond between the cysteine thiol groups was introduced. We obtained two cyclic analogues with quite different biological profiles. © 2005 Elsevier Ltd. All rights reserved.
• Ndungu, J. M., Cain, J. P., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2006). Synthesis of constrained analogues of cholecystokinin/opioid chimeric peptides. Tetrahedron Letters, 47(13), 2233-2236.
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  Abstract: In our ongoing research on the synthesis of constrained analogues of CCK/opioid chimeric peptides, a bicyclic dipeptide mimetic for Nle-Asp was designed and synthesized. Starting from β-allyl substituted aspartic acids, the terminal double bond was oxidized resulting in spontaneous cyclization to form racemic hemiaminals. Allylation of the hemiaminals afforded 5-allyl substituted proline analogues, which on oxidation, Horner-Emmons olefination, asymmetric hydrogenation, and bicyclization afforded bicyclic dipeptide mimetics for Nle-Asp. Constrained CCK/opioid peptide analogues containing bicyclic dipeptide mimetics for Nle-Gly, Nle-Asp, and homoPhe-Gly were then synthesized and analyzed at both the CCK and opioid receptors. © 2006 Elsevier Ltd. All rights reserved.
• Petrov, R. R., Vardanyan, R. S., Lee, Y. S., Ma, S., Davis, P., Begay, L. J., Lai, J. Y., Porreca, F., & Hruby, V. J. (2006). Synthesis and evaluation of 3-aminopropionyl substituted fentanyl analogues for opioid activity. Bioorganic and Medicinal Chemistry Letters, 16(18), 4946-4950.
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  PMID: 16828552;PMCID: PMC1783977;Abstract: An enkephalin analogue coupled to 'aminofentanyl' has been synthesized and tested for biological activities at the μ and δ opioid receptors. Aminofentanyl which represents a structural derivative of fentanyl has been synthesized by acylation of 1-(2-phenethyl)-4-(N-anilino)piperidine with phthaloyl protected β-alaninyl chloride in the presence of DIPEA, followed by deprotection with hydrazine hydrate. Aminofentanyl has also been successfully acylated with ethyl isocyanate, various acid anhydrides, to further investigate structure-activity relationships of these new fentanyl derivatives. Among the new derivatives compound 7 which carries a Tyr-d-Ala-Gly-Phe opioid message sequence showed good opioid affinity (1 nM at both δ and μ opioid receptors) and bioactivity (34.9 nM in MVD and 42 nM in GPI/LMMP bioassays). © 2006 Elsevier Ltd. All rights reserved.
• Petrov, R. R., Vardanyan, R. S., Nichol, G. S., Carducci, M. D., Ma, S., Lai, J. Y., & Hruby, V. J. (2006). 4-{N-[2-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-3-phenylpropionyl]anilino} -1-phenethylpiperidinium chloride methanol disolvate. Acta Crystallographica Section E: Structure Reports Online, 62(7), o2815-o2816.
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  Abstract: The structure of the title compound, a fentanyl derivative with formula C36H36N3O3+· Cl-·CH3OH, crystallizes as a racemic mixture. The organic cation has an extended conformation and the structure displays O - H⋯-O, O - H⋯Cl and N -H⋯Cl hydrogen bonding. © 2006 International Union of Crystallography. All rights reserved.
• Vagner, J., Handl, H. L., Monguchi, Y., Jana, U., Begay, L. J., Mash, E. A., Hruby, V. J., & Gillies, R. J. (2006). Rigid linkers for bioactive peptides. Bioconjugate Chemistry, 17(6), 1545-1550.
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  PMID: 17105235;PMCID: PMC2743982;Abstract: Rigid linkers of variable length were used to connect two high-affinity Nle4-D-Phe7-α-melanocyte stimulating hormone (NDP-α-MSH) or two low-affinity MSH(4) ligands. The linked peptides were synthesized by solid-phase methods. Control experiments indicate there is little or no effect of these linkers on NDP-α-MSH or MSH-(4) binding to the human melanocortin 4 receptor (hMC4R). Tethering two high-affinity ligands gave no binding enhancement, while tethering two low-affinity ligands resulted in binding enhancement that decreased with increased linker length. Furthermore, for the low-affinity ligands, the enhancement of affinity is inversely proportional to the estimated molecular moments of inertia. These results are consistent with a model wherein binding is enhanced when the rate of ligand reattachment to the receptor is fast relative to the rate of ligand diffusion. © 2006 American Chemical Society.
• Vardanyan, R. S., & Hruby, V. J. (2006). Synthesis of Essential Drugs. Synthesis of Essential Drugs.
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  Abstract: This book represents an effort to express a long overdue need of compiling information which has been accumulated over the course of more than 30 years of our work in the area of the synthesis of medicinal drugs. Our effort fills obvious gaps that exist in the literature of drug synthesis. The synthesis of various groups of drugs are presented in an order similar to that traditionally presented in a pharmacology curriculum. This was done with a very specific goal in mind - to harmonize the chemical aspects with the pharmacology curriculum in a manner useful to chemists. Practically every chapter begins with an accepted brief definition and description of a particular group of drugs, proposes their classification, and briefly explains the present model of their action. This is followed by a detailed description of methods for their synthesis. Of the thousands of drugs existing on the pharmaceutical market, we mainly discuss generic drugs that are included in the WHO's "Essential List of Drugs." For practically all of the 700+ drugs described in the book, references (around 2350) to the methods of their synthesis are given along with the most widespread synonyms. This book will provide the kind of information that will be of interest to those who work, or plan to begin work in the captivating areas of biologically active compounds and the synthesis of medicinal drugs. * Provides a brief description of methods of synthesis, activity and implementation of all drug types * Includes synonyms * Includes over 2300 references. © 2006 Elsevier B.V. All rights reserved.
• Ying, J., Xuyuan, G. u., Cai, M., Dedek, M., Vagner, J., Trivedi, D. B., & Hruby, V. J. (2006). Design, synthesis, and biological evaluation of new cyclic melanotropin peptide analogues selective for the human melanocortin-4 receptor. Journal of Medicinal Chemistry, 49(23), 6888-6896.
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  PMID: 17154518;PMCID: PMC1764620;Abstract: Intensive efforts have been made to develop potent and selective ligands for certain human melanocortin receptors as possible treatments for obesity and sexual dysfunction due to the role of these receptors in feeding behavior, energy homeostasis, sexual function, etc. A number of novel α-MSH analogues were designed and synthesized primarily on the basis of our previous MTII NMR structure. In these peptide analogues, a disulfide or lactam bridge between residues at positions 5 and 8 was used as a conformational constraint to enhance the β-turn spanning His6 and D-Phe7, while the pharmacophore group in Arg8 was mimicked via N α-alkylation of residues 8 or 9 with the guanidinylbutyl group. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues have good binding affinity for the hMC4R (0.7-4.1 nM), but have no binding affinity up to 10 μM at the other three melanocortin receptors. Interestingly, the three hMC4R selective analogues display only 50% binding efficiency, suggesting there is allosteric modulation of the melanocortin-4 receptor. These analogues were found to act as antagonists of the hMC4R. This result represents a discovery of very selective peptide-based antagonists for the hMC4R. The high selectivity may be due to the strong conformational constraint via ring contraction as compared to MTII, and the rigid conformation preferred by these new ligands allows them to recognize only the hMC4R, but not to activate the second messenger. The MTII NMR structure-based design thus not only examined the structural model of melanocortin ligands, but also yielded new biologically unique α-MSH analogues. © 2006 American Chemical Society.
• Alves, I. D., Ciano, K. A., Boguslavski, V., Varga, E., Salamon, Z., Yamamura, H. I., Hruby, V. J., & Tollin, G. (2005). Erratum: Selectivity, cooperativity, and reciprocity in the interactions between the δ-opioid receptor, its ligands, and G-proteins (Journal of Biological Chemistry (2004) 279 (44673-44682)). Journal of Biological Chemistry, 280(48), 40384-.
• Alves, I. D., F., G., Salamon, Z., Brown, M. F., Tollin, G., & Hruby, V. J. (2005). Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy. Biophysical Journal, 88(1), 198-210.
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  PMID: 15501933;PMCID: PMC1304998;Abstract: Flash photolysis studies have shown that the membrane lipid environment strongly influences the ability of rhodopsin to form the key metarhodopsin II intermediate. Here we have used plasmon-waveguide resonance (PWR) spectroscopy, an optical method sensitive to both mass and conformation, to probe the effects of lipid composition on conformational changes of rhodopsin induced by light and due to binding and activation of transducin (Gt). Octylglucoside-solubilized rhodopsin was incorporated by detergent dilution into solid-supported bilayers composed either of egg phosphatidylcholine or various mixtures of a nonlamellar-forming lipid (dioleoylphosphatidylethanolamine; DOPE) together with a lamellar-forming lipid (dioleoylphosphatidylcholine; DOPC). Light-induced proteolipid conformational changes as a function of pH correlated well with previous flash photolysis studies, indicating that the PWR spectral shifts monitored metarhodopsin II formation. The magnitude of these effects, and hence the extent of the conformational transition, was found to be proportional to the DOPE content. Our data are consistent with previous suggestions that lipids having a negative spontaneous curvature favor elongation of rhodopsin during the activation process. In addition, measurements of the G t/rhodopsin interaction in a DOPC/DOPE (25:75) bilayer at pH 5 demonstrated that light activation increased the affinity for Gt from 64 nM to 0.7 nM, whereas Gt affinity for dark-adapted rhodopsin was unchanged. By contrast, in DOPC bilayers the affinity of Gt for light-activated rhodopsin was only 18 nM at pH 5. Moreover exchange of GDP for GTPγS was also monitored by PWR spectroscopy. Only the light-activated receptor was able to induce this exchange which was unaffected by DOPE incorporation. These findings demonstrate that nonbilayer-forming lipids can alter functionally linked conformational changes of G-protein-coupled receptors in membranes, as well as their interactions with downstream effector proteins.
• Alves, I. D., Park, C. K., & Hruby, V. J. (2005). Plasmon resonance methods in GPCR signaling and other membrane events. Current Protein and Peptide Science, 6(4), 293-312.
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  PMID: 16101432;PMCID: PMC1357018;Abstract: The existence of surface guided electromagnetic waves has been theoretically predicted from Maxwell's equations and investigated during the first decades of the 20th century. However, it is only since the late 1960's that they have attracted the interest of surface physicists and earned the moniker of "surface plasmon". With the advent of commercially available instruments and well established theories, the technique has been used to study a wide variety of biochemical and biotechnological phenomena. Spectral response of the resonance condition serves as a sensitive indicator of the optical properties of thin films immobilized within a wavelength of the surface. This enhanced surface sensitivity has provided a boon to the surface sciences, and fosters collaboration between surface chemistry, physics and the ongoing biological and biotechnological revolution. Since then, techniques based on surface plasmons such as Surface Plasmon Resonance (SPR), SPR Imaging, Plasmon Waveguide Resonance (PWR) and others, have been increasingly used to determine the affinity and kinetics of a wide variety of real time molecular interactions such as protein-protein, lipid-protein and ligand-protein, without the need for a molecular tag or label. The physical-chemical methodologies used to immobilize membranes at the surface of these optical devices are reviewed, pointing out advantages and limitations of each method. The paper serves to summarize both historical and more recent developments of these technologies for investigating structure-function aspects of these molecular interactions, and regulation of specific events in signal transduction by G-protein coupled receptors (GPCRs). © 2005 Bentham Science Publishers Ltd.
• Alves, I. D., Salamon, Z., Hruby, V. J., & Tollin, G. (2005). Ligand modulation of lateral segregation of a G-protein-coupled receptor into lipid microdomains in sphingomyelin/phosphatidylcholine solid-supported bilayers. Biochemistry, 44(25), 9168-9178.
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  PMID: 15966741;PMCID: PMC1440485;Abstract: A growing body of evidence supports the idea that the plasma membrane bilayer is characterized by a laterally inhomogeneous mixture of lipids, having an organized structure in which lipid molecules segregate into small domains or patches. Such microdomains are characterized by high contents of sphingolipids that form thicker liquid-ordered regions that are resistant to extraction with nonionic detergents. The existence of lipid lateral segregation has been demonstrated in both model and biological membranes, although its role in protein sorting and membrane function still remains unclear. In these studies, plasmon-waveguide resonance (PWR) spectroscopy was employed to investigate the properties of microdomains in a model system consisting of a solid-supported lipid bilayer composed of a 1:1 mixture of palmitoyloleoylphosphatidylcholine (POPC) and brain sphingomyelin (SM), and their influence on the partitioning and functioning of the human delta opioid receptor (hDOR), a G-protein coupled receptor (GPCR). Resonance signals corresponding to two microdomains (POPC-rich and SM-rich) were observed in such bilayers, and the sorting of the receptor into each domain was highly dependent on the type of ligand that was bound. When no ligand was bound, the receptor was incorporated preferentially into the POPC-rich domain; when an agonist or antagonist was bound, the receptor was incorporated preferentially into the SM-rich component, although with a 2-fold greater propensity for this microdomain in the case of the agonist. Binding of G-protein to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than binding to the receptor in the PC-rich domain. The binding of the agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich to the SM-rich component. Since the SM-rich domain is thicker than the PC-rich domain, and previous studies with the hDOR have shown that the receptor is elongated upon agonist activation, we propose that hydrophobic matching between the receptor and the lipid is a driving force for receptor trafficking to the SM-rich component. © 2005 American Chemical Society.
• Alves, I. D., Salamon, Z., Varga, E., Yamamura, H. I., Tollin, G., & Hruby, V. J. (2005). Erratum: Direct observation of G-protein binding to the human δ-opioid receptor using plasmon-waveguide resonance spectroscopy (Journal of Biological Chemistry (2003) 278 (48890-48897)). Journal of Biological Chemistry, 280(52), 43280-.
• Cai, M., Mayorov, A. V., Cabello, C., Stankova, M., Trivedi, D., & Hruby, V. J. (2005). Novel 3D pharmacophore of α-MSH/γ-MSH hybrids leads to selective human MC1R and MC3R analogues. Journal of Medicinal Chemistry, 48(6), 1839-1848.
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  PMID: 15771429;Abstract: To further evaluate elements that could contribute to the 3D topographical structure of γ-MSH, we have systematically designed a group of linear γ-MSH analogues and evaluated their biological activities: without a N-terminal acetyl, with and without a C-terminal amide, with Nle3, with L- or D-Phe6 or D-Nal(2′)6, and with D-Trp 8 or D-Nal(2′)8. It was found that changing the C-terminal acid in γ-MSH to an amide and replacing Met with Nle leads to increased binding affinities at all four subtypes of melanocortin receptors (10-100 fold). Substitution of Trp8 with D-Nal(2′)8 and Phe6 with D-Phe6 in γ-MSH-NH2 forms a selective antagonist for the hMC3R, whereas, substitution of Phe6 with D-Nal(2′)6 and replacing Trp8 with D-Trp 8 at γ-MSH-NH2 yields a selective partial agonist for the hMC1R. Finally, substitution of His5 with Pro5 and Trp8 with D-Nal(2′)8 in γ-MSH-NH2 leads to a highly potent and selective agonist for the hMC1R. Molecular modeling showed that, at the C-terminal of Nle3-γ-MSH-NH2, there is a reverse-turn-like structure, suggesting that there might be a secondary binding site involved in ligand - receptor interaction for γ-MSH analogues that may explain the enhanced binding affinities of the Nle 3-γ-MSH-NH2 analogues. Our results indicate that increasing the hydrophobicity and replacing Phe6 and Trp8 with bulkier aromatic amino acid residues is very important for selectivity of α-MSH/γ-MSH hybrids for hMCRs. © 2005 American Chemical Society.
• Cai, M., Mayorov, A. V., Ying, J., Stankova, M., Trivedi, D., Cabello, C., & Hruby, V. J. (2005). Design of novel melanotropin agonists and antagonists with high potency and selectivity for human melanocortin receptors. Peptides, 26(8), 1481-1485.
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  PMID: 15876475;Abstract: α-MSH and γ-MSH are the natural endogenous hormones for the human melanocortin-1, 3, 4 and 5 receptors (hMC1R, hMC3R, hMC4R and hMC5R). These and more potent, stable and prolonged acting analogues such as NDP-α-MSH, MT-II and SHU-9119 are not very receptor selective. To develop potent and selective agonist and antagonist ligands for the melanocortin receptors we have used state-of-the-art biophysical studies, computational chemistry, and design of conformational and topographical constraints with novel templates. © 2005 Elsevier Inc. All rights reserved.
• Devanathan, S., Salamon, Z., Nagar, A., Narang, S., Schleich, D., Darman, P., Hruby, V., & Tollin, G. (2005). Subpicomolar sensing of δ-opioid receptor ligands by molecular-imprinted polymers using plasmon-waveguide resonance spectroscopy. Analytical Chemistry, 77(8), 2569-2574.
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  PMID: 15828795;Abstract: Here we report, for the first time, the formation of a biomimetic covalentiy imprinted polymeric sensor for a target ligand, the δ-opioid G-protein coupled receptor agonist DPDPE, which reproducibly exhibits subpicomolar binding affinity in an aqueous environment. In addition to having a well-defined and homogeneous binding site, the imprinted polymer template is quite stable to storage in both the dry and wet states and has at least 6 orders of magnitude higher affinities than exhibited by similar peptide-based molecular-imprinted polymers (MIPs) thus far. A highly sensitive optical detection methodology, plasmon-waveguide resonance spectroscopy, was employed, capable of measuring binding in real time and discriminating between ligand molecules, without requiring labeling protocols (fluorophores or radioisotopes). The DPDPE-imprinted polymer showed a broad structure-activity relationship profile, not unlike that found for protein receptors. Such sensitivity and robustness of MIPs suggests potential applications ranging from biowarfare agent detection to pharmaceutical screening. © 2005 American Chemical Society.
• Dhanasekaran, M., Palian, M. M., Alves, I., Yeomans, L., Keyari, C. M., Davis, P., Bilsky, E. J., Egleton, R. D., Yamamura, H. I., Jacobsen, N. E., Tollin, G., Hruby, V. J., Porreca, F., & Polt, R. (2005). Glycopeptides related to β-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers. Journal of the American Chemical Society, 127(15), 5435-5448.
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  PMID: 15826181;Abstract: A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (KD) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis). © 2005 American Chemical Society.
• Fung, S., & Hruby, V. J. (2005). Design of cyclic and other templates for potent and selective peptide α-MSH analogues. Current Opinion in Chemical Biology, 9(4), 352-358.
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  PMID: 16023401;PMCID: PMC1820743;Abstract: For over three decades, the design of linear peptide ligands often has incorporated cyclic constraints to improve potency, receptor selectivity, proteolytic stability and biodistribution. Its importance has been so well established that modern day schemes for ligand-based drug design often start with cyclization of linear peptides to rigidify peptide structure, to limit its conformational possibilities, and to find key pharmacophore elements in three-dimensional space. In the past several years, cyclic constraints have been used to develop ligands with improved efficacy, binding affinity, biostability and receptor selectivity for α-melanocyte-stimulating hormone (α-MSH). Furthermore, potent cyclic α-MSH analogues, such as MT-II and SHU-9119, have made structure-activity relationship studies and molecular modeling more useful for creating new three-dimensional, topographical pharmacophore templates. © 2005 Elsevier Ltd. All rights reserved.
• Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J., & Gillies, R. J. (2005). Development of a lanthanide-based assay for detection of receptor-ligand interactions at the δ-opioid receptor. Analytical Biochemistry, 343(2), 299-307.
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  PMID: 16004955;Abstract: A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested five peptide ligands for the δ-opioid receptor that were modified with a europium (Eu)-containing chelate. These labeled ligands were tested for their binding affinities and compared with the unlabeled parental ligands. The Eu-diethylenetriaminepentaacetic acid (DTPA)-[d-Pen2,l-Cys 5] enkephalin (DPLCE) ligand bound to Chinese hamster ovary (CHO) cells overexpressing the human δ-opioid receptor with affinity similar to the unlabeled ligand. This ligand was used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. These lanthanide-based assays provide superior results with higher throughput and eliminate the need for radioactive waste disposal; hence, they are appropriate for high-throughput screening of ligand libraries. © 2005 Elsevier Inc. All rights reserved.
• Hruby, V. J. (2005). On the topic of peptide research and drug discovery. Journal of Peptide Research, 66(6), 309-310.
• Hruby, V. J., & Lawson, S. (2005). Where are the science heroes? [3] (multiple letters). Scientist, 19(17), 8-.
• Hsu, R., Taylor, J. R., Newton, S. S., Alvaro, J. D., Haile, C., Han, G., Hruby, V. J., Nestler, E. J., & Duman, R. S. (2005). Blockade of melanocortin transmission inhibits cocaine reward. European Journal of Neuroscience, 21(8), 2233-2242.
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  PMID: 15869520;PMCID: PMC2694749;Abstract: Melanocortins and the melanocortin-4 receptor (MC4-R) are enriched in the nucleus accumbens, a brain region that has been implicated in the rewarding action of cocaine and other drugs of abuse. In the present study we use a number of rat behavioral models to show that infusion of a melanocortin peptide antagonist into the nucleus accumbens blocks the reinforcing, incentive motivational, and locomotor sensitizing effects of cocaine. We also show that locomotor responses to repeated cocaine exposure are completely blocked in MC4-R null mutant mice and reduced in Agouti mice that overexpress an endogenous inhibitor of melanocortins in the brain. The results also demonstrate that cocaine administration increases the expression of MC4-R in the nucleus accumbens and striatum, and that MC4-R is co-localized with prodynorphin in medium spiny neurons in the nucleus accumbens. Together, these findings indicate that the behavioral actions of cocaine are dependent on activation of MC4-R, and suggest that upregulation of this receptor by drug exposure may contribute to sensitization of these behavioral responses. Modulation of cocaine reward is a novel action of the melanocortin-MC4-R system and could be targeted for the development of new medications for cocaine addiction. © Federation of European Neuroscience Societies.
• King, T., Gardell, L. R., Wang, R., Vardanyan, A., Ossipov, M. H., Malan Jr., T. P., Vanderah, T. W., Hunt, S. P., Hruby, V. J., Lai, J., & Porreca, F. (2005). Role of NK-1 neurotransmission in opioid-induced hyperalgesia. Pain, 116(3), 276-288.
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  PMID: 15964684;PMCID: PMC1440305;Abstract: Opiates are among the most important drugs for treatment of moderate to severe pain and prolonged opiate administration is often required to treat chronic pain states. We investigated the neurobiological actions of sustained opiate administration revealing paradoxical pronociceptive adaptations associated with NK-1 receptor function. Sustained morphine delivered over 6 days elicited hyperalgesia in rats and mice during the period of opiate delivery. Sustained morphine administration increased substance P (SP) and NK-1 receptor expression in the spinal dorsal horn. Sustained morphine treatment also enhanced capsaicin-evoked SP release in vitro, and increased internalization of NK-1 receptors in response to noxious stimulation. While NK-1 receptor internalization was observed primarily in the superficial laminae of placebo-treated rats, NK-1 receptor internalization was seen in both superficial and deep lamina of the dorsal horn in morphine-treated animals. Morphine-induced hyperalgesia was reversed by spinal administration of an NK-1 receptor antagonist in rats and mice, and was observed in wildtype (NK-1 +/+), but not NK-1 receptor knockout (NK-1-/-), mice. These data support a critical role for the NK-1 receptor in the expression of sustained morphine-induced hyperalgesia. Additionally, these data indicate that sustained opiate administration induces changes reminiscent of those associated with inflammatory pain. These opiate-induced changes might produce unintended deleterious actions in the course of pain treatment in patients. Understanding of sustained morphine-induced neurochemical changes will help identify approaches that limit the deleterious actions of opiates. © 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
• Lin, J., Liao, S., & Hruby, V. J. (2005). Syntheses of optically pure, conformationally constrained, and highly hydrophobic unusual amino acids: 2-Amino-3, 3-diarylpropionic acids. Journal of Peptide Research, 65(1), 105-112.
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  PMID: 15686541;Abstract: A series of optically pure, conformationally constrained, and highly hydrophobic unusual aromatic amino acids, 2-amino-3,3-diarylpropionic acids, were synthesized via asymmetric 1,4-Michael addition reaction/azidation reactions in seven steps with overall yields of 20-30%.
• Liu, J., Ying, J., T., V., Hruby, V. J., & Satyanarayanajois, S. D. (2005). Structure-activity studies of peptides from the "hot-spot" region of human CD2 protein: Development of peptides for immunomodulation. Journal of Medicinal Chemistry, 48(20), 6236-6249.
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  PMID: 16190751;PMCID: PMC1351114;Abstract: CD2 is a cell surface protein belonging to the immunoglobulin superfamily (IgSF) that plays a key role in mediating adhesion between human T-lymphocytes and target cells. The interaction between cell-adhesion molecules CD2 and CD58 is critical for immune response. Modulation or inhibition of these interactions has been shown to be therapeutically useful. Synthetic 12-mer linear and cyclic peptides and cyclic hexapeptides from the β-turn and β-strand region (hot spot) of human CD2 protein were designed to modulate CD2-CD58 interaction. The 12-amino acid synthetic cyclic peptides effectively blocked the interaction between CD2 and CD58 proteins as demonstrated by E-rosetting and heterotypic adhesion assays. NMR and molecular modeling studies indicated that these cyclic peptides exhibit β-turn structure in solution and closely mimic the β-turn structure of the surface epitopes of CD2 protein. The designed cyclic peptides with β-turn structure have the ability to modulate CD2-CD58 interaction. © 2005 American Chemical Society.
• Mollica, A., Davis, P., Ma, S., Lai, J., Porreca, F., & Hruby, V. J. (2005). Synthesis and biological evaluation of new biphalin analogues with non-hydrazine linkers. Bioorganic and Medicinal Chemistry Letters, 15(10), 2471-2475.
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  PMID: 15863299;Abstract: Biphalin is a potent opioid peptide agonist, with a palandromic structure, composed of two enkephalin-like active fragments connected tail to tail by a hydrazine linker (Tyr-D-Ala-Gly-Phe-NH-NH
• Monguchi, Y., Vagner, J., Handl, H. L., Jana, U., Begay, L. J., Hruby, V. J., Gillies, R. J., & Mash, E. A. (2005). Design, synthesis, and validation of rigid linkers for bioactive peptides. Tetrahedron Letters, 46(44), 7589-7592.
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  Abstract: Rigid linkers of variable length were synthesized and used to connect two NDP-α-MSH ligands. The linkers were incorporated by solid-phase synthesis. Biological evaluations indicate that there is virtually no effect of these linkers on ligand binding to the human melanocortin 4 receptor. © 2005 Elsevier Ltd. All rights reserved.
• Smith, E. A., Coym, J. W., Cowell, S. M., Tokimoto, T., Hruby, V. J., Yamamura, H. I., & Wirth, M. J. (2005). Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins. Langmuir, 21(21), 9644-9650.
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  PMID: 16207048;PMCID: PMC1456321;Abstract: The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-D-penicillamine, 5-D-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D 1 = 2.0(±1.2) × 10 -7 and D 2 = 1.2(±0.5) × 10 -6 cm 2/s) and also the largest electrophoretic mobility for the transmembrane protein (3 × 10 -8 cm 2/V·s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface. © 2005 American Chemical Society.
• Soloshonok, V. A., Cai, C., Yamada, T., Ueki, H., Ohfune, Y., & Hruby, V. J. (2005). Michael addition reactions between chiral equivalents of a nucleophilic glycine and (S)- or (R)-3-[(E)-enoyl]-4-phenyl-1,3-oxazolidin-2-ones as a general method for efficient preparation of β-substituted pyroglutamic acids. Case of topographically controlled stereoselectivity. Journal of the American Chemical Society, 127(43), 15296-15303.
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  PMID: 16248672;PMCID: PMC1851941;Abstract: This paper describes a systematic study of addition reactions between the chiral Ni(II) complex of the Schiff base of glycine with (S)-o[N-(N- benzylprolyl)amino]benzophenone and (S)- or (R)-3-[(E)-enoyl]-4-phenyl-1,3- oxazolidin-2-ones as a general and synthetically efficient approach to β-substituted pyroglutamic acids and relevant compounds. These reactions were shown to occur at room temperature in the presence of nonchelating organic bases and, most notably, with very high (>98% diastereomeric excess (de)) Stereoselectivity at both newly formed stereogenic centers. The stereochemical outcome of the reactions was found to be overwhelmingly controlled by the stereochemical preferences of the Michael acceptors, and the chirality of the glycine complex influenced only the reaction rate. Thus, in the reactions of both the (S)-configured Ni(II) complex and the Michael acceptors, the reaction rates were exceptionally high, allowing preparation of the corresponding products with virtually quantitative (>98%) chemical and stereochemical yields. In contrast, reactions of the (S)-configured Ni(II) complex and (R)-configured Michael acceptors proceeded at noticeably lower rates, but the addition products were obtained in high diastereo- and enantiomeric purity. To rationalize the remarkably high and robust Stereoselectivity observed in these reactions, we consider an enzyme-substrate-like mode of interaction involing a topographical match or mismatch of two geometric figures. Excellent chemical and stereochemical yields, combined with the simplicity and operational convenience of the experimental procedures, render the present method of immediate use for preparing various β-substituted pyroglutamic acids and related compounds. © 2005 American Chemical Society.
• Subramaniam, V., Alves, I. D., F., G., Lau, P., Wysocki Jr., R. J., Salamon, Z., Tollin, G., Hruby, V. J., Brown, M. F., & Saavedra, S. S. (2005). Rhodopsin reconstituted into a planar-supported lipid bilayer retains photoactivity after cross-linking polymerization of lipid monomers. Journal of the American Chemical Society, 127(15), 5320-5321.
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  PMID: 15826160;Abstract: Transmembrane proteins (TMPs), particularly ion channels and receptors, play key roles in transport and signal transduction. Many of these proteins are pharmacologically important and therefore targets for drug discovery. TMPs can be reconstituted in planar-supported lipid bilayers (PSLBs), which has led to development of TMP-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects TMP structure and activity. In this study, we have examined the effects of UV polymerization of bis-Sorbylphosphatidylcholine (bis-SorbPC) on the photoactivation of reconstituted bovine rhodopsin (Rho), a model G-protein-coupled receptor. Plasmon-waveguide resonance spectroscopy (PWR) was used to compare the degree of Rho incorporation and activation in fluid and poly(lipid) PSLBs. The results show that reconstitution of Rho into a supported lipid bilayer composed only of bis-SorbPC, followed by photoinduced lipid cross-linking, does not measurably diminish protein function. Copyright © 2005 American Chemical Society.
• Vardanyan, R. S., Hruby, V. J., Danagulyan, G. G., & Mkrtchyan, A. D. (2005). Isomerization/recyclization of some 5-ethoxycarbonyl-pyrimidines. Journal of Heterocyclic Chemistry, 42(4), 557-562.
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  Abstract: This communication reports on the investigation of a new recyclization conversion of a pyrimidine ring, which can be referred to as C-C recyclization. In this reaction the nucleophile cleaves the pyrimidine ring at the N(3)-C(4) bond, and following rotation around the single C(5)-C(6) bond the new cyclization takes place. This type of recyclization has general applicability, and takes place upon alkali treatment of substituted 4-methyl-5-ethoxycarbonyl- and 4-amino-5-ethoxycarbonyl-pyrimidines (1) which are transformed respectively to 4-hydroxy-5-acetyl- and 4-hydroxy-5-carbamoylpyrimidines (2). The obtained pyrimidyl-ketones can be readily converted to their hydrazones 7-12.
• Vessey, K. A., Lencses, K. A., Rushforth, D. A., Hruby, V. J., & Stell, W. K. (2005). Glucagon receptor agonists and antagonists affect the growth of the chick eye: A role for glucagonergic regulation of emmetropization?. Investigative Ophthalmology and Visual Science, 46(11), 3922-3931.
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  PMID: 16249465;PMCID: PMC1483902;Abstract: PURPOSE. In chicks, plus defocus retards eye growth, thickens the choroid, and activates glucagonergic amacrine cells, probably releasing glucagon. Glucagon receptor antagonists (expected to inhibit compensation to plus defocus) and agonists (expected to block myopia induction by form deprivation) were administered to eyes of chicks, to test the hypothesis that glucagon mediates the induction of changes in eye growth by plus defocus. METHODS. Seven-day-old (P7) chick eyes were injected intravitreally with peptides at concentrations of ∼10-9 to 10-5 in 20 μL (injection volume). The glucagon-receptor antagonists [des-His1,des- Phe6,Glu 9]-glucagon-NH2 (des-Phe6-antagonist) and [des-His1,Glu9]-glucagon-NH2 (Phe 6-antagonist) were administered daily for 4 to 5 days to plus-defocused eyes. Agonists (porcine glucagon-[1-29] and [Lys 17,18,Glu21]-glucagon-NH2) were monocularly administered daily for 5 days to form-deprived eyes. The contralateral eye remained open and received saline. After treatment, eyes were refracted, measured, and examined for histologic changes. MESULTS. The Phe 6-antagonist at 10-5 M (in the syringe) inhibited changes in both refractive error and axial length compensation induced by +7-D lens wear; however, des-Phe6-antagonist (10-5 M) had weak, inconsistent effects and did not antagonize the action of exogenous glucagon. Glucagon prevented ocular elongation and myopia and induced choroidal thickening in form-deprived eyes. [Lys17,18,Glu21]-glucagon-NH 2 had little effect at 10-7 M, but at 10-6 to 10-5 M altered rod structure and inhibited eye growth. CONCLUSIONS. Exogenous glucagon inhibited the growth of form-deprived eyes, whereas Phe 6-antagonist inhibited compensation to plus defocus, as might be expected if glucagon is an endogenous mediator of emmetropization. The reason for the failure of des-Phe6-antagonist to counteract the effects of exogenous glucagon requires further investigation. Copyright © Association for Research in Vision and Ophthalmology.
• Xuyuan, G. u., Xuyuan, G. u., Ying, J., Ying, J., Min, B., Min, B., Cain, J. P., Cain, J. P., Davis, P., Davis, P., Willey, P., Willey, P., Navratilova, E., Navratilova, E., Yamamura, H. I., Yamamura, H. I., Porreca, F., Porreca, F., Hruby, V. J., & Hruby, V. J. (2005). Parallel synthesis and biological evaluation of different sizes of bicyclo^[2,3]-Leu-enkephalin analogues. Biopolymers - Peptide Science Section, 80(2-3), 151-163.
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  PMID: 15660379;Abstract: Parallel synthesis of peptides and peptidomimetics has been an important approach to search for biologically active ligands. A novel systematic synthesis of different size bicyclic dipeptide mimetics was developed on solid-phase supports. By taking advantage of the enantioselective synthesis of ω-unsaturated amino acids and their N-methylated derivatives, the hemiaminal problem was prevented in the pathway to thiazolidine formation. The bicyclic dipeptide was generated on the solid-phase support in three steps by an unconventional method. By inserting this bicyclic scaffold into the synthesis of a larger bioactive peptide, 11 different sizes of bicyclo[2,3]- Leu-enkephalin analogues were synthesized in a fast and efficient way. Modeling studies show that a reversed turn structure at positions 2-3 was favored when an L- and L-bicyclic scaffold was used, and that an extended conformation at the N-terminal was favored when a D- and L-bicyclic scaffold was inserted. Binding affinities and bioassay studies show ligands with micromolar binding affinities and antagonist bioactivities for the [6,5]- and [7,5]-bicyclo-Leu-enkephalin analogues. © 2005 Wiley Periodicals, Inc.
• Alves, I. D., Ciano, K. A., Boguslavski, V., Varga, E., Salamon, Z., Yamamurat, H. I., Hruby, V. J., & Tollin, G. (2004). Selectivity, cooperativity, and reciprocity in the interactions between the δ-opioid receptor, its ligands, and G-proteins. Journal of Biological Chemistry, 279(43), 44673-44682.
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  PMID: 15317820;Abstract: A better understanding of signal transduction mechanisms is of critical importance. Methodologies that allow studies to be done while receptors are incorporated into lipid bilayers are advantageous. One such technique is plasmon-waveguide resonance (PWR) spectroscopy, which can follow changes in conformation accompanying protein-ligand, protein-protein, and protein-lipid interactions occurring in G-protein-coupled receptors in real time with high sensitivity and without the need for molecular labeling. Here we investigated several aspects of human δ-opioid receptor (hDOR)-G-protein interactions: 1) the effect of different types of agonists on the interaction with individual G-protein subtypes; 2) the affinities of the separate G-protein α and βγ subunits to different ligand-occupied states of the receptor; and 3) the effect of the presence of the G-protein on the interactions of the ligand with the receptor. To accomplish this we have incorporated the receptor into a solid supported lipid bilayer in the presence of ligand or G-protein and monitored the PWR spectral changes induced by the reciprocal G-protein or ligand interactions. We found a high degree of selectivity in the interactions of different agonist-bound states of the receptor with the different G-protein subtypes. This has important implications for agonist-directed trafficking and selective drug design. Studies with the separated α and βγ subunits show that cooperativity exists in these interactions. The high affinities of the separated subunits to the receptor point to the possibility of independent promotion of specific signaling events. The presence of G-proteins increased the affinity of agonists to the hDOR, and caused faster binding kinetics and different ligand-induced conformational changes. Because ligand also influences G-protein binding, reciprocity exists between these two binding processes.
• Alves, I. D., Cowell, S. M., Salamon, Z., Devanathan, S., Tollin, G., & Hruby, V. J. (2004). Different structural states of the proteolipid membrane are produced by ligand binding to the human δ-opioid receptor as shown by plasmon-waveguide resonance spectroscopy. Molecular Pharmacology, 65(5), 1248-1257.
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  PMID: 15102953;Abstract: Understanding structure-function relationships and mechanisms of signal transduction in G-protein-coupled receptors (GPCRs) is becoming increasingly important, both as a fundamental problem in membrane biology and as a consequence of their central role as pharmacological targets. Their integral membrane nature and rather low natural abundance present many challenging problems. Using a recently developed technique, plasmon-waveguide resonance (PWR) spectroscopy, we investigated the structural changes accompanying the binding of ligands to the human δ-opioid receptor (hDOR) immobilized in a solid-supported lipid bilayer. This highly sensitive technique can directly monitor changes in mass density, conformation, and orientation occurring in such thin proteolipid films. Without requiring labeling protocols, PWR allows the direct determination of binding constants in a system very close to the receptor's natural environment. In the present study, conformational changes of a proteolipid membrane containing the hDOR were investigated upon binding of a variety of peptide and nonpeptide agonists, partial agonists, antagonists, and inverse agonists. Distinctly different structural states of the membrane were observed upon binding of each of these classes of ligands, reflecting different receptor conformational states, and the formation of each state was characterized by different kinetic properties. Binding constants, obtained by quantifying the extent of conformational change as a function of the amount of ligand bound, were in good agreement with published values determined by radiolabeling methods. The results provide new insights into ligand-induced GPCR functioning and illustrate a powerful new protocol for drug development.
• Alves, I., Cowell, S., Lee, Y. S., Tang, X., Davis, P., Porreca, F., & Hruby, V. J. (2004). A novel 3-step enantioselective synthesis of pyrenylalanine with subsequent incorporation into opioid, CCK, and melanotropin ligands. Biochemical and Biophysical Research Communications, 318(2), 335-340.
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  PMID: 15120606;Abstract: Pyrene possesses unique spectroscopic properties such as a high quantum yield, a long half-life in the excited state, and the ability to form excimers when in proximity to each other in the excited state. These properties allow pyrenylalanine, which is a pyrene moiety incorporated into an amino acid, to be used as a fluorescent probe in peptides and proteins [J. Chem. Soc. Perkin Trans. 2 (1995) 1133; J. Am. Chem. Soc. 111 (1989) 6790; J. Chem. Soc. Perkin Trans. 2 (1997) 517]. The common route for the synthesis of pyrenylalanine involves 5 steps [Macromolecules 18 (1985) 882], with subsequent separation of the two isomers by recrystallization. This paper reports a novel 3-step asymmetric synthesis of pyrenylalanine with high enantioselectivity, good yields, and facile isomer purification. After synthesis, pyrenylalanine was incorporated into a series of opioid, CCK, and melanotropin peptide ligands in order to study the effects of aromaticity, lipophilicity, and steric properties on their potency and efficacy at their corresponding biological receptors. The change in binding and efficacy of the labeled ligands as compared to the unlabeled ligands demonstrates the possible role of lipophilicity/aromaticity in the binding and signal transduction of the ligand-receptor interaction. © 2004 Elsevier Inc. All rights reserved.
• Cai, C., Yamada, T., Tiwari, R., Hruby, V. J., & Soloshonok, V. A. (2004). Application of (S)- and (R)-methyl pyroglutamates as inexpensive, yet highly efficient chiral auxiliaries in the asymmetric Michael addition reactions. Tetrahedron Letters, 45(37), 6855-6858.
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  Abstract: Methyl N-(E-enoyl)pyroglutamates, derived from inexpensive and readily available in both enantiomeric forms pyroglutamic acid were found to be an efficient Michael acceptors in the addition reactions with nucleophilic glycine equivalent allowing for an efficient practical asymmetric synthesis of β-substituted pyroglutamic acids and related compounds. © 2004 Elsevier Ltd. All rights reserved.
• Cai, M., Cai, C., Mayorov, A. V., Xiong, C., Cabello, C. M., Soloshonok, V. A., Swift, J. R., Trivedi, D., & Hruby, V. J. (2004). Biological and conformational study of β-substituted prolines in MT-II template: Steric effects leading to human MC₅ receptor selectivity. Journal of Peptide Research, 63(2), 116-131.
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  PMID: 15009533;Abstract: To investigate the molecular basis for the interaction of the χ-constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β-substituted proline analogs were synthesized and incorporated into the Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]- NH2 (MT-II) template at the His6 and D-Phe7 positions. It was found that the binding affinities generally diminished as the steric bulk of the p-substituents of the 3-phenylproline residues increased. From (25, 3R)-3-phenyl-Pro6 to (2S, 3R)-3-(p-methoxyphenyl)-Pro 6 analogs the binding affinity decreased 23-fold at the human melanocortin-3 receptor (hMC3R), 17-fold at the hMC4R, and eight-fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the D-Phe7 residue with a (2R, 3S)-3-phenyl-Pro resulted in greatly reduced binding affinity (103-105) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS-AA force field simulations revealed that both MT-II and SHU-9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of D-Phe7/D-Nal (2′)7 in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His6 position (analogs 2-6) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β-modified Pro6 analogs of MT-II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.
• Cai, M., Stankova, M., J., S., Mayorov, A. V., Perry, J. W., Yamamura, H. I., Trivedi, D., & Hruby, V. J. (2004). Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy. Journal of the American Chemical Society, 126(23), 7160-7161.
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  PMID: 15186137;Abstract: Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled β-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon β-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes. Copyright © 2004 American Chemical Society.
• Cowell, S. M., Lee, Y. S., Cain, J. P., & Hruby, V. J. (2004). Exploring Ramachandran and Chi space: Conformationally constrained amino acids and peptides in the design of bioactive polypeptide ligands. Current Medicinal Chemistry, 11(21), 2785-2798.
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  PMID: 15544476;Abstract: Ligand binding and concomitant changes in receptor structure provide the means to target signal transduction pathways. With appropriate refinement of the ligand's interaction with the " receptor," one in theory could produce ligands that have greater therapeutic benefits. This review will discuss how, when these ligands are amino acids and peptides, the introduction of appropriate conformational constraints provides a powerful strategy for improved drug design. This review will discuss how various constraints on amino acids can provide a powerful tool for ligand design, determination of the three dimensional pharmacophore and new insights into receptor systems and information transduction. Through the use of constrained ligands, new information regarding their interaction with their " receptor" systems, and further refinement of the use of constraints, scientists can produce more beneficial drugs for mankind. © 2004 Bentham Science Publishers Ltd.
• Elmagbari, N. O., Egleton, R. D., Palian, M. M., Lowery, J. J., Schmid, W. R., Davis, P., Navratilova, E., Dhanasekaran, M., Keyari, C. M., Yamamura, H. I., Porreca, F., Hruby, V. J., Polt, R., & Bilsky, E. J. (2004). Antinociceptive structure-activity studies with enkephalin-based opioid glycopeptides. Journal of Pharmacology and Experimental Therapeutics, 311(1), 290-297.
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  PMID: 15166257;Abstract: Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth and subsequent amino acid residues (Ser or Thr) and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono- or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared with their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55°C tail-flick test. The calculated A50 values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A50 values ranging from 2.2 to 46.4 μmol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis- and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.
• Han, G., Haskell-Luevano, C., Kendall, L., Bonner, G., Hadley, M. E., Cone, R. D., & Hruby, V. J. (2004). De Novo Design, Synthesis, and Pharmacology of α-Melanocyte Stimulating Hormone Analogues Derived from Somatostatin by a Hybrid Approach. Journal of Medicinal Chemistry, 47(6), 1514-1526.
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  PMID: 14998337;Abstract: A number of α-melanotropin (α-MSH) analogues have been designed de novo, synthesized, and bioassayed at different melanocortin receptors from frog skin (fMC1R) and mouse/rat (mMC1R, rMC3R, mMC4R, and mMC5R). These ligands were designed from somatostatin by a hybrid approach, which utilizes a modified cyclic structure (H-D-Phe-c[Cys-Cys]-Thr-NH2) related to somatostatin analogues (e.g. sandostatin) acting at somatostatin receptors, CTAP which binds specifically to μ opioid receptors, and the core pharmacophore of α-MSH (His-Phe-Arg-Trp). Ligands designed were H-D-Phe-c[XXX-YYY-ZZZ-Arg-Trp-AAA]-Thr-NH2 [XXX and AAA = Cys, D-Cys, Hcy, Pen, D-Pen; YYY = His, His(1′-Me), His(3′-Me); ZZZ = Phe and side chain halogen substituted Phe, D-Phe, D-Nal(1′), and D-Nal(2′)]. The compounds showed a wide range of bioactivities at the frog skin MC1R; e.g. H-D-Phe-c[Hcy-His-D-Phe-Arg-Trp-Cys]-Thr-NH2 (6, EC50 = 0.30 nM) and H-D-Phe-c[Cys-His-D-Phe-Arg-Trp-D-Cys]-Thr-NH2 (8, EC50 = 0.10 nM). In addition, when a lactam bridge was used as in H-D-Phe-c[Asp-His-D-Phe-Arg-Trp-Lys]-Thr-NH2 (7, EC50 = 0.10 nM), the analogue obtained is as potent as α-MSH in the frog skin MC1R assay. Interestingly, switching the bridge of 6 to give H-D-Phe-c[Cys-His-D-Phe-Arg-Trp-Hcy]-Thr-NH2 (5, EC50 = 1000 nM) led to a 3000-fold decrease in agonist activity. An increase in steric size in the side chain of D-Phe7 reduced the bioactivity significantly. For example, H-D-Phe-c[Cys-His-D-Nal(1′ )-Arg-Trp-D-Cys]-Thr-NH2 (24) is 2000-fold less active than 9. On the other hand, H-D-Phe-c[Cys-His-D-Phe(p-I)-Arg-Trp-D-Cys]-Thr-NH2 (23) lost all agonist activity and became a weak antagonist (IC50 = 1 × 10-5 M). Furthermore, the modified CTAP analogues with a D-Trp at position 7 all showed weak antagonist activities (EC50 = 10-6 to 10-7 M). Compounds bioassayed at mouse/rat MCRs displayed intriguing results. Most of them are potent at all four receptors tested (mMC1R, rMC3R, mMC4R, and mMC5R) with poor selectivities. However, two of the ligands, H-D-Phe-c[Cys-His-D-Phe-Arg-Trp-Pen]-Thr-NH2 (9, EC50 = 6.9 × 10-9 M, 6.4 × 10-8 M, 2.0 × 10-8 M, and 1.4 × 10-10 M at mMC1R, rMC3R, mMC4R, and mMC5R, respectively) and H-D-Phe-c[Cys-His(3′ -Me)-D-Phe-Arg-Trp-Cys]-Thr-NH2 (16, EC50 = 3.5 × 10-8 M, 3.1 × 10-8 M, 8.8 × 10-9 M, and 5.5 × 10-10 M at mMC1R, rMC3R, mMC4R, and mMC5R, respectively) showed significant selectivities for the mMC5R. Worthy of mention is that neither of these two ligands is potent in the frog skin MC1R assay (EC50 = 10-7 M for 9 and EC50 = 10 -5 M for 16). These results clearly demonstrated that binding behaviors in rodent MCRs are quite different from those in the classical frog skin (R pipiens) assay.
• Handl, H. L., Vagner, J., Han, H., Mash, E., Hruby, V. J., & Gilles, R. J. (2004). Hitting multiple targets with multimeric ligands. Expert Opinion on Therapeutic Targets, 8(6), 565-586.
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  PMID: 15584863;Abstract: Multimeric ligands consist of multiple monomeric ligands attached to a single backbone molecule, creating a multimer that can bind to multiple receptors or targets simultaneously. Numerous examples of multimeric binding exist within nature. Due to the multiple and simultaneous binding events, multimeric ligands bind with an increased affinity compared to their corresponding monomers. Multimeric ligands may provide opportunities in the field of drug discovery by providing enhanced selectivity and affinity of binding interactions, thus providing molecular-based targeted therapies. However, gaps in our knowledge currently exist regarding the quantitative measures for important design characteristics, such as flexibility, length and orientation of the inter-ligand linkers, receptor density and ligand sequence. In this review, multimeric ligand binding in two separate phases is examined. The prerecruitment phase describes the binding of one ligand of a multimer to its corresponding receptor, an event similar to monomeric ligand binding. This results in transient increases in the local concentration of the other ligands, leading to apparent cooperativity. The postrecruitment phase only occurs once all receptors have been aligned and bound by their corresponding ligand. This phase is analogous to DNA-DNA interactions in that the stability of the complex is derived from physical orientation. Multiple factors influence the kinetics and thermodynamics of multimeric binding, and these are discussed. © 2004 Ashley Publications Ltd.
• Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J., & Gillies, R. J. (2004). Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions. Analytical Biochemistry, 330(2), 242-250.
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  PMID: 15203329;Abstract: A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for 125I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-α-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-α-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries. © 2004 Elsevier Inc. All rights reserved.
• Lipkowski, A. W., Carr, D. B., Hruby, V. J., & Yoshikawa, M. (2004). Is RNA-laden p24 capsid protein a vector for HIV?. Medical Hypotheses, 62(6), 919-921.
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  PMID: 15142648;Abstract: The consensus HIV infection pathway, although well supported by abundant experimental evidence, fails to account for infection of cells (e.g., neurons) that lack CD4 and/or coreceptors. We present the hypothesis that p24 capsid protein laden with RNA may be an alternative virulent agent. A novel mechanism of HIV infection may involve binding of the capsid with membrane receptors, followed by internalization of the capsid-receptor complex. This new pathway suggests novel strategies for antiviral pharmacotherapy. © 2004 Elsevier Ltd. All rights reserved.
• Ndungu, J. M., Xuyuan, G. u., Gross, D. E., Cain, J. P., Carducci, M. D., & Hruby, V. J. (2004). Synthesis of bicyclic dipeptide mimetics for the cholecystokinin and opioid receptors. Tetrahedron Letters, 45(21), 4139-4142.
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  Abstract: The cholecystokinin C-terminal octapeptide analogue H-Asp-Tyr-D-Phe-Gly- Trp-(N-Me)-Nle-Asp-Phe-NH2 (SNF 9007) is a potent and selective ligand for both the CCK-B and δ-opioid receptors. To constrain the peptide into the biologically active conformation(s), bicyclic dipeptide mimetics for Nle-Gly and homoPhe-Gly were designed and synthesized from β-substituted aspartic acids. Alkylation of L-aspartic acid using lithium bis(trimethylsilyl) amide (LHMDS) in the presence of hexamethylphosphoramide (HMPA) gave β-substituted aspartic acids, with the major product being the (2S,3R) isomer. Additional isomers of Nle-Gly bicyclic dipeptide mimetic were obtained via the Kazmaier-Claisen rearrangement reaction. The stereochemistries of the bicyclic dipeptide mimetics were assigned by X-ray and NMR. © 2004 Elsevier Ltd. All rights reserved.
• Ndungu, J. M., Xuyuan, G. u., Gross, D. E., Ying, J., & Hruby, V. J. (2004). A simple and efficient synthesis of an Asp-Gly dipeptide mimetic. Tetrahedron Letters, 45(16), 3245-3247.
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  Abstract: Alkylation of Nα-Boc protected aspartic acid with allyl bromide in the presence of lithium bis(trimethylsilyl)amide (LHMDS) and hexamethylphosphoramide (HMPA) afforded chiral β-allyl substituted aspartic acid in good yields. After deprotection of the Nα-Boc group and reprotection as a trifluoroacetamide, the terminal alkene was oxidized to an aldehyde. The aldehyde was then coupled with L-cysteine through a cascade three-bond formation process to afford aspartic acid-glycine bicyclic dipeptide mimetics. © 2004 Elsevier Ltd. All rights reserved.
• Ossipov, M. H., Lai, J., King, T., Vanderah, T. W., Malan Jr., T. P., Hruby, V. J., & Porreca, F. (2004). Antinociceptive and nociceptive actions of opioids. Journal of Neurobiology, 61(1), 126-148.
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  PMID: 15362157;Abstract: Although the opioids are the principal treatment options for moderate to severe pain, their use is also associated with the development of tolerance, defined as the progressive need for higher doses to achieve a constant analgesic effect. The mechanisms which underlie this phenomenon remain unclear. Recent studies revealed that cholecystokinin (CCK) is upregulated in the rostral ventromedial medulla (RVM) during persistent opioid exposure. CCK is both antiopioid and pronociceptive, and activates descending pain facilitation mechanisms from the RVM enhancing nociceptive transmission at the spinal cord and promoting hyperalgesia. The neuroplastic changes elicited by opioid exposure reflect adaptive changes to promote increased pain transmission and consequent diminished antinociception (i.e., tolerance). © 2004 Wiley Periodicals, Inc.
• Soloshonok, V. A., Ueki, H., Tiwari, R., Cai, C., & Hruby, V. J. (2004). Virtually complete control of simple and face diastereoselectivity in the Michael addition reactions between achiral equivalents of a nucleophilic glycine and (S)- or (R)-3-(E-enoyl)-4-phenyl-1,3-oxazolidin-2-ones: Practical method for preparation of β-substituted pyroglutamic acids and prolines. Journal of Organic Chemistry, 69(15), 4984-4990.
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  PMID: 15255725;Abstract: This study demonstrates a new strategy for controlling the stereochemical outcome of the Michael addition reactions between nucleophilic glycine equivalents and α,β-unsaturated carboxylic acid derivatives: The addition reactions between achiral Ni(II)-complex of the Schiff base of glycine with o-[N-α-pycolylamino]acetophenone and (S)- or (R)-3-(E-enoyl)-4- phenyl-1,3-oxazolidin-2-ones were shown to occur at room temperature in the presence of nonchelating organic bases and, most notably, with very high stereoselectivity at both newly formed stereogenic centers. Thus, the chiral 4-phenyl-1,3-oxazolidin-2-one moiety was found to control efficiently both face diastereoselectivities of the glycine derived enolate and the C,C double bond of the Michael acceptor. The new strategy developed in this work is methodologically superior to previous methods, most notably in terms of generality and synthetic efficiency. Excellent chemical yields and diastereoselectivities, combined with the simplicity of the experimental procedures, render the present method of immediate use for preparing various 3-substituted pyroglutamic acids and related amino acids (glutamic acids, glutamines, prolines, etc.) available via conventional transformations of the former.
• Vagner, J., Handl, H. L., Gillies, R. J., & Hruby, V. J. (2004). Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: Homooligomers of α-MSH. Bioorganic and Medicinal Chemistry Letters, 14(1), 211-215.
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  PMID: 14684330;Abstract: Homooligomers constructed with 4- and 6-amino acid fragments of melanocortin (α-MSH) bind with higher affinity and with apparent cooperativity to melanocortin receptor, compared to their constituent monomers. Individual ligands were tethered with various spacers of different length and rigidity and the influence of spacers on binding was studied. Binding assays were performed on cells transfected with the melanocortin receptor, hMC4R. There is a 5-7-fold decrease in the EC50 with the addition of each subunit, going from monomer to trimer. The Hill coefficient increases from 0.76 for the monomer to 1.12 for the dimer and 1.35 for the trimer. These data show a general trend of increasing avidity with increasing number of ligands in oligomers. © 2003 Elsevier Ltd. All rights reserved.
• Xuyuan, G. u., Ndungu, J. M., Qiu, W., Ying, J., Carducci, M. D., Wooden, H., & Hruby, V. J. (2004). Large scale enantiomeric synthesis, purification, and characterization of ω-unsaturated amino acids via a Gly-Ni(II)-BPB-complex. Tetrahedron, 60(37), 8233-8243.
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  Abstract: The enantiomeric syntheses of ω-unsaturated amino acids and β-substituted ω-unsaturated amino acids were accomplished by using Gly-Ni-2[N-(N′-benzylprolyl)amino]benzophenone (BPB) as a chiral auxiliary. The synthesis provides excellent yields and high diastereoselectivities. The product crystallization followed by isomer epimerization strategy makes the reaction practical and useful for large-scale preparations. Dialkylation of the Ni(II)-complex, which was designed for mechanistic considerations, revealed that high diastereoselectivity is obtained due to the thermodynamic conformational stability of the Ni(II)-complex. The assignment of absolute configuration was accomplished by NMR, which is supported by corresponding X-ray structure and optical rotation data. Both enantiomerically pure amino acids can be synthesized in this alkylation-hydrolysis two-step strategy in multi gram scales. © 2004 Elsevier Ltd. All rights reserved.
• Xuyuan, G. u., Ying, J., Agnes, R. S., Navratilova, E., Davis, P., Stahl, G., Porreca, F., Yamamura, H. I., & Hruby, V. J. (2004). Novel design of bicyclic β-turn dipeptides on solid-phase supports and synthesis of [3.3.0]-bicyclo^[2,3]-leu-enkephalin analogues. Organic Letters, 6(19), 3285-3288.
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  PMID: 15355033;Abstract: (Chemical Equation Presented) External bicyclic β-turn dipeptide mimetics provide an excellent design approach that can offer a rich chiral ensemble of structures with different backbone conformations. We report herein a novel design of a convergent combinatorial synthetic methodology, which is illustrated by the solid-phase synthesis of a series of [3.3.0]-bicyclo [2,3]-Leu-enkephalin analogues. The reactions were optimized and the epimeric configurations were determined by 2D NMR spectroscopy. Biological assays show that these analogues have more potent δ binding affinity and bioactivity for δ vs μ opioid receptor, which may be related to the different conformations preferred by these analogues in our modeling studies.
• Alves, I. D., Salamon, Z., Varga, E., Yamamura, H. I., Tollin, G., & Hruby, V. J. (2003). Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy.. The Journal of biological chemistry, 278(49), 48890-48897.
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  PMID: 14522991;Abstract: Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791-2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain delta-opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist ( approximately 10 nm); the lowest when receptor was bound to an antagonist ( approximately 500 nm); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of approximately 60 nm. Furthermore, GTP binding to the receptor.G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the Galphao (Kd approximately 20 nm) and lowest affinity toward the Galphai2 ( approximately 590 nm) subtypes, whereas the agonist-bound state had highest affinity for the Galphao and Galphai2 subtypes (Kd approximately 9 nm and approximately 7 nm, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.
• Balse-Srinivasan, P., Grieco, P., Cai, M., Trivedi, D., & Hruby, V. J. (2003). Structure-Activity Relationships of γ-MSH Analogues at the Human Melanocortin MC3, MC4, and MC5 Receptors. Discovery of Highly Selective hMC3R, hMC4R, and hMC5R Analogues. Journal of Medicinal Chemistry, 46(23), 4965-4973.
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  PMID: 14584947;Abstract: It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in α-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in γ-MSH, two different series of γ-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native γ-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr-Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH2 with Gly4) is more important for selectivity. The 2nd series is made of D-2-naphthylalanine (D-Nal(2′)) scan of the γ-MSH sequence at position 6 and 8 and the replacement of His5 with Pro (analogue 13). Analogue 12, H-Tyr-Val-Nle-Gly-His-Phe-Arg-D-Nal(2′ )-Asp-Arg-Phe-Gly-NH2, is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-D-Nal(2′ )-Asp-Arg-Phe-Gly-NH2, is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr-Val-Nle-Gly-Pro-D-Nal(2′ )-Arg-Trp-Asp-Arg-Phe-Gly-NH2, which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores.
• Balse-Srinivasan, P., Grieco, P., Cai, M., Trivedi, D., & Hruby, V. J. (2003). Structure-activity relationships of novel cyclic α-MSH/β-MSH hybrid analogues that lead to potent and selective ligands for the human MC3R and human MC5R. Journal of Medicinal Chemistry, 46(17), 3728-3733.
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  PMID: 12904077;Abstract: It has been shown by extensive studies that α-MSH bioactivity is critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, however with poor selectivity for the human MC3R-MC5R. The structure-activity relationships study here is aimed at identifying lead structures or templates of this core sequence by the use of different conformational constraints that might impart changes in its topography and thus promote differences in potency and selectivity at these receptors. Our peptide library consists of a novel series of cyclic α-MSH analogues that have disulfide bridges between Cys of Cys-like residues at positions 4 and 10, giving rise to 23-membered rings fused at the C-terminal end with the C-terminal fragment of β-MSH (Pro-Pro-Lys-Asp). While such constraints of the peptide backbone with disulfide bridges of different chirality affect potency and selectivity at, these receptors, further changes in the hydrophobicity at position 7 with either a D-Phe or D-Nal(2′) and replacement of a His with a Pro in position 6 cause additional effects. Thus, the most interesting lead compounds that emerged from this study are (1) compound 5, Ac-c[Cys-Glu-His-D-Phe-Arg-Trp-D-Cys]-Pro-Pro-Lys-Asp-NH 2 (IC50 = 10 nM), which is the first potent and highly selective antagonist ligand for the hMC5R (560-fold vs the MC3R and 1000-fold,vs the MC4R); (2) compound 7, Ac-c[Cys-Glu-Pro-D-Nal(2′)-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH2 (IC50 = 31 nM), which is a highly selective antagonist analogue for the MC3R (560-fold vs the hMC4R and about 3000-fold vs the hMC5R; and (3) compound 9, Ac-c[Pen-Glu-His-D-Nal(2′)-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH2 (IC50 = 3 nM), which is more potent than 7 at the MC3R but not as selective.
• Boguslavsky, V., Hruby, V. J., O'Brien, D., Misicka, A., & Lipkowski, A. W. (2003). Effect of peptide conformation on membrane permeability. Journal of Peptide Research, 61(6), 287-297.
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  PMID: 12753376;Abstract: The effect of peptide conformational constraint on the peptide permeation across the model membranes was examined by determining the permeability of pairs of cyclic and acyclic peptides related to C[D-Pen2, D-Pen5] enkephalin (DPDPE). The peptides were cyclized by formation of an intramolecular disulfide bridge between the second and fifth residues composed of either D-penicillamine or cysteine. In each case the acyclic peptide was three to seven times more permeable than corresponding cyclic peptide. The possibility that the differences in permeability of cyclic and acyclic peptides is based on the greater conformational freedom of the acyclic peptides in the presence of membrane was examined in more detail by isothermal titration calorimetric studies of Trp6-DPDPE and its acyclic analog. The membrane binding of the acyclic peptide is a more exothermic process than binding of its cyclic Trp6-DPDPE. The transfer of acyclic peptide from water to membrane is an enthalpy driven process, whereas the transfer of the cyclic peptide is driven by entropy.
• Cowell, S. M., Xuyuan, G. u., Vagner, J., & Hruby, V. J. (2003). Intelligent Design in Combinatorial Chemistry: Use of Designed Peptide Libraries to Explore Secondary and Tertiary Structures in Peptides and Proteins. Methods in Enzymology, 369, 288-297.
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  PMID: 14722960;
• Gillies, R. J., & Hruby, V. J. (2003). Expression-driven reverse engineering of targeted imaging and therapeutic agents. Expert Opinion on Therapeutic Targets, 7(2), 137-139.
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  PMID: 12667092;Abstract: Cancer is a particularly daunting disease to treat because, in its most insidious forms, cells are disseminated throughout the body, rendering surgery and local therapies ineffective. Hence, systemic disease must be targeted by its metabolic, physiological or molecular phenotype rather than by its location. Both tumour metabolism and physiology have been exploited to target therapies with some success. There is every reason to expect that these approaches will continue to advance therapies that discriminate tumour metabolism from that of normat tissues. As an alternative, molecular phenotyping (pharmacogenomics) is a relatively new science, and holds great promise for development of novel therapies and approaches. The discipline of pharmacogenomics has, to date, involved segmentation of patients according to their protein expression patterns in order to direct existing therapies to those populations who stand to benefit the most. In this communication, the authors propose a further application of this technology to develop agents that are reverse-engineered to explicitly target a patient's expressed protein patterns.
• Grieco, P., Balse-Srinivasan, P., Han, G., Weinberg, D., MacNeil, T., Van, L., & Hruby, V. J. (2003). Extensive structure - Activity studies of lactam derivatives of MT-II and SHU-9119: Their activity and selectivity at human melanocortin receptors 3, 4, and 5. Journal of Peptide Research, 62(5), 199-206.
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  PMID: 14531843;Abstract: The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His 6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nM) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-11 and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nM, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.
• Han, G., Quillan, J. M., Carlson, K., Sadée, W., & Hruby, V. J. (2003). Design of novel chimeric melanotropin-deltorphin analogues. Discovery of the first potent human melanocortin 1 receptor antagonist. Journal of Medicinal Chemistry, 46(5), 810-819.
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  PMID: 12593660;Abstract: A number of novel α-melanotropin (α-MSH) analogues have been designed, synthesized, and assayed for bioactivity at the melanocortin-1 (MC1) receptor from Xenopus frog skin, and selected potent analogues were examined at recombinant human MC1, MC3, and MC4 receptors expressed in human embryonic kidney (HEK) cells. These ligands were designed from Deltorphin-II, by a new hybrid approach, which incorporates the hydrophobic tail and the address sequence of Deltorphin-II (Glu-Val-Val-Gly-NH2) and key pharmacophore elements of melanotropins. Some of the ligands designed, c[Xxx-Yyy-Zzz-Arg-Trp-Glu]-Val-Val-Gly-NH2 {XXX = nothing, Gly, β-Ala, γ-Abu, 6-Ahx; YYY = His, His(3-Bom), (S)-cyclopentylglycine (Cpg); ZZZ = Phe, D-Phe; D-Nal(2′)}, show high potency at melanocortin receptors. One ligand, GXH-32B-c[β-Ala-His-D-Nal(2′)-Arg-Trp-Glu]-Val-Val- Gly-NH2, the most potent of the chimeric analogues tested, displayed agonist activity at each of the MC receptor subtypes analyzed, with an EC50 of 2 nM at the amphibian MC1 receptor. In contrast, GXH-38B-c[Gly-Cpg-D-Nal-(2′)-Arg-Trp-Glu]-Val-Val-Gly-NH2 (Cpg = cyclopentyl glycine) was an antagonist with a IC50 of 43 nM at the amphibian receptor, and among the human subtypes tested, was the most potent at the MC1 receptor subtype where it also acted as an antagonist (Ki = 53 nM), which is the first potent antagonist discovered for the human MC1 receptor. These results provide strong evidence supporting our hypothesis that ligand scaffolds for different G-protein coupled receptors (GPCRs) can be used to design ligands for other GPCRs and to design more potent ligands to treat diseases associated with the human MC1 receptor.
• Horais, K., Hruby, V., Rossi, S., Cizkova, D., Meschter, C., Dorr, R., & Yaksh, T. L. (2003). Effects of chronic intrathecal infusion of a ∂ opioid agonist in dogs. Toxicological Sciences, 71(2), 263-275.
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  PMID: 12563112;Abstract: To define the effects of chronic spinal exposure to a highly selective ∂ opioid agonist c[DPen2, DPen5]enkephalin (DPDPE), adult beagles were prepared with chronic lumbar intrathecal catheters. Groups of dogs received intrathecal infusions (100 μl/h) of saline (vehicle), DPDPE 3 mg/ml or 6 mg/ml for 28 days. Over the 28-day period, saline or 3 mg/ml showed minimal changes in neurological function, whereas in the 6 mg/ml animals, prominent hind limb dysfunction evolved over the 28-day interval. Histopathology in control animals displayed a modest pericatheter reaction considered normal for this model. Dogs receiving DPDPE (three of four at 6 mg/ml and one of four at 3 mg/ml) but not saline (zero of four) developed large inflammatory masses (granulomas) in the intrathecal space located proximal to the catheter tip. In these masses, severe chronic inflammatory changes in combination with necrosis and fibrosis was detected. Occasional focal destruction of neuropil was detected also in the adjacent spinal cord parenchyma. These masses contained extensive accumulation of mouse antihuman macrophages (MAC)-positive inflammatory cells expressing tumor necrosis factor-α (TNF-α), revealing infiltration of macrophages, granulocytes, and monocytes. In separate animals, prepared with dual intrathecal catheters, lumbar CSF was sampled at specified time points following intrathecal bolus (3 mg/ml) and 24 h DPDPE infusion (3 mg/ml and 6 mg/ml). Steady-state cerebrospinal fluid (CSF) DPDPE levels were 18.6 ± 1.0 and 22.6 ± 4.0 μg/ml for 3 mg/ml and 6 mg/ml infusions respectively. These results indicate that this ∂ opioid agonist DPDPE produces a concentration and time-dependent formation of an intrathecal inflammatory mass.
• Hosohata, K., Varga, E. V., Alfaro-Lopez, J., Tang, X., Vanderah, T. W., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2003). (2S,3R)β-methyl-2′,6′-dimethyltyrosine-L- tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] is a potent, selective δ opioid receptor antagonist in mouse brain. Journal of Pharmacology and Experimental Therapeutics, 304(2), 683-688.
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  PMID: 12538822;Abstract: The constrained opioid peptide (2S,3R)β-methyl-2′,6′-dimethyltyrosine-L- tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] exhibits high affinity and selectivity for the δ-opioid receptors (Liao et al., 1997). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5′-O-(3-[35S]thiotriphosphate) ([35S]GTPγS) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. δ-(SNC80; (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl) -3-methoxy-benzyl]-N,N-diethyl-benzamide) or μ- (DAMGO; [D-Ala2, Me-Phe4, Gly(ol)5]enkephalin) selective opioid full agonists stimulated [35S]GTPγS binding in mouse brain membranes 150 ± 4.5% and 152 ± 5.7% over the basal level, respectively. (2S,3R)TMT-L-Tic-OH did not influence basal [35S]GTP±S binding in mouse brain membranes but dose dependently shifted the dose-response curve of SNC80 to the right, with a Ke value of 3.6 ± 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the dose-response curve of the μ-selective opioid agonist, DAMGO. Warm water (55°C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin II- but not DAMGOmediated antinociception (40 ± 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral δ-opioid antagonist in mouse brain.
• Hruby, V. J. (2003). Peptide science: Exploring the use of chemical principles and interdisciplinary collaboration for understanding life processes. Journal of Medicinal Chemistry, 46(20), 4215-4231.
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  PMID: 13678399;
• Hruby, V. J., Agnes, R. S., Davis, P., Ma, S. -., Lee, Y. S., Vanderah, T. W., Lai, J., & Porreca, F. (2003). Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain. Life Sciences, 73(6), 699-704.
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  PMID: 12801591;Abstract: Disease states such as neuropathic pain offer special challenges in drug design due to the system changes which accompany these diseases. In this manuscript we provide an example of a new approach to drug design in which we have modified a potent and selective peptide ligand for the CCK-2 receptor to a peptide which has potent agonist binding affinity and bioactivity at delta and mu opioid receptors, and simultaneous antagonist activity at CCK receptors. De novo design based on the concept of overlapping pharmacophores was a central hypothesis of this design, and led to compounds such as H-Tyr-DPhe-Gly-DTrp-NMeNle-Asp-Phe-NH2 (i.e., RSA 601) which have the designed properties. © 2003 Elsevier Science Inc. All rights reserved.
• Hruby, V. J., Cai, M., Grieco, P., Han, G., Kavarana, M., & Trivedi, D. (2003). Exploring the stereostructural requirements of peptide ligands for the melanocortin receptors. Annals of the New York Academy of Sciences, 994, 12-20.
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  PMID: 12851293;Abstract: The melanotropin peptides α-MSHH, γ-MSH, and β-MSH are believed to be the natural ligands for the four melanocortin receptors, MC1R, MC3R, MC4R, and MC5R. However, these peptides generally have low selectivity for these receptors. We report on some approaches to the development of selective agonists and antagonists peptide ligands for these receptors.
• Kövér, K. E., Batta, G., & Hruby, V. J. (2003). Solution-phase chemical shift anisotropy as a promising tool to probe intermolecular interactions and peptide bond geometry: A case study on ¹⁵N-labeled Nα-t-Boc-L-valine. Magnetic Resonance in Chemistry, 41(10), 828-836.
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  Abstract: Geometry-dependent chemical shift anisotropy (CSAg) values of 1H and 15N nuclei have been determined in solution for 15N-labeled, Nα-t-Boc-L-valine by measurements of CSA/dipole-dipole cross-correlated relaxation rates using longitudinal variants of the recently proposed one-dimensional cross-correlation experiments. We demonstrate that solvent dependence of the CSAg is an invaluable tool for monitoring intermolecular H-bonding interactions. In addition, enhanced temperature dependence was observed for CSAg, which indicates that the anisotropy of chemical shift is more sensitive to subtle changes in the electronic environment of the nucleus than the motionally averaged isotropic chemical shift. 15N CSAg values have been determined in cyclosporin A at natural isotope abundance using the proposed 1H-detected pulse schemes. A remarkable correlation was observed between the measured 15N CSAg and the peptide ω angle, taken from the X-ray structure of cyclosporin A. Copyright © 2003 John Wiley & Sons, Ltd.
• Mogil, J. S., Wilson, S. G., Chesler, E. J., Rankin, A. L., V., K., Lariviere, W. R., Groce, M. K., Wallace, M. R., Kaplan, L., Staud, R., Ness, T. J., Glover, T. L., Stankova, M., Mayorov, A., Hruby, V. J., Grisel, J. E., & Fillingim, R. B. (2003). The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans. Proceedings of the National Academy of Sciences of the United States of America, 100(8), 4867-4872.
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  PMID: 12663858;PMCID: PMC153647;Abstract: Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from κ-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mclr) gene mediates K-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered κ-opioid analgesia. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the κ-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human.
• Okura, T., Varga, E. V., Hosohata, Y., Navratilova, E., Cowell, S. M., Rice, K., Nagase, H., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2003). Agonist-specific down-regulation of the human δ-opioid receptor. European Journal of Pharmacology, 459(1), 9-16.
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  PMID: 12505529;Abstract: Down-regulation of the δ-opioid receptor contributes to the development of tolerance to δ-opioid receptor agonists. The involvement of the carboxy terminus of the mouse δ-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human δ-opioid receptor by structurally distinct δ-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human δ-opioid receptors were incubated with various δ-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [3H]naltrindole saturation binding. Each δ-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all δ-opioid receptor agonists, except SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen2-D-Pen5]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human δ-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of Gi/o protein activation and subsequent downstream signaling. © 2002 Elsevier Science B.V. All rights reserved.
• Tollin, G., Salamon, Z., Cowell, S., & Hruby, V. J. (2003). Plasmon-waveguide resonance spectroscopy: A new tool for investigating signal transduction by G-protein coupled receptors. Life Sciences, 73(26), 3307-3311.
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  PMID: 14572873;Abstract: Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned δ-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems. © 2003 Elsevier Inc. All rights reserved.
• Tollin, G., Tollin, G., Salamon, Z., Salamon, Z., Hruby, V. J., & Hruby, V. J. (2003). Techniques: Plasmon-waveguide resonance (PWR) spectroscopy as a tool to study ligand-GPCR interactions. Trends in Pharmacological Sciences, 24(12), 655-659.
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  PMID: 14654307;Abstract: Plasmon-waveguide resonance (PWR) spectroscopy can be applied to integral membrane proteins incorporated into a supported lipid bilayer without the need for labeling. With high sensitivity and wide dynamic range, this technique can be used to characterize the kinetics and thermodynamics of conformational events associated with the binding of ligands to G-protein-coupled receptors (GPCRs), and to directly examine the interactions of GPCRs with G proteins and other downstream effectors in signal transduction. This allows an easy distinction to be made between agonists, antagonists and inverse agonists, and provides a powerful new tool for studying membrane signaling and for drug development.
• Varga, E. V., Rubenzik, M. K., Stropova, D., Sugiyama, M., Grife, V., Hruby, V. J., Rice, K. C., Roeske, W. R., & Yamamura, H. I. (2003). Converging protein kinase pathways mediate adenylyl cyclase superactivation upon chronic δ-opioid agonist treatment. Journal of Pharmacology and Experimental Therapeutics, 306(1), 109-115.
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  PMID: 12660310;Abstract: Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic δ-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 ± 91, 399 ± 2, and 433 ± 73% after chronic treatment with the δ-opioid agonists (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- methoxy-benzyl]-N,N-diethyl benzamide (SNC 80), [D-Pen2,D-Pen5]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 μM, 4-h) treatment augmented 32p incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 ± 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic δ-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.
• Wessells, H., Hruby, V. J., Hackett, J., Han, G., Balse-Srinivasan, P., & Vanderah, T. W. (2003). Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ induces penile erection via brain and spinal melanocortin receptors. Neuroscience, 118(3), 755-762.
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  PMID: 12710982;Abstract: Penile erection induced by α-melanocyte-stimulating hormone and melanocortin receptors (MC-R) in areas of the spinal cord and periphery has not been demonstrated. To elucidate sites of the proerectile action of melanocortin peptides, in awake male rats we administered the MC-R agonist Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 (MT-II) i.c.v., intrathecal (i.th.) and i.v. and scored penile erection and yawning. Injection of the MC-R antagonist Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2 (SHU-9119) i.c.v. or i.th. in combination with i.th. MT-II differentiated spinal from supraspinal effects. To exclude a site of action in the penis, we recorded intracavernous pressure responses to intracavernosal injection of MT-II in the anesthetized rat. I.c.v., i.th., and i.v. MT-II induced penile erections in a dose-dependent fashion. Yawning was observed with i.c.v. and i.v. MT-II, while spinal injection did not produce this behavior. Intrathecal delivery of MT-II to the lumbosacral spinal cord was more efficacious in inducing erections than i.c.v. or i.v. administration; SHU-9119 blocked the erectile responses to i.th. MT-II when injected i.th. but not i.c.v. Intracavernosal MT-II neither increased intracavernous pressure nor augmented neurostimulated erectile responses. We confirmed the central proerectile activity of MT-II and demonstrated that in addition to a site of action in the brain, the distal spinal cord contains melanocortin receptors that can initiate penile erection independent of higher centers. These results provide new insight into the central melanocortinergic pathways that mediate penile erection and may allow for more efficacious melanotropin-based therapy for erectile dysfunction. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
• Wessells, H., Hruby, V. J., Hackett, J., Han, G., Balse-Srinivasan, P., & Vanderah, T. W. (2003). MT-II induces penile erection via brain and spinal mechanisms. Annals of the New York Academy of Sciences, 994, 90-95.
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  PMID: 12851302;Abstract: α-Melanocyte-stimulating hormone induces penile erection via melanocortin (MC) receptors in areas surrounding the third ventricle, but spinal and peripheral mechanisms have not been demonstrated. We used pharmacological strategies to localize the site of the proerectile action of the melanocortin receptor agonist MT-II. We administered MT-II intracerebroventribularly (i.c.v.), intrathecally (i.th.), and intravenously (i.v.) and scored penile erection and yawning for 90 min in awake male rats. In some animals i.c.v. or i.th. SHU-9119 was injected 10 minutes before i.c.v. and i.th. MT-II to confirm the MC receptor action of the agonist and to distinguish spinal from supraspinal effects. To exclude a site of action in the penis, we recorded intracorporal pressure responses to intracavernosal injection of MT-II in the anesthetized rat. MT-II induced penile erections in a dose-dependent fashion, with optimal response at 1 μg for both i.c.v. and i.th. routes. Supraspinal MT-II-induced erections were completely suppressed by 1 μg SHU-9119 i.c.v. Yawning was observed with i.c.v. and i.v. MT-II, whereas spinal injection did not produce this behavior. SHU-9119 blocked the erectile responses to i.th. MT-II when injected i.th. but not i.c.v. Intracavernosal MT-II neither increased intracorporal pressure nor augmented neurostimulated erectile responses. The lumbosacral spinal cord contains MC receptors that can initiate penile erection independent of higher centers. We confirmed the supraspinal proerectile action of MT-II. These results provide insight into the central melanocortinergic pathways that mediate penile erection and may allow for more efficacious melanotropin-based therapy for erectile dysfunction.
• Xiong, C., Zhang, J., Davis, P., Wang, W., Ying, J., Porreca, F., & Hruby, V. J. (2003). Stereoselective synthesis of individual isomers of Leu-enkephalin analogues containing substituted β-turn bicyclic dipeptide mimetics. Chemical Communications, 9(13), 1598-1599.
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  Abstract: Novel constrained β-turn dipeptide mimetics, 8-phenyl thiaindolizidinone amino acids 3, have been synthesized stereoselectively and incorporated into Leu-enkephalin peptides as a replacement of dipeptide Gly3-Phe4 to afford four individual isomers of Leu-enkephalin analogues 6.
• Xuyuan, G. u., Cowell, S., Ying, J., Tang, X., & Hruby, V. J. (2003). Synthesis of β-phenyl-δ,ε-unsaturated amino acids and stereoselective introduction of side chain groups into [4,3,0]-bicyclic β-turn dipeptides. Tetrahedron Letters, 44(31), 5863-5866.
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  Abstract: (2S,3S)- and (2R,3R)-2-amino-3-phenyl-5-hexenoic acids have been synthesized in large scale by using Ni(II)-complexes as a template. The amino acids were used in the synthesis of [4,3,0]-bicyclic β-turn mimetics by a convergent methodology. The unique advantage of this strategy is the convenience of introducing side chain groups with predetermined chiralities on both the five- and six-membered heterocyclic rings. © 2003 Elsevier Ltd. All rights reserved.
• Ying, J., Ahn, J., Jacobsen, N. E., Brown, M. F., & Hruby, V. J. (2003). NMR solution structure of the glucagon antagonist [desHis¹, desPhe⁶, Glu⁹]glucagon amide in the presence of perdeuterated dodecylphosphocholine micelles. Biochemistry, 42(10), 2825-2835.
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  PMID: 12627948;Abstract: Glucagon, a 29-residue peptide hormone, plays an important role in glucose homeostasis and in diabetes mellitus. Several glucagon antagonists and agonists have been developed, but limited structural information is available to clarify the basis of their biological activity. The solution structure of the potent glucagon antagonist, [desHis1, desPhe6, Glu9]glucagon amide, was determined by homonuclear 2D NMR spectroscopy at pH 6.0 and 37 °C in perdeuterated dodecylphosphocholine micelles. The overall backbone root-mean-square deviation (rmsd) for the structured portion (residues 7-29, glucagon numbering) of the micelle-bound 27-residue peptide is 1.36 Å for the 15 lowest-energy structures, after restrained molecular dynamics simulation. The structure consists of four regions (segment backbone rmsd in Å): an unstructured N-terminal segment between residues 2 and 5 (1.68), an irregular helix between residues 7 and 14 (0.79), a hinge region between residues 15 and 18 (0.54), and a well-defined α-helix between residues 19 and 29 (0.33). The two helices form an L-shaped structure with an angle of about 90° between the helix axes. There is an extended hydrophobic cluster, which runs along the inner surface of the L-structure and incorporates the side chains of the hydrophobic residues of each of the amphipathic helices. The outer surface contains the hydrophilic side chains, with two salt bridges (D15-R18 and R17-D21) implied from close approach of the charged groups. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. The relationship of the two helical structural elements may have important implications for the biological activity of the glucagon antagonist.
• Ying, J., Ying, J., Kövér, K. E., Kövér, K. E., Xuyuan, G. u., Xuyuan, G. u., Han, G., Han, G., Trivedi, D. B., Trivedi, D. B., Kavarana, M. J., Kavarana, M. J., Hruby, V. J., & Hruby, V. J. (2003). Solution Structures of Cyclic Melanocortin Agonists and Antagonists by NMR. Biopolymers - Peptide Science Section, 71(6), 696-716.
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  PMID: 14991679;Abstract: The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets for drugs to treat obesity, sexual dysfunction, etc. Understanding the conformational basis of the receptor-ligand interactions is crucial to the design of potent and selective ligands for these receptors. The solution structures of the cyclic melanocortin agonists, partial agonist, and antagonists MTII, VJH085, SHU9II9, MK5, and MK9 were determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy at pH 4.5 and 25 °C in water (90% H2O/10% D2O). The overall backbone structures of these cyclic α-melanocyte-stimulating hormone (α-MSH) analogues around the message sequence (His6-D-Phe 7/D-Nal(2′)7-Arg8-Trp9) were similar and reasonably well defined. β-Turns spanning His6 and D-Phe7/D-Nal(2′)7 were identified in all analogues, and an amphiphilic molecular surface was obtained for the message sequence residues in most structures within the NMR ensembles. The β-turn, which most closely resembles a type II β-turn, leads to stacking between the aromatic rings of His6 and D-Phe7 in MTII and VJH085. However, no aromatic stacking between His6 and D-Nal(2′) 7 was found in structures of the D-Nal(2′) 7-containing analogues. The difference in the side-chain dispositions of His6 and D-Nal(2′)7 may be responsible for the reduced potency or antagonist activity of the D-Nal(2′)7-containing analogues. In addition, our results suggest that the side-chain orientations may also modulate the receptor selectivity. The information found in this study will be useful for the further design of ligands for melanocortin receptors. © 2003 Wiley Periodicals, Inc.
• Zhang, J., Wang, W., Xiong, C., & Hruby, V. J. (2003). Efficient and stereoselective synthesis of novel cis-4-substituted proline analogues. Tetrahedron Letters, 44(7), 1413-1415.
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  Abstract: A series of novel cis-4-substituted proline analogues were designed and synthesized. Highly stereoselective alkylations at the γ-position of glutamic ester 2 were achieved, followed by reduction, mesylation, and cyclization to afford the title compounds 1 in good yields and high diastereoselectivity. © 2003 Elsevier Science Ltd. All rights reserved.
• Zhang, J., Xiong, C., Ying, J., Wang, W., & Hruby, V. J. (2003). Stereoselective synthesis of novel dipeptide β-turn mimetics targeting melanocortin peptide receptors. Organic Letters, 5(17), 3115-3118.
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  PMID: 12916995;Abstract: (Matrix presented) The novel dipeptide β-turn mimetic, 4,8-disubstituted azabicyclo[4.3.0]nonane amino acid ester (15), has been synthesized to serve as a peptide mimetic of the dipeptide Phe-Arg, which contains two important pharmacophore elements in melanotropin peptides. Introduction of side-chain functionality at C-8 was achieved by using β-functionalized pyroglutamate (8) as a synthetic precursor. The side chain at C-4 was introduced by bromination of dehydroamino acid intermediate (10) followed by Suzuki cross-coupling.
• Flippen-Anderson, J., Deschamps, J. R., George, C., Hruby, V. J., Misicka, A., & Lipkowski, A. W. (2002). Crystal structure of biphalin sulfate: A multireceptor opioid peptide. Journal of Peptide Research, 59(3), 123-133.
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  PMID: 11985706;Abstract: Biphalin is a dimeric opioid peptide, composed of two tetrapeptides connected 'tail-to-tail', that exhibits a high affinity for all three opioid receptor types (i.e. μ, δ and κ). This study presents the X-ray crystal structure of biphalin sulfate and compares it to other opioids that interact with the same biological targets. Both halves of the molecule have a folded backbone conformation but differ significantly from one another. Residues 1-4 in biphalin, which compare well with the δ selective opioid peptide DADLE, fold into a random coil. Residues 5-8, which can be fit to the μ selective peptide D-TIPP-NH2, exhibit a fairly normal type III′ β bend. Biphalin also exhibits structural similarities with two naltrexone analogs, naltrexonazine and norbinaltorphamine, that are specific to μ and κ receptor sites.
• Grieco, P., Balse-Srinivasan, P., Han, G., Hruby, V. J., Weinberg, D., MacNeil, T., & Van, L. (2002). Synthesis and biological evaluation on hMC₃, hMC₄ and hMC₅ receptors of γ-MSH analogs substituted with L-alanine. Journal of Peptide Research, 59(5), 203-210.
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  PMID: 11966977;Abstract: To elucidate the molecular basis of the interaction of the native dodecapeptide γ-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced L-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of γ-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when L-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in γ-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with L-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of γ-MSH.
• Grieco, P., Han, G., Weinberg, D., MacNeil, T., Van, L., & Hruby, V. J. (2002). Design and synthesis of highly potent and selective melanotropin analogues of SHU9119 modified at position 6. Biochemical and Biophysical Research Communications, 292(4), 1075-1080.
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  PMID: 11944925;Abstract: The melanocortin receptors are involved in several important physiological functions. The potent and enzymatically stable analogues MT-II (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and SHU9119 (Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2) are important ligands of these receptors but are relatively nonselective. To differentiate between the physiological functions of these receptors, agonists, and antagonists with improved receptor selectivities are needed. We report here analogues of the well-characterized antagonist SHU9119 in which we replaced His6 with conformationally constrained amino acids. By this structure-activity study we discovered two important compounds, PG-901 (Ac-Nle4-c[Asp5-Pro6-DNal(2′) 7-Arg8-Trp9-Lys10]-NH 2) and PG-911 (Ac-Nle4-c[Asp5-Hyp6-DNal(2′) 7-Arg8-Trp9-Lys10]-NH 2), characterized to be full agonists at the hMC5R (EC50 = 0.072 nM and 0.031 nM, respectively), but full antagonists at the hMC3R and the hMC4R. We also demonstrated that the relative stereochemistry of the amino acid at the 6-position is critical for activity, and could play an important role in potency as well as in selectivity for the melanocortin receptors. © 2002 Elsevier Science (USA).
• Grieco, P., Lavecchia, A., Cai, M., Trivedi, D., Weinberg, D., MacNeil, T., Van, L., & Hruby, V. J. (2002). Structure-activity studies of the melanocortin peptides: Discovery of potent and selective affinity antagonists for the hMC3 and hMC4 receptors. Journal of Medicinal Chemistry, 45(24), 5287-5294.
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  PMID: 12431055;Abstract: We have designed and synthesized several novel cyclic SHU9119 analogues (Ac-Nle4-[Asp5-His6-DNal (2′)7-Arg8-Trp9- Lys10]-NH2) modified in position 6 with nonconventional amino acids. SHU9119 is a high affinity nonselective antagonist at hMC3R and hMC4R with potent agonist activity at hMC1R and hMC5R. We measured the binding affinity and agonist potency of the novel analogues at cloned hMC3R, hMC4R, and hMC5R receptors and identified several selective, high affinity hMC3R and hMC4R antagonists. Compound 4 containing Che substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.48 nM) with 100-fold selectivity over hMC3R antagonist. Analogue 7 with a Cpe substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.51 nM) with a 200-fold selectivity vs the hMC3R. Interestingly, analogue 9 with an Acpc residue in position 6 is a high affinity hMC3R antagonist (IC50 = 2.5 nM) with 100-fold selectivity vs the hMC4R antagonist based on its binding affinities. This compound represents the first cyclic lactam antagonist with high selectivity for the hMC3R vs hMC4R. To understand the possible structural basis responsible for selectivity of these peptides at hMCR3 and hMCR4, we have carried out a molecular modeling study in order to examine the conformational properties of the cyclic peptides modified in position 6 with conformationally restricted amino acids.
• Hruby, V. J. (2002). Designing peptide receptor agonists and antagonists. Nature Reviews Drug Discovery, 1(11), 847-858.
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  PMID: 12415245;Abstract: The most ubiquitous mode for controlling and modulating cellular function, intercellular communication, immune response and information-transduction pathways is through peptide-protein non-covalent interactions. Hormones, neurotransmitters, antigens, cytokines and growth factors represent key classes of such peptide ligands. These ligands might either be processed fragments of larger precursor proteins or surface segments of larger proteins. Athough there are numerous exceptions, such as insulin, oxytocin and calcitonin, most ligands are not used directly as drugs, and often the most useful ligands for therapy would be analogues that act as antagonists of the native ligands. A search for systematic structure-based or ligand-based approaches to designing such ligands has been an important concern. Today, a robust strategy has been developed for the design of peptides as drugs, drug candidates and biological tools. This strategy includes structural, conformational, dynamic and topographical considerations.
• Hruby, V. J. (2002). NIDA/NIH symposium on 'Structural Biology and Structural Genomics/Proteomics'. Journal of Peptide Research, 60(6), 305-306.
• Kavarana, M. J., Trivedi, D., Cai, M., Ying, J., Hammer, M., Cabello, C., Grieco, P., Han, G., & Hruby, V. J. (2002). Novel cyclic templates of α-MSH give highly selective and potent antagonists/agonists for human melanocortin-3/4 receptors. Journal of Medicinal Chemistry, 45(12), 2644-2650.
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  PMID: 12036373;Abstract: In an effort to develop highly selective and potent agonists and/or antagonists for the hMC3 and hMC4 receptors, a new approach involving the use of linker arms and a backbone to side chain cyclization strategy was employed. Three key analogues were identified to have the required selectivity and potency at the hMC3 or hMC4 receptors, implicated to play pivotal roles in energy homeostasis and other biological effects. The novel cyclic peptide (O)C-CH2-CH2-C(O)-c-[His6-D- Phe7-Arg8-Trp9-Lys10] -NH2 (1) was found to be a highly selective and potent agonist of the hMC4 receptor. Structure-activity studies have shown that replacing the succinyl linker arm of 1 by an o-phthalic acid group and substituting a D-Nal(2′)7 residue in place of D-Phe7 results in a potent antagonist 7 at the hMC4 receptor. Furthermore, increasing the 23-membered lactam ring of 1 by one carbon atom (succinyl → glutaric acid linker) gives a highly selective and potent antagonist 9 for the hMC3 receptor. Analogues 1, 7, and 9 therefore represent the first examples of a class of cyclic melanotropin ligands with high selectivity and defined biological activities at the physiologically important hMC3 and hMC4 receptors.
• Lipkowski, A. W., Misicka, A., Kosson, D., Kosson, P., Lachwa-From, M., Brodzik-Bienkowska, A., & Hruby, V. J. (2002). Biological properties of a new fluorescent biphalin fragment analogue. Life Sciences, 70(8), 893-897.
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  PMID: 11853227;Abstract: Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH←X, where X = Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X = DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both μ and δ opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation. © 2002 Elsevier Science Inc. All rights reserved.
• Moens, K., Berger, V., Ahn, J., Schravendijk, C. V., Hruby, V. J., Pipeleers, D., & Schuit, F. (2002). Assessment of the role of interstitial glucagon in the acute glucose secretory responsiveness of in situ pancreatic β-cells. Diabetes, 51(3), 669-675.
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  PMID: 11872665;Abstract: Glucagon is a potent stimulator of insulin release in the presence of a permissive glucose concentration, activating β-cells in vitro via both glucagon- and glucagon-like peptide-1 (GLP-1)-receptors. It is still unclear whether locally released glucagon amplifies the secretory responsiveness of neighboring β-cells in the intact pancreas. The present study investigates this question in the perfused pancreas by examining the effects of antagonists for glucagon receptors ([des-His1,des-Phe6, Glu9]glucagon-NH2, 10 μmol/l) and GLP-1-receptors [exendin-(9-39)-NH2, 1 μmol/l] on the insulin secretory response to glucose. The specificity of both antagonists was demonstrated by their selective interaction with glucagon-receptor signaling in rat hepatocytes and GLP-1-receptor signaling in Chinese hamster lung (CHL) fibroblasts. In purified rat β-cells, the glucagon-receptor antagonist (10 μmol/l) inhibited the effect of 1 nmol/l glucagon upon glucose-induced insulin release by 78 ± 6%. In the perfused rat pancreas, neither of these antagonists inhibited the potent secretory response to 20 mmol/l glucose, although they effectively suppressed the potentiating effect of, respectively, an infusion of glucagon (1 nmol/l) or GLP-1 (1 nmol/l) on insulin release. When endogenous glucagon release was enhanced by isoproterenol (100 nmol/l), no amplification was seen in the simultaneous or subsequent insulin secretory response to glucose. It is concluded that, at least under the present selected conditions, the glucose-induced insulin release by the perfused rat pancreas seems to occur independent of an amplifying glucagon signal from neighboring α-cells.
• Paizs, B., Suhai, S., Hargittai, B., Hruby, V. J., & Somogyi, Á. (2002). Ab initio and MS/MS studies on protonated peptides containing basic and acidic amino acid residues - I. Solvated proton vs. salt-bridged structures and the cleavage of the terminal amide bond of protonated RD-NH₂. International Journal of Mass Spectrometry, 219(1), 203-232.
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  Abstract: The results of a detailed ab initio investigation on one of the simplest model peptides, RD-NH2, containing both basic (R) and acidic (D) residues are presented here. The ab initio (B3LYP/6-31+G(d,p)) relative energies of several internally solvated (IS) and salt-bridged (SB) structures are in the range of 0-33kcal/mol. Upon ion activation in a tandem mass spectrometer, the conversion of IS into SB structures is energetically feasible but very probably kinetically controlled. Several theoretical pathways are suggested for the NH3 loss from the amide terminus of protonated RD-NH2. The loss of NH3 is proposed to occur either via "four-center-one-step" (FCOS) processes or an "oxazolone ring" formation (OX). The FCOS pathways indicate that the formation of a SB structure is not a prerequisite for the loss of NH3 from the amide terminus. The ab initio results clearly show the complexity of the potential energy surface of even such a small protonated peptide that is manifested in different protonated structures and pathways for a "simple" NH3 loss. Low-energy CID MS2 and MS3 spectra of the singly charged RD-NH2 have also been recorded and discussed in conjunction with the theoretical results. A brief discussion on the limitations of our current model to the fragmentation behavior of larger peptides containing both basic and acidic amino acids is also presented. © 2002 Elsevier Science B.V. All rights reserved.
• Salamon, Z., Hruby, V. J., Tollin, G., & Cowell, S. (2002). Binding of agonists, antagonists and inverse agonists to the human δ-opioid receptor produces distinctly different conformational states distinguishable by plasmon-waveguide resonance spectroscopy. Journal of Peptide Research, 60(6), 322-328.
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  PMID: 12464110;Abstract: Structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned δ-opioid receptor from human brain immobilized in a solid-supported lipid bilayer were monitored using plasmon-waveguide resonance (PWR) spectroscopy. Agonist (e.g. deltorphin II) binding causes an increase in membrane thickness because of receptor elongation, a mass density increase due to an influx of lipid molecules into the bilayer, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (e.g. TIPPpsi) binding produces no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter thought to be due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) [Salamon et al. (2000) Biophys. J. 79, 2463-2474]. In addition, we now find that an inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand-induced G-protein-coupled receptor functioning. The potential utilization of this new biophysical method to examine structural changes both parallel and perpendicular to the membrane normal for GPCRs is emphasized.
• Soloshonok, V. A., Ueki, H., Jiang, C., Cai, C., & Hruby, V. J. (2002). A convenient, room-temperature-organic base protocol for preparing chiral 3-(enoyl)-1,3-oxazolidin-2-ones. Helvetica Chimica Acta, 85(11), 3616-3623.
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  Abstract: In this study, we developed a new protocol for the preparation of the chiral 3-[(E)-enoyl]-1,3-oxazolidin-2-ones under the ultimately simple reaction conditions starting with the corresponding enoyl chlorides and 1.3-oxazolidin-2-ones with Et3N/LiCl at room temperature. The method generally allows efficient preparation of various derivatives regardless of the steric and electronic nature of the substituents on both the enoyl or the oxazolidinone sites. Excellent yields, combined with the simplicity of the experimental procedures, render the present method immediately useful for preparing the target compounds.
• Wang, W., Cai, M., Xiong, C., Zhang, J., Trivedi, D., & Hruby, V. J. (2002). Design and synthesis of novel χ²-constrained phenylalanine, naphthylalanine, and tryptophan analogues and their use in biologically active melanotropin peptides. Tetrahedron, 58(36), 7365-7374.
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  Abstract: A series of novel hydrophobic, bulky χ2-constrained phenylalanine, naphthylalanine, and tryptophan derivatives was designed and synthesized. The key steps involved asymmetric hydrogenations of α-enamides using Burk's DuPHOS-based Rh(I) catalysts to give high enantiomerically pure α-amino acid derivatives. The subsequent Suzuki cross couplings of the amino acid analogues with boronic acid derivatives afforded these aromatic substituted amino acids in high yields and with high enantioselectivity. The incorporation of these novel χ2-constrained amino acids into peptides and peptidomimetics provides fruitful information in the development of peptide and peptidomimetic ligands of melanotropins and an understanding of the interactions between ligands and receptors/acceptors. © 2002 Elsevier Science Ltd. All rights reserved.
• Wang, W., Xiong, C., Yang, J., & Hruby, V. J. (2002). An efficient synthesis of (2S, 6S)- and meso-diaminopimelic acids via asymmetric hydrogenation. Synthesis, 94-98.
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  Abstract: An efficient synthesis of the title compounds 1 and 2 has been successfully developed. The key step is the asymmetric hydrogenation of dehydroamino acid 7 using [Rh(I)(COD)-(S,S) or - (R,R)-Et-DuPHOS)]+OTf- to produce the optically active, protected amino acid derivatives in high ee (>95%). The approach also can be used for the synthesis of other isomers and analogues.
• Wang, W., Xiong, C., Zhang, J., & Hruby, V. J. (2002). Practical, asymmetric synthesis of aromatic-substituted bulky and hydrophobic tryptophan and phenylalanine derivatives. Tetrahedron, 58(15), 3101-3110.
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  Abstract: Aromatic ring substituted tryptophans and phenylalanines can provide valuable tools in developing highly potent and selective peptide ligands with specific structural features in addition to providing a large lipophilic surface for binding to receptors and for crossing membrane barriers. An efficient method for the synthesis of these novel amino acids has been developed. In the approach, asymmetric hydrogenations of α-enamides using Burk's DuPHOS-based Rh (I) catalysts generated high enantiomerically pure α-amino acid derivatives, which subsequently underwent Suzuki cross couplings with boronic acid derivatives to afford these aromatic substituted amino acids in high yields and high enantioselectivity. The method can allow for the preparation of such amino acids in large scales for extensive structure-activity studies. © 2002 Elsevier Science Ltd. All rights reserved.
• Wang, W., Yang, J., Ying, J., Xiong, C., Zhang, J., Cai, C., & Hruby, V. J. (2002). Stereoselective synthesis of dipeptide β-turn mimetics: 7-Benzyl and 8-phenyl substituted azabicyclo[4.3.0]nonane amino acid esters. Journal of Organic Chemistry, 67(18), 6353-6360.
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  PMID: 12201753;Abstract: A stereoselective method has been developed for the synthesis of 7- and 8-substituted dipeptide β-turn mimetic azabicyclo[4.3.0]nonane amino acid esters. The allyl groups were introduced in high diastereoselectivity, controlled by 3-phenyl or 4-benzyl groups in pyroglutamic acid derivatives 3 or 9, respectively. The precursors, dehydroamino acids 7 and 13 derived from 5 or 11, underwent asymmetric hydrogenations with Burk's DuPHOS Rh(I)-based catalysts to furnish α-amino acid derivatives in high stereoselectivity. The resulting amino acids 8 and 14 were converted to the β-turn mimetics 6,5-bicyclic lactams 1a-d in high yields.
• Wang, W., Zhang, J., Xiong, C., & Hruby, V. J. (2002). Design and synthesis of hydrophobic, bulky χ²-constrained phenylalanine and naphthylalanine derivatives. Tetrahedron Letters, 43(12), 2137-2140.
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  Abstract: A series of novel hydrophobic, bulky χ2-constrained phenylalanine and naphthylalanine derivatives were designed and synthesized. Asymmetric hydrogenations of α-enamides using Burk's DuPHOS-based Rh(I) catalysts generated high enantiomerically pure α-amino acid derivatives, which subsequently underwent Suzuki cross couplings with boronic acid derivatives to afford these aromatic substituted amino acids in high yields and with high enantioselectivity. © 2002 Elsevier Science Ltd. All rights reserved.
• Xiong, C., Wang, W., & Hruby, V. J. (2002). A general asymmetric synthesis of syn- and anti-β-Substituted cysteine and serine derivatives. Journal of Organic Chemistry, 67(10), 3514-3517.
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  PMID: 12003570;Abstract: A stereodivergent synthetic route has been developed to make the optically pure anti- and syn-β-substituted cysteine and serine derivatives. In this approach, the key intermediates, > 94% enantiomerically pure cyclic sulfates 3 and aziridines 7, were prepared from α,β-unsaturated esters 1, employing the Sharpless asymmetric dihydroxylation. The high regio- and stereoselective ring-opening reactions of cyclic sulfates and aziridines provided enantiomerically pure β-substituted cysteine and serine derivatives.
• Xiong, C., Wang, W., Cai, C., & Hruby, V. J. (2002). Regioselective and stereoselective nucleophilic ring opening reactions of a phenyl-substituted aziridine: Enantioselective synthesis of β-substituted tryptophan, cysteine, and serine derivatives. Journal of Organic Chemistry, 67(4), 1399-1402.
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  PMID: 11846696;Abstract: The asymmetric synthesis of β-phenyl-substituted cysteine, tryptophan, and serine derivatives was successfully developed. In this approach, the key intermediate, enantiomerically pure 3-phenylaziridine-2-carboxylic ester 7, was prepared from α,β-unsaturated ester 1 by employing the Sharpless asymmetric dihydroxylation. The aziridine 7 was treated with 4-methoxybenzylthiol, indole, and acetic acid to give β-phenyl-substituted cysteine, tryptophan, and serine, respectively, in a clean SN2 type ring opening at the C3 position. This general approach can be used to synthesize a variety of β-substituted novel amino acids.
• Xuyuan, G. u., Tang, X., Cowell, S., Ying, J., & Hruby, V. J. (2002). A novel strategy toward [6,5]-bicyclic β-turn dipeptide. Tetrahedron Letters, 43(37), 6669-6672.
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  Abstract: A novel strategy toward the syntheses of [6,5]-bicyclic β-turn dipeptides has been developed starting from δ,ε-unsaturated amino acids. This is the first example showing that this scaffold can be synthesized from a terminal alkene using a trifluoroacetyl protected amino acid. Both enantiomers of the δ,ε-unsaturated amino acid were synthesized by a modified method using Ni(II)-complexes. © 2002 Elsevier Science Ltd. All rights reserved.
• Zhang, J., Xiong, C., Wang, W., Ying, J., & Hruby, V. J. (2002). Stereoselective bromination - Suzuki cross-coupling of dehydroamino acids to form novel reverse-turn peptidomimetics: Substituted unsaturated and saturated indolizidinone amino acids. Organic Letters, 4(23), 4029-4032.
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  PMID: 12423078;Abstract: (matrix presented) A general and efficient methodology has been developed to prepare the C4-substituted dipeptide reverse-turn mimetics unsaturated (9a, 10a) and saturated (11a) azabicyclo[4.3.0] alkane amino acid derivatives. The side chain was introduced by bromination of dehydroamino acid intermediates followed by Suzuki coupling. Hydrogenation of the bicyclic dehydroamino acid 9a afforded saturated bicyclic lactam 11a. This approach can be further explored for the synthesis of a variety of such β-turn mimetics with aryl and alkyl side chain functionalities.
• Ahn, J. -., Gitu, P. M., Medeiros, M., Swift, J. R., Trivedi, D., & Hruby, V. J. (2001). A new approach to search for the bioactive conformation of glucagon: Positional cyclization scanning. Journal of Medicinal Chemistry, 44(19), 3109-3116.
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  PMID: 11543679;Abstract: In search for the bioactive conformation of glucagon, "positional cyclization scanning" was used to determine secondary structures of glucagon required for maximal interaction with the glucagon receptor. Because glucagon is flexible in nature, its bioactive conformation is not known except for an amphiphilic helical conformation at the C-terminal region. To understand the conformational requirement for the N-terminal region that appears to be essential for signal transduction, a series of glucagon analogues conformationally constrained by disulfide or lactam bridges have been designed and synthesized. The conformational restrictions via disulfide bridges between cysteine i and cysteine i + 5, or lactam bridges between lysine i and glutamic acid i + 4, were applied to induce and stabilize certain corresponding secondary structures. The results from the binding assays showed that all the cyclic analogues with disulfide bridges bound to the receptor with significantly reduced binding affinities compared to their linear counterparts. On the contrary, glucagon analogues containing lactam bridges, in particular, c[Lys5, Glu9]glucagon amide (10) and c[Lys17, Glu21]glucagon amide (14), demonstrated more than 7-fold increased receptor binding affinities than native glucagon. These results suggest that the bioactive conformation of glucagon may adopt a helical conformation at the N-terminal region as well as the C-terminal region, which was not evident from earlier biophysical studies of glucagon.
• Ahn, J. -., Medeiros, M., Trivedi, D., & Hruby, V. J. (2001). Development of potent truncated glucagon antagonists. Journal of Medicinal Chemistry, 44(9), 1372-1379.
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  PMID: 11311060;Abstract: In pursuit of truncated glucagon analogues that can interact with the glucagon receptor with substantial binding affinity, 23 truncated glucagon analogues have been designed and synthesized. These truncated analogues consist of several fragments of glucagon with 11 or 12 amino acid residues (1-4), conformationally constrained analogues containing the sequence of the middle region of glucagon (5-15), and truncated analogues containing the sequence of the C-terminal region (16-23). Biological assays of these analogues showed that the truncated glucagon analogues with the sequence of the C-terminal region possess significantly better binding affinity compared to the truncated analogues with the sequence of the middle region, and these analogues (17-23) demonstrated potent antagonistic activity (pA2 values between 6.5 and 7.5). On the basis of these results, it can be suggested that glucagon interacts with its receptor with two hydrophobic patches located in the middle and the C-terminal regions of glucagon, and both hydrophobic patches are necessary for significant receptor recognition. These two hydrophobic binding motifs, located in two different regions of glucagon, appear to be the reason why the earlier attempts to obtain truncated analogues with good binding affinity did not result in any success. Long peptide hormones such as glucagon seem to require more than one binding pocket on the receptors for maximal interaction.
• Ahn, J. M., Medeiros, M., Trivedi, D., & Hruby, V. J. (2001). Development of potent glucagon antagonists: Structure-activity relationship study of glycine at position 4. Journal of Peptide Research, 58(2), 151-158.
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  PMID: 11532074;Abstract: We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis1, Glu9]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly4 in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly4 residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis1, Glu9]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis1, Glu9]glucagon amide. These potent glucagon antagonists have among the highest pA2 values of any glucagon analogs reported to date.
• Cai, C., Soloshonok, V. A., & Hruby, V. J. (2001). Michael addition reactions between chiral Ni(II) complex of glycine and 3-(trans-enoyl)oxazolidin-2-ones. A case of electron donor - Acceptor attractive interaction-controlled face diastereoselectivity. Journal of Organic Chemistry, 66(4), 1339-1350.
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  PMID: 11312965;Abstract: This study has demonstrated that the readily available and inexpensive 3-(trans-3′-alkyl/arylpropenoyl)oxazolidin-2-ones, featuring high electrophilicity and conformational homogeneity, are synthetically superior Michael acceptors over the conventionally used alkyl enoylates, allowing for a remarkable improvement in reactivity and, in most cases, diastereoselectivity of the addition reactions with a Ni(II) complex of the chiral Schiff base of glycine with (S)-o-[N-(N-benzylprolyl)-amino]benzophenone. Kinetically controlled diastereoselectivity in the corresponding Michael addition reactions between the Ni(II) complex of glycine and the oxazolidin-2-ones was systematically studied as a function of steric, electronic, and position effects of the substituents on the starting Michael acceptor. In both aliphatic and aromatic series the simple diastereoselectivity was found to be virtually complete, affording the products via the corresponding TSs with the approach geometry like. The face diastereoselectivity of the reactions between the Ni(II) complex of glycine and the 3-(trans-3′-alkylpropenoyl)oxazolidin-2-ones was found to depend exclusively on the steric bulk of the alkyl group on the starting Michael acceptor. In contrast, the face diastereoselectivity in the reactions of aromatic oxazolidin-2-ones with the Ni(II) complex of glycine was shown to be controlled predominantly by the electronic properties of the aryl ring. In particular, the additions of the Ni(II) complex of glycine with 3-(trans-3′-arylpropenoyl)oxazolidin-2-ones, bearing electron-withdrawing substituents on the phenyl ring, afforded the (2S,3R)-configured products with synthetically useful diastereoselectivity and in quantitative chemical yields, thus allowing for an efficient access to the sterically constrained β-aryl-substituted pyroglutamic and glutamic acids.
• Cowell, S. M., Balse-Srinivasan, P., Ahn, J. -., & Hruby, V. J. (2001). Design and synthesis of peptide antagonists and inverse agonists for G protein-coupled receptors. Methods in Enzymology, 343, 49-72.
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  PMID: 11665587;
• Grieco, P., Gitu, P. M., & Hruby, V. J. (2001). Preparation of 'side-chain-to-side-chain' cyclic peptides by Allyl and Alloc strategy: Potential for library synthesis. Journal of Peptide Research, 57(3), 250-256.
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  PMID: 11298927;Abstract: Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side-chain-to-side-chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibé method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh3)4/PhSiH3)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium-mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.
• Han, G., & Hruby, V. J. (2001). A study of conjugate addition to a γ,δ-dioxolanyl-α,β-unsaturated ester. Tetrahedron Letters, 42(26), 4281-4283.
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  Abstract: Diastereoselective phenylcuprate addition to (S)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-trans-2-propenoate has been achieved in the presence of lithium bromide, copper(I) cyanide, trimethylsilyl chloride and dimethylsulfide cocatalysts. © 2001 Elsevier Science Ltd.
• Han, G., Lewis, A., & Hruby, V. J. (2001). Synthesis of (2S,3S)-β-methyltryptophan. Tetrahedron Letters, 42(28), 4601-4603.
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  Abstract: Nα-Fmoc-protected (2S,3S)-β-methyltryptophan has been synthesized by asymmetric synthesis through the aid of an oxazolidinone auxiliary. Coupling of the mixed anhydride of Nin-Boc-protected indoleacrylic acid to the oxazolidinone auxiliary was achieved by lithium bromide and N,N-dimethylpyridine (DMAP). This reaction is milder and more convenient to run than the traditional conditions using butyl lithium. © 2001 Elsevier Science Ltd.
• Han, G., Tamaki, M., & Hruby, V. J. (2001). Fast, efficient and selective deprotection of the tert-butoxycarbonyl (Boc) group using HCL/dioxane (4 M). Journal of Peptide Research, 58(4), 338-341.
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  PMID: 11606219;Abstract: Fast, efficient and selective deprotection of the tert-butoxycarbonyl (Boc) group of various amino acids and peptides was achieved by using hydrogen chloride (4 M) in anhydrous dioxane solution for 30 min at room temperature. In the cases studied in our laboratory, this protocol provided superior selectivity to deprotect Nα-Boc groups in the presence of tert-butyl esters and tert-butyl ethers, including thio-tert-butyl ethers, but not phenolic tert-butyl ethers.
• Hosohata, Y., Varga, E. V., Stropova, D., Xiaoping, L. i., Knapp, R. J., Hruby, V. J., Rice, K. C., Nagase, H., Roeske, W. R., & Yamamura, H. I. (2001). Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation. Life Sciences, 68(19-20), 2233-2242.
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  PMID: 11358332;Abstract: Intrinsic activities of different δ opioid agonists were determined in a [35S]GTPγS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other δ selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPγS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPγS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that δ opioid agonists of different chemical classes use specific conformations for G protein activation. © Elsevier Science Inc.
• Hruby, V. J. (2001). Design in topographical space of peptide and peptidomimetic ligands that affect behavior. A chemist's glimpse at the mind-body problem. Accounts of Chemical Research, 34(5), 389-397.
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  PMID: 11352717;Abstract: Efforts to determine the bioactive conformations of peptide ligands for membrane-bound proteins such as G-protein-coupled receptors (GPCRs) have been particularly challenging due to the flexibility of the ligands and the lack of 3D structural information (X-ray, NMR, etc.) for integral membrane proteins. An approach to determining these conformations by conformational constraint of the backbone template (φ and ψ angles) and by topographical constraint (χ1, χ2, etc. constraint) is outlined. Special attention is given to peptide neurotransmitter ligands that affect critical behaviors (feeding, sexual, addiction, pain, etc.). It is demonstrated that small changes in structure or a single torsional angle are sufficient to dramatically modify complex behaviors.
• Hruby, V. J. (2001). Journal of Peptide Research: Editorial. Journal of Peptide Research, 58(6), 443-.
• Hruby, V. J. (2001). Vincent duvigneaud 1901-1978: A personal tribute. Journal of Peptide Research, 58(3), 191-192.
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  PMID: 11576324;
• Hruby, V. J., & Soloshonok, V. A. (2001). Asymmetric synthesis of novel sterically constrained amino acids. Tetrahedron, 57(30), ix.
• Hruby, V. J., Agnes, R. S., & Cai, C. (2001). Design of peptide agonists. Methods in Enzymology, 343, 73-91.
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  PMID: 11665596;
• Hruby, V. J., Qiu, W., Okayama, T., & Soloshonok, V. A. (2001). Design of nonpeptides from peptide ligands for peptide receptors. Methods in Enzymology, 343, 91-123.
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  PMID: 11665597;
• Liao, S., Shenderovich, M., Zhang, Z., Hruby, V. J., Kövér, K., Hosohata, K., Davis, P., Porreca, F., & Yamamura, H. I. (2001). Synthesis, biology, NMR and conformation studies of the topographically constrained δ-opioid selective peptide analogs of [β-iPrPhe³]deltorphin I. Journal of Peptide Research, 57(4), 257-276.
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  PMID: 11328484;Abstract: Replacement of Phe3 in the endogenous δ-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β-isopropylphenylalanine (β-iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand-binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [β-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50=2 nM), bioassay potency (IC50=1.23 nM in MVD assay) and exceptional selectivity for the δ-opioid receptor over the μ-opioid receptor (30 000). Further conformational studies based on two-dimensional NMR and computer-assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr1 and (2S, 3R)-β-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted β-turn conformation.
• Lipkowski, A. W., Misicka, A., Hosohata, K., Davis, P., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2001). Biological properties of Phe°-opioid peptide analogues. Life Sciences, 68(8), 969-972.
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  PMID: 11213367;Abstract: Biological properties of new analogues, which represent Phe°-propeptides of a variety of opioid peptides, are described. All Phe°-opioid analogues expressed both receptor binding affinities and in vitro biological activities at least at the level of the primary opioid peptides. Surprisingly, some of the propeptides expressed slightly higher activity than the primary opioid peptides. Nevertheless, no significant shift in receptor selectivity was observed, which indicate that these Phe°-analogues undoubtedly are propeptides. The possible role of membrane proteolytic enzymes associated with opioid receptors in transformation of propeptides is discussed. © 2001 Elsevier Science Inc.
• Mitchell, S. A., Pratt, M. R., Hruby, V. J., & Polt, R. (2001). Solid-phase synthesis of O-linked glycopeptide analogues of enkephalin. Journal of Organic Chemistry, 66(7), 2327-2342.
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  PMID: 11281773;Abstract: The synthesis of 18 N-α-FMOC-amino acid glycosides for solid-phase glycopeptide assembly is reported. The glycosides were synthesized either from the corresponding O'Donnell Schiff bases or from N-α-FMOC-amino protected serine or threonine and the appropriate glycosyl bromide using Hanessian's modification of the Koenigs-Knorr reaction. Reaction rates of D-glycosyl bromides (e.g., acetobromoglucose) with the L- and D-forms of serine and threonine are distinctly different and can be rationalized in terms of the steric interactions within the two types of diastereomeric transition states for the D/L and D/D reactant pairs. The N-α-FMOC-protected glycosides [monosaccharides Xyl, Glc, Gal, Man, GlcNAc, and GalNAc; disaccharides Gal-β(1-4)-Glc (lactose), Glc-β(1-4)-Glc (cellobiose), and Gal-α(1-6)-Glc (melibiose)] were incorporated into 22 enkephalin glycopeptide analogues. These peptide opiates bearing the pharmacophore H-Tyr-c[DCys-Gly-Phe-DCys]- were designed to probe the significance of the glycoside moiety and the carbohydrate-peptide linkage region in blood-brain barrier (BBB) transport, opiate receptor binding, and analgesia.
• Qiu, W., Xuyuan, G. u., Soloshonok, V. A., Carducci, M. D., & Hruby, V. J. (2001). Stereoselective synthesis of conformationally constrained reverse turn dipeptide mimetics. Tetrahedron Letters, 42(2), 145-148.
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  Abstract: Peptide side chains play critical roles in the event of molecular recognition. In order to study the bioactive conformation of parent peptides, a concise and straightforward five-step synthesis of [5.5]-bicyclic reverse turn dipeptide mimetic scaffolds with side chain functionality at the i + 1 and i + 2 positions has been developed. In the bicyclic structure, two dihedral angles (ψ2 and φ3) are greatly restricted. © 2000 Elsevier Science Ltd.
• Soloshonok, V. A., Tang, X., & Hruby, V. J. (2001). Large-scale asymmetric synthesis of novel sterically constrained 2′,6′-dimethyl- and α,2′,6′-trimethyltyrosine and -phenylalanine derivatives via alkylation of chiral equivalents of nucleophilic glycine and alanine. Tetrahedron, 57(30), 6375-6382.
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  Abstract: Asymmetric synthesis of (S)-2′,6′-dimethyltyrosine (DMT), (S)-2′,6′-dimethylphenylalanine (DMP), (S)-α,2′,6′-trimethyltyrosine (α-TMT) and (S)-α,2′,6′-trimethylphenylalanine (α-TMP) via reactions of 4′-benzyloxy-2′,6′-dimethylbenzyl bromide or 2′,6′-dimethylbenzylbromide with Ni(II)-complexes of the chiral Schiff base of glycine or alanine with (S)-o-[N-(N-benzylprolyl)amino]benzophenone were developed. Inexpensive and readily available reagents and solvents, a recyclable chiral auxiliary, simplicity of the experimental procedures and high chemical yields make this method synthetically attractive for preparing the target amino acids on a multi-gram scale. © 2001 Elsevier Science Ltd. All rights reserved.
• Soloshonok, V. A., Tang, X., Hruby, V. J., & Meervelt, L. V. (2001). Asymmetric Synthesis of α,β-Dialkyl-α-phenylalanines via Direct Alkylation of a Chiral Alanine Derivative with Racemic α-Alkylbenzyl Bromides. A Case of High Enantiomer Differentiation at Room Temperature. Organic Letters, 3(3), 341-343.
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  PMID: 11428009;Abstract: (Matrix Presented) This study demonstrates that the direct alkylation of a Ni(ll)-complex of the chiral Schiff base of alanine with (S)-o-[N-(N-benzylprolyl)amino]-benzophenone, with racemic α-alkylbenzyl bromides, is a synthetically feasible and methodologically advantageous approach to the target α,β-dialkylphenylalanines over previously reported methods. For the first time we report and rationalize a case of a high enantiomer differentiation process at room temperature.
• Tamaki, M., Han, G., & Hruby, V. J. (2001). Practical and efficient synthesis of orthogonally protected constrained 4-guanidinoprolines. Journal of Organic Chemistry, 66(3), 1038-1042.
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  PMID: 11430070;
• Tamaki, M., Han, G., & Hruby, V. J. (2001). Synthesis of 4-cis-Phenyl-l-proline via Hydrogenolysis. Journal of Organic Chemistry, 66(10), 3593-3596.
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  PMID: 11348150;
• Wang, W., Xiong, C., & Hruby, V. J. (2001). An efficient approach to asymmetric synthesis of dipeptide β-turn mimetics: Indolizidinone amino acids. Tetrahedron Letters, 42(18), 3159-3161.
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  Abstract: Azabicyclo[X.Y.0] alkane amino acids are rigid dipeptide β-turn mimetics with great potential applications for drug discovery. The lack of efficient methods to synthesize these compounds is a major bottleneck in this field. Herein we report an efficient approach to the enantiopure synthesis of (3S,6S,9S) and (3R,6R,9R) methyl 2-oxo-3-[N-(Boc/Cbz)amino]-1-azabicyclo[4.3.0]nonane-9-carboxylates 1. In this approach, the key intermediates 5a and 5b with different stereochemical configurations were efficiently constructed from the same precursor in high stereoselectivity via asymmetric hydrogenations using (S,S) or (R,R) Et-DUPHOS, Rh(I)-based catalysts. The process, starting from inexpensive diethyl 1,3-acetonedicarboxylate 2, can allow for the practical synthesis of this class of compounds. © 2001 Elsevier Science Ltd.
• Wang, W., Xiong, C., Yang, J., & Hruby, V. J. (2001). Practical, asymmetric synthesis of aromatic-substituted bulky and hydrophobic tryptophan derivatives. Tetrahedron Letters, 42(44), 7717-7719.
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  Abstract: An efficient method for the synthesis of novel aromatic substituted, bulky and hydrophobic tryptophan derivatives has been developed. Asymmetric hydrogenations of α-enamide 5 using Burk's DuPHOS-based catalysts generated high enantiomerically pure D- and L-α-amino acid derivatives 6, which subsequently underwent Suzuki cross couplings with boronic acid derivatives to afford aromatic substituted tryptophan derivatives 7 and 8 in high yields. The method can allow for the preparation of such amino acids in large-scales for extensive structure-activity studies. © 2001 Elsevier Science Ltd. All rights reserved.
• Wang, Z., Gardell, L. R., Ossipov, M. H., Vanderah, T. W., Brennan, M. B., Hochgeschwender, U., Hruby, V. J., Malan Jr., T. P., Lai, J., & Porreca, F. (2001). Pronociceptive actions of dynorphin maintain chronic neuropathic pain. Journal of Neuroscience, 21(5), 1779-1786.
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  PMID: 11222667;Abstract: Whereas tissue injury increases spinal dynorphin expression, the functional relevance of this upregulation to persistent pain is unknown. Here, mice lacking the prodynorphin gene were studied for sensitivity to non-noxious and noxious stimuli, before and after induction of experimental neuropathic pain. Prodynorphin knock-out (KO) mice had normal responses to acute non-noxious stimuli and a mild increased sensitivity to some noxious stimuli. After spinal nerve ligation (SNL), both wild-type (WT) and KO mice demonstrated decreased thresholds to innocuous mechanical and to noxious thermal stimuli, indicating that dynorphin is not required for initiation of neuropathic pain. However, whereas neuropathic pain was sustained in WT mice, KO mice showed a return to baselines by post-SNL day 10. In WT mice, SNL upregulated lumbar dynorphin content on day 10, but not day 2, after injury. Intrathecal dynorphin antiserum reversed neuropathic pain in WT mice at post-SNL day 10 (when dynorphin was upregulated) but not on post-SNL day 2; intrathecal MK-801 reversed SNL-pain at both times. Opioid (μ, δ, and κ) receptor density and G-protein activation were not different between WT and KO mice and were unchanged by SNL injury. The observations suggest (1) an early, dynorphin-independent phase of neuropathic pain and a later dynorphin-dependent stage, (2) that upregulated spinal dynorphin is pronociceptive and required for the maintenance of persistent neuropathic pain, and (3) that processes required for the initiation and the maintenance of the neuropathic pain state are distinct. Identification of mechanisms that maintain neuropathic pain appears important for strategies to treat neuropathic pain.
• Balse, P. M., Kim, H. J., Han, G., & Hruby, V. J. (2000). Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis. Journal of Peptide Research, 56(2), 70-79.
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  PMID: 10961541;Abstract: The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base- labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with N(α)-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, γ-melanocyte-stimulating hormone (γ- MSH), its [Nle3]-analogue, and a cyclic α-MSH/β-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The γ-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-γ- MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.
• Bilsky, E. J., Egleton, R. D., Mitchell, S. A., Palian, M. M., Davis, P., Huber, J. D., Jones, H., Yamamura, H. I., Janders, J., Davis, T. P., Porreca, F., Hruby, V. J., & Polt, R. (2000). Enkephalin glycopeptide analogues produce analgesia with reduced dependence liability. Journal of Medicinal Chemistry, 43(13), 2586-2590.
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  PMID: 10891118;Abstract: Endogenous peptides (e.g. enkephalins) control many aspects of brain function, cognition, and perception. The use of these neuroactive peptides in diverse studies has led to an increased understanding of brain function. Unfortunately, the use of brain-derived peptides as pharmaceutical agents to alter brain chemistry in vivo has lagged because peptides do not readily penetrate the blood-brain barrier. Attachment of simple sugars to enkephalins increases their penetration of the blood-brain barrier and allows the resulting glycopeptide analogues to function effectively as drugs. The δ- selective glycosylated Leu-enkephalin amide 2, H2N-TyrD-Thr-Gly-Phe-Leu- Ser(β-D-Glc)-CONH2, produces analgesic effects similar to morphine, even when administered peripherally, yet possesses reduced dependence liability as indicated by naloxone-precipitated withdrawal studies. Similar glycopeptide- based pharmaceuticals hold forth the promise of pain relief with improved side-effect profiles over currently available opioid analgesics.
• Bilsky, E. J., Qian, X., Hruby, V. J., & Porreca, F. (2000). Antinociceptive activity of [β-methyl-2',6'-dimethyltyrosine¹]- substituted cyclic [D-Pen²,D-Pen⁵]enkephalin and [D-Ala²,Asp⁴]deltorphin analogs. Journal of Pharmacology and Experimental Therapeutics, 293(1), 151-158.
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  PMID: 10734164;Abstract: Research in our laboratories involves the development of selective opioid agonists and antagonists as: 1) pharmacological tools to elucidate the mechanisms of opioid antinociception, and 2) potential analgesics that possess therapeutic advantages over currently available drugs. We hypothesized that the selectivity of peptide agonists toward the opioid receptor types and subtypes is topographically dependent. The current results assess the antinociceptive activity and opioid receptor selectivity of a series of β-methyl-2',6'-dimethyltyrosine (TMT)-substituted cyclic [D- Pen2,B-Pen5]enkephalin (DPDPE) and [D-Ala2,Asp4]deltorphin (DELT I) analogs. Compounds were injected via the intracerebroventricular route into male ICR mice, and antinociception was assessed using the 55°C warm water tail-flick test. Antinociceptive A50 values ranged from 0.35 to 17 nmol for the DELT I analogs and from 7.05 to > 100 nmol for the DPDPE analogs. To test for receptor selectivity, mice were treated with selective μ- and δ-opioid antagonists. In general, μ [β-funaltrexamine (β-FNA)]- and δ1 ([D- Ala2,Leu5,Cys6] enkephalin)-antagonists blocked the antinociceptive actions of [TMT1]DPDPE analogs, whereas the antinociceptive actions of [TMT1]DELT I analogs were more sensitive to antagonism by the δ2-selective antagonist [Cys4]deltorphin and the μ-antagonist β-FNA. The antinociceptive actions of the [(2R,3S)TMT1]DELT I analog was suppressed by both [D-Ala2,Leu5, Cys6]enkephalin and β-FNA. These results are in contrast to those found with the parent molecules DPDPE (primarily a δ1 agonist) and DELT I (a mixed δ1/δ2 agonist). These results demonstrate that topographical modification in position 1 of the DPDPE and DELT I peptides affects antinociceptive potency and opioid receptor selectivity.
• Bonner, G. G., Davis, P., Stropova, D., Edsall, S., Yamamura, H. I., Porreca, F., & Hruby, V. J. (2000). Opiate aromatic pharmacophore structure-activity relationships in CTAP analogues determined by topographical bias, two-dimensional NMR, and biological activity assays. Journal of Medicinal Chemistry, 43(4), 569-580.
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  PMID: 10691683;Abstract: Topographically constrained analogues of the highly μ-opioid-receptor- selective antagonist CTAP (H-D-Phe-c[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH2, 1) were prepared by solid-phase peptide synthesis. Replacement of the D-Phe residue with conformationally biased β-methyl derivatives of phenylalanine or tryptophan (2R,3R; 2R,3S; 2S,3R; 2S,3S) yielded peptides that displayed widely varying types of biological activities. In an effort to correlate the observed biological activities of these analogues with their structures, two- dimensional 1H NMR and molecular modeling was performed. Unlike the parent (1), which is essentially a pure μ antagonist with weak δ agonist activities in the MVD bioassay, the diastereomeric β-MePhe1-containing peptides exhibited simultaneous δ agonism and μ antagonism by the (2R,3R)- containing isomer 2; μ antagonism by the (2R,3S)-containing isomer 3; weak μ agonism by the (2S,3R)-containing isomer 4; and δ agonism by the (2S,3S)- containing isomer 5. Incorporation of β-MeTrp isomers into position i led to peptides that were μ antagonists (2R,3R), 8; (2R,3S), 9, or essentially inactive (
• Dorr, R. T., Dvorakova, K., Brooks, C., Lines, R., Levine, N., Schram, K., Miketova, P., Hruby, V., & Alberts, D. S. (2000). Increased eumelanin expression and tanning is induced by a superpotent melanotropin [Nle⁴-D-Phe⁷]-α-MSH in humans. Photochemistry and Photobiology, 72(4), 526-532.
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  PMID: 11045725;Abstract: Seven normal volunteers (six males and one female) with tanning skin types III or IV (Fitzpatrick scale) were given 10 daily subcutaneous injections of a superpotent synthetic analog of alpha-melanocyte stimulating hormone (α-MSH) over two weeks. This agent, [Nle4-D-Phe7]α-MSH, also called Melanotan-I (MT-I), was administered at a dose of 0.16 mg/kg/day (Monday-Friday), over a two week period. Tanning was measured serially using computerized light reflectance. This regimen induced tanning at 3 of 8 anatomic sites including the face, neck and forearm by comparison of baseline to (1) the end of the daily dosing period, (day 14), and (2) one week later, (day 21). Shave biopsies of the forearm taken at baseline and day 21 were analyzed by high performance liquid chromatography for eumelanin content which was measured as the permanganate oxidation product, pyrrole-2,3,5-tricarboxylic acid or PTCA. Pheomelanin content was measured as the hydroiodic acid digestion product, amino-hydroxyphenylalanine (AHP). Eumelanin was also measured in the forehead skin samples of three subjects. The HPLC results show that mean (±SD) baseline eumelanin (PTCA) levels in forehead skin (n = 3) averaged 1.38 (±0.87) ng/mg of wet skin tissue weight. Higher mean baseline levels of PTCA were detected in forearm skin (2.06 ± 0.28 ng/mg wet weight, n = 7). One week after MT-I treatments ended, there was a mean (SD) 49% (±17.6%) increase in forehead skin PTCA levels compared to baseline (P = 0.019, n = 3, by paired sample T-test). The mean (SD) increase in forearm skin PTCA levels was 98% (±25.4%) over the same period (P = 0.003). In contrast, forearm pheomelanin expression following MT-I treatment did not significantly change from baseline. Overall, the MT-I regimen increased the eumelanin: pheomelanin ratio in forearm skin from 51:1 at baseline to 86:1 following MT-I (P = 0.054 by paired sample T-test). These results show that the tanning induced by MT-I in the face and forearm is associated with a significant increase in the eumelanin content of the human skin.
• Egleton, R. D., Mitchell, S. A., Huber, J. D., Janders, J., Stropova, D., Polt, R., Yamamura, H. I., Hruby, V. J., & Davis, T. P. (2000). Improved bioavailability to the brain of glycosylated Met-enkephalin analogs. Brain Research, 881(1), 37-46.
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  PMID: 11033091;Abstract: The blood-brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [D-Cys2,5,Ser6,Gly7] enkephalin. The peptide was glycosylated on the Ser6 via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague-Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the δ-opioid receptor, and mixed effect on binding to the μ-opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C. Copyright (C) 2000 Elsevier Science B.V.
• Grieco, P., Balse, P. M., Weinberg, D., MacNeil, T., & Hruby, V. J. (2000). D-amino acid scan of γ-melanocyte-stimulating hormone: Importance of Trp⁸ on human MC3 receptor selectivity. Journal of Medicinal Chemistry, 43(26), 4998-5002.
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  PMID: 11150170;Abstract: In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide γ-melanocyte-stimulating hormone (γ-MSH). The native γ-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the γ-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp8 analogue, there is an increase in binding affinity by about 1 order of magnitude (IC50 = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe6 analogue is about 10-fold more potent (IC50 = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, MC5R revealed that the native peptide shows potent activity at the MC3R (EC50 = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg10 (EC50 = 35 nM) and the DPhe11 (EC50 = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp8 compound (EC50 = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr1 peptide is selective for the MC3R (EC50 = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg10, but not Arg7, result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of γ-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg10 (but not Arg7), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.
• Haskell-Luevano, C., Lim, S., Yuan, W., Cone, R. D., & Hruby, V. J. (2000). Structure activity studies of the melanocortin antagonist SHU9119 modified at the 6, 7, 8, and 9 positions. Peptides, 21(1), 49-57.
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  PMID: 10704719;Abstract: The melanocortin system is involved in the regulation of several diverse physiological pathways, including energy homeostasis. Several synthetic peptide analogs have been designed, synthesized, and pharmacologically characterized at the mouse melanocortin receptor subtypes MC1R, MC3R, MC4R, and MC5R. These peptides incorporate modifications of the melanocortin core amino acids His-Phe-Arg-Trp by using the cyclic lactam templates of the lead structures MTII and SHU9119. Analogs containing DNal(2') at position 7 resulted in partial agonist and antagonistic activities at the mMC3R while possessing full antagonistic activities at the mMC4R. Recently, the melanocortin-5 receptor (MC5R) has been demonstrated to have a role in the regulation of exocrine gland function. This study has characterized the following analogs of SHU9119 that possess antagonist activity at the MC5R: Ac-Nle-c[Asp-(1-Me)His6-DNal(2')7-Arg-Trp-Lys]-NH2, pA2 = 7.1; Ac-Nle- c[Asp-(1-Me)His6-DNal(2')7-Arg-Nal(2')9-Lys]-NH2, pA2 = 7.2; and Ac-Nle- c[Asp-Trp6-DNal(2')7-Arg-Nal(2')9-Lys]-NH2, pA2 = 6.6. (C) 2000 Elsevier Science Inc.
• Hohmann, J. G., Teal, T. H., Clifton, D. K., Davis, J., Hruby, V. J., Han, G., & Steiner, R. A. (2000). Differential role of melanocortins in mediating leptin's central effects on feeding and reproduction. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 278(1 47-1), R50-R59.
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  PMID: 10644621;Abstract: Leptin serves as a humoral link coupling the status of energy reserves to the functional activity of the reproductive system. Leptin is thought to act through melanocortinergic pathways in the brain to regulate ingestive behaviors; however, whether melanocortins mediate leptin's actions on the neuroendocrine-reproductive axis is unknown. We tested this hypothesis first by determining whether the effects of leptin on feeding behavior and reproduction in the ob/ob mouse could be blocked by the melanocortin receptor (MC-R) antagonist SHU9119 and second, by examining the effects of the MC-R agonist MTII on feeding and the endocrine-reproductive system. Administered by intracerebroventricular injections, leptin inhibited food intake, raised plasma gonadotropin levels, and increased seminal vesicle weights compared with controls; SHU9119 (intracerebroventricularly) attenuated leptin's effects on food intake and body weight but did not alter leptin's stimulatory effect on the reproductive axis. MTII (intracerebroventricularly and intraperitoneally) decreased food intake and increased body temperature compared with controls but had no effect on the reproductive-endocrine axis. These results suggest that although leptin acts centrally through melanocortinergic pathways to inhibit ingestive behaviors and stimulate metabolism, leptin's activational effect on the reproductive axis is likely to be mediated by other, unknown neuroendocrine circuits.
• Hosohata, K., Logan, J. K., Varga, E., Burkey, T. H., Vanderah, T. W., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (2000). The role of the G protein γ₂ subunit in opioid antinociception in mice. European Journal of Pharmacology, 392(3), R9-R11.
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  PMID: 10762674;Abstract: We examined the role of the γ2 subunit of G proteins (Gγ2) in the antinociception produced by c[D-Pen2,D-Pen5]enkephalin (DPDPE) in mice. DPDPE produced 84.0±9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Gγ2 subunit, DPDPE-mediated antinociception decreased to 24.4±7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1±10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4±23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P
• Hosohata, Y., Vanderah, T. W., Burkey, T. H., Ossipov, M. H., Kovelowski, C. J., Sora, I., Uhl, G. R., Zhang, X., Rice, K. C., Roeske, W. R., Hruby, V. J., Yamamura, H. I., Lai, J., & Porreca, F. (2000). δ-Opioid receptor agonists produce antinociception and [³⁵S]GTPγS binding in μ receptor knockout mice. European Journal of Pharmacology, 388(3), 241-248.
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  PMID: 10675732;Abstract: We examined the effects of [D-Pen2,D-Pen5]enkephalin (DPDPE), [D- Ala2,Glu4]deltorphin (DELT), and (+)-4-[(αR)-α((2S,5R)-4-Allyl-2,5- dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) on [35S]GTPγS binding in brain membranes prepared from μ-opioid receptor knockout (-/-) mice. The potency and maximal response (E(max)) of these agonists were unchanged compared to control mice. In contrast, while the potency of [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE) was not significantly different, the E(max) was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in -/- mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in -/- compared to control mice, respectively. Reduced DPDPE antinociceptive potency in -/- mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from -/- mice, demonstrate that these agonists require μ-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between -/- and wild type mice, we conclude that the μ-opioid receptor is not a critical component of δ-opioid receptor function. (C) 2000 Elsevier Science B.V.
• Hruby, V. J. (2000). Peptide science - Whither thou goest?. Journal of Peptide Research, 56(1), 1-2.
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  PMID: 10917451;
• Hruby, V. J., & Balse, P. M. (2000). Conformational and topographical considerations in designing agonist peptidomimetics from peptide leads. Current Medicinal Chemistry, 7(9), 945-970.
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  PMID: 10911024;Abstract: The design of peptidomimetic ligands with agonist biological activities in vitro and in vivo has been challenging. Lofty goals have been set for this research including high potency, high receptor type selectivity, high stability in vitro and in vivo, and high efficacy in vitro and in vivo for agonists. A systematic stepwise strategy has been developed to accomplish these goals. These include determining the primary amino acid side chain residues required for molecular recognition and, in the case of agonist activity, those required for information transduction. In addition to determining the preferred backbone conformation which can serve as a template for the bioactive conformation (an (α-helix, β-turn, β-sheet, etc.), a strategy has been developed to examine and determine the preferred side chain conformations in chi space (chi1, ch2, etc.). These include specific covalent and non-covalent constraints which can place the constrained side chains at highly preferred gauche (-), or gauche (+), or trans conformations. Examples are provided that illustrate this methodology and provide insight into the topographical requirements for ligand receptor interactions. Often, at this juncture one can obtain a quite precise 3D pharmacophore for the ligand, as well as high stability to agonist biodegradation and good bioavailability including the ability to cross membrane barriers. If a non- peptide ligand is desired, efforts are in progress to develop templates, and aspects of conformational design that permit assembling of all components necessary for molecular recognition and transduction. Here the proper choice of template that can place the key side chain residue in 3D space is still difficult, and thus only partial success has been achieved in terms of potent and selective ligands. A few of these approaches are presented and discussed in some detail.
• Ko, M. C., Willmont, K. J., Burritt, A., Hruby, V. J., & Woods, J. H. (2000). Local inhibitory effects of dynorphin A-(1-17) on capsaicin-induced thermal allodynia in rhesus monkeys. European Journal of Pharmacology, 402(1-2), 69-76.
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  PMID: 10940359;PMCID: PMC2850053;Abstract: Although dynorphin A-(1-17) has been characterized in vitro as a high efficacy κ-opioid receptor agonist, functional studies of dynorphin A-(1-17) following central or systemic administration indicate the involvement of both opioid and non-opioid components. The aim of this study was to investigate whether local administration of dynorphin-related analogs can attenuate capsaicin (8-methyl-N-vanillyl-6-nonenamide)-induced nociception and what type of opioid receptor mediates the local action of dynorphin A-(1-17) in monkeys. Capsaicin (100 μg) was used to evoke a nociceptive response, thermal allodynia, which was manifested as a reduced tail-withdrawal latency in normally innocuous 46°C warm water. Co-administration of dynorphin A-(1- 17) (0.3-10 μg) with capsaicin in the tail dose-dependently inhibited thermal allodynia; however, both non-opioid fragments dynorphin A-(2-17) (10- 300 μg) and dynorphin A-(2-13) (10-300 μg) were ineffective. Local antiallodynia of dynorphin A-(1-17) was antagonized by a small dose (100 μg) of an opioid receptor antagonist, quadazocine, applied s.c. in the tail. Pretreatment with a selective κ-opioid receptor antagonist, nor- binaltorphimine (nor-BNI), s.c. 320 μg in the tail also reversed local antiallodynia of dynorphin A-(1-17). Both locally effective doses of antagonists, when applied s.c. in the back, did not antagonize local dynorphin A-(1-17), indicating that peripheral κ-opioid receptors selectively mediated the local action of dynorphin A-(1-17) in the tail. In addition, a much larger dose of dynorphin A-(1-17) (1000 μg), when administered s.c. in the back or i.m. in the thigh, did not cause sedative or diuretic effects. These results suggest that in vivo opioid actions of dynorphin-related peptides can be differentiated locally in this procedure. They also indicate that local application of peptidic ligands may be a useful medication for localized pain. (C) 2000 Elsevier Science B.V.
• Kriss, C. T., Lou, B., Szabò, L. Z., Mitchell, S. A., Hruby, V. J., & Polt, R. (2000). Enkephalin-based drug design: Conformational analysis of O-linked glycopeptides by NMR and molecular modeling. Tetrahedron Asymmetry, 11(1), 9-25.
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  Abstract: Glycosylation provides an effective means of enhancing penetration of the blood-brain barrier by pharmacologically active peptides. Glycosylated enkephalin analogues demonstrate much greater analgesic effects than their unglycosylated counterparts when administered peripherally. The solution conformations of glycopeptide enkephalin analogues with the sequences H-Tyr-c-[D-Cys-Gly-Phe-D-Cys]-Ser(β-O-Glcp)-Gly-NH2, 2, and H-Tyr-c-[D-Cys-Gly-Phe-D-Cys]-Ser(α-O-Glcp)-Gly-NH2, 3, have been determined by NMR and molecular modeling, and were compared to the unglycosylated peptide H-Tyr-c-[D-Cys-Gly-Phe-D-Cys]-Ser-Gly-NH2, 1, to determine the impact of glycosylation on peptide conformation. The only observed conformational effects were on the residue of attachment, Ser6, and on the adjacent Gly7-amide. This has important implications in peptide-based drug design in that strategically placed glycosylation can improve transport without destruction of the receptor selectivity of a pre-existing non-glycosylated peptide pharmacophore. Copyright (C) 2000 Elsevier Science Ltd.
• Okayama, T., Burritt, A., & Hruby, V. J. (2000). 4-alkoxy-2-hydroxybenzaldehyde (AHB): A versatile aldehyde linker for solid-phase synthesis of C-terminal modified peptides and peptidomimetics. Organic Letters, 2(13), 1787-1790.
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  PMID: 10891158;Abstract: (Formula presented) A new and versatile 4-alkoxy-2-hydroxybenzaldehyde (AHB) linker for solid-phase syntheses is described. Acylation of the polymer-bound secondary amine obtained from reductive amination of the aldehyde in the AHB linker showed good reactivity. Following acylation of the phenolic hydroxyl group, the resulting carboxamide resin was stable to treatment with 95% TFA. The O-acyl functional group was removed with 20% piperidine and the desired compound was cleaved from the resin by TFA treatment.
• Okura, T., Cowell, S. M., Varga, E., Burkey, T. H., Roeske, W. R., Hruby, V. J., & Yamamura, H. I. (2000). Differential down-regulation of the human δ-opioid receptor by SNC80 and [D-Pen²,D-Pen⁵]enkephalin. European Journal of Pharmacology, 387(2), R11-R13.
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  PMID: 10650167;Abstract: We examined the contribution of the human δ-opioid receptor carboxyl terminal tail to (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1- piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)- and cyclic[D- Pen2,D-Pen5]enkephalin (DPDPE)-mediated receptor down-regulation. Both SNC80 and DPDPE mediated down-regulation of an epitope tagged human δ-opioid receptor. Truncation of the human δ-opioid receptor after Gly338 blocked DPDPE-mediated down-regulation. However, SNC80 mediated significant down- regulation of the truncated receptor. These findings suggest that SNC80- mediated down-regulation involves receptor domains in addition to the carboxyl terminal tail. (C) 2000 Elsevier Science B.V.
• Qiu, W., Soloshonok, V. A., Cai, C., Tang, X., & Hruby, V. J. (2000). Convenient, large-scale asymmetric synthesis of enantiomerically pure trans-cinnamylglycine and -α-alanine. Tetrahedron, 56(17), 2577-2582.
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  Abstract: Asymmetric syntheses of (S)-trans-cinnamylglycine and (S)-α-trans- cinnamyl-α-alanine via reactions of cinnamyl halides (Cl, Br) with Ni(II)- complexes of the chiral Schiff base of glycine or alanine with (S)-o-[N-(N- benzylprolyl)amino]benzophenone were developed. Inexpensive and readily available reagents and solvents were used, including an easily recyclable chiral auxiliary. The simplicity of the experimental procedures and the high stereochemical outcome make this method synthetically attractive for preparing the target amino acids on multi-gram scales. (C) 2000 Elsevier Science Ltd.
• Salamon, Z., Cowell, S., Varga, E., Yamamura, H. I., Hruby, V. J., & Tollin, G. (2000). Plasmon resonance studies of agonist/antagonist binding to the human δ-opioid receptor: New structural insights into receptor-ligand interactions. Biophysical Journal, 79(5), 2463-2474.
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  PMID: 11053123;PMCID: PMC1301131;Abstract: Structural changes accompanying the binding of ligands to the cloned human δ-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.
• Shenderovich, M. D., Liao, S., Qian, X., & Hruby, V. J. (2000). A three-dimensional model of the δ-opioid pharmacophore: Comparative molecular modeling of peptide and nonpeptide ligands. Biopolymers, 53(7), 565-580.
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  PMID: 10766952;Abstract: A comparative molecular modeling study of δ-opioid ligands was performed under the assumption that potent peptide and nonpeptide agonists may have common three-dimensional (3D) arrangement of pharmacophore groups upon binding to the δ-receptor. Low-energy conformations of the agonists 7- spiroindanyloxymorphone (SlOM) and 2-methyl-4a-α-(3-hydroxyphenyl)- 1,2,3,4,4a, 5,12,12a-α-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67), and a partial agonist oxomorphindole (OMI) were determined by high- temperature molecular dynamics (MD). A good spatial overlap was found for the pharmacophore groups of SlOM, TAN-67, and OMI, including the basic nitrogen, phenol hydroxyl, and two aromatic ring. Based on this overlap we proposed a 3D pharmacophore model for nonpeptide δ-opioid agonists with a distance of 7.0 ± 1.3 Å between the two aromatic rings and of 8.2 ± 1.0 Å between the nitrogen and phenyl ring. The potent and highly δ-opioid receptor selective agonist [(2S, 3R)-TMT1]DPDPE, which shares global backbone constraints of the 14-membered disulfide cycle and a strong preference for the trans rotamer of the TMT-1 side chain, was chosen as a peptide template of the δ-opioid pharmacophore. Extensive MD simulations at 300 K with the AMBER force field were performed for [(2S, 3R)-TMT1]DPDPE and the less potent [(2S,3S)- TMT1]DPDPE analogue. Multiple MD trajectories were collected for each peptide starting from the x-ray structures of DPDPE and [L-Ala3]DPDPE and from models proposed in the literature. Low-energy MD conformations were filtered by the nonpeptide pharmacophore query and then directly superimposed with SIOM, OMI, and TAN-67. Two conformers of [(2S,3R)-TMT1]DPDPE that showed the best overlap with the nonpeptide pharmacophore (rms deviation ≤ 1.0 Å for N,O atoms and centroids of two aromatic rings) were selected as possible δ-receptor binding conformations. These conformations have similar backbone structures, and trans rotamers of the TMT1 side-chain group. They are reasonably close to the crystal structure of [L-Ala3]DPDPE, and differ significantly from the crystal structure of DPDPE. The conformer with a gauche(-) rotamer of Phe4 is most consistent with structure-activity relationships of δ-opioid peptides. The proposed 3D models were used for rational design of new nonpeptide δ-receptor ligands. (C) 2000 John Wiley and Sons, Inc.
• Soloshonok, V. A., Cai, C., & Hruby, V. J. (2000). (S)- or (R)-3-(E-enoyl)-4-phenyl-1,3-oxazolidin-2-ones: Ideal Michael acceptors to afford a virtually complete control of simple and face diastereoselectivity in addition reactions with glycine derivatives. Organic Letters, 2(6), 747-750.
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  PMID: 10754676;Abstract: (figure presented) Enantiomerically pure (S)- or (R)-3-(E-enoyl)-4-phenyl-1,3-oxazolidin-2-ones were found to serve as ideal Michael acceptors in addition reactions with achiral Ni(II) complexes of glycine Schiff bases. Virtually complete control of simple and face diastreoselectivity, observed in these reactions, combined with quantitative chemical yields renders this methodology synthetically superior to the previous methods.
• Soloshonok, V. A., Cai, C., & Hruby, V. J. (2000). A practical asymmetric synthesis of enantiomerically pure 3-substituted pyroglutamic acids and related compounds. Angewandte Chemie - International Edition, 39(12), 2172-2175.
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  Abstract: DBU-catalyzed Michael addition reactions were shown to occur at room temperature between a nickel(II) complex of the Schiff base of glycine 1 and (S)- or (R)-N-(E-enoyl)-4-phenyl-3-oxazolidin-2-ones (2, see scheme). This reaction, which has an almost completely stereoselective outcome, provides a practical and generalized approach to a family of glutamic/pyroglutamic acids. R = alkyl, aryl.
• Soloshonok, V. A., Cai, C., & Hruby, V. J. (2000). A unique case of face diastereoselectivity in the Michael addition reactions between Ni(II)-complexes of glycine and chiral 3-(E-enoyl)-1,3-oxazolidin-2-ones. Tetrahedron Letters, 41(49), 9645-9649.
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  Abstract: The origin of virtually complete face diastereoselectivity in the organic base-catalyzed, room temperature Michael addition reactions between Ni(II)-complexes of Schiff bases of glycine and chiral 3-(E-enoyl)-4-substituted-1,3-oxazolidin-2-ones was shown to stem from the unusual mode of steric interactions in determining the corresponding transition state. (C) 2000 Elsevier Science Ltd.
• Soloshonok, V. A., Cai, C., & Hruby, V. J. (2000). Toward design of a practical methodology for stereocontrolled synthesis of χ-constrained pyroglutamic acids and related compounds. Virtually complete control of simple diastereoselectivity in the Michael addition reactions of glycine Ni(II) complexes with N-(enoyl)oxazolidinones. Tetrahedron Letters, 41(2), 135-139.
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  Abstract: A Ni(II) complex of the Schiff base of glycine with o-[N-α- picolylamino]benzophenone or -acetophenone as a nucleophilic glycine equivalent, and N-trans-enoyloxazolidinones, as a derivative of an α,β- unsaturated carboxylic acid, were found to be the substrates of choice in the corresponding Michael addition reactions. The reactions proceed at room temperature in the presence of catalytic amounts of DBU to afford quantitatively a virtually diastereocomplete formation of the corresponding addition products with (2R*,3R*) or (2R*,3S*) relative configuration, depending on the nature of the starting N-enoyloxazolidinones, Acidic decomposition of the products followed by treatment of the reaction mixture with NH4OH gives rise to the corresponding diastereomerically pure 3- substituted pyroglutamic acids.
• Soloshonok, V. A., Cai, C., Hruby, V. J., Meervelt, L. V., & Yamazaki, T. (2000). Rational design of highly diastereoselective, organic base-catalyzed, room-temperature Michael addition reactions. Journal of Organic Chemistry, 65(20), 6688-6696.
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  PMID: 11052120;Abstract: Via the rational design of a single-preferred transition state, stabilized by electron donor-acceptor-type attractive interactions, structural and geometric requirements for the corresponding starting compounds have been determined. The Ni(II) complex of the Schiff base of glycine with o-[N-α-picolylamino]acetophenone, as a nucleophilic glycine equivalent, and N-(trans-enoyl)oxazolidin-2-ones, as derivatives of an α,β-unsaturated carboxylic acid, were found to be the substrates of choice featuring geometric/conformational homogeneity and high reactivity. The corresponding Michael addition reactions were found to proceed at room temperature in the presence of catalytic amounts of DBU to afford quantitatively the addition products with virtually complete diastereoselectivity. Acidic decomposition of the products followed by treatment of the reaction mixture with NH4OH gave rise to the diastereomerically pure 3-substituted pyroglutamic acids.
• Tang, X., Soloshonok, V. A., & Hruby, V. J. (2000). Convenient, asymmetric synthesis of enantiomerically pure 2',6'- dimethyltyrosine (DMT) via alkylation of chiral equivalent of nucleophilic glycine. Tetrahedron Asymmetry, 11(14), 2917-2925.
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  Abstract: Asymmetric synthesis of (S)-2',6'-dimethyltyrosine (DMT) via reactions of 4'-benzyloxy-2',6'-dimethylbenzyl bromide with Ni(II)-complexes of the chiral Schiff base of glycine with (S)-o-[N-(N-benzylprolyl)amino]- benzophenone was developed. Inexpensive and readily available reagents and solvents involved, including recyclable chiral auxiliary, simplicity of the experimental procedures and high chemical yields, make this method synthetically attractive for preparing the target amino acids on a multi-gram scale. (C) 2000 Elsevier Science Ltd.
• Trivedi, D., Lin, Y., Ahn, J., Siegel, M., Mollova, N. N., Schram, K. H., & Hruby, V. J. (2000). Design and synthesis of conformationally constrained glucagon analogues. Journal of Medicinal Chemistry, 43(9), 1714-1722.
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  PMID: 10794689;Abstract: Glucagon was systematically modified by forming lactam bridges within the central region of the molecule to give conformationally constrained cyclic analogues. Six cyclic glucagon analogues have been designed and synthesized. They are c[Asp9,Lys12][Lys17,18,Glu21]glucagon-NH2 (1), c[Asp9,Lys12]glucagon-NH2 (2), c[Lys12,Asp15]glucagon-NH2 (3), c[Asp15,Lys18]glucagon-NH2 (4), [Lys17-c[Lys18,Glu21]glucagon-NH2 (5), and c[Lys12,Asp21]glucagon-NH2 (6). The receptor binding potencies and receptor second messenger activities were determined by radio-receptor binding assays and adenylate cyclase assays, respectively, using rat liver plasma membranes. Most interestingly, analogues 1, 2, 3, and 4 were antagonists of glucagon stimulated adenylate cyclase activity, whereas analogues 5 and 6 were partial agonists in the functional assay. All of the cyclic analogues were found to have reduced binding potencies relative to glucagon. The structural features that might be responsible for these effects were studied using circular dichroism spectroscopy and molecular modeling. These results demonstrated the significant modulations of both receptor binding affinity and transduction (adenylate cyclase activity) that can accompany regional conformational constraints even in larger polypeptide ligands. These studies suggest that the entire molecular conformation, including the flexible middle portion, is important for molecular recognition and transduction at the hepatic glucagon receptor.
• Wang, S., Tang, X., & Hruby, V. J. (2000). First stereoselective synthesis of an optically pure β-substituted histidine: (2s,3s)-β-methylhistidine. Tetrahedron Letters, 41(9), 1307-1310.
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  Abstract: We report the first example of the asymmetric synthesis of the β- substituted histidine, (2S,3S)-β-methylhistidine. A key in the synthesis is the use of the protecting group, 2-mesitylenesulfonyl (Mts-), for the imidazole ring to minimize epimerization during synthesis. (C) 2000 Elsevier Science Ltd.
• Wessells, H., Gralnek, D., Dorr, R., Hruby, V. J., Hadley, M. E., & Levine, N. (2000). Effect of an alpha-melanocyte stimulating hormone analog on penile erection and sexual desire in men with organic erectile dysfunction. Urology, 56(4), 641-646.
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  PMID: 11018622;Abstract: Objectives. To assess the safety, erectogenic properties, and effect on sexual desire of Melanotan II, a synthetic melanotropic initiator of erection, in men with erectile dysfunction and organic risk factors. Methods. Ten subjects were enrolled in a double-blind, placebo-controlled, crossover study. Melanotan II (0.025 mg/kg) and vehicle were each administered twice by subcutaneous injection; real-time RigiScan monitoring and a visual analog were used to quantify the erections during a 6-hour period. The level of sexual desire and side effects were recorded with a questionnaire. Results. Melanotan II initiated subjectively reported erections in 12 of 19 injections versus only 1 of 21 doses of placebo. The mean rigidity score of the responders was 6.9 on a scale of 0 to 10. The mean duration of tip rigidity greater than 80% was 45.3 minutes with Melanotan II versus 1.9 for placebo (P = 0.047). The level of sexual desire after injection was significantly higher after Melanotan II administration than after placebo. Nausea and stretching/yawning occurred more frequently with Melanotan II, and 4 of 19 injections were associated with severe nausea. Conclusions. The erectogenic properties of Melanotan II are not limited to cases of psychogenic erectile dysfunction; men with a variety of organic risk factors developed penile erections. The finding of increased sexual desire warrants further investigation of centrally acting agents on disorders of sexual desire. Copyright (C) 2000 Elsevier Science Inc.
• Wessells, H., Levine, N., Hadley, M. E., Dorr, R., Hruby, V., Broderick, ., & Nehra, . (2000). Melanocortin receptor agonists, penile erection, and sexual motivation: Human studies Melanotan II. International Journal of Impotence Research, 12(SUPPL. 4), S74-S79.
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  PMID: 11035391;Abstract: We review our experience with Melanotan II, a non-selective melanocortin receptor agonist, in human subjects with erectile dysfunction (ED). Melanotan II was administered to 20 men with psychogenic and organic ED using a double-blind placebo-controlled crossover design. Penile rigidity was monitored for 6 h using RigiScan. Level of sexual desire and side effects were reported with a questionnaire. In the absence of sexual stimulation, Melanotan II led to penile erection in 17 of 20 men. Subjects experienced a mean of 41 min Rigiscan tip rigidity > 80%. Increased sexual desire was reported after 13/19 (68%) doses of Melanotan II vs 4/21 (19%) of placebo (P < 0.01). Nausea and yawning were frequently reported side effects due to Melanotan II; at a dose of 0.025 mg/kg, 12.9% of subjects had severe nausea. We conclude that Melanotan II is a potent initiator of penile erection in men with erectile dysfunction. Our findings warrant further investigation of melanocortin agonists and antagonists on penile erection.
• Witt, K. A., Slate, C. A., Egleton, R. D., Huber, J. D., Yamamura, H. I., Hruby, V. J., & Davis, T. P. (2000). Assessment of stereoselectivity of trimethylphenylalanine analogues of δ-opioid [D-Pen²,D-Pen⁵]-enkephalin. Journal of Neurochemistry, 75(1), 424-435.
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  PMID: 10854288;Abstract: [D-Pen2,D-Pen5]-Enkephalin (DPDPE) is an enzymatically stable δ- opioid receptor-selective peptide, which was modified by the trimethylation of the Phe4 residue to give β-methyl-2',6'-dimethylphenylalanine (TMP), resulting in four conformations: (2S,3S)-β-Phe-DPDPE, (2R,3R)-β-Phe-DPDPE, (2R,3S)-β-Phe-DPDPE, and (2S,3R)-β-Phe-DPDPE. Synthesis was by solid-phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (δ- versus μ- opioid receptor) were evaluated by radioreceptor binding in rat brain, with a μ/δ ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)-TMP. In situ vascular perfusion in anesthetized rats showed a 1.6- fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)-TMP and a 1.5-fold (p < 0.01) decrease in uptake for (2R,3R)-TMP. Saturability of (2S,3S)-TMP was shown (p < 0.01) against 100 μM unlabeled DPDPE, showing a shared nondiffusionary transport system. P-glycoprotein affinity was shown in situ for the parent and (2S,3S)-TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin- Ringer showed (2R,3S)-TMP and (2S,3R)-TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)-TMP and (2R,3R)-TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)-TMP, (2R,3S)-TMP, and (2S,3R)-TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P-glycoprotein affinity, and blood-brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.
• Alfaro-Lopez, J., Okayama, T., Hosohata, K., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1999). Exploring the structure-activity relationships of [1-(4-(4- tert-butyl-3'-hydroxy)benzhydryl-4-benzylpiperazine] (SL-3111), a high-affinity and selective δ-opioid receptor nonpeptide agonist ligand. Journal of Medicinal Chemistry, 42(26), 5359-5368.
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  PMID: 10639279;Abstract: SL-3111 [1-(4-tert-butyl-3'-hydroxy)benzhydryl-4- benzylpiperazine] is a de novo designed, high-affinity and selective nonpeptide peptidomimetic agonist of the δ-opioid receptor. In a previous report we had described the unique biological characteristics of this ligand and also a need for further structural evaluation. To pursue this, we have introduced a completely different heterocyclic template (2 and 3), which, based on molecular modeling studies, may present the required structural features to properly orient the pharmacophore groups. We also have made more subtle changes to the original piperazine scaffold (5 and 11). The biological activities of these compounds revealed an important participation of the scaffold in the ligand-receptor interaction. To further explore functional diversity on the scaffold, we have maintained the original piperazine ring and introduced four different functionalities at position 2 of the heterocyclic ring (15a-d; a = CH2-O-CH2-Ph; b = Me; c = CH2Ph; d = CH2OH). The biological activities observed for these compounds showed a very interesting trend in terms of the steric effects of the groups introduced at this position. A decrease of almost 2000-fold in affinity and potency at the δ- receptor was observed for 15c compared with 15b. This difference may be explained if we postulate that the bioactive conformation of these peptidomimetics is close to the minimal energy conformations calculated in our study. On the basis of these findings we have realized the importance of this position to further explore and simplify the structure of future generations of peptidomimetic ligands.
• Bartosz-Bechowski, H., Davis, P., Slaninova, J., Malatynska, E., Stropova, D., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1999). Cyclic enkephalin analogs that are hybrids of DPDPE-related peptides and Met-enkephalin-Arg-Gly-Leu: Prohormone analogs that retain good potency and selectivity for δ opioid receptors. Journal of Peptide Research, 53(3), 329-336.
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  PMID: 10231722;Abstract: We report here on the binding affinity and bioassay results of cyclic enkephalin analogs comprising a cyclic moiety and C-terminal fragment of MERGL, where ME denotes methionine enkephalin. MERGL (YGGFMRGL) has been suggested to be cleaved enzymatically by membrane-bound enkephalinase 24.11 to leave ME and the tripeptide RGL. In our study we have synthesized hybrids of DPDPE or DPLCE and the C-terminal tripeptide RGL in order to mimic a prohormone able to cross the blood-brain barrier. The study has shown that of the homologs presented here, analogs of DPLCE often are more potent at delta opioid receptors both in binding affinity and in bioactivity at the MVD, than DPDPE. Our hypothesis that hybrids (consisting of the drug and the spacer for the carrier) could be designed which would either have no opioid activity or, alternatively, be by themselves very active, has been verified.
• Gentry, C. L., Egleton, R. D., Gillespie, T., Abbruscato, T. J., Bechowski, H. B., Hruby, V. J., & Davis, T. P. (1999). The effect of halogenation on blood-brain barrier permeability of a novel peptide drug. Peptides, 20(10), 1229-1238.
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  PMID: 10573295;Abstract: The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [D-Pen2, L-Pen5, Phe6] Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood-brain barrier permeability of a novel enkephalin analog DPLPE- Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe- NH2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe4]DPLPE-Phe-OH. p- [Cl-Phe4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood-brain barrier permeability, providing evidence for improved delivery to the central nervous system.
• Hau, V. S., Campos, C., Hoshata, K., Lipokowski, A., Yamamura, H. I., Hruby, V. J., & Davis, T. P. (1999). Guanidino cationization effects on the blood-brain barrier permeability, in vitro stability and receptor binding of endomorphins. Journal of Investigative Medicine, 47(2), 34A.
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  Abstract: Cationization has been shown to improve the blood-brain barrier (BBB) passage of peptides via adsorptive endocytosis. Guanidino (GU) addition to the amino group of tyrosine is one way to cationize peptides containing tyrosine. Endomorphins (END), a possible endogenous ligand for the μ opioid receptor have an N-terminal tyrosine and thus can be used to study the effect of cationization. Structural relationships of opioids in drug delivery will direct future directions in analgesia research. In this study we investigated GU effects on in vitro BBB transport, stability and receptor binding of END II, [Pro4] END II by comparing non-GU peptides and their GU analogs. An in vitro bovine brain microvessel endothelial cell model (BBMEQ was used to study BBB permeability. In vitro stability was studied via incubating peptide with mouse serum and mouse brain homogenate resuspended at 15% protein. Receptor binding was done using rat whole brain tissue incubated at 25°C for 180 minutes. BBMEC: BBMEC confluent monolayer membrane filters were placed in side-by-side diffusion chambers. Peptides were placed in the huminal chamber and sampling done from the abluminal chamber over time-points (0-120 min). GU addition to END II increased BBB permeability from 31.9 ± 0.90 x 10-4 to 32.5 ± 0.53 x 10-4 while GU addition of [Pro4] END II increased BBB permeability significantly from 30.6 ± 1.2 x 1-4 to 40.2 ± 0.5 x 104.In vitro stability: Peptides were added to samples time-course incubated at 37°C and analyzed by HPLC. END II half-life increased from 13.2 min to 140.2 min in brain and from 3.2 min to 26.6 min in serum with GU addition. [Pro4] END II half-life increased from 24.2 min to 149.09 min in brain and from 2.92 min to 34.33 min in serum with GU addition. Receptor Binding: END II was found to have an IC50 δ/μ ratio of 0.71 whereas both GU END II and GU [PrO4] END II increased to >300. These data provide evidence that increased cationization with GU modification improves both stability and permeability and increases 1C50 δ/μ ratios.
• Hosohata, K., Burkey, T. H., Alfaro-Lopez, J., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1999). (2S,3R)TMT-L-Tic-OH is a potent inverse agonist at the human δ-opioid receptor. European Journal of Pharmacology, 380(1), R9-R10.
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  PMID: 10513562;Abstract: We examined the pharmacologic effect of β-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid ((2S,3R)TMT-L-Tic-OH) on G protein activation in membranes prepared from Chinese Hamster Ovary cells transfected with cDNA of the human δ-opioid receptor. (2S,3R)TMT-L-Tic-OH inhibited G protein activation to 58% of basal with an EC50 of 0.72 nM as determined by [35S]GTPγS binding. These findings suggest that (2S,3R)TMT-L-Tic-OH is a highly potent inverse agonist at the human δ-opioid receptor. Copyright (C) 1999 Elsevier Science B.V.
• Hruby, V. J. (1999). Editorial: A call for greater participation in The Journal of Peptide Research. Journal of Peptide Research, 54(6), 459-.
• Hruby, V. J. (1999). The Journal of Peptide Research: Editorial. Journal of Peptide Research, 53(4), 353-.
• Hruby, V. J. (1999). The Journal of Peptide Research: Editorial. Journal of Peptide Research, 54(3), 175-176.
• Hruby, V. J., & Agnes, R. S. (1999). Conformation-activity relationships of opioid peptides with selective activities at opioid receptors. Biopolymers - Peptide Science Section, 51(6), 391-410.
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  PMID: 10797229;Abstract: The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (μ, δ, and κ) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for μ, δ, and κ receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have δ agonist, μ antagonist, and κ agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies. (C) 2000 John Wiley and Sons, Inc.
• Huang, Q., Hruby, V. J., & Tatro, J. B. (1999). Role of central melanocortins in endotoxin-induced anorexia. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 276(3 45-3), R864-R871.
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  PMID: 10070149;Abstract: Inflammation and microbial infection produce symptoms, including fever, anorexia, and hypoactivity, that are thought to be mediated by endogenous proinflammatory cytokines. Melanocortins are known to act centrally to suppress effects on fever and other sequelae of proinflammatory cytokine actions in the central nervous system, but the roles of melanocortins in anorexia and hypoactivity occurring during the acute phase response are unknown. The present study was designed to determine the effects of exogenous and endogenous α-melanocyte stimulating hormone (α-MSH) on lipopolysaccharide (LPS)-induced anorexia in relation to their effects on fever. Rats were fasted overnight to promote feeding behavior, then injected intraperitoneally with LPS (100 μg/kg ip), followed 30 min later by intracerebroventricular injection of either α-MSH or the melanocortin receptor subtype 3/subtype 4 (MC3-R/MC4-R) antagonist SHU-9119. Food intake, locomotor activity, and body temperature (Tβ) were monitored during the ensuing 24-h period. Each of two intracerebroventricular doses of α-MSH (30 and 300 ng) potentiated the suppressive effects of LPS on food intake and locomotion, despite the fact that the higher dose alleviated LPS-induced fever. In control rats that were not treated with LPS, only the higher dose of α-MSH significantly inhibited food intake, and T(b) and locomotor activity were unaffected. To assess the roles of endogenous central melanocortins, LPS-treated rats received intracerebroventricular SHU-9119 (200 ng). Central MC3-R/MC4-R blockade did not affect T(b) or food intake in the absence of LPS treatment, but it reversed the LPS-induced reduction in 24-h food intake and increased LPS-induced fever without altering the LPS- induced suppression of locomotion. Taken together, the results suggest that exogenous and endogenous melanocortins acting centrally exert divergent influences on different aspects of the acute phase response, suppressing LPS- induced fever but contributing to LPS-induced anorexia and hypoactivity.
• Kovelowski, C. J., Bian, D., Hruby, V. J., Lai, J., Ossipov, M. H., & Porreca, F. (1999). Selective opioid δ agonists elicit antinociceptive supraspinal/spinal synergy in the rat. Brain Research, 843(1-2), 12-17.
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  PMID: 10528105;Abstract: A multiplicative antinociceptive interaction of morphine activity at supraspinal and spinal sites has been clearly established and is thought to be responsible, in part, for the clinical utility of this compound in normal dose-ranges. While synergistic actions of μ-opioid receptor agonists have been shown, it is unclear whether a similar interaction exists for opioid agonists acting via δ-opioid receptors. Responses to acute nociception were determined with the 52°C hot plate, 52°C warm-water tail-flick and the Hargreaves paw-withdrawal tests. The peptidic opioid δ1 agonist [D-Pen2,D- Pen5]enkephalin (DPDPE) or δ2 agonist [D-Ala2,Glu4]deltorphin (DELT) were given into the rostral-ventral medulla (RVM), intrathecally (i.th.) or simultaneously into both the RVM and i.th. (1:1 fixed ratio). Both of the opioid δ agonists produced dose-dependent antinociception in all tests. With the exception of DPDPE in the hot plate test, isobolographic analysis revealed that the supraspinal/spinal antinociceptive interaction for both DPDPE and DELT were synergistic in all nociceptive tests. These data suggest that opioid δ agonists exert a multiplicative antinociceptive interaction between supraspinal and spinal sites to acute noxious stimuli and suggest possibility that compounds acting through δ-opioid receptors may have sufficient potency for eventual clinical application.
• Kovelowski, C. J., Ossipov, M. H., Hruby, V. J., & Porreca, F. (1999). Lesions of the dorsolateral funiculus block supraspinal opioid delta receptor mediated antinociception in the rat. Pain, 83(2), 115-122.
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  PMID: 10534582;Abstract: Previous experiments have demonstrated that [D-Ala2,Glu4]deltorphin (DELT) produces δ-receptor mediated antinociceptive effects when microinjected into the rat lateral ventricle and ventral medial medullary reticular formation (MRF), but not in the periaqueductal grey region (PAG). The present experiments were undertaken to further characterize the role of δ opioid agonists microinjected into the MRF and to explore the possibility of a descending pain modulatory system which might be linked to supraspinal δ opioid receptors. Rats received formalin into the dorsum of the right hindpaw and flinching responses were recorded. DELT given intracerebroventricularly (i.c.v.), intrathecally (i.th.) or into the MRF before formalin produced a dose-dependent and δ opioid receptor-mediated attenuation of both the first and second phases of the formalin-induced foot flinch response. DELT given i.c.v., i.th., or into the MRF also blocked formalin-induced increase in Fos-like immunoreactivity (FLI) in the dorsal horn of lumbar spinal cord ipsilateral to the formalin injection. Unilateral lesioning of the ipsilateral dorsolateral funiculus (DLF) did not alter nociceptive responses to formalin alone, but blocked the antinociceptive effect of DELT administered into the MRF; DELT was fully active in sham-DLF lesioned rats. Additionally, rats with DLF lesions did not show decreases in formalin-induced FLI in the ipsilateral lumbar spinal cord after injection of DELT into the MRF. These data suggest that δ opioid receptors in the MRF may be involved in activation of a descending inhibitory pain pathway projecting through the DLF to modulate tonic nociceptive input at the spinal level. Copyright (C) 1999 International Association for the Study of Pain. Published by Elsevier Science B.V.
• Lipkowski, A. W., Misicka, A., Davis, P., Stropova, D., Janders, J., Lachwa, M., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1999). Biological activity of fragments and analogues of the potent dimeric opioid peptide, biphalin. Bioorganic and Medicinal Chemistry Letters, 9(18), 2763-2766.
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  PMID: 10509931;Abstract: The synthesis and biological activity of two fragments of the very potent opioid peptide biphalin, showed that Tyr-D-Ala-Gly-Phe-NH-NH
• Prabhu, N. V., Perkyns, J. S., Pettitt, B. M., & Hruby, V. J. (1999). Structure and dynamics of α-MSH using DRISM integral equation theory and stochastic dynamics. Biopolymers, 50(3), 255-272.
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  PMID: 10397788;Abstract: The structural and dynamical features of the hormone α-MSH in solution have been examined over a 100 ns time scale by using free energy molecular mechanics models at room temperature. The free energy surface has been modeled using methods from integral equation theory and the dynamics by the Langevin equation. A modification of the accessible surface area friction drag model was used to calculate the atomic friction coefficients. The molecule shows a stable β-turn conformation in the message region and a close interaction between the side chains of His6, Phe7, and Trp9. A salt bridge between Glu5 and Arg8 was found not to be a preferred interaction, whereas a Glu5 and Lys11 salt bridge was not sampled, presumably due to relatively high free energy barriers. The message region was more conformationally rigid than the N-terminal region. Several structural features observed here agree well with experimental results. The conformational features suggest a receptor-hormone interaction model where the hydrophobic side chains of Phe7 and Trp9 interact with the transmembrane portion of the MC1 receptor. Also, the positively charged side chain of Arg8 and the imidazole side chain of His6 may interact with the negatively charged portions of the receptor which may even be on the receptor's extracellular loops.
• Soloshonok, V. A., Cai, C., & Hruby, V. J. (1999). Asymmetric Michael addition reactions of chiral Ni(II)-complex of glycine with (N-trans-enoyl)oxazolidines: Improved reactivity and stereochemical outcome. Tetrahedron Asymmetry, 10(22), 4265-4269.
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  Abstract: Application of the (N-trans-enoyl)oxazolidines as Michael acceptors in the kinetically controlled additions with a Ni(II)-complex of the chiral Schiff base of glycine with (S)-o-[N-(N-benzylprolyl)amino]benzophenone 1 was shown to be synthetically advantageous over the alkyl enoylates, allowing for remarkable improvement in reactivity and, in most cases, diastereoselectivity of the reactions. While the stereochemical outcome of the Michael additions of the aliphatic (N-trans-enoyl)oxazolidines with complex 1 depended on the steric bulk of the alkyl group on the starting oxazolidines, the diastereoselectivity of the aromatic (N-trans-enoyl)oxazolidines reactions was found to be controlled by the electronic properties of the aryl ring. In particular, the additions of complex 1 with (N-cinnamoyl)oxazolidines, bearing electron-withdrawing substituents on the phenyl ring, afforded the (2S,3R)-configured products with synthetically useful selectivity and in quantitative chemical yield, thus allowing an efficient access to sterically constrained β-substituted pyroglutamic acids and related compounds. (C) 1999 Elsevier Science Ltd.
• Soloshonok, V. A., Cai, C., Hruby, V. J., & Meervelt, L. V. (1999). Asymmetric synthesis of novel highly sterically constrained (2S,3S)-3- methyl-3-trifluoromethyl- and (2S,3S,4R)-3-trifluoromethyl-4- methylpyroglutamic acids. Tetrahedron, 55(41), 12045-12058.
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  Abstract: Asymmetric synthesis of the novel highly sterically constrained (2S,3S)- 3-methyl-3-trifluoromethyl-and (2S,3S,4R)-3-trifluoromethyl-4- methylpyroglutamic acids has been developed via diastereoselective Michael addition reactions between a Ni(II) complex of the chiral non-racemic Schiff base of glycine with (S)-o-[N-(N-benzylprolyl)amino]benzophenone (BPB) and the corresponding trifluoromethyl-containing crotonates. Of particular synthetic interest is the reaction of the glycine Ni-complex with ethyl 3- trifluoromethyl crotonate featuring excellent diastereoselectivity (>98% de) as a result of complete stereochemical discrimination between the methyl and trifluoromethyl groups. A mechanistic rationale for the observed kinetically controlled stereochemical outcome is discussed.
• Soloshonok, V. A., Cai, C., Hruby, V. J., Meervelt, L. V., & Mischenko, N. (1999). Stereochemically defined C-substituted glutamic acids and their derivatives. 1. An efficient asymmetric synthesis of (2S,3S)-3-methyl- and - 3-trifluoromethylpyroglutamic acids. Tetrahedron, 55(41), 12031-12044.
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  Abstract: An efficient asymmetric synthesis of biologically important (2S,3S)-3- methyl- and (2S,3S)-3-trifluoromethylpyroglutamic acid has been developed. The method consists of diastereoselective Michael addition reaction between ethyl crotonate or ethyl 4,4,4-trifluorocrotonate and a Ni(II) complex of the chiral non-racemic Schiff base of glycine with (S)-o-[N-(N- benzylprolyl)amino]benzophenone (BPB) followed by decomposition of the addition products by aq. HCl and treatment of the resultant glutamic acid derivatives with NH4OH to afford the target pyroglutamic acids along with recovery of the chiral auxiliary BPB. The stereochemical outcome of the addition reactions was found to be subjected to kinetic control. A mechanistic rationale for the observed stereochemical preferences is discussed.
• Tang, Q., Gandhoke, R., Burritt, A., Hruby, V. J., Porreca, F., & Lai, J. (1999). High-affinity interaction of (des-tyrosyl)dynorphin A(2-17) with NMDA receptors. Journal of Pharmacology and Experimental Therapeutics, 291(2), 760-765.
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  PMID: 10525097;Abstract: The opioid peptide dynorphin A elicits non-opioid receptor-mediated, neurotoxic response in vivo, which is blocked by pretreatment with MK-801, a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist. In the present study, we examined the possible direct interaction of dynorphin A on the NMDAR. A nonopioid dynorphin A analog, 125I-(des-tyrosyl) dynorphin A(2-17), was used in radioligand binding analysis on rat cortical brain membranes. This radioligand exhibited a saturable, specific binding at high affinity with a K(d) value of 9.4 ± 1.6 nM and maximal binding of 2.4 ± 0.6 pmol/mg protein. This binding site was associated with the NMDAR complex because it was modulated by a number of NMDAR ligands. Transient expression of the rat NR1a/NR2A complex in human embryonic kidney 293 cells confirmed a coexpression of 125I-(des-tyrosyl) dynorphin A(2-17), [3H]CGP39,653, and [3H]MK-801 binding. These data provide direct evidence of the presence of a high-affinity binding site for dynorphin A on the NMDAR. The modulatory effect of the various NMDAR-selective ligands on dynorphin A binding suggests that dynorphin A may bind preferentially to the closed/desensitized state of the NMDAR. The physiological role of dynorphin A binding to the NMDAR remains to be established.
• Al-Obeidi, F., Hruby, V. J., & Sawyer, T. K. (1998). Peptide and peptidomimetic libraries: Molecular diversity and drug design. Applied Biochemistry and Biotechnology - Part B Molecular Biotechnology, 9(3), 205-223.
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  PMID: 9718581;Abstract: Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split- couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.
• Al-Obeidi, F., O'Connor, S. D., Job, C., Hruby, V. J., & Pettitt, B. M. (1998). NMR and quenched molecular dynamics studies of superpotent linear and cyclic α-melanotropins. Journal of Peptide Research, 51(6), 420-431.
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  PMID: 9650716;Abstract: Conformational searching, computer simulations, synthesis and NMR are used on a variety of α melanocyte-stimulating hormone (α-MSH) analogues to understand the physical characteristics required for biological potency. Peptides I (Ac-[Nle4,Asp5,D-Phe7,Lys10]α-MSH(4-10)-NH2), II (Ac- c[Nle4,Asp5,D-Phe7,Lys10]α-MSH(4-10)-NH2) and III (Ac-[Nle4,Asp5,D- Phe7,Dap10]α-MSH(4-10)-NH2 all show very similar conformational properties (backbone and side-chain torsional angles), and all-display high biological potencies. The modeling results for these compounds are supported by the NMR data. Peptide IV (Ac-c[Nle4,Asp5,D-Phe7,Dap10]α-MSH(4-10)- NH2) appears to have a markedly different conformation and has decreased biological potency.
• Alfaro-Lopez, J., Yuan, W., Phan, B. C., Kamath, J., Lou, Q., Lam, K. S., & Hruby, V. J. (1998). Discovery of a novel series of potent and selective substrate-based inhibitors of p60(c-src) protein tyrosine kinase: Conformational and topographical constraints in peptide design. Journal of Medicinal Chemistry, 41(13), 2252-2260.
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  PMID: 9632358;Abstract: On the basis of the efficient substrate for p60(c-src) protein tyrosine kinase (PTK) YIYGSFK-NH2 (1) (K(m) = 55 μM) obtained by combinatorial methods, we have designed and synthesized a series of conformationally and topographically constrained substrate-based peptide inhibitors of this enzyme, which showed IC50 values in the low-micromolar range (1-3 μM). A 'rotamer scan' was performed by introducing the four stereoisomers of β- Me(2')Nal in the postulated interaction site of the peptide inhibitor 23 (IC50 = 1.6 μM). This substitution led to selective and potent inhibitors of p60(c-src) PTK; however, no substantial difference in potency was observed among them. This and the results of the 'stereochemical scan' performed at residues 2 and 7 of 3 (peptides 19-21), which form the disulfide bond, may suggest that the enzyme active site does not have rigid topographic requirements and thus is able to achieve important conformational changes to bind the ligand as long as the pharmacophore pattern in the inhibitor is conserved. Two new potent iodo-containing nonphosphorylatable tyrosine analogues were also incorporated into our lead inhibitory sequence 23, producing the most potent inhibitors for p60(c-src) PTK identified thus far in our studies. Compounds 29 and 30 exhibit IC50 values of 0.13 and 0.54 μM, respectively. Peptide 29 is 420-fold more potent than the parent peptide 1. Selectivity studies of peptides 23-30 toward p60(c-src), Lyn, and Lck PTK showed in general high Lyn/Src and moderate Lck/Src selectivity ratios. We found that the χ1 space constraints of the specialized amino acids, introduced at position 3 of the peptide lead 23, were not as important as the configuration of the C(α) of that residue to recognize the subtle chemical environment surrounding the active site of Src and Lck PTK, as reflected on the obtained Lck/Src selectivity ratios.
• Burkey, T. H., Ehlert, F. J., Hosohata, Y., Quock, R. M., Cowell, S., Hosohata, K., Varga, E., Stropova, D., Li, X., Slate, C., Nagase, H., Porreca, F., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1998). The efficacy of δ-opioid receptor-selective drugs. Life Sciences, 62(17-18), 1531-1536.
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  PMID: 9585131;Abstract: δ-Opioid receptor-selective drugs may provide an alternative to μ- opioid-selective drugs currently used for the relief of pain. To develop improved δ-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new δ-opioid receptor-selective drugs.
• Hadley, M. E., Hruby, V. J., Blanchard, J., Dorr, R. T., Levine, N., Dawson, B. V., al-Obeidi, F., & Sawyer, T. K. (1998). Discovery and development of novel melanogenic drugs. Melanotan-I and -II.. Pharmaceutical biotechnology, 11, 575-595.
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  PMID: 9760697;
• Han, Y., & Hruby, V. J. (1998). Erratum: Lewis acid promoted conjugate addition of vinylmagnesium bromide to chiral α,β-unsaturated N-acyl oxazolidinones (Tetrahedron Letters (1997) 38 (7317)) PII: S0040403997017772. Tetrahedron Letters, 39(47), 8561-.
• Hosohata, K., Burkey, T. H., Alfaro-Lopez, J., Varga, E., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1998). Endomorphin-1 and endomorphin-2 are partial agonists at the human μ- opioid receptor. European Journal of Pharmacology, 346(1), 111-114.
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  PMID: 9617760;Abstract: Recently two tetrapeptide ligands that bind preferentially to the μ- opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human μ-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPγS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the μ-selective agonist [D-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the μ-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.
• Hruby, V. J. (1998). Chapter 16 Glucagon: Molecular biology and structure-activity. Principles of Medical Biology, 10(C), 387-401.
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  Abstract: The actions of glucagon at hepatic and fat cell receptors is critical for the control of glucose levels and glucose homeostasis in normal and diabetic states. Its interactions with insulin in maintaining homeostasis in normal animals is understood in considerable detail and tools are now available to obtain an even deeper understanding. In the diabetic state, the interplay of glucagon and insulin (and other factors) in hyperglycemia, ketoses, and other manifestations of diabetes is less clear, and provides a continuing challenge for biochemists, physiologists, endocrinologists, and molecular biologists. Fortunately, new tools in the form of potent receptor agonists and antagonists, cloned receptors, assays with enhanced sensitivity, and increased understanding of receptor transduction mechanism should greatly aid in obtaining these needed new insights and understanding. © 1998 Elsevier B.V. All rights reserved.
• Hruby, V. J. (1998). Editorial. Journal of Peptide Research, 52(5), 329-.
• Hruby, V. J., Han, G., & Hadley, M. E. (1998). Design and bioactivities of melanotropic peptide agonists and antagonists: Design based on a conformationally constrained somatostatin template. Letters in Peptide Science, 5(2-3), 117-120.
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  Abstract: α-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (α-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained α-melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the μ opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.
• Huang, Q., Hruby, V. J., & Tatro, J. B. (1998). Systemic α-MSH suppresses LPS fever via central melanocortin receptors independently of its suppression of corticosterone and IL-6 release. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 275(2 44-2), R524-R530.
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  PMID: 9688689;Abstract: Systemically administered α-melanocyte-stimulating hormone (α-MSH) inhibits endotoxin (lipopolysaccharide; LPS)-or interleukin (IL)-1-induced fever and adrenocortical activation, but the sites of these actions and the mechanisms involved are unknown. The aims of this study were, first, to determine whether melanocortin receptors (MCR) located within the central nervous system mediate the suppressive effects of peripherally administered α-MSH on LPS-induced fever and activation of the pituitary-adrenal axis and, second, to determine whether systemic α-MSH suppresses the LPS-induced rise in plasma IL-6 levels, potentially contributing to its antipyretic effect. Male rats received Escherichia coli LPS (25 μg/kg ip). Core body temperatures (T(b)) were determined hourly by radiotelemetry (0-8 h), and blood was withdrawn via venous catheters for plasma hormone immunoassays (0- 2 h) and IL-6 bioassay (0-8 h). α-MSH (100 μg/kg ip) completely prevented the onset of LPS-induced fever during the first 3-4 h after LPS and suppressed fever throughout the next 4 h but did not affect T(b) in afebrile rats treated with intraperitoneal saline rather than LPS. Intraperitoneal α- MSH also suppressed the LPS-induced rise in plasma IL-6, ACTH, and corticosterone (CS) levels. Intracerebroventricular injection of SHU-9119, a potent melanocortin-4 receptor (MC4-R)/MC3-R antagonist, completely blocked the antipyretic effect of intraperitoneal α-MSH during the first 4 h after LPS but had no effect on α-MSH-induced suppression of LPS-stimulated plasma IL-6 and CS levels. Taken together, the results indicate that the antipyretic effect of peripherally administered α-MSH during the early phase of fever is mediated by MCR within the brain. In contrast, the inhibition of LPS-induced increases in plasma CS and IL-6 levels by intraperitoneal α-MSH appears to be mediated by a different mechanism(s), and these effects do not contribute to its antipyretic action.
• Kövér, K. E., Uhrín, D., & Hruby, V. J. (1998). Gradient- and Sensitivity-Enhanced TOCSY Experiments. Journal of Magnetic Resonance, 130(2), 162-168.
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  PMID: 9500895;Abstract: A pulsed field gradient version of the sensitivity-enhanced 2D TOCSY experiment is proposed which yields high-quality spectra with improved sensitivity and a minimum of two scans per t1 increment. For rapid acquisition of ID TOCSY spectra, the 1D DPFGSE-TOCSY experiment was modified to include phase-encoded multiple-selective excitation followed by a simple spectral editing. Combination of these two building blocks is used in a sensitivity-enhanced 2D analog of the 3D TOCSY-TOCSY experiment which provides an efficient tool for resolving severely overlapped signals of oligomers in short experimental time. © 1998 Academic Press.
• Liao, S., Alfaro-Lopez, J., Shenderovich, M. D., Hosohata, K., Lin, J., Xiaoping, L. i., Stropova, D., Davis, P., Jernigan, K. A., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1998). De novo design, synthesis, and biological activities of high-affinity and selective non-peptide agonists of the δ-opioid receptor. Journal of Medicinal Chemistry, 41(24), 4767-4776.
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  PMID: 9822547;Abstract: On the basis of the structure-activity relationships of δ-opioid- selective peptide ligands and on a model of the proposed bioactive conformation for a potent and selective, conformationally constrained δ- opioid peptide ligand [(2S,3R)-TMT1]DPDPE, a series of small organic peptide mimetic compounds targeted for the δ-opioid receptor have been designed, synthesized, and evaluated in radiolabeled ligand binding assays and in vitro bioassays. The new non-peptide ligands use piperazine as a template to present the most important pharmacophore groups, including phenol and phenyl groups and a hydrophobic moiety. This hydrophobic group was designed to mimic the hydrophobic character of the D-Pen residues in DPDPE, which has been found to be extremely important for increasing the binding affinity and selectivity of these non-peptide ligands for the δ-opioid receptor over the μ-opioid receptor. Compound 6f (SL3111) showed 8 nM binding affinity and over 2000-fold selectivity for the δ-opioid receptor over the μ-opioid receptor. Both enantiomers of SL-3111 were separated, and the (-)-isomer was shown to be the compound with the highest affinity for the δ-opioid receptor found in our study (IC50 = 4.1 nM), with a selectivity very similar to that observed for the racemic compound. The phenol hydroxyl group of SL-3111 turned out to be essential to maintain high affinity for the δ-opioid receptor, which also was observed in the case of the δ-opioid-selective peptide ligand DPDPE. Binding studies of SL-3111 and [p-C]Phe4]DPDPE on the cloned wild-type and mutated human δ-opioid receptors suggested that the new non-peptide ligand has a binding profile similar to that of DPDPE but different from that of (+)-4-[((αR)-α(2S,5R)-4-allyl-2,5dimethyl-1- piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), another δ- opioid-selective non-peptide ligand.
• Liao, S., Shenderovich, M. D., Zhang, Z., Maletinska, L., Slaninova, J., & Hruby, V. J. (1998). Substitution of the side-chain-constrained amino acids β-methyl-2',6'- dimethyl-4'-methoxytyrosine in position 2 of a bicyclic oxytocin analogue provides unique insights into the bioactive topography of oxytocin antagonists [24]. Journal of the American Chemical Society, 120(29), 7393-7394.
• Lin, J., Liao, S., & Hruby, V. J. (1998). Asymmetric syntheses of highly hydrophobic chimeric aromatic amino acids: 2-Amino-3,3'-diarylpropionic acids. Tetrahedron Letters, 39(20), 3117-3120.
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  Abstract: Four conformationally constrained, highly hydrophobic, and enantiomerically pure chimeric aromatic amino acids have been asymmetrically synthesized in 7 steps with overall yields of 20-30%.
• Ni, X., Kesterson, R. A., Sharma, S. D., Hruby, V. J., Cone, R. D., Wiedemann, E., & Humphreys, M. H. (1998). Prevention of reflex natriuresis after acute unilateral nephrectomy by melanocortin receptor antagonists. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 274(4 43-4), R931-R938.
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  PMID: 9575953;Abstract: γ-Melanocyte-stimulating hormone (γ-MSH), atrial natriuretic peptide (ANP), and oxytocin have been identified as candidate hormonal mediators of the reflex natriuresis that follows acute unilateral nephrectomy (AUN). Pharmacological characterization of the third melanocortin receptor (MC3-R) indicates that it uniquely responds to physiological concentrations of γ- MSH. We tested the roles of γ-MSH, ANP, and oxytocin in the postnephrectomy natriuresis by carrying out AUN during continuous intrarenal infusion of specific antagonists for their cognate receptors. In anesthetized Sprague- Dawley rats, urinary sodium excretion (U(Na)V) increased from 0.34 ± 0.04 to 1.12 ± 0.11 μeq/min 90 min after AUN (P < 0.001). No change in U(Na)V occurred in rats undergoing a sham AUN procedure. Plasma immunoreactive γ- MSH concentration was 53 ± 8 fmol/ml after sham AUN but 112 ± 17 fmol/ml after AUN (P < 0.01). SHU-9119 and SHU-9005 are substituted derivatives of α-MSH with potent antagonism at the MC3-R in vitro. Infusion of these compounds at 5 pmol/min completely blocked the natriuretic response to AUN despite a similar elevation in plasma γ-MSH (111 ± 12 vs. 49 ± 8 fmol/ml in sham rats, P < 0.01). Intrarenal infusion of the ANP receptor antagonist A-71915 (5 pmol/min) or the oxytocin receptor antagonist [d(CH2)51, Tyr(Me)2,Orn8] vasotocin (10 pmol/min) effectively inhibited the natriuresis induced by intravenous infusion of ANP or oxytocin (each at i pmol/min), respectively, but did not block the natriuresis after AUN. Plasma immunoreactivity of these peptides was not increased after AUN. These results indicate that reflex natriuresis after AUN is accompanied by an increase in plasma γ-MSH but not ANP or oxytocin concentration and is prevented by intrarenal infusion of receptor antagonists with selectivity for MC3-R. The data indicate that γ-MSH or a closely related peptide mediates postnephrectomy natriuresis and provide further support for the possibility that γ-MSH may play a wider role in sodium homeostasis.
• Nikiforovich, G. V., Sharma, S. D., Hadley, M. E., & Hruby, V. J. (1998). Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues. Biopolymers, 46(3), 155-167.
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  PMID: 9699465;Abstract: Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10- Lys11-Pro12-Val13-NH2) and [D-Phe7]αMSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly10 residue, as substitutions for L-Ala, D- Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH (4-11) and α-MSH (5-11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly10 residue with L-Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp 'message' sequence within the sequences of α-MSH and [D- Phe7]α-MSH. In such conformations the aromatic moieties of the side chains of the His6, L/D-Phe7, and Trp9 residues form a continuous hydrophobic 'surface,' presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D-Phe7]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations.
• Olczak, J., Kaczmarek, K., Maszczyńska, I., Lisowski, M., Stropova, D., Hruby, V. J., Yamamura, H. I., Lipkowski, A. W., & Zabrocki, J. (1998). Consequences of cis-amide bond simulation in opioid peptides. International Journal of Peptide Research and Therapeutics, 5(5-6), 437-440.
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  Abstract: Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity. © 1998 Kluwer Academic Publishers.
• Olczak, J., Kaczmarek, K., Maszczyńska, I., Lisowski, M., Stropova, D., Hruby, V. J., Yamamura, H. I., Lipkowski, A. W., & Zabrocki, J. (1998). Consequences of cis-amide bond simulation in opioid peptides. Letters in Peptide Science, 5(5-6), 437-440.
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  Abstract: Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.
• Slaninova, J., Appleyard, S. M., Misicka, A., Lipkowski, A. W., Knapp, R. J., Weber, S. J., Davis, T. P., Yamamura, H. I., & Hruby, V. J. (1998). [¹²⁵I-Tyr¹]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes. Life Sciences, 62(14), 199-204.
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  PMID: 9570343;Abstract: Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I- Tyr1]biphalin to the δ and μ opioid receptors are not independent. [125I-Tyr1]Biphalin binds to δ receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to δ receptors in rat brain membranes was hardly evident and μ receptor binding predominated or at least was much more readily detectable in this preparation.
• Sturm, N. S., Lin, Y., Burley, S. K., Krstenansky, J. L., Ahn, J., Azizeh, B. Y., Trivedi, D., & Hruby, V. J. (1998). Structure-function studies on positions 17, 18, and 21 replacement analogues of glucagon: The importance of charged residues and salt bridges in glucagon biological activity. Journal of Medicinal Chemistry, 41(15), 2693-2700.
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  PMID: 9667960;Abstract: We have designed and synthesized eight compounds 2-9 which incorporate various amino acid residues in positions 17, 18, and 21 of the glucagon molecule: 2, [Lys17]glucagon amide; 3, [Lys18]glucagon amide; 4, [Nle17,Lys18,Glu21]glucagon amide; 5, [Orn17,18 Glu21]glucagon amide; 6, [D-Arg17]glucagon; 7, [D-Arg18]glucagon; 8, [D- Phe17]glucagon; and 9, [D-Phe18]glucagon. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have binding affinity IC50 values (in nM) of 0.7, 4.1, 1.0, 2.0, 5.0, 25.0, 43.0, and 32.0, respectively. When these compounds were tested for their ability to stimulate adenylate cyclase (AC) activity, they were found to be full or partial agonists having maximum stimulation values of 100, 100, 100, 100, 87, 78, 94, and 100%, respectively. On the basis of the X-ray crystal structure of [Lys17,18,Glu21]glucagon amide reported here, the ability to form a salt bridge between Lys18 and Glu21 is probably key to their increased binding and second messenger activities. Among the eight analogues synthesized here, only analogue 4 preserves the ability to form a salt bridge between Lys18 and Glu21. However, since these modifications are minor they do not seem to change the amphiphilic character of the C-terminus, allowing these analogues to reach 78-100% stimulation in the adenylate cyclase assay. Biological data from analogues 6-9 supports the idea that position 18 of glucagon may influence binding only, while position 17 may influence both receptor recognition and transduction.
• Uhrín, D., Batta, G., Hruby, V. J., Barlow, P. N., & Kövér, K. E. (1998). Sensitivity- and Gradient-Enhanced Hetero (ω₁) Half-Filtered TOCSY Experiment for Measuring Long-Range Heteronuclear Coupling Constants. Journal of Magnetic Resonance, 130(2), 155-161.
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  PMID: 9515088;Abstract: An enhanced version of the X(ω1) half-filtered TOCSY experiment for measurement of long-range heteronuclear coupling constants is proposed which yields high-quality spectra with substantially increased sensitivity and resolution. The modified method features gradient-enhanced X filtering sequences, broadband homonuclear decoupling during t1, optional 1JXII scaling in the F1 domain, and gradient coherence selection in combination with the sensitivity-enhanced protocol for the TOCSY transfer. These modifications extend the applicability of the method - coupling constants can be measured accurately for natural abundance samples at low concentrations and for compounds yielding complex spectra. Computer-aided analysis of E.COSY-type multiplets is applied for the determination of heteronuclear long-range coupling constants. © 1998 Academic Press.
• Vanderah, T. W., Raffa, R. B., Lashbrook, J., Burritt, A., Hruby, V., & Porreca, F. (1998). Orphanin-fq/nociceptin: Lack of antinociceptive, hyperalgesic or allodynic effects in acute thermal or mechanical tests following intracerebroventricular or intrathecal administration to mice or rats. European Journal of Pain, 2(3), 267-280.
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  PMID: 15102387;Abstract: A recent review calls attention to the discrepant results resulting from studies that have examined the nociceptive or antinociceptive properties of orphanin-FQ/nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys- Leu-Ala-Asn-Gln; OFQ/N), the heptadecapeptide isolated from rat (nociceptin) and pig (orphanin FQ) brain that binds with high affinity to the opioid 'orphan' receptor (a seven transmembrane protein with sequence homology to opioid receptors), but exhibits only low affinity binding with conventional opioid ligands. Some of the discrepancy might result from differences in species, test, route of administration or time-course. We undertook a comprehensive examination of the effects of spinal (i.t.) or supraspinal (i.c.v.) administration of OFQ/N in mice and rats. Mice treated with OFQ/N either i.t. or i.c.v. demonstrated no significant nociceptive effect in the hot plate, warm-water or radiant heat tail-flick tests (except for the highest and most sedative dose of 10 nmol i.c.v. in the mouse warm-water tail-flick test). Pretreatment with the opioid antagonist naloxone or with peptidase inhibitors did not enhance the nociceptive effects of OFQ/N peptide in the warm-water tail-flick test. The motor activity in mice administered OFQ/N i.c.v. decreased significantly compared to controls. Rats administered i.c.v. or i.t. OFQ/N displayed no significant difference from vehicle- treated animals in similar noxious stimulus tests and OFQ/N-treated rats did not exhibit allodynia in a paw-withdrawal test. Overall, OFQ/N was ineffective in significantly altering response to noxious stimuli, regardless of whether the peptide was given at supraspinal or spinal sites in mice or in rats. In addition, i.c.v. or i.t. application of antisense or mismatch ODN to the orphan receptor did not modify tail-flick latency in either mice or rats, arguing against a tonic nociceptive tone mediated via the OFQ/N receptor.
• Wessells, H., Fuciarelli, K., Hansen, J., Hadley, M. E., Hruby, V. J., Dorr, R., & Levine, N. (1998). Synthetic melanotropic peptide initiates erections in men with psychogenic erectile dysfunction: Double-blind, placebo controlled crossover study. Journal of Urology, 160(2), 389-393.
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  PMID: 9679884;Abstract: Purpose: We evaluated the erectogenic properties of a new cyclic α-melanocyte-stimulating hormone analogue, Melanotan-II, to treat men with psychogenic erectile dysfunction. Materials and Methods: Ten men with erectile dysfunction of no known organic cause were entered in a double-blind, placebo controlled crossover study in which the erectogenic properties of Melanotan- II and a vehicle placebo were compared using real-time RigiScan monitoring. The presence, duration and rigidity of erections were recorded during a 6-hour period. Results: In 8 of 10 men treated with Melanotan-II clinically apparent erections developed. Mean duration of tip rigidity greater than 80% was 38.0 minutes with Melanotan-II and 3.0 with placebo (p = 0.0045). Transient side effects of nausea, stretching and yawning, and decreased appetite were reported more frequently after injections of Melanotan-II than placebo but none required treatment. Conclusions: Melanotan- II is a potent initiator of erections in men with psychogenic erectile dysfunction and has manageable side effects at a dose of 0.025 mg./kg.
• Wessells, H., Fuciarelli, K., Hansen, J., Hadley, M. E., Hruby, V. J., Dorr, R., & Levine, N. (1998). Synthetic melanotropic peptide initiates erections in men with psychogenic erectile dysfunction: Double-blind, placebo controlled crossover study. Journal of Urology, 160(2), 393-.
• Xi-Ping, N., Kesterson, R. A., Sharma, S. D., Hruby, V. J., Cone, R. D., Wiedemann, E., & Humphreys, M. H. (1998). Prevention of reflex natriuresis after acute unilateral nephrectomy by melanocortin receptor antagonists. American Journal of Physiology, 274(4 PART2), R931-R938.
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  Abstract: Mclanocyte-stimu-lating hormone (γ-MSH), atrial natriuretic peptide (ANP), and oxytocin have been identified as candidate hormonal mediators of the reflex natriuresis that follows acute unilateral nephrectomy (AUN). Pharmacological characterization of the third melanocortin receptor (MC3-R) indicates that it uniquely responds to physiological concentrations of γ-MSH. We tested the roles of γ-MSH, ANP, and oxytocin in the postnephrectomy natriuresis by carrying out AUN during continuous intrarenal infusion of specific antagonists for their cognate receptors. In anesthetized Sprague-Dawley rats, urinary sodium excretion (UNaV) increased from 0.34 ± 0.04 to 1.12 ±0.11 μeq/min 90 min after AUN (P < 0.001). No change in UNaV occurred in rats undergoing a sham AUN procedure. Plasma immunoreactive 7-MSH concentration was 53 ±8 fmol/ml after sham AUN but 112 ±17 fmoyml after AUN (P < 0.01). SHU-9119 and SHU-9005 are substituted derivatives of a-MSH with potent antagonism at the MC3-R in vitro. Infusion of these compounds at 5 pmol/min completely blocked the natriuretic response to AUN despite a similar elevation in plasma 7-MSH (111 ±12 vs. 49 ±8 fmol/ml in sham rats, P < 0.01). Intrarenal infusion of the ANP receptor antagonist A-71915 (5 pmol/min) or the oxytocin receptor antagonist [dCCH2)51, Tyr(Me)2,Orn8] vasotocin (10 pmol/min) effectively inhibited the natriuresis induced by intravenous infusion of ANP or oxytocin (each at 1 pmol/min), respectively, but did not block the natriuresis after AUN. Plasma immunoreactivity of these peptides was not increased after AUN. These results indicate that reflex natriuresis after AUN is accompanied by an increase in plasma γ-MSH but not ANP or oxytocin concentration and is prevented by intrarenal infusion of receptor antagonists with selectivity for MC3-R. The data indicate that γ-MSH or a closely related peptide mediates postnephrectomy natriuresis and provide further support for the possibility that γ-MSH may play a wider role in sodium homeostasis. Copyright ©1998 the American Physiological Society.
• Abbruscato, T. J., Thomas, S. A., Hruby, V. J., & Davis, T. P. (1997). Blood-brain barrier permeability and bioavailability of a highly potent and/μ-selective opioid receptor antagonist, CTAP: Comparison with morphine. Journal of Pharmacology and Experimental Therapeutics, 280(1), 402-409.
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  PMID: 8996221;Abstract: D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) is a cyclic, penicillamine-containing octapeptide that is structurally similar to somatostatin and displays greater antagonist potency and selectivity for μ- opioid receptors, compared with the classical μ-selective antagonist D-Phe- Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. The aim of this study was to determine whether CTAP can enter the central nervous system (CNS) by crossing either the blood-brain barrier or the blood-cerebrospinal fluid barrier (CSF) and to characterize the mechanism of CNS entry. CNS entry of [3H]CTAP was compared with that of the vascular space marker [14C]inulin and the μ-agonist [3H]morphine. By using an in situ brain perfusion technique coupled to high- performance liquid chromatographic analysis, greater amounts of radioactivity were detected in the brain or CSF at most time points for [3H]CTAP, compared with [14C]inulin. [3H]CTAP was found to remain predominantly intact in the brain after a 20-min rat brain perfusion (62.8%). CTAP was also stable in the blood and serum of rats (T(1/2) > 500 min), showing that the structure of this peptide offers enzymatic resistance. Additionally, [3H]CTAP was found to be extensively protein-bound to albumin in the perfusion medium (68.2%) and to proteins in rat serum (84.2%). Entry into the brain and CSF was not inhibited by the addition of unlabeled CTAP to the perfusion medium, suggesting that passage into the CNS is most likely through diffusion across the membranes that comprise the blood-brain barrier, rather than by saturable transport. Also, greater amounts of [3H]morphine entered both the brain and CSF after a 20-min brain perfusion, compared with [3H]CTAP. The increased CNS penetration observed for [3H]morphine, compared with [3H]CTAP, is likely due to the increased lipophilicity of morphine, as shown by its higher octanol/saline partition coefficient. Based on the pharmacokinetic profile, CTAP may be a promising μ-selective antagonist that can be used as a treatment for opiate overdose or addiction and also as a pharmacological tool to further understand opioid neurobiology.
• Abbruscato, T. J., Thomas, S. A., Hruby, V. J., & Davis, T. P. (1997). Brain and spinal cord distribution of biphalin: Correlation with opioid receptor density and mechanism of CNS entry. Journal of Neurochemistry, 69(3), 1236-1245.
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  PMID: 9282948;Abstract: Biphalin [(Tyr-D-Ala-Gly-Phe-NH)2] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [125I-Tyr1]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [125I-Tyr1]-biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ- and μ-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [125I-Tyr1] biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [125I-Tyr1]biphalin was decreased with naloxone, D-Phe-Cys-Tyr-D-Trp- Arg-Thr-Pen-Thr-NH2, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ- and μ-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [125I-Tyr1]biphalin, which could be described by Michaelis-Menten kinetics with a K(m) of 2.6 ± 4.8 μM, V(max) of 14.6 ± 2.89 pmol-1 · min-1 · g-1 and K(d) of 0.568 ± 0.157 μl · min-1 · g-1. Brain entry of [125-Tyr1]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and L- phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.
• Azizeh, B. Y., A., B., Trivedi, D., & Hruby, V. (1997). Pure glucagon antagonists: Biological activities and camp accumulation using phosphodiesterase inhibitors. Peptides, 18(5), 633-641.
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  PMID: 9213355;Abstract: Five new glucagon analogues have been designed, synthesized, characterized and their biological activities tested. The investigation was centered on modifications in the N-terminal region in particular, residues at Thr5, Phe6 and Tyr10 positions, with the goal of obtaining pure glucagon antagonists in our newly developed high sensitivity cAMP accumulation assay. The structures of the designed compounds are: [des-His1, des-Phe6. Glu9 ]glucagon-NH2 (1); [des-His1, des-Phe6, Glu9, Phe10] glucagon-NH2 (2): [des-His1, Tyr5, des-Phe6, Glu9] glucagon NH2 (3): [des-His1, Phe5, des-Phe6, Glu9]glucagon-NH2 (4) and [des-His1, des-Phe6, Glu9, D- Arg18]glucagon-NH2 (5). The binding potencies IC50 values in (nM) were 48.0, 27.4, 26.0, 20.0 and 416.0, respectively. All of these analogues when tested in the classical adenylate cyclase assay demonstrate antagonist properties, and in competition experiments, all caused a right-wardshift of the glucagon stimulated adenylate cyclase dose-response curve. The pA2 values for these analogues were 8.20 (1); 6.25 (2): 6.10 (3); 6.25 (4); and 6.08 (5), respectively. A newly revised assay has been developed to determine the intracellular cAMP accumulation levels in hepatocytes at the highest possible sensitivity. Four of the five glucagon analogues in this report (analogues 1, 2, 4 and 5), did not activate the adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 μM, and thus are pure antagonists.
• Azizeh, B. Y., Ahn, J., Caspari, R., Shenderovich, M. D., Trivedi, D., & Hruby, V. J. (1997). The role of phenylalanine at position 6 in glucagon's mechanism of biological action: Multiple replacement analogues of glucagon. Journal of Medicinal Chemistry, 40(16), 2555-2562.
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  PMID: 9258362;Abstract: Extensive evidence gathered from structure-activity relationship analysis has identified and confirmed specific positions in the glucagon sequence that are important either for binding to its receptor or for signal transduction. Fifteen glucagon analogues have been designed and synthesized by incorporating structural changes in the N-terminal region of glucagon, in particular histidine-1, phenylalanine-6, and aspartic acid-9. This investigation was conducted to study the role of phenylalanine at position 6 on the glucagon mechanism of action. These glucagon analogues have been made by either deleting or substituting hydrophobic groups, hydrophilic groups, aromatic amino acids, or a D-phenylalanine residue at this position. The structures of the new analogues are as follows: [des-His1,des- Phe6,Glu9]glucagon-NH2 (1); [des-His1,Ala6Glu9]glucagon-NH2 (2); [des- His1,Tyr6,Glu9]glucagon-NH2 (3); [des-His1,Trp6,Glu9]-glucagon-NH2 (4); [des-His1,D-Phe6,Glu9]glucagon-NH2 (5); [des- His1,Nl36,Glu9]glucagon-NH2 (6); [des-His1,Asp6,Glu9]glucagon-NH2 (7); [des-His1,des-Gly4,Glu9]glucagon-NH2 (8); [des-Phe6,- Glu9]glucagon-NH2 (9); [des-Phe6]glucagon-NH3 (10); [des-His1-,des- Phe6]glucagon-NH2 (11); [des-His1,des-Phe6,Glu9]glucagon (12); [des- Phe6,Glu9]glucagon (13); des-Phe6]glucagon (14); and [des-His1,des- Phe6]glucagon (15). The receptor binding potencies IC50 values are 48 (1), 126 (2), 40 (3), 19 (4), 100 (5), 48 (6), 2000 (7), 52 (8), 113 (9), 512 (10, 128 (11) 1000 (12), 2000 (13), 500 (14), and 200 nM (15). All analogues were found to be antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10-4 M except for analogues 6 and 8, which were found to be weak partial agonists/partial antagonists with maximum stimulation between 6-12%. In competitive inhibition experiments, all the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 8.20 (1), 6.40 (2), 6.20 (3), 6.25 (4), 6.30 (5), 6.30 (7), 6.05 (8), 6.20 (9), 6.30 (10), 6.25 (11), 6.10 (12), 6.20 (13), 6.20 (14), and 6.35 (15).
• Bonner, G. G., Davis, P., Stropova, D., Ferguson, R., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1997). Opioid peptides: Simultaneous δ agonism and μ antagonism in somatostatin analogues. Peptides, 18(1), 93-100.
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  PMID: 9114458;Abstract: Four isomers of the Somatostatin analogue H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were made with β-MePhe in position 1 and assayed for opioid binding in rat brain, biological activity in MVD and GPI bioassays, and antinociception in mouse warm-water tail flick assays. These analogues displayed varying potencies and biological activities including: simultaneous δ receptor agonism/μ receptor antagonism, μ receptor antagonism, and δ receptor agonism. These analogues demonstrated that the N-terminal residue is important for receptor potency/selectivity and signal transduction. These analogues may represent leads to therapeutic agents that yield analgesia via δ agonist effects, yet lack side effects associated with μ activity.
• Fan, W., Boston, B. A., Kesterson, R. A., Hruby, V. J., & Cone, R. D. (1997). Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. FASEB Journal, 11(3), A174.
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  Abstract: The melanocortinergic neurons located in the arcuate nucleus and the nucleus of the solitary tract project to a number of hypothalamic nuclei, including paraventricular nucleus, lateral hypothalamic area, dorso- and ventromediate hypothalamic nucleus, which are known to be important in regulation of feeding behavior. The melanocortinergic receptor MC3-R and MC4-R have also be identified at these sites. These neuroanatomic data suggest that the melanocortinergic pathways are involved in the regulation of feeding. The agouti obesity syndrome is caused by expression of the agouti peptide outside its normal site in the skin; agouti is also a potent antagonist of the MC4-R. The "melanocortin hypothesis" proposed that agouti causes obesity by antagonism of the hypothalamic MC4-R. To test this, we centrally administered the cyclic melanocortin analogues. MTII a potent agonist of MC4-R (EC50=0.057±0.024 nM) and MC3-R (EC50=0.27±0.23 nM), SHU9119, a potent antagonist of MC4-R (IC50=0.36±0.13 nM) and less potent antagonist of MC3-R (IC50=4.5±2.1 nM), and observed their effects on feeding behavior. Intracerebroventricular (ICV) administration of MTII produced a dose-responsive inhibition of food intake in 16 hr fasted C57BL/6J mice with an IC50 of 0.6 nmol at the 2 hr point after drug administration. This inhibition of feeding could be completely blocked by coadministration of SHU9119. MTII also inhibited feeding in three other models of hyperphagia, ob/oh, A, and Neuropeptide Y-injected mice. ICV injection of γ2-MSH, which only activated MC3-R, failed to inhibit feeding in fasted C57BL/6J mice. Furthermore, ICV administration of SHU9119 enhanced nocturnal feeding in mice fed ad libitum, and daytime feeding in fasted animals. The data show that melanocortinergic neurons may exert a tonic inhibition of feeding through MC4-R. Chronic disruption of this inhibitory signal is a likely explanation of the agouti obesity syndrome. (Funded by NIDDK & NICHD. RDC. NIDDK. VJH, BAB. RAK.).
• Fan, W., Boston, B. A., Kesterson, R. A., Hruby, V. J., & Cone, R. D. (1997). Role of melanocortlnergic neurons in feeding and the agouti obesity syndrome. Nature, 385(6612), 165-168.
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  PMID: 8990120;Abstract: DOMINANT alleles at the agouti locus (A) cause an obesity syndrome in the mouse, as a consequence of ectopic expression of the agouti peptide. This peptide, normally only found in the skin, is a high-affinity antagonist of the melanocyte-stimulating hormone receptor (MC1-R), thus explaining the inhibitory effect of agouti on eumelanin pigment synthesis. The agouti peptide is also an antagonist of the hypothalamic melanocortin-4 receptor (MC4-R). To test the hypothesis that agouti causes obesity by antagonism of hypothalamic melanocortin receptors, we identified cyclic melanocortin analogues that are potent agonists or antagonists of the neural MC3 (refs 11, 12) and MC4 receptors. Intracerebroventricular administration of the agonist, MTII, inhibited feeding in four models of hyperphagia: fasted C57BL/6J, ob/ob, and A(Y) mice, and mice injected with neuropeptide Y. Co- administration of the specific melanocortin antagonist and agouti-mimetic SHU9119 completely blocked this inhibition. Furthermore, administration of SHU9119 significantly enhanced nocturnal feeding, or feeding stimulated by a prior fast. Our data show that melanocortinergic neurons exert a tonic inhibition of feeding behaviour. Chronic disruption of this inhibitory signal is a likely explanation of the agouti obesity syndrome.
• Haaseth, R. C., Zalewska, T., Davis, P., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1997). Para-substituted phenylalanine-4 analogues of [L-Ala³]DPDPE: Highly selective δ opioid receptor ligands. Journal of Peptide Research, 50(3), 171-177.
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  PMID: 9309580;Abstract: Several para-substituted Phe4 analogues of the δ1-selective antagonist [L-Ala3]DPDPE (DPADPE) were prepared and evaluated for their brain-binding and in vitro pharmacological effects. Unlike the p-haloPhe4 analogues of DPDPE and the deltorphins, similar analogues of DPADPE with electron-withdrawing groups substituted at the para-position of the Phe4 aromatic ring did not all have increased potency and selectivity for δ opioid receptors, but all retained high potency and selectivity for δ opioid receptors greater than DPDPE.
• Han, Y., & Hruby, V. J. (1997). Lewis acid promoted conjugate addition of vinylmagnesium bromide to chiral α,β-unsaturated N-acyl oxazolidinones. Tetrahedron Letters, 38(42), 7317-7320.
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  Abstract: Lewis acid promoted conjugated additions of vinylmagnesium bromide to chiral α,β-unsaturated N-acyl oxazolidinones are described. A series of enatiomerically pure β-branched 4-pentenoic acid derivatives have been synthesized with high diastereoselectivity.
• Han, Y., Liao, S., Qiu, W., Cai, C., & Hruby, V. J. (1997). Total asymmetric synthesis of highly constrained amino acids β-isopropyl-2',6'-dimethyl-tyrosines. Tetrahedron Letters, 38(29), 5135-5138.
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  Abstract: All four stereoisomers of the highly constrained aromatic α-amino acid β-isopropyl-2',6'-dimethyltyrosine have been asymmetrically synthesis on a large scale. A catalytic asymmetric Michael addition of an organocuprate to a chiral α,β-unsaturated acyloxazolidinone and subsequent direct or indirect stereoselective electrophilic azidation of the α-position of the resulting product was followed by hydrolysis, hydrogenation and finally deprotection of the phenol group to afford the desired amino acids. The reactions generally proceeded in good stereoselectivities (75-95% ee/de) and yields (70-90%), making these optically pure amino acids available in large scale practical for the synthesis of peptides and other studies.
• Haskell-Luevano, C., Hendrata, S., North, C., Sawyer, T. K., Hadley, M. E., Hruby, V. J., Dickinson, C., & Gantz, I. (1997). Discovery of prototype peptidomimetic agonists at the human melanocortin receptors MC1R and MC4R. Journal of Medicinal Chemistry, 40(14), 2133-2139.
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  PMID: 9216831;Abstract: [Nle4,DPhe7]-α-MSH (NDP-MSH), a highly potent analogne of α- melanocyte-stimulating hormone (α-MSH), possesses nanomolar efficacies at all the melanocortin receptor subtypes except the MC2R. Evaluation of the melanocortin 'message' sequence of [Nle4,DPhe7]-α-MSH was performed on the human melanocortin receptor subtypes designated hMC1, hMC3R, hMC4R, and hMC5R. Tetrapeptides and tripeptides were stereochemically modified to explore topochemical preferences at these receptors and to identify lead peptides possessing agonist activity and subtype selectivity. Four peptides were discovered to only bind to the hMC1 and hMC4 receptor subtypes. The tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 (1) possessed 0.6μM binding affinity at the hMC1R, 1.2μM binding affinity at the hMC4R, and agonist activity at both receptors. The tripeptides Ac-DPhe-Arg-Trp-NH2 (6) and Ac-DPhe-Arg- DTrp-NH2 (7) possessed 2.0 and 9.1 μM binding affinities, respectively, only at the hMC4R, and both compounds effected agonist activity. The tetrapeptide Ac-His-Phe-Arg-DTrp-NH2 (4) possessed 6.3μM affinity and full agonist activity at the hMC1R, while only binding 7% at the hMC3R, 36% at the hMC4R, and 11% at the hMC5R at a maximal concentration of 10 μM. These data demonstrate that the His-Phe-Arg-Trp message sequence of the melanocortin peptides does not bind and stimulate each melanocortin receptor in a similar fashion, as previously hypothesized. Additionally, this study identified the simplest structural agonists for the hMC1R and hMC4R receptors reported to date.
• Haskell-Luevano, C., Nikiforovich, G., Sharma, S. D., Yang, Y., Dickinson, C., Hruby, V. J., & Gantz, I. (1997). Biological and conformational examination of stereochemical modifications using the template melanotropin peptide, Ac-Nle-c[Asp-His-Phe- Arg-Trp-Ala-Lys]-NH₂, on human melanocortin receptors. Journal of Medicinal Chemistry, 40(11), 1738-1748.
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  PMID: 9171884;Abstract: Examination of conformationally constrained melanotropin peptides (Ac- Nle4-c[Asp5-His-Phe7 Arg-Trp9-Ala-Lys]-NH2) on four human melanotropin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) resulted in identifying the importance of ligand stereochemistry at positions 5, 7, and 9 for agonist binding affinity and receptor selectivity. A trend in ligand structure- activity relationships emerged for these peptides, with the hMC1R and hMC4R possessing similar tendencies, as did the hMC3R and hMC5R. α-MSH (Ac-Ser- Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), NDP-MSH (Ac-Ser- Tyr-Ser-Nle4-Glu-His-D-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), and MTII (Ac- Nle4-c[Asp5,D-Phe7,Lys10]-α-MSH(4-10)-NH2) were also examined at each of these melanocortin receptors. Interestingly, the linear NDP-MSH possessed greater binding affinity for the hMC3R and hMC5R than did the cyclic analogue MTII. The peptide Ac-Nle-c[Asp-His-Phe-Arg-D-Trp9-Ala-Lys]-NH2 demonstrated the greatest differentiation in binding affinity between the hMC1R and hMC4R (78-fold). Analogue Ac-Nle-c[Asp-His-Phe7-Arg-Trp-Ala-Lys]-NH2 resulted in micromolar binding affinity (or greater) at the hMC3R and hMC5R, demonstrating the importance of D-Phe7 for ligand binding potency at these receptors. Ac-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2 resulted in loss of binding affinity at the hMC5R, implicating the importance of Nle4 (or a hydrophobic residue in this position) for binding to this receptor. Ac-Nle-c[D-Asp5- His-Phe-Arg-Trp-Ala-Lys]-NH2 was unable to competitively displace [125I]NDP-MSH binding at micromolar concentrations on the hMC3R and hMC5R, suggesting the importance of chirality of Asp5 either for ligand-receptor interactions or for orientation of the side chain lactam bridge and the structural integrity of the peptide conformation. Energy calculations performed for these peptides resulted in the identification of a low-energy ligand conformer family that is common to all the ligands. The differences in ligand binding affinities observed in this study are postulated to be a result of different ligand-receptor complexed interactions and not solely to the ligand structure.
• Haskell-Luevano, C., Toth, K., Boteju, L., Job, C., Maria, A., Hadley, M. E., & Hruby, V. J. (1997). β-methylation of the Phe⁷ and Trp⁹ melanotropin side chain pharmacophores affects ligand-receptor interactions and prolonged biological activity. Journal of Medicinal Chemistry, 40(17), 2740-2749.
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  PMID: 9276019;Abstract: Topographically modified melanotropin side chain pharmacophore residues Phe7 and Trp9 in a cyclic peptide template (Ac-Nle4-c[Asp-His-Xaa7-Arg- Yaa9-Lys]-NH2) and Phe7 in a linear peptide template (Ac-Ser-Tyr-Ser- Nle4-Glu-His-Xaa7-Arg-Trp-Giy-Lys-Pro-Val-NH2) result in differences in potency and prolonged biological activity in the frog and lizard skin bioassays. These topographic modifications included the four isomers of β- methylphenylalanine (β-MePhe)7 and β-methyltryptophan (β-MeTrp)9 and the two isomers of 1,2,3,4-tetrahydro-β-carboline (Tca)9. Modifications in the cyclic template resulted in up to a 1000-fold difference in potency for the β-MePhe7 stereoisomeric peptides; up to a 476-fold difference in potency resulted for the β-MeTrp9 peptides, and about a 50-fold difference between the Tca9-containing peptides. Up to a 40-fold difference in potency resulted for the β-MePhe7 stereoisomeric peptides using the linear template in these assays. The relative potency ranking for modifications 'in the cyclic template of β-MePhe7 were 2R,3S > 2S,3S = 2S,3R > 2R,3R in the frog assay and 2S,3R > 2R,3S > 2S,3S > 2R,3R in the lizard assay. The relative potencies for modifications in the cyclic template of β-MeTrp9 were 2R,3S > 2R,3R > 2S,3S >> 2S,3R in the frog assay and 2S,3S = 2R,3R > 2R,3S > 2S,3R in the lizard assay. The relative potencies for modifications in the cyclic template of Tca9 were DTca > LTca in both assays. Significant differences in prolonged (residual) activities were also observed for these modified peptides and were dependent upon stereochemistry of the β-methyl amino acid, peptide template, and bioassay system. Furthermore, comparisons of β- MeTrp9 stereoisomeric peptides on the frog, lizard, and human MC1 receptors suggest that structure-activity relationships on both the classical frog and lizard skin bioassays do not necessarily predict corresponding SAR profiles for the human melanocortin receptors, indicating a remarkable species specificity of the MC1 receptor requirements.
• Hruby, V. J. (1997). Prospects for peptidomimetic drug design. Drug Discovery Today, 2(5), 165-167.
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  Abstract: Peptidomimetic drug design offers many exciting opportunities and challenges for the basic research scientist interested in understanding the chemical and physical principles critical for drug action, and for medicinal chemists, biochemists, molecular biologists and others to apply these principles to the next-generation of drugs. Considerable success already has been achieved, and it is likely that continued research in peptide and protein structural chemistry, coupled with the development of novel scaffolds and templates that can predictably mimic the stereoelectronic, stereostructural and dynamic properties of peptides and proteins, will provide the tools for successful de novo peptidomimetic design.
• Hruby, V. J., Ahn, J., & Liao, S. (1997). Synthesis of oligopeptide and peptidomimetic libraries. Current Opinion in Chemical Biology, 1(1), 114-119.
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  PMID: 9667835;Abstract: The elaboration of peptide libraries prepared by either chemical or biological methods in a format useful for discovery of peptides with specific biological activities was first introduced in the 1980s. A virtual explosion of activity in this area has occurred recently, and the basic approaches have been applied to a wide variety of chemistries and for all manner of biological, chemical and physical targets. Recent advances include new synthetic methodologies, new analytical methods, new design methods and new assay procedures.
• Hruby, V. J., Bartosz-Bechowski, H., Davis, P., Slaninova, J., Zalewska, T., Stropova, D., Porreca, F., & Yamamura, H. I. (1997). Cyclic enkephalin analogues with exceptional potency and selectivity for δ-opioid receptors. Journal of Medicinal Chemistry, 40(24), 3957-3962.
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  PMID: 9397176;Abstract: Superpotent and highly δ-opioid receptor selective cyclic peptides of the general formula H-Tyr-c[D-Pen-Gly-Phe(p-X)-Pen]-Phe-OH (where X = hydrogen or halogen) have been synthesized. In the binding assays the most selective and most potent compound is the p-bromophenyl-alanine-4 analogue (IC50 value = 0.19 nM, selectivity ratio = 21 000 for δ vs μ). In the GPI and MVD bioassays the most selective and most potent analogue is the p- fluoro-substituted analogue Tyr-[D-Pen-Gly-Phe(p-F)-Pen]-Phe-OH. In the MVD assay it has an exceptionally low IC50 value of 0.016 nM and a δ vs μ selectivity ratio of 45 000.
• Hruby, V. J., Davis, T. P., O'Brien, D. F., Porreca, F., & Yamamura, H. I. (1997). Design of peptides and peptidomimetics that are selective, stable and can cross membrane barriers. Protein Engineering, 10(3333), 76-.
• Hruby, V. J., Li, G., Haskell-Luevano, C., & Shenderovich, M. (1997). Design of peptides, proteins, and peptidomimetics in chi space. Biopolymers - Peptide Science Section, 43(3), 219-266.
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  PMID: 9277134;Abstract: Peptide and protein biological activities depend on their three dimensionals structures in the free state and when interacting with their receptors/acceptors. The backbone conformations such as α-helix, β-sheet, β-turn, and so forth provide critical templates for the three-dimensional structure, but the overall shape and intrinsic stereoelectronic properties of the peptide or protein important for molecular recognition, signal transduction, enzymatic specificity, immunomodulation, and other biological effects depend on arrangement of the side chain groups in three-dimensional chi space (their χ1,χ2, etc. torsional angles). In this paper we explore approaches to the de novo design of polypeptides and peptidomimetics with biased or specific conformational/topographical properties in chi space. We consider computational and experimental methods that can be used to examine the effects of specific structural modifications in constraining side chain groups of amino acid residues and their similarities in chi space to the natural amino acids to evaluate what sort of mimetics are likely to minic normal amino acids. We then examine some of the asymmetric synthetic methods that are being developed to obtain the amino acid mimetics. Finally, we consider selected examples in the literature where these specialized amino acids have been incorporated in biologically active peptides and the specific insights they have provided regarding the topographical requirements for bioactive peptide potency, selectivity, and other biochemical and pharmacological properties. Constraints in chi space show great promise as useful tools in peptide, protein, and peptidomimetic de novo design of structures and pharmacophores with specific stereostructural, biochemical and biological properties.
• Huang, Q., Entwistle, M. L., Alvaro, J. D., Duman, R. S., Hruby, V. J., & Tatro, J. B. (1997). Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever. Journal of Neuroscience, 17(9), 3343-3351.
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  PMID: 9096167;Abstract: Bacterial infection causes fever, an adaptive but potentially self- destructive response, in the host. Also activated are counterregulatory systems such as the pituitary-adrenal axis. Antipyretic roles have also been postulated for certain endogenous central neuropeptides, including the melanocortins (α-MSH-related peptides). To test the hypothesis that endogenous central melanocortins have antipyretic effects mediated by central melanocortin receptors (MCRs), we determined the effect of intracerebroventricular injection of a synthetic MCR antagonist, Ac-Nle4,c- [Asp5,DNal(2')7,Lys10]α-MSH(4-10)-NH2 (SHU-9119) in endotoxin- challenged rats. The efficacy and specificity of SHU-9119 as an MCR antagonist in the rat was first validated in vitro and in vivo. In vitro, in heterologous cells expressing either rat MC3-R or MC4-R, the major MCR subtypes expressed in brain, SHU-9119 showed no intrinsic agonism, but it inhibited α-MSH-induced cAMP accumulation (IC50 = 0.48 ± 0.19 and 0.41 ± 0.28 nM, respectively) and [126I]-[Nle4,DPhe7]-α-MSH binding (IC50 = 1.0 ± 0.1 and 0.9 ± 0.3 nM, respectively). In vivo exogenous α-MSH (180 pmol) inhibited fever in rats when administered intracerebroventricularly 30 min after Escherichia coli lipopolysaccharide (LPS) (25 μg/kg, i.p.). When co-injected with α-MSH, SHU-9119 (168 pmol, i.c.v.) prevented the antipyretic action of exogenous α-MSH. In contrast, neither α-MSH nor SHU- 9119, alone or in combination, affected body temperatures in afebrile rats. In LPS-treated rats, intracerebroventricular injection of SHU-9119 significantly increased fever, whereas intravenous injection of the same dose of SHU-9119 had no effect. Neither intracerebroventricular nor intravenous SHU-9119 significantly affected LPS-stimulated plasma ACTH or corticosterone levels. The results indicate that endogenous central melanocortins exert an antipyretic influence during fever by acting on MCRs located within the brain, independent of any modulation of the activity of the pituitary- adrenal axis.
• Jiang, J., Sharma, S. D., Hruby, V. J., Fink, J. L., & Hadley, M. E. (1997). Human epidermal melanocyte and keratinocyte melanotropin receptors: Visualization by melanotropic peptide conjugated macrospheres (polyamide beads). Experimental Dermatology, 6(1), 6-12.
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  PMID: 9067701;Abstract: The objectives of this research were to determine whether melanotropin receptors are characteristic membrane markers of human epidermal melanocytes. Methodologies were developed to visualize these receptors by light microscopy. Multiple copies (up to a thousand) of [Nle4,D-Phe7]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromolecular carrier, large polyamide beads (macrospheres). Incubation in the presence of the I conjugated macrospheres resulted in binding of human epidermal melanocytes to the macrospheres. Specificity of the binding of melanocytes to the melanotropin-conjugated macrospheres was demonstrated by several studies: (i) Binding of melanocytes to the conjugate was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; (ii) The macrospheres after removal of the bound ligand did not bind to the melanocytes; (iii) Another peptide hormone ligand (e.g., a substance-P analog) attached to the macrospheres failed to bind to the melanocytes; (iv) B16/F10 mouse melanoma cells known to express melanotropin receptors bound to the macrospheres; (v) Cells of nonmelanocyte origin (e.g., mammary cancer cells, lung cancer cells, fibroblasts) did not bind to the macrospheres. One exception was that human epidermal keratinocytes also expressed melanotropin receptors as determined by all the criteria established for epidermal melanocytes. Thus, cell specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.
• Kramer, T. H., Bartosz-Bechowski, H., Davis, P., Hruby, V. J., & Porreca, F. (1997). Extraordinary potency of a novel delta opioid receptor agonist is due in part to increased efficacy. Life Sciences, 61(2), 129-135.
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  PMID: 9217271;Abstract: A new cyclic opioid peptide of sequence Tyr-D-Pen-Gly-Phe-Cys-Phe (HBP2) was examined in the mouse isolated vas deferens (MVD) bioassay. Studies with receptor-selective opioid antagonists showed the peptide to be highly selective for σ opioid receptors. HBP2 and the standard σ agonist DPDPE were simultaneously compared using the technique of partial irreversible receptor blockade; data were analyzed using the operational model of pharmacologic agonism. HBP2 was approximately 160 times as potent as DPDPE; estimation of the affinity and efficacy of the two peptides revealed that the potency increase was due to a 5.3-fold increase in efficacy, as well as a 37-fold increase affinity. This contrasts with our previous findings with other cyclic enkephalin analogs, in which increased affinity was achieved without a change in apparent efficacy. Analysis of concentration-response curve shape suggested in addition the possibility of heterogeneity in transduction mechanism for MVD σ receptors.
• Kunos, G., Li, S. -., Varga, K., Archer, P., Kesterson, R. A., Cone, R. D., Hruby, V. J., & Sharma, S. D. (1997). Novel neural pathways of cardiovascular control by α- and γ-MSH. Fundamental and Clinical Pharmacology, 11(SUPPL. 1), 44s-48s.
• Kövér, K. E., Hruby, V. J., & Uhrín, D. (1997). Sensitivity- and Gradient-Enhanced Heteronuclear Coupled/Decoupled HSQC-TOCSY Experiments for Measuring Long-Range Heteronuclear Coupling Constants. Journal of Magnetic Resonance, 129(2), 125-129.
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  PMID: 9441876;Abstract: A pulsed field gradient version of the sensitivity-enhanced 2D HSQC-TOCSY experiment is proposed for measurement of long-range heteronuclear coupling constants. The coupling constants are obtained by computer-aided analysis of mixed-phase multiplets with and without the heteronuclear splitting. Generation of pure phase data is not required. Since large 1JXH and JHH couplings are used for coherence transfer, small nJXH can be measured accurately, which could be difficult to obtain from purely heteronuclear polarization transfer experiments. © 1997 Academic Press.
• Liao, S., Lin, J., Shenderovich, M. D., Han, Y., Hasohata, K., Davis, P., Qiu, W., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1997). The stereochemical requirements of the novel δ-opioid selective dipeptide antagonist TMT-Tic. Bioorganic and Medicinal Chemistry Letters, 7(23), 3049-3052.
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  Abstract: Five conformationally constrained dipeptide TMT-L-Tic analogues have been synthesized and evaluated for their bioactivity using in vitro bioassays. The most potent and selective analogue (2S,3R)-TMT-L-Tic showed 9 nM binding affinity and 4000-fold selectivity for the δ vs μ opioid receptor. The lowest-energy conformation of (2S,3R)-TMT-L-Tic is suggested to be bioactive one in which the X, torsional angle is trans for TMT and gauche (+) for Tic.
• Liao, S., Shenderovich, M. D., Lin, J., & Hruby, V. J. (1997). Synthesis and conformational features of topographically constrained designer chimeric amino adds: The β-isopropyl phenylalanines. Tetrahedron, 53(49), 16645-16662.
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  Abstract: All four optically pure isomers of the highly conformationally constrained novel chimeric amino acid, β-isopropylphenylalanine or β- phenylleucine, were asymmetrically synthesized in five to six steps in 20 - 25% overall yield. Computer-assisted molecular modeling revealed that the β- isopropyl group in these chimeric amino acids plays the dominant role in determining the most favorable side chain conformations.
• Lin, J., Liao, S., Han, Y., Qiu, W., & Hruby, V. J. (1997). Asymmetric synthesis of all four isomers of topographically constrained novel amino acids: β-isopropyltyrosines. Tetrahedron Asymmetry, 8(19), 3213-3221.
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  Abstract: All four stereoisomers of the highly constrained novel amino acid, β-isopropyltyrosine, have been synthesized with high stereoselectivities (>90% de) and in 40-50% overall yields by using the optically pure 4-phenyloxazolidinone as a chiral auxiliary via asymmetric Michael addition, direct or indirect azidation, hydrogenolysis and demethylation reactions.
• Maria, A., Luisa, A., Al-Obeidi, F. A., Hadley, M. E., Hruby, V. J., Staples, D. J., & Sawyer, T. K. (1997). Comparative biological activities of α-MSH antagonists in vertebrate pigment cells. General and Comparative Endocrinology, 105(3), 410-416.
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  PMID: 9073503;Abstract: We have previously reported that melatonin was an effective lightening agonist in the teleost Synbranchus marmoratus, the amphibians Rana pipiens and Bufo ictericus, and in the lizard Anolis carolinensis. The hormone, previously applied to the preparations, effectively inhibited α-MSH darkening activity in a dose-independent manner, and was also able to reverse MSH-induced darkening. We presently describe the inhibitory effect of the indoleamine on the murine melanoma cell proliferation. Interestingly, the hormone also stimulated tyrosinase activity, with a correlated increase in melanin content. We also demonstrate that in a diverse lizard species, Urosaurus ornatus, the indoleamine was totally ineffective. The competitive MSH antagonistic activity of H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH2 has been demonstrated previously in R. pipiens and U. ornatus. Herein, its inhibitory activity is also reported in another lizard species, A. carolinensis. However, this MSH analogue was inactive in S. marmoratus, and in murine melanoma cells. On the other hand, the 7 thru 10 α-MSH fragment, Ac-Phe- Arg-Trp-Gly-NH2, although ineffective in S. marmoratus and R. pipiens, was an α-MSH antagonist in A. carolinensis. Surprisingly, in the melanoma cell line, the MSH fragment exhibited no agonist or antagonist activity, but dramatically potentiated the MSH-induced increase in tyrosinase activity. These data might suggest that the fragment is participating either in the process of facilitation or in positive cooperativity. The present results, taken together with our previously reported data, demonstrate a major interspecies diversity of the MC1 subtype of melanocortin receptor, and point out the relevance of the membrane microenvironment for the final receptor configuration.
• Misicka, A., Cavagnero, S., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1997). Synthesis and biological properties of β-MePhe³ analogues of deltorphin I and dermenkephalin: Influence of biased χ¹ of Phe³ residues on peptide recognition for δ-opioid receptors. Journal of Peptide Research, 50(1), 48-54.
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  PMID: 9273887;Abstract: Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I and dermenkephalin containing each of the four stereoisomers (2S,3S; 2S,3R; 2R,3S; 2R,3R) of the unusual amino acid β-methylphenylalanine in position three. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (μ-ligand) and [3H][p-ClPhe4]DPDPE (δ-ligand), and by bioassay using the mouse vas deferens (δ-receptor assay) and guinea pig ileum (μ-receptor assay) assays. The substitution of a β-MePhe for Phe3 in deltorphin I and dermenkephalin has a large and variable effect on the bioactivities of the synthesized analogues. The synthesized analogues are somewhat less potent than the native peptides. Both [(2S,3R)-β- MePhe3]deltorphin and [(2S,3R)-β-MePhe3]dermenkephalin are more selective, however, and interact essentially specifically with the receptor in the binding assays and bioassays. The bioassay data in vitro of the synthesized analogues of deltorphin I and dermenkephalin follow the same general trends as the receptor binding data. These results demonstrate that topographical modifications of the side-chain conformation of critical structural moieties in a ligand can significantly modulate both the potency and receptor selectivity for ligands that have multiple sites of biological activity, and they illustrate that this approach has general application to peptide and peptidomimetic ligand design.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1997). Structure-activity relationship of biphalin. The synthesis and biological activities of new analogues with modifications in positions 3 and 4. Life Sciences, 60(15), 1263-1269.
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  PMID: 9096243;Abstract: New analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2] with modifications of amino acid residues in positions 3,3' and 4,4' have been synthesized. The potency and selectivity of these analogues were evaluated by competitive radioreceptor binding assay in the rat brain using [3H]CTOP (mu ligand) and [3H][p-Cl-Phe4]DPDPE (delta ligand) as ligands, and by bioassay in the mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The symmetrical substitution of phenylalanine in positions 4 and 4' with p-fluorophenylalanine or p-nitrophenylalanine resulted in an enhancement of the affinity at both delta and mu receptors, with some increase of the selectivity for delta opioid receptors. The analogue containing p-chlorophenylalanine in positions 4 and 4' is the most selective to the delta receptors in this series, with a selectivity ratio about 5. The symmetrical substitution of the glycine-3 residue with phenylalanine resulted in a decrease of binding affinities and biological potencies at both μ and δ receptors.
• Patel, D., McKinley, B. D., Davis, T. P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1997). Peptide targeting and delivery across the blood-brain barrier utilizing synthetic triglyceride esters: Design, synthesis, and bioactivity. Bioconjugate Chemistry, 8(3), 434-441.
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  PMID: 9177851;Abstract: As an approach to the development of therapeutically useful peptide pharmaceuticals that can penetrate the blood-brain barrier, we have designed and demonstrated the application of a carrier-targeting system. We have developed a prodrug design strategy that is designed to utilize membrane-bound enzymes whereby release of a bioactive peptide from a highly lipophilic triglyceride peptide-carrier is achieved in situ, thus attaining high localized concentrations of the bioactive peptide. Following localization of such a system, normal peptidase and lipase action is utilized to release the active peptide (deltorphin II) intact and in high concentration. At present, the exact mechanisms are unclear, but the observed results in which analgesia is observed following peripheral administration suggest that the active peptide is able to cross the blood brain barrier and sustain prolonged periods of analgesia as determined by antinociception tests by release of the bioactive peptide. In vitro tests of binding and bioactivity by the peptide conjugate show essentially no potency in either target or control analogues, but potent antinociceptive effects are observed following peripheral administration.
• Quock, R. M., Hosohata, Y., Knapp, R. J., Burkey, T. H., Hosohata, K., Zhang, X., Rice, K. C., Nagase, H., Hruby, V. J., Porreca, F., Roeske, W. R., & Yamamura, H. I. (1997). Relative efficacies of 6-opioid receptor agonists at the cloned human δ-opioid receptor. European Journal of Pharmacology, 326(1), 101-104.
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  PMID: 9178661;Abstract: The present study was conducted to determine the relative efficacies of the selective δ-opioid receptor agonists SNC80((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1- piperazinyl)-3- S,5 R) -4-allyl-2,5-dimethyl-1-piherazinyl)-3-methoxybenzyl]-N,N- diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahyhro-quinolino -[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPγS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human δ-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/E(maxi))(A'/A+1)x 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of(-)-TAN67.
• Schiö, H. B., Muceniece, R., Mutulis, F., Prusis, P., Lindeberg, G., Sharma, S. D., Hiruby, V. J., & E., J. (1997). Selectivity of cyclic [D-Nal⁷] and [D-Phe⁷] substituted MSH analogues for the melanocortin receptor subtypes. Peptides, 18(7), 1009-1013.
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  PMID: 9357059;Abstract: The binding of the 2 cyclic lactam MSH (4-10) analogues (MTII, SHU9119), and 5 cyclic [Cys4, Cys10]α-MSH analogues were tested on cells transiently expressing the human MC1, MC3, MC4 and MC5 receptors. The results indicate a differential importance of the C-terminal (Lys-Pro-Val) and N-terminal (Ser-Tyr-Ser) of cyclic [Cys4, Cys10]α-MSH analogues in binding to the MC receptor subtypes. Substitution of D-Phe7 by D-Nal(2')7 in both the cyclic lactam MSH (4-10) and the cyclic disulphide MSH (4-10) analogues resulted in a shift in favour of selectivity for the MC4 receptor; the disulphide analogue, [Cys4, D-Nal(2')7 Cys10]α-MSH (4-10) (HS9510), showing the highest selectivity for the MC4 receptor among all the substances tested. However, the cyclic lactams displayed an over all higher affinity for the MC receptors, than any of the cyclic disulphide MSH (4-10) analogues.
• Schiöth, H. B., Muceniece, R., Szardenings, M., Prusis, P., Lindeberg, G., Sharma, S. D., Hruby, V. J., & Wikberg, J. E. (1997). Characterisation of D117A and H260A mutations in the melanocortin 1 receptor. Molecular and Cellular Endocrinology, 126(2), 213-219.
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  PMID: 9089659;Abstract: Recent site directed mutagenesis studies on the melanocortin 1 (MC1) receptor have indicated the importance of D117 and H260 amino acid residues for the binding of α-MSH (melanocyte stimulating hormone). Here, we report the testing of 12 cyclic and linear MSH peptides on the D117A and H260A mutant receptors. Moreover, we constructed a double mutant which displayed a major loss in affinity for [Nle4, D-Phe7]α-MSH. Our new data of His6 and Phe7 substituted MSH peptides are compared with previous results and the hypothesis of putative interactions of D117 and H260 with single amino acids in the MSH peptide. Our conclusions are that the D117A and the H260A mutations may cause conformational changes in the receptor which can not be linked to any specific amino acid in the MSH-peptides.
• Shenderovich, M. D., Kövér, K. E., Wilke, S., Collins, N., & Hruby, V. J. (1997). Solution conformations of potent bicyclic antagonists of oxytocin by nuclear magnetic resonance spectroscopy and molecular dynamics simulations. Journal of the American Chemical Society, 119(25), 5833-5846.
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  Abstract: The solution conformations of two potent bicyclic antagonists of oxytocin (H-Cys1-Tyr2-Ile3-Gln4- Asn5-Cys6-Pro7-Leu8-Gly9-NH2, OT). [Mpa1,cyclo(Glu4,Lys8)]OT, and [dPen1, cyclo(Glu4,Lys8)]OT were studied by a combined use of 1H and 13C NMR spectroscopy in DMSO and molecular dynamics (MD) simulations. NMR data have suggested a model for the three- dimensional (3D) structure of the bicyclic analogues of OT (OT-BC) with a β- turn at the Tyr2 and Ile3 residues, and with a cis amide bond between Cys6 and Pro7. A 3D structure containing a type III β-turn at Tyr2-Ile3 has been shown to be consistent with NMR data. This structure was proposed as a model of the solution conformation of OT-BC and extensively tested by MD simulations with the AMBER force field. MD simulations at 300 K with NMR derived distance and Φ torsion angle constraints demonstrated the consistency of this model with NMR data, and its stability was further demonstrated by non-constrained MD simulations. Dynamic properties of the 3D structure were explored by high-temperature MD at 500 K. Conformational transitions induced by a constrained rotation around the S-S bond revealed relatively low potential energy barriers 130 to 50 kJ/mol) between equilibrium left-handed and right-handed conformers of the disulfide bridge in OT-BC. A dynamic model of the solution structure of OT-BC with the relatively stable backbone conformation and a fast conformational equilibrium in the disulfide bridge and lactam bridge moieties was proposed as a result of the extensive MD simulations. The solution structure of OT-BC is consistent with structure-activity relations of peptide and non-peptide antagonists of OT. In particular, a β-turn at Tyr2-Ile3 seems to be the common feature responsible for antagonist interaction with the uterine receptor of OT. On the other hand, the 3D structure of OT-BC differs considerably from the crystal and solution structures of OT analogues with agonist activity. Therefore, this study supports the hypothesis of different modes of receptor binding for agonists and antagonists of OT. The model of 3D structure of OT-BC proposed in this study may be used as a template for the rational design of peptide and non-peptide antagonists of oxytocin.
• Sturm, N. S., Hutzler, A. M., David, C. S., Azizeh, B. Y., Trivedi, D., & Hruby, V. J. (1997). Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon. Journal of Peptide Research, 49(4), 293-299.
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  PMID: 9176812;Abstract: We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis1,Val9,Ile11,16] glucagon amide, (3) [desHis1,Val9,11,16]glucagon amide, (4) [desHis1,Val9,Leu11,16]glucagon amide, (5) [desHis1,Nle9,Ile11,16]glucagon amide, (6) [desHis1,Nle9,Val11,16]glucagon amide, (7) [desHis1,- Nle9,Leu11,16]glucagon amide. (8) [desHis1,Val9,Leu11,16,Lys17,18,Glu21]glucagon amide and (9) [desHis1,Nle9,Leu11,16,Lys17,18,Glu21]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have IC50 values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with pA2 values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively.
• Tessmer, M. R., Meyer, J. -., Hruby, V. J., & Kallick, D. A. (1997). Structural model of a cyclic dynorphin a analog bound to dodecylphosphocholine micelies by NMR and restrained molecular dynamics. Journal of Medicinal Chemistry, 40(14), 2148-2155.
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  PMID: 9216833;Abstract: The compound c[Cys5,11]dynorphin A-(1-11)- NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the κ- opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 ω bond contains either a β-turn from residues Arg6 to Arg9 or an α-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 ω bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III β-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a β-turn from residues Cys5 and Arg8. In this report we describe the generation of three- dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as κ-opioid agonists.
• Thomas, S. A., Abbruscato, T. J., Hau, V. S., Gillespie, T. J., Zsigo, J., Hruby, V. J., & Davis, T. P. (1997). Structure-activity relationships of a series of [D-Ala²]deltorphin I and II analogues; in vitro blood-brain barrier permeability and stability. Journal of Pharmacology and Experimental Therapeutics, 281(2), 817-825.
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  PMID: 9152390;Abstract: [D-Ala2]deltorphins are enzymatically stable, amphibian heptapeptides that have a higher affinity and selectivity for deltaopioid receptors than any endogenous mammalian compound known. This study investigated the in vitro blood-brain barrier permeability, using primary bovine brain microvessel endothelium culture, and the resistance to enzymatic degradation, in mouse 15% brain membrane homogenates and 100% plasma, of [D-Ala2]deltorphin I, [D- Ala2]deltorphin II and several analogues. Derivatives were designed with the addition of N-terminal neutral and basic amino acids or with alterations of the amino acids present within the deltorphin sequences. The results indicated that the N-terminal sequence and the amino acids in position 4 and 5 are critical to deltorphin analogue BBB permeability and biological stability, i.e., t 1/4 brain; 4.8 hr- [D-Ala2]deltorphin I; >15 hr- [D-Ala2, Ser4, D-Ala5]deltorphin. Although, no analogue was found to increase the BBB permeability coefficient (PC; x 10-4 cm/min) of the parent compounds ([D-Ala2]deltorphin II, PC = 23.49 ± 2.42) analogues were identified: [Arg0, D-Ala2]deltorphin II, PC = 19.06 ± 3.73 and [Pro-1, Pro0, D- Ala2]deltorphin II, PC = 22.22 ± 5.93; which had similar permeability coefficients, even though they had larger molecular weights and, in the case of the cationic pro-drug, a significantly lower lipophilicity. These analogues provide directions in the development of future pro-drugs for the treatment of pain and this study further clarifies the structure-activity relationship of the deltorphins.
• Thomas, S. A., Abbruscato, T. J., Hruby, V. J., & Davis, T. P. (1997). The entry of [D-penicillamine^2,5]enkephalin into the central nervous system: Saturation kinetics and specificity. Journal of Pharmacology and Experimental Therapeutics, 280(3), 1235-1240.
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  PMID: 9067309;Abstract: The delta opioid receptor-selective, enzymatically stable peptide [D- Penicillamine2,5]enkephalin (DPDPE) has recently acquired special significance with the identification of a saturable uptake system for this analgesic into the CNS. The aim of the present study was to characterize further the entry of [3H]DP-DPE into the brain and CSF by means of a bilateral in situ brain perfusion method. Initial experiments revealed a saturable [3H]DPDPE uptake into the brain that followed Michaelis-Menten type kinetics with a K(m) value of 45.5 ± 27.6 μM, a V(max) value of 51.1 ± 13.2 pmol · min · g-1 and a K(d) value of 0.6± 0.3 μl · min-1 · g-1. Uptake of [3H]DPDPE into the CSF could not be inhibited (K(d) = 0.9 ± 0.1 μl · min-1 · g-1). Entry of [3H]DPDPE into the CNS was not inhibited in the presence of 10 mM 2-aminobicyclo-[2,2,1]-heptane-2- carboxylic acid (BCH) or 50 μM ICI 174,864, which suggests that the saturable mechanism does not involve the large neutral amino acid transporter or binding to opioid receptors. It would also appear that [3H]D-PDPE is not in competition with either poly-L-lysine or insulin to enter the CNS. However, both of these substances significantly increased the CNS entry of [3H]DPDPE but not that of the vascular space marker [14C]sucrose, and this may have valuable clinical implications. It is not known at present which saturable uptake mechanism is responsible for the CNS entry of [3H]DPDPE, but overall the results suggest a carrier-mediated transport system.
• Ugwu, S. O., Blanchard, J., Dorr, R. T., Levine, N., Brooks, C., Hadley, M. E., Aickin, M., & Hruby, V. J. (1997). Skin pigmentation and pharmacokinetics of melanotan-I in humans. Biopharmaceutics and Drug Disposition, 18(3), 259-269.
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  PMID: 9113347;Abstract: A comparative pharmacokinetic trial was performed with a superpotent synthetic melanotropic peptide, [Nle4-D-Phe7]-α-MSH1-13 (melanotan-I or MT-I) given by three routes of administration. Plasma levels were measured by RIA and tanning was quantitated using serial reflectometry. Doses of 0.16 mg kg-1 were administered intravenously (IV) and orally (PO), and doses from 0.08 to 0.21 mg kg-1 subcutaneously (SC), in a randomized crossover fashion to three male volunteers over five consecutive days for 2 weeks (ten doses). The results indicate that the SC dose is completely bioavailable compared to the IV dose. No detectable drug levels were observed following PO dosing. The plasma half-lives following SC dosing ranged from 0.07 to 0.79 h for the absorption phase and from 0.8 to 1.7 h for the β-phase. Clearance ranged from 0.12 to 0.19 L kg-1 h-1 and 3.9% or less of the dose was recovered in the urine. Side-effects were minimal, consisting of occasional gastrointestinal upset and facial flushing. Significant tanning of the forehead, arms, and neck was noted following IV or SC dosing. This effect peaked at 1 week following drug administration but was still present 3 weeks after completing the ten-dose regimen. It is concluded that SC administration is an efficacious method of delivering melanotan-I.
• Yuan, W., & Hruby, V. J. (1997). Asymmetric synthesis of unusual amino acid: Synthesis of four isomers of β-methyl-3-(2'-naphthyl)alanine. Tetrahedron Letters, 38(22), 3853-3856.
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  Abstract: The synthesis of all four individual isomers of 3-methyl-3-(2'-naphthyl)analine was accomplished using asymmetric conjugate 1,4-addition followed by direct or indirect azidation using an Evans-type chiral auxiliary (4-phenyl-2-oxazolidinone).
• A., B., Azizeh, B. Y., Trivedi, D., Phelps, J. R., Houslay, M. D., Johnson, D. G., & Hruby, V. J. (1996). Low level cyclic adenosine 3',5'-monophosphate accumulation analysis of [des-His¹,des-Phe⁶,Glu⁹]glucagon-NH₂ identifies glucagon antagonists from weak partial agonists/antagonists. Endocrinology, 137(8), 3316-3322.
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  PMID: 8754757;Abstract: [des-His1,des-Phe6,Glu9]Glucagon-NH2 is a newly designed glucagon antagonist. This analog has a binding IC50 of 48 nM (compared to glucagon IC50 of 1.5 nM) and demonstrates pure antagonism in an adenylate cyclase assay. Although the number of glucagon antagonists has grown rapidly recently, closer examination suggested that many of these antagonists retained very low, almost imperceptible levels of cAMP accumulation that were sufficient to elicit an in vivo biological response. To investigate more carefully this secondary biological signal, we measured cAMP accumulation in a revised assay using isolated hepatocytes in the presence of the phosphodiesterase (PDE) inhibitor Rolipram. The PDE inhibitors Rolipram and isobutyl-1-methylxanthine (IBMX) increased the sensitivity of the cAMP accumulation assay from approximately 10-fold for the native hormone to 35- fold above basal levels. On the other hand, amrinone, another PDE inhibitor, did not affect the cAMP accumulation caused by glucagon. The use of PDE inhibitors indicated that three glucagon analogs that had previously been reported to have strong antagonist properties in classical adenylate cyclase assays were actually weak partial agonists in this new assay system. [N(α)- Trinitrophenyl-His1,homo-Arg12]glucagon, [des-amino-His1,D- Phe4,Tyr5,Arg12, Lys17,18,Glu21]glucagon, and [des- His1,Glu9]glucagon-NH2 demonstrated 233%, 21%, and 5.5% cAMP accumulation relative to the native hormone in the presence of 25 μM Rolipram. On the other hand, [des-His1,des-Phe6,Glu9]glucagon-NH2, a newly designed glucagon antagonist, did not activate adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 μM, indicating that it was a pure antagonist of glucagon-induced adenylate cyclase activity and also the first one in this class. This compound and others were tested in a glycogen phosphorylase assay. As [des-His1,des-Phe6,Glu9]glucagon-NH2 did not activate phosphorylase activity, it was chosen as our candidate for in vivo testing in streptozotocin-induced diabetic rats. An initial dose of 0.75 mg/kg was found to cause the greatest lowering of blood glucose levels (to 63% of the initial levels in 15 min) when the bolus was followed by continuous infusion of 25 μg/kg·min for 1 h.
• Abbruscato, T. J., Williams, S. A., Misicka, A., Lipkowski, A. W., Hruby, V. J., & Davis, T. P. (1996). Blood-to-central nervous system entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives. Journal of Pharmacology and Experimental Therapeutics, 276(3), 1049-1057.
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  PMID: 8786534;
• Azizeh, B. Y., Shenderovich, M. D., Trivedi, D., Guigen, L. i., Sturm, N. S., & Hruby, V. J. (1996). Topographical amino acid substitution in position 10 of glucagon leads to antagonists/partial agonists with greater binding differences. Journal of Medicinal Chemistry, 39(13), 2449-2455.
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  PMID: 8691441;Abstract: The role of position 10 in the β-turn region of glucagon was investigated by substituting chiral constrained amino acids and other modifications in the N-terminal region. A series of glucagon analogues have been designed and synthesized by incorporating β-methylphenylalanine isomers (2S,3S, 2S,3R, 2R,3R, and 2R,3S) at position 10 in order to explore the structural and topographical requirements of the glucagon receptor, and, in addition, utilizing previous studies which indicated that antagonism could be enhanced by modifications (des-His1, Glu9) and a bulky group at position 5. The structures of the new analogues are as follows: [des-His1,- Tyr5,Glu9]glucagon-NH2 (II), [des-His1,Tyr5,Glu9,Phe10]glucagon-NH2 (III), [des-His1,Tyr5,Glu9 Ala10]glucagon-NH2 (IV), [des- His1,Tyr5,Glu9,(2S,3R)-β-MePhe10]glucagon-NH2 (V), [des-His1,- Tyr5,Glu9,(2S,3S)-β-MePhe10]glucagon-NH2 (VI), [des- His1,Tyr5,Glu9,D-Tyr10]glucagon-NH2 (VII), [des-His1,Tyr5,Glu9,D- Phe10]glucagon-NH2 (VIII), [des-His1,Tyr5,Glu9,D-Ala10]glucagon-NH2 (IX), [des-His1,Tyr5,Glu9,(2R,3R)-β-MePhe10]glucagon-NH2 (X), and [des-His1,Tyr5,Glu9,(2R,3S)-β-MePhe10]glucagon-NH2 (XI). These analogues led to dramatically different changes in in vitro binding affinities for glucagon receptors. Their receptor binding potencies IC50 values (nM) are 2.3 (II), 4.1 (III), 395.0 (IV), 10.0 (V), 170.0 (VI), 74.0 (VII), 34.5 (VIII), 510.0 (IX), 120.0 (X), and 180.0 (XI). Analogues II, III, V, VI, and XI were found to be weak partial agonists/partial antagonists with maximum stimulation between 5%-9%, while the other compounds (IV and VII-X) were antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10-5 M. In competition experiments, all of the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 6.60 (II), 6.85 (III), 6.20 (IV), 6.20 (V), 6.10 (VI), 6.50 (VII), 6.20 (VIH), 5.85 (IX), 6.20 (X), and 6.00 (XI). Putative topographical requirements of the glucagon receptor for the aromatic side chain conformation in position 10 of glucagon antagonists are discussed.
• Bilsky, E. J., Bernstein, R. N., Hruby, V. J., Rothman, R. B., Lai, J., & Porreca, F. (1996). Characterization of antinociception to opioid receptor selective agonists after antisense oligodeoxynucleotide-mediated 'knock-down' of opioid receptors in vivo. Journal of Pharmacology and Experimental Therapeutics, 277(1), 491-501.
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  PMID: 8613959;Abstract: Pharmacological studies in vivo and in vitro have suggested the existence of subtypes of the delta opioid receptor termed delta1 and delta2 (δ1 and δ2). The hypothesis of subtypes of δ receptors was further explored by assessing the effects of administration of antisense or mismatch oligodeoxynucleotides (ODN) in vivo to the cloned DOR, or to a conserved region of the cloned opioid receptors, on the antinociceptive responses elicited by selective μ, κ and δ opioid receptor agonists in mice. Additionally, the density of opioid δ receptors in brain after δ opioid receptor (DOR) ODN treatment was investigated. Repeated twice daily intracerebroventricular (i.c.v.) administration of DOR antisense, but not mismatch, ODN, produced a dose- and time-related blockade of i.c.v. [D- Ala2, Glu4]deltorphin (δ2 agonist), but not [D-Pen2, D-Pen5]enkephalin (δ1 agonist), antinociception. The antinociceptive responses to selective μ and κ opioid agonists were unaffected by DOR antisense or mismatch ODN treatments. The antinociceptive effect of an A90 dose of [D-Ala2, Glu4]deltorphin was significantly reduced by the third day of DOR antisense ODN administration and persisted over a treatment period of 6 days with recovery by the third posttreatment day. Saturation studies in mouse whole brain preparations with the selective δ-radioligand [3H]naltrindole showed that DOR antisense, but not mismatch, ODN treatment produced a significant time-related reduction in B(max) values of approximately 30 to 40% by day 6, without changing the K(d) value. The reduction in DOR density was reversible and returned to control levels within 3 days after cessation of antisense ODN treatment. The i.c.v. administration of an antisense, but not mismatch, ODN directed to a conserved region of the cloned opioid receptors, termed common opioid receptor antisense ODN, inhibited the antinociceptive effects of i.c.v. μ, κ and δ agonists, including [D-Pen2, D-Pen5]enkephalin. These data further support the hypothesis of subtypes of opioid receptors.
• Bilsky, E. J., Inturrisi, C. E., Sadée, W., Hruby, V. J., & Porreca, F. (1996). Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective μ or δ opioid agonists in mice. Pain, 68(2-3), 229-237.
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  PMID: 9121809;Abstract: N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (μ) and delta (δ) agonists, however, has not been reported. In these experiments, selective μ and δ receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm-water tail-flick test. Repeated i.c.v. injections of μ agonists including morphine, fentanyl, [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) and Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) or [D-Ala2, Glu4]deltorphin, a δ agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose-response curves compared to controls. Single injections or repeated administration of MK801 (a non-competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [D-Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold-water swim-stress episode, previously shown to be a δ-opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective μ and δ opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to 'opiates' in general.
• Boteju, L. W., Nikiforovich, G. V., Haskell-Luevano, C., Fang, S., Zalewska, T., Stropova, D., Yamamura, H. I., & Hruby, V. J. (1996). The use of topographical constraints in receptor mapping: Investigation of the topographical requirements of the tryptophan 30 residue for receptor binding of Asp-Tyr-D-Phe-Gly-Trp-(N-Me)Nle-Asp-Phe-NH₂ (SNF 9007), a cholecystokinin (26-33) analogue that binds to both CCK-B and δ-opioid receptors. Journal of Medicinal Chemistry, 39(20), 4120-4124.
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  PMID: 8831778;Abstract: The cholecystokinin (26-33) [CCK (26-33)] octapeptide analog Asp-Tyr-D- Phe-Gly-Trp(N-Me)-Nle-Asp-Phe-NH2 (SNF 9007) is a potent and selective ligand for both the CCK-B and δ-opioid receptors. Pharmacological studies of SNF 9007 suggest a relationship between the ligand requirements of CCK-B and δ-opioid receptors, which further implies a possible structural relationship between these receptors. We have utilized topographical constrainment of the important Trp30 residue to investigate structural features of SNF 9007 that would distinguish between binding requirements in this region for the CCK-B and δ-opioid receptors. Thus, the four optically pure isomers of β-MeTrp were substituted for L-Trp30 of SNF 9007. Receptor binding results suggest that the preferred topography of the Trp30 residue for CCK-B receptor binding may be the 2S,3S (erythro-L) configuration whereas for the δ-opioid receptor it may be the 2S,3R(threo-L)configuration. Molecular modeling studies of these ligands further support the recently revised receptor-bound model for CCK-B octapeptide ligands (Kolodziej et al. J. Med. Chem. 1995, 38, 137-149) and are in good agreement with the DPDPE-δ opioid receptor 'template' model (Nikiforovich et al. Biopolymers 1991, 31,941-955).
• Collins, N., Flippen-Anderson, J. L., Haaseth, R. C., Deschamps, J. R., George, C., Kövér, K., & Hruby, V. J. (1996). Conformational determinants of agonist versus antagonist properties of [D-Pen²,D-Pen⁵]enkephalin (DPDPE) analogs at opioid receptors. Comparison of X-ray crystallographic structure, solution ¹H NMR data, and molecular dynamic simulations of [L-Ala³]DPDPE and [D-Ala³]DPDPE. Journal of the American Chemical Society, 118(9), 2143-2152.
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  Abstract: c-[D-Pen2,D-Pen5]enkephalin (DPDPE, 1) is a cyclic, constrained, highly potent, δ opioid receptor selective peptide agonist. Substitution of Gly3 with L-Ala in DPDPE to give [L-Ala3]DPDPE (2) has been shown to produce a peptide with much greater δ receptor binding selectivity than DPDPE itself. However [L-Ala3]DPDPE is only a partial agonist in in vivo antinociception and actually was found to potently antagonize the antinociceptive effects of DPDPE at δ receptors in the brain. In comparison, [D-Ala3]DPDPE (3) is a weak and poorly selective δagonist. In an effort to correlate the biological profiles of these peptides with secondary structure, [L-Ala3]DPDPE and [D-Ala3]-DPDPE were studied by X-ray crystallography and 1H and 13C NMR in DMSO solution. Crystals of both peptides were obtained using vapor diffusion techniques. [L-Ala3]DPDPE crystallizes in the monoclinic space group C2 with cell dimensions a = 36.35(1) Å, b = 19.737(4) Å, c = 28.16(1) Å, β = 129.07(2)°, and V = 15688(9) Å3. The asymmetric unit contains four peptide molecules and approximately 20 water molecules, giving a calculated density of 1.274 g cm-3. The conformation of all four independent [L-Ala3]DPDPE molecules is essentially the same. [D-Ala3]-DPDPE crystallizes in the monoclinic space group P21 with cell dimensions a = 12.271(2) Å, b = 9.600(a) Å, c = 18.750(4) Å, β = 103.56(2)°, and V = 2147.2(7) Å3. The asymmetric unit contains one peptide molecule and 10 molecules of water, giving a calculated density of 1.298 g cm-3. Comparison of these X-ray structures with the crystal structure previously reported for DPDPE indicates that there are differences in the disulfide bond region for all three peptides. ROEs determined about the disulfide regions of 1-3 in solution are indicative of a high degree of conformational interconversion, while heteronuclear coupling constants between the D-Pen2.3 Hα and Cγ,γ + ′ carbons indicate a strong preference for a gauche (+) χ1 angle in 2. The backbone conformations of DPDPE and [D-Ala3]-DPDPE in the X-ray structures are virtually identical, while in [L-Ala3]DPDPE, there is a rotation of approximately 160° about both ψ2 and φ3 compared to DPDPE which has the effect of rotating this amide group approximately 180° relative to DPDPE. The solution NMR data for the peptide backbone conformations of 2 and 3 are mainly consistent with their X-ray structures. However, MD simulation of all three compounds, starting with the geometries of their X-ray structures, indicates that by comparison of observed and predicted ROE intensities an equilibrium between these conformations is likely in solution. The "DPDPE-like" conformation for [L-Ala3]DPDPE is however significantly higher in energy than the X-ray structure reported here and, thus, is predicted to be less populated in solution and in receptor binding. It is concluded that the X-ray structure of DPDPE represents an agonist conformation for this peptide at the δ opioid receptor and that the corresponding X-ray structure of [L-Ala3]DPDPE represents an antagonist conformation due to the differences in conformation between positions 2 and 3. Considerations on the structural implications of this conformational difference on receptor binding are discussed.
• Dorr, R. T., Lines, R., Levine, N., Brooks, C., Xiang, L., Hruby, V. J., & Hadley, M. E. (1996). Evaluation of melanotan-II, a superpotent cyclic melanotropic peptide in a pilot phase-I clinical study. Life Sciences, 58(20), 1777-1784.
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  PMID: 8637402;Abstract: A pilot phase I study was conducted with a cyclic heptapeptide analog of α-melanocyte stimulating hormone (α-MSH). The lactam-bridged molecule, called Melanotan-II (MT-II), has the structure Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10α-MSH4-10-NH2 (MT-II) and has superpotent melanotropic activity in vitro. A single-blind, alternating day (saline or MT-II), placebo-controlled trial was conducted in 3 normal male volunteers at the starting dose of 0.01 mg/kg of MT-II. Subcutaneous injections of MT-II or saline were given daily (Monday-Friday) for 2 consecutive weeks. Two subjects were escalated by 0.005 mg/kg increments to 0.03 mg/kg and one to 0.025 mg/kg. The 0.03 mg/kg dose produced Grade II somnolence and fatigue in one of two subjects (WHO standards). Mild nausea, not requiring antiemetic treatment, was reported at most MT-II dose levels. A stretching and yawning complex appeared to correlate with the onset of spontaneous, penile erections which were intermittently experienced for 1-5 hours after MT-II dosing, depending on the MT-II dose. Two subjects had increased pigmentation in the face, upper body and buttock, as measured by quantitative reflectance and by visual perception 1 week after MT-II dosing ended. These results demonstrate that MT-II has tanning activity in humans given only 5 low doses every other day by subcutaneous injection. The recommended single MT-II dose for future Phase I studies is 0.025 mg/kg/day.
• Greene, D. L., Hau, V. S., Abbruscato, T. J., Bartosz, H., Misicka, A., Lipkowski, A. W., Hom, S., Gillespie, T. J., Hruby, V. J., & Davis, T. P. (1996). Enkephalin analog prodrugs: Assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability. Journal of Pharmacology and Experimental Therapeutics, 277(3), 1366-1375.
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  PMID: 8667199;Abstract: To improve the blood-brain barrier penetration of the δ-opioid receptor peptides [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2) to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3) to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe(o)-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe(o)-DPDPE from 3.9 min to >450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe(o)-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.
• Hadley, M. E., Hruby, V. J., Jiang, J., Sharma, S. D., Fink, J. L., Haskell-Luevano, C., Bentley, D. L., Al-Obeidi, F., & Sawyer, T. K. (1996). Melanocortin receptors: Identification and characterization by melanotropic peptide agonists and antagonists. Pigment Cell Research, 9(5), 213-234.
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  PMID: 9014208;Abstract: Hormones are chemical messengers released from cells to act on and control the activity of other cells. Hormonal ligands initiate their actions by interacting with receptive substances (Langley, 1906) of the target cells. These receptors are proteins that are either integral components of the cell membrane or are localized cytoplasmically within cells. Ligand-receptor interaction results in either the stimulation or inhibition of cellular activity. Since most hormones bind rather specifically to receptors possessed by their target cells, labeling of hormonal ligands can be utilized to identify and localize cells within an animal. In this report we discuss what is presently known about melanocortin receptors (MCRs) as studied by the use of labeled melanotropic peptide ligands.
• Haskell-Luevano, C., Miwa, H., Dickinson, C., Hadley, M. E., Hruby, V. J., Yamada, T., & Gantz, I. (1996). Characterizations of the unusual dissociation properties of melanotropin peptides from the melanocortin receptor, hMC1R. Journal of Medicinal Chemistry, 39(2), 432-435.
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  PMID: 8558511;Abstract: Variation in the degree of prolonged (residual) biological activity of the melanotropin peptides α-MSH (α-melanocyte-stimulating hormone, Ac-Ser-Tyr- Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and the superpotent analogues [Nle4,DPhe7]α-MSH (MT-I) and Ac-[Nle4,Asp5,DPhe7,Lys10]α- MSH(4-10)-NH2 (MT-II) has stimulated considerable interest regarding this biological phenomena. We have examined the differences in their relative dissociation rates from the melanocortin receptor, hMC1R, to try and correlate peptide dissociation rates with the observations of prolonged biological activity. Interestingly, these studies revealed that α-MSH remained 25% bound, MT-I 65% bound, and MT-II 86% bound 6 h after the ligand had been removed from the assay medium. The relative dissociation rate of MT- II was 4 times slower than that for α-MSH and 2 times slower than that for MT-I, which was 2 times slower than that for α-MSH. These data suggest that slow dissociation kinetics (hours) may contribute to the prolonged biological activities observed for both MT-I and MT-II peptides in vitro and in vivo. The prolonged binding, biological activities, and enzymatic stability of MT- I and MT-II make them putative candidates for clinical uses such as external scintigraphy for the localization of tumors (i.e., melanoma).
• Haskell-Luevano, C., Sawyer, T. K., Hendrata, S., North, C., Panahinia, L., Stum, M., Staples, D. J., M., A., Hadley, M. E., & Hruby, V. J. (1996). Truncation studies of α-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity. Peptides, 17(6), 995-1002.
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  PMID: 8899819;Abstract: Systematic analysis of fragment derivatives of the superpotent α-MSH analogue, Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) 'active site.' The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereochemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His6 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.
• Haskell-Luevano, C., Sawyer, T. K., Trumpp-Kallmeyer, S., Bikker, J. A., Humblet, C., Gantz, L., & Hruby, V. J. (1996). Three-dimensional molecular models of hMC1R melanocortin receptor: Complexes with melanotropin peptide agonists. Drug Design and Discovery, 14(3), 197-211.
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  PMID: 9017363;
• Hruby, V. J., Shenderovich, M., Lam, K. S., & Lebl, M. (1996). Design considerations and computer modeling related to the development of molecular scaffolds and peptide mimetics for combinatorial chemistry. Molecular Diversity, 2(1-2), 46-56.
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  PMID: 9238633;Abstract: A critical issue in drug discovery utilizing combinatorial chemistry as part of the discovery process is the choice of scaffolds to be used for a proper presentation, in a three-dimensional space, of the critical elements of structure necessary for molecular recognition(binding) and information transfer (agonist/ antagonist). In the case of polypeptide ligands, considerations related to the properties of various backbone structures (α-helix, β-sheets, etc.; φ, ψ space) and those related to three-dimensional presentation of side-chain moieties (topography; χ (chi) space) must be addressed, although they often present quite different elements in the molecular recognition puzzle. We have addressed aspects of this problem by examining the three-dimensional structures of chemically different scaffolds at various distances from the scaffold to evaluate their putative diversity. We find that chemically diverse scaffolds can readily become topographically similar. We suggest a topographical approach involving design in chi space to deal with these problems. © 1996 ESCOM Science Publishers B.V.
• Jiang, J., Sharma, S. D., Fink, J. L., Hadley, M. E., & Hruby, V. J. (1996). Melanotropic peptide receptors: Membrane markers of human melanoma cells. Experimental Dermatology, 5(6), 325-333.
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  PMID: 9028794;Abstract: The objectives of this research were to determine whether melanotropin receptors are characteristic (constant) membrane markers of human melanoma cells. Methodologies were developed to visualize these receptors by fluorescence microscopy. Multiple copies (10-20) of both [Nle4,D-Phe7]αMSH), a superpotent analog of α-melanocyte stimulating hormone (α-MSH), and a fluorophore, were conjugated to polyvinyl alcohol (PVA). Incubation in the presence of the multivalent macromolecular conjugate (FITC-PVA-MSH) resulted in binding of human epidermal melanocytes and keratinocytes and human melanoma cells (bath melanotic and amelanotic) to the fluorescent conjugate. Binding of the conjugate to the cells exhibited a unique cluster pattern (capping) suggesting a receptor internalization related phenomenon. Most importantly, every cell of every melanoma cell line, melanotic or amelanotic, possessed receptors as visualized by fluorescence microscopy. Since the cells were not synchronized, some binding apparently took place during all phases of the cell cycle. Therefore, receptor expression appears not to be cell-cycle dependent. Specificity of binding of FITC-PVA-MSH was demonstrated by several studies. (i) Binding of the conjugate to melanoma cells could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; [Nle4,D-Phe7]α-MSH. (ii) The macromolecular conjugate lacking bound ligand (FITC-PVA) did not bind to the melanoma cells. (iii) Another peptide, a substance-P analog, attached to the substrate (FITC-PVA-SP) failed to bind to the cells. (iv) With the exception of keratinocytes, other cells of nonmelanocyte origin (e.g., fibroblasts, spleen, liver, kidney cells, and mammary cancer cells, lung cancer cells) did not bind to the conjugate. Thus, cell-specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes, keratinocytes, and melanoma cells. In several human melanoma cell lines these receptors appeared to be functional since [Nle4,D-Phe7]α-MSH stimulated tyrosinase activity. Fluorescent melanotropin conjugates might prove useful in determining whether all human melanoma (primary and metastatic) tumors possess such receptors. These receptors might then provide targets for melanotropic peptides for the identification, localization, and chemotherapy of melanoma.
• Jiang, J., Sharma, S. D., Hruby, V. J., Bentley, D. L., Fink, J. L., & Hadley, M. E. (1996). Human epidermal melanocyte and keratinocyte melanocortin receptors: Visualization by melanotropic peptide conjugated microspheres (latex beads). Pigment Cell Research, 9(5), 240-247.
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  PMID: 9014210;Abstract: The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle4,D-Phe7]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromolecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) microspheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.
• Li, S., Varga, K., Archer, P., Hruby, V. J., Sharma, S. D., Kesterson, R. A., Cone, R. D., & Kunos, G. (1996). Melanocortin antagonists define two distinct pathways of cardiovascular control by α- and γ-melanocyte-stimulating hormones. Journal of Neuroscience, 16(16), 5182-5188.
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  PMID: 8756446;Abstract: Melanocortin peptides and at least two subtypes of melanocortin receptors (MC3-R and MC4-R) are present in brain regions involved in cardiovascular regulation. In urethane-anesthetized rats, unilateral microinjection of α-melanocyte-stimulating hormone (MSH) into the medullary dorsal-vagal complex (DVC) causes dose-dependent (125-250 pmol) hypotension and bradycardia, whereas γ-MSH is less effective. The effects of α-MSH are inhibited by microinjection to the same site of the novel MC4-R/MC3-R antagonist SHU9119 (2-100 pmol) but not naloxone (270 pmol), whereas the similar effects of intra-DVC injection of β-endorphin (1 pmol) are inhibited by naloxone and not by SHU9119. Hypotensive and bradycardic responses to electrical stimulation of the arcuate nucleus also are inhibited by ipsilateral intra-DVC microinjection of SHU9119. γ-MSH and ACTH(4-10), but not α-MSH, elicit dose-dependent (0.1-12.5 nmol) pressor and tachycardic effects, which are much more pronounced after intracarotid than after intravenous administration. The effects of γ-MSH (1.25 nmol) are not inhibited by the intracarotid injection of SHU9119 (1.25-12.5 nmol) or the novel MC3-R antagonist SHU9005 (1.25-12.5 nmol). We conclude that the hypotension and bradycardia elicited by the release of α-MSH from arcuate neurons is mediated by neural melanocortin receptors (MC4-R/MC3-R) located in the DVC, whereas the similar effects of β-endorphin, a peptide derived from the same precursor, are mediated by opiate receptors at the same site. In contrast, neither MC3-R nor MC4-R is involved in the centrally mediated pressor and tachycardic actions of γ-MSH, which, likely, are mediated by an as yet unidentified receptor.
• Liao, S., & Hruby, V. J. (1996). Asymmetric synthesis of optically pure β-isopropylphenylalanine: A new β-branched unusual amino acid. Tetrahedron Letters, 37(10), 1563-1566.
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  Abstract: All four optically pure isomers of a highly conformationally constrained unusual amino acid, β-isopropylphenylalanine, have been asymmetrically synthesized.
• Liao, S., Han, Y., Qiu, W., Bruck, M., & Hruby, V. J. (1996). Syntheses of highly constrained β-aryl isohexanoic acid derivatives via asymmetric Michael addition. Tetrahedron Letters, 37(44), 7917-7920.
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  Abstract: A series of enantiomerically pure highly sterically hindered β-branched isohexanoic acid derivatives have been synthesized with high diastereoselectivity via asymmetric Michael addition. The X-ray crystal structure of (4S,3'S)-3-[3'-(2,6-dimethylphenyl)isohexanyl]-4-phenyl-2-oxazolidinon e demonstrated that the β-configuration was induced from the Si-face, and that the torsional angle χ2 was restricted by the bulky β-isopropyl group to the range expected from molecular modeling.
• Liwo, A., Tempczyk, A., Oldziej, S., Shenderovich, M. D., Hruby, V. J., Talluri, S., Ciarkowski, J., Kasprzykowski, F., Lankiewicz, L., & Grzonka, Z. (1996). Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven monte carlo and molecular dynamics methods. Biopolymers, 38(2), 157-175.
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  PMID: 8589250;Abstract: Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine-vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration-shell model, and by simulated-annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low-energy conformations obtained for both hormones were classified using the minimal-tree clustering algorithm and characterized according to the locations of β-turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a β-turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a β-turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The latter difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x-ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr-derived distance constraints. Generally, the low-energy conformations obtained with the hydration-shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low-energy conformations do not satisfy all of the nmr-derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with β-turns at residues 2,3, 3,4, and 4,5. The low-energy structures of OT with a β-turn at residues 2,3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M. D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921-927], although the native hormone differs from the bicyclic analogue by the conformation of the C-terminal tripeptide. This finding confirms the hypothesis of different receptor-bound conformations of agonists and antagonists of OT. ©1996 John Wiley & Sons, Inc.
• Lung, F. T., Collins, N., Stropova, D., Davis, P., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1996). Design, synthesis, and biological activities of cyclic lactam peptide analogues of dynorphin A(1-11)-NH₂¹. Journal of Medicinal Chemistry, 39(5), 1136-1141.
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  PMID: 8676350;Abstract: We previously have reported four possible binding conformations of dynorphin A (Dyn A) for the central κ opioid receptors, induced by the address sequence, using a molecular mechanics energy minimization approach. The lowest energy conformation was found to exhibit an α-helical conformation in the cyclized address sequence. It was suggested that an α-helical conformation in the cyclized address sequence or a helical conformation induced by the conformational characteristics of the message sequence may be important for binding potency and κ opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i + 4 positions have been shown to stabilize α-helical conformations. Thus, a series of cyclic lactam analogues of dynorphin A(1-11)-NH2 have been designed, synthesized and evaluated by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate the conformational analysis prediction and, further, to investigate the conformational requirements for high potency and selectivity for κ opioid receptors. Positions 2-6, 3-7, and 5-9 were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between D-Asp2 and Lys6 in c[D-Asp2,Lys6]Dyn A(1-11)-NH2 led to an analogue with pronounced potency and selectivity enhancement for the μ opioid receptor, whereas cyclization between D-Asp3 and Lys7 in c[D-Asp3,Lys7]Dyn A(1-11)-NH2 led to a potent ligand (IC50 4.9 nM) with κ receptor selectivity. The other analogues in the series proved to be less selective. The biological results led to the suggestion that the binding conformation for the κ receptor may have structural requirements that are distinct from those of μ and δ receptors. Interestingly, analogues with a D-Asp at position 2, 3, or 9 were found to be more potent for the κ receptor than analogues with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position 2, 3, or 9 of Dyn A(1-11)-NH2 may have stereochemical and conformational effects on the nearby amino acids which can help discriminate the preference between κ, μ, and δ receptors.
• Lung, F. T., Meyer, J. -., Lou, B., Xiang, L., Guigen, L. i., Davis, P., A., I., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1996). Effects of modifications of residues in position 3 of dynorphin A(1-11)-NH₂ on κ receptor selectivity and potency. Journal of Medicinal Chemistry, 39(13), 2456-2460.
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  PMID: 8691442;Abstract: Tyrosine1 and phenylalanine4 in dynorphin A (Dyn A) have been reported to be important residues for opioid agonist activity and for potency at κ receptors. The glycine residues in the 2 and 3 positions of dynorphin A may affect the relative orientation of the aromatic rings in positions 1 and 4, but their flexibility precludes careful analysis. To examine these effects on dynorphin A, we previously have synthesized the linear analogues [D-Ala3]Dyn A(1-11)-NH2 (2) and [Ala3]Dyn A(1-11)-NH2 (3) and reported their biological activities. Analogues 2 and 3 displayed affinities for the central κ opioid receptor (IC50 = 0.76 and 1.1 nM, respectively) similar to that of Dyn A(1-11)-NH2 (1) (IC50 = 0.58 nM) and greatly enhanced selectivities for κ vs μ and κ vs δ receptors (IC50 ratios of 350 and 1300 for 2, and 190 and 660 for 3, respectively). These results suggest that the structure and lipophilicity of the amino acid present in position 3 of Dyn A(1-11)-NH2 as well as the conformational changes they induce in the message sequence of dynorphin have important effects on potency and selectivity for κ opioid receptors. To further investigate structure-activity relationships involving the residue at the 3 position of Dyn A(1-11)-NH2, a series of Dyn A analogues with aromatic, charged, and aliphatic side chain substitutions at the 3 position was designed, synthesized, and evaluated for their affinities for κ, μ, and δ opioid receptors. It was found that analogues with lipophilic amino acids at the 3 position of Dyn A(1-11)-NH2 generally displayed higher affinity but similar selectivities for the κ receptor than analogues with charged residues at the same position. It is suggested that the structural, configurational, and steric/lipophilic effects of amino acids at position 3 of Dyn A(1-11)-NH2 may play an important role in potency and selectivity for the κ receptor.
• Malatynska, E., Wang, Y., Knapp, R. J., Waite, S., Calderon, S., Rice, K., Hruby, V. J., Yamamura, H. I., & Roeske, W. R. (1996). Human delta opioid receptor: Functional studies on stably transfected Chinese hamster ovary cells after acute and chronic treatment with the selective nonpeptidic agonist SNC-80. Journal of Pharmacology and Experimental Therapeutics, 278(3), 1083-1089.
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  PMID: 8819489;Abstract: The SNC-80 series of nonpeptidic agonists for the δ-opioid receptor are being developed as potential analgesic drugs. It is important to understand their acute and chronic effects at human δ-opioid receptors. Thus, we measured the ability of SNC-80 and [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin to inhibit forskolin-stimulated adenylyl cyclase activity in recombinant Chinese hamster ovary cells stably expressing the cloned human δ-opioid receptor. The calculated EC50 values for [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin and SNC-80 were 0.6 ± 0.1 nM and 6.3 ± 0.1 nM, respectively. Pretreatment of these cells with SNC-80 (100 nM) for 24 hr produced 1) a time-dependent reduction of δ receptor density, as measured by radioligand binding studies with [3H]naltrindole; 2) a shift in the EC50 value of SNC-80 from 7.7 ± 4.2 nM to 44.1 ± 12 nM, as measured by the cyclic AMP assay; 3) a reduction in the maximum inhibition of adenylyl cyclase activity from 86% to 48%; 4) a marked increase in the forskolin stimulation of basal cyclic AMP accumulation by nearly 100% (from 442 pmol/mg of protein to 824 pmol/mg of protein); and 5) a 5-fold increase in forskolin-stimulated cyclic AMP accumulation after addition of naltrindole. These studies showed that SNC-80 produced desensitization and down-regulation of human δ-opioid receptors in recombinant Chinese hamster ovary cells after chronic treatment and that this effect was associated with an increase in adenylyl cyclase activity.
• Misicka, A., Maszczynska, I., Lipkowski, A. W., Stropova, D., Yamamura, H. I., & Hruby, V. J. (1996). Synthesis and biological properties of gamma-glutamyl-dermorphin, a prodrug. Life Sciences, 58(11), 905-911.
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  PMID: 8786696;Abstract: The possibility of using the gamma-glutamyl-transpeptidase system for transformation of inactive propeptide, gamma-glutamyl-neuropeptides into active neuropeptides has been tested on dermorphin and its gamma-glutamyl analogue. Gamma-glutamyl-dermorphin 2 showed little affinity for opioid receptors. Nonetheless, systemic (intraperitoneal (i.p.), or intravenous (i.v.)) application of this compound induced significant antinociceptive effects, although ten to twenty-fold higher doses were required compared to the parent dermorphin 1. On the other hand, the analogue 2 showed high, antinociceptive activity when injected intrathecally (i.t.). When compared to dermorphin, 2 was one third as potent, but did show a significant prolonged duration of the effect. These results suggest that in the periphery, the peptidase metabolism which results in degradation of bioactivity, is offset by gammaglutamyl transpeptidase (GGTP) activity that liberates bioactive peptide 2. On the other hand, in the central nervous system, the activity of gamma-glutamyl transpeptidase system seems to be more effective than other peptidase systems, resulting in formation of active peptide 2 in a significant amount. These data suggests that gamma-glutamyl analogues of neuropeptides can be considered as potential prodrugs, especially for synthetic analogues which themselves are resistant to peptidase action.
• Qian, X., Liao, S., Stropova, D., Yamamura, H. I., & Hruby, V. J. (1996). Novel scaffolds for non-peptide mimetics of δ opioid receptor agonists based on peptide leads. Regulatory Peptides, 65(1), 79-82.
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  PMID: 8876039;
• Qian, X., Shenderovich, M. D., Kover, K. E., Davis, P., Horvath, R., Zalewska, T., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1996). Probing the stereochemical requirements for receptor recognition of δ opioid agonists through topographic modifications in position 1. Journal of the American Chemical Society, 118(31), 7280-7290.
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  Abstract: A series of side-chain constrained tyrosine derivatives, 2',6'-dimethyl-β-methyltyrosines (TMT), has been designed and incorporated into position 1 of the highly selective δ opioid agonists DPDPE (Tyr-D-Pen2-Gly-Phe-D-Pen5-OH) and deltorphin I (DELT I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2). Molecular mechanics calculations on isolated TMT residues and nuclear magnetic resonance (NMR) studies of the TMT1-containing peptides in DMSO showed that each of the four stereoisomers of TMT favors one particular rotamer of the side-chain χ1 torsional angle. Therefore, substitution of four TMT isomers for Tyr1 allows us to perform a systematic conformational scan through three staggered rotamers of the aromatic side chain, gauche (-), trans, and gauche (+), and to explore specific binding requirements of the receptor in relation to the side chain conformation. The potency and selectivity of four isomers of [TMT1]DPDPE and four isomers of [TMT1]DELT I were evaluated by radioreceptor binding assays in the rat brain using μ- and δ-selective radiolabeled ligands and by bioassays with guinea pig ileum (GPI, μ receptor) and mouse vas deferens (MVD, δ receptor). In the DPDPE series only one isomer, [(2S,3R)-TMT1 ]DPDPE showed high potency and selectivity for the δ opioid receptors. The favorable side-chain rotamers found for this analogue, i.e., the trans rotamer of TMT1 and the gauche (-) rotamer of Phe4, were proposed as the most probable δ receptor-binding conformations of DPDPE analogues. Two [TMT1]DELT I isomers possessed considerable δ receptor potencies. The (2S,3R)-TMT1 isomer appeared to be a superpotent, but moderately δ-selective agonist, while the (2S,3S)-TMT1 isomer showed the highest selectivity for the δ receptors in this series. Surprisingly, [(2R,3R)TMT1]DELT I also was moderately potent at the δ receptor. These results suggest that the δ receptor requirements for the linear DELT I analogues may be satisfied with two different modes of binding of the (2S,3S)- and (2S,3R)TMT1 isomers. This study provides important guidance for the design of peptide and non-peptide ligands selective for the δ opioid receptor.
• Roerig, S. C., Williams, C. L., Hruby, V. J., Burks, T. F., & Rosenfeld, G. C. (1996). Inhibition of adenylyl cyclase activity by the cholecystokinin analog SNF 9007 in neuroblastoma × glioma NG108-15 hybrid cells. Regulatory Peptides, 61(1), 51-56.
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  PMID: 8701027;Abstract: The effect of the cholecystokininB (CCKB) receptor-selective cholecystokinin octapeptide (CCK-8) analog SNF 9007 on forskolin-stimulated adenylyl cyclase activity in NG108-15 hybrid cells was measured. The activity of SNF 9007 was compared to the δ opioid agonists D-Pen2 - D-Pen5-enkephalin (DPDPE, δ1 receptor-selective) and Tyr - D-Ala - Phe - Glu - Val - Val - Gly - NH2, (D-Ala2-deltorphin II, δ2-receptor-selective) because SNF 9007 binds with moderate affinity to δ opioid receptors. SNF 9007 inhibited forskolin-stimulated adenylyl cyclase activity with efficacy similar to DPDPE. IC50 determinations showed that D-Ala2-deltorphin II was the most potent, followed by DPDPE, then SNF 9007 (IC50 values = 0.013, 0.21 and 4.8 μM, respectively). CCK-8 had no effect on adenylyl cyclase activity. The δ1 receptor-selective antagonist 7-benzylidenenaltrexone hydrochloride (BNTX, 10 nM) had no effect on the activity of any of these agonists, but the δ2 receptor-selective antagonist naltriben methanesulfonate (NTB, 10 nM) increased IC50 values of all the agonists. Combinations of BNTX and NTB (10 nM each) increased the D-Ala2-deltorphin II IC50 value 12-fold, the DPDPE IC50 value 18-fold and the SNF 9007 IC50 value 26-fold. The effect of the combined δ antagonists on SNF 9007 activity was different from the effect on DPDPE or D-Ala2-deltorphin II activity. These data suggest that the interaction of the CCK-8 analog SNF 9007 with opioid receptors in NG108-15 hybrid cells is different from the interaction of opioid peptides with these receptors.
• Sharma, S. D., Jiang, J., Hadley, M. E., Bentley, D. L., & Hruby, V. J. (1996). Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors. Proceedings of the National Academy of Sciences of the United States of America, 93(24), 13715-13720.
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  PMID: 8943000;PMCID: PMC19401;Abstract: We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone (MSH) receptors of melanoma cells. Multiple copies of [Nle-4,D-Phe-7]-α-MSH, a potent analog of α-MSH, were conjugated to microspheres (latex beads) or macrospheres (polyamide beads) through a thioether or disulfide bond. Binding between the beads and mouse and human melanoma cells was examined by scanning electron microscopy and by light microscopy. Each mouse and human melanoma cell (of all cell lines) evinced binding to the beads. Binding of the melanotropin conjugates was not restricted to any one phase of the cell cycle. Specificity of binding was demonstrated by several studies. Negative controls included cell types of nonmelanocyte origin (e.g., mammary cancer cells) and beads that lacked the melanotropic ligand or had other attached ligands. Beads with a disulfide-linked melanotropin analog served as a direct control. Treatment of these beads with DTT during or before incubation of the beads with melanoma cells (resulting in release of the MSH analog from the beads) eliminated binding of the beads to melanoma cells. Binding interactions between melanoma cells and melanotropin-bound beads also could be abolished by prior incubation with unconjugated MSH analog. During these experiments, certain membrane receptor-hormone associated phenomena, such as capping (aggregation) of the receptor-ligand complex, also were observed. These results provide visual evidence that MSH receptors are a property common to melanoma cells. Normal human epidermal melanocytes and keratinocytes were also shown to express melanotropin receptors by the same criteria established for melanoma cells.
• Shenderovich, M. D., Kövér, K. E., Nikiforovich, G. V., Jiao, D., & Hruby, V. J. (1996). Conformational analysis of βmethyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen², D-Pen⁵]enkephalin by NMR spectroscopy and conformational energy calculations. Biopolymers, 38(2), 141-156.
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  PMID: 8589249;Abstract: Solution conformations of β-methyl-para-nitrophenylalanine4 analogues of the potent δ-opioid peptide cyclo[ D-Pen2, D-Pen5 ] enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and 3JHNCαH coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of [β-Me-pNO2Phe4]DPDPE in DMSO were compared with low energy conformer obtained by energy minimization in the Empirical Conformational Energy Program for Peptides (ECEPP/2) force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homo-nuclear (3JHαHβ) and heteronuclear (3JHαCγ) coupling constants and 13C chemical shifts, show that the β-methyl substituent eliminates one of the three staggered rotamers of the torsion angle X1for each stereoisomer of the β-Me-p-NO2Phe4. Similar solution conformations were suggested for the L-Phe4-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe4-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main δ-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [β-Me-pNO2Phe4]DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [ β-Me-p-NO2Phe4]DPDPE in the δ-receptor-bound state may assume a conformation different from their favorable conformations in DMSO. ©1996 John Wiley & Sons, Inc.
• Tourwé, D., Verschueren, K., Frycia, A., Davis, P., Porreca, F., Hruby, V. J., Toth, G., Jaspers, H., Verheyden, P., & Binst, G. V. (1996). Conformational restriction of Tyr and Phe side chains in opioid peptides: Information about preferred and bioactive side-chain topology. Biopolymers, 38(1), 1-12.
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  PMID: 8679939;Abstract: The side chain of Tyr and Phe was fixed into the gauche (-) or gauche (+) conformation by using the Tic or Htc structures, and into the trans conformation by using an aminobenzazepine-type (Aba) structure. When incorporated into dermorphin or deltorphin II, the Tic and Htc analogues all showed a large decrease in both μ and δ affinities and activities. Fixation of Phe3 in the trans rotamer resulted in a large increase in δ affinity in the dermorphin analogue, whereas in the [Aba3-Gly4] deltorphin II analogue, good δ affinity is maintained despite the removal of the Glu side chain. Whereas several authors propose a gauche (-) preferred conformation for the Phe3 side chain, these results suggest a trans conformation at the δ receptor. The use of these conformationally constrained residues for evaluating the preferred solution conformation in the flexible N-terminal tripeptide Tyr-D-Ala-Phe is illustrated. The 1H-nmr parameters - chemical shift, temperature dependence, and nuclear Overhauser effects to the D-Ala2 methyl protons in the different analogues - provide direct evidence to confirm the proposed sandwich conformation in the native peptides. © 1996 John Wiley & Sons, Inc.
• Vanderah, T. W., Bernstein, R. N., Yamamura, H. I., Hruby, V. J., & Porreca, F. (1996). Enhancement of morphine antinociception by a CCK(B) antagonist in mice is mediated via opioid delta receptors. Journal of Pharmacology and Experimental Therapeutics, 278(1), 212-219.
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  PMID: 8764354;Abstract: This study investigated the possible involvement of opioid δ receptors in the modulation of morphine antinociceptive potency produced by L365,260, a CCK(B) antagonist. Intracerebroventricular (i.c.v.), intrathecal (i.th.) or subcutaneous (s.c.) L365,260 alone did not produce any antinociceptive actions in the mouse warm-water tail-flick test. Treatment with L365,260 by any of these routes produced a leftward shift of the corresponding morphine dose-effect curve that was blocked by pretreatment with a receptor-selective dose of s.c. naltrindole, an opioid δ receptor antagonist. Pretreatment with i.c.v. antisera to [Leu5]enkephalin also blocked the leftward displacement of the i.c.v. morphine dose-effect curve resulting from L365,260 but did not directly alter the i.c.v. morphine dose-effect curve; antisera to [Met5]enkephalin did not alter the effects of morphine or the modulation of morphine antinociception produced by L365,260. Repeated pretreatment with L365,260 resulted in a progressive decrease in the magnitude of the morphine modulatory action (i.e., L365,260 'tolerance'). In these 'L365,26O-tolerant' mice, the dose-effect curve for i.c.v. [D-Ala2, Glu4]deltorphin (a selective δ agonist) was displaced to the right by approximately 8.2-fold. The i.c.v. administration of [Leu5]enkephalin produced a leftward displacement of the i.c.v. morphine dose-effect curve that diminished after repeated administration (i.e., [Leu5]enkephalin 'tolerance'). In '[Leu5]enkephalin-tolerant' mice, L365,260 failed to produce the leftward shift of the morphine dose-effect curve seen in control animals. That is, two-way antinociceptive cross-tolerance was observed between an opioid δ agonist and a CCK(B) receptor antagonist. Intracerebroventricular thiorphan, a peptidase inhibitor, did not elicit antinociception directly. Co- administration of thiorphan with L365,260 elicited significant antinociception that was blocked by naltrindole or antisera to [Leu5]enkephalin; antisera to [Met5]enkephalin had no effect. Repeated administration of i.c.v. [D-Ala2, Glu4]deltorphin resulted in a progressively decreasing antinociceptive effect (i.e., [D-Ala2, Glu4]deltorphin 'tolerance'). In '[D-Ala2, Glu4]deltorphin-tolerant' mice, the thiorphan/L365,260 antinociceptive effect was inhibited. Collectively, these data suggest that CCK interacts at the CCK(B) receptor to inhibit tonically the release and/or availability of an endogenous substance acting at opioid δ receptors. The subsequent enhancement of morphine antinociceptive potency may reflect the well-known modulation of morphine antinociception produced by opioid δ receptor agonists. In this case, the latter may be [Leu5]enkephalin or a [Leu5]enkephalin-like substance.
• Varga, E. V., Xiaoping, L. i., Stropova, D., Zalewska, T., Landsman, R. S., Knapp, R. J., Malatynska, E., Kawai, K., Mizusura, A., Nagase, H., Calderon, S. N., Rice, K., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1996). The third extracellular loop of the human δ-opioid receptor determines the selectivity of δ-opioid agonists. Molecular Pharmacology, 50(6), 1619-1624.
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  PMID: 8967985;Abstract: In the present study, we replaced the third extracellular loop of the human δ-opioid receptor with that of the human μ-opioid receptor. A modified polymerase chain reaction overlap extension method was used to achieve the exact splicing in the chimera to show the importance of the extracellular loop in ligand binding without interference from transmembrane substitutions. The replacement of the third extracellular loop did not alter the affinity of [3H]diprenorphine but caused a dramatic decrease in the affinity of both the δ-selective peptide agonists cyclo[D-Pen2,4'Cl- Phe4,D-Pen5]enkephalin and deltorphin II and the δ-selective nonpeptide agonists SNC 121 and (-)TAN 67. The affinities of the μ-selective peptide agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin and the μ-preferring nonpeptide agonist morphine were not affected. Site-directed mutagenesis studies show that the mechanism of ligand recognition might be different for each structural class of opioid ligands.
• Williams, S. A., Abbruscato, T. J., Hruby, V. J., & Davis, T. P. (1996). Passage of a δ-opioid receptor selective enkephalin, [D-penicillamine2,5] enkephalin, across the blood-brain and the blood-cerebrospinal fluid barriers. Journal of Neurochemistry, 66(3), 1289-1299.
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  PMID: 8769896;Abstract: [D-Penicillamine2,5] enkephalin (DPDPE) is an enzymatically stable, 6-opioid receptor-selective peptide, which produces analgesia when given intracerebroventricularly. However, because only modest analgesic effects were seen after subcutaneous administration of DPDPE, it has been inferred that it does not cross the blood-brain barrier well. In this present study, a vascular brain perfusion technique in anesthetized rats was used to measure directly whether [3H] DPDPE could cross the blood-brain and/or the blood-CSF barriers. The results indicated that the brain uptake of [3H] DPDPE was significantly greater than that of [14C] sucrose, a vascular marker (p < 0.01), and than that of [3H] DPDPE into the CSF (p < 0.01). Furthermore, HPLC analysis confirmed the integrity of the 3H to DPDPE and demonstrated that intact [3H] DPDPE entered the brain. Although 1 mM leucine-enkephalin failed to inhibit uptake of [3H] DPDPE, unlabeled DPDPE (100 μM) caused a significant inhibition of the brain uptake (p < 0.01) but not the CSF uptake of [3H] DPDPE. These data provide evidence that intact [3H] DPDPE enters the CNS of anesthetized rats by saturable and nonsaturable mechanisms. In addition, the saturable mechanism is likely to be found at the blood-brain barrier, with the blood-CSF barrier playing only a minor role in the brain uptake of this peptide.
• Azizeh, B. Y., A., B., Sturm, N. S., Hutzler, A. M., David, C., Trivedi, D., & Hruby, V. J. (1995). [des His¹, des Phe⁶, Glu⁹]glucagon amide: A newly designed "pure" glucagon antagonist. Bioorganic and Medicinal Chemistry Letters, 5(16), 1849-1852.
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  Abstract: We report the synthesis and biological activity of a new glucagon analog that was designed as a glucagon receptor antagonist by appropriate modifications in the N-terminal region of glucagon. The structure of the new analog is [des His1, des Phe6, Glu9]glucagon amide, and its binding potency IC50 value of 48 nM. The compound was found to be a pure antagonist in a new much more sensitive assay for glucagon stimulated cAMP accumulation activity and showed a pA2 value of 8.20 in this assay. We report the sythesis and biological activity of a new glucagon analog that was designed as a glucagon receptor antagonist. The new analog, [des His1, des Phe6, Glu9]glucagon, amide, was found to be a pure antagonist in a new more sensitive assay for partial agonist activity, with a binding potency IC50 of 48 nM and a pA2 valueof 8.20. © 1995.
• Bilsky, E. J., Calderon, S. N., Wang, T., Bernstein, R. N., Davis, P., Hruby, V. J., McNutt, R. W., Rothman, R. B., Rice, K. C., & Porreca, F. (1995). SNC 80, a selective, nonpeptidic and systemically active opioid delta agonist. Journal of Pharmacology and Experimental Therapeutics, 273(1), 359-366.
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  PMID: 7714789;Abstract: The present study has investigated the pharmacology of SNC 80, a nonpeptidic ligand proposed to be a selective δ agonist in vitro and in vivo. SNC 80 was potent in producing inhibition of electrically induced contractions of mouse vas deferens, but not in inhibiting contractions of the guinea pig isolated ileum (IC50 values of 2.73 nM and 5457 nM, respectively). The δ selective antagonist ICI 174,864 (1 μM) and the μ selective antagonist CTAP (1 μM) produced 236- and 1.9-fold increases, respectively, in the SNC 80 IC50 value in the mouse vas deferens. SNC 80 preferentially competed against sites labeled by [3H]naltrindole (δ receptors) rather than against those labeled by [3H]DAMGO (μ receptors) or [3H]U69, 593 κ receptors) in mouse whole-brain assays. The ratios of the calculated K(i) values for SNC 80 at μ/δ and κ/δ sites were 495- and 248- fold, respectively, which indicates a significant degree of δ selectivity for this compound in radioligand binding assays. SNC 80 produced dose- and time-related antinociception in the mouse warm-water tail-flick test after i.c.v., i.th. and i.p. administration. The calculated A50 values (and 95% C.I.) for SNC 80 administered i.c.v., i.th. and i.p. were 104.9 (63.7-172.7) nmol, 69 (51.8-92.1) nmol and 57 (44.5-73.1) mg/kg, respectively. The i.c.v. administration of SNC 80 also produced dose- and time-related antinociception in the hot-plate test, with a calculated A50 value (and 95% C.I.) of 91.9 (60.3-140.0) nmol. Intraperitoneal SNC 80 antinociception was antagonized by pretreatment with i.c.v. naloxone (3 nmol), with i.c.v. or i.th. N,N-diallyl- Tyr-(Aib)2-Phe-Leu-OH(Aib=α-amino isobutyric acid) (4.4 nmol) or with i.p. naltrindole (20 mg/kg), but not i.c.v. or i.th. β-FNA (18.8 nmol at -24 hr). Furthermore, the antinociceptive effects of i.c.v. SNC 80 were antagonized by i.c.v. pretreatment with either [D-Ala2,Leu5,Cys6]enkephalin (a putative δ1 antagonist) or [D-Ala2, Cys4]deltorphin (a putative δ2 antagonist), but not by β-funaltrexamine (a μ antagonist). This suggests that the antinociceptive actions of SNC 80 are produced via both opioid δ1 and δ2, but not μ, receptors. On the basis of its profile in vivo and in vitro, SNC 80 is perhaps the first highly selective, nonpeptidic and systemically active opioid δ agonist. SNC 80 promises to be a useful compound for the exploration of opioid δ-receptor pharmacology and provides a basis for the further identification of selective nonpeptidic δ ligands.
• Craft, R. M., Henley, S. R., Haaseth, R. C., Hruby, V. J., & Porreca, F. (1995). Opioid antinociception in a rat model of visceral pain: Systemic versus local drug administration. Journal of Pharmacology and Experimental Therapeutics, 275(3), 1535-1542.
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  PMID: 8531126;Abstract: Antinociceptive effects of systemically or locally administered opioid μ, κ and δ agonists were evaluated in a rat model of visceral pain. Resiniferatoxin (RTX, 3 nmol), a capsaicin-like irritant, produced abdominally directed grooming behavior after direct administration into the urinary bladder (intravesical, i.ves.) by indwelling cannula. Systemic (s.c. or i.p.) pretreatment with the μ agonists morphine or [D-Ala2, NMePhe4, Gly-ol]enkephalin (Damgo), the κ agonists trans-3,4-dichloro-N-methyl-N-[2- (1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) or [5R-(5,7,8-β)]-N- methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro-[4,5]dec-8-yl]-4-benzofuranacetamide (CI-977), or the nonpeptidic δ agonist (±)-4-((α-R*)-α-((2S*,5R(*)-4- Allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N, N-diethylbenzamide (BW373U86) dose-dependently decreased RTX-induced abdominal licking; such antinociception was selectively blocked by the appropriate receptor-selective antagonists β-funaltrexamine (μ), norbinaltorphimine (κ) and naltrindole (δ). Local (i.ves.) BW373U86, [D-Ala2, Glu4]deltorphin (DELT II) and CI- 977 also significantly decreased RTX-induced licking. Intracerebroventricular quaternary naloxone partially blocked the effects of systemic morphine, but not that of CI-977 or BW373U86. Intraperitoneal quaternary naloxone blocked the effect of local and systemic BW373U86 but not that of local or systemic CI-977; systemic morphine was partially blocked. Thus, systemic μ, κ and δ agonists all produced antinociception against a novel visceral chemical stimulus in the rat. Local CI-977 also produced antinociception, but the only compound clearly acting at peripheral opioid receptors was BW373U86, a δ agonist. This study suggests that opioid δ receptors may be present on bladder nociceptive afferents and may be activated for production of peripheral analgesia.
• Davis, T. P., Abbruscato, T. J., Brownson, E., & Hruby, V. J. (1995). Conformationally constrained peptide drugs targeted at the blood-brain barrier. NIDA Research Monograph Series, 47-60.
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  PMID: 8606749;
• Haskell-Luevano, C., Boteju, L. W., & Hruby, V. J. (1995). Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions. Letters in Peptide Science, 1(4), 163-170.
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  Abstract: Nitrogen indole protection of the β-methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for β-methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions. © 1995 ESCOM Science Publishers B.V.
• Haskell-Luevano, C., Boteju, L. W., Miwa, H., Dickinson, C., Gantz, I., Yamada, T., Hadley, M. E., & Hruby, V. J. (1995). Topographical modification of melanotropin peptide analogues with β-methyltrytophan isomers at position 9 leads to differential potencies and prolonged biological activities. Journal of Medicinal Chemistry, 38(23), 4720-4729.
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  PMID: 7473600;Abstract: We have introduced topographical constraints at the 9 position of a superpotent cyclic α-melanotropin analogue, Ac-Nle4-Asp5-His6-DPhe7-Arg 8-Trp9-Lys10-NH2, by incorporating a methyl group at the β-carbon of Trp9. These studies were performed on the Trp side chain pharmacophore to identify the bioactive topography of the indole moiety with melanocortin MC1 receptors. The four β-MeTrp9 isomers, in addition to the stereochemical controls L- and DTrp9, were used to probe differential receptor molecular recognition of the tryptophan moiety in two bioassay systems. Approximately a 460-fold difference in potency was observed-between the diastereoisomeric peptides in the frog skin bioassay, with only 33- and- 10-fold efficacy differences observed in binding and intracellular cAMP accumulation, respectively, on the human melanocortin receptor, hMC1R. The relative orders of potencies in the frog skin bioassay were 2R,3S > 2S,3S = 2R,3R ≫ 2S,3R and for the hMC1R were 2S,3S > 2R,3R > 2R,3S ≫ 2S,3R. Of particular interest is the ability of these topographically constrained to differentially affect prolonged biological activity. The 2R,3R diastereoisomeric peptide possessed superprolonged activity, whereas the 2S,3S peptide lacked any residual activity in the frog skin bioassay. However, on the melanocortin receptor, the 2S,3S diastereoisomeric peptide maintained slow dissociation rates (t1/2 = 7 h), while the other diastereoisomeric peptides possessed dissociation t1/2 rates of ca. 2 h. These data strongly implicate ligand-receptor interactions and kinetics as contributing to the observed prolonged biological activities and clearly illustrate topographical recognition differences between these two peripheral MC1 receptors involved in skin pigmentation. This study also demonstrates that topographical modifications of pharmacophore side chain residues, in addition to identifying preferential side chain orientation, can be a useful strategy for the design of peptides to increase the duration of biological activity, relative to the native ligand. © 1995 American Chemical Society.
• Haskell-Luevano, C., Shenderovich, M. D., Sharma, S. D., Nikiforovich, G. V., Hadley, M. E., & Hruby, V. J. (1995). Design, synthesis, biology, and conformations of bicyclic α-melanotropin analogues. Journal of Medicinal Chemistry, 38(10), 1736-1750.
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  PMID: 7752197;Abstract: Seven side chain-constrained bicyclic α-melanotropin (α-MSH) analogues were designed and synthesized, their conformations analyzed, and their biological properties examined in the frog skin and lizard skin bioassays. The structure of these analogues is based on the central sequence Ac-Cys4-Xaa5-His6-DPhe7-Arg 8-Trp9-Cys10-Lys11-NH2 (Xaa5 = Asp or Glu) and has been extended on the N-terminal with the amino acids Ser1-Tyr2-Ser3 and on the C-terminal with Pro12-Val13 to more closely resemble the native hormone α-MSH. The analogue Ac-Cys4-Asp5-His6-DPhe7-Arg 8-Trp9-Lys10-Cys11-NH2 also was synthesized, and its conformational and biological properties were examined. Design of these analogues was based upon the previously identified superpotent monocyclic peptides [Cys4,DPhe7,Cys10]α-MSH(4-10)-NH 2 and [Nle4,Asp5,DPhe7,Lys 10]α-MSH(4-10)-NH2 with the rationale of increasing conformational constraints to restrict the available backbone conformations as a means to identify the conformations that facilitate biological activity. Computer-assisted conformational analysis of the central tetrapeptide residues 6-9 identified β-turns which varied with respect to the residue in the i + 1 position. Each highly constrained peptide contains D-Phe7 and a 23-membered ring which has previously been identified as crucial to produce prolonged acting peptides with superagonistic activities. The bicyclic peptides reported in this study are full agonists and are 25-400-fold less potent than α-MSH in the frog and lizard skin bioassays. © 1995 American Chemical Society.
• Hruby, V. J., Danho, W., & Yamashiro, D. (1995). Johannes Arnold Meienhofer. 1929-1993.. International journal of peptide and protein research, 46(3-4), 193-194.
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  PMID: 8537169;
• Hruby, V. J., Dongsi, L. u., Sharma, S. D., L., A., Kesterson, R. A., Al-Obeidi, F. A., Hadley, M. E., & Cone, R. D. (1995). Cyclic lactam α-melanotropin analogues of Ac-Nle⁴-cyclo[Asp⁵,D-Phe⁷,Lys¹⁰] α-melanocyte-stimulating hormone-(4-10)-NH₂ with bulky aromatic amino acids at position 7 show high antagonist potency and selectivity at specific melanocortin receptors. Journal of Medicinal Chemistry, 38(18), 3454-3461.
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  PMID: 7658432;Abstract: The cloning of the melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) receptors (MC1-R and MC2-R, respectively) recently has led to the identification of three additional melanocortin receptors, MC3-R, MC4-R, and MC5-R. The MC2 receptor primarily recognizes only ACTH peptides, but the other four receptors all recognize a-melanocyte-stimulating hormone (α-MSH) and potent α-MSH agonists such as [Nle4,D-Phe7]α-MSH-NH2 and Ac-Nle4-c[Asp5,D-Phe7,Lys 10]α-MSH-(4-10)-NH2 as well as ACTH. The absence of any known physiological role for these new receptors, expressed both in the brain (MC3-R and MC4-R) and throughout a number of peripheral tissues (MC5-R), has necessitated a search for potent and receptor selective agonists and antagonists. We report here that analogues of the superpotent cyclic agonist analogue Ac-Nle4-c[Asp5,D-Phe7,Lys 10]a-MSH-(4-10)-NH2, in which a bulky aromatic amino acid is substituted in the 7-position, can produce potent and selective antagonists for melanocortin receptors. Thus, the D-p-iodophenylalanine7-containing analogue Ac-Nle4-c[Asp5,D-Phe(pI)7,Lys 10]α-MSH-(4-10)-NH2 is a potent antagonist (pA2 = 10.3) in the classical frog skin (Rana pipiens) assay (MC1-R), as is the D-2′-naphthylalanine7 (D-Nal(2)7)-containing analogue Ac-Nle4-c[Asp5,D-Nal(2)7, Lys10]α-MSH-(4-10)-NH2 (pA2 > 10.3). Interestingly, the D-p-chloro- and D-p-fluorophenylalanine7-containing analogues lacked antagonist activities at all melanotropin receptors, and both exhibited full agonist potency in the frog skin assay. The activity of these analogues also was examined at four mammalian melanocortin receptors. Interestingly, Ac-Nle4-c[Asp5,(D-Nal(2)7, Lys10]α-MSH-(4-10)-NH2 was found to be a potent antagonist of the MC4-R (pA2 = 9.3) with minimal agonist activity, a less potent antagonist of the MC3-R (pA2 = 8.3) with minimal agonist activity, and a full agonist of the MC1 and MC5 receptors. Surprisingly, Nle4-c[Asp5,D-Phe(pI)7,Lys 10]α-MSH was found to be a potent agonist at the cloned human MC1-R (EC50 = 0.055 nM) and mouse MC1-R (EC50 = 0.19 nM) but had potent antagonist activities at the human MC4-R (pA2 = 9.7) and human MC3-R (pA2 = 8.3) with significant partial agonist activities (EC50 = 0.57 and 0.68 nM, respectively) as well. Thus, highly potent and receptor selective antagonist analogues can arise from substitution of the D-Phe7 residue with a bulky aromatic amino acid. These analogues can be used to help determine the functional roles of these receptors. © 1995 American Chemical Society.
• Hruby, V. J., Yamamura, H. I., & Porreca, F. (1995). Molecular organization of receptors. Efficacy, agonists, and antagonists. Annals of the New York Academy of Sciences, 757, 7-22.
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  PMID: 7611710;
• Jiang, J., Sharma, S. D., Nakamura, S., Lai, J. Y., Fink, J. L., Hruby, V. J., & Hadley, M. E. (1995). The melanotropic peptide, [Nle4,D-Phe7] alpha-MSH, stimulates human melanoma tyrosinase activity and inhibits cell proliferation.. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 8(6), 314-323.
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  PMID: 8789740;Abstract: Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle4,D-Phe7] alpha-MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called "amelanotic" (tyrosinase-negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle4,D-Phe7] alpha-MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and "amelanotic" cell lines incubated with [Nle4,D-Phe7] alpha-MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle4,D-Phe7] alpha-MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.
• Kazmierski, W. M., Ferguson, R. D., Lipkowski, A. W., & Hruby, V. J. (1995). A topographical model of μ-opioid and brain somatostatin receptor selective ligands. NMR and molecular dynamics studies. International Journal of Peptide and Protein Research, 46(3-4), 265-278.
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  PMID: 8537180;Abstract: We have refined the 1H NMR-based conformations of the μ-opioid receptor selective peptides related to somatostatin of general formula Xxx-Yyy1-Cys-Zzz-D-Trp-Lys(Orn)5-Thr-Pen-Thr8-NH2, where Xxx, Yyy, Zzz are 0, D-Phe and Tyr for 1; 0, D-Tic and Tyr for 2; Gly, D-Tic and Tyr for 3; and 0, D-Phe and Tic for 4, respectively, (Kazmierski et al., J. Am. Chem. 113, 2275-2283), using a molecular-dynamics approach. We present evidence that the NMR data are compatible with βII'-, γ- and γ'-turns for the central tetrapeptide Tyr-D-Trp-Lys/Orn-Thr. Based on detailed structural and topographical considerations, we suggest that the μ-opioid receptor selectivity of 2 is due to a particular spatial arrangement of aromatic side chains of D-TiC1 and Tyr3 (7.5 Å), and that the opioid receptor recognition domain is located in the N-terminal part of the peptide while the somatostatin receptor recognition domain is determined by the central, turn forming part of this class of Gyclic peptides. A model for a μ-opioid selective ligand has emerged from these studies that shows excellent structural similarities to rigid opioid alkaloids.
• Knapp, R. J., Landsman, R., Waite, S., Malatynska, E., Varga, E., Haq, W., Hruby, V. J., Roeske, W. R., Nagase, H., & Yamamura, H. I. (1995). Properties of TAN-67, a nonpeptide δ-opioid receptor agonist, at cloned human δ-and μ-opioid receptors. European Journal of Pharmacology: Molecular Pharmacology, 291(2), 129-134.
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  PMID: 8566162;Abstract: 2-methyl-4a α(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a α-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67) is a nonpeptidic δ-opioid receptor agonist. This report describes its receptor binding affinity and agonist potency at human and mouse δ and μ-opioid receptors. The binding affinities of TAN-67 and the cyclic enkephalin analog, [D-Pen2, 4′-Cl-Phe4, D-Pen5]enkephalin (pCl-DPDPE) were measured by the radioligand binding inhibition studies at mouse and human variants of the δ and μ-opioid receptor using [3H]Naltrindole and [3H]D-Phe-Cys Tyr D Trp Orn Thr Pen-Thr-NH2, respectively. TAN-67 showed high binding affinity (Ki = 0.647 nM) at the human δ-opioid receptor and high δ-opioid receptor binding selectivity (> 1000-fold) relative to the human μ-opioid receptor. TAN-67 also showed high potency (EC50 = 1.72 nM) for the inhibition of forskolin-stimulated cAMP accumulation at human δ-opioid receptors expressed by intact Chinese hamster ovary cells but low potency (EC50 = 1520 nM) at human μ-opioid receptors expressed by intact B82 mouse fibroblast cells. The results show that TAN-67 has similar binding affinities, selectivity and potencies as pCl-DPDPE at human δ and μ-opioid receptors. These results combined with the nonpeptidic structure of TAN-67 suggest that this compound has therapeutic potential as a δ-opioid receptor agonist. © 1995.
• Knapp, R. J., Malatynska, E., Collins, N., Fang, L., Wang, J. Y., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1995). Molecular biology and pharmacology of cloned opioid receptors. FASEB Journal, 9(7), 516-525.
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  PMID: 7737460;Abstract: The cloning and expression of DNA for the three major opioid receptor types (μ, δ, and κ) present new research opportunities for the characterization of opioid drugs and their interactions with these receptors. Genomic and cDNA clones for opioid receptors exist for several animal species including mouse, rat, guinea pig, and human. These include clones for all three human opioid receptor types. The receptor proteins consist of about 400 amino acids and have the characteristic seven transmembrane domain structure of G-protein-coupled receptors. There is about 60% amino acid identity between opioid receptor types and about 90% identity between a receptor type cloned from different animal species. All opioid receptor types mediate the inhibition of adenylyl cyclase in response to agonist binding. Radioligand binding and functional studies using the cloned receptors tend to support current conclusions on opioid drug receptor selectivity and activity. Investigations of opioid receptor chimeras and single amino acid mutants are providing information on the ligand recognition sites of these receptors and essential support for the development of computational opioid receptor models. A molecular model of the human δ opioid receptor is included in this review.
• Lipkowski, A. W., Misicka, A., Porreca, F., Davis, P., Yamamura, H. I., Stropova, D., & Hruby, V. J. (1995). Benzomorphan alkaloids: Natural peptidomimetics of opioid peptide pharmacophores. Letters in Peptide Science, 2(3-4), 177-181.
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  Abstract: The synthesis of hybrid analogues of alkaloid nalindol acid and C-terminal fragments of opioid peptides aims to provide the final proof of the proposed topographical relation between opioid peptides and benzomorphan skeleton-containing alkaloids during interaction with receptors. The biological data indicate that the topographical requirements for Tyr1 and Phe4 in opioid peptides are similar for interactions with both δ and κ receptor types. © 1995 ESCOM Science Publishers B.V.
• Lou, B., Guigen, L. i., Lung, F., & Hruby, V. J. (1995). NMR investigation of asymmetric conjugate additions using chiral 4-phenyloxazolidinone as a mechanistic probe. The Journal of Organic Chemistry, 60(17), 5509-5514.
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  Abstract: The asymmetric conjugate addition of organocopper(I) reagents to α,β-unsaturated N-acyl-4-phenyl-2-oxazolidinones has been studied by 1H- and 13C-NMR spectroscopy using the Evans-type 4-phenyloxazolidinone auxiliary as a mechanistic probe. Three chiral intermediates were observed directly. The two metal methyl groups in the olefin-copper(I) complex 2, which is crucial for asymmetric induction, were assigned by both chemical shift and kinetic analysis. The dihedral angle of the α- and β-protons of the metallo enolate 3 was measured as 150° which provided valuable information for examining the stereochemical effects of the β position on the chirality of the α center. Enolates 3 and 4 are reversibly temperature dependent. Enolate 3 is the major component at 253 K, while enolate 4 becomes the major component at 293 K. Therefore, temperatures lower than ∼253 K are required for high stereoselectivity in the electrophilic bromination of the resulting enolate to build an a-chiral center. The free energy of activation (ΔG†) and rate constant (kc) of the equilibrium were measured as 15.0 kcal/mol and 1.4 × 102 s-1, respectively. © 1995 American Chemical Society.
• Lung, F. -., Li, G., Lou, B. -., & Hruby, V. J. (1995). A new strategy for the synthesis of four individual isomers of β-methylphenylalanine. Synthetic Communications, 25(1), 57-61.
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  Abstract: The application of an allylic strain effect in boron enolates and asymmetric Michael-like addition/electrophilic bromination reactions is reported for the asymmetric synthesis of the individual isomers of unusual constrained amino acids. For β-substituted α-amino acids, all of the final optically pure products were identical to authentic samples, which provided further and unequivocal evidence to confirm the assignments of stereochemical control of the new methods in this report.
• Lung, F. T., Meyer, J., Guigen, L. i., Lou, B. -., Stropova, D., Davis, P., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1995). Highly κ receptor-selective dynorphin A analogues with modifications in position 3 of dynorphin A(1-11)-NH₂. Journal of Medicinal Chemistry, 38(4), 585-586.
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  PMID: 7861405;
• Meyer, J., Davis, P., Lee, K. B., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1995). Synthesis using a Fmoc-based strategy and biological activities of some reduced peptide bond pseudopeptide analogues of dynorphin A. Journal of Medicinal Chemistry, 38(18), 3462-3468.
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  PMID: 7658433;Abstract: Eight analogues of Dyn A(1-11)-NH2 incorporating the enzymatically stable ψ(CH2-NH) isosteric peptide bond replacement were synthesized and tested for binding affinity at the central opioid μ, δ, and κ receptors in guinea pig brain (GPB) homogenates and for activity at the peripheral κ (and μ) receptors in the guinea pig ileum (GPI). The peptidic analogues were synthesized by solid phase techniques using a Fmoc/tert-butyl strategy, and the ψ(CH2-NH) bond, or reduced bond, was introduced via reductive alkylation of the N-terminal amino group of the growing peptide with a Fmoc-Nα-protected amino aldehyde. The synthesis of Fmoc-Nα-protected amino aldehydes also is described. Several other peptides have been previously synthesized incorporating this modification and showed for instance increased enzymatic stability and antagonist properties. Results obtained in the GPB show that modifications of the peptide bond in the address site (analogues 4-9) do not affect the binding at the κ receptor and, with a few exceptions, at the μ and δ receptors. On the other hand, analogues 2 and 3, modified in the message segment of Dyn A(1-11)-NH2, show a decrease in binding affinity at all three receptors. In the GPI, the results are more varied as the influence of the peptide bond modification seems to be more important than in the GPB. Finally, selected analogues were tested with no indication for antagonist activity at the κ peripheral receptor. © 1995 American Chemical Society.
• Meyer, J., Gillespie, T. J., Hom, S., Hruby, V. J., & Davis, T. P. (1995). In vitro stability of some reduced peptide bond pseudopeptide analogues of dynorphin A. Peptides, 16(7), 1215-1219.
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  PMID: 8545241;Abstract: Eight analogues of DYN A(1-11)-NH2 incorporating the nonhydrolyzable Ψ[CH2-NH] peptide bond surrogate were tested for their in vitro enzymatic stability in mouse brain homogenates. Results show that the Leu5-Arg6 and to a lesser extent the Arg7-Ile8 and Ile8-Arg9 peptide bonds are the more susceptible to enzymatic cleavage in the native peptide. (Leu5Ψ[CH2-NH]Arg6)DYN A(1-11)-NH2 exhibits an almost complete resistance to enzymatic cleavage with a half-life greater than 500 min in brain, compared to 42 min for the standard peptide, DYN A(1-11)-NH2. © 1995.
• Misicka, A., Lipkowski, A. W., Slaninova, J., Davis, P., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1995). The synthesis and opioid receptor binding affinities of analogues of dermorphin and its N-terminal tetrapeptide fragment with dibasic acids in position 2. Life Sciences, 57(18), 1633-1640.
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  PMID: 7475903;Abstract: Analysis of possible μ opioid receptor active conformations for dermorphin suggested that the topographical location of the tyramine moiety of the N-tenninal tyrosine can be simulated with the phenol of tyrosine1 or desamino-tyrosine1 (4-hydroxyphenylpropionic acid) and a basic group located on the side chain of a dibasic acid residue located in position 2. The biological properties of respective analogs with D- or L-arginine, and D- or L-lysine in the position 2 of dermorphin or desaminodermorphin and their N-terminal tetrapeptide fragments, has provided evidence in support of this prediction, and questions the dogma that an N-terminal tyrosine is a necessary element for opioid agonist peptides. © 1995.
• Misicka, A., Lipkowski, A. W., Stropova, D., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1995). Amino acids with amphiphilic side chains: Deltorphin analogues with Phe³ replaced by all β-hydroxyphenylalanine diastereoisomers. Letters in Peptide Science, 2(3-4), 203-205.
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  Abstract: Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I containing each of the four stereoisomers of the unusual amphiphilic amino acid β-hydroxyphenylalanine in position 3. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays and by bioassay in MVD and GPI. The results show that introducing a hydrophilic group into the β-carbon of Phe3 decreases the affinity and biological activity of δ-opioid receptors, which strongly depend on the chirality of the α-carbon, but not on that of the β-carbon. © 1995 ESCOM Science Publishers B.V.
• Ossipov, M. H., Kovelowski, C. J., Nichols, M. L., Hruby, V. J., & Porreca, F. (1995). Characterization of supraspinal antinociceptive actions of opiod delta agonists in the rat. Pain, 62(3), 287-293.
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  PMID: 8657428;Abstract: Supraspinally mediated antinociception has been clearly established for agonists acting via both μ- and δ-opioid receptors. The present experiments were undertaken to further characterize the role of supraspinal opioid δ receptors in the mediation of antinociception in rats and to examine the possible role of putative δ1- and δ2-opioid receptors in the antinociceptive effect. Cannulae directed at the right lateral ventricle, the periaqueductal gray (PAG), or the medullary reticular formation (MRF) were implanted in adult male, Sprague-Dawley rats for the microinjection of [d-Ala2, Glu4]deltorphin (δ2 agonist), [d-Pen2,d-Pen5]enkephalin (DPDPE, δ1 agonist), [d-Ser2,Leu5,Thr6]enkephalin (DSLET, mixed δ μ agonist) or morphine (reference μ-opioid). Pretreatments (24 h prior to agonist microinjection) were made with the putative δ1 and δ2 antagonists, [d-Ala2,Leu5,Cys6]enkephalin (DALCE) and [d-Ala2,Cys4]deltorphin (Cys-DELT) and antinociception was measured in the 55°C hot plate (HP) and 52°C and 55°C (low and high intensity) warm-water tail-flick (TF) tests. Data were converted to percent maximal possible effect (%MPE). Intracerebroventricular (i.c.v.) administration of DPDPE produced less than a 50% MPE in the HP test whereas [d-Ala2,Glu4]deltorphin produced Cys-DELT sensitive antinociception of up to 92% MPE. Neither i.c.v. agonist was effective in the TF assays, and both agonists were without effect in the PAG. [d-Ala2,Glu4]deltorphin microinjected into the MRF produced Cys-DELT sensitive antinociception of 60 and 47% MPE in the HP and low-intensity TF tests, respectively, but was not effective in the 55°C TF test; DPDPE did not produce antinociception when microinjected at this site. Microinjection of DSLET in the MRF produced significant antinociception in all three assays. Morphine produced antinociception following i.c.v. administration or microinjection into the PAG in all tests. Microinjection of morphine into the MRF produced antinociception in the HP and 52°C, but not 55°C, TF tests. Morphine antinociception was not antagonized by either DALCE or Cys-DELT. These data demonstrate that supraspinal δ-opioid receptors can be activated to elicit antinociception in the rat and that opioid δ2 receptors predominate in this effect. Further, these effects may occur predominately via ibhibition of supraspinally organized behavior without activation of descending systems such as those mediating the TF response in the rat. © 1995.
• Qian, X., Russell, K., Boteju, L. W., & Hruby, V. J. (1995). Stereoselective total synthesis of topographically constrained designer amino acids: 2′, 6′-dimethyl-β-methyltyrosines. Tetrahedron, 51(4), 1033-1054.
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  Abstract: The constrained aromatic α-amino acid 2′, 6′-dimethyl-β-methyl tyrosine (Figure 1) was designed to provide specific local constraints to peptides or peptide mimetics. We report here methods for the total asymmetric synthesis of all four stereoisomers. The precursors used were α,β-unsaturated acid derivatives, (2E) 3-(4′)-methoxy-2′,6′-dimethyl-2-propenoic acid (5) and crotonyl chloride (6). In order to introduce chirality at both the α- and the β-positions of the amino acids, optically pure 4-phenyl-2-oxazolidinones (Xc) were coupled to 5 and 6. The key steps for the synthesis were: (1) a Michael type addition using either methylmagnesium bromide/copper (I) bromide-dimethyl sulfide complex or 4-methoxy-2,6-dimethylphenylmagnesium bromide/copper (I) bromide-dimethyl sulfide complex as nucleophiles; (2) an asymmetric bromination of the α-position of the N-acyloxazolidinones using di(n-butyl)boron triflate/DIEA/NBS as reagents at low temperature. In both cases, the stereoselectivies and yields were excellent; (3) amination was achieved in nearly quantative yield by treating the bromides with azide exchange resin via an SN2 mechanism. (Electrophilic azidation using 2,4,6-triisopropylsulfonyl azide also was achieved). The excellent stereoselectivity (80-98% ee/de) and overall yield (30-60%) made these optically pure amino acids available in amounts practical for peptide synthesis and further conformational and structure-activity relationship studies of various peptide analogues. © 1995.
• Slaninova, J., Knapp, R. J., Weber, S. J., Davis, T. P., Fang, S., Hruby, V. J., & Yamamura, H. I. (1995). [¹²⁵I]SNF 8702: A selective radioligand for CCK_B receptors. Peptides, 16(2), 221-224.
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  PMID: 7784252;Abstract: The CCK-8 analogue, SNF 8702, was radioiodinated. [125I]SNF 8702 showed high-affinity specific binding for both guinea pig and mouse brain tissues, whereas no specific binding was seen for guinea pig pancreatic tissue. The properties of the site labeled by [125I]SNF 8702 were characterized by binding inhibition studies for a series of CCKA and CCKB receptor ligands. The binding selectivity profile corresponded to that for the CCKB receptor. The labeled compound is stable for more than 6 weeks during storage at -20°C. © 1995.
• Xiang, L., Huiwei, W. u., & Hruby, V. J. (1995). Stereoselective synthesis of all individual isomers of β-methyl-2′,6′-dimethylphenylalanine. Tetrahedron: Asymmetry, 6(1), 83-86.
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  Abstract: Asymmetric synthesis of all four isomers of β-methyl-2′,6′-dimethylphenylalanine was accomplished with complete stereoselectivities and high yields by using the Evans-like auxiliary 4-phenyl-oxazolidinone as a chiral auxiliary and as a chiral resolution reagent. © 1994.
• Bartosz-Bechowski, H., Davis, P., Zalewska, T., Slaninova, J., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Cyclic enkephalin analogs with exceptional potency at peripheral δ opioid receptors. Journal of Medicinal Chemistry®, 37(1), 146-150.
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  PMID: 8289188;Abstract: A series of super potent and δ-opioid-receptor-selective cyclic hexapeptides of the general formula H-Tyr-D-Pen-Gly-Phe(p-X)-Cys-Phe-OH (where X is hydrogen or halogen) has been synthesized. The unsubstituted hexapeptide H-Tyr-D-Pen-Gly-Phe-Cys-Phe-OH (HB-P2, [Phe6]DPLCE) has extremely high potency at peripheral δ opioid receptors (IC50 value in the MVD assay is 0.016 nM) and in bioassays is the most selective compound in this series. The introduction of halogens in the phenyl ring of phenylalanine at position 4 led to significant changes in the selectivity and affinities at peripheral and central opioid receptors. In the binding studies, the most potent compound is the p-fluoro analog, whereas the most selective analog is the p-iodo-substituted peptide. © 1994 American Chemical Society.
• Bilsky, E. J., Bernstein, R. N., Pasternak, G. W., Hruby, V. J., Patel, D., Porreca, F., & Lai, J. (1994). Selective inhibition of [D-ALA², GLU⁴]deltrophin antinociception by supraspinal, but not spinal, administration of an antisense oligodeoxynucleotide to an opioid delta receptor. Life Sciences, 55(2), PL37-PL43.
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  PMID: 8015351;Abstract: Evidence in vivo has suggested the existence of subtypes of the δ opioid receptor (DOR), which have been termed δ1 and δ2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D- Pen5]enkephalin (DPDPE, δ1) and [D-Ala2, Glu4]deltorphin (δ2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, μ agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonist selective for δ, μ and κ receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4deltorphin but not of i.th. DAMGO or U69, 593 (κ agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, seletively inhibited the anitinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as δ2 and further, suggest that this δ receptor subtype may play a major role in eliciting spinal δ-mediated antinociception. © 1994.
• Boteju, L. W., Wegner, K., Qian, X., & Hruby, V. J. (1994). Asymmetric synthesis of unusual amino acids: Synthesis of optically pure isomers of N-indole- (2-mesitylenesulfonyl)-β-methyltryptophan. Tetrahedron, 50(8), 2391-2404.
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  Abstract: We have developed methods for the synthesis of the four optically pure isomers of β-methyltryptophan with the 2-mesitylenesulfonyl indole protecting group for peptide synthesis. Starting from 3-indoleacrylic acid, the β-methyl function was generated by a chiral auxiliary-directed asymmetric conjugate 1,4-addition. Asymmetric bromination was achieved via a tandem addition of N-bromosuccinimide to the enolate formed by the conjugate addition. Displacement of the bromide by azide, hydrolysis of the chiral auxiliary and then reduction, led to the two erythro isomers. Chiral imide enolate azidation of the conjugate adduct, hydrolysis of the chiral auxiliary and reduction yielded the two threo isomers in high optical purity. © 1994.
• Brownson, E. A., Abbruscato, T. J., Gillespie, T. J., Hruby, V. J., & Davis, T. P. (1994). Effect of peptidases at the blood brain barrier on the permeability of enkephalin. Journal of Pharmacology and Experimental Therapeutics, 270(2), 675-680.
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  PMID: 7915319;Abstract: The blood brain barrier (BBB) presents an enzymatic barrier to the passage of peptides from blood to brain. The studies presented here used a well established in vitro model of the BBB to measure the presence of peptidases and the permeability of two opioid peptides. The in vitro BBB model consisted of confluent monolayers of bovine brain microvessel endothelial cells (BMECs). Enkephalin metabolizing enzymes, total aminopeptidase, aminopeptidase M (APM), angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) activities were measured in BMEC monolayers. The effect of specific inhibitors of APM, ACE and NEP on the permeability of [Met5]enkephalin (Met-Enk) and a conformationally constrained and enzymatically stable analog, DPDPE, also was determined. High levels of membrane-associated enzyme activity were measured for total aminopeptidase, APM and ACE. Interestingly, the permeability coefficient of Met-Enk was increased 4-fold in the presence of specific inhibitors of APM and ACE. Low levels of NEP activity were measured in BMEC monolayers and inhibition of NEP had no effect on Met-Enk permeability. The permeability coefficient for DPDPE was not increased with enzyme inhibitors but was 4-fold greater than Met-Enk alone. In the presence of APM or ACE inhibitors, there was no difference in the permeability of DPDPE and Met-Enk. These experiments demonstrate the presence of specific peptidases in BMECs and that the presence of inhibitors to Met-Enk inactivating peptidases significantly increased permeability of this biologically active peptide.
• Collins, N., & Hruby, V. J. (1994). Prediction of the conformational requirements for binding to the κ-opioid receptor and its subtypes. I. Novel α-helical cyclic peptides and their role in receptor selectivity. Biopolymers - Peptide Science Section, 34(9), 1231-1241.
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  PMID: 7948735;Abstract: A conformational search of two similar κ-selective cyclic Dynorphin A (Dyn A) analogues is presented. [Cys5, Cys11] Dyn A1-11-NH2(1) and [Cys5, D-Ala8, Cys11] Dyn A1-11-NH2 (2) are not only highly potent κ-selective peptides but they also exhibit exceptional selectivity for κ receptors in the central (brain) vs. the peripheral (ileum) systems. Molecular mechanics systematic searching of the conformational preferences of the cyclic moieties of 1 and 2 produced 741 and 1003 starting ring structures, which were minimized at two dielectric constants of 2.0 and 80.0 in the AMBER force field. By rms superimposition, these low energy structures were grouped into conformational families for each ring system minimized at each dielectric. Comparison of the lowest energy structure of each of these families demonstrated that two (labeled A and B) were found as low energy ring systems for both 1 and 2 after minimization at either dielectric constant. These two structures are thus predicted to be the putative binding conformations for Dynorphin A at receptors in the brain. Interestingly, one of these putative binding structures exhibited an α-helical conformation in the disulfide bridged ring that has not been observed for small cyclic peptides of this nature before. Molecular dynamics simulation of the helical binding structures indicated that the helical configuration in 2 is lower in energy and is more conformationally stable than that of 1. We correlate this with the increased selectivity and potency of 2 for κ receptors in the brain compared to the periphery, implying that this may be due to an α-helical conformation in the cyclized address or helical induction in the message sequence.
• Fang, L., Knapp, R. J., Horvath, R., Matsunaga, T. O., Haaseth, R. C., Hruby, V. J., Porreca, F., & Yamamura, H. I. (1994). Characterization of [³H]naltrindole binding to delta opioid receptors in mouse brain and mouse vas deferens: Evidence for delta opioid receptor heterogeneity. Journal of Pharmacology and Experimental Therapeutics, 268(2), 836-846.
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  PMID: 8113996;Abstract: Naltrindole (NTI) is a potent and selective nonpeptide delta opioid receptor antagonist. This study reports on the binding characteristics of [3H]NTI (specific activity = 30.5 Cl/mmole) for mouse brain and vas deferens (MVD) tissues. In brain, [3H]NTI had unusually high specific binding to delta receptors (80% at its Kd concentration) relative to other selective delta receptor radioligands. Saturation Kd values with 95% confidence intervals for mouse brain and MVD tissue preparations were 56.2 (41.8-75.7) and 104 (25.8-420) pM, respectively. These Kd values were significantly different (P = .028) and [3H]NTI binding to both tissues was best fit by a one-site model. Receptor densities were 83.9 (66.8-106) fmol/mg of protein for mouse brain and 14.8 (7.03-31.2) fmol/mg of protein for the MVD. Binding inhibition studies showed that NTI and the delta opioid receptor agonists [4'-Cl-Phe4]DPDPE and [D-Ala2, Glu4]deltorphin had high affinity for the sites labeled by [3H]NTI in both tissue preparations whereas mu [Tyr-Pro- ψ-MePhe-D-Pro-NH2 (PL-17)] and kappa (U-69593) agonists had micromolar affinity. Both agonists recognized multiple sites in mouse brain under control (with 5 mM Mg++) and treatment (with 50 μM guanylyl-5'- imidodiphosphate and 100 mM NaCl) conditions but only single-site binding was observed for MVD (only control condition tested) [D-Ala2, Glu4]deltorphin showed about 6.5-fold selectivity for a portion (≃33%) of mouse brain sites (Ki = 130 pM) compared to sites labeled by [3H]NTI in MVD (Ki = 1200 pM) under control conditions. No significant difference was observed for [4'-Cl- Phe4]DPDPE binding affinity to both tissues (Ki = 450-680 pM) under control conditions. The affinity of opioid agonists, but not antagonists at [3H]NTI binding sites in mouse brain, was substantially reduced by the presence of guanylyl-5'-imidodiphosphate and sodium ions consistent with guanine nucleotide binding protein regulation of the delta receptors. The portions of high- and low-affinity sites recognized by [4'-Cl-Phe4]DPDPE and [D-Ala2, Glu4]deltorphin in mouse brain labeled by [3H]NTI under treatment conditions were not significantly different (each subtype represented ≃50% of the total population) suggesting delta receptor heterogeneity in this tissue. It is concluded that [3H]NTI binds to delta opioid receptor affinity states and subtypes with equal affinity and can be used for their characterization in conjunction with different treatment conditions and ligands.
• Flippen-Anderson, J. L., Hruby, V. J., Collins, N., George, C., & Cudney, B. (1994). X-ray structure of [D-Pen²,D-Pen⁵]enkephalin, a highly potent, δ opioid receptor-selective compound: Comparisons with proposed solution conformations. Journal of the American Chemical Society, 116(17), 7523-7531.
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  Abstract: [D-Pen2,D-Pen5]enkephalin (DPDPE), a cyclic, constrained, highly potent, and δ opioid receptor-selective analogue of enkephalin, has been obtained from an aqueous solution in a crystalline form suitable for X-ray analysis. It crystallizes in the triclinic space group P1. The unit cell contains three conformationally distinct molecules of DPDPE which are located with approximate 3-fold symmetry about a water channel made up of approximately 24 disordered and one ordered water molecules. There are also 13 ordered water molecules which form an intricate network of hydrogen bonds which hold the peptide molecules together in the crystal. The conformation of the 14-membered ring is essentially identical for all three molecules; however, the Tyr-1 residue is conformationally different in each case. Comparison of the conformations found in the crystal with those previously determined by NMR methods in conjunction with energy calculations indicates that the most favorable conformation of the 14-membered ring in aqueous solution is similar to that in the crystal. This was interpreted to be due to the cyclic constraint in DPDPE and the high degree of solvation in the crystal structure. In addition, low-energy conformations previously determined by computational methods in attempts to determine the binding conformations of DPDPE gave conformations of the 14-membered rings which were generally similar to those found in the crystal structure. These results and previous structure-activity relationships suggest that the solid-state conformations are a useful starting point for understanding the bioactive conformation important for biological activity and δ receptor selectivity of cyclic enkephalin analogues.
• Fox-Threlkeld, J., Daniel, E. E., Christinck, F., Hruby, V. J., Cipris, S., & Woskowska, Z. (1994). Identification of mechanisms and sites of actions of mu and delta opioid receptor activation in the canine intestine. Journal of Pharmacology and Experimental Therapeutics, 268(2), 689-700.
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  PMID: 8113980;Abstract: Perfusion with ([N-Me-Phe3,D-Pro4]morphiceptin (PL017)), [D- Pen2,5]enkephalin (DPDPE) and Met5 and Leu5 enkephalin induced circular muscle contractions and decreased immunoreactive vasoactive intestinal polypeptide (VIP) venous output in canine ileal segments. Motility and VIP responses to PL017 were abolished by the mu antagonist CTAP (D-Phe-Cys-Tyr- D-Trp-Arg-Thr-Pen-Thr-NH2) and unchanged by the delta antagonist ICI 174,864 ([N,N-dially-Tyr1,Aib2,3]Leu-enkephalin) which abolished DPDPE motility and VIP responses. The VIP response to DPDPE was unchanged by CTAP, which reduced motility responses, suggesting a DPDPE interaction with endogenous mu opioids, at a mu/delta(complexed) receptor. ICI 174,864 abolished Met5 and Leu5 enkephalin motility responses and Leu5 enkephalin VIP responses while CTAP was ineffective on Leu5 enkephalin motility responses or on both enkephalin VIP responses. CTAP increased Met5 enkephalin motility responses suggesting mu actions to inhibit excitatory nerves. ICI 174,864 reduced Met5 enkephalin VIP output decrements requiring CTAP addition for abolition, suggesting actions at mu/delta(complexed) receptors. Inhibition of nitric oxide synthase with N-ω-L-arginine methyl ester (L-NAME) abolished delta opioid and reduced by 30% mu opioid motility responses, leaving the VIP response intact. Hexamethonium and atropine abolished tonic VIP output, leaving intact motility responses to PL017 and DPDPE. Subsequently L-NAME eliminated delta opioid and reduced by 1/3 mu opioid motility responses. All opioids reduced the NO-mediated IJPs in myenteric plexus-free ileal circular muscle. Thus mu or delta opioids inhibit both NO and VIP release but removal of NO, not VIP, disinhibits circular muscle motility.
• Guigen, L. i., Patel, D., & Hruby, V. J. (1994). 1, 2-Asymmetric cis induction and its application to the asymmetric synthesis of precursors of β-branched unusual amino acids. Tetrahedron Letters, 35(15), 2301-2304.
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  Abstract: A new method for the asymmetric synthesis of key intermediates of unusual amino acids has been established by utilizing β-carbon chirality for asymmetric induction in allylic-strained boron enolates. © 1994.
• Guigen, L. i., Patel, D., & Hruby, V. J. (1994). Exploration for large-scale stereoselective synthesis of unusual amino acids by using 4-phenyloxazolidin-2-one as a new chiral resolution reagent. Journal of the Chemical Society, Perkin Transactions 1, 3057-3059.
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  Abstract: Individual isomers of β-branched α-amino acids have been stereoselective and asymmetrically synthesized in high yield by a new method which uses 4-phenyloxazolidin-2-one as a novel chiral resolution reagent acting simultaneously as the auxiliary.
• Haaseth, R. C., Horan, P. J., Bilsky, E. J., Davis, P., Zalewska, T., Slaninova, J., Yamamura, H. I., Weber, S. J., Davis, T. P., Porreca, F., & Hruby, V. J. (1994). [L-Ala³]DPDPE: A new enkephalin analog with a unique opioid receptor activity profile. Further evidence of δ-opioid receptor multiplicity. Journal of Medicinal Chemistry, 37(11), 1572-1577.
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  PMID: 8201592;Abstract: To investigate δ-opioid receptor topography near the 3-position of [D-Pen2,D-Pen5]enkephalin (DPDPE), a series of small-group 3-position analogs of DPDPE have been synthesized and assayed for binding potencies and in vitro biological activities. L-Amino acid substitutions at this position are highly favored over D-amino acid substitutions, with the smallest, [L-Ala3]DPDPE (DPADPE), being the most favored in the series investigated. [L-Ala3] DPDPE is nearly as δ-potent and more δ-selective in both rat brain binding (18 nM vs [3H] [p-ClPhe4]DPDPE and μ/δ = 610) and peripheral bioassays (12 nM in the MVD and GPI/MVD = 4500) when compared to DPDPE (8.5 nM, μ/δ = 73 and 4.1 nM, GPI/MVD = 1800, respectively). Whereas DPDPE is a potent analgesic when given icv, [L-Ala3] DPDPE is only a weak analgesic. However, [L-Ala3] DPDPE has been found to antagonize DPDPE, but not Deltorphin II, in a moderately potent (pA2 = 5.7) and selective fashion in vivo. Thus, [L-Ala3] DPDPE is a fairly potent agonist at peripheral δ receptors and is a moderately potent (mixed) antagonist of δ1 receptors in the brain. It appears that [L-Ala3] DPDPE does not interact in any significant manner with δ2 or μ receptors in the brain. © 1994 American Chemical Society.
• Haskell-Luevano, C., Miwa, H., Dickinson, C., Hruby, V. J., Yamada, T., & Gantz, I. (1994). Binding and cAMP studies of melanotropin peptides with the cloned human peripheral melanocortin receptor, hMC1R. Biochemical and Biophysical Research Communications, 204(3), 1137-1142.
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  PMID: 7980588;Abstract: Binding and stimulation of cAMP by the melanotropin peptides α-MSH (α-melanocyte-stimulating hormone) and its superpotent analogues [Nle4, DPhe7] α-MSH (MT-I) and Ac-[Nle4, Asp5, DPhe7, Lys10] α-MSH4-10-NH2 (MT-II) were undertaken to examine their respective properties on the human peripheral melanocyte melanocortin receptor, hMC1R. α-MSH was found to possess a binding IC50 value of 6.5 ± 0.9 x 10-9 M and cAMP EC50 value of 2.0 ± 10-9 M. MT-I possesses a binding IC50 value of 1.2 ± 0.3 x 10-9 M and cAMP EC50 of 0.5 ± 0.03 x 10-9 M. MT-II possesses a binding IC50 of 0.57 ± 0.08 x 10-9 M and cAMP EC50 value of 0.20 ± 0.05 x 10-9 M.
• Hruby, V. J., & Bonner, G. G. (1994). Design of novel synthetic peptides including cyclic conformationally and topographically constrained analogs.. Methods in molecular biology (Clifton, N.J.), 35, 201-240.
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  PMID: 7894602;
• Hruby, V. J., & Qian, X. (1994). Approaches to the asymmetric synthesis of unusual amino acids.. Methods in molecular biology (Clifton, N.J.), 35, 249-286.
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  PMID: 7894604;
• Hruby, V. J., Collins, N., Lung, F. -., Meyer, J. -., Davis, T. P., Yamamura, H. I., & Porreca, F. (1994). Design of peptides and peptidomimetics for delta and kappa opioid receptor subtypes. Regulatory Peptides, 54(1), 123-124.
• Hruby, V. J., Misicka, A., Lipkowski, A. W., Haaseth, R., Bartosz, H., Qian, X., Collins, N., Meyer, J. -., Szabo, L., Polt, R., Porreca, F., Davis, T., & Yamamura, H. I. (1994). New opioid compounds in analgesia. Regulatory Peptides, 53(SUPPL. 1), S71-S72.
• Hruby, V. J., Wilke, S., Al-Obeidi, F., Jiao, D., & Lin, Y. (1994). Strategies for cyclizations of novel peptides on solid supports. Reactive Polymers, 22(3), 231-241.
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  Abstract: Synthetic strategies and methods for the preparation of cyclic peptides on solid supports are very important for peptide synthesis in general, and for the preparation of conformationally constrained biologically active peptides. In this paper we report synthetic methods for the preparation on solid supports of the cyclic and bicyclic biologically active peptides, Ac-[Nle4, Asp5, d-Phe, Lys10]α-melanotropin(4-10)-NH2, [Lys17, Lys18, Asp21]glucagon-NH2, [β-Mpa1, Glu4, Cys6, Lys8]oxytocin and cyclo[GlyTrpNMeNleAspPhe] (cyclic CCK-5 analogue). © 1994.
• Jean-Philippe, M., Collins, N., Feng-Di, L., Davis, P., Zalewska, T., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Design, synthesis, and biological properties of highly potent cyclic dynorphin a analogues. Analogues cyclized between positions 5 and 11¹. Journal of Medicinal Chemistry, 37(23), 3910-3917.
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  PMID: 7966152;Abstract: We have recently reported the synthesis of several cyclic disulfide bridge-containing peptide analogues of dynorphin A (Dyn A), which were conformationally constrained in the putative address segment of the opioid ligand. Several of these analogues, bridged between positions 5 and 11 of Dyn A1-11-NH2, exhibited unexpected selectivities for the κ and μ receptors of the central over the peripheral nervous systems. In order to further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, we have synthesized a systematic series of Dyn A1-11-NH2 analogues incorporating the sulfydryl containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11, thus producing 16 cyclic peptides. In addition, Dyn A1-11-NH2, [D-Leu5]Dyn A1-11-NH2, and [D-Lys11]Dyn A1-11NH2 were synthesized as standards. Several of these cyclic analogues, especially c[Cys5, D-Cys11] Dyn A1-11-NH2) c[Cys5, L- or D-Pen11]Dyn A1-11-NH2, c[Pen5, L-Pen11]Dyn A1-11-NH2 and c[Pen5, L- or D-Cys11]Dyn A1-11-NH2, retained the same affinity and selectivity (vs theμ and δ receptors) as the parent compound Dyn A1-11-NH2 in the guinea pig brain (GPB). These same analogues and most others exhibited a much lower activity in the guinea pig ileum (GPI), thus leading to centrally vs peripherally selective peptides, but showed a different structure-activity relationship than found previously. In a wider scope, this series of analogues also provided new insights into which amino acids (and their configurations) may be used in positions 5 and 11 of Dyn A analogues for high potency and good selectivity at κ opioid receptors. The results obtained in the GPB suggest that requirements for binding are not the same for the κ, μ, or δ central receptors. © 1994 American Chemical Society.
• Johnson, P. D., Dawson, B. V., Dorr, R. T., Hadley, M. E., Levine, N., & Hruby, V. J. (1994). Coat color darkening in a dog in response to a potent melanotropic peptide.. American Journal of Veterinary Research, 55(11), 1593-1596.
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  PMID: 7879984;Abstract: Analogues of a melanocyte-stimulating hormone (alpha-MSH) have been documented to be effective in inducing integumental melanogenesis in several species. These melanotropin analogues are more potent than the natural hormone and have prolonged biological activity, without apparent teratogenic or other toxic effects, at least in rodents. In a pilot study, a cyclic alpha-MSH analogue, Ac-[Nle4, Asp5, D-Phe7, Lys10] alpha-MSH4-10-NH2, was administered SC to a dog at a dose of 1 mg of analogue in 1 ml of 0.9% NaCl for 3 weeks, without noticeable adverse effects. There was gradual and extensive darkening of the coat, which originally was predominantly tan, with tips of black. Initially, the darkening involved face and extremities, then gradually expanded to include the trunk and tail hair. Visual pigmentation peaked approximately 2 months after injections were completed. As new hair growth continued subsequent to the injections, the original tan color appeared at the proximal end of the hair shaft, leaving a dark terminal band on all affected hairs. These observations clearly indicated that follicular melanogenesis can be induced in dogs by treatment with a melanotropic peptide.
• Kazmierski, W. M., Urbanczyk-Lipkowska, Z., & Hruby, V. J. (1994). New amino acids for the topographical control of peptide conformation: Synthesis of all the isomers of α,β-dimethylphenylalanine and α,β-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid of high optical purity. Journal of Organic Chemistry, 59(7), 1789-1795.
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  Abstract: The synthesis of all four diastereoisomers of α,β-dimethylphenylalanine (4) as well as those of α,β-dimethyl-1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid (5 and 6) have been accomplished in high yield and high optical purity. Molecular mechanics calculations on the Nα-acetyl and N-methylcarboxamide derivatives of (3R,4R)-6 and (3R,4S)-5 indicate large and moderate energy stabilization for the gauche(-) but not the gauche(+) side-chain conformers of (3R,4S)-5 and (3R,4R)-6, respectively. By symmetry rules, the same holds for (3S,4R)-5 and (3S,4S)-6, respectively. Thus, these amino acids are potential building blocks for the topographical design of peptides (Kazmierski et al., J. Am. Chem. Soc. 1991, 113, 2275-2283) by providing acylated 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives in which a gauche(-) and not a gauche(+) side-chain conformation is energetically more stable for the L amino acid. Synthetic details and implications of these new amino acids for peptide and protein design are discussed. © 1994 American Chemical Society.
• Knapp, R. J., Malatynska, E., Fang, L., Xiaoping, L. i., Babin, E., Nguyen, M., Santoro, G., Varga, E. V., Hruby, V. J., Roeske, W. R., & Yamamura, H. I. (1994). Identification of a human delta opioid receptor: Cloning and expression. Life Sciences, 54(25), PL463-PL469.
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  PMID: 8201839;Abstract: The δ opioid receptor is an important target for analgesic drug development. This report describes the identification of δ opioid receptor clones from human cDNA libraries and the preparation of a human δ receptor cDNA in the pcDNA3 expression vector for transfection studies. The cDNA encodes a 372 amino acid protein that has 93% amino acid identity to mouse and rat δ receptors. COS-7 cells transfected with this clone express over 1.0 pmole receptor/mg protein when measured by saturation binding with [3H]naltrindole. The δ receptor selective ligands NTB, BNTX, [4′-Cl-Phe4]DPDPE and [D-Ala2, Glu4]deltorphin all have Ki values under 10 nM while the affinities of the μ and κ opioid receptor ligands CTAP and U-69593, respectively, are over 4.0 μM. Agonists show binding to multiple affinity states of the receptor consistent with the presence of G-protein coupled and uncoupled forms of the expressed receptor. The 8-fold higher affinity of NTB relative to BNTX suggests that the human δ receptor is of the δ2 subtype. © 1994.
• Knapp, R. J., Malatynska, E., Hashimoto, S., Fang, S., Hunt, M., Wamsley, J. K., Peterson, P., Zalewska, T., Hruby, V. J., & Yamamura, H. I. (1994). [³H]SNF 8702 autoradiography of CCK-B receptors in guinea pig brain and studies with a cloned rat CCK-B receptor. Annals of the New York Academy of Sciences, 713, 380-383.
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  PMID: 8185194;
• Knapp, R. J., Malatynska, E., Peterson, P., Zalewska, T., Fang, S., Hruby, V. J., Smith, T. L., & Yamamura, H. I. (1994). [N-methylnorleucine-(28,31)]cholecystokinin-(26-33) (SNF 8702) activity at a cloned rat CCK_B receptor. European Journal of Pharmacology: Molecular Pharmacology, 269(2), 133-138.
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  PMID: 7851490;Abstract: [N-methyl-Nle 28,31)]cholecystokinin-(26-33) (SNF 8702) is a highly ligand for the CCKB type of receptor present in the vertebrate central nervous system. Radioligand binding data showing that SNF 8702 binding affinity is reduced by the GTP analog guanylyl-imidodiphosphate suggest that SNF 8702 is an agonist but the ability of SNF 8702 to activate CCKB receptors has not been demonstrated. The present study shows that SNF 8702 is a highly potent agonist at rat CCKB receptors expressed on COS-7 cells and that these receptors are coupled to the mobilization of intracellular calcium. The A50 measured for SNF 8702-induced calcium mobilization (66 pM) is over 6-fold less than that of cholecytstokinin octapeptide (420 pM). Data are also presented showing that SNF 8702 has high binding affinity for these receptors with a Kd value (760 pM) consistent with previous measurements using guinea pig brain tissue preparations. © 1994.
• Kover, K. E., Jiao, D., Uhrin, D., Forgo, P., & Hruby, V. J. (1994). One-Dimensional z-Filtered Relay Experiments for Measurement of Homo- and Heteronuclear Coupling Constants. Journal of Magnetic Resonance, Series A, 106(1), 119-122.
• Kövér, K. E., Jiao, D., Fang, S., & Hruby, V. J. (1994). Conformational properties of the unnatural amino acid β-methylphenylalanine in a linear octapeptide system; correlations of ¹³C-NMR chemical shifts with the side-chain stereochemistry of these amino acid residues. Journal of Organic Chemistry, 59(5), 991-998.
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  Abstract: Conformational properties of the four stereoisomers ([2S,3S], [2S,3R], [2R,3S], and [2R,3R]) of a synthetic amino acid, β-methylphenylanaline (β-MePhe), in a bioactive octapeptide sequence of CCK, H-Asp1-Tyr2-β-MePhe3-Gly 4-Trp5-Nle6-Asp7-Phe 8-NH2, have been studied by using 1H and 13C-2D NMR spectroscopy. β-MePhe3 residues introduce significant perturbations to the side-chain conformations. On the basis of the rotamer populations determined by a combination of homonuclear and heteronuclear vicinal coupling constants, each of the four different stereoisomers of β-MePhe residues virtually eliminates one of the three staggered side-chain conformations, trans for (2S,3S)-and (2R,3R)-β-MePhe, gauche(+) for (2S,3R)-β-MePhe, and gauche(-) for (2R,3S)-β-MePhe, respectively. It also was revealed that the side-chain rotamer populations of the Tyr2 residue are influenced by different configurations of the β-carbon in the adjacent β-MePhe3 residues. An empirical correlation between the 13C chemical shifts of the β-CH3 and the stereochemistry of β-methylphenylalanine side chains has been established, i.e., the δC of the β-MePhe in (2S,3S)- and (2R,3R)-isomers is at lower field by ca. 3 ppm relative to those in (2S,3R)- and (2R,3S)-isomers. This correlation can be rationalized on the basis of the γ-substituent effect in 13C-NMR chemical shift, and it may become a useful probe for side-chain conformations of similar molecules. Furthermore, these β-methylphenylalanine amino acids will provide useful side-chain conformational constraints in peptide and mimetic design. © 1994 American Chemical Society.
• Lan, E. -., Ugwu, S. O., Blanchard, J., Fang, X., Hruby, V. J., & Sharma, S. (1994). Preformulation studies with melanotan-II: A potential skin cancer chemopreventive peptide. Journal of Pharmaceutical Sciences, 83(8), 1081-1084.
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  PMID: 7983590;Abstract: Melanotan-II (1) is a cyclic heptapeptide analogue of α-melanocyte- stimulating hormone (α-MSH) which tans the skin and is currently being evaluated for the prevention of sunlight-induced skin cancers. The dissociation constants of 1 were determined using potentiometric titration and ultraviolet spectrophotometry. The pK(a1) (histidine) and pK(a2) (arginine) were estimated to be 6.54 and 11.72, respectively. The apparent partition coefficient (PC) was measured at three pH values using both n- octanol and isooctane as the nonpolar phase. The PC(octanol) and Δlog PC at pH 7.35 were 2.82 and 1.05, respectively. These data, together with the observance of a bioavailability of 4.6% in the rat, indicate that 1 may be a suitable candidate for oral delivery. The data presented here are useful in developing an appropriate dosage form for 1.
• Matsunaga, T. O., Collins, N., Ramaswami, V., Yamamura, S. H., O'Brien, D., & Hruby, V. J. (1994). Erratum: Comparison of the membrane-bound states of two structurally similar δ-selective opioid peptides by transferred nuclear overhauser effect spectroscopy and molecular modeling (Biochemistry (December 7, 1993) 32:48 (13180-13189)). Biochemistry, 33(17), 5356-.
• McBride, R. B., Beckwith, B. E., Swenson, R. R., Sawyer, T. K., Hadley, M. E., Matsunaga, T. O., & Hruby, V. J. (1994). The actions of melanin-concentrating hormone (MCH) on passive avoidance in rats: A preliminary study. Peptides, 15(4), 757-759.
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  PMID: 7937355;Abstract: Melanin-concentrating hormone (MCH) is a hepadecapeptide hormone that is synthesized in the CNS and is responsible for melanosome aggregation in the teleost fish. Recent evidence suggests that this peptide hormone has a unique distribution in the mammalian brain, which leads to the speculation that it may serve as a neuromodulator. The present study was undertaken to explore the comparative effects of MCH to those of α-melanocyte-stimulating Hormone (MSH) (a neuropeptide that is known to influence learning) on the rate of extinction of a passive avoidance response in rats. Both MCH and MSH were administered SC at 10 μg per animal. Treatment with MCH appeared to hasten, whereas treatment with MSH appeared to delay, extinction of the passive avoidance response. © 1994.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Delta opioid receptor selective ligands; DPLPE-deltorphin chimeric peptide analogues. International Journal of Peptide and Protein Research, 44(1), 80-84.
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  PMID: 7718035;Abstract: Further efforts to correlate the topography of the bioactive structures of DPDPE and the deltorphins, two δ-opioid receptor active peptide families, are reported. A number of DPLPE-deltorphin chimeric peptides have been synthesized in which the C-terminal dipeptide δ-address of the deltorphins (-Val-GlyNH2, -Nle-GlyNH2) have been linked to the highly δ-opioid selective cyclic peptides DPDPE or DPLPE. These studies demonstrate that a major structural feature determining high potency of hybrid analogues is the chirality of the amino acid residue in position 5. The radioligand binding assays have revealed a decrease in potency (compared to DPDPE) at δ- receptors when the C-terminal dipeptides were added to DPDPE. On the other hand, chimeric peptides of DPLPE with these same C-terminal dipeptides retained high δ-selectivity and affinity. Similar results were obtained using the mouse vas deferens (MVD) and guinea pig ileum (GPI), bioassays. The importance of the hydrophilicity of amino acids in positions 2 and 5 for δ- selectivity is consistent with the previous finding for DPLPE and DPDPE. On the other hand, the replacement of phenylalanine-4 with p- chlorophenylalanine-4 did not increase δ-selectivity as in DPDPE. These findings suggest that the δ-receptor interacts with hybridized enkephalins and deltorphins somewhat differently than with DPDPE.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Delta opioid receptor selective ligands; DPLPE-deltorphin chimeric peptide analogues.. International Journal of Peptide and Protein Research, 44(6), 607-.
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  PMID: 7705984;
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Erratum: Delta opioid receptor selective ligands; DPLPE-deltorphin chimeric peptide analogues (International Journal of Peptide and Protein Research Vol. 44 (1994) (80-84)). International Journal of Peptide and Protein Research, 44(6), 607-.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Structure-activity relationships of analogues of higly potent opioid peptide, biphalin. Regulatory Peptides, 53(SUPPL. 1), S131-S132.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Topographic model of delta opioid selective ligands; DPLPE-deltorphin hybrid analogues. Regulatory Peptides, 53(SUPPL. 1), S129-S130.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Yamamura, H. I., Porreca, F., & Hruby, V. J. (1994). Design of cyclic deltorphins and dermenkephalins with a disulfide bridge leads to analogues with high selectivity for δ-opioid receptors. Journal of Medicinal Chemistry®, 37(1), 141-145.
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  PMID: 8289187;Abstract: We earlier suggested that the low receptor selectivity observed for previously synthesized constrained analogues of deltorphin I (DT I) was the result of a reduction in the lipophilic surface of the C-terminal of the peptide. To confirm this prediction and to further test a previously proposed conformational model for bioactivity at delta opioid receptors, we have synthesized several new cyclic analogues with the general structure [D-Xaa2,Yaa5] deltorphin I and II in which Xaa2 is D-cysteine or D-penicillamine (D-Pen), and Yaa5 is an L- or D-penicillamine residue. Additional substitutions at positions 4, 6, and 7 also were examined. The analogues were tested for binding to μ- and δ-opioid receptors and in mouse vas deferens and guinea pig ileum biological assays. The introduction of a lipophilic L-Pen in position 5 and D-Cys or D-Pen in position 2 resulted in a highly δ-selective series of analogues, which fully confirmed our prediction. The cyclic analogues [D-Pen2,Pen5]DT I and [D-Pen2,Pen5,Nle6]DT I are among the most δ-selective analogues described thus far. © 1994 American Chemical Society.
• Polt, R., Porreca, F., Szabò, L. Z., Bilsky, E. J., Davis, P., Abbruscato, T. J., Davis, T. P., Horvath, R., Yamamura, H. I., & Hruby, V. J. (1994). Glycopeptide enkephalin analogues produce analgesia in mice: Evidence for penetration of the blood-brain barrier. Proceedings of the National Academy of Sciences of the United States of America, 91(15), 7114-7118.
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  PMID: 8041755;PMCID: PMC44349;Abstract: Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl β-D-glucoside analogues of [Met5]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted μ- and δ-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.
• Qian, X., Kövér, K. E., Shenderovich, M. D., Lou, B., Misicka, A., Zalewska, T., Horváth, R., Davis, P., Bilsky, E. J., Porreca, F., Yamamura, H. I., & Hruby, V. J. (1994). Newly discovered stereochemical requirements in the side-chain conformation of δ opioid agonists for recognizing opioid δ receptors. Journal of Medicinal Chemistry, 37(12), 1746-1757.
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  PMID: 8021915;Abstract: Topographic design of peptide ligands using specialized topographically constrained amino acids can provide new insights into the stereochemical requirements for δ opioid receptors. A highly constrained tyrosine derivative, (2S,3S)-β-methyl-2′,6′-dimethyltyrosine [(25,3S)-TMT], was prepared by asymmetric synthesis and incorporated in [D-Pen2,D-Pen5]enkephalin (δ1) and Deltorphin I (δ2). The results of binding assays and bioassays showed that the two analogues (3 and 4) acted very differently at δ opioid receptors. Further pharmacological evaluations suggested that they actually interact primarily with the δ1 and δ2 receptor subtypes, respectively. These results, and conformational studies using NMR and computer-assisted modeling, provided insights into the different stereochemical requirements for these two δ opioid ligands to recognize the δ opioid receptor and its subtypes. © 1994 American Chemical Society.
• Rao, R. K., Levenson, S., Fang, S. -., Hruby, V. J., Yamamura, H. I., & Porreca, F. (1994). Characterization of SNF 9007, a novel cholecystokinin/opioid ligand in mouse ileum in vitro: Evidence for involvement of cholecystokinin(A) and cholecystokinin(B) receptors in regulation of ion transport. Journal of Pharmacology and Experimental Therapeutics, 268(2), 1003-1009.
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  PMID: 8113956;Abstract: The effects of cholecystokinin (CCK) fragments and Asp-Tyr-D-Phe-Gly-Trp- [N-Me]Nle-Asp-Phe-NH2 1(SNF 9007), a synthetic CCK analog which binds with high affinity to CCK(B) and opioid delta receptors, were evaluated in isolated sheets of mouse ileum mounted in Ussing flux chambers. Serosal, but not mucosal, administration of cholecystokinin octapeptide-sulfated [CCK8(s)] and cholecystokinin tetrapeptide (30-33) [CCK4(30-33)] produced a brief, concentration-related increase in short circuit current (I(sc)) without changing tissue conductance. Serosal, but not mucosal, SNF 9007 produced a similar concentration-related increase in I(sc) which was followed by an immediate concentration-related and sustained decrease in I(sc); no decrease in I(sc) was observed for either CCK8 or CCK4(30-33). The increase and subsequent decrease in the SNF 9007 I(sc) response were respectively classified as phase I (i.e., CCK-like) and phase II (opioid-like) activity. CCK8(s) and SNF 9007 (phase I) were active at low nanomolar concentrations, whereas CCK4(30-33) was active only at high nanomolar concentrations: the rank order of potencies to increase I(sc) was CCK8(s) > SNF 9007 > CCK4(30- 33). Devazepide (L364,718), a selective antagonist of CCK(A) receptors, effectively blocked the action of CCK8(s), but not that of CCK4(30-33) or SNF 9007 (phase I). In contrast, 3R[+]-N-[2,3-dihydro-1-methyl-2-oxo-5- phenyl-1H-benzodiazepin-3-yl]-N'-[3-methyl-phenyl]urea (L365,260), a selective CCK(B) receptor antagonist, blocked the action of CCK4(30-33) and SNF 9007 (phase I), and also antagonized CCK(B)(s), though to a lesser degree. The phase II response of SNF 9007 was antagonized by N,N-diallyl- Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), a selective opioid delta receptor antagonist; this opioid antagonist did not influence the phase I response. Neither L364,718 or L365,260 influenced the SNF 9007 phase II response. Serosal pretreatment of tissues with tetrodotoxin, or the ganglionic blocker chlorisondamine, significantly blocked the actions of CCK8(s) and CCK4(30- 33), and both phase I and phase II responses to SNF 9007. Further, these peptides produced no significant response in mucosal preparations of ileum physically stripped of the enteric ganglia and muscularis externa. The data suggest that ileal ion-transport can be modulated by the activation of neural CCK(A) or CCK(B) receptors which are located partly preganglionically and that these receptors can be selectively activated by derivatives or analogs of CCK. CCK8(s) appears to produce its effects predominately, but not exclusively, at the CCK(A) receptor, whereas SNF 9007 and CCK4(30-33) selectively activate CCK(B) receptors in mouse ileum; SNF 9007 (phase I) is several-fold more potent than CCK4(30-33) in influencing ion transport at the CCK(B) receptor. Finally, SNF 9007 has the unusual profile of acting at opioid delta receptors to produce a subsequent decrease in I(sc). These data demonstrate the importance of both CCK(A) and CCK(B), as well as opioid delta, receptors in the regulation of ion transport in the same intestinal segment.
• Rao, R. K., Levenson, S., Fang, S. -., Hruby, V. J., Yamamura, H. I., & Porreca, F. (1994). Role of substance P in the regulation of ion transport by CCK(A) and CCK(B) receptors in mouse ileum. Annals of the New York Academy of Sciences, 713, 420-421.
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  PMID: 7514373;
• Roerig, S. C., Williams, C. L., Hruby, V. J., Burks, T. F., & Rosenfeld, G. C. (1994). Cholecystokinin (CCK) analog SNF9007-induced inhibition of adenylyl cyclase activity in NG108-16 hybrid cells and SH-SY5Y cells shows differential activation of d opioid receptors. Regulatory Peptides, 54(1), 247-248.
• Sharma, S. D., Granberry, M. E., Jiang, J., P., S., Hadley, M. E., & Hruby, V. J. (1994). Multivalent melanotropic peptide and fluorescent macromolecular conjugates: New reagents for characterization of melanotropin receptors. Bioconjugate Chemistry, 5(6), 591-601.
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  PMID: 7873662;Abstract: Radioreceptor binding studies have documented the presence of melanotropin receptors on some but not all of the various human melanoma cell lines that have been studied. Using a newly developed class of multivalent fluorescent melanotropin-macromolecular conjugates, we have demonstrated for the first time the presence of specific melanotropin receptors on all of the melanoma cell lines, both mouse and human, melanotic as well as amelanotic, that were investigated. The conjugates developed by us consisted of multiple copies of both a potent melanotropin analogue and a fluorophore, both arranged in a pendent fashion on a biologically inert macromolecule. While the multivalency of these conjugates may have established stronger binding with the melanotropin receptors on the cell surface (perhaps by establishing simultaneous multiple interactions), the presence of multiple copies of the fluorophore also greatly increased the level of detection in fluorescence labeling experiments. Membrane receptor-hormone-associated phenomena, such as capping and internalization of the receptor-ligand complex, also were observed. The details of these methods are described using B-16 mouse melanoma cells as a model system. The demonstration of MSH receptors as a common marker for melanoma suggests that this methodology might be employed for early clinical detection and anatomical localization of melanoma. These results also offer the possibility that substitution of the fluorophore in these conjugates by a chemical agent of (chemo-)therapeutic relevance may provide a powerful tool for site specific (tumor) targeting and cytotoxicity. © 1994 American Chemical Society.
• Ugwu, S. O., Lan, E., Sharma, S., Hruby, V., & Blanchard, J. (1994). Kinetics of degradation of a cyclic lactam analog of α-melanotropin (MT-II) in aqueous solution. International Journal of Pharmaceutics, 102(1-3), 193-199.
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  Abstract: The kinetics of degradation of MT-II in aqueous buffered solution was studied in order to facilitate the formulation of a stable oral dosage form. A stability-indicating high-performance liquid Chromatographic (HPLC) assay was used to measure the concentrations of MT-II remaining at various time periods. The rate of degradation of MT-II was studied as a function of pH, phosphate buffer concentration, temperature and ionic strength. Results indicated that the degradation of MT-II followed apparent first-order kinetics. The pH-rate profile showed that MT-II was most stable at approximately pH 5.0. Data obtained from this study also indicated that the degradation rate of this peptide was directly proportional to phosphate buffer concentration and temperature. The shelf-life of MT-II in aqueous buffer solutions at 25°C was 27 h. The activation energy was 7.5 kcal/mol. The degradation rate of MT-II appeared to be independent of the ionic strength of the aqueous buffered solution. © 1994.
• Vanderah, T., Takemori, A. E., Sultana, M., Portoghese, P. S., Mosberg, H. I., Hruby, V. J., Haaseth, R. C., Matsunaga, T. O., & Porreca, F. (1994). Interaction of [d-Pen², d-Pen⁵]enkephalin and [d-Ala², Glu⁴]deltorphin with δ-opioid receptor subtypes in vivo. European Journal of Pharmacology, 252(2), 133-137.
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  PMID: 8157053;Abstract: The interaction of [d-Pen2, d-Pen5]enkephalin (DPDPE) and [d-Ala2, Glu4]deltorphin with δ-opioid receptor subtypes was investigated. Pretreatment of mice with the δ1-opioid receptor antagonists, [d-Ala2, Leu5, Cys6]enkephalin (DALCE), produced a virtually complete antagonism of the antinociceptive actions of DPDPE, but had no effect on those of [d-Ala2, Glu4]deltorphin. In DALCE pretreated mice (i.e., δ1-opioid receptors blocked), DPDPE was able to significantly antagonize the antinociceptive effects of [d-Ala2, Glu4]deltorphin. Pretreatment of mice with the δ2-opioid receptor antagonist, na naltrindole-5′-isothiocynate (5′-NTII) produced a virtually complete antagonism of the antinociceptive effects of [d-Ala2, Glu4]deltorphin, but had no effect on the antinociception produced by DPDPE. In 5′-NTII pretreated mice (i.e., δ2-opioid receptors blocked), [d-Ala2, Glu4]deltorphin had no effect on the antinociception produced by DPDPE. These data suggest that [d-Ala2, Glu4]deltorphin is highly selective for the δ2-opioid receptor in vivo, and that neither agonist nor antagonist actions can be demonstrated at δ1-opioid receptors for this peptide. In contrast, under appropriate conditions, DPDPE can be shown to interact with both δ1- and δ2-opioid receptor subtypes; DPDPE may have limited efficacy (i.e., is a partial agonist) at the δ2-opioid receptor. © 1994.
• Williams, C. L., Rosenfeld, G. C., Dafny, N., Fang, S. -., Hruby, V. J., Bowden, G., Cullinan, C. A., & Burks, T. F. (1994). SNF9007: A novel analgesic that acts simultaneously at delta₁, delta₂ and mu opioid receptors. Journal of Pharmacology and Experimental Therapeutics, 269(2), 750-755.
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  PMID: 8182541;Abstract: Intracerebroventricular administration of the synthetic cholecystokinin analog SNF9007 (Asp-Tyr-D-Phe-Gly-Trp-[NMe]-Nle-Asp-Phe-NH2) produced antinociception in the mouse hot-plate and warm water tail-flick tests. The mechanisms of its analgesic actions were assessed by administering antagonists selective for CCK (cholecystokinin octapeptide, sulfated)-A and CCK-B receptors, as well as specific antagonists for the mu opioid receptor (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, 1 μg i.c.v.), the delta-1 opioid receptor [D-Ala2-Leu5, Cys6)enkephalin, 4.57 nmol i.c.v., 24 hr pretreatment), the delta-2 opioid receptor (naltrindole benzofuran, 25 pmol i.c.v.) and the kappa opioid receptor (nor-binaltorphimine, 10 mg/kg s.c.). The antinociceptive activity of SNF9007 was not a result of the activation of CCK receptors, as treatment with either CCK-A or CCK-B receptor antagonist was ineffective in blocking SNF9007 antinociception. Nor-binaltorphimine and naltrindole benzofuran were completely ineffective in blocking SNF9007 antinociception when administered alone or in combination. However, co- administration of delta-1 or delta-2 opioid receptor antagonists with the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 resulted in a dramatic reduction in analgesic response to SNF9007. Furthermore, the co-administration of mu + delta-1 + delta-2 opioid receptor antagonists resulted in an even greater inhibition of SNF9007 antinociception (>10-fold shift). We conclude that SNF9007 acts simultaneously at brain delta-1, delta- 2 and mu opioid receptors to induce antinociceptive effects in mice.
• Boteju, L. W., & Hruby, V. J. (1993). Tryptophan-containing 1,5-tetrazole dipeptide analogs: Synthesis of Trpψ[CN₄]Nle as a cis amide bond surrogate.. Tetrahedron Letters, 34(11), 1757-1760.
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  Abstract: The 1,5-disubstituted tetrazole dipeptide analog Z-Trp(NinZ)ψ[CN4]Nle-OBzl was synthesized in good yield and high optical purity using a modified procedure of Zabrocki et al. This dipeptide analog can be selectively deprotected at either the N or the C terminal for further incorporation into peptides. © 1993.
• Boteju, L. W., & Hruby, V. J. (1993). Typtophan-containing 1,5-tetrazole dipeptide analogs:synthesis of Trp0P[CN^p4]Nle as a cis amide bond surrogate. Tetrahedron Letters, 34(47), 7498-.
• Boteju, L. W., Zalewska, T., Yamamura, H. I., & Hruby, V. J. (1993). Tryptophan-norleucine 1,5-disubstituted tetrazoles as cis peptide bond mimics: Investigation of the bioactive conformation of a potent and selective peptide for the cholecystokinin-B receptor. Bioorganic and Medicinal Chemistry Letters, 3(10), 2011-2016.
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  Abstract: It has been suggested that the cis conformation about the TrpNMeNle amide bond is important in conferring high affinity and selectivity to CCK-B receptor ligands. Substitution of the cis amide bond mimic Trpψ(CH4)Nle into the peptide Gly-Trp-N-(Me)Nle-Asp-PheNH2 causes it to lose activity, suggesting that other structural effects of N-(Me)Nle incorporation may be important for high affinity selectivity for the CCK-B receptor. © 1993.
• Dharanipragada, R., Trivedi, D., Bannister, A., Siegel, M., Tourwe, D., Mollova, N., Schram, K., & Hruby, V. J. (1993). Synthetic linear and cyclic glucagon antagonists. International Journal of Peptide and Protein Research, 42(1), 68-77.
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  PMID: 8396562;Abstract: The synthesis and biological activities of seven new glucagon analogues are reported. The design of compounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8, related to a linear in vivo antagonist [Glu9]glucagon, reported by Merrifield (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4083-4087). The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (2); [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon amide (3); [3-(4-nitrobenzyl)His1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21] glucagon (4); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (5); [dHis1,Glu9]glucagon (6); (desHis1)[Glu9,Lys12]glucagon amide (7); (desHis1)[Glu9,Lys12,Asp15]glucagon amide (8). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding are 2.6 (2), 0.12 (3), 0.8 (4), 0.8 (5), 2.2 (6). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC50 values for 7 at the high and low affinity sites are 1.5 and 167 nM, respectively (IC50 of glucagon = 1.3 nM). The IC50 values for 8 at the high and low affinity sites are 4.7 and 3451 nM, respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2, 3 and 5-8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary α-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.
• Guigen, L. i., Jarosinski, M. A., & Hruby, V. J. (1993). Diastereospecific tandem Michael-like addition / electrophilic bromination: A one-pot tandem asymmetric synthesis of precursors of unusual amino acids. Tetrahedron Letters, 34(16), 2561-2564.
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  Abstract: A systematic series of key intermediates of unusual β-methyl-amino acids have been synthesized by using a modified Evans auxiliary in an asymmetric Michael-like reaction followed by direct bromination in a one-pot reaction. © 1993.
• Guigen, L. i., Pattel, D., & Hruby, V. J. (1993). Asymmetric synthesis of (2R, 3S) and (2S, 3R) precursors of β-methyl-histidine, -phenylalanine and -tyrosine. Tetrahedron: Asymmetry, 4(11), 2315-2318.
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  Abstract: A systematic series of (2S, 3R) and (2R, 3S) precursors to β-methyl-histidine, -phenylalanine and -tyrosine, which are of significant importance in the design of peptide and protein ligands, have been synthesized in high optically purity and yield. © 1993.
• Guigen, L. i., Russell, K. C., Jarosinski, M. A., & Hruby, V. J. (1993). Asymmetric synthesis of unusual amino acids : An enantioselective synthesis of the four isomers of D- and L-O-Methyl-2′, β-Dimethyltyrosine. Tetrahedron Letters, 34(16), 2565-2568.
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  Abstract: The four individual isomers of D- and L-O-methyl-2′, β-dimethyltyrosines have been synthesized in high optical purity. The evidence for asymmetric induction was cofirmed by the X-ray analysis of one of the key intermediates. © 1993.
• Guigm, L. i., Patel, D., & Hruby, V. J. (1993). An efficient procedure for the demethylation of aryl-methyl ethers in optically pure unusual amino acids. Tetrahedron Letters, 34(34), 5393-5396.
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  Abstract: An efficient procedure was developed for the removal of methyl groups from aryl methyl ethers, without racemization, in derivatives of unusual amino acids that are of significant importance in the design of highly selective peptide protein ligands with specicific conformational topographical features. Demethylation of aromatic amino acids can result in an appreciable increase in receptor affinity, hence the new mild procedure which have been developed represents a facile practical method for demethylation of Tyr (OMe) derivatives, including novel sidechain ring or C-3 modified analogs. © 1993.
• Hadley, M. E., Sharma, S. D., Hruby, V. J., Levine, N., & Dorr, R. T. (1993). Melanotropic peptides for therapeutic and cosmetic tanning of the skin. Annals of the New York Academy of Sciences, 680, 424-439.
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  PMID: 8390162;Abstract: Several authors have expressed the view that MSH may not play a role in the control of human skin pigmentation. For example, 'there is little evidence that it has any importance as a pigmentary hormone in man'. α-MSH alone may be of limited importance for the normal regulation of pigmentation in vivo. More recently, the same group stated that it seems unlikely that α-MSH is able to increase the synthesis of tyrosinase in human melanocytes. Finally, most recently it was stated that 'apparent major differences between regulation of human and rodent melanocytes have been identified with α-MSH playing a major role for the latter but apparently not the former. These negative views on a role of MSH in human skin pigmentation were based on in vitro studies using skin or isolated melanocytes of animals and man. As noted earlier, several investigators have clearly documented the pigmentogenic actions of melanotropins when administered to humans. In addition, the cardinal symptom of Addison's disease is hyperpigmentation of the skin due to the actions of one or more melanotropic peptides released in excess by the pituitary gland. As documented here, we have shown that melanotropic peptides are potent stimulators of melanogenesis when administered to humans. It is possible that a melanotropic peptide may become commercially available for therapeutic and cosmetic tanning of the skin. Depletion of the ozone layer may necessitate the use of such a peptide for protection of the skin from the damaging effects of increased UV radiation.
• Horan, P. J., Costa, B. D., Rice, K., Haaseth, R. C., Hruby, V. J., & Porreca, F. (1993). Differential antagonism of bremazocine- and U69,593-induced antinociception by quadazocine: Further functional evidence of opioid κ receptor multiplicity in the mouse. Journal of Pharmacology and Experimental Therapeutics, 266(2), 926-933.
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  PMID: 8394923;Abstract: In these studies, the antagonistic actions of (-)-1-Cyclopentyl-5- (1,2,3,4,5,-hexahydro-8-hydroxy-3,6,11-trimethyl-2,6-methyano-3-benzazocin- 11-yl)-3-pentanone methanesulfonate (quadazocine) were evaluated against the κ-receptor-mediated antinociceptive effects of i.c.v. (5α,7α,8β)-(+)-N- methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzeneacetamide (U69,593) or bremazocine in the mouse warm water tail-flick test. Quadazocine produced no antinociceptive effects alone, and it selectively antagonized the actions of bremazocine, but not U69,593, in a dose- and time-related fashion, supporting previous suggestions of differences in κ receptors mediating the antinociceptive effects of these agonists. Quadazocine, however, also antagonized the antinociceptive effects of both DAMGO (opioid μ agonist) and DPDPE (opioid δ agonist) at doses approximately 3-fold less than those needed to attenuate significantly the effects of bremazocine. The structurally diverse κ opioids (±)-trans-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzo[b]-thiopene-4-acetamide (PD 117,302), ethylketocyclazocine (EKC) and tifluadom were studied under κ-selective conditions, and the sensitivity of their effects to 1S,2S-trans-2-isothiocyanato-4,5-dichloro-N- [2(1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(-)-UPHIT] (κ1 antagonist) or quadazocine (κ2 antagonist) was determined. On this basis PD 117,302, EKC and tifluadom were classified as acting at opioid κ1, κ1, and κ2 receptors, respectively; EKC and tifluadom were also shown to have significant activity at opioid μ, but not δ, receptors. These data demonstrating two-way differential antagonism of U69,593 and bremazocine by quadazocine and (-)-UPHIT provide strong functional evidence of opioid κ receptor subtypes mediating supraspinal antinociception in the mouse. Additionally, the κ-subtype classification of κ agonists of different structures begins to provide a basis for structure-activity relationships of opioids acting at κ1 and κ2 receptors.
• Horan, P. J., Mattia, A., Bilsky, E. J., Weber, S., Davis, T. P., Yamamura, H. I., Malatynska, E., Appleyard, S. M., Slaninova, J., Misicka, A., Lipkowski, A. W., Hruby, V. J., & Porreca, F. (1993). Antinociceptive profile of biphalin, a dimeric enkephalin analog. Journal of Pharmacology and Experimental Therapeutics, 265(3), 1446-1454.
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  PMID: 8389867;Abstract: The dimeric enkephalin biphalin (Tyr-D-Ala-Gly-Phe-NH)2 was evaluated in mice using antinociceptive, gastrointestinal and physical dependence paradigms and compared with that of morphine (reference μ agonist) and etorphine (ultrapotent opioid agonist). Intracerebroventricular biphalin was 6.7- and 257-fold more potent than etorphine or morphine in eliciting antinociception. When administered i.t., biphalin produced only a 60% maximal antinociceptive effect in the tail-flick test even when given at doses up to 3 orders of magnitude higher than those effective i.c.v.; morphine was equipotent in this assay when given i.c.v. or i.t. Both morphine and biphalin were equipotent after i.p. administration. In spite of its antinociceptive effectiveness after i.p. administration, only a small fraction of [125I]biphalin was shown to penetrate to the brain (0.051 ± 0.011%, at 20 min). After i.c.v. administration, biphalin antinociception was antagonized by receptor selective doses of β-funaltrexamine (μ antagonist), naloxonazine (μ1 antagonist), ICI 174,864 (δ antagonist) and [D- Ala2,Cys4]deltorphin (δ2 antagonist), but not by [D- Ala2,Leu5,Cys6]enkephalin (δ1 antagonist) or nor-binaltorphimine (κ antagonist), whereas etorphine antinociception was significantly antagonized only by β-funaltrexamine and naloxonazine. Intracerebroventricular biphalin inhibited gastrointestinal propulsion at doses 8-fold higher than those producing i.c.v. antinociception; i.c.v. morphine showed a similar antinociceptive and gastrointestinal propulsion A50. Intraperitoneal biphalin, but not i.p. morphine, showed little, if any, physical dependence, but both biphalin and morphine produced significant physical dependence when equiantinociceptive doses were infused i.c.v. These results demonstrate an unusual profile for biphalin which suggests a potentially novel mechanism which may involve, in part, the putative opioid receptor complex of physically or functionally interacting μ and δ2 opioid receptors. Biphalin may thus represent the first in a series of such compounds which may lead to significant therapeutic advantages.
• Horan, P. J., Wild, K. D., Kazmierski, W. W., Ferguson, R., Hruby, V. J., Weber, S. J., Davis, T. P., Fang, L., Knapp, R. J., Yamamura, H. I., Kramer, T. H., Burks, T. F., Bowen, W. D., Takemori, A. E., & Porreca, F. (1993). Unexpected antinociceptive potency of cyclic [D-Tca¹]CTAP: potential for a novel mechanism of action. European Journal of Pharmacology, 233(1), 53-62.
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  PMID: 8386089;Abstract: This study tested the hypothesis that compounds which may bind simultaneously to δ and μ receptors may be more potent antinociceptive agents than would be predicted from their bindings affinities at individual μ and δ opioid receptors. D-Tca-Cys- Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 ([D-Tca1]CTAP) where D-Ta is a cyclic D-trytophan analogue) was synthesized and evaluated in radioligand competition assays, opioid bioassays, and in an antinociceptive assay (the tail-flick test in mice). Additionally, the metabolic stability of [D-Tca1]CTAP was evaluated in striatal and cerebellar tissue slices. In the rat brain in vitro, [D-Tca1]CTAP competed weakly for sites labelled by [3H]D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH2 ([3H]CTOP) (μ-ligand), and [3H][D-Pen2, pCl-Phe5]enkephalin (δ-ligand); [D-Pen2, D-Pen5]enkephalin (DPDPE) (δ-agonist) was 6.5-fold less and 230-fold more potent, respectively, against these ligands. Additionally, in mouse isolated vas deferens and guinea pig isolated ileum smooth muscle preparations, [D-Tca1]CTAP proved to be weak as either a δ(IC50 of approximately 2 μM) or μ (IC50 > 8 μM) receptor agonist. Surprisingly, however, i.c.v. [D-Tca1]CTAP produced antinociception with potency no similar to DPDPE. The antinociceptive actions of [D-Tca1]CTAP were apparently not due to a metabolite or the release of endogeneous opiods, as this compound proved stable in both striatal and cerebellar tissue slices and its antinociceptive actions were not enhance by the 'enkephalinase' inhibitor thiorphan. The suggestion that [D-Tca1]CTAP might be acting by binding simultaneously go μ and δ receptors to produced its antinociceptive effect is supported by the demonstrated antagonism resulting from μ receptor blockade with either β-funaltrexamine (β-FNA) or naloxonazine, or by δ receptor blockade by ICI 174,864 ([N,N-dially-Tyr1, Aib2,3,Leu5] enkephalin). Furthermore, the antinociceptive properties of [D-Tca1]CTAP were antagonized by (naltrindole-5′-isothiocyanate) (5′-NTII), an antagonist at the δ2 opioid receptor subtype, but not by the δ1 antagonist [D-Ala2, D-Leu5, Cys6]enkephalin (DALCE). Additionally, no antagonism was produced by nor-binaltorphimine (nor-BNI), a κ antagonist. From these data, [D-Tca1]CTAP appears to bind to μ, and 5′-NTII-sensitive δ2, opioid receptors, and may represent the first of a class of compounds which may act at an opioid recptor complex via 'self-potentiation'. © 1993.
• Horan, P. J., Wild, K. D., Misicka, A., Lipkowski, A., Haaseth, R. C., Hruby, V. J., Weber, S. J., Davis, T. P., Yamamura, H. I., & Porreca, F. (1993). Agonist and antagonist profiles of [D-Ala², Glu⁴]deltorphin and its [Cys⁴]- and [Ser⁴]-substituted derivatives: Further evidence of opioid delta receptor multiplicity. Journal of Pharmacology and Experimental Therapeutics, 265(2), 896-902.
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  PMID: 8388462;Abstract: Pharmacological evidence has suggested the presence of two supraspinal opioid delta receptor subtypes in the mouse, termed delta-1 and delta-2. [D- Pen2, D-Pen5]enkephalin (DPDPE) is thought to be primarily an agonist at the opioid delta-1 subtype, whereas H2N-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 ([D-Ala2, Glu4] deltorphin) is a selective agonist at the delta-2 subtype. Based on previous reports suggesting that a receptor sulfhydryl group may be critical for ligand binding to the opioid delta receptor, the present investigation has attempted to discover whether this concept extends to the opioid delta-2 receptor. For this purpose, a cysteine-substituted deltorphin was synthesized and the potential agonist and antagonist properties of this compound, H2N-Tyr-D-Ala-Phe-Cys-Val-Val-Gly-NH2 ([D-Ala2, Cys4]deltorphin), were evaluated in an antinociceptive assay after i.c.v. administration to mice and stability in mouse brain was determined. As a control, a serine-substituted deltorphin was also prepared and the potential agonist and antagonist properties of this compound, H2N-Tyr-D-Ala-Phe-Ser- Val-Val-Gly-NH2 ([D-Ala2, Ser4]deltorphin, as well as those of the parent deltorphin, [D-Ala2, Glu4]deltorphin, were evaluated. Acutely, [D-Ala2, Cys4]deltorphin, [D-Ala2, Ser4]deltorphin and [D-Ala2, Glu4]deltorphin each produced dose-related antinociceptive effects. Pretreatment with a single i.c.v. injection of [D-Ala2, Cys4]deltorphin, [D-Ala2, Ser4]deltorphin or [D-Ala2, Glu4]deltorphin blocked the antinociceptive effects of [D-Ala2, Glu4]deltorphin (delta-2 agonist) for up to 24 hr, but not the antinociceptive effects of DPDPE (delta-1 agonist). Pretreatment with a single dose [D-Ser2, Leu5, Thr6]enkephalin (DSLET) (delta-2 agonist) failed to affect [D-Ala2, Glu4]deltorphin-mediated antinociception, suggesting that the antagonistic actions of the deltorphin derivatives were not due to the production of acute tolerance. From these data, we postulate that an intact thiol group is apparently not critical for the binding of these ligands at the opioid delta-2 receptor.
• Hristova-Kazmierski, M. K., Horan, P., Davis, P., Yamamura, H. I., Kramer, T., Horvath, R., Kazmierski, W. M., Porreca, F., & Hruby, V. J. (1993). A new approach to enhance bioavailability of biologically active peptides: conjugation of a δ opioid agonist to β-cyclodextrin. Bioorganic and Medicinal Chemistry Letters, 3(5), 831-834.
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  Abstract: The cyclic δ opioid against [p-I-Phe4]DPDPE, 1, was conjugated to mono-6-amino-permethyl-β-cyclodextrin at the C-terminus to improve the bioavailability of 1. In the rat brain building assay, the conjugate 8 showed an IC50 = 134 nM vs. a δ ligand and IC50 > 10 μM at the μ receptor, making it less potent and selective than 1. However, 8 shows antinociceptive properties (i.v.) in the mouse tail flick test and prolonged activity. © 1993.
• Hruby, V. J. (1993). Comparison of the membrane-bound states of two structurally similar δ-selective opioid peptides by transferred nuclear overhauser effect spectroscopy and molecular modeling. Biochemistry®, 32(48), 13180-13189.
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  PMID: 8241172;Abstract: NMR spectroscopic, peptide-membrane conformational studies on [ D-Pen2, D-Pen5] -enkephalin (DPDPE), an opioid receptor selective peptide, and an acyclic analog of DPDPE (DPDPE reduced at the disulfide bond) were conducted. The N MR method of transferred nuclear Overhauser effect (TRNOE) was used to obtain NOE profiles of the free and membrane bound forms of DPDPE and acyclic DPDPE. After comparison of the profiles of both peptides in the free and membrane-bound states, we hypothesize that the cyclic DPDPE undergoes little if any conformational change upon interaction with the membrane. However, for the acyclic analog, large changes in the NOE profile associated with backbone and side-chain groups were observed after interaction with the membrane. Results of computerized molecular modeling studies also were consistent with our theory that the free and membrane-bound forms of cyclic DPDPE have very similar free and membrane-bound states. The free acyclic DPDPE has a reverse turn conformation with sidechains situated so that hydrophobic surface exposure to aqueous solution is minimized. After membrane interaction, the acyclic DPDPE has an extended conformation near the carboxy terminus with aromatic sidechains widely separated. We propose that the interaction of the acyclic DPDPE with the membrane surface is mediated by the amino terminus. We further propose that the interaction of the cyclic DPDPE with the membrane surface is limited because the D-Pen2 side chain is covalently bonded and the aromatic side chains and backbone are only slightly altered after membrane contact. Permeability studies by Ramaswami et al. [(1992) Biochim. Biophys. Acta 1109(2), 195-202] demonstrated that the acyclic DPDPE permeated through membranes at a rate 4 times greater than cyclic DPDPE. We conclude that conformational and topographical flexibility may be critical factors in peptide-membrane interactions and permeability of bilayer membranes to opioid peptides. © 1993 American Chemical Society.
• Hruby, V. J. (1993). Conformational and topographical considerations in the design of biologically active peptides. Biopolymers, 33(7), 1073-1082.
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  PMID: 8102072;Abstract: An outline of the basic considerations that are under development for the rational design of biologically active peptides and peptidomimetics is given. The necessary interplay of biophysical, chemical, and biological considerations is emphasized. The importance of properly designed biological assays to provide chemical information analogous to that from biophysical studies is discussed. The development of asymmetric synthesis in conjunction with conformational considerations for the preparation of specialized amino acids and amino acid mimetics is a critical aspect of the approach. The overall approach is illustrated with three examples from our laboratory: (1) the redesign of somatostatin to a highly potent and selective μ-opioid receptor antagonist using conformational and topographical considerations in design and for obtaining insights into the pharmacophor; (2) the use of topographical considerations for obtaining oxytocin antagonists; and (3) the application of designer amino acids prepared by asymmetric synthesis to obtain insight into the topographical requirements at δ-opioid receptors.
• Hruby, V. J., Boteju, L., & Guigen, L. i. (1993). Explosion with sodium azide [1]. Chemical and Engineering News, 71(41), 2-.
• Hruby, V. J., Gysin, B., Trivedi, D., & Johnson, D. G. (1993). New glucagon analogues with conformational restrictions and altered amphiphilicity: Effects on binding, adenylate cyclase and glycogenolytic activities. Life Sciences, 52(10), 845-855.
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  PMID: 8445980;Abstract: In an effort to obtain highly potent glucagon antagonists, we have investigated glucagon (1) structure-function relationships utilizing the following design principles: (1) structural changes known to lead to partial agonist activities; (2) conformational restrictions; (3) changes in the conformational probabilities of the primary sequence; and (4) increased amphiphilicity. In this report we present the total synthesis, purification, receptor binding, adenylate cyclase activity, in vivo glycogenolytic activity and CD spectrum of the following four glucagon analogues: [Ahx17,18]glucagon (2), [D-Phe4,Tyr5, 3,5-diiodo- Tyr10,Arg12,Lys17,18,Glu21]glucagon (3), [Asp9,Lys17,18,Glu21]glucagon 4, and [Glu15,Lys17,18]glucagon 5. Compound 2 binds exclusively to the high affinity receptor and compound 3 was a highly potent antagonist with respect to adenylate cyclase activity. Analog 4 showed distinct biphasic binding (IC50 5.6 nM and 630 nM), with only the low affinity binding leading to adenylate cyclase activity. Furthermore in analogue 5 receptor binding and adenylate cyclase activity were dissociated by a factor of 5. The results are consistent with a multistep binding mechanism in which glucagon interacts first nonspecifically with the anisotropic interphase of the cell membrane, followed by a conformational transition which occurs in the sequences 10-14 and 15-18 when the membrane bound peptide binds to its receptor. © 1993.
• Hruby, V. J., Lam, K. S., Lebl, M., Kazmierski, W., Hersh, E. M., & Salmon, S. E. (1993). Preparation of large peptide libraries with one peptide per bead and their use for the discovery of peptides that bind to acceptors.. NIDA research monograph, 134, 75-83.
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  PMID: 8289888;
• Hruby, V. J., Sharma, S. D., Toth, K., Jaw, J. Y., Al-Obeidi, F., Sawyer, T. K., & Hadley, M. E. (1993). Design, synthesis, and conformation of superpotent and prolonged acting melanotropins. Annals of the New York Academy of Sciences, 680, 51-63.
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  PMID: 8390180;
• Ito, A. S., M., A., Hruby, V. J., Hadley, M. E., Krajcarski, D. T., & Szabo, A. G. (1993). Structure-activity correlations of melanotropin peptides in model lipids by tryptophan fluorescence studies. Biochemistry, 32(45), 12264-12272.
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  PMID: 8218305;Abstract: Steady-state and time-resolved fluorescence spectroscopy were employed in the study of the structure and interactions of α-MSH (α-melanocyte-stimulating hormone) and its analogs, [Nle4,D-Phe7]-α-MSH (MSH-I) and Ac-[Nle4,Asp5,D-Phe7,Lys 10]-α-MSH(4-10)-NH2 (MSH-II). In aqueous buffer, the fluorescence parameters of the single tryptophan of α-MSH and MSH-I were similar and did not allow any distinction between these molecules. On the other hand, the tryptophan fluorescence of MSH-II was notably different, reflecting its cyclic lactam turn structure. In the presence of acidic lipid vesicles, the fluorescence properties of the peptides were different, indicating structural changes on incorporation of the peptide into the liquid-crystalline phase of the lipid. No evidence of interaction was observed in the presence of the neutral lipid dimyristoylphosphatidylcholine (DMPC). The association constants for lipid-peptide interactions were compared for binding isotherms which either neglected or accounted for electrostatic effects through Gouy-Chapman potential functions. The relative order of association constants in either treatment was MSH-II > MSH-I > α-MSH. These results parallel the reported biological activities that show increased potencies and prolongation of response for the analogs, MSH-II and MSH-I, as compared to the native hormone, α-MSH. Time-resolved fluorescence results showed that the fluorescence decay of melanotropins is best described by triple-exponential kinetics. In the lipid-peptide complex, there was a change in the relative concentrations of the components, with the intermediate-lifetime component predominating compared to those in solution. The results are consistent with a model where the peptides are initially attracted electrostatically to the vesicle surface and are then incorporated into the lipid phase, due to hydrophobic forces with accompanying conformational changes, to form a compact reversed turn structure.
• Jiao, D., Barfield, M., & Hruby, V. J. (1993). Ab initio IGLO study of the φ- and ψ-angle dependence of the ¹³C chemical shifts in the model peptide N-acetyl-N′-methylglycinamide. Journal of the American Chemical Society, 115(23), 10883-10887.
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  Abstract: The ab initio IGLO (individual gauge for localized orbitals) method was used to examine the conformational dependencies of the isotropic 13C chemical shifts in the model peptide N-acetyl-N′-methylglycinamide. A surface plot of the calculated 13C isotropic chemical shifts for the Ca carbon was constructed at 30° grid intervals of the φ and ψ angles. These data are used to examine the relationship between chemical shifts and protein secondary structure. The Ca carbons in α-helix and β-sheet conformations are calculated to be shifted 2.3 ppm downfield and 2.9 ppm to high field, respectively, of the random coil value. Considering the spread in experimental values, especially for the β-sheet conformations, these secondary shifts are in reasonable agreement with the average experimental values of 3.2 and -1.2 ppm, respectively, for glycyl residues in peptides and proteins. The smaller differences predicted for other types of secondary structures are also consistent with the experimental results. Thus, for the Cα carbon it is not necessary to include interresidue hydrogen-bonding effects to explain the major chemical shift trends. An analysis of the localized MO contributions (LMOC) shows that all four bonds directly connected to the Ca carbon are important to the total shift but each of these has a different (φ, ψ) angle dependence. The LMOC from the Cα-C′ bond provides the largest contribution to the chemical shift difference between the α-helix and the β-sheet conformations.
• Jiao, D., Russell, K. C., & Hruby, V. J. (1993). Erratum: Locally constrained tyrosine analogues with restricted side chain dynamics (Tetrahedron, 1993, 49, 3511-3520). Tetrahedron, 49(22), 4759-.
• Jiao, D., Russell, K. C., & Hruby, V. J. (1993). Locally constrained tyrosine analogues with restricted side chain dynamics. Tetrahedron, 49(17), 3511-3520.
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  Abstract: While investigating synthetic locally constrained analogues of amino acids, we have found that a series of tyrosine analogues, such as the 2′,6′-dimethyl-β-methyl-tyrosines, exhibit restricted rotations about their CβCγ bonds which can be detected in the 1H-NMR spectra of these compounds. The dynamic properties of these tyrosine analogues and their synthetic intermediates are described in this paper. The potential use of these tyrosine analogues in the study of the roles of side chain dynamics played in peptide-receptor interactions and protein functions also is discussed. © 1993.
• Kawasaki, A. M., Knapp, R. J., Kramer, T. H., Walton, A., Wire, W. S., Hashimoto, S., Yamamura, H. I., Porreca, F., Burks, T. F., & Hruby, V. J. (1993). Design and synthesis of highly potent and selective cyclic dynorphin A analogs. 2. New analogs. Journal of Medicinal Chemistry, 36(6), 750-757.
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  PMID: 8096246;Abstract: We have designed and synthesized several cyclic disulfide-containing peptide analogs of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogs exhibit unexpected apparent selectivities for the κ and μ opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogs has resulted in the κ/μ opioid receptor ligands [L-Pen5,Cys11]Dyn A1-11-NH2 (4), [Cys5,Cys10]Dyn A1-11-NH2 (5), [Cys5,Cys9]Dyn A1-11-NH2(6), and [Cys4,Cys9,Arg10] Dyn A1-11-NH2 (7). All of these analogs possess high κ and μ opioid receptor affinities for the central receptor (guinea pig brain), but effect only weak potency at peripheral κ and μ opioid receptors (GPI). In fact cyclic dynorphin A analog 4 shows >19 000-fold differences between central κ opioid affinity and potency in the guinea pig ileum (GPI). Additionally analog 4 is not an antagonist in the GPI, suggesting possible receptor differences between these sites. Substitution of Tyr1 by Phe1 in the cyclic 1-11 series gave the analog [Phe1,Cys5,Cys11]Dyn A1-11-NH2 (1) that was surprisingly potent in the guinea pig brain binding assay (IC50 = 15.1 nM) at the κ receptor, but was inactive in the GPI and mouse vas deferens bioassays. D-Ala2 and Tic4 analogs of 1 had lower affinity at brain κ receptors and had very weak potencies in the GPI and MVD bioassays. On the other hand, [Cys6,Cys10]Dyn A1-11-NH2 (8), [Cys8,D-Cys13]Dyn A1-13-NH2 (9), [D-Cys8,D-Cys12]Dyn A1-13-NH2(10), and [D-Pro10,Cys5,Cys13]-Dyn A1-13-NH2 (11) were surprisingly potent in the GPI bioassay, though considerable apparent selectivity for central receptors is still retained. The apparent lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays, particularly with 1 and 4, may suggest the existence of different subtypes of the κ and μ opioid receptors in the brain and peripheral systems. © 1993 American Chemical Society.
• Kawasaki, A. M., Knapp, R. J., Walton, A., Wire, W. S., Zalewska, T., Yamamura, H. I., Porreca, F., Burks, T. F., & Hruby, V. J. (1993). Syntheses, opioid binding affinities, and potencies of dynorphin A analogues substituted in positions 1, 6, 7, 8 and 10. International Journal of Peptide and Protein Research, 42(5), 411-419.
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  PMID: 7906258;Abstract: Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A(1-11)-NH2 analogues at opioid receptors were explored by substitution of Tyr1, Arg6, Arg7, Ile8 and Pro10 with other amino acid residues. Interestingly, substitution of Tyr1 with N(α)-Ac-Tyr1, D-Tyr1, Phe1 or p-BrPhe1 led to analogues that were quite potent and κ opioid receptors, and additional substitution of Ile8 with D-Ala8 and/or Pro10 with D-Pro10 retained high potency in brain binding assay: [N(α)-Ac-Tyr1]-(1), [D-Tyr1]-(2) [Phe1]-(3), [Phe1,D-Ala8]-(5), [p-BrPhe1, D-ala8]-(6), [Phe1, D-Pro10]-(7) and [Phe1,D-Ala8, D-Pro10]-Dyn A(1-11)-NH2 (8) had IC50 (nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe1 analogue 4, however was only weakly active (610 nM). All of the analogues except 4 were modestly selective for k vs. μ guinea pig brain opioid receptor (11-88-fold) and quite selective for k vs. δ receptors (65-576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as β-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reducedd potency and biological activity were obtained (compounds 12-14). It had previously been suggested that the Arg6 and Arg7 residues were critical for biological activity. However, when we replace either one of these residues, [Nle6]Dyn A(1-11) (9) and [Nle7]Dyn A(1-11)-NH2 (10) were both highly potent binders in κ receptor binding studies (IC50 = 0.95 and 0.43 nM, respectively), and interestingly also were potent in μ and δ binding studies. Furthermore, both of the analogues were modestly potent in the GPI and MVD assays (94, 65 nM; 31, 81 nM, respectively). These results demonstrate that basic residues at positions 6 and 7 in dynorphin are not very important for binding to κ opioid receptors. Finally, many of the compounds reported here showed high selectivity for central vs. peripheral κ opioid receptors, with compound 4 being the most selective (63,000-fold).
• Kover, K. E., Prakash, O., & Hruby, V. J. (1993). z-Filtered Heteronuclear Coupled-HSQC-TOCSY Experiment as a Means for Measuring Long-Range Heteronuclear Coupling Constants. Journal of Magnetic Resonance, Series A, 103(1), 92-96.
• Kramer, T. H., Davis, P., Hruby, V. J., Burks, T. F., & Porreca, F. (1993). In vitro potency, affinity and agonist efficacy of highly selective delta opioid receptor ligands. Journal of Pharmacology and Experimental Therapeutics, 266(2), 577-584.
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  PMID: 8394911;Abstract: The purpose of these investigations was to estimate the relative potency, receptor affinity, and agonist efficacy of several selective delta opioid agonist peptides of diverse structure, including cyclic [D-Pen2,D- Pen5]enkephalin and its p-Phe4 halogen-substituted analogs, [D-Ser2-O- tBu,Leu5,Thr6]enkephalin. Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II) in functional bioassays. The mouse-isolated vas deferens (MVD) and guinea pig-isolated ileum longitudinal muscle/myenteric plexus bioassay preparations were used; selectivity for delta opioid receptors was quantified by the relative agonist activity of the various peptides in the guinea pig-isolated ileum and MVD assays; agonist affinity and efficacy were determined using the technique of partial irreversible receptor inactivation in the MVD. Data from these experiments were analyzed both by the traditional null method and by use of the operational model of pharmacologic agonism; a comparison of these two methods, which were found to be similar, was performed. Potency determinations in MVD for the various peptides essentially matched those determined in other investigations; the relative affinity of the peptides correlated with the results of radioligand binding studies performed in other laboratories. The relative efficacies of the peptides studied were indistinguishable except for the peptide deltorphin I, which demonstrated efficacy several-fold lower than the remaining seven. All peptides were sufficiently efficacious to appear as full agonists under control conditions. These results suggest that the principle factor determining increases in potency of novel delta receptor ligands to date is an increase in receptor affinity. Efficacy should be incorporated as a drug design consideration for delta opioid agonists.
• Lam, K. S., Hruby, V. J., Lebl, M., Knapp, R. J., Kazmierski, W. M., Hersh, E. M., & Salmon, S. E. (1993). The chemical synthesis of large random peptide libraries and their use for the discovery of ligands for macromolecular acceptors. Bioorganic and Medicinal Chemistry Letters, 3(3), 419-424.
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  Abstract: A method is outlined for preparing large, diverse peptide libraries (105-107 peptides) such that there is one peptide per bead. These libraries can be used to screen for binding to macromolecular receptor (acceptor) molecules and to determine the structure of the peptides that bind. © 1993.
• Lebl, M., Patek, M., Kocis, P., Krchnak, V., Hruby, V. J., Salmon, S. E., & Lam, K. S. (1993). Multiple release of equimolar amounts of peptides from a polymeric carrier using orthogonal linkage-cleavage chemistry. International Journal of Peptide and Protein Research, 41(2), 201-203.
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  PMID: 8458693;Abstract: Peptides generated on polymeric beads that are attached to the support via cleavable linkers are released into solution in equimolar amounts in several independent steps and screening of the 'peptide libraries' can be performed in the usual manner used for testing peptides in solution.
• Lippens, G., Hallenga, K., Belle, D. V., Wodak, S. J., Nirmala, N. R., Hill, P., Russell, K. C., Smith, D. D., & Hruby, V. J. (1993). Transfer nuclear overhauser effect study of the conformation of oxytocin bound to bovine neurophysin I. Biochemistry®, 32(36), 9423-9434.
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  PMID: 8369312;Abstract: This study reports the structure of the peptide hormone oxytocin bound to its carrier protein, neurophysin I, obtained by nuclear magnetic resonance techniques. At the pH value of 2.1 in our experiments, the ligand is in fast exchange with its carrier protein, allowing the use of transfer-NOE methods. The number of distance constraints for the peptide being limited, considerable attention has been paid to an accurate distance determination. The resulting accurate distance limits were used as input for a distance geometry calculation followed by a restrained molecular dynamics run. Convergence to a well-defined family of structures for oxytocin in its bound state was reached. Both the backbone and the side-chain conformations differ between the bound form and the crystal structure of free oxytocin [Wood, S. P., et al. (1986) Science 232, 633]. These differences, as well as other structural features of the bound form, are discussed in terms of interactions made with the carrier protein. Transfer-NOE experiments at low peptide protein ratios provide direct experimental evidence for contacts between the oxytocin Tyr2 residue and an aromatic residue of neurophysin. The resonance assignments of the aromatic groups [Whittaker, B. A., et al. (1985) Biochemistry 24, 2782] together with the recently published X-ray structure of the neurophysin II protein complexed with a dipeptide [Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4240] allow us to assign the aromatic signal on the protein to the neurophysin Phe22 residue. © 1993 American Chemical Society.
• Miller, C. L., Hruby, V. J., Matsunaga, T. O., & Bickford, P. C. (1993). A-MSH and MCH are functional antagonists in a central nervous system auditory gating paradigm. Annals of the New York Academy of Sciences, 680, 571-574.
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  PMID: 8390195;
• Miller, C. L., Hruby, V. J., Matsunaga, T. O., & Bickford, P. C. (1993). Alpha-MSH and MCH are functional antagonists in a CNS auditory gating paradigm. Peptides, 14(3), 431-440.
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  PMID: 8392716;Abstract: The peptides alpha-melanocyte stimulating hormone (alpha-MSH) and melanin concentrating hormone (MCH; rat and salmon sequence) were administered to anesthetized rats by intracerebroventricular infusion. Depth recordings were carried out in the dorsal hippocampus, and auditory gating was assessed. Auditory gating in this paradigm refers to the decrease in amplitude of the second of two tone-evoked CNS potentials that can be measured when pairs of identical tones are presented 500 ms apart. Alpha-MSH increases auditory gating, whereas MCH has the opposite effect. When MCH was administered prior to alpha-MSH, the ability of alpha-MSH to increase auditory gating was blocked. Thus, the two peptides appear to be functional antagonists. © 1993.
• Mollova, N. N., Schram, K. H., Lin, Y., Dharanipragada, R., & Hruby, V. J. (1993). Characterization of linear and cyclic glucagon analogs by fast atom bombardment mass spectrometry. Biological Mass Spectrometry, 22(5), 267-276.
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  PMID: 8507672;Abstract: Fast atom bombardment mass spectral mapping of endoproteinase Asp-N digest mixtures is used for characterization of new synthetic linear and cyclic glucagon analogs. The results allow rapid identification of sequence modifications in linear glucagon analogs. For the cyclic compounds, the technique allows confirmation of the presence and position of the cyclic amide bond, as well as verification of the sequence of the modified glucagon analogs. The specificity of the Asp-N enables differentiation of isometric glucagon analogs which differ only in the position of the cyclic amide bond. Important information concerning the purity of the synthetic analogs is also available.
• Nicolás, E., Russell, K. C., & Hruby, V. J. (1993). Asymmetric 1,4-addition of organocuprates to chiral α,β-unsaturated N-acyl-4-phenyl-2-oxazolidinones: A new approach to the synthesis of chiral β-branched carboxylic acids. Journal of Organic Chemistry, 58(3), 766-770.
• Nicolás, E., Russell, K. C., Knollenberg, J., & Hruby, V. J. (1993). Efficient method for the total asymmetric synthesis of the isomers of β-methyltyrosine. Journal of Organic Chemistry, 58(26), 7565-7571.
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  Abstract: α-Amino acids modified at the β-carbon atom can provide topographical constraints when incorporated into a peptide. Such modifications can modulate the physical, chemical, and biological properties of the compound. In order to properly evaluate the effect of such modifications, large-scale asymmetric syntheses of the isomers are needed. A method for the stereoselective large-scale synthesis of all four stereoisomers of β-methyltyrosine is described in this paper. The stereochemistry of both the α- and β-stereocenters was set using 4-phenyl-2-oxazolidinone as a chiral auxiliary. The key reactions were an asymmetric Michael-like addition of an organocuprate to a chiral α,β-unsaturated acyloxazolidinone (β center) and subsequent stereoselective electrophuic bromination of the resulting product (α center). Conversion of the bromide to the azide, catalyzed hydrolysis to the azido acid with simultaneous recovery of the chiral auxiliary, reduction of the azide, and final deprotection of the phenol group afforded the desired amino acids. In general, the reactions were performed in yields over 80%, and the isomers were obtained in enantiomeric purities of 98:2 to 99:1. © 1993 American Chemical Society.
• Nikiforovich, G. V., & Hruby, V. J. (1993). Models for the A- and B-receptor-bound conformation of CCK-8. Biochemical and Biophysical Research Communications, 194(1), 9-16.
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  PMID: 8333874;Abstract: Energy calculations were performed for CCK-8 (Asp26-Tyr(SO3)27-Met28-Gly29-Trp30-Met31-Asp32-Phe33 -NH2, I) and [desaminoTyr(SO3)27, Nle28,31]CCK-7 (II), which are nonselective ligands of CCK receptors, and for the CCK-A selective analog [desaminoTyr(SO3)27, Nle28,31, N-Me-Asp32]CCK-7 (III) and the CCK-B selective analog [desaminoTyr(SO3)27, Nla28, N-Me-Leu31]CCK-7 (IV). The geometrical shapes of the obtained low energy backbone conformers were then compared with each other, searching for similar spatial arrangements of specific atomic centers. The comparisons were performed separately for peptides with high affinity towards CCK receptors of the A type (compounds I, II and III) and for peptides with high affinity towards CCK receptors of the B type (compounds I, II and IV). Possible models for CCK 'A'- and 'B'-receptor-bound conformations were then developed. The proposed CCK 'B-conformation' has a distorted β-III turn at the C-terminal Gly-Trp-Met-Asp fragment, the Phe33 residue and the C-terminal amide being directed outward from the turn. The CCK 'A-conformation' has two reversals of the peptide chain so that the C(α)-atoms of the C-terminal pentapeptide appear at the corners of a nearly regular pentagon, and a distinct β-II turn is centered at the N-terminal Tyr-Met-Gly-Trp fragment, the planes of the turn and the pentagon being almost perpendicular. The proposed models are consistent with the results of biological testing for CCK related peptides including cyclic analogs and CCK-A selective tetrapeptides.
• Nikiforovich, G. V., Prakash, O. M., Gehrig, C. A., & Hruby, V. J. (1993). Solution conformation of the peptide backbone for DPDPE and its β-MePhe⁴-substituted analogs. International Journal of Peptide and Protein Research, 41(4), 347-361.
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  PMID: 8496017;Abstract: The solution structures of DPDPE, a conformationally restricted pentapeptide with the sequence H-Tyr1-D-Pen2-Gly3-Phe4-D-Pen5-OH, and its four β-MePhe4-substituted analogs were examined by a combined approach including the NMR measurements in DMSO and water as well as independent energy calculations. It was concluded that several low energy conformers of DPDPE backbone satisfy the NMR data obtained in this study as well as in previous studies by other authors. These possible solution conformers of DPDPE in both DMSO and water share virtually the same type of cyclic backbone structure, with the Gly3 residue in a conformation close to a γ-turn, and the Phe4 residue in a conformation close to α-helical torsion angles. They differ in the space arrangements of the flexible Tyr1 moiety. The solution structures of the β-MePhe4-substituted analogs of DPDPE are interesting. For analogs with an S-configuration at the C(α) atom in the Phe4-residue, the cyclic backbone conformations resemble those of DPDPE itself, whereas for analogs with an R-configuration at the C(α) atom, the backbone conformation is somewhat different. This observation is in line with the high biological potencies and selectivities displayed by the former compounds but not by the latter ones. It was noted also that as far as the peptide backbone conformers are concerned, some of the possible DPDPE-conformers in water are similar to the previously suggested model for the δ-receptor-bound conformation of DPDPE, becoming virtually identical to this conformation by rotating the side chains of the Tyr1 and the Phe4 residues.
• Nikiforovich, G. V., Prakash, O., Gehrig, C. A., & Hruby, V. J. (1993). Conformations of the dermenkephalin backbone in DMSO solution by a new approach to the solution conformations of flexible peptides. Journal of the American Chemical Society, 115(9), 3399-3406.
• Nikiforovich, G. V., Prakash, O., Gehrig, C. A., & Hruby, V. J. (1993). Conformations of the dermenkephalin backbone in DMSO solution by a new approach to the solution conformations of flexible peptides. Journal of the American Chemical Society, 115(9), X-3402.
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  Abstract: Dermenkephalin (DRE, H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2), a natural peptide found in frog skin, has high potency and receptor selectivity for δ opioid receptors and has potent in vivo analgesic activity. Structurally the compound is related to both the μ opioid receptor selective dermorphin and the δ opioid receptor selective deltorphins. Interestingly, the N-terminal tetrapeptide of DRE is potent and selective for the μ opioid receptor. Efforts to understand the conformational properties of DRE and their relationships to biological activity are of great importance. We report here a novel approach to analyze conformations of short linear peptides in solution to determine the possible solution conformations of DRE. We have combined extensive NMR studies with comprehensive conformational energy calculations, including extensive Monte Carlo sampling and statistical evaluations of the results, to obtain the statistical weight estimations for DRE low-energy backbone conformations that are consistent with all of the NMR data. A random search of conformer statistical weights was performed to satisfy the condition of statistical indistinguishability between the experimental values and the weighted sum of calculated values for each measured parameter. From these studies, two low-energy conformers were found to be essential for matching the energy calculation results with the NMR data. At least one of them should be present among the DRE solution conformers with a significant statistical weight. Except for the rotamers of the side chain groups of the Tyr1 and Phe3 residues, the conformations of the N-terminal tripeptide fragment match in detail a previously suggested topographical model for the conformation responsible for interaction with the δ opioid receptor. This suggests that the δ selectivity of DRE, which is a linear flexible peptide, might be due to pre-existence in solution of a specific conformer for its N-terminal tripeptide. The combined approach employed in this study offers a useful methodology to aid in conformational analysis of linear, conformationally flexible peptides that are active at receptors and other biological important acceptors.
• Sawyer, T. K., Castrucci, A. M., Staples, D. J., Affholter, J. A., Vaux, A. D., Hruby, V. J., & Hadley, M. E. (1993). Structure-activity relationships of [Nle⁴, D-Phe⁷]α-MSH. Discovery of a tripeptidyl agonist exhibiting sustained bioactivity. Annals of the New York Academy of Sciences, 680, 597-599.
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  PMID: 8390200;
• Vanderah, T. W., Wild, K. D., Takemori, A. E., Sultana, M., Portoghese, P. S., Bowen, W. D., Hruby, V. J., Mosberg, H. I., & Porreca, F. (1993). Modulation of morphine antinociception by swim-stress in the mouse: Involvement of supraspinal opioid delta-2 receptors. Journal of Pharmacology and Experimental Therapeutics, 267(1), 449-455.
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  PMID: 8229774;Abstract: The present study evaluated the effect of a brief exposure of mice to cold-water swim-stress (CWSS) on the antinociceptive potency of i.c.v. given morphine. No significant antinociceptive response could be demonstrated in the warm-water tail-flick test, 10 min after a 30-sec exposure of mice to water at 5°C. However, the i.c.v. morphine dose-response curve in mice exposed to CWSS was displaced significantly to the left when compared to that obtained in control (i.e., non-CWSS-exposed) mice. Although coadministration of the delta antagonist, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH 1 (ICI 174,864), with i.c.v. morphine did not produce antagonism of the antinociceptive action of this mu opiate, the leftward displacement of the i.c.v. morphine dose- response curve seen in CWSS-exposed mice was blocked in ICI 174,864-treated mice suggesting involvement of opioid delta receptors in the modulatory effect. Pretreatment of mice with the delta-1 antagonist, [D-Ala2, Leu5, Cys6] enkephalin, did not antagonize the antinociception of morphine and further did not antagonize the leftward displacement produced by exposure to CWSS. Pretreatment of mice with the delta-2 antagonist, 5'-isothiocyanate, also did not antagonize the antinociceptive effects of morphine but blocked the leftward displacement in the morphine dose-response curve associated with CWSS, suggesting involvement of an opioid delta-2 receptor in this effect. Pretreatment of mice with the mu antagonist, β-funaltrexamine, produced a significant antagonism of the morphine antinociceptive effect as seen by a rightward displacement of the morphine dose-effect curve. Further, in these β-funaltrexamine-pretreated mice, exposure to CWSS did not result in an enhancement of morphine potency. Finally, i.c.v. pretreatment of mice with antibodies raised against [Leu5]enkephalin, but not antibodies against [Met5]enkephalin, blocked the modulatory action produced by exposure to CWSS; both antibodies to [Met5]enkephalin or [Leu5]enkephalin did not alter the morphine response directly. These data suggest that modulation of morphine potency by exposure to CWSS involves activation of an opioid delta- 2 receptor which may be functionally and/or physically associated with opioid mu receptors. Additionally, the modulatory action appears to be produced in the brain by release of a [Leu5]enkephalin-like molecule. Finally, these observations may provide, in part, a physiological basis for the observed increase in analgesic effectiveness of morphine in situations of stress.
• Weber, S. J., Abbruscato, T. J., Brownson, E. A., Lipkowski, A. W., Polt, R., Misicka, A., Haaseth, R. C., Bartosz, H., Hruby, V. J., & Davis, T. P. (1993). Assessment of an in vitro blood-brain barrier model using several [Met⁵]enkephalin opioid analogs. Journal of Pharmacology and Experimental Therapeutics, 266(3), 1649-1655.
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  PMID: 8371162;Abstract: Confluent monolayers of primary and continuous passaged cultures of bovine brain microvessel endothelial cells (BMEC) have been suggested to model the blood-brain barrier (BBB). Increased lipophilicity has been previously suggested to increase BBB penetration. The intent of this study was to examine the effect that structural modifications of the [Met5]enkephalin analog DPDPE had on lipophilicity and passage across the BMEC. The BMEC consisted of a monolayer of confluent primary BMEC grown on polycarbonate (10 μm) filters. Permeability coefficients were calculated on the basis of the diffusion of peptides across the BMEC in a Side-Bi-Side® diffusion chamber. Lipophilicity of the peptides examined was determined by using reversed- phase HPLC and calculating the capacity factor (k). Diffusion across the BMEC (for all peptides examined) was linear from 15 to 120 min; therefore, these time points were used to calculate permeability coefficients. Permeability coefficients ranged from 14.34 to 92.00 cm/min (x 10-4), with [ρ- CIPhE(4,4')]biphalin the highest. Analysis of variance coupled with the Newman-Keuls test showed significantly greater (P < .01) passage of select peptide analogs across the BMEC, including [ρ-CIPhe(4,4')]biphalin, [ρ- CIPHe4]DPDPE and reduced DPDPE. Interestingly, upon passage across the confluent monolayer, reduced DPDPE was converted to cyclized DPDPE. Calculated HPLC capacity factors ranged from 3.82 to 12.50. The most lipophilic peptide (highest) examined was acetylated Phe0-DPDPE. Analysis of the regression line of permeability coefficients plotted against capacity factors yielded a correlation coefficient of 0.745 (P < .01). The data provided in this study offer strong evidence that increasing peptide lipophilicity enhances passage across the BMEC. The greatest BMEC permeability coefficients, though not the greatest capacity factors, were obtained with peptides having a chlorohalogenation at the Phe4 residue, suggesting that factors other than lipophilicity may play a role in BMEC passage. Comparison of the permeability coefficients obtained from the BMEC system with those obtained from in vivo BBB studies suggest that the BMEC system may be very useful in predicting peptide (analog) passage across the in vivo BBB.
• Wild, K. D., Carlisi, V. J., Mosberg, H. I., Bowen, W. D., Portoghese, P. S., Sultana, M., Takemori, A. E., Hruby, V. J., & Porreca, F. (1993). Evidence for a single functional opioid delta receptor subtype in the mouse isolated vas deferens. Journal of Pharmacology and Experimental Therapeutics, 264(2), 831-838.
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  PMID: 8382281;Abstract: The identification of opioid δ receptor subtypes in mouse brain led to the investigation of the nature of the opioid δ receptors in the mouse isolated vas deferens in vitro. Noncumulative concentration-effect curves were constructed for DPDPE (δ1 agonist) and [D-Ala2, Glu4]deltorphin (δ2 agonist) in control tissues, or in tissues which had been incubated with either [D-Ala2, Leu5, Cys6] enkephalin (DALCE) (noncompetitive δ1 antagonist) or 5'-naltrindole isothiocyanate (5'-NTII) (noncompetitive δ2 antagonist). Incubation of the tissues with DALCE, under either oxygenated or nonoxygenated conditions, did not alter the concentration-effect curves for either agonist. In contrast, incubation of the tissues with 5'-NTII resulted in a significant rightward displacement of the concentration-effect curves of both DPDPE and [D-Ala2, Glu4]deltorphin. Additionally, naltriben, a selective and competitive δ2 antagonist, showed no significant difference in its ability to antagonize a fixed, submaximal concentration of either DPDPE or [D-Ala2, Glu4]deltorphin. Furthermore, there was no significant difference in the affinity of naloxone (i.e., pA2) at the receptor(s) acted upon by either DPDPE or [D-Ala2, Glu4]deltorphin. Tolerance to DPDPE or [D- Ala2, Glu4]deltorphin was produced by incubation of the tissues with these agonists; construction of the [D-Ala2, Glu4]deltorphin concentration- effect curve in DPDPE-tolerant tissues demonstrated cross-tolerance between these agonists and, conversely, construction of DPDPE concentration-effect curves in [D-Ala2, Glu4]deltorphin-tolerant tissues revealed cross- tolerance between these agonists. Thus, the present data provide support for one subtype of opioid δ receptor in the mouse isolated vas deferens based on 1) the lack of antagonism of the effects of both agonists selective for δ1 and δ2 receptor subtypes by DALCE, a δ1 antagonist, 2) the antagonism of δ1 and δ2 agonists by 5'-NTII or naltriben (δ2 antagonists), 3) the similar antagonist potency of NTB against either DPDPE or [D-Ala2, Glu4]deltorphin, 4) the lack of significant difference in the naloxone pA2 against either δ agonist and 5) the demonstration of two-way cross-tolerance between the effects of DPDPE and [D-Ala2, Glu4]deltorphin in this tissue.
• Wild, K. D., Horan, P. J., Misicka, A., Lipkowski, A., Haaseth, R. C., Matsunaga, T. O., Hruby, V. J., Toth, G., Borsodi, A., Yamamura, H. I., & Porreca, F. (1993). Pseudoirreversible binding characteristics of [D-Ala², Glu⁴]deltorphin and its Cys⁴ substituted derivative to δ-opioid receptors. European Journal of Pharmacology: Molecular Pharmacology, 246(1), 25-31.
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  PMID: 8394818;Abstract: Following the identification of [D-Ala2,Glu4]deltorphin as a selective δ2-opioid receptor agonist in vivo, we synthesized the Cys4-substituted analogue as a potential ligand which might bind 'irreversibly' at this site through a proposed thil-disulfide exchange mechanism. Previous studies showed that intracerebroventricular (i.c.v.) pretreatment with [D-Ala2,Cys4]deltorphin, 24 h prior to antinociceptive testing, produced a selective antagonism of [D-Ala2,Glu4]deltorphin-induced antinociception in mice. Surprisingly, however, the Ser4-analogue (synthesized as a control) and even the parent molecule, [D-Ala2,Glu4]deltorphin, had the same antagonistic effect following pretreatment in vivo, while pretreatment with an equiantinociceptive dose of [D-Ser2,Leu5,Thr6]-enkephalin, a structurally unrelated δ2-opioid receptor agonist did not exhibit long-lasting antinociceptive actions. These data raised questions regarding the mechanism of the antagonism observed in vivo with the deltorphins; the present studies have attempted to explore these issues using radioligand binding techniques. The results demonstrate a decrease in the Bmax of [tyrosyl-3′,5′-3H,D-Pen2,p-Cl-Phe4,D-Pen5]-enkephalin ([3H]p-Cl-DPDPE) (δ-opioid receptor ligand) following i.c.v. pretreatment of mice (at -24 h) with [D-Ala2,Cys4]deltorphin or [D-Ala2,Glu4]deltorphin, but not with [D-Ala2,Ser4] deltorphin, suggesting a difference in mechanism of antagonism seen in vivo with these compounds. Incubation of mouse whole brain homogenates in vitro with [D-Ala2,Cys4]deltorphin or with [D-Ala2,Glu4]deltorphin, also resulted in a decrease in the radioligand binding of [3H]p-Cl-DPDPE, but this effect was not prevented by coincubation with dithiothreitol, a thiol-reducing agent. Direct evaluation of binding using [3H][D-Ala2,Glu4]deltorphin (5 nM) showed that a portion of this ligand (i.e., about 10% of all specific binding) remained specifically and 'irreversibly' bound to mouse brain membranes following incubation in vitro and extensive washing. The 'irreversibly', specifically bound [3H][D-Ala2,Glu4]deltorphin could be removed, however, by brief exposure of the membranes to a low pH (2.5), high-salt (0.5 M NaCl) solution. These data suggest that [D-Ala2,Cys4] deltorphin and [D-Ala2,Glu4]deltorphin bind in a 'pseudoirreversible' (no-covalent) manner to an δ-opioid receptor via a mechanism that apparently does not involve thiol-disulfide exchange. © 1993.
• Al-Obeidi, F., Hruby, V. J., Yaghoubi, N., Marwan, M. M., & Hadley, M. E. (1992). Synthesis and biological activities of fatty acid conjugates of a cyclic lactam α-melanotropin. Journal of Medicinal Chemistry, 35(1), 118-123.
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  PMID: 1732518;Abstract: Four fatty acid conjugates of a cyclic lactam-bridged α-MSH fragment analogue were synthesized and their potencies and biological activities compared in several melanotropin bioassays. Palmitoyl, myristoyl, decanoyl, and hexanoyl conjugates of H-Asp-His-D-Phe-Arg-Trp-Lys-NH2 were prepared. In the in vitro mouse melanoma cell assay, each of the conjugates was 10-100 times more potent than α-MSH or the substrate peptide in elevating tyrosinase activity. The shorter conjugates of hexanoic and decanoic acid were as potent as α-MSH in the lizard skin bioassay, whereas the longer myristoyl and pahnitoyl analogues were about 100 times less potent. The potency of the myristoyl and palmitoyl conjugates increased with time in contact with the skins. These observations may be related to the more lipid-like nature of these peptide-fatty acid conjugates. Each of the conjugates exhibited prolonged melanotropic activity in the lizard skin bioassays and in the mouse 891 melanoma tyrosinase bioassay, since the biological response continued following removal of the conjugates from the incubation media. The prolonged residual melanotropic activity resulted from conjugation of the fatty acids to the MSH fragment analogue since the analogue itself did not exhibit prolonged activity. © 1992 American Chemical Society.
• Altszuler, N., Rosenberg, C. R., Winkler, B., Fuchs, A. R., Hill, P. S., & Hruby, V. J. (1992). The metabolic effects of oxytocin are mediated by a uterine type of receptor and are inhibited by oxytocin antagonist and by arginine vasopressin in the dog. Life Sciences, 50(10), 739-746.
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  PMID: 1310790;Abstract: Infusion of oxytocin (OT) into normal dogs, in doses which produced plasma levels of OT in the physiological range, has been shown to increase plasma levels of glucose, insulin and glucagon and increase rates of glucose production and uptake. This study sought to determine whether there was a correlation between these metabolic effects and the oxytocic potency of four less potent oxytocic analogues when infused into normal dogs. The rank order of oxytocic potency of all 4 correlated well with the rise in plasma glucose levels, and in 3 of the 4 with the rise in plasma insulin levels. An antagonist of the oxytocic effect of OT suppressed the usual OT-induced rise in plasma glucose, insulin and glucagon as well as the increased glucose production and uptake. Arginine vasopressin (AVP) infusion, which by itself did not produce any metabolic effects, blocked completely the effects of OT infusion to raise plasma glucose and insulin levels and increase glucose production and uptake. The data suggest that the metabolic effects of OT in the dog are mediated by OT receptors that are similar to those producing the oxytocic effects. Whether the inhibition by AVP of the metabolic and hormonal effects of OT occurs at the receptor or post receptor level or via other mechanisms remains to be determined. © 1992.
• Boteju, L. W., Wegner, K., & Hruby, V. J. (1992). Asymmetric synthesis of unusual amino acids: Synthesis of the optically pure isomers of indole-protected β-methyltryptophan suitable for peptide synthesis.. Tetrahedron Letters, 33(49), 7491-7494.
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  Abstract: The four isomers of N-indole-(2-mesitylenesulfonyl)-β-methyltryptophan have been synthesized in high optical purity using in part, asymmetric conjugate 1, 4-additions followed by chiral imide enolate azidation and reduction. © 1992.
• Búzás, B., Tóth, G., Cavagnero, S., Hruby, V. J., & Borsodi, A. (1992). Synthesis and binding characteristics of the highly delta-specific new tritiated opioid peptide, [³H] deltorphin II. Life Sciences, 50(14), PL75-PL78.
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  PMID: 1313131;Abstract: A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species. © 1992.
• Dharanipragada, R., Bruck, M., & Hruby, V. J. (1992). The absolute configuration of an intermediate in the asymmetric synthesis of unusual amino acids.. Acta Crystallographica, Section C: Crystal Structure Communications, 48, Pt 7/-.
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  PMID: 1418812;Abstract: (4R)-3-[(2'R,3'R)-2'-Bromo-3'-(phenylbutanoyl)]-4-(phenylmethyl)-2 - oxazolidinone, C20H20Br-NO3, M(r) = 402.30, monoclinic, P2(1), a = 11.542 (2), b = 7.625 (1), c = 11.667 (1) A, beta = 113.97 (1) degrees, V = 938.2 (2) A3, Z = 2, Dx = 1.42 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 21.8 cm-1, F(000) = 412, T = 296 +/- 1 K, final R = 0.028 for 2369 observed reflections. Since a D-chiral auxiliary was used the configuration at the alpha-carbon was R as expected. The two carbonyls are aligned in opposite directions to each other to overcome van der Waals repulsions.
• Dharanipragada, R., VanHulle, K., Bannister, A., Bear, S., Kennedy, L., & Hruby, V. J. (1992). Asymmetric synthesis of unusual amino acids: An efficient synthesis of optically pure isomers of β-methylphenylalanine.. Tetrahedron, 48(23), 4733-4748.
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  Abstract: Substitution of the diastereotopic β-hydrogens of many α-amino acids provides an approach to the three dimensional topographic control of peptide structure. Asymmetric synthesis of the desired amino acids is needed to facilitate these studies. All four individual isomers of β-methylphenylalanine, (2S,3S)-, (2R,3R)-, (2S,3R)- and (2R,3S)-β-methylphenylalanine have been synthesized in high optical purity. The stereochemistry at the β-center was set by the choice of starting material, either (+)- or (-)-3-phenylbutyric acid. These acids were attached to the appropriate D- or L-auxiliary (a 4-phenylmethyl-2-oxazolidinone) to give a 3′-phenylbutanoyl-4-phenylmethyl-2-oxazolidinone. Asymmetric bromination was accomplished via the ciral imide enolate bromination methodology of Evans and co-workers (J. Am. Chem. Soc. 1990 112, 4011-40). Evidence for asymmetric induction was obtained from the X-ray structure of one of the intermediate bromides. The bromide was converted to the diastereoisomeric azide by SN2 displacement using tetramethylguanidinium azide. After recovery of the chiral auxiliary by catalyzed hydrolysis, the chiral amino acid was obtained by catalytic hydrogenation over 10% Pd/C. All four isomers were obtained in enantiomeric purities of 95:5 to 99:1. © 1992.
• Fang, L., Knapp, R. J., Matsunaga, T., Weber, S. J., Davis, T., Hruby, V. J., & Yamamura, H. I. (1992). Synthesis of [d-ALA², 4′-¹²⁵I-PHE³, GLU⁴] deltorphin and characterization of its δ opioid receptor binding properties. Life Sciences, 51(20), PL189-PL193.
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  PMID: 1331636;Abstract: Both [d-Ala2, Glu4]Deltorphin and [d-Ala2, 4′-I-Phe3, Glu4]Deltorphin are highly selective ligands for δ, relative to μ, opioid receptors. Radiolabeled [d-Ala2, 4′-125I-Phe3, Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [d-Ala2, 4′-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for μ, δ, and κ opioid receptors is consistent with binding by the radioligand to a single site having the properties of a δ opioid receptor. The results of these studies are in good agreement with those obtained by structurally different δ opioid receptor ligands. The similarity between the δ receptor site labeled by [125I]Deltorphin and those labeled by other δ receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed. © 1992.
• Horan, P., Tallarida, R. J., Haaseth, R. C., Matsunaga, T. O., Hruby, V. J., & Porreca, F. (1992). Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: Supra- and sub-additivity. Life Sciences, 50(20), 1535-1541.
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  PMID: 1315897;Abstract: In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the δ agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of δ agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid μ and δ receptors and a potential regulatory role for the endogenous ligands of the opioid δ receptor. © 1992.
• Hruby, V. J. (1992). Strategies in the development of peptide antagonists. Progress in Brain Research, 92, 215-224.
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  PMID: 1363847;
• Jiao, D., Barfield, M., Combariza, J. E., & Hruby, V. J. (1992). Ab initio molecular orbital studies of the rotational barriers and the ³³S and ¹³C chemical shieldings for dimethyl disulfide. Journal of the American Chemical Society, 114(10), 3639-3643.
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  Abstract: A series of ab initio molecular orbital calculations were carried out for dimethyl disulfide as a model for the disulfide bridges in proteins and peptides. The potential energy profile for rotation around the S-S bond was obtained at the HF/6-31G* level with full geometry optimization. Cis- and trans-barrier heights were estimated to be 11.40 and 6.27 kcal/mol, respectively, on the basis of fourth-order Møller-Plesset perturbation theory and 6-311G** basis sets. Calculations of the torsion angle dependence of the isotropic 33S and 13C NMR shieldings were based on the method of individual gauge for localized orbitals (IGLO). These are of interest for NMR studies of the disulfide bond in peptides and proteins. The minimum in the plot of 13C shielding as a function of torsion angle occurs for a C1-S1-S2-C2 angle close to 110°, which is an optimum arrangement for lone pair back-bonding. An analysis of the paramagnetic bond contributions to the 13C shielding at C1, for example, shows that the conformational dependence is dominated by the paramagnetic contributions to the C1-H1 bond, which points away from a lone pair on S2.
• Kazmierski, W. M., Ferguson, R. D., Knapp, R. J., Lui, G. K., Yamamura, H. I., & Hruby, V. J. (1992). Reduced peptide bond cyclic somatostatin based opioid octapeptides. Synthesis, conformational properties and pharmacological characterization. International Journal of Peptide and Protein Research, 39(5), 401-414.
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  PMID: 1358847;Abstract: The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the μ opioid receptor selective antagonist D-Phe1-Cys2-Tyr3-D-Trp4-Orn5-Thr6-Pen7-Thr8-NH2 (IV), have been investigated. Peptide analogues that contain [CH2NH] and [CH2N] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinoline carboxylate (Tic) and of D-Trp4 with D-1,2,3,4-tetrahydro-β-carboline(D-Tca4), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for μ opioid receptors. Conformational analysis based on extensive NMR investigations demonstrated that the backbone conformations of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the μ opioid receptor. Peptide bond modifications were made including (Tyr3-ψ[CH2NH]-D-Trp4), 3; (Tyr3-ψ[CH2N]-D-Tca4), 2; and (Cys2-ψ[CH2N]-Tic3), 6. These analogues showed decreases in their μ opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 1H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between μ and δ opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility.
• Kövér, K. E., Prakash, O., & Hruby, V. J. (1992). Efficient purging sequence for ¹H-detected heteronuclear shift correlation experiments. Journal of Magnetic Resonance (1969), 99(2), 426-432.
• Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmeierski, W. M., & Knapp, R. J. (1992). Erratum: A new type of synthetic peptide library for identifying ligand-binding activity (Nature 354, 82-84 (1991)). Nature, 358(6385), 434-.
• Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmeierski, W. M., & Knapp, R. J. (1992). Erratum: A new type of synthetic peptide library for identifying ligand-binding activity (Nature 354, 82-84 (1991)). Nature, 360(6406), 768-.
• Lebl, M., Toth, G., Slaninova, J., & Hruby, V. J. (1992). Conformationally biased analogs of oxytocin. International Journal of Peptide and Protein Research, 40(2), 148-151.
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  PMID: 1446971;Abstract: Four diastereomeric analogs of oxytocin containing substituted phenylalanine in position 2 were synthesized. This modified phenylalanine side chain contained one methyl group attached to the β-carbon and the second one at the 2' position of the aromatic ring. All analogs were found to be inhibitors of uterotonic activity of oxytocin with pA2 values ranging from 6.0 to 8.3; the most potent one (pA2 = 8.3) contained dimethylphenylalanine of the D-erythro configuration.
• Maria, A., Visconti, M. A., Matsunaga, T. O., Hadley, M. E., & Hruby, V. J. (1992). Enzymological studies of melanin concentrating hormone (MCH) and related analogues. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 103(2), 317-320.
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  PMID: 1424563;Abstract: 1. 1. Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide possessing the following primary structure: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val. 2. 2. In the fish, Synbranchus marmoratus, skin bioassay MCH5-15 is equipotent to MCH whereas MCH5-14, which comprises only the ring structure, is about 100-fold less active. 3. 3. MCH and two fragment analogues, MCH5-15 and MCH5-14, were studied to determine their relative stability in the presence of fish serum and purified proteolytic enzymes, trypsin and alpha-chymotrypsin. 4. 4. After 4 hr incubation in fish serum, MCH5-15 retained 1/100, MCH5-14 1/1000 and MCH only 6/1000 of the potency of the native hormone. 5. 5. The three peptides were also very resistant to degradation by purified proteolytic enzymes involving the following relative order of resistance: MCH5-14 > MCH5-15 > MCH. MCH5-14 potency was not altered after a 1 hr incubation in either enzyme whereas MCH retained 1/10 and 4/100 of its original potency, and MCH5-15 retained 1/10 and 8/10 of its original potency, after 1 hr in trypsin and alpha-chymotrypsin, respectively. © 1992.
• Matsunaga, T. O., Hruby, V. J., Lebl, M., Maria, A., & Hadley, M. E. (1992). Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone. Life Sciences, 51(9), 679-685.
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  PMID: 1501512;Abstract: Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide. MCH stimulates perinuclear aggregation of melanosomes within integumental melanocytes of teleost fishes resulting in skin blanching. MCH contains a disulfide bridge forming a 10-residue ring (H-Asp-Thr-Met-Arg-Cyss-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH). It has been proposed that the ring is necessary for maintenance of potency. In order to test this proposal, we have synthesized two pseudo-isosteric analogues of MCH that cannot cyclize. They differed only in the polarity of the side chain group of positions 5 and 14. Serine was substituted for Cys5 and Cys14 in one peptide and L α-aminobutyrate (Abu) was the substitution at the two positions in the other peptide. Using a fish skin bioassay we determined that these analogues exhibit less than 1/10,000th the potency of the native hormone. These results suggest that the disulfide bridge is necessary to maintain the correct conformational and topographical features of the hormone for receptor binding and transmembrane signal transduction. © 1992.
• Misicka, A., Lipkowski, A. W., Horvath, R., Davis, P., Kramer, T. H., Yamamura, H. I., & Hruby, V. J. (1992). Topographical requirements for delta opioid ligands: Common structural features of dermenkephalin and deltorphin. Life Sciences, 51(13), 1025-1032.
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  PMID: 1326067;Abstract: We propose a common topographical model for the bioactive conformation of deltorphin and dermenkephalin at the delta opioid receptor. In this model a hydrophilic surface from the N- to C-termini is surrounded by lipophilic residues ("hot dog" structure). The important element that orients the N-terminal tyramine is the interaction of the N-terminal amino group, with the carboxyl group of Asp4 in deltorphin I and with Asp7 through His4 (as a triad) in dermenkephalin. The biological properties of synthetic analogues designed to test this model demonstrate that the hydrophilic amino acid residues of these peptides are interchangeable. In addition, incorporation of Aib residues that change the lipophilic topography of these molecule, strongly reduces affinity for the delta opioid receptor. © 1992.
• Misicka, A., Nikiforovich, G., Lipkowski, A. W., Horvath, R., Davis, P., Kramer, T. H., Yamamura, H. I., & Hruby, V. J. (1992). Topographical requirements for delta opioid ligands: The synthesis and biological properties of a cyclic analogue of deltorphin I. Bioorganic and Medicinal Chemistry Letters, 2(6), 547-552.
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  Abstract: A cyclic constrained analogue of deltorphin, {A figure is presented} has been proposed on the basis of an energetically favored model of a delta-selective conformation for deltorphin. The biological properties of this synthetic analogue demonstrate that incorporation of a disulfide bridge into Deltorphin I does not affect its high affinity for delta opioid receptors. The analogue shows low receptor selectivity as a result of a large increase in its affinity for mu receptors when compared to the parent deltorphin. © 1992.
• Polt, R., Szabó, L., Treiberg, J., Yushun, L. i., & Hruby, V. J. (1992). General methods for α- Or β-O-Ser/Thr glycosides and glycopeptides. Solid-phase synthesis of O-glycosyl cyclic enkephalin analogues. Journal of the American Chemical Society, 114(26), 10249-10258.
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  Abstract: O-Linked glycopeptides have been efficiently synthesized using the highly nucleophilic α-imino esters (O'Donnell's Schiff bases) derived from L-serine (3a-c), L-threonine (4a,b), and a dipeptide ester (5). General methodology has been developed which can provide β-glycosides of β-hydroxy-α-amino acid derivatives 6-16 in excellent yield (63-94%) and excellent selectivity (>20:1) using Hanessian's modification or Helferich's modification of the Koenigs-Knorr reaction. Likewise, selective α-glycosylation has been achieved using the in situ anomerization methodology of Lemieux (28, 30). The increased nucleophilicity of the serine/threonine hydroxyl has been shown to be due to intramolecular hydrogen bonding to the N=CPh2 moiety. Deprotection of the intermediate Schiff bases has been demonstrated, and the glycosides have been incorporated into fully deprotected O-linked glycopeptides in high yield using either solution-phase peptide methods or solid-phase Fmoc-based technology. The potent glycosylenkephalin analogue 38 has been prepared using the solid-phase methodology.
• Ramaswami, V., Haaseth, R. C., Matsunaga, T. O., Hruby, V. J., & O'Brien, D. (1992). Erratum: Opioid peptide interactions with lipid bilayer membranes (BBAMEM 75731) (Biochimica et Biophysica Acta, 1109 (1992) 195-202). Biochimica et Biophysica Acta - Biomembranes, 1111(1), 143-.
• Ramaswami, V., Haaseth, R. C., Matsunaga, T. O., Hruby, V. J., & O'Brien, D. F. (1992). Opioid peptide interactions with lipid bilayer membranes. BBA - Biomembranes, 1109(2), 195-202.
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  PMID: 1520696;Abstract: The interaction of the δ-opioid receptor selective peptides, cyclic [d-Pen2, d-Pen5]-enkephalin [DPDPE] and its acyclic analog, DPDPE(SH)2, with neutral phospholipid bilayer membranes was examined by permeability and calorimetry measurements. The permeabilities were accomplished by entrapping either peptide inside of unilamellar liposomes (composed of a mixture of a molar ratio 65:25:10 phosphatidylcholine/prosphatidylethanolamine/cholesterol) then monitoring the peptide efflux through the bilayer. The initial permeability of DFDPE (first 12 h) averaged over four experiments was (0.91 ± 0.47) · 10-12 cm s-1. In contrast the average permeability of the acyclic DPDPE(SH)2 was (4.26 ± 0.23) · 10-12 cm s-1. The effect of these peptides on the phase transition, Tm, of 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by high sensitivity differential scanning calorimetry. The Tm, the calorimetric enthalpy, and the van 't Hoff enthalpy of DPPC were not significantly altered by the presence of DPDPE, whereas the calorimetric data for DPPC with DPDPE(SH)2 showed a small, yet significant, increase (0.2°C) in the Tm with a 30% decrease in the cooperative unit. Both the permeability and calorimetry data reveal a stronger peptide-membrane interaction in the case of the more flexible acyclic peptide. © 1992.
• Schoffelmeer, A. N., Vries, T. D., Hogenboom, F., Hruby, V. J., Portoghese, P. S., & Mulder, A. H. (1992). Opioid receptor antagonists discriminate between presynaptic mu and delta receptors and the adenylate cyclase-coupled opioid receptor complex in the brain. Journal of Pharmacology and Experimental Therapeutics, 263(1), 20-24.
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  PMID: 1328606;Abstract: The present study addressed the question as to whether or not interacting mu and delta opioid receptors, which may constitute an opioid receptor complex-inhibitory coupled to adenylate cyclase in rat neostriatum, display different antagonistic properties than the classical (noncomplexed) mu and delta receptors. In concentrations that antagonized the presynaptic inhibitory effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) on [3H]norepinephrine release from rat neocortical slices, the cyclic somatostatin-related mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn- Thr-Pen-Thr-NH2 did not affect the inhibition of dopamine-sensitive adenylate cyclase caused by DAMGO in neostriatal slices. The delta opioid receptor antagonist naltrindole appeared to be about 200-fold more effective as an antagonist against inhibitory effect of [D-Ser2(O-tert- butyl),Leu5]enkephalyl-Thr6 on [14C]acetylcholine release from neostriatal slices than against the inhibitory effect of DAMGO on [3H]norepinephrine release from neocortical slices, in agreement with the involvement of presynaptic delta and mu receptors; respectively. However, regarding the inhibitory effect of DAMGO and [D-Ser2(O-tert- butyl),Leu5]enkephalyl-Thr6 on adenylate cyclase activity in neostriatal slices, naltrindole not only displayed a very low affinity but also only 10- fold delta-selectivity. In striking contrast to D-Phe-Cys-Tyr-D-TRP-ORN-THR- PEN-THR-NH2 and naltrindole, naloxone did not discriminate between the neurotransmitter release- and adenylate cyclase-inhibitory effects of DAMGO and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6. These data strongly suggest that interacting mu and delta receptors in rat neostriatum display pharmacological characteristics that are clearly different from (presynaptic) mu, delta and kappa receptors in the brain, and indicate that the putative opioid receptor complex may represent a pharmacologically unique type of opioid receptor.
• Sharma, S. D., Toth, G., & Hruby, V. J. (1992). Erratum: A simple general method for (radio)iodination of a phenylalanine residue in peptides: Preparation of [D-Pen²,4'-¹²⁵I-Phe⁴, D-Pen⁵] enkephalin, a peptide with extraordinary selectivity for δ-opioid receptors (Journal of Organic Chemistry, page 4981 (1992)). Journal of Organic Chemistry, 57(13), 3750-.
• Smith, D. D., Slaninova, J., & Hruby, V. J. (1992). Structure-activity studies of a novel bicyclic oxytocin antagonist. Journal of Medicinal Chemistry, 35(9), 1558-1563.
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  PMID: 1578481;Abstract: In this report, we describe structure-activity studies of the bicyclic oxytocin antagonist [Mpa1,cyclo(Glu4,Lys8)oxytocin. The monocylic analogue [dPen1,Glu4,Lys8)]oxytocin was a weak oxytocin antagonist with a pA2 value of 5.8 in the uterotonic assay. Bicyclization of this analogue yielded [dPen1,cyclo(Glu4,Lys8)]oxytocin, a potent antagonist of oxytocin in the uterotonic assay (pA2 8.74) with a potency 3 times greater than that of [Mpa1,cyclo(Glu4,Lys8)]oxytocin. [dPen1,cyclo(Glu4,Lys8]oxytocin also was a weak antagonist in the pressor assay with a pA2 of 6.3. To establish if the potent antagonistic effects of these bicyclic compounds was because of the lactam ring or merely the result of obtaining an optimal degree of lipophilicity of the side chains in positions 4 and 8, we synthesized a series of analogues containing neutral and/or charged groups on these side chains. Monocyclic derivatives of [Mpa1,Gln4,Lys(CHO)8]oxytocin were moderate to weak agonists of oxytocin all following classical structure-activity profiles of oxytocin. The monocyclic derivatives of [dPen1,Gln4,Lys(CHO)8]oxytocin were antagonists of oxytocin which was attributed to the dPen1 substitution. However, the potency of all of these latter derivatives was at least 1 order of magnitude less than [dPen1,cyclo(Glu4,Lys8)]oxytocin. These results suggest that the potent antagonistic properties of the bicyclic analogues [Mpa1,cyclo(Glu4,Lys8)]oxytocin and [dPen1,cyclo(Glu4,Lys8)]oxytocin can be attributed to the effect of the lactam bridge on the conformational flexibility and topographical properties of the analogues, rendering them more favorable for binding to the receptor in such a manner as to prevent transduction of a biological response. © 1992 American Chemical Society.
• Tam, J. P., & Hruby, V. J. (1992). Editorial. International Journal of Peptide and Protein Research, 40(3-4), 161-.
• Toth, G., Russell, K. C., Landis, G., Kramer, T. H., Fang, L., Knapp, R., Davis, P., Burks, T. F., Yamamura, H. I., & Hruby, V. J. (1992). Ring substituted and other conformationally constrained tyrosine analogues of [D-Pen²,D-Pen⁵]enkephalin with δ opioid receptor selectivity. Journal of Medicinal Chemistry, 35(13), 2384-2391.
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  PMID: 1320122;Abstract: The conformationally restricted, cyclic disulfide-containing δ opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (DPDPE) was modified by 2′ (CH3) and 3′ (I, OCH3, NO2, NH2) ring substitutions and by β-methyl conformationally constrained β-methyltyrosine derivatives in the 1 position. The potency and selectivity of these analogues were evaluated by bioassay in the mouse vas deference (MVD, δ receptor assay) and guinea pig ileum (GPI, μ receptor assay) assays and by radioreceptor binding assays in the rat brain using [3H]CTOP (μ ligand) and [3H][p-ClPhe4]DPDPE (δ ligand). The analogues showed highly variable potencies in the binding assays and in the bioassays. Aromatic ring substituents with positive Hammett constants had decreased potency, while substituents with negative Hammett constants has increased potency for the opioid receptor. The most potent and most selective compound based on the binding was [2′-MeTyr1]DPDPE (IC50 = 0.89 nM and selectivity ratio 1310 in the binding assays). The 6-hydroxy-2-aminotetralin-2-carboxylic acid-containing analogue, [Hat1]DPDPE, also was highly potent and selective in both assays, demonstrating that significant modifications of tyrosine in enkephalins are possible with maintenance of high potency and δ opioid receptor selectivity. Of the β-methyl-substituted Tyr1 analogues, [(2S,3R)-β-MeTyr1]DPDPE was the most potent and δ receptor selective. The results with substitution of β-MeTyr or Hat instead of Tyr also demonstrate that topographical modification in a conformationally restricted ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity. © 1992 American Chemical Society.
• Tourwé, D., Verschueren, K., Binst, G. V., Davis, P., Porreca, F., & Hruby, V. J. (1992). Dermorphin sequence with high δ-afinity by fixing the phe sidechain to trans at ξ₁. Bioorganic and Medicinal Chemistry Letters, 2(10), 1305-1308.
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  Abstract: The Phe sidechain in dermorphin was fixed into the trans conformation by linking the aromatic ring to the Gly nitrogen through a methylene bridge. The compound has high μ- and δ-opioid activities. © 1992.
• Valle, G., Kazmierski, W. M., Crisma, M., Bonora, G. M., Toniolo, C., & Hruby, V. J. (1992). Constrained phenylalanine analogues. Preferred conformation of the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue. International Journal of Peptide and Protein Research, 40(3-4), 222-232.
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  PMID: 1335996;Abstract: Three Tic-containing (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) model peptides were synthesized to assess the tendency of this constrained Phe analogue to fold into a β-bend and a helical structure, and to adopt a preferred side-chain disposition. The results of the solution conformational analysis, performed by using Fourier transform infrared absorption and 1H nuclear magnetic resonance, indicate that in chloroform the -Aib-D-Tic-Aib-, -(Aib)2-D-Tic-(Aib)2-, and -L-Pro-D-Tic- sequences fold into intramolecularly H-bonded forms to a great extent. An X-ray diffraction analysis on p-BrBz-(Aib)2-DL-Tic-(Aib)2-OMe monohydrate and p-BrBz-L-Pro-D-Tic-NHMe allows us to conclude that, while the pentapeptide methylester forms an incipient (distorted) 310-helix, the dipeptide methylamide adopts a type-II β-bend conformation. In both cases, the D-Tic side-chain conformation is D, gauche(-). The implications for the use of the Tic residue in designing conformationally restricted analogues of bioactive peptides are briefly discussed.
• Verschueren, K., Toth, G., Tourwe, D., Lebl, M., Binst, G. V., & Hruby, V. (1992). A facile synthesis of 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid, a conformationally constrained tyrosine analogue. Synthesis, 458-460.
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  Abstract: A rapid synthesis of 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid is given. Pictet-Spengler reaction on diiodo- or dibromo-substituted tyrosine (3-(3,5-dihalo-4-hydroxyphenyl)-2-aminopropanoic acid), followed by catalytic dehalogenation gives the desired compound in high optical purity.
• Weber, S. J., Greene, D. L., Hruby, V. J., Yamamura, H. I., Porreca, F., & Davis, T. P. (1992). Whole body and brain distribution of [³H]cyclic [D-Pen²,D-Pen⁵] enkephalin after intraperitoneal, intravenous, oral and subcutaneous administration. Journal of Pharmacology and Experimental Therapeutics, 263(3), 1308-1316.
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  PMID: 1469637;Abstract: The route of administration of a given drug can have a significant influence upon whole body distribution. The present study examined whole body distribution of the δ opioid receptor-selective peptide [3H]DPDPE in male CD1 mice after administration by several routes. Additionally, we describe regional brain distribution of [3H]DPDPE after i.v. administration with and without pretreatment with naloxone or the selective δ receptor antagonist naltrindole. Finally, characterization of the inherent enzymatic stability of DPDPE was also examined. Intravenous administration results in a significantly large amount of [3H]DPDPE in the small intestine and flush at 15 and 30 min postadministration, suggesting rapid biliary excretion. The highest level in the brain after i.v. administration occurred at 60 min (0.08%). After i.p. and s.c. administration, large amounts of [3H]DPDPE were found in the small intestine and flush, but not until 60 min postadministration, suggesting a slower rate of absorption from the site of administration. The i.p. and s.c. groups' brain levels peaked at 120 min (0.07 and 0.09%, respectively). The highest levels in the brain after p.o. administration were seen at 240 min (0.03%). Examination of regional brain distribution data showed no significant difference in the levels of [3H]DPDPE between brain regions at any time point studied. However, naloxone pretreatment resulted in significant reductions of [3H]DPDPE in all brain regions at 5 and 10 min. Naltrindole pretreatment resulted in significant reductions in the frontal cortex and striatum at 5 and/or 10 min postadministration, but had no effect on [3H]DPDPE levels in cerebellum, hippocampus or brain stem. The incubation of DPDPE and its noncyclized, reduced form in mouse serum revealed that the presence of the disulfide bridge between D-Pen2 and D-Pen5 is an extremely important determinant of DPDPE's resistance to enzymatic degradation. The results from this study suggest that the route of administration can play a significant role in the time course distribution of [3H]DPDPE to the small intestine and the brain, and the inherent stability of DPDPE is due to its cyclized nature resulting from the presence of a disulfide bridge.
• Yamamura, M. S., Horvath, R., Toth, G., Otvos, F., Malatynska, E., Knapp, R. J., Porreca, F., Hruby, V. J., & Yamamura, H. I. (1992). Characterization of [³H]naltrindole binding to delta opioid receptors in rat brain. Life Sciences, 50(16), PL119-PL124.
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  PMID: 1313133;Abstract: [3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25°C measured an equilibrium dissociation constant (Kd) value of 37.0 ± 3.0 pM and a receptor density (Bmax) value of 63.4 ± 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (δ) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (μ) and kappa (κ) opioid receptors were only effective inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors. © 1992.
• Abrão, M., Castrucci, A. M., Hadley, M. E., & Hruby, V. J. (1991). Protein-kinase C mediates MCH signal transduction in teleost, synbranchus marmoratus, melanocytes.. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 4(2), 66-70.
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  PMID: 1946211;Abstract: MCH (melanin concentrating hormone) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which stimulates melanosome (melanin granule) aggregation to a perinuclear position within teleost fish integumental melanocytes, resulting in lightening of the skin. The mechanisms of action of MCH are unknown. Drugs that affect the diacylglycerol/inositol triphosphate pathway were used to investigate the possible roles of this pathway in the mechanisms of action of MCH on Synbranchus marmoratus (teleost) melanocytes. The shift of the dose-response curve to MCH in the presence of various concentrations of 4-bromophenacyl bromide and neomycin sulphate, phospholipase C inhibitors, suggests that phospholipase C is stimulated after MCH receptor activation. Low concentrations (10(-9) to 10(-8) M) of the phorbol ester TPA exhibited MCH-like activity, eliciting a dose-dependent melanosome aggregation. Higher doses, however, displaced to the right the dose-response curve to MCH, as did the protein kinase C inhibitors, dibucaine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). These results support the assumption that protein kinase C mediates the pigment aggregating activity of MCH. Both MCH and norepinephrine lightening actions were abolished by beta-glycerophosphate, a phosphatase inhibitor, suggesting that a protein dephosphorylation occurs during melanosome aggregation, and is, therefore, a common event triggered by MCH and norepinephrine, although both agonists act through separate receptors and exhibit different transduction mechanisms.
• Ayres, E. A., Parkhurst, D. N., Fang, S., Kramer, T. H., Hruby, V. J., & Burks, T. F. (1991). Antinociceptive and gastrointestinal transit effects of cholecystokinin (CCK-8) and related analogs of CCK-8 in the mouse. Proceedings of the Western Pharmacology Society, 34, 477-484.
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  PMID: 1788334;
• Cavagnero, S., Misicka, A., Knapp, R. J., Davis, P., Fang, L., Burks, T. F., Yamamura, H. I., & Hruby, V. J. (1991). Delta opioid receptor-selective ligands: [ ] enkephalin-dermenkephalin-dermenkephalin chimeric peptides. Life Sciences, 49(7), 495-503.
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  PMID: 1650414;Abstract: A number of DPDPE-dermenkephalin chimeric peptides have been synthesized in which the putative C-terminal delta-address of dermenkephalin has been linked to the highly delta opioid selective cyclic peptide {A figure is presented} enkephalin (DPDPE). Asp, Met-Asp and Leu-Met-Asp have been added to the C-terminus of DPDPE and both the carboxyl terminal and the carboxamide terminal series have been prepared. The bioassays using the mouse vas deferens and guinea pig ileum preparations have revealed a steady decrease in potency (compared to DPDPE) at delta and mu receptors as the dermenkephalin sequences were added. Some of the analogues, however, retained high delta selectivity. Similar results were obtained using radioligand binding assays. These findings suggest that the C-terminal amino acid sequence of dermenkephalin plays a role of delta-address which is specific to dermenkephalin itself, and is not additive with another delta selective ligand such as DPDPE. © 1991.
• Chan, W. Y., Berezin, I., Daniel, E. E., Russell, K. C., & Hruby, V. J. (1991). Effects of inactivation of oxytocin receptor and inhibition of prostaglandin synthesis on uterine oxytocin receptor and gap junction formation and labor in the rat. Canadian Journal of Physiology and Pharmacology, 69(9), 1262-1267.
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  PMID: 1661637;Abstract: Normal term labor is associated with a surge in myometrial oxytocin receptor formation and gap junction development. We have previously shown that inhibition of prostaglandin synthesis by naproxen sodium, 2.0 mg/day, suppressed oxytocin receptor formation but not gap junction formation and prolonged gestation. In this study, we investigated the effects of a specific oxytocin antagonist on oxytocin receptor formation, gap junction formation, and labor in the rat. [Pen1,Phe(Me)2,Thr4,Orn8]oxytocin, a specific oxytocin antagonist, was infused subcutaneously during the last 3 days of pregnancy at 300 μg/day. Measurements of myometrial oxytocin receptor concentrations and gap junction formation on days 21 and 22 and days 22-23 (in labor) pregnant uteri showed no significant differences in the B(max) and K(d) values between the control and the treated group. Gestation period was not prolonged by the oxytocin antagonist. However, in a separate group of day 23 pregnant rats, the uterine contractile response to 60 mU of oxytocin i.v. was found completely blocked by 10 μg of the oxytocin antagonist. These findings suggest that although functional oxytocin receptors did not appear to be essential for the initiation of labor, oxytocin antagonists may still be effective in the prevention of premature contractions. We also examined the effects of a higher dose of naproxen sodium, 5.0 mg/day, on gap junction formation. At this dose, naproxen sodium suppressed both oxytocin receptor and gap junction formation, prolonged gestation, and delayed parturition by 24 h or longer. Prostaglandin appears to be an important regulator or mediator of oxytocin receptor and gap junction formation and plays a critical role in the initiation of labor.
• Gehrig, C. A., Prakash, O., Toth, G., & Hruby, V. J. (1991). Design and synthesis of opioid receptor selective enkephalin analogues.. NIDA research monograph, 105, 378-379.
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  PMID: 1652078;
• Hadley, M. E., al-Obeidi, F., Hruby, V. J., Weinrach, J. C., Freedberg, D., Jiang, J. W., & Stover, R. S. (1991). Biological activities of melanotropic peptide fatty acid conjugates.. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 4(4), 180-185.
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  PMID: 1667821;Abstract: Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the alpha-MSH fragment analog, H-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a "creeping" potency in the lizard skin bioassay-that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10-100 times more active than alpha-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Asp5, D-Phe7, Lys10]alpha-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.
• Hill, P. S., Chan, W. Y., & Hruby, V. J. (1991). Oxytocin antagonists with changes in the Asn⁵ position shed light on hormone-oxytocin receptor interactions. International Journal of Peptide and Protein Research, 38(1), 32-37.
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  PMID: 1657804;Abstract: Since oxytocin agonists and antagonists have different structure-activity relationships, we have investigated the stereostructural and stereoelectronic requirements of the Asn5 residue in oxytocin antagonists by the synthesis of four analogues of the potent, prolonged acting oxytocin antagonist [Pen1,D-Phe2,Thr4,Orn8]-oxytocin (I) in which Asn5 was replaced respectively with Thr (II), Leu5 (III), Asp5 (IV) and Tyr5 (V). These analogues had pA2 values in the antioxytocic in vitro rat uterine assay of 7.23 (I), 7.16 (II), 6.67 (III), 7.21 (IV), and 6.76 (IV), respectively. All were also found to be weakly potent in the in vivo anti-vasopressor assay in the rat. These studies demonstrate very different structural and stereoelectronic requirements for oxytocin agonists and antagonists when they interact with the oxytocin uterine receptor.
• Hruby, V. J. (1991). Editorial. International Journal of Peptide and Protein Research, 37(1), 1-.
• Hruby, V. J., & Sharma, S. D. (1991). Designing peptide and protein ligands for biological receptors. Current Opinion in Biotechnology, 2(4), 599-605.
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  PMID: 1369346;Abstract: A clearer understanding of structure-function relationships of protein hormone-receptor systems is emerging from the increased use of molecular biology approaches. On the other hand, the introduction of rationally designed conformational constraints into peptide hormones and neurotransmitters is leading to the development of highly receptor-selective ligands that allow further investigations into the topographic modulation of their bioactivities and rational design principles for peptide antagonists.
• Hruby, V. J., Prakash, O., Kazmierski, W., Gehrig, C., & Matsunaga, T. O. (1991). Conformational analysis of opioid receptor-selective peptides using nuclear magnetic resonance and theoretical calculations.. NIDA research monograph, 112, 198-217.
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  PMID: 1661378;
• Hruby, V. J., Toth, G., Gehrig, C. A., Kao, L., Knapp, R., Lui, G. K., Yamamura, H. I., Kramer, T. H., Davis, P., & Burks, T. F. (1991). Topographically designed analogues of [D-Pen,D-Pen⁵]enkephalin. Journal of Medicinal Chemistry, 34(6), 1823-1830.
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  PMID: 1648137;Abstract: The conformationally restricted, cyclic disulfide-containing δ opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (1, DPDPE) was systematically modified topographically by addition of a methyl group at either the pro-S or pro-R position of the β carbon of an L-Phe4 or D-Phe4 residue to give [(2S,3S)-β-MePhe4]DPDPE (2), [(2R,3R)-β-MePhe4]DPDPE (3), [(2S,3R)-β-MePhe4]DPDPE (4), and [(2R,3S)-β-MePhe4]DPDPE (5). The four corresponding isomers were prepared in which the β-methylphenylalanine residue was p-nitro substituted, that is with a β-methyl-p-nitrophenylalanine (β-Me-p-NO2Phe) residue, to give [(2S,3S)-β-Me-p-NO2Phe4]DPDPE (6), [(2R,3R)-β-Me-p-NO2Phe4]DPDPE (7), [(2S,3R)-β-Me-p-NO2Phe4]DPDPE (8), and [(2R,3S)-β-Me-p-NO2Phe4]DPDPE (9), respectively. The potency and selectivity (δ vs μ opioid receptor) were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (μ ligand) and [3H]DPDPE (δ ligand) and by bioassay with mouse vas deferens (MVD, δ receptor assay) and guinea pig ileum (GPI, μ receptor assay). The eight analogues of DPDPE showed highly variable binding and bioassay activities particularly at the δ opioid receptor (4 orders of magnitude), but also at the μ opioid receptor, which led to large differences (3 orders of magnitude) in receptor selectivity. For example, [(2S,3S)-β-MePhe4]DPDPE (2) is 1800-fold selective in binding to the δ vs μ receptor, making it one of the most selective δ opioid receptor liganda in the enkephalin series as assessed by the rat brain binding assay, whereas the corresponding (2R,3R)-β-Me-p-NO2Phe-containing analogue 9 is only 4.5-fold selective (nonselective) in this same assay. On the other hand, in the bioassay systems, [(2S,3S)-β-Me-p-NO2Phe4]DPDPE (5) is more potent than DPDPE and 8800-fold selective for the MVD (δ receptor) vs the GPI (μ receptor), making it the most highly selective ligand in this series for the δ opioid receptor on the basis of these bioassays. In these assay systems, the (2R,3S)-β-MePhe4-containing analogue 5 had very weak potency and virtually no receptor selectivity (4.4-fold). These results demonstrate that topographical modification alone in a conformationally restricted peptide ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity and suggest that this approach may have general application to peptide ligand design. © 1991 American Chemical Society.
• Kazmierski, W. M., & Hruby, V. J. (1991). Asymmetric synthesis of topographically constrained amino acids: synthesis of the optically pure isomers of α,β-dimethyl-phenylalanine and α,β-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Tetrahedron Letters, 32(41), 5769-5772.
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  Abstract: All four isomers of α,β-dimethylphenylalanine and α,β-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid have been synthesized in high optical purity for use in the design of topographically constrained peptides. © 1991.
• Kazmierski, W. M., Yamamura, H. I., & Hruby, V. J. (1991). Topographic Design of Peptide Neurotransmitters and Hormones on Stable Backbone Templates: Relation of Conformation and Dynamics to Bioactivity. Journal of the American Chemical Society, 113(6), 2275-2283.
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  Abstract: We have proposed that development of methods for controlling the side-chain topography of amino acid residues in peptides and proteins provides a new approach to the topographical design of biologically active peptides. An example of this approach is the use of the 1,2,3,4-tetrahydroisoquinolinecarboxylic acid (Tic) residue, which favors a gauche (-) side-chain conformation when in the N-terminal position, whereas in its acylated form (internal position), the most stable side-chain conformation is gauche (+). This approach has been tested by incorporating D-Tic or Tic at different positions of μ opioid receptor specific octapeptides such as D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP, 1), examination of the biological consequences of these modifications, and detailed 1H NMR based conformational analysis. The compounds prepared and their biological activities were as follows: D-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (2; gauche (-), δ/μ = 7800, IC50 μ = 1.2 nM); Gly-D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (3; gauche (+), δ/μ = 19, IC50 μ = 278.7 nM); and D-Phe-Cys-Tic-D-Trp-Orn-Thr-Pen-Thr-NH2 (4; gauche (+), δ/μ = ∼7, IC50 μ = 1439.0 nM). In the absence of a geminal pair of protons suitable for distance calibration, a new technique (Davis, D. G. J. Am. Chem. Soc. 1987, 109, 3471-3472) of transverse and longitudinal cross-relaxation rate measurements has been utilized in conjunction with other 2D NMR methods in order to determine the three-dimensional solution conformations for the peptides 1-4, with subsequent application of restrained molecular dynamics (GROMOS). The average backbone conformations in peptides 1-4 were very similar, but the side-chain conformational preferences in the analogues differed, suggesting that the different affinities and selectivities for μ opioid receptors were primarily due to differences in the side-chain conformations of Tic (D-Tic), and thus due to differences in the topographies of these peptides, and not the backbone conformations. A detailed analysis of these relationships is presented.
• Knapp, R. J., Sharma, S. D., Toth, G., Duong, M. T., Fang, L., Bogert, C. L., Weber, S. J., Hunt, M., Davis, T. P., Wamsley, J. K., Hruby, V. J., & Yamamura, H. I. (1991). [D-Pen², 4'-¹²⁵I-Phe⁴, D-Pen⁵]Enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity¹. Journal of Pharmacology and Experimental Therapeutics, 258(3), 1077-1083.
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  PMID: 1653834;Abstract: [D-Pen2, 4'-125I-Phe4, D-Pen5]enkephalin ([125I]DPDPE) is a highly selective radioligand for the δ opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [125I]DPDPE binds to a single site in rate brain membranes with an equilibrium dissociation constant (K(d)) value of 421 ± 67 pM and a receptor density (B(max)) value of 36.4 ± 2.7 fmol/mg protein. The high affinity of this site for δ opioid receptor ligands and its low affinity for μ or k receptor-selective ligands are consistent with its being a δ opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of δ opioid receptors. Association and dissociation binding kinetics of 1.0 nM [125I] DPDPE are monophasic at 25°C. The association rate (k+1= 5.80 ± 0.88 x 107 M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM [3H]DPDPE and 0.8 nM [3H][D-Pen2, 4'-Cl-Phe4, D-Pen5]enkephalin, respectively. The dissociation rate of [125I]DPDPE (0.917 ± 0.117 x 10-2 min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [125I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [125I]DPDPE a valuable new radioligand for studies of δ opioid receptors.
• Kramer, T. H., Toth, G., Haaseth, R. C., Matsunaga, T. O., Davis, P., Hruby, V. J., & Burks, T. F. (1991). Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro. Life Sciences, 48(9), 881-886.
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  PMID: 1847736;Abstract: Several peptides of diverse structure, reported to possess high affinity and selectivity for the δ opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5]enkephalin, the cyclic enkephalin analogs [D-Pen2, D-Pen5] enkephalin (DPDPE) and [D-Pen2, p-F-Phe4, D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2, D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5, Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 X 10-10 molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type. © 1991.
• Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierskit, W. M., & Knappt, R. J. (1991). A new type of synthetic peptide library for identifying ligand-binding activity. Nature, 354(6348), 82-84.
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  PMID: 1944576;Abstract: Our aim was to improve techniques for drug development by facilitating the identification of small molecules that bind with high affinity to acceptor molecules (for example, cell-surface receptors, enzymes, antibodies) and so to mimic or block their interaction with the natural ligand1,2. Previously such small molecules have been characterized individually on a serial basis. The systematic synthesis and screening of peptide libraries of defined structure represents a new approach. For relatively small libraries, predetermined sequence variations on solid-phase supports have been used3,4, and large libraries have been produced using a bacteriophage vector into which random oligodeoxynucleotide sequences have been introduced5-8, but these techniques have severe limitations. Here we investigate an alternative approach to synthesis and screening of peptide libraries. Our simple methodology greatly enhances the production and rapid evaluation of random libraries of millions of peptides so that acceptor-binding ligands of high affinity can be rapidly identified and sequenced, on the basis of a 'one-bead, one-peptide' approach.
• Lebl, M., Fang, S., & Hruby, V. J. (1991). High-performance liquid chromatography of peptides at reduced temperatures: Separation of isomers. Journal of Chromatography A, 586(1), 145-148.
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  PMID: 1806549;Abstract: Cholecystokinin analogues containing N-methyl amino acids were studied by reversed-phase high-performance liquid chromatography at reduced temperatures. A reduction in temperature to - 17°C led to lower efficiency, but at the same time separations of cis and trans isomers (and some impurities) were achieved. The velocity constants for cis-trans equilibria were calculated. © 1991.
• Levine, N., Sheftel, S. N., Eytan, T., Dorr, R., Hadley, M. E., Weinrach, J. C., Ertl, G. A., Toth, K., McGee, D. L., & Hruby, V. J. (1991). Induction of skin tanning by subcutaneous administration of a potent synthetic melanotropin. Journal of the American Medical Association, 266(19), 2730-2736.
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  PMID: 1658407;Abstract: Objective.-To determine the efficacy of short-term administration of a synthetic analogue of α-melanotropin, [Nle4D-Phe7] (NDP)-α-melanocyte-stimulating hormone (MSH), in darkening (tanning) human skin. Design. - Randomized, placebo-controlled, double-blind clinical trial. Setting.-Clinical research unit of a university medical center. Subjects. - Twenty-eight healthy white men with a history of either poor tanning (skFn type I or II) or good tanning (skin type III or IV) recruited from advertisements and paid to participate in the study. Methods. - Each subject received 10 subcutaneous injections of either a purified NDP preparation or saline over 12 days. They were followed up for 7 weeks after therapy was completed. All subjects used a high-potency sunscreen during the the trial. Main Outcome Measure. - Skin darkening was quantified by serial chromaticity measurements prior to, during, and after therapy. Results. - A significant parabolic curve of skin darkening activity was noted in subjects with skin type I or II (P
• Misicka, A., Lipkowski, A. W., Fang, L., Knapp, R. J., Davis, P., Kramer, T., Burks, T. F., Yamamura, H. I., Carr, D. B., & Hruby, V. J. (1991). Topographical requirements for delta opioid ligands: Presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity. Biochemical and Biophysical Research Communications, 180(3), 1290-1297.
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  PMID: 1659410;Abstract: To investigate the role of the carboxyl group in deltorphin molecules, we have synthesized three new analogues in which the acidic amino acid residues in position 4 of the deltorphins were replaced by non-acidic but hydrophilic amino acids residues. The three analogues, [Ser4]-, [Gln4]-, and [Cys4]-deltorphin, all are as potent or more potent than either deltorphin I or II at delta opioid receptors and possess good delta selectivities. The excellent correlation between their in vitro delta receptor potencies and their intrathecal antinociception activity forms a strong argument for involvement of those receptors in spinal nociceptive modulation in the rats. © 1991 Academic Press, Inc.
• Mulder, A. H., Wardeh, G., Hogenboom, F., Kazmierski, W., Hruby, V. J., & Schoffelmeer, A. N. (1991). Cyclic somatostatin analogues as potent antagonists at μ-, but not δ- and κ-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the brain. European Journal of Pharmacology, 205(1), 1-6.
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  PMID: 1687463;Abstract: The opioid receptor antagonist properties of four conformaiionally constrained cyclic octapeptidc analogues of somatostatin were investigated using in vitro functional paradigms of μ-, δ- and κ-opioid receptors in the rat brain. The analogues examined were D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pcn-Thr-NH2 (CTAP), D-Tic-CTOP (TCTOP) and D-Tic-CTAP (TCTAP). Activation of μ-receptors by the enkephalin analogue Tyr-D-Ala-Gly-(NMc)Phc-Gly-ol (DAGO) inhibited the (electrically evoked) release of [3H]noradrenalinc (NA) from superfused cortical slices and this inhibitory effect was antagonized in a competitive fashion by all of the octapeptides tested (pA2 values: CTOP and CTAP 7.9-8.0, TCTOP and TCTAP 8.7-8.8). Selective activation of κ-opioid receptors by the cyclohcxylbenzeneacctamidc U69593 (0.02 μM) inhibited (by 40-45%) the release of [3H]dopamine (DA) from striatal slices, whereas selective activation of δ-opioid receptors by [D-Scr2O-t-butyl),Leu5]enkephalyl-Thr6 (DSTBULET; 0.1 μM) caused an inhibition (by 38-46%) of striatal [14C]acctylcholine (ACh) release. However, these inhibitory effects were not affected by any of the octapeptides in concentrations that caused full antagonism of the inhibitory effect (55-65%) of 0.1 μM DAGO on cortical [3H]NA release. Thus, the cyclic octapcptide somatostatin analogues CTOP, CTAP, TCTOP and TCTAP are potent and highly selective antagonists at the μ-opioid receptors mediating presynaptic inhibition of NA release in the brain. The μ-rceeptor affinity of the most potent of these antagonists, TCTOP and TCTAP, appears to be similar to that of naloxone but these antagonists have a much greater selectivity than the latter. © 1991.
• Nikiforovich, G. V., & Hruby, V. J. (1991). Erratum: ''Examination of the conformational meaning of 'δ-address' in the dermenkephalin sequence,'' (Volume 173, Number 2, pages 521-527). Biochemical and Biophysical Research Communications, 174(2), 1053-.
• Nikiforovich, G. V., Hruby, V. J., Prakash, O., & Gehrig, C. A. (1991). Topographical requirements for δ-selective opioid peptides. Biopolymers, 31(8), 941-955.
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  PMID: 1782355;Abstract: The conformational possibilities of three different δ-selective opioid peptides, which are DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen), DCFPE (Tyr-D-Cys-Phe-D-Pen), and DRE (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, dermenkephalin), were explored using energy calculations. Sets of low-energy conformers were obtained for each of these peptides. The sets consisted of 61 structures for DPDPE, 32 for DCFPE, and 38 for DRE, including various types of rotamers of the Tyr and Phe side-chain groups. Comparison of the geometrical shapes of the conformers was performed for these sets using topographical considerations, i.e., examination of the mutual spatial arrangement of the N-terminal α-amino group, and of the Tyr and Phe side-chain groups. The results obtained suggest a model for the δ-receptor-bound conformer(s) for opioid peptides. The model suggests the placement of the Phe side chain in a definite position in space corresponding to the g- rotamer of Phe for peptides containing Phe4 and to the t rotamer for peptides containing Phe3. The position of the Tyr1 side chain cannot be specified so precisely. The proposed model is in a good agreement with the results of biological testing of β-Me-Phe4-substituted DPDPE analogues that were not considered in the process of model construction.
• Olson, B. R., Drutarosky, M. D., Chow, M., Hruby, V. J., Stricker, E. M., & Verbalis, J. G. (1991). Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats. Peptides, 12(1), 113-118.
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  PMID: 1646995;Abstract: Intracerebroventricular administration of oxytocin (OT) and an OT agonist significantly decreased food intake in a dose-related manner in fasted rats. Central administration of an OT antagonist by itself (up to doses of 8 nmol) did not potentiate deprivation-induced food intake, but pretreatment with the OT receptor antagonist prevented the expected inhibition of food intake produced by OT and the OT agonist. Once-daily ICV injections of OT led to the development of tolerance to the inhibitory effects on food intake by the third day of treatment, but daily pretreatment with the OT antagonist prevented the development of this tolerance. In addition to causing decreased food intake, ICV administration of OT significantly increased grooming behavior but produced no dyskinesias. The inhibitory effect of OT on food intake was characterized by decreased amounts of food intake but a normal pattern of ingestion. The anorexia produced was central in nature and was not associated with altered plasma levels of hormones involved in caloric homeostasis or with changes in blood glucose. The OT agonist had relatively little effect on water intake when given in doses that significantly inhibited food intake. These results support the hypothesis that specific OT receptors within the central nervous system participate in the inhibition of feeding under certain conditions in rats. © 1991.
• Pettitt, B. M., Matsunaga, T., Al-Obeidi, F., Gehrig, C., Hruby, V. J., & Karplus, M. (1991). Dynamical search for bis-penicillamine enkephalin conformations. Biophysical Journal, 60(6), 1540-1544.
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  PMID: 1777571;PMCID: PMC1260211;Abstract: Quenched molecular dynamics is used as a conformational search technique for the constrained cyclic analog [D-Pen2,D-Pen5]enkephalin (DPDPE) in a continuum solvent. The results show a Gaussianlike distribution of conformations as a function of energy, unlike the distributions found for simple liquids which have sharp bands for different crystal forms and broad glasslike states are found. The lowest energy conformers have structural features in common with those obtained from constrained searches based on energy minimization. (Hruby, V. J., L.-F. Kao, B. M. Pettitt, and M. Karplus. 1988. J. Am. Chem. Soc. 110:3351-3359.) Many of the low energy configurations are amphiphilic with the carbonyl groups on one surface and the hydrophobic groups on the other. This supports the conclusions from the previous modeling study, which yielded amphiphilic structures as the most probable conformations of DPDPE when NOE data were included.
• Sharma, S. D., Toth, G., & Hruby, V. J. (1991). A simple general method for (radio)iodination of a phenylalanine residue in peptides: Preparation of [D-Pen ²,4′-¹²⁵I-Phe⁴,D-Pen ⁵]enkephalin, a peptide with extraordinary selectivity for δ-opioid receptors. Journal of Organic Chemistry, 56(16), 4981-4983.
• Slaninova, J., Knapp, R. J., Jinji, W. u., Su-Nan, F., Kramer, T., Burks, T. F., Hruby, V. J., & Yamamura, H. I. (1991). Opioid receptor binding properties of analgesic analogues of cholecystokinin oceapeptide. European Journal of Pharmacology, 200(1), 195-198.
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  PMID: 1663041;Abstract: Four analogues of cholecystokinin (CCK) octapeptide having analgesic activity after i.c.v. administration and high affinity for CCK-B receptors were studied for their ability to displace specific ligands,[3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. [3H]D-Pen2, 4'-Cl-Phe4,D-Pen5]enkephalin and [3H]U-69,593. for μ-, δ- and κ-opioid receptors, respectively. None of the analogues tested have high affinity for either μ- or κ-receptors (IC50 values > 0.7 μM), but their IC50 values for δ-receptors range from 29 to 1023 nM. The results suggest a relationship between the ligand requirements of CCK-B and δ-opioid receptors which further implies a possible structural relationship between these receptors. © 1991.
• Weber, S. J., Greene, D. L., Sharma, S. D., Yamamura, H. I., Kramer, T. H., Burks, T. F., Hruby, V. J., Hersh, L. B., & Davis, T. P. (1991). Distribution and analgesia of [³H][D-Pen²,D-Pen⁵]enkephalin and two halogenated analogs after intravenous administration. Journal of Pharmacology and Experimental Therapeutics, 259(3), 1109-1117.
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  PMID: 1762065;Abstract: To improve pharmacological characteristics of the delta-selective, cyclic peptide [D-Pen2,D-Pen5]enkephalin (DPDPE), modification by halogenation at the Phe4 residue was undertaken. The present study was to determine the extent [3H]DPDPE, [3H][p-Cl-Phe4]DPDPE and [p-125IPhe4]DPDPE crosses the blood-brain barrier, elicits analgesia and to characterize selective organ distribution and stability after i.v. administration. A significantly greater percentage of total [3H][p-Cl-Phe4]DPDPE reached the brain after 10, 20 and 40 min as compared to [3H]DPDPE and both peptides were significantly displaced by pretreatment with naloxone or naltrindole. The amount of [3H]DPDPE detected in the brain was greater than that of [p-125IPhe4]DPDPE. Distribution results revealed large amounts of the administered peptides were sequestered rapidly in the gall bladder and secreted into the small intestine. Hot-plate antinociception tests 5 min after i.v. administration (30 and 60 mg/kg) revealed [p-Cl-Phe4]DPDPE to elicit a much greater analgesic effect as compared to DPDPE or [p-125IPhe4]DPDPE. These results provide evidence that [p-Cl-Phe4]DPDPE has a greater apparent distribution to the brain and has a greater effect on the antinociception threshold as tested on the hot-plate than DPDPE or [p-125IPhe4]DPDPE. Stability of unlabeled and tritiated DPDPE and [p-Cl-Phe4]DPDPE was determined both in vitro and in vivo; both unlabeled and tritiated DPDPE and [p-Cl-Phe4]DPDPE remain intact.
• Wire, W. S., Fang, S. N., Hruby, V. J., Finch, S. T., Kramer, T. H., & Burks, T. F. (1991). The effects of a centrally selective cholecystokinin analog on male, Sprague Dawley rats in the non-fasted food intake model. Proceedings of the Western Pharmacology Society, 34, 453-459.
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  PMID: 1788328;
• Zechel, C., Trivedi, D., & Hruby, V. J. (1991). Synthetic glucagon antagonists and partial agonists. International Journal of Peptide and Protein Research, 38(2), 131-138.
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  PMID: 1664420;Abstract: This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18, Glu21]glucagon, a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucaon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10-5 M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve. Hence these compounds are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase system.
• Al-Obeidi, F., Hruby, V. J., Hadley, M. E., Sawyer, T. K., & Castrucci, A. D. (1990). Design, synthesis, and biological activities of a potent and selective α-melanotropin antagonist. International Journal of Peptide and Protein Research, 35(3), 228-234.
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  PMID: 2162330;
• Al-Obeidi, F., Mulcahy, M., Pitt, V. S., Begay, V., Hadley, M. E., & Hruby, V. J. (1990). Synthesis and actions of a melanotropin conjugate, Ac-[Nle⁴, Glu(gamma-4'-hydroxyanilide)⁵, D-Phe⁷]α-MSH_4-10-NH₂, on melanocytes and melanoma cells in vitro. Journal of Pharmaceutical Sciences, 79(6), 500-504.
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  PMID: 2168479;Abstract: L-Glutamic acid (γ-4'-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4'-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, D-Phe7]α-MSH4-10-NH2. The melanotropin:anilide conjugate, Ac-[Nle4, Glu(γ-4'-hydroxyanilide)5, D-Phe7]α-MSH4-10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than α-MSH or Ac-[Nle4, D-Phe7]α-MSH4-10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.
• Al-Obeidi, F., Sanderson, D. G., & Hruby, V. J. (1990). Synthesis of β- and γ-fluorenylmethyl esters of respectively N(α)-Boc-L-aspartic acid and N(α)-Boc-L-glutamic acid. International Journal of Peptide and Protein Research, 35(3), 215-218.
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  PMID: 1972376;
• Benson, B., Ebels, I., & Hruby, V. J. (1990). Isolation and structure elucidation of bovine pineal arginine vasopressin: Arginine vasotocin not identified. International Journal of Peptide and Protein Research, 36(2), 109-121.
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  PMID: 2272747;
• Burks, T. F., Peterson, J. M., Hruby, V. J., & Kramer, T. H. (1990). Differential effects of intracerebroventricular μ and δ selective opioids on small and large intestinal motility and transit in the rat. European Journal of Pharmacology, 183(1), 139-140.
• Burks, T. F., Peterson, J. M., Hruby, V. J., & Kramer, T. H. (1990). Differential effects of intracerebroventricular μ and δ selective opioids on small and large intestinal motility and transit in the rat. European Journal of Pharmacology, 183(6), 2335-2336.
• Bushfield, M., Murphy, G. J., Lavan, B. E., Parker, P. J., Hruby, V. J., Milligan, G., & Houslay, M. D. (1990). Hormonal regulation of G(i)2 α-subunit phosphorylation in intact hepatocytes. Biochemical Journal, 268(2), 449-457.
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  PMID: 2114093;PMCID: PMC1131453;Abstract: Hepatocytes contain the G(i)2 and G(i)3 forms of the 'G(i)-family' of guanine-nucleotide-binding proteins (G-proteins), but not G(i)1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate G(i)2 and G(i)3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein G(s). Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-α-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the α-subunit of immunoprecipitated G(i)2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[βγ-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('G(i)'-function). The immunoprecipitation of phosphorylated G(i)-2α-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of G(z) (a 'G(i)-like' G protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the G(i) family), in the immunoprecipitation assay. No labelling of the α-subunits of either G(i)3 or G(s) was observed. α-G(i)2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of α-G(i)2 was markedly biphasic where the loss of G(i) function paralleled the high-affinity component of the labelling of α-G(i)2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of α-G(i)2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 μM) or 8-bromo-cyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated α-G(i)2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [γ-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated α-G(i)2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of α-G(i)2 in intact cells. We suggest that there are two possible sites for the phosphorylation of α-G(i)2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.
• Caldwell, J. D., Barakat, A. S., Smith, D. D., Hruby, V. J., & Pedersen, C. A. (1990). A uterotonic antagonist blocks the oxytocin-induced facilitation of female sexual receptivity. Brain Research, 512(2), 291-296.
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  PMID: 2354364;Abstract: The nonapeptide oxytocin (OXT) has been shown to facilitate female sexual receptivity when infused into the cerebral ventricles or the basal forebrain. Various selective antagonists have been used to block other behavioral effects of centrally administered OXT. In this study we compared the effects of equal doses of uterotonic, antidiuretic (V2) or vasopressor (V1) antagonists in blocking the facilitative effects of a simultaneous infusion of OXT into the basal forebrain. Ovariectomized (OVXed) animals were implanted with chronic cannulas in the basal forebrain. All animals were then given 0.5 μg estradiol benzoate daily for 3 days before testing. On the fourth day animals were tested to 8-10 mounts with a sexually vigorous male before and 20, 40 and 90 min after infusions of 500 ng OXT alone or in combination with a uterotonic, V2 or a V1 antagonist analogue. OXT significantly increased lordosis responding 20 and 40 min after its infusion into the medial preoptic area and anterior hypothalamus when compared to the receptivity of normal saline vehicle infused animals. The uterotonic antagonist significantly blocked the facilitation seen after OXT. The V1 and V2 antagonist at equal doses had no effect on the OXT-induced facilitation of lordosis postures. The V1 antagonist itself facilitated sexual receptivity 90 min after infusion. The facilitative effect of OXT on receptivity appears to be mediated by central uterotonic receptors, while central vasopressor receptors may serve an inhibitory role. © 1990 Elsevier Science Publishers B. V. (Biomedical Division).
• Chan, W. Y., Cao, L., Hill, P. S., & Hruby, V. J. (1990). Oxytocin- and vasopressin-binding sites in the rat uterus: Competition binding and inhibitory pA₂ studies with oxytocin and oxytocin antagonists. Endocrinology, 126(4), 2095-2101.
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  PMID: 2318158;Abstract: Recent reports have presented evidence suggesting that there are distinct oxytocin (OT) and vasopressin (VP) receptors in the human and rabbit myometrium. In this study we have investigated whether OT and arginine vasopressin (AVP) activate the same or two different receptor systems in the rat uterus in producing their uterotonic action and whether the myometrial OT/VP receptors are similar to the V1 receptors in the vascular smooth muscle cells. We compared the dose-response characteristics of OT and AVP by the in vitro cumulative dose-response curve technique. We determined the ligand-receptor binding characteristics of [3H]OT and [3H]AVP on uterine membrane fractions from nonpregnant and pregnant rats. Specific OT antagonists were used in competition receptor binding assays and in antioxytocic pA2 bioassays against OT and AVP to determine whether OT antagonists can discriminate between OT- and AVP-binding sites in the myometrium. We also compared the in vitro antioxytocic (OT receptor-mediated action) and the in vivo antivasopressor (V1 receptor-mediated action) potencies of a series of six OT antagonists. Our results show that OT- and AVP-binding sites in the nonpregnant rat uterus have similar binding characteristics and cannot be distinguished by the dose-response study, radioligand receptor binding assays, or OT antagonists in the competition binding and pA2 assays. However, in the term pregnant parturient uterus, the two binding sites can be clearly differentiated. OT receptor density, but not AVP, was markedly increased at term pregnancy. All six OT antagonists studied in this investigation were more potent in antagonizing the uterotonic response to OT than the vasopressor response to AVP. The antioxytocic:antivasopressor potency ratios, however, were different between the antagonists, ranging from nearly equal (0.91) to low (0.1). The results above suggest that there are distinct OT- and AVP-binding sites in the rat myometrium. The myometrial OT/ AVP receptors are similar to but not the same as the V1 receptors in the vascular smooth muscle cells.
• Dawson, B. V., Hadley, M. E., Levine, N., Kreutzfeld, K. L., Don, S., Eytan, T., & Hruby, V. J. (1990). In vitro transdermal delivery of a melanotropic peptide through human skin. Journal of Investigative Dermatology, 94(4), 432-435.
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  PMID: 2155969;Abstract: A superpotent analogue of α-MSH, (Nle4, D-Phe7)-α-MSH, when applied topically to mice induces darkening of follicular melanocytes throughout the skin. In vitro studies have demonstrated delivery of the peptide across mouse but not rat skin. This variation in permeability of skin of animal models prompted us to use human skin in vitro. The melanotropin was applied to the surface of human skin samples through a permeation apparatus and allowed to penetrate for 24 h at 36°C. Passage of the analogue was shown by both bioassay and radioimmunoassay. These assays correlated well and demonstrated both the presence and the biologic integrity of the peptide after transdermal passage. Regional differences were noted in the degree of transdermal penetration. In addition, split thickness skin allowed greater penetration suggesting dermal binding of the hormone. This study is the first to show that a melanotropic peptide can be delivered transdermally through human skin in vitro. This has potential importance in the development of therapies for hypopigmentary disorders and for the stimulation of skin tanning without ultraviolet light. © 1990.
• De, A., Sawyer, T. K., Al-Obeidi, F., Hruby, V. J., & Hadley, M. E. (1990). Melanotropic peptide antagonists: Recent discoveries and biomedical implications. Drugs of the Future, 15(1), 41-55.
• Fric, I., Hlavacek, J., Rockway, T. W., Chan, W. Y., & Hruby, V. J. (1990). Effects of conformational constraint in 2- and 8-cycloleucine analogues of oxytocin and [1-pencillamine] oxytocin examined by circular dichroism and biosassay. Journal of Protein Chemistry, 9(1), 9-15.
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  PMID: 2340080;Abstract: The analogues of oxytocin and [1-pencicillamine]oxytocin, containing a cycloleucine (CLe) residue in position 2 or 8, were investigated by means of circular dichroism measurements in different solvents, and the results examined in terms of their biological activities. A cycloleucine residue in position 2 substantially reduces the free conformational space of the hormone 20-membered ring moiety (including the disulfide group), and stabilizes a conformation which is close to one of the possible conformations of oxytocin and involves a β-turn. In position 8, the Cle residue affects the conformation of the Tyr2 side chain, apparently forcing it away from the space above the 20-membered disulfide ring. However, it does not appear that the Cle residue has any significant effect on the overall backbone conformation of the hormone. The steric effect of the penicillamine residue in position 1 on the conformation of the disulfide group and Tyr2 side chain from previous investigations is further confirmed. The synthesis and biological potency of [1-penicillamine, 8-cycloleucine]oxytocin is described. This analogue exhibits a strong inhibitory effect on the uterotonic activity of oxytocin in vitro. It also inhibited the vasopressor response to vasopressin.
• García-Sáinz, J. A., Macías-Silva, M., Hernández-Sotomayor, S. T., Torres-Márquez, M. E., Trivedi, D., & Hruby, V. J. (1990). Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism. Cellular Signalling, 2(3), 235-243.
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  PMID: 2169291;Abstract: Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.
• Gehrig, C. A., Prakash, O., Toth, G., & Hruby, V. J. (1990). Design and synthesis of opioid receptor selective enkephalin analogues. NIDA Research Monograph Series, 378-379.
• Hill, P. S., Smith, D. D., Slaninova, J., & Hruby, V. J. (1990). Bicyclization of a weak oxytocin agonist produces a highly potent oxytocin antagonist. Journal of the American Chemical Society, 112(8), 3110-3113.
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  Abstract: [Mpa1,Glu4,Cys6,Lys8]oxytocin was prepared and found to be a very weak agonist in the oxytocic assay with about 1 /1400 the potency of the native hormone. Bicyclization of this compound via a lactam bridge between the Glu4 δ-carboxyl group and the ε-amino group of Lys8 led to the bicyclic analogue 4,8-cyclo[Mpa1,Glu4,Cys6,Lys 8]oxytocin. This bicyclic peptide acts as a very potent antagonist of oxytocin in the rat uterus assay with an in vitro pA2 value of 8.2. In the in vivo oxytocic assay the pA2 value was 6.45. The peptide displays mixed agonist/antagonist character in the in vivo galactogogic assay. The implications of these results to hormone agonist and antagonist activity are discussed with respect to earlier studies on constrained oxytocin antagonists and the results from the X-ray crystal structure of deaminooxytocin.
• Hruby, V. J. (1990). Career choices [2]. Chemical and Engineering News, 68(52), 2-.
• Hruby, V. J., Al-Obeidi, F., & Kazmierski, W. (1990). Emerging approaches in the molecular design of receptor-selective peptide ligands: Conformational, topographical and dynamic considerations. Biochemical Journal, 268(2), 249-262.
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  PMID: 2163604;PMCID: PMC1131425;
• Hruby, V. J., Chow, M., & Smith, D. D. (1990). Conformational and structural considerations in oxytocin-receptor binding and biological activity. Annual Review of Pharmacology and Toxicology, 30(1), 501-534.
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  PMID: 2160792;
• Hruby, V. J., Fang, S., Knapp, R., Kazmierski, W., Lui, G. K., & Yamamura, H. I. (1990). Cholecystokinin analogues with high affinity and selectivity for brain membrane receptors. International Journal of Peptide and Protein Research, 35(6), 566-573.
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  PMID: 2401597;
• Hruby, V. J., Kazmierski, W., Pelton, J. T., Yamamura, H. I., & Burks, T. F. (1990). Conversion of somatostatin to an opioid peptide: design using a conformational template and topographical constraints. European Journal of Pharmacology, 183(1), 54-.
• Husain, J., Blundell, T. L., Cooper, S., Pitts, J. E., Tickle, I. J., Wood, S. P., Hruby, V. J., Buku, A., Fischman, A. J., & Wyssbrod, H. R. (1990). The conformation of deamino-oxytocin: X-ray analysis of the 'dry' and 'wet' forms.. Philosophical transactions of the Royal Society of London. Series B: Biological sciences, 327(1243), 625-654.
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  PMID: 1972289;Abstract: Two crystal structures of (1 beta-mercaptopropionic acid) deamino-oxytocin are reported. The 'dry form' in space group C2 has cell dimensions a = 27.08 +/- 0.03, b = 9.06 +/- 0.01, c = 22.98 +/- 0.02 A, beta = 102.06 +/- 0.03 with one deamino-oxytocin and six water molecules per asymmetric unit. The 'wet form' in space group P2(1) has cell dimensions a = 27.27 +/- 0.02, b = 9.04 +/- 0.01, c = 23.04 +/- 0.02 A, beta = 102.24 +/- 0.02, with two deamino-oxytocin and 13 water molecules per asymmetric unit. A local twofold parallel to the monoclinic axis gives a pseudo C2 packing. Initial phases of the 'dry form' were calculated by the heavy-atom method from the isomorphous and anomalous difference Pattersons and anomalous difference Fouier synthesis. The structure was refined by using restrained least-squares at 1.2 A resolution to a crystallographic R = 0.10. The molecular replacement method yielded the P2(1) structure that was refined with geometric restraints to R less than 0.09, by using all data to 1.09 A resolution. Deamino-oxytocin consists of a cyclic tocin ring formed by six amino acids, closed by a disulphide bridge, S1-S6, and held by two trans-annular hydrogen bonds N2-O5 and N5-O2 with a type II turn at residues 3 and 4. A flexible tripeptide tail has a loosely hydrogen-bonded type I beta-turn between N9 and O6. The sulphur of cysteine at position 1 is disordered in all the molecules leading to alternative hands of disulphide. The conformational flexibility of Ile 3, Asn 5, Pro 7 side chains and the disulphide bridge is consistent with previous models of oxytocin in which flexibility is necessary for biological activity.
• Kawasaki, A. M., Knapp, R. J., Kramer, T. H., Wire, W. S., Vasquez, O. S., Yamamura, H. I., Burks, T. F., & Hruby, V. J. (1990). Design and synthesis of highly potent and selective cyclic dynorphin A analogues. Journal of Medicinal Chemistry, 33(7), 1874-1879.
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  PMID: 1972964;Abstract: We have designed and synthesized several cyclic disulfide-containing peptide analogues of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogues exhibit unexpected selectivities for the κ and μ opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogues has resulted in the κ/μ opioid receptor ligands [Cys5,Cys11]Dyn A1-11-NH2 (1) and [Cys5,Cys11,D-Ala8]Dyn A1-11-NH2 (2), which possess high κ and μ opioid receptor affinities centrally (guinea pig brain, GPB), but only weak activity at peripheral κ and μ opioid receptors (guinea pig ileum, GPI). On the other hand, [Cys8,Cys13]Dyn A1-13-NH2 and [D-Cys8,D-Cys13]Dyn A1-13-NH2 (5) display high κ potencies and selectivities at the peripheral (GPI) but not at the central (GPB) κ opioid receptor. The lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays suggests the existence of different subtypes of the κ and μ opioid receptors in the brain and peripheral nervous systems. © 1990 American Chemical Society.
• Knapp, R. J., Vaughn, L. K., Fang, S. -., Bogert, C. L., Yamamura, M. S., Hruby, V. J., & Yamamura, H. I. (1990). A new, highly selective CCK-B receptor radioligand ([³H][N-methyl-Nle^28,31]CCK_26-33): Evidence for CCK-B receptor heterogeneity. Journal of Pharmacology and Experimental Therapeutics, 255(3), 1278-1286.
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  PMID: 2262906;Abstract: [N-methyl-Nle28,31]CCK26-33 (SNF 8702) is a nonsulfated cholecystokinin octapeptide analog that is highly selective for cholecystokinin-B (CCK-B) receptors. Inhibition studies using [125I]Bolton-Hunter-labeled CCK-8 show that SNF 8702 has over 4,000-fold greater affinity for CCK receptors in guinea pig cortex relative to those in guinea pig pancreas. SNF 8702 was tritium-labeled to a specific activity of 23.7 Ci/mmol and its binding properties characterized for guinea pig brain membrane preparations. [3H]SNF 8702 binds to a single site with high affinity (K(d) = 0.69-0.90 nM) in guinea pig cortex, cerebellum, hippocampus and pons-medulla. Of these four tissues, the highest receptor density was measured in the cortex (86 fmol/mg of protein) and the lowest in the pons-medulla (22 fmol/mg of protein). In contrast to findings of single-site binding in some brain regions, evidence for CCK-B receptor heterogeneity is observed under other conditions. [3H]SNF 8702 binding to membranes prepared from whole guinea pig brain shows biphasic association kinetics at a concentration of 2.0 nM consistent with the presence of binding site heterogeneity. Binding site heterogeneity is consistently observed for [3H]SNF 8702 binding to guinea pig whole brain membranes in saturation studies where a high-affinity site (K(d) = 0.31 nM) is distinguished from a low-affinity site (K(d) = 3.3 nM). Binding site heterogeneity is also observed for the midbrain-thalamic region. CCK-B receptor heterogeneity is suggested by the effect of the guanyl nucleotide analogue, guanylyl-imidodiphosphate (Gpp(NH)p), on [3H]SNF 8702 binding to CCK-B receptors in the cerebellum. The addition of Gpp(NH)p to cerebellar membranes reveals the presence of a low concentration of high-affinity (Gpp(NH)p insensitive sites and a high concentration of low-affinity (Gpp(NH)p sensitive) sites. Cortical and hippocampal CCK-B receptors have reduced binding affinity for [3H]SNF 8702 in the presence of Gpp(NH)p but this effect is smaller than for cerebellar Gpp(NH)p sensitive sites and additional high affinity binding sites are not observed. The data show that SNF 8702 is a highly selective ligand for CCK-B receptors. Regional variations in the observed binding properties of [3H]SNF 8702 suggest the presence of CCK-B receptor subtypes.
• Kramer, T. H., Toth, G., Ayres, E. A., Hruby, V. J., & Burks, T. F. (1990). Antinociceptive actions of novel delta opioid receptor agonists in the mouse.. Progress in clinical and biological research, 328, 473-476.
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  PMID: 2154807;
• Kramer, T. H., Wild, K. D., Toth, G., Davis, P., Hruby, V. J., Burks, T. F., & Porreca, F. (1990). Potency, affinity and efficacy of currently available delta opioid agonists. New leads in opioid research: proceedings of the International Narcotics Research Conference. ICS914, 222-.
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  Abstract: We compared several peptides currently used as selective ligands for δ-opioid receptors with respect to their potency, receptor affinity and transduction efficacy. The compounds differed markedly in terms of receptor affinity, whereas variation in efficacy was minimal.
• Lebl, M., Hill, P., Kazmierski, W., Karaszova, L., Slaninova, J., Fric, I., & Hruby, V. J. (1990). Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation. International Journal of Peptide and Protein Research, 36(4), 321-330.
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  PMID: 2079387;
• Lenzen, R., Hruby, V. J., & Tavoloni, N. (1990). Mechanism of glucagon choleresis in guinea pigs. American Journal of Physiology - Gastrointestinal and Liver Physiology, 259(5 22-5), G736-G744.
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  PMID: 2173415;Abstract: The present studies were carried out to clarify the mechanism of glucagon choleresis in guinea pigs. At the infusion rate of 1.4 nmol·min-1·kg-1, glucagon increased bile flow from 206.6 ± 14.3 to 302.6 ± 35.0 μl·min-1·kg-1 and bicarbonate biliary concentration from 63.7 ± 4.2 to 75.5 ± 5.9 meq/l. Measurements of bile acid excretion in bile, the biliary tree volume, and of the hormone choleretic effect in guinea pigs with proliferated bile ductules/ducts induced by α-naphthylisothiocyanate feeding indicated that glucagon, unlike secretin, stimulated canalicular bile flow. Inhibition of prostaglandin synthesis by indomethacin administration (5 mg·kg-1·h-1) did not modify the choleretic effect of glucagon, and infusion of a glucagon analogue (TH-glucagon, 1.4 nmol·min-1·kg-1), which did not increase hepatic formation of adenosine 3'5'-cyclic monophosphate (cAMP), failed to stimulate bile flow. Like the parent hormone, however, TH-glucagon augmented plasma glucose levels and stimulated formation of inositol phosphates. Colchicine pretreatment (0.5 mg/kg ip) almost entirely prevented the choleretic effect of glucagon but did not modify spontaneous and bile acid-induced bile flow and the stimulatory effect of the hormone on glucose release and on hepatic formation of cAMP and inositol phosphates. Finally, glucagon produced a large increase in the biliary entry of horseradish peroxidase, even though this effect was transient and was not coupled to the increase in bile flow. These results indicate that glucagon choleresis in the guinea pig is not secondary to prostaglandin release, is canalicular in origin, involves bicarbonate secretion, is mediated by cAMP, and requires an intact microtubular system.
• Matsunaga, T. O., Gehrig, C. A., & Hruby, V. J. (1990). H-NMR assignments and conformational studies of melanin concentrating hormone in water using two-dimensional NMR. Biopolymers - Peptide Science Section, 30(13-14), 1291-1295.
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  PMID: 1964810;Abstract: The question of a structural link between MCH and MSH has led us to study the conformation of MCH in solution by nmr spectroscopy. Peptides or linear fragments of proteins of this size and magnitude are not as disordered as originally believed, and actually can have a good deal of relatively stable secondary structure. The presence of a cyclic disulfide ring in MCH enhances the probability of a structurally defined peptide. Using recent advances in two-dimensional (2D) methodology including phase-sensitive nuclear Overhauser enhanced spectroscopy (NOESY), and phase-sensitive correlated spectroscopy (COSY), we have been able to ascertain considerable conformational information about MCH.
• Nikiforovich, G. V., & Hruby, V. J. (1990). Examination of the conformational meaning of "δ-address" in the dermenkephalin sequence. Biochemical and Biophysical Research Communications, 173(2), 521-527.
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  PMID: 1979733;Abstract: Comprehensive energy calculations were applied to four opioid-related peptides with different receptor selectivities, namely the δ-selective dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, DRE), the μ-selective dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DRM) and their "hybrid" peptides DRM/DRE (Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2) and DRE/DRM (Tyr-D-Met-Phe-His-Tyr-Pro-Ser-NH2). It was shown that the N-terminal tripeptide "μ-messages" in the δ-selective ligands DRE and DRM/DRE can possess similar low energy space arrangements of their functionally important elements (the N-terminal α-amino group and the aromatic moieties of Tyr and Phe), but that these are different from the space arrangement of these moieties in μ-selective DRM and DRE/DRM. These results suggest that the C-terminal tripeptide "δ-address" in DRE may influence the conformation of the "μ-message" in DRM. A refined model for the δ-receptor-bound conformation of DRE is proposed based on these calculations which is similar to that previously suggested for the cyclic δ-selective peptide [D-Pen2, D-Pen5]enkephalin (DPDPE). This model also has partial correspondence with the structure of the δ-selective alkaloid naltrindole. © 1990 Academic Press, Inc.
• Sawyer, T. K., Staples, D. J., Castrucci, A. M., Hadley, M. E., Al-Obeidi, F. A., Cody, W. L., & Hruby, V. J. (1990). α-Melanocyte stimulating hormone message and inhibitory sequences: Comparative structure-activity studies on melanocytes. Peptides, 11(2), 351-357.
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  PMID: 2162535;Abstract: We investigated the structure-activity relationships of α-MSH (α-melanocyte stimulating hormone) fragment derivatives of the generic formulae Ac-α-MSH(x-13)-NH2 and Ac-α-MSH(6-x)-NH2. The minimal C-terminal sequences required for melanotropic activity were 8-13 and 7-13, respectively, in the frog and lizard skin bioassays. The Arg8-Trp9 sequence appears to be a fundamental component of the minimal message sequences found to date such as α-MSH(6-9), α-MSH(8-13) and α-MSH(7-13). We discovered that Ac-α-MSH(7-10)-NH2 was a weak and selective α-MSH antagonist on the lizard skin bioassay. Analysis of α-MSH(7-10) analogues of the generic formula Ac-Xaa-Arg-Trp-Yaa-NH2 led to Ac-[D-Trp7,D-Phe10]α-MSH(7-10)-NH2, a moderately potent, specific and competitive inhibitor of α-MSH in both the frog and the lizard skin bioassays. © 1990.
• Spencer, R. L., Hruby, V. J., & Burks, T. F. (1990). Alteration of thermoregulatory set point with opioid agonists. Journal of Pharmacology and Experimental Therapeutics, 252(2), 696-705.
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  PMID: 2313595;Abstract: This paper focuses on the behavioral thermoregulatory effects of i.c.v. administration of [D-Ala2-MePhe4,Gly5-ol]enkephalin (DAMGO), cyclic [D-Pen2,D-Pen5]enkephalin (DPDPE) and U-50,488H, selective agonists for the mu, delta and kappa opioid receptors, respectively. Rats were tested in a thermally graded tunnel (thermocline) which allowed for simultaneous measurement of body temperature, ambient temperature selection and general ambulatory activity levels. DAMGO (0.3 μg) caused an increase in body temperature which was facilitated by the selection of a warm ambient temperature. Both DPDPE (30 μg) and U-50,488H (100 μg) caused decreases in body temperature which were accompanied by a selection of a cool ambient temperature. In each case there was evidence for a regulated change in body temperature, with DAMGO increasing thermoregulatory set point and DPDPE and U-50,488H decreasing set point. DAMGO and U-50,488H produced a depression of activity levels for the first 15 min after injection. DAMGO and DPDPE produced increases in activity levels which peaked after body temperature had returned toward base-line levels. These data characterize further the differentiable profiles of physiological effects produced by these three compounds. The central modulation of the control of body temperature by these opioid receptor agonists may reflect a role of endogenous opioids in thermoregulatory control.
• Toth, G., Kramer, T. H., Knapp, R., Lui, G., Davis, P., Burks, T. F., Yamamura, H. I., & Hruby, V. J. (1990). [D-Pen²,D-Pen⁵]enkephalin analogues with increased affinity and selectivity for δ opioid receptors. Journal of Medicinal Chemistry, 33(1), 249-253.
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  PMID: 2153205;Abstract: The conformationally restricted, cyclic disulfide-containing enkephalin analogue [D-Pen2,D-Pen5]enkephalin (DPDPE) was modified by halogenation (F, Cl, Br, I) of the phenylalanine-4 residue in the para position. The potency and selectivity of these analogues for the δ opioid receptor was greater than that of the parent peptide. The analogues possessed greater potency and affinity for the δ receptors than DPDPE in the mouse vas deferens assay and in radioreceptor assays (against [3H]DPDPE), respectively. [p-ClPhe4]DPDPE was the most selective in the radioligand binding assays (IC50(μ)/IC50(δ) = 574), being about 5-fold more δ opioid receptor selective than DPDPE in this assay, whereas [p-IPhe4]DPDPE was the most selective in the classical bioassay systems using the mouse vas deferens and guinea pig ileum assays (IC50(GPI)/IC50(MVD) = 17 374), making it nearly 9-fold more selective than DPDPE in direct comparisons using the same assay conditions. © 1989 American Chemical Society.
• Toth, G., Lebl, M., & Hruby, V. J. (1990). Chiral thin-layer chromatographic separation of phenylalanine and tyrosine derivatives. Journal of Chromatography A, 504(C), 450-455.
• Vaughn, L. K., Wire, W. S., Davis, P., Shimohigashi, Y., Toth, G., Knapp, R. J., Hruby, V. J., Burks, T. F., & Yamamura, H. I. (1990). Differentiation between rat brain and mouse vas deferens δ opioid receptors. European Journal of Pharmacology, 177(1-2), 99-101.
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  PMID: 2160370;Abstract: Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine ((2R,3S)-{down triangle, open}EPhe), have been shown to have high affinity for brain δ opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between δ opioid receptors in brain and mouse was deferens, the ability of a selective δ opioid compound. [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-{down triangle, open}EPhe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferences binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens δ opioid receptors may be distinct from brain δ opioid receptors. © 1990.
• Wire, W. S., Fang, S. N., Hruby, V. J., Farmer, S. C., Riviere, P., Kramer, T. H., & Burks, T. F. (1990). Evaluation of a food intake model as a tool for studying cholecystokinin (CCK) and derivatives of CCK in male Sprague-Dawley rats. Proceedings of the Western Pharmacology Society, 33, 65-68.
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  PMID: 2274547;
• Al-Obeidi, F., Hadley, M. E., Pettitt, B. M., & Hruby, V. J. (1989). Design of a new class of superpotent cyclic α-melanotropins based on quenched dynamic simulations. Journal of the American Chemical Society, 111(9), 3413-3416.
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  Abstract: A new highly potent, receptor-selective and prolonged-acting cyclic lactam analogue of α-melanotropin (α-MSH) has been designed and synthesized. Molecular dynamics simulations and molecular mechanics calculations were used in conjunction with results from previous conformational structure-biological activity studies to design a new class of linear and cyclic α-melanotropin (α-MSH) analogues. Examination of these properties of α-MSH and [Nle4,D-Phe7]α-MSH led to the design of the potent linear fragment analogue Ac-[Nle4,Asp5,D-Phe7,Lys 10]α-MSH4-10-NH2, in which the Gly10 residue of α-MSH4-10 was replaced by Lys10 as the major novel change from previous investigations. This in turn led to the synthesis of the cyclic lactam analogue Ac-[Nle4,Asp5,D-Phe7,Lys 10]α-MSH4-10-NH2, which was exceptionally potent in the lizard skin (90 times that of α-MSH) and mammalian melanoma tyrosinase (100 times that of α-MSH) assays and in addition exhibited prolonged biological activity. © 1989 American Chemical Society.
• Al-Obeidi, F., Hruby, V. J., M., A., & Hadley, M. E. (1989). Design of potent linear α-melanotropin 4-10 analogues modified in positions 5 and 10. Journal of Medicinal Chemistry, 32(1), 174-179.
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  PMID: 2535874;Abstract: α-Melanocyte stimulating hormone (α-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that has diverse physiological functions in addition to its reversible darkening of amphibian skins by stimulating melanosome dispersion within melanophores. On the basis of theoretical and experimental results from our laboratory and others, we have designed a group of 1-13, 4-13, and especially 4-10 analogues related to the superpotent analogue [Nle4,D-Phe7]α-MSH in which the Glu5 has been replaced with Asp5, and the Gly10 has been replaced with Lys10 and other basic amino acid residues in the 4-10 analogues, and in which Gly10 and Lys11 were interchanged in the longer peptide analogues. In the 1-13 and 4-13 series the Lys10, Gly11 analogues generally retained superpotency for the D-Phe7-containing analogues. Most interestingly, synthesis of Ac-[Nle4,Xxx5,Yyy7,Zzz 10]α-MSH4-10-NH2 analogues where Xxx = Asp or Glu, Yyy = Phe or D-Phe, and Zzz = basic amino acids (Lys, Orn, α,γ-diaminobutyric acid (Dab), and α,β-diaminopropionic acid (Dpr)) provided melanotropins with potencies up to 10 times that of the native hormone in stimulating frog (Rana pipiens) skin darkening and 8-50 times more potent than α-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. To our knowledge, Ac-[Nle4,Asp5,D-Phe7,Dab 10]α-MSH4-10-NH2, the most potent analogue, is the most potent melanotropin obtained thus far for the Anolis assay system. These results provide new insights into the structural and conformational requirements for biological potency of α-MSH and the differential structural and conformational requirements of α-MSH and its analogues at two different types of pigment cell receptors. © 1988 American Chemical Society.
• Al-Obeidi, F., M., A., Hadley, M. E., & Hruby, V. J. (1989). Potent and prolonged acting cyclic lactam analogues of α-melanotropin: Design based on molecular dynamics. Journal of Medicinal Chemistry, 32(12), 2555-2561.
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  PMID: 2555512;Abstract: Utilizing results from previous structure-activity relationships and theoretical studies of α-melanotropin (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and its related superpotent analogues, Ac-[Nle4,D-Phe7]-α-MSH and Ac-[Cys4,Cys10]-α-MSH, we have designed a new class of α-MSH4-13 and α-MSH4-10 cyclic lactam fragment analogues of α-melanotropin. The cyclic peptides have the following general structures: Ac-[Nle4,Xxx5,D-Phe7,Yyy10,Gly 11]-α-MSH4-13-NH2 and Ac-[Nle4,Xxx5,D-Phe7,Yyy 10]-α-MSH4-10-NH2, where Xxx = Glu or Asp and Yyy = Lys, Orn, Dab, or Dpr. Formation of the lactam bridge between the side-chain groups Xxx and Yyy was performed either in solution or on a solid-phase support. Seven cyclic peptides were prepared and bioassayed for their melanotropic potency by using standard frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. Relative to α-MSH (relative potency = 1), the potencies of the cyclic peptides in the lizard skin bioassay were as follows: α-MSH (1); Ac-[Nle4,Glu5,D-Phe7,Lys10,Gly 11]-α-MSH4-13-NH2 (6); Ac-[Nle4,Asp5,D-Phe7,Lys10,Gly 11]-α-MSH4-13-NH2 (100); Ac-[Nle4,Glu5,D-Phe7,Lys 10]-α-MSH4-10-NH2 (9); Ac-[Nle4,Asp6,D-Phe7,Lys 10]-α-MSH4-10-NH2 (90); Ac-[Nle4,Asp5D-Phe7,Orn 10]-α-MSH4-10-NH2 (20); Ac-[Nle4,Asp5,D-Phe7,Dab 10]-α-MSH4-10-NH2 (5); Ac-[Nle4,Asp5,D-Phe7,Dpr 10]-α-MSH4-10-NH2 (5). Similar results were obtained in the frog skin bioassay, but the analogues were much less potent. Cyclic melanotropins with 23-membered rings exhibited 100-fold higher melanotropic potency than α-MSH with selectivity for the lizard melanocyte receptors over the frog melanocyte receptors. Increasing or decreasing the ring size of these cyclic melanotropins from 23 diminishes the biological potency of the resulting cyclic peptide. The 23- and 24-membered ring analogues showed prolonged (residual) biological activities in both biological assays, but the smaller ring systems (20, 21, 22) did not. These results provide new insights into the structural and conformational requirements of α-MSH and its analogues at two different types of pigment cell (melanocyte) receptors. © 1989 American Chemical Society.
• Ayres, E. A., Lemcke, P. K., Kramer, T. H., Toth, G., Hruby, V. J., & Burks, T. F. (1989). A comparison among antinociceptive bioassays for μ and δ agonists. Proceedings of the Western Pharmacology Society, 32, 167-171.
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  PMID: 2550941;
• Beckwith, B. E., Tinius, T. P., Hruby, V. J., Al-Obeidi, F., Sawyer, T. K., & Affholter, J. A. (1989). The effects of structure-conformation modifications of melanotropin analogs on learning and memory: D-amino acid substituted linear and cyclic analogs. Peptides, 10(2), 361-368.
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  PMID: 2547204;Abstract: Alpha-MSH has a wide variety of putative biological activities in addition to its classical melanocyte dispersing activity. Since each of these activities appears to be mediated by a discrete receptor, this peptide is an excellent candidate for exploring conformational restrictions which determine the chemical-physical basis for hormone action on specific activities. Experiments One and Two evaluated several cyclic and linear analogs of alpha-MSH on retrieval of memory during the reactivation of memory for a passive avoidance response following hypothermia-induced amnesia. Three of the cyclic analogs appear to have enhanced the peptide's ability to serve as a reactivation agent. One of the linear Nle4,D-Phe7 analogs antagonized whereas three others enhanced reactivation. The D-Phe7 substitution in cyclic analogs did not affect reactivation. Another group of animals were trained on a step-through passive avoidance task and tested 25 days later. The cyclic analog enhanced memory whereas the D-Phe7 analog and alpha-MSH had no effect. Finally, two analogs were tested on a black-white discrimination. Although the cyclic analog had no effect on either acquisition or reversal of this learning, the Nle4,D-Phe7 analog significantly impaired reversal learning. The results from these preliminary studies suggest that structural modifications of alpha-MSH do alter its potency and pattern of actions in learning and memory situations. © 1989.
• Burks, T. F., Kramer, T. H., Davis, P., Toth, G., & Hruby, V. J. (1989). Novel [D-Pen2,D-Pen5]enkephalin derivatives with increased sigma receptor potency and selectivity: Potential tools for opioid pharmacology. NIDA Research Monograph Series, 291-292.
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  PMID: 2577035;
• Castrucci, A. M., Hadley, M. E., Sawyer, T. K., Wilkes, B. C., Al-Obeidi, F., Staples, D. J., Vaux, A. d., Dym, O., Hintz, M. F., Riehm, J. P., Rao, K. R., & Hruby, V. J. (1989). α-melanotropin: The minimal active sequence in the lizard skin bioassay. General and Comparative Endocrinology, 73(1), 157-163.
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  PMID: 2537778;Abstract: α-Melanotropin (α-melanocyte-stimulating hormone, α-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The minimal sequence of α-MSH required for agonism in the lizard (Anolis carolinensis) skin bioassay was determined to be Ac-His-Phe-Arg-Trp-NH2 (Ac-α-MSH6-9-NH2). Smaller fragments of this sequence (Ac-α-MSH6-8-NH2, Ac-α-MSH6-7-NH2, Ac-α-MSH7-9-NH2, and Ac-α-MSH7-8-NH2) were devoid of melanotropic activity. The tetrapeptide, Ac-α-MSH7-10-NH2, was also inactive, thus again demonstrating the importance of His at position 6 for minimal activity. The important potentiating amino acids were found to be Met-4, Lys-11, and Pro-12, since Ac-α-MSH4-10-NH2 was about 100 times more potent than Ac-α-MSH5-10-NH2, and Ac-[Nle4]-α-MSH4-11-NH2 was about 40 times more potent than Ac-α-MSH4-10-NH2 or Ac-[Nle4]-α-MSH4-10-NH2. Ac-α-MSH4-12-NH2 and Ac-[Nle4]-α-MSH4-12-NH2 were equipotent and about six times more potent than α-MSH. Since [Nle4]-α-MSH and Ac-[Nle4]-α-MSH4-13-NH2 were both equipotent but about sixfold less active than Ac-[Nle4]-α-MSH4-12-NH2, it is clear that valine at position 13 does not contribute to the potency of α-MSH, except possibly in a negative way. The minimal message sequence for equipotency to α-MSH appears to be Ac-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-NH2, since the analog, Ac-[Nle4]-α-MSH4-11-NH2, was as active as the native hormone. Ser-1, Tyr-2, Ser-3, Glu-5, and Val-13 are not important for melanotropic potency since Ac-α-MSH4-12-NH2 was more potent than α-MSH, and Ac-α-MSH5-10-NH2 and Ac-α-MSH6-10-NH2 were equipotent, being about 4,000 times less active than α-MSH. © 1989.
• Dharanipragada, R., Nicolas, E., Toth, G., & Hruby, V. J. (1989). Asymmetric synthesis of unusual amino acids: Synthesis of optically pure isomers of β-methylphenylalanine. Tetrahedron Letters, 30(49), 6841-6844.
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  Abstract: All the four individual isomers of β-methylphenylalanine have been synthesized in very high optical purities by utilizing in part the chiral imide enolate bromination methodology of Evans and co-workers. © 1989.
• Froimowitz, M., & Hruby, V. J. (1989). Conformational analysis of enkephalin analogs containing a disulfide bond. Models for delta- and mu-receptor opioid agonists. International Journal of Peptide and Protein Research, 34(2), 88-96.
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  PMID: 2553632;
• Hadley, M. E., Marwan, M. M., al-Obeidi, F., Hruby, V. J., & Castrucci, A. M. (1989). Linear and cyclic alpha-melanotropin [4-10]-fragment analogues that exhibit superpotency and residual activity.. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 2(6), 478-484.
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  PMID: 2557603;Abstract: Two analogues of alpha-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2), Ac-[Nle4, Asp5, D-Phe7, Lys10]alpha-MSH4-10NH2 and Ac-[Nle4, Asp5, D-Phe7, Lys10] alpha-MSH4-10-NH2, were synthesized, and the melanotropic activities of the peptides were compared in several bioassays. Potencies were determined in the in vitro frog and lizard skin bioassays and in the S91 melanoma cell tyrosinase assay. Both analogues were equipotent or more potent than alpha-MSH in all bioassays, and the activities of the analogues were prolonged compared to alpha-MSH. The two analogues were very resistant to inactivation by purified proteolytic enzymes (alpha-chymotrypsin, trypsin, and pepsin). The two peptides could be topically applied and transdermally delivered across the skin of mice in vivo, resulting in a shift from pheomelanogenesis to eumelanogenesis within follicular melanocytes. The cyclic analogue exhibited greater potency, prolonged activity, and stability against enzyme inactivation than did the linear peptide. The significance of the findings for the further design of melanotropin analogues is discussed, as in the possible relevance of these melanotropin analogues for use in biomedical studies.
• Hawkins, K. N., Knapp, R. J., Lui, G. K., Gulya, K., Kazmierski, W., Wan, Y. -., Pelton, J. T., Hruby, V. J., & Yamamura, I. (1989). [³H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂] ([³H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain. Journal of Pharmacology and Experimental Therapeutics, 248(1), 73-80.
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  PMID: 2563293;Abstract: The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 108 M-1 and 2.49 x 108 M-1 min-1 at 25 and 37°C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10-2 min-1 and 1.03 x 10-1 min-1 at 25 and 37°C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent K(d) values of 0.16 and 0.41 nM at 25 and 37°C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37°C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2, D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands. Putative mu opioid receptor ligands such as naltrexone, naloxone, CTOP and Tyr-D-Ala-Gly-N-MePhe-Gly-ol inhibited specific [3H]CTOP binding with high affinity. Other ligands, including delta and kappa opioids, somatostatin, substance P, alpha and beta adrenergic, dopaminergic as well as cholinergic ligands were much less potent or were ineffective. The density of [3H]CTOP binding sites in different regions of the central nervous system is consistent with reports of the regional distribution of mu receptors in the rat brain. These data show that [3H]CTOP is a potent and selective ligand and may be useful for further characterization of mu opioid receptors.
• Hruby, V. J. (1989). Designing molecules: Specific peptides for specific receptors. Epilepsia, 30(SUPPL. 1), S42-S50.
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  PMID: 2570689;Abstract: Peptides are the largest class of mediators of intercellular communication in the central nervous system. These molecules pose special problems in design for potential medical applications because of the high degree of flexibility, lack of high receptor selectivity, and ready biodegradation or clearance. The global and local use of conformational constraints has overcome these difficulties. Structure-biological activity relationships, molecular modeling, conformational analysis, conformational calculations, and molecular dynamics simulations can all be used to derive suitable lead structures and conformational models. Often, a single, constrained peptide analogue can be designed, which will have many of the desired biological and biophysical properties, and will serve as a template. Peptide analogues with high potency, exquisite receptor selectivity, and biological stability can be obtained. The approach is illustrated by the design of cyclic enkephalin-agonist analogues with exceptional δ-opioid-receptor selectivity, and of constrained somatostatin analogues that have become opioid peptides and possess potent opioid antagonist activities and exceptional selectivity for μ-opioid receptors.
• Hruby, V. J., & Gehrig, C. A. (1989). Recent developments in the design of receptor specific opioid peptides. Medicinal Research Reviews, 9(3), 343-401.
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  PMID: 2547125;
• Knapp, R. J., Kazmierski, W., Hruby, V. J., & Yamamura, H. I. (1989). Structural characteristics of two highly selective opioid peptides.. BioEssays, 10(2-3), 58-61.
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  PMID: 2541696;Abstract: The demonstration of opioid receptors by radioligand binding and the discovery of their endogenous peptide ligands has provided a new class of compounds that can be used for the development of novel opioids. The number of potential receptor targets for such opioids has been expanded by the identification of multiple opioid receptor types. The development of highly selective opioid peptides using the principles of conformational restriction permits the analysis of the structure-activity requirements of each receptor type, and is facilitating the elucidation of the functional properties of the different opioid receptors.
• Kramer, T. H., Shook, J. E., Kazmierski, W., Ayres, E. A., Wire, W. S., Hruby, V. J., & Burks, T. F. (1989). Novel peptidic Mu opioid antagonists: Pharmacologic characterization in vitro and in vivo. Journal of Pharmacology and Experimental Therapeutics, 249(2), 544-551.
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  PMID: 2566679;Abstract: A series of six synthetic octapeptides, structurally related to somatostatin, demonstrate high affinity and selectivity for mu opioid receptors in radioligand binding assays. The compounds, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), D-tetrahydroisoquinoline carboxylic acid (D-Tic)-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (Tic-CTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (D-Tic-CTOP) and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (D-Tic-CTAP), were tested in vitro and in vivo for agonist and antagonist potency and selectivity. In vitro bioassays included the guinea pig ileum, mouse vas deferens and rabbit vas deferens. In vivo tests included hotplate antinociception and gastrointestinal transit inhibition, performed in mice. In vitro, all six derivatives were competitive, highly selective mu antagonists (pA2 values from 6.4-7.9). The compounds demonstrated varying degrees of intrinsic agonist activity especially in the mouse vas deferens, the least active being CTAP and D-Tic-CTAP, which showed no mu or kappa agonist actions, and delta activity only at very high (>3 μM) concentrations. In vivo, none of these compounds showed antinociceptive actions when administered i.c.v. in mice. All were competitive mu antagonists in the hotplate antinociception test. Antagonist potency for all the derivatives was similar and approximately 10-fold greater than naloxone in this in vivo test (pA2 values from 11.2-11.9). CTAP and CTOP gave comparable results (competitive mu antagonism, lack of agonist activity) when administered intraspinally in the gastrointestinal transit inhibition test with apparent antagonist potency also approximately 10-fold greater than naloxone. (pA2 values of 11.4 and 11.3, respectively). At present, these compounds represent the most selective and potent mu opioid antagonists yet synthesized.
• Landis, G., Lui, G., Shook, J. E., Yamamura, H. I., Burks, T. F., & Hruby, V. J. (1989). Synthesis of highly μ and δ opioid receptor selective peptides containing a photoaffinity group. Journal of Medicinal Chemistry, 32(3), 638-643.
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  PMID: 2537426;Abstract: A series of cyclic, conformationally constrained photolabile peptides related to the enkephalins and to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the δ and μ opioid receptors. The following new peptides were prepared and tested for their δ opioid receptor potency and selectivity in the guinea pig ileum assay, the mouse vas deferens assay, and the rat brain binding assay: H-Tyr-D-Pen-Gly-p-NH2Phe-D-Pen-OH (1, [p-NH2Phe4]DPDPE) and H-Tyr-D-Pen-Gly-p-N3Phe-D-Pen-OH (2, [p-N3Phe4]-DPDPE). The following new peptides were prepared and tested for their μ opioid receptor potency and selectivity in the same assays: H-D-Phe-Cys-p-NH2Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (3, [p-NH2Phe3]CTP) and D-Phe-Cys-p-N3Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (4, [p-N3Phe3]CTP). The δ selective photoaffinity peptide 2 displayed both high affinity (IC50 = 9.5 nM) and good selectivity (IC50 μ/IC50 δ = 1053) as an agonist at δ opioid receptors in bioassays, and 2 also displayed moderate affinity (33 nM) and excellent selectivity (IC50 μ/IC50 δ = 110) for rat brain δ opioid receptors. The μ selective photoaffinity peptide 4 displayed very weak affinity (8% contraction at 300 nM) at μ opioid receptors in bioassays, but good affinity (IC50 = 48.6 nM) and excellent selectivity (IC50 δ/IC50 μ = 412) for the rat brain μ opioid receptors. These conformationally constrained cyclic photoaffinity peptides may be useful tools to investigate the pharmacology of δ and opioid receptors. © 1989 American Chemical Society.
• Lebi, M., Hruby, V. J., M., A., & Hadley, M. E. (1989). Melanin concentrating hormone analogues: Contraction of the cyclic structure. II. Antagonist activity. Life Sciences, 44(7), 451-457.
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  PMID: 2784530;Abstract: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, melanin concentrating hormone (MCH), is a cyclic hormone possessing both MCH-like (melanin granule aggregating effect) and melanocyte stimulating hormone (MSH)-like (melanin granule dispersing effect) activities. Nine ring-contracted analogues were synthesized and characterized for their melanotropic activity on the fish (Synbranchus marmoratus) and frog (Rana pipiens) bioassays. In most cases, these analogues were totally devoid of MCH-like agonist activity, demonstrating the essential role of the disulfide bridge between residues 5 and 14 of the hormone. [Ala5, Cys10]MCH, for example, was totally devoid of MCH-like activity. This analogue, like α-MSH, however, antagonized the melanosome aggregating actions of MCH on fish melanocytes. The antagonistic activity of the analogue, like that of α-MSH, was Ca2+-dependent. Evidence suggested that this antagonism of MCH activity was related to the intrinsic MSH-like activity of the analogue. These results suggest that MCH and α-MSH may be structurally and, therefore, evolutionarily related. © 1989.
• Maria, A., Hadley, M. E., Lebl, M., Zechel, C., & Hruby, V. J. (1989). Melanocyte stimulating hormone and melanin concentrating hormone may be structurally and evolutionarily related. Regulatory Peptides, 24(1), 27-35.
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  PMID: 2544929;Abstract: Two melanotropic peptides, melanin concentrating hormone (MCH) and α-melanocyte stimulating hormone (α-MSH), exert opposing actions on melanosome (melanin granule) movements within teleost pigment cells, melanocytes (melanophores). MCH stimulates melanosome aggregation to the cell center whereas α-MSH stimulates pigment organelle dispersion out into the dendritic processes of the melanocytes. The actions of α-MSH are dependent upon extracellular calcium (Ca2+), whereas those of MCH are actually enhanced in the absence of the cation. At high concentrations (10-5-10-8 M) MCH also exhibits MSH-like activity (autoantagonism), an effect which is abolished in the absence of Ca2+. Therefore, MCH exhibits MCH-like as well as MSH-like activity depending on the presence or absence of extracellular Ca2+. An analogue of MCH, [Ala5, Cys10]MCH, has been synthesized which is totally devoid of MCH activity but still exhibits MSH-like activity. These results suggest that the two melanotropic peptides share some component of structural similarity and may be evolutionarily related. © 1989.
• Maria, A., Hadley, M. E., Wilkes, B. C., Hruby, V. J., & Sawyer, T. K. (1989). Melanotropin structure-activity studies on melanocytes of the teleost fish, Synbranchus marmoratus. General and Comparative Endocrinology, 74(2), 209-214.
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  PMID: 2714625;Abstract: The minimal sequence of α-MSH required for full agonism on fish (Synbranchus marmoratus) melanocytes was determined to be Ac-α-MSH5-10-NH2 since Ac-α-MSH6-10-NH2 and Ac-α-MSH6-9-NH2 were inactive. The N-terminal tripeptide sequence, Ser-Tyr-Ser, lacked any contribution to potency since the 4-13 (Ac-[Nle4]-α-MSH4-13-NH2) sequence was equipotent to α-MSH. The important potentiating amino acids were found to be Met at position 4 of the amino terminus and Val at position 13 of the carboxy terminus of the hormone, since Ac-α-MSH4-10-NH2 was about 100 times more potent than the Ac-α-MSH5-10-NH2 sequence, and Ac-[Nle4]-α-MSH4-13-NH2 was about 10 times more active than Ac-[Nle4]-α-MSH4-12-NH2. The minimal sequence for equipotency to α-MSH was demonstrated to be Ac-[Nle4]-α-MSH4-13-NH2. [Nle4, d-Phe7]-α-MSH was about 10 times more active than α-MSH. Unexpectingly, several conformationally restricted cyclic melanotropins were either partial agonists ([Cys4, Cys10]-α-MSH) or totally inactive (Ac[Cys4, Cys10]-α-MSH4-10-NH2) on fish melanocytes. These results point out some rather remarkable differences between S. marmoratus and tetrapod melanophores relative to structural requirements for MSH receptor recognition and signal transduction. © 1989.
• Maria, A., Lebl, M., Hruby, V. J., Matsunaga, T. O., & Hadley, M. E. (1989). Melanin concentrating hormone (MCH): The message sequence. Life Sciences, 45(13), 1141-1148.
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  PMID: 2796600;Abstract: Melanin concentrating hormone (MCH) is a heptadecapeptide synthesized by the hypothalamus and secreted by the neurohypophysis of the teleost pituitary gland. MCH stimulates melanosome aggregation within teleost melanocytes but also exhibits MSH-like (melanosome dispersing) activity on tetrapod (frog and lizard) melanocytes. We have synthesized a number of MCH analogues to determine the essential features of the primary structure necessary to stimulate either melanosome aggregation or dispersion in fish or tetrapod melanocytes, respectively. An analysis of the potencies and actions of these analogues on vertebrate melanocytes is provided and demonstrates that the two activities have different structural requirements. © 1989.
• Matsunaga, T. O., Hruby, V. J., Lebl, M., Maria, A., & Hadley, M. E. (1989). Melanin concentrating hormone (MCH): Structure-function aspects of its melanocyte stimulating hormone-like (MSH-like) activity. Peptides, 10(4), 773-778.
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  PMID: 2587419;Abstract: Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the brain and secreted from the pars nervosa of teleost fish. This hormone stimulates melanosome (melanin granule) aggregation within integumental melanocytes of fishes but, in contrast, stimulates melansome dispersion within tetrapod (frog and lizard) melanocytes. We determined the message sequence of the primary structure of MCH which is responsible for its MSH-like component of activity. Removal of the N-terminal amino acid results in an almost total loss of MSH-like activity. The C-terminal amino acid is also essential for full MSH-like activity since the analogue, MCH(1-16), is about 100 times less active than MCH. Therefore, the entire heptadecapeptide sequence of MCH appears to contribute to the MSH-like activity of MCH. Ring-contracted analogues (e.g., [Ala5,Cys10]MCH) of MCH are almost devoid of any melanosome aggregating (MCH-like) activity but generally possess considerable or as great an MSH-like activity as MCH. Racemization of MCH by heat-alkali treatment drastically reduces the MCH-like activity of MCH, but does not enhance the MSH-like activity of the hormone. © 1989.
• Matsunaga, T. O., Maria, A., Hadley, M. E., & Hruby, V. J. (1989). Melanin concentrating hormone (MCH): Synthesis and bioactivity studies of MCH fragment analogues. Peptides, 10(2), 349-354.
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  PMID: 2755875;Abstract: Nineteen analogues of melanin concentrating hormone (MCH) were synthesized and tested for their skin-lightening activities in the in vitro eel skin (Synbranchus marmoratus) bioassay. All the analogues synthesized were fragments of the native sequence: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val with sequential elimination of substituents from both the carboxy- and amino-termini. All the analogues that contained trytophan in position 15 were found to be full agonists and equipotent to MCH. In the absence of Trp15, full agonist activity was maintained but potency was reduced ten-fold or more. The minimal fragment analogue possessing equipotency to the parent peptide, MCH, was the MCH(5-15) sequence. These observations coupled with results from work reported previously by our laboratories suggest the importance of the Trp15 residue for interaction with the MCH receptor in this assay system. © 1989.
• Nicolas, E., Dharanipragada, R., Toth, G., & Hruby, V. J. (1989). Asymmetric synthesis of unusual amino acids: Synthesis of optically pure isomers of β-methyltyrosine. Tetrahedron Letters, 30(49), 6845-6848.
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  Abstract: Synthesis of optically pure isomers of β-methyltyrosine has been accomplished. © 1989.
• Patterson, T. A., Schulteis, G., Alvarado, M. C., Martinez Jr., J. L., Bennett, E. L., Rosenzweig, M. R., & Hruby, V. J. (1989). Influence of Opioid Peptides on Learning and Memory Processes in the Chick. Behavioral Neuroscience, 103(2), 429-437.
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  PMID: 2539840;Abstract: Several experiments were conducted to examine the effects of intracranial injection of opioid peptides and antagonists on learning and memory in the chick. Pretraining injection of [leu5]enkephalin and the selective delta receptor agonist [D-Pen2, L-Pen5]enkephalin (DPLPE) into the intermediate medial hyperstriatum ventrale (IMHV) produced impairment. ICI 174,864, a delta-selective antagonist, reversed the impairment produced by either [leu5]enkephalin or DPLPE, results indicating that delta receptors may play a role in learning in the chick and suggesting that the impairment produced by [leu5]enkephalin is mediated through delta opioid receptors. β-endorphin produced a naloxone-reversible impairment in performance, which suggests that this impairment is mediated by opioid receptors. Bilateral injection of β-endorphin into the IMHV produced impairment, as did unilateral injection into the right, but not left, IMHV. Only bilateral injections into IMHV of [leu5]enkephalin were effective. These results suggest that the effects of β-endorphin are centrally mediated whereas the effects of [leu5]enkephalin may be localized to other brain regions or are peripherally mediated. These initial results suggest that opioids are associated with learning and memory in the chick.
• Shook, J. E., Lemcke, P. K., Gehrig, C. A., Hruby, V. J., & Burks, T. F. (1989). Antidiarrheal properties of supraspinal mu and delta and peripheral mu, delta and kappa opioid receptors: Inhibition of diarrhea without constipation. Journal of Pharmacology and Experimental Therapeutics, 249(1), 83-90.
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  PMID: 2540324;Abstract: We evaluated the ability of mu [morphine, Tyr-Pro-N-MePhe-D-Pro-NH2 (PLO17)], delta (Tyr-D-Pen-Gly-Phe-D-Pen) (DPDPE) and kappa [U50,488H, (trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclo- hexyl)benzeneacetamine)] opioid receptor selective agonists to inhibit diarrhea induced by castor oil (0.6 ml p.o.) in mice after supraspinal (i.c.v.) and peripheral (s.c.) administration. The antidiarrheal potency of each compound was compared to its analgesic and gastrointestinal antitransit potency when given by the same route of administration. When administered i.c.v. morphine, PLO17 and DPDPE inhibited diarrhea in a dose-related fashion. The mu agonists, morphine and PLO17, given i.c.v., inhibited diarrhea at doses much lower than those needed to produce analgesia or to inhibit gastrointestinal transit. DPDPE (i.c.v.) was equipotent in inhibiting diarrhea and in eliciting analgesia, but did not effect the rate of transit. U50,488H (i.c.v.) inhibited diarrhea only at extremely high doses which also caused profound postural-motor incapacitance. U50,488H given i.c.v. had no effect on transit at any dose. When given peripherally, morphine, PLO17, DPDPE and U50,488H all inhibited diarrhea in a dose-related fashion. All four compounds inhibited diarrhea at doses much below those needed to cause analgesia. Morphine s.c. and PLO17 s.c. both inhibited diarrhea at doses lower than those required to inhibit transit. DPDPE s.c. and U50,488H s.c. had no effect on transit at any dose. The antidiarrheal effects of i.c.v. morphine, i.c.v. PLO17 and i.c.v. DPDPE were antagonized by pretreatment with 1 μg i.c.v. of naltrexone. Likewise, the antidiarrheal actions of s.c. PLO17, s.c. DPDPE and s.c. U50,488H were all blocked by 1 mg/kg s.c. of naltrexone. These findings indicate that supraspinal mu and delta, and peripheral mu, delta and kappa opioid receptors all possess antidiarrheal activity. Each compound prevented diarrhea without slowing the rate of transit, indicating an antisecretory-related mechanism of antidiarrheal action.
• Vaughn, L. K., Knapp, R. J., Toth, G., Wan, Y. -., Hruby, V. J., & Yamamura, H. I. (1989). A high affinity, highly selective ligand for the delta opioid receptor: [³H]-[D-PEN², pCl-PHE⁴, D-PEN⁵]enkephalin. Life Sciences, 45(11), 1001-1008.
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  PMID: 2552241;Abstract: Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25°C determined a dissociation constant (Kd) of 328 ± 27 pM and a receptor density (Bmax) of 87.2 ± 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 × 108 ± 2.5 × 108 and 0.147 ± 108 ± 0.014 × 108 M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 × 10-3 ± 0.25 × 10-1 min-1. The average Kd values determined by these kinetic studies were 8.4 ± 2.7 pM and 201 ± 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor. © 1989.
• Visconti, M. A., Castrucci, A. M., Hadley, M. E., & Hruby, V. J. (1989). Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores.. Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 2(3), 213-217.
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  PMID: 2771878;Abstract: Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.
• Wynants, C., Tourwe, D., Kazmierski, W., Hruby, V. J., & Binst, G. V. (1989). Conformation of two somatostatin analogues in aqueous solution. Study by NMR methods and circular dichroism. European Journal of Biochemistry, 185(2), 371-381.
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  PMID: 2573530;Abstract: Cyclic-disulfide-containing analogues of somatostatin, Xaa1-Cys2-Xaa3-DTrp4-Lys6-Thr5-Xaa7-Xaa8 [Xaa1 = H or DPhe; Xaa3 = Phe or Tyr; Xaa7 = Cys, Me2DCys or Me2DCys; Xaa8 = OH, Thr8 (OH) or Thr8NH2], were examined in aqueous solution by 1H-NMR spectroscopy and circular dichroism. The influence of the helical nature of the disulfide bridge and the presence of exocyclic residues on biological activity were investigated with particular care.
• Ayres, E. A., Villar, R., Kramer, T. H., Kazmierski, W., Hruby, V. J., & Burks, T. F. (1988). Highly selective mu opioid antagonist peptides block spinal mu opioid inhibition of gastrointestinal transit. Proceedings of the Western Pharmacology Society, 31, 41-43.
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  PMID: 2905467;
• Chan, W. Y., & Hruby, V. J. (1988). Natriuretic action of neurohypophysial peptides: Effects of agonists and antagonists and implication of natriuretic receptor. Journal of Pharmacology and Experimental Therapeutics, 246(2), 597-602.
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  PMID: 2969979;Abstract: Neurohypophysial peptides possess natriuretic activity. Although it has been shown that the natriuretic action of these peptides can be dissociated from their antidiuretic activity (a V2-receptor mediated response), it is not known whether the V1-receptor or yet a third receptor type mediates the natriuretic response. Also, it has not been studied what effects V1- and V2-antagonists may have on urinary sodium excretion. To define this, we have studied the effects of four oxytocin (OT) agonists: arginine-vasopressin, OT, [Leu4)OT and [cyclo-Leu8]OT; two V1-receptor antagonists: [penicillamine1,Phe(Methyl)2,Thr4,Orn8)OT and [penicillamine1,D-Phe)Ethyl)2,Thr4,Orn8]OT and one V2-receptor antagonist: d-(CH2)5[D-Ile2,α-aminobutyric acid4]arginine-vasopressin on renal excretion of water and electrolytes in anesthetized rats under water diuresis. We also studied the effects of the antagonists on the OT-induced antidiuretic and natriuretic responses. Only the agonists, but not the antagonists, were found to have natriuretic activity. The natriuretic potency, was not related to the peptide's antidiuretic activity, but was in the same rank order as their oxytocic activity (a V1-agonist effect). The effects of the antagonists on the OT-induced renal responses were studied at two dose levels, representing a strong and near maximal of their respective V1 and V2 inhibitory doses. The V1-antagonist had no effect on the antidiuretic response to OT but inhibited the natriuretic response in a dose-dependent manner. The antinatriuretic effect was also long-lasting as its antioxytocic activity. The V2-antagonist inhibited the antidiuretic response to OT in a dose-dependent manner but only the high dose inhibited the natriuretic response. These results indicate that the natriuretic action of OT was not mediated by V2-receptors and antinatriuresis was not specific for V1-antagonist. This suggests that a V1-subtype or a third receptor type may be involved.
• Dawson, B. V., Hadley, M. E., Kreutzfeld, K., Dorr, R. T., Hruby, V. J., Al-Obeidi, F., & Don, S. (1988). Transdermal delivery of a melanotropic peptide hormone analogue. Life Sciences, 43(14), 1111-1117.
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  PMID: 2845208;Abstract: We previously reported that topical application of [Nle4,D-Phe7]alpha-MSH, a superpotent analogue of alpha-melanocyte stimulating hormone, to mice induces a darkening of follicular melanocytes throughout the skin. We now report that the melanotropin analogue can be delivered across mouse but not rat skin in an in vitro model system. Passage of the analogue from the topically applied vehicle (polyethylene glycol) across the skin into a subcutaneous receiving vessel was demonstrated by both bioassay as well as by radioimmunoassay. The bioassay data demonstrate that percutaneous absorption of the melanotropin did not result in loss of biological activity of the peptide. The differential penetration of the peptide across rodent skin reveals that one cannot predict percutaneous absorption of a substance across the stratum corneum from studies on a single species. The present results are the first to demonstrate, by direct quantitative measurements, that a bioactive peptide can be delivered across the vertebrate integument in vitro. These studies point out the potential of a topically applied melanotropin for tanning of the skin and possibly for treatment of certain hypopigmentary disorders. © 1988.
• Dorr, R. T., Dawson, B. V., Al-Obeidi, F., Hadley, M. E., Levine, N., & Hruby, V. J. (1988). Toxicologic studies of a superpotent α-melanotropin, [Nle⁴, D-Phe⁷]α-MSH. Investigational New Drugs, 6(4), 251-258.
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  PMID: 2852652;Abstract: A toxicology study was performed in mice given a superpotent α melanocyte stimulating hormone (MSH) analog. This 13 amino acid derivative, [Nle4, D-Phe7]α-MSH or NDP-MSH, is a melanotropin which is very slowly biodegraded in vivo and is active at 1/1,000 the concentration of natural α-MSH. Mice were administered up to 2 mg/kg of the analog daily and weekly over 4 or 12 weeks by both topical application (in 90% DMSO) or by IP injections (in physiologic saline). At the end of this period, no toxic effects were observed in various organs, on hematologic indices, or on weight gain. A slight increase in triglyceride and platelet levels were noted in mice given the analog weekly for 12 weeks. There was no evidence of an effect on behavior nor ACTH-like endocrine actions such as elevated serum cortisol levels. Transdermal drug delivery studies performed in vitro showed reproducible diffusion of the NDP-MSH analog through full-thickness mouse skin. Approximately 0.002% to 0.05% of a 10-4M preparation was transdermally delivered using a DMSO/water solution or a PEG/alcohol cream base, respectively. This superpotent analog is now entering a Phase I clinical trial with possible therapeutic applications for the treatment of hypomelanotic disorders such as vitiligo and for pharmacologic tanning without the need for sunlight exposure. © 1988 Kluwer Academic Publishers.
• Hadley, M. E., de, A., & Hruby, V. J. (1988). Melanin concentrating hormone (MCH) mechanisms of action.. Progress in clinical and biological research, 256, 531-545.
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  PMID: 3368499;
• Hawkins, K. N., Knapp, R. J., Gehlert, D. R., Lui, G. K., Yamamura, M. S., Roeske, L. C., Hruby, V. J., & Yamamura, H. I. (1988). Quantitative autoradiography of [³H]CTOP binding to mu opioid receptors in rat brain. Life Sciences, 42(25), 2541-2551.
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  PMID: 2898716;Abstract: [3H]-HDPhetsqbCysTyrDTrpOrnThrPenThrNH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69,593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding. © 1988.
• Hruby, V. J. (1988). In memoriam Choh Hao Li, April 21, 1913-November 28, 1987.. International Journal of Peptide and Protein Research, 31(3), 253-254.
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  PMID: 3286549;
• Hruby, V. J., & Schwyzer, R. (1988). Preface. Tetrahedron, 44(3), xi.
• Hruby, V. J., Kao, L., Pettitt, B. M., & Karplus, M. (1988). The conformational properties of the delta opioid peptide [D-Pen²,D-Pen⁵]enkephalin in aqueous solution determined by NMR and energy minimization calculations. Journal of the American Chemical Society, 110(11), 3351-3359.
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  Abstract: The conformational properties of the highly potent delta opioid receptor selective cyclic peptide [D-Pen2,D-Pen5]enkephalin (DPDPE) have been investigated by use of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, molecular modeling based on the NMR results, and molecular mechanics energy minimization. A new method for elimination of H2O (HOD) signals was used which, in conjunction with 2D methods, made possible a complete assignment of all hydrogen atoms in DPDPE. Additional computer simulations allowed an accurate determination of all 3J and 2J coupling constants. NOESY experiments gave direct evidence for transannular interactions of the Tyr1 and Phe4 aromatic rings with the β,β-dimethyl groups of D-Pen2. Utilizing NMR parameters in conjunction with model building, extensive energy minimization studies led to two pairs of very similar energy-minimized conformations for DPDPE. Each pair of conformations primarily differed by the sign of the disulfide helicity. One pair was of lower energy and satisfied all of the NMR criteria. Its conformation is distinguished by an amphiphilic conformation with a type IV β-turn and transannular interactions between the aromatic side chains of Tyr1 and Phe4 with the β,β-dimethyl groups of D-Pen2. The factors which stabilize this conformation are discussed, and the possible relationship of this conformation to the high delta opioid receptor selectivity is suggested. © 1988 American Chemical Society.
• Hussin, A. H., Allan, C. J., Hruby, V. J., & Skett, P. (1988). The effects of glucagon and TH-glucagon on steroid metabolism in isolated rat hepatocytes. Molecular and Cellular Endocrinology, 55(2-3), 203-207.
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  PMID: 3128458;Abstract: Glucagon decreases the activity of steroid-metabolising enzymes in isolated rat liver cells at physiological concentrations. Higher concentrations are less effective. TH-glucagon (1-N-α-trinitrophenylhistidine-12-homoarginine-glucagon) also reduces enzyme activity but does not lose activity at higher concentrations. The effects of the two hormones mimic closely their reported effects on phosphatidylinositol-4,5-bisphosphate breakdown. It is, thus, likely that the effect of glucagon on steroid metabolism is mediated via breakdown of this phospholipid. The calcium ionophore, A23187, had no effect on steroid metabolism whereas the phorbol ester 4β-phorbol-12-myristate-13-acetate (PMA) mimicked the effect of glucagon, showing that activation of protein kinase C but not Ca2+ mobilization may be involved in glucagon's action on hepatic steroid metabolism. © 1988.
• Kazmierski, W., & Hruby, V. J. (1988). A new approach to receptor ligand design: synthesis and conformation of a new class of potent and highly selective μ opioid antagonists utilizing tetrahydroisoouinoline carroxylic acid. Tetrahedron, 44(3), 697-710.
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  Abstract: Investigations of the physiological functions of opioid receptors (μ,δ,κ and others) require potent and selective receptor ligands. Conformational constraint provides a useful approach to increase receptor selectivity of flexible peptides. This approach reduces the set of low energy conformations accessible for the ligand and thus can provide insight into topologica1 features that may be responsible for high affinity to a particular receptor subtype. Using this approach, we describe a new class of potent and selective μ opioid receptor antagonists and demonstrate a new approach for the design of receptor specific ligands by which a low affinity, "non-physiological" activity of a natural peptide hormone is converted to a high potency, receptor selective ligand for that receptor, and, at the same time, eliminates the activity at the natural receptors for the peptide. Recently we reported the design and synthesis of a new class of μ opioid receptor selective antagonists, of which {A figure is presented}(CTP) was found to be among the most potent and selective, and {A figure is presented}(PCTP) exhibited a sharp decrease of affinity for μ receptors ( 100 foTd) and a modest increase ( 3 fold) in affinity for δ receptors. We now present 1h NMR evidence which suggests a more folded conformation for the latter compound. This result led to the design of further constrained analogues in which a methylene bridge is inserted between the α-amino group and the 2'position of the aromatic ring of n-phe1 in CTP. This analogue {A figure is presented}(TCTP) was found to be the most μ vs. δ receptor selective ligand known (> 9000 fold selective), with very litt1e somatostatin-like activity. NMR investigations have revealed that the side chain of D-Tic residue exists exclusively in a g- conformation. Disconnection of this methylene bridge via synthesis of {A figure is presented} gave an analogue that exhibited low affinity for the μ opioid receptor and greatly reduced selectivity. NMR investigations have uncovered a large participation of g+ and trans side chain conformations for the aromatic ring in the D-N-MePhe residue, and a more folded overall conformation. These results illustrate how constraint of side chain moieties of critical amino acid residues to a specific or "biased" conformation can provide important insights into the topological requirements for peptide-receptor interactions and can contribute to design of ligands for receptor mapping. © 1988.
• Kazmierski, W., Wire, W. S., Lui, G. K., Knapp, R. J., Shook, J. E., Burks, T. F., Yamamura, H. I., & Hruby, V. J. (1988). Design and synthesis of somatostatin analogues with topographical properties that lead to highly potent and specific μ opioid receptor antagonists with greatly reduced binding at somatostatin receptors. Journal of Medicinal Chemistry, 31(11), 2170-2177.
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  PMID: 2903246;Abstract: A series of conformationally restricted, cyclic octapeptides containing a conformationally stable tetrapeptide sequence related to somatostatin, -Tyr-D-Trp-Lys-Thr-, as a template, were designed and synthesized with the goal of developing highly potent and selective μ opioid antagonists with minimal or no somatostatin-like activity. Three distinct structures of the peptide became targets of chemical modifications and constraints; the N- and C-terminal amino acids and the cyclic 20-membered ring moiety. Based on the conformational analysis of active and inactive analogues of the parent peptide D-Phe1-Cys2-Tyr3-D-Trp4-Lys 5-Thr6-Pen7-Thr8-NH2, CTP (Kazmierski, W.; Hruby, V. J. Tetrahedron 1988, 44, 697-710), we designed analogues to include the tetrahydroisoquinolinecarboxylate (Tic) moiety as the N-terminal amino acid instead of D-Phe, since Tic can exist only as a gauche (-) or a gauche (+) conformer. In this series, the following peptides were synthesized and pharmacologically evaluated: D-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (TCTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (TCTOP), and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (TCTAP). In rat brain membrane opioid radioligand binding assays, all three peptides displayed high affinity for μ opioid receptors (IC50 = 1.2, 1.4, 1.2 nM, respectively), and exceptional μ vs δ opioid receptor selectivity: 7770, 11 396, and 1060, respectively. TCTOP and TCTAP also possess exceptional μ vs somatostatin receptor selectivity: 14 574 and 28 613, respectively. In the peripheral in vitro GPI bioassay, TCTP, TCTOP, and TCTAP were highly effective antagonists of the potent μ opioid receptor agonist PL017, with pA2 = 8.69 for TCTAP, 8.10 for TCTP, and 7.38 for TCTOP. Our results show that a 10-fold higher affinity and selectivity for μ opioid receptors (in both central and peripheral studies) over δ and somatostatin receptor was gained as a result of the D-Tic1 substitution. These three peptides, TCTP, TCTOP, and TCTAP, are the most potent and selective μ opioid antagonists known. CTP has been shown to possess prolonged biological action, much longer than that of naloxone. This renders these analogues potentially useful ligands for investigating the physiological functions of the μ opioid receptor. Analogues of TCTP in which the 20-membered disulfide ring was contracted by deletion of D-Trp4, and/or Lys5, and/or Thr6 led to compounds with greatly reduced potency at the μ opioid receptor. Furthermore, modification of the Thr8 residue with Val, Ser, Asn, and Asp or by simple deletion of the residue led to analogues with greatly reduced potency and reduced μ opioid receptor selectivity. © 1988 American Chemical Society.
• Kramer, T., Kazmierski, W., Shook, J., Hruby, V., & Burks, T. (1988). A novel peptidic mu opioid antagonist with exceptional potency and specificity. NIDA Research Monograph Series, 47-.
• Krstenansky, J. L., Zechel, C., Trivedi, D., & Hruby, V. J. (1988). Importance of the C-terminal α-helical structure for glucagon's biological activity. International Journal of Peptide and Protein Research, 32(6), 468-475.
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  PMID: 2854536;
• Landis, G. C., Eckman, D., Hruby, V. J., & Kreulen, D. L. (1988). Temperature dependence of the effect of tachykinins on whole guinea pig ileum in vitro. Proceedings of the Western Pharmacology Society, 31, 29-32.
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  PMID: 2463638;
• Lebl, M., Hruby, V. J., M., A., Visconti, M. A., & Hadley, M. E. (1988). Melanin concentrating hormone analogues: Contraction of the cyclic structure. 1. Agonist activity. Journal of Medicinal Chemistry, 31(5), 949-954.
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  PMID: 3258925;Abstract: Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which is synthesized in the hypothalamus and secreted by the neurohypophysis of teleost fishes. This hormone exhibits both MCH-like as well as α-MSH (α-melanocyte stimulating hormone) like activity. We have examined the role of the disulfide bond for the two contrasting melanotropic activities of MCH. Nine analogues of the parent peptide were synthesized and characterized for biological activity. The disulfide ring was contracted from the 5-14 to the 7-14, 8-14, and 10-14 residues with concomitant substitution of alanine for Cys at position 5 in each of the heptadecapeptides. Similar substitutions were made in a series of MCH5-17 analogues. In addition, the following cyclic peptides also were synthesized: [Cys7]MCH7-17, [Cys8]MCH8-17, and [Cys10]MCH10-17. The fish-skin bioassay is sensitive to MCH at a concentration of 10-12 M. All ring-contracted analogues were inactive at 10-6 M or lower concentrations; less than 1/1 000 000 compared to MCH (1.0) except [Ala5,Cys8]MCH5-17 (0.0008; 1/1250), [Cys10]MCH10-17 (0.00009; 1/10 000), and [Cys8]MCH8-17 (0.000001; 1/1 000 000). In the frog-skin bioassay, [Ala5,Cys10]MCH, although lacking MCH-like activity in the fish-skin bioassay, was equipotent to MCH in its α-MSH-like component of activity. Most other analogues were either inactive or much less active than MCH in stimulating melanosome dispersion. These results demonstrate that the disulfide bond between positions 5 and 14 is essential for the MCH-like activity since contraction of the ring generally leads to inactive peptides. Contraction of the disulfide bridge does not, however, have as great an effect on the MSH-like activity of MCH. © 1988 American Chemical Society.
• Levine, N., Hadley, M. E., & Hruby, V. J. (1988). Reply. Journal of Investigative Dermatology, 91(6), 606-.
• Mckee, R. L., Hruby, V. J., Trivedi, D. B., Johnson, D. G., Gandolfi, A., Krumdieck, C. L., & Brendel, K. (1988). Perifused precision-cut liver slice system for the study of hormone-regulated hepatic glucose metabolism. Journal of Pharmacological Methods, 19(4), 339-354.
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  PMID: 2840533;Abstract: A nonrecirculatory perifusion system for precision-cut rat liver slices has been developed and utilized for investigating hormone-regulated hepatic glucose metabolism. In this system, slices are cultured in a highly controlled environment and exhibit excellent retention of viability as judged by their maintenance of intracellular potassium and glycogen contents. Using this system, the complex physiological phenomenon of hormone-regulated glycogenolysis was investigated at both extra- and intracellular sites. Specifically, the sensitive responses of intracellular cyclic AMP (cAMP) production, activation of cyclic AMP-dependent protein kinase, and production of glucose upon glucagon stimulation have been measured. The maximal responses observed for these parameters were either equal to or greater than those previously reported for either isolated hepatocytes or perfused livers, demonstrating the sensitivity of this technique. Upon doseresponse examination of glucagon challenge, it was observed that high doses of glucagon (>16 nM) stimulate glucose production by activating the cAMP-second messenger cascade. In contrast, low doses (
• Mulder, A. H., Wardeh, G., Kazmierski, W., & Hruby, V. J. (1988). Antagonist activity of the cyclic somatostatin analogue CTP at μ- but not δ- and κ;;-opioid receptors involved in presynaptic inhibition of neurotransmitter release. European Journal of Pharmacology, 157(1), 109-114.
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  PMID: 2906879;Abstract: In the present study the opioid receptor antagonist properties of the confomationally constrained cyclic octapeptide D-Phe-{A figure is presented}-Thr-NH2 (CTP), which is derived from somatostatin, were investigated, using in vitro functional paradigms of central μ-, δ- and κ-opioid receptors. Activation of μ-opioid receptors by the enkephalin analogues DADLE or DAGO resulted in a strong inhibition (by 60-70%) of the (electrically evoked) release of [3H]noradrenaline (NA) from superfused cortical slices. This inhibitory effect was antagonized by CTP in a competitive fashion (pA2 value 7.7-7.9). Activation of κ-opioid receptors by bremazocine selectively inhibited (by 45-50%) the release of [3H]dopamine (DA) from striatal slices, whereas activation of δ-opioid receptors by DADLE caused an inhibition (by 55-60%) of striatal [14C]acetylcholine (ACh) release, but neither of these inhibitory effects was affected by CTP. By itself, CTP inhibited cortical [3H]NA release (by 35-40%), but it did not affect the release of [3H]DA nor that of [14C]ACh from striatal slices. The inhibitory effect of CTP was not antagonized by naloxone. The data indicate that CTP selectively antagonizes μ-opioid receptors, involved in presynaptic inhibition of NA release in the brain. In addition, the peptide by itself causes an inhibition of NA release via a non-opioid receptor-mediated process. © 1988.
• Pelton, J. T., Whalon, M., Cody, W. L., & Hruby, V. J. (1988). Conformation of D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂ (CTP-NH₂), a highly selective mu-opioid antagonist peptide, by ¹H and ¹³C n.m.r.. International Journal of Peptide and Protein Research, 31(2), 109-115.
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  PMID: 2896638;Abstract: The 1H and 13C n.m.r. spectral parameters of CTP-NH2 [D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2], a potent, highly selective μ-opiate antagonist, were measured in aqueous solution and a possible conformation has been deduced from the spectral data. The data are consistent with a type II' β-turn for the tetrapeptide sequence -Tyr3-D-Trp4-Lys5-Thr6-. Solvent shielding of the Cys2 amide proton, observed in variable temperature experiments, suggestsan orientation of this amide proton toward the gem dimethyls of Pen7 with possible hydrogen bonding to the Thr6 carbonyl oxygen, and a dihedral angle of -110° for the disulfide bond. Partially relaxed Fourier transform 13C relaxation studies confirm a constrained cyclic system, with the Cα carbons in the 'hinge' of the β-turn having the shortest t1 times. Segmental motion was observed for the side chain of Lys5.
• Sawyer, T. K., Hadley, M. E., Hruby, V. J., De, A., Staples, D. J., Farah, J., & O'Donohue, T. (1988). α-Melanocyte-stimulating hormone structure-activity studies: Comparative analysis of melanotropic and CNS bioactivities. Synapse, 2(3), 288-292.
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  PMID: 2850630;Abstract: The structure-activity relationships in vitro of α-MSH (α-melanocyte-stimulating hormone, α-melanotropin) analogs as determined on normal and transformed (melanoma cell) melanocyte bioassays are summarized. Specifically, the characterization of potent and metabolically stable melanotropic agonist analogs and a newly discovered antagonist of α-MSH are highlighted. Comparison of these data versus the known structure-activity relationships of α-MSH related to CNS bioactivities suggests the existence of nonclassical α-MSH receptor-mediated pathways or, perhaps, a yet undefined endogenous neuropeptidergic pathway(s) having different selectivities for α-MSH analogs. In summary, several of the α-MSH analogs reported here may be useful molecular probes in future strategies aimed at the identification and systematic characterization of both peripheral and central α-MSH receptors.
• Schulteis, G., Martinez Jr., J. L., & Hruby, V. J. (1988). Stimulation and Antagonism of Opioid δ-Receptors Produce Opposite Effects on Active Avoidance Conditioning in Mice. Behavioral Neuroscience, 102(5), 678-686.
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  PMID: 2848536;Abstract: The effects of opioid δ-receptor activation on conditioning in a one-way active avoidance paradigm were investigated in mice. Peripheral administration of 30 and 100 μg/kg of [Leu5]enkephalin (LE) and 11.6 μg/kg of [D-Pen2, D-Pen5]enkephalin (DPDPE), a synthetic enkephalin analog with high δ-selectivity, impaired acquisition of avoidance responding. The dose response functions for both peptides were U-shaped. Deficits in responding were also present 24 hours after training, which suggests that the impaired performance observed during training was not an impairment in the ability of the mice to perform. Also, neither LE nor DPDPE decreased footshock sensitivity or open-field activity, which rules out analgesia and decreased activity levels as likely explanations for the deficits in conditioning. Finally, antagonizing endogenous ligands with the δ-selective antagonist ICI 154,129 enhanced acquisition, an effect opposite that produced by LE and DPDPE. The results suggest that activation of δ-receptors is normally involved in the modulation of active avoidance learning.
• Shook, J. E., Kazmierski, W., Wire, W. S., Lemcke, P. K., Hruby, V. J., & Burks, T. F. (1988). Opioid receptor selectivity of β-endorphin in vitro and in vivo: Mu, delta and epsilon receptors. Journal of Pharmacology and Experimental Therapeutics, 246(3), 1018-1025.
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  PMID: 2843623;Abstract: The relative contributions of mu and delta opioid receptors in the response to Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr- Leu-Phe-Lys-Asn-Ala-Ileu-Ileu-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu (B-endorphin) were assessed as reductions in B-endorphin potency in the presence of mu and delta receptor selective antagonists in the guinea pig ileum, mouse vas deferens, rat vas deferens and in analgesic and gastrointestinal transit time tests in mice. We used the nonselective antagonist naloxone, the mu antagonist D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) and the delta antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864) in each test system at concentrations that effectively antagonized the respective mu and delta agonists, Tyr-Pro-N-MePhe-D-Pro-NH2 and Tyr-D-Pen-Gly-Phe-D-Pen. In the guinea pig ileum, the inhibitory effects of 1 μM B-endorphin were blocked by 1 μM CTP and 1 μM naloxone, but not by 1 μM ICI 174,864. In the mouse vas deferens, B-endorphin (0.2 μM) was antagonized by 1 μM CTP, 1 μM ICI 174,864 and by 1 μM naloxone. In contrast, in the rat vas deferens, B-endorphin (0.01-1 μM) produced potent inhibitory actions that were blocked by 1 μM naloxone, but not by 1 μM-CTP or by 1 μM ICI 174,864. The mu agonist Tyr-Pro-N-MePhe-D-Pro-NH2 (0.1-10 μM), like B-endorphin, also had inhibitory actions in the rat vas deferens, but its effects were blocked by 1 μM CTP. The delta agonist Tyr-D-Pen-Gly-Phe-D-Pen (1-10 μM) caused weak inhibition at very high concentrations and, like B-endorphin, was blocked by 1 μM naloxone, but not by 1 μM CTP or 1 μM ICI 174,864. When tested in vivo, centrally administered B-endorphin (0.1-5 μg i.c.v.) produced analgesia and slowed the rate of gastrointestinal transit. Both effects were inhibited by 1 μg of i.c.v. CTP, 3 μg of i.c.v. ICI and 1 μg of i.c.v. naloxone. These findings indicate that B-endorphin enlists both mu and delta receptors to produce its effects in vitro and in vivo, and that B-endorphin in the rat vas deferens acts at another non-mu, non-delta opioid receptor, which may be the putative epsilon receptor. In addition to the epsilon receptor, the rat vas deferens also contains mu, but not delta, opioid receptors.
• Shook, J., Kazmierski, W., Hruby, V., & Burks, T. (1988). Precipitation of spinally mediated withdrawal signs by intrathecal administration of naloxone and the mu-receptor antagonist CTP in morphine-dependent mice. NIDA Research Monograph Series, 143-148.
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  PMID: 2900467;
• Spencer, R. L., Hruby, V. J., & Burks, T. F. (1988). Body temperature response profiles for selective mu, de4lta and kappa opioid agonists in restrained and unrestrained rats. Journal of Pharmacology and Experimental Therapeutics, 246(1), 92-101.
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  PMID: 2839673;Abstract: In many cases, body temperature is altered in response to opioid agonists, but the direction, magnitude and time course of alteration vary with a number of factors. Body temperature may be subject to differential modification by different opioid receptor types. The authors examined the effect (i.c.v.) of the selective mu, delta and kappa opioid agonists, [D-Ala2,MePhe4,Gly5-ol] enkephalin (DAGO), [D-Pen2,D-Pen5] enkephalin and U50488H, respectively, on the body temperature of restrained and unrestrained rats. Each of the three opioid agonists produced a differentiable profile of body temperature changes. DAGO caused a primary decrease in body temperature of restrained rats and an increase in body temperature of unrestrained rats. The pretreatment dose of naloxone necessary to attenuate the hyperthermic response to DAGO of unrestrained rats was 10 times higher than that required to block the hypothermic response to DAGO in restrained rats. Low doses of both [D-Pen2,D-Pen5]enkephalin and U50488H caused a decrease in body temperature of both restrained and unrestrained rats. Hypothermic responses to U50488H were not blocked by naloxone, whereas hypothermic responses to [D-Pen2-D-Pen5]enkephalin in unrestrained rats were potentiated by naloxone. The results indicate that the three compounds modified body temperature by different means, suggesting activation of different opioid, and perhaps nonopioid, receptors. This may reflect a differential modulation of body temperature by endogenous opioids depending on the specific peptide release and the receptor type activated. Besides the physiologic implications, body temperature responses provided a sensitive pharmacologic measure for distinguishing the in vivo activity of different selective opioid agonists.
• Stivers, J. A., Kaltwasser, M. T., Hill, P. S., Hruby, V. J., & Crawley, J. N. (1988). Ventral tegmental oxytocin induces grooming. Peptides, 9(SUPPL. 1), 223-231.
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  PMID: 2856647;Abstract: Bilateral microinjection of oxytocin (OXY) into the ventral tegmental area (VTA) of rat brain produced a significant increase in grooming behaviors at doses from 100 pg to 400 ng. Sites in the caudal region of the VTA were sensitive to lower doses of OXY than sites in the rostral region of the VTA. The time course of action of OXY in the grooming paradigm indicated onset beginning immediately after injection, and termination at 60-75 minutes after injection. Comparison of OXY-induced grooming in male, female, and ovariectomized, estrogen-treated female rats showed no differences in potency for OXY among these groups, suggesting that the grooming effects of OXY are not regulated by sex steroids. Analysis of locomotor activity in rats microinjected with OXY 200 ng bilaterally into the caudal VTA revealed that OXY had no effect on ambulatory locomotion, suggesting that this peptide may activate neurons within the VTA which mediate grooming but not locomotion. The OXY receptor antagonist, [Pen1, pMePhe2, Thr4, Orn8]-OT, blocked OXY-induced grooming when both were simultaneously microinjected into the VTA. The dopamine D-2 receptor antagonist, haloperidol, and the D-1 receptor antagonist, SCH 23390, when microinjected into the VTA five minutes before microinjection of OXY into the VTA, did not block OXY-induced grooming, suggesting that OXY is not working through a dopamine autoreceptor on the VTA neurons. Systemic pretreatment with haloperidol and SCH 23390 effectively blocked grooming induced by OXY in the VTA, suggesting that OXY is directly stimulating OXY receptors on VTA neurons to release dopamine at postsynaptic sites regulating grooming behaviors. © 1988.
• Sugg, E. E., Maria, A., Hadley, M. E., Binst, G. V., & Hruby, V. J. (1988). Cyclic lactam analogues of Ac-[Nle⁴]α-MSH_4-11-H₂. Biochemistry, 27(21), 8181-8188.
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  PMID: 2852955;Abstract: Two side-chain cyclic lactam analogues of the 4-11 fragment of α-melanocyte-stimulating hormone (α-MSH), Ac-[Nle4,D-Orn5,Glu8]α-MSH 4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu 8]α-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of Nα-Boc and Nα-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of α-MSH, Ac-[Nle4]α-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]α-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]α-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (≥4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10000 times less potent than linear Ac-[Nle4,D-Phe7]α-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]α-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10000-fold less potent than both Ac-[Nle4]α-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]α-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible. More importantly, these results suggest that melanotropic potency may be correlated with a close spatial relationship between the side chains of His6, Phe7, and Trp9. © 1988 American Chemical Society.
• Sugg, E. E., Serra, M., Shook, J. E., Yamamura, H. I., Burks, T. F., Korc, M., & Hruby, V. J. (1988). Cholecystokinic activity of N(α)-hydroxysulfonyl-[Nle^28,31]CCK_26-33 analogues modified at the C-terminal residue. International Journal of Peptide and Protein Research, 31(6), 514-519.
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  PMID: 2457563;Abstract: Three new analogues of N(α)-hydroxysulfonyl-[Nle28,31]CCK26-33 are reported in which the C-terminal L-Phe33 residue has been replaced by L-Leu, D-Phe or N-methyl-L-Phe. Biological evaluation in a series of binding and bioassays demonstrates that both L-stereochemistry and an aromatic side chain at position-33 are essential for full agonist activity. While the L-Leu33 and D-Phe33 analogues had reduced potencies in stimulating contraction of the guinea pig ileum or gall bladder, the D-Phe33 analogue was foufold selective for the ileum. This latter analogue also exhibited apparent partial agonism in the rat pancreatic amylase release assay. The N-methyl-L-Phe33 analogue was almost equipotent to the parent analogue in all bioassays, suggesting that this modification might be useful for introducing enzymatic stability in CCK analogues.
• Sugg, E. E., Tourwe, D., Kazmierski, W., Hruby, V. J., & Binst, G. V. (1988). Proton n..m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system op opioid receptor selective somatostatin derivatives. International Journal of Peptide and Protein Research, 31(2), 192-200.
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  PMID: 2896639;Abstract: Three cyclic disulfide analogs related to somatostatin, D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Thr6-Xxx7-Thr8NH2 (where Xxx = L-Pen 1; L-Cys 3; or D-Pen 4) were examined in DMSO-d6 by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a β II' turn solution conformation for the core tetrapeptide-Tyr3-D-Trp4-Lys5-Thr6-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.
• de, A., Visconti, M. A., Hadley, M. E., Hruby, V. J., Oshima, N., & Fujii, R. (1988). Melanin concentrating hormone (MCH) control of chromatophores.. Progress in clinical and biological research, 256, 547-557.
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  PMID: 3368500;
• Barth, T., Slaninova, J., Lebl, M., & Hruby, V. J. (1987). Effect of threonine in position 4 in oxytocin and vasotocin analogs on the time course of uterotonic response. Endocrinologia Experimentalis, 21(3), 191-197.
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  PMID: 3117517;
• Brendel, K., McKee, R. L., Hruby, V. J., Johnson, D. G., Gandolfi, A. J., & Krumdieck, C. L. (1987). Precision cut tissue slices in culture: a new tool in pharmacology.. Proceedings of the Western Pharmacology Society, 30, 291-293.
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  PMID: 3306695;
• Caldwell, J. D., Mason, G. A., Stanley, D. A., Jerdack, G., Hruby, V. J., Hill, P., Prange Jr., A. J., & Pedersen, C. A. (1987). Effects of nonapeptide antagonists on oxytocin- and arginine-vasopressin-induced analgesia in mice. Regulatory Peptides, 18(3-4), 233-241.
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  PMID: 3671785;Abstract: Several peptides, including arginine-vasopressin (AVP), neurotensin, and substance P, produce analgesia that is not mediated by opiate systems. Using the hot plate test, we studied the analgesic effects of intracisternal (i.c.) administration of various doses of the nonapeptide oxytocin (OXY) in Swiss-Webster mice. We found that OXY (1-4 μg) significantly increased the latency of animals to jump or lick their paws after placement on a hot plate. This effect was not blocked by naloxone pretreatment, which suggests that it is not opiate dependent. Using the hot plate test, we confirmed that AVP (1 and 4 μg) also produces analgesia. We then studied the analgesia produced by OXY and by AVP using 3 nonapeptide analogues with antagonist properties: [Pen1, LpMePhe2, Thr4, Orn8]OXY (PLMPTO-OXY) that has anti-oxytocic properties in the uterine contraction assay, d(CH2)5Tyr(Me)AVP (dTM-AVP) which antagonizes the antidiuretic properties of AVP and d(CH2)5d-Ile2,Abu4-AVP (dIA-AVP) which antagonizes the vasopressor effects of AVP. Simultaneous administration of PLMPTO-OXY completely blocked the analgesia produced by OXY whereas the antidiuretic antagonist dIA-AVP partially blocked OXY-induced analgesia and dTM-AVP had no effect. None of the antagonists used blocked AVP-induced analgesia. We concluded that the neural systems mediating the analgesic effects of i.c. OXY differ from those for AVP. © 1987.
• Chan, W. Y., Rockway, T. W., & Hruby, V. J. (1987). Long-acting oxytocin antagonists: Effects of 2-D-stereoisomer substitution on antagonistic potency and duration of action (42533). Proceedings of the Society for Experimental Biology and Medicine, 185(2), 187-192.
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  PMID: 3575333;Abstract: Recently we reported the discovery of a series of 2-O-alkyltyrosine- (or 2-p-alkyl-phenylalanine), 4-threonine-, and 8-ornithine-substituted analogs of [1-penicillamine]oxytocin ([Pen1]OT) which possess prolonged anti-OT activity. In this study, we attempt to improve the potency and the duration of action of this series of OT antagonists by exploring the effects of D-stereoisomer substitution in the 2 position. We compare the in vitro anti-OT potency, expressed in pA2 values, and the duration of in vivo inhibitory action, expressed in recovery t(1/2) of [Pen1]OT, [Pen1,Orn8]OT, [Pen1Thr4]OT, [Pen1,Tyr(OMe)2,Thr4,Orn8]OT, [Pen1,Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,D-Tyr(Oet)2,Thr4,Orn8]OT, [Pen1,Phe2,Thr4]OT, [Pen1,Phe(Me)2,Thr4,Orn8]OT, [Pen1,D-Phe(Me)2,Thr4,Orn8]OT, [Pen1,Phe(Et)2,Thr4,Orn8]OT, and [Pen1,D-Phe(Et)2,Thr4,Orn8]OT. The results show that modifications of the amino acid in position 2 by alkylation of the aromatic ring and use of D-stereoisomerism produce nonparallel effects on the in vitro potency and duration of action of OT antagonists. Time-action curve determinations show that long-acting OT antagonists exhibit delayed peak inhibitory action. Long action is not coupled with high potency in all cases. This dissociation between potency and duration of action gives support to our hypothesis that the potency and duration of action of these peptides may each have different conformational structure requirements.
• Cody, W. L., & Hruby, V. J. (1987). Unnatural amino acids: synthesis and biological significance.. Acta Universitatis Palackianae Olomucensis Facultatis Medicae, 116, 495-516.
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  PMID: 2962463;
• Grady, T., Fickova, M., Tager, H. S., Trivedi, D., & Hruby, V. J. (1987). Stimulation and inhibition of cAMP accumulation by glucagon in canine hepatocytes. Journal of Biological Chemistry, 262(32), 15 514-15 520.
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  Abstract: We have examined, by use of isolated canine hepatocytes and selected hormone analogs, the mechanisms by which glucagon modifies the accumulation of cellular cAMP. Low concentrations of glucagon (≤ 3 nM) enhanced the accumulation of hepatocyte cAMP, whereas higher concentrations of the hormone diminished the effectiveness of lower ones. This biphasic concentration dependence was observed as well for some glucagon analogs, but not for others, and was apparent for cells incubated in the presence or absence of theophylline. Glucagon at high concentrations (≥ 10 nM) also inhibited the accumulation of cAMP induced by isoproterenol. The inhibitory effect of glucagon in both of these systems was reversed or attenuated by cell incubations involving the use of pertussis toxin (islet-activating protein) or a peptide antagonist of the glucagon-adenylyl cyclase system. We conclude that (a) glucagon, through its interaction with high and low affinity binding sites, can either stimulate or inhibit the production of hepatocyte cAMP; (b) the inhibitory action of the hormone appears to arise from interactions of ligand with a subset of these binding sites and to require structural characteristics in addition to those that determine receptor binding affinity per se; and (c) the glucagon and adrenergic systems involved in stimulating cAMP accumulation are linked, at least with regard to the negative effect induced by high concentrations of glucagon.
• Grady, T., Fickova, M., Tager, H. S., Trivedi, D., & Hruby, V. J. (1987). Stimulation and inhibition of cAMP accumulation by glucagon in canine hepatocytes.. Journal of Biological Chemistry, 262(32), 15514-15520.
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  PMID: 2824463;Abstract: We have examined, by use of isolated canine hepatocytes and selected hormone analogs, the mechanisms by which glucagon modifies the accumulation of cellular cAMP. Low concentrations of glucagon (less than or equal to 3 nM) enhanced the accumulation of hepatocyte cAMP, whereas higher concentrations of the hormone diminished the effectiveness of lower ones. This biphasic concentration dependence was observed as well for some glucagon analogs, but not for others, and was apparent for cells incubated in the presence or absence of theophylline. Glucagon at high concentrations (greater than or equal to 10 nM) also inhibited the accumulation of cAMP induced by isoproterenol. The inhibitory effect of glucagon in both of these systems was reversed or attenuated by cell incubations involving the use of pertussis toxin (islet-activating protein) or a peptide antagonist of the glucagon-adenylyl cyclase system. We conclude that (a) glucagon, through its interaction with high and low affinity binding sites, can either stimulate or inhibit the production of hepatocyte cAMP; (b) the inhibitory action of the hormone appears to arise from interactions of ligand with a subset of these binding sites and to require structural characteristics in addition to those that determine receptor binding affinity per se; and (c) the glucagon and adrenergic systems involved in stimulating cAMP accumulation are linked, at least with regard to the negative effect induced by high concentrations of glucagon.
• Gysin, B., Johnson, D. G., Trivedi, D., & Hruby, V. J. (1987). Synthesis of two glucagon antagonists: Receptor binding, adenylate cyclase, and effects on blood plasma glucose levels. Journal of Medicinal Chemistry, 30(8), 1409-1415.
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  PMID: 3039134;Abstract: In diabetes mellitus, hyperglycemia is often associated with elevated levels of glucagon in the blood. This suggests that glucagon (1) is a contributing factor in the metabolic abnormalities of diabetes mellitus. A glucagon-receptor antagonist would provide direct evidence for glucagon's role in diabetes mellitus. On the basis of careful consideration of conformational, amphiphilic, and structural factors, we have synthesized two new glucagon analogues with antagonist biological activities by using solid-phase methodology. These two new analogues, [Asp3 D-Phe4,Ser5,Lys17,18,Glu21]glucagon (2) and [D-Phe4,Tyr5,3,5-I2-Tyr10,Arg 12,Lys17,18,Glu21]glucagon (3), had IC50 values 5.4% and 50% those of glucagon, respectively, and showed no measurable adenylate cyclase activity. When tested in normal rats, 2 lowered plasma glucose levels and suppressed glucagon-mediated hyperglycemia 105 ± 8%, back to basal levels. Analogue 3, which lowered the basal adenylate cyclase activity in rat liver plasma membranes, increased plasma glucose levels at very high concentration in vivo and inhibited glucagon-mediated hyperglycemia in normal rats by 50%. However, neither of the new glucagon antagonists lowered the plasma glucose levels of diabetic animals. The data would suggest these new glucagon-receptor antagonists may have two actions: (a) in normal rats they can act as standard glucagon-receptor inhibitors of glucagon-mediated glycogenolysis; (b) in diabetic rats, however, because of the low levels of glycogen in the liver, the antagonists apparently have little or no antagonist effect or enhancement on glucagon-mediated glucose production. © 1987 American Chemical Society.
• Hadley, M. E., Wood, S. H., Lemus-Wilson, A. M., Dawson, B. V., Levine, N., Dorr, R. T., & Hruby, V. J. (1987). VII. Topical application of a melanotropic peptide induces systemic follicular melanogenesis. Life Sciences, 40(19), 1889-1895.
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  PMID: 3573985;Abstract: We determined the relative effectiveness of α-MSH and a highly potent melanotropin analogue, [Nle4, D-Phe7]-α-MSH, in stimulating a shift from pheomelanogenesis to eumelanogenesis within hair bulbs of mice. The analogue proved to be at least a hundred times more effective than the native hormone when injected subcutaneously. The two melanotropins were then incorporated into an ointment base and topically applied to a shaved area of the skin on the back of a yellow strain of mice (C57BL/6JAY). Within 24-48 hours eumelanin production was visible within hair bulb melanocytes in both treated and untreated areas of animals. The presence of melanized organelles (eumelanosomes) within melanocytes was confirmed by electron microscopy. These results document the delivery of a peptide hormone through the skin and into the systematic circulation. This is the first demonstration of the delivery of a peptide hormone by percutaneous absorption and may provide a model for a similar route of delivery of other peptide hormones. The hormone analogue has also been delivered across human skin in vitro. Delivery of a melanotropin by a transdermal route may prove to be clinically useful in the treatment of some integumental hypopigmentary disorders in humans. © 1987.
• Hadley, M. E., Zechel, C., Wilkes, B. C., M., A., Visconti, M. A., Pozo-Alonso, M., & Hruby, V. J. (1987). Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone. Life Sciences, 40(12), 1139-1145.
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  PMID: 3494178;Abstract: {A figure is presented} melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH were synthesized as described herein and the melanotropic activities of the peptides were determined. In the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays, the 5-17 and 5-14 fragments of MCH were inactive (at concentrations of 10-5M or less), whereas the 1-14 sequence exhibited minimal (about 10%) MSH-like activity compared to MCH, which, as reported previously, was about 600 times less active than α-MSH. In the teleost (fish) skin bioassay, the MCH5-17 analogue was equipotent to MCH, whereas the 1-14 analogue was 10-30 times and the cyclic N- and C- terminal truncated analogue, MCH5-14, was about 300 times less active than MCH. These results suggest that the N-terminal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH, particularly in the C-terminal, are more related to MCH activity in teleosts. © 1987.
• Hagopian, W. A., Tager, H. S., Gysin, B., Trivedi, D., & Hruby, V. J. (1987). Interactions of glucagon and glucagon analogs with isolated canine hepatocytes. Journal of Biological Chemistry, 262(32), 15 506-15 513.
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  Abstract: We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 106-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites.
• Hagopian, W. A., Tager, H. S., Gysin, B., Trivedi, D., & Hruby, V. J. (1987). Interactions of glucagon and glucagon analogs with isolated canine hepatocytes.. Journal of Biological Chemistry, 262(32), 15506-15513.
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  PMID: 2824462;Abstract: We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 10(6)-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites.
• Hruby, V. J. (1987). Implications of the X-ray structure of deamino-oxytocin to agonist/antagonist-receptor interactions. Trends in Pharmacological Sciences, 8(9), 336-339.
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  Abstract: Abstract: A recent Science article demonstrating the X-ray structure of an oxytocin analog indicates low-energy interconversion of the disulfide between a right-handed and left-handed helicity, as well as conformational flexibility for the side-chain Asn5 and Ile3 residues. These results and other corresponding biological data support a hypothesis which, Victor Hruby explains, suggests that oxytocin antagonist analogs interact with the oxytocin receptor in a different manner to the interaction of oxytocin agonists. This may have general implications for agonist/antagonist interactions and may aid rational analog design. © 1987 Elsevier Publication.
• Hruby, V. J., Wilkes, B. C., Hadley, M. E., Al-Obeidi, F., Sawyer, T. K., Staples, D. J., E., A., Dym, O., Maria, A., Hintz, M. F., Riehm, J. P., & Rao, K. R. (1987). α-Melanotropin: The minimal active sequence in the frog skin bioassay. Journal of Medicinal Chemistry, 30(11), 2126-2130.
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  PMID: 2822931;Abstract: The minimal sequence required for biological activity of α-MSH (α-melanotropin, α-melanocyte stimulating hormone) was determined in the frog (Rana pipiens) skin bioassay. The sequence required to elicit measurable biological activity was the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 (Ac-α-MSH6-9-NH2), which was about 6 orders of magnitude less potent than the native tridecapeptide. Smaller fragments of this sequence (Ac-His-Phe-NH2, Ac-Phe-Arg-NH2, Ac-His-Phe-Arg-NH2) were devoid of melanotropic activity at concentrations as high as 10-4 M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2 (Ac-α-MSH7-10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2 (Ac-α-MSH11-13-NH2). We prepared a series of fragment analogues of α-MSH in an attempt to determine the contribution of each individual amino acid to the biological activity of the native hormone. The minimal potency of Ac-α-MSH6-9-NH2 could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-α-MSH6-10-NH2, (Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-α-MSH5-10-NH2, which was only slightly more potent than Ac-α-MSH6-10-NH2, indicating that position 5 contributes little to the biological potency of α-MSH in this assay. Addition of methionine to the N-terminus of Ac-α-MSH5-10-NH2 resulted in the heptapeptide, Ac-α-MSH4-10-NH2, which was only about 4-fold more potent than Ac-α-MSH5-10-NH2. Addition of lysine and proline to the C-terminal of the Ac-α-MSH4-10-NH2 sequence yielded the peptide, Ac-α-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-α-MSH4-10-NH2. This peptide was only about 6-fold less potent than α-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-α-MSH4-10-NH2 was about 4 times more potent than Ac-α-MSH4-10-NH2. Ac-[Nle4]-α-MSH4-11-NH2 also was about 4 times more potent than Ac-α-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of α-MSH on the frog skin melanocyte receptor. However, addition of proline-12 to this fragment, yielding Ac-[Nle4]-α-MSH4-12-NH2, resulted in about a 90-fold increase in relative potency of the melanotropin. Addition of the final C-terminal amino acid, valine-13, provided the decapeptide, Ac [Nle4]-α-MSH4-13-NH2, which showed only a small further increase in potency. This analogue was, however, only about 2-3-fold less active than α-MSH. Addition of the N-terminal tripeptide Ac-Ser-Tyr-Ser to yield the tridecapeptide [Nle4]-α-MSH resulted in an analogue that was 3 times more potent than α-MSH. The importance of the amino acids in the primary structure of α-MSH in contributing to the biological activity of α-MSH in the frog skin bioassay can be summarized as follows: (1) the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 represents the minimum chain length for observable biological activity; (2) the active sequence of α-MSH is contiguous in that no two structurally noncontiguous fragment sequences were found to have biological activity (3) Met-4, Gly-10, and Pro-12 are important potentiating amino acids and contribute significantly to the biopotency of α-MSH; and (4) Ser-1 and -3, Tyr-2, Glu-5, Lys-11, and Val-13 apparently contribute only minimally to the biological potency of α-MSH at the frog melanocyte skin receptor. © 1987 American Chemical Society.
• Landis, G. C., Eckman, D. R., Hruby, V. J., & Kreulen, D. L. (1987). A time dose-response desensitization study of substance P in the guinea-pig ileum. Proceedings of the Western Pharmacology Society, Vol. 30, 163-165.
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  PMID: 2442770;
• Lebl, M., Sugg, E. E., & Hruby, V. J. (1987). Proton n.m.r. spectroscopic evidence for sulfur-aromatic interactions in peptides.. International Journal of Peptide and Protein Research, 29(1), 40-45.
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  PMID: 3570653;Abstract: The downfield shift of the tyrosyl proton resonances and an increased chemical shift difference between the resonances for the 2',6' and 3',5' hydrogens in a series of deamino-oxytocin analogs modified in the disulfide bridge provide evidence for aromatic-sulfur interactions in d6-dimethylsulfoxide solutions.
• Lebl, M., Sugg, E. E., Binst, G. v., Elst, P. V., Tourwé, D., Slaninová, J., & Hruby, V. J. (1987). Analogs of oxytocin containing a modified peptide bond. International Journal of Peptide and Protein Research, 30(3), 318-322.
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  PMID: 3692679;Abstract: Analogs of deamino-oxytocin and deamino-oxypressin containing a CH2-NH group instead of an amide bond between positions 8 and 9 were synthesized. All tested compounds exhibit significantly lowered biological activities.
• Lemcke, P. K., Sugg, E., Malarchik, M., Lui, G., Serra, M., Yamamura, H. I., Hruby, V. J., Shook, J. E., & Burks, T. F. (1987). N-terminal cyclization of a nonsulfated CCK analogue exhibits opioid antagonist activity in vitro.. Proceedings of the Western Pharmacology Society, 30, 223-226.
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  PMID: 3628282;
• Levine, N., Lemus-Wilson, A., Wood, S. H., A., Z., Al-Obeidi, F., Hruby, V. J., & Hadley, M. E. (1987). Stimulation of follicular melanogenesis in the mouse by topical and injected melanotropins. Journal of Investigative Dermatology, 89(3), 269-273.
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  PMID: 3624899;Abstract: The effects of melanocyte-stimulating hormone (α-MSH) and related analogs on follicular melanogenesis in the mouse (C57BL/6JAγ) were studied. [Nle4, D-Phe7]-α-MSH and the related fragment analogues Ac-[Nle4, D-Phe7]-α-MSH4-11-NH2 and Ac-[Nle4, D-Phe7]-α-MSH4-10-NH2, stimulated the conversion of pheomelanogenesis to eumelanogenesis when subcutaneously injected at concentrations 100-fold lower than the native hormone, α-MSH. In addition, the melanotropin analogs stimulated follicular eumelanogenesis when applied topically to the skin of mice. The melanotropins were transdermally delivered to the systemic circulation as evidenced by the fact that eumelanogenesis was stimulated in hair follicles in areas distant from the site of topical application. These results demonstrate that peptide hormone analogs can be transported across the skin. The unique actions of the melanotropin analogs may relate to the fact that these peptides are nonbiodegradable and thus exert prolonged actions on melanocytes. These compounds may prove important for studies on normal integumental melanogenesis and for the treatment of hypopigmentary disorders in humans. © 1987.
• Maria, A., Hadley, M. E., & Hruby, V. J. (1987). A teleost skin bioassay for melanotropic peptides. General and Comparative Endocrinology, 66(3), 374-380.
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  PMID: 3609709;Abstract: A teleost (the eel, Synbranchus marmoratus) skin bioassay for melanotropic peptides and other agonists is described. Unlike previous teleost assays that generally monitor or observe individual melanophores, this objective assay utilizes large intact, pieces-of-skin and quantitative photoreflectance methods. Since melanosomes within most teleost melanophores are generally dispersed, the present assay provides a method for measuring the response of integumental melanophores to melanosome-aggregating agents such as MCH, a putative melanin-concentrating hormone. This bioassay is sensitive to MCH at a concentration as low as 10-12M. Because of the magnitude of this lightening response, four-point dose-response curves can be obtained. Skins lightened by MCH can then be used for bioassay of melanotropins or other melanosome-dispersing agents, such as β-adrenoceptor agonists. This bioassay is unique in providing a method for determining the biological activities of melanotropic peptides with opposing actions. © 1987.
• Maria, A., Hadley, M. E., Wilkes, B. C., Zechel, C., & Hruby, V. J. (1987). I. Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores. Life Sciences, 40(19), 1845-1851.
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  PMID: 3573981;Abstract: AspThrMetArgCysMetValGlyArgValTyrArgProCysTrpGluVal (melanin concentrating hormone, MCH) and several fragment analogs (MCH1-14, MCH5-17, MCH5-14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH1-17 (10-12M) >< MCH5-17 (10-12M) > MCH1-14 (10-11M) > MCH5-14 (2 × 10-10M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of ≺-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH1-14 fragment analog. The MCH activities of the MCH5-17 and MCH5-14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self- antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone. © 1987.
• Murphy, C. J., Hruby, V. J., Trivedi, D., Wakelam, M. J., & Houslay, M. D. (1987). The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism. Biochemical Journal, 243(1), 39-46.
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  PMID: 3038085;PMCID: PMC1147811;Abstract: Treatment of intact hepatocytes with glucagon, TH-glucagon ([1-N-α-trinitrophenylhistidine, 12-homo-arginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/logg of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein G(s), and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.
• Porreca, F., Nunan, L., Kazmierski, W., & Hruby, V. J. (1987). Effects of a novel opioid peptide antagonist on rat bladder motility in vivo. Peptides, 8(4), 625-632.
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  PMID: 2888099;Abstract: The agonist, and opioid antagonist, effects of intracerebroventricularly (ICV) given D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), a cyclic analogue of somatostatin octapeptide, were evaluated using the micturition reflex of the anesthetized rat as the endpoint. Antagonist effects were evaluated against equieffective doses of selective mu [D-Ala2,NMPhe4,Gly-ol]enkephalin (DAGO) and delta [D-Pen2,D-Pen5] enkephalin (DPDPE) opioid agonists. At low ICV doses, CTP preferentially antagonized DPDPE rather than DAGO; increasing the dose of CTP further effectively antagonized both mu and delta agonists, while even higher doses showed an agonist effect alone which was not blocked by adrenergic, cholinergic or opioid antagonists. Selective opioid antagonist doses of CTP failed to block the inhibition of the micturition reflex produced by pentobarbital. Possible residual somatostatin like properties of CTP were tested by using somatostatin as a possible antagonist of equieffective doses of DPDPE and DAGO; somatostatin did not antagonize these agonists. Repeated exposure to CTP resulted in the development of acute tolerance to the agonist effect, and also prevented the inhibition of the reflex by high doses of somatostatin, with the converse experiment showing a similar pattern; thus, repeated somatostatin resulted in tolerance and subsequent cross-tolerance to the agonist effects of CTP. In animals tolerant to somatostatin, CTP nevertheless behaved as an opioid antagonist. The present results indicate that CTP possesses opioid antagonist properties in vivo which are pharmacological in nature but nevertheless retains residual somatostatin-like activity at higher doses. © 1987.
• Shook, J. E., Pelton, J. T., Hruby, V. J., & Burks, T. F. (1987). Peptide opioid antagonist separates peripheral and central opioid antitransit effects. Journal of Pharmacology and Experimental Therapeutics, 243(2), 492-500.
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  PMID: 2824748;Abstract: The purpose of these investigations was to determine 1) whether peripherally located mu, delta and kappa opioid receptors can inhibit the rate of gastrointestinal transit and, if so, 2) do peripheral opioid receptors mediate the constipation caused by systemic morphine? and 3) whether constipation can be separated from analgesia on the basis of different sites of action. We studied the effects of peripherally administered (s.c.) mu, delta and kappa opioid receptor selective agonists on the rate of gastrointestinal transit in mice. We used peptidergic agonists with high peripheral selectivity (limited ability to cross the blood-brain barrier) including [MePhe3,D-Pro4]morphiceptin (PL017) (mu), [D-Pen2,D-Pen5]enkephalin (DPDPE) (delta) and Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg (dynorphin 1-9) (kappa). As peripheral selectivity is dose-related, we included the hot-plate test as an index of that dose at which each compound lost its peripheral selectivity and entered the central nervous system. When given s.c., [MePhe3,D-Pro4]morphiceptin inhibited transit (IC50 = 0.37 mg/kg s.c.) at doses much lower than those needed to produce analgesia (A50 = 30 mg/kg s.c.), indicating that peripheral mu receptors can inhibit transit independently of central mu receptors. The independence of peripheral mu antitransit receptors from central receptors was demonstrated further as the lack of antagonism of s.c. [MePhe3,D-Pro4]morphiceptin antitransit effects by simultaneous i.c.v. administration of the mu receptor antagonist D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) (1 μg i.c.v.). [D-Pen2,D-Pen5]enkephalin and Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg did not inhibit transit when given s.c., even at very high doses, indicating that peripheral delta and kappa receptors do not mediate transit. Morphine given s.c. inhibited transit (IC50 = 1 mg/kg s.c.) at doses similar to those which also caused analgesia (A50 = 4 mg/kg s.c.). Effects of coadministration of CTP (1 μg i.c.v. or 1 mg/kg s.c.) with s.c. morphine varied with the dose of morphine: at subanalgesic doses of morphine (≤ 1 mg/kg s.c.), its antitransit effects were blocked totally by s.c. CTP, but not i.c.v. CTP, whereas at analgesic doses (> 1 mg/kg s.c.), it was blocked partially, but nearly equally, by both i.c.v. and s.c. CTP. These results suggest that at subanalgesic doses, systemic morphine acts at peripheral mu receptors (not delta or kappa) to inhibit transit, and at analgesic doses, it acts through both central and peripheral opioid receptors. It therefore seems unlikely that the antitransit effects of morphine can be separated from analgesia on the basis of different (peripheral vs. central) sites of action.
• Shook, J. E., Pelton, J. T., Kazmierski, W., Lemcke, P. K., Villar, R. G., Hruby, V. J., & Burks, T. F. (1987). A cyclic somatostatin analog that precipitates withdrawal in morphine-dependent mice.. NIDA research monograph, 76, 295-301.
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  PMID: 2893981;Abstract: We evaluated the ability of the mu selective, peptidic, opioid antagonist CTP to precipitate withdrawal in morphine-dependent mice after intracerebroventricular (i.c.v.) and subcutaneous (s.c.) administration. The withdrawal syndrome evoked by i.c.v. CTP was different in some respects from that observed after i.c.v. naloxone. Naloxone, given i.c.v., produced shakes and tremors, defecation, diarrhea, wet dog shakes, jumping and weight loss. In contrast, the prominent signs following i.c.v. CTP were grooming, tremors and shakes, defecation, wet dog shakes and weight loss. CTP treated mice exhibited a greatly reduced incidence of jumping behaviors and diarrhea. While s.c. naloxone evoked similar effects to i.c.v. naloxone, CTP given s.c. stimulated defecation and modest weight loss only. The differences in the profile of withdrawal signs between naloxone and CTP may be related to their differences in receptor selectivity or possibly to their respective alkaloidal and peptidic natures. The relative lack of behavioral effects seen after s.c. CTP probably reflects the inability of CTP to pass through the blood brain barrier, and indicates that although the majority of withdrawal signs are mediated by centrally located opioid receptors, the gastrointestinal tract can be withdrawn independently of the central nervous system.
• Shook, J. E., Pelton, J. T., Lemcke, P. K., Porreca, F., Hruby, V. J., & Burks, T. F. (1987). Mu opioid antagonist properties of a cyclic somatostatin octapeptide in vivo: Identification of mu receptor-related functions. Journal of Pharmacology and Experimental Therapeutics, 242(1), 1-7.
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  PMID: 2886635;Abstract: We have shown previously that D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) produces selective antagonism of mu, but not delta or kappa, opioid receptor-selective ligands in the guinea pig ileum and mouse vas deferens bioassays, and in radioligand binding assays using homogenized rat brains. In the present study we characterized the agonist and opioid antagonist profile of CTP in analgesic (hot-plate test, abdominal stretch test) and in gastrointestinal assays (transit time test) in mice. CTP was a potent antagonist of the supraspinal and spinal analgesic effects of the mu selective agonist [MePhe3, D-Pro4]morphiceptin (PL017) in both assays. The gastrointestinal antitransit actions of PL017 were also antagonized by CTP at both supraspinal and spinal sites. CTP did not alter the effects of the kappa agonist trans-3,4-dichloro-N-methyl-N-(1-pyrrolidinyl)cyclohexyl)benz eneacetamine in any test. Surprisingly, CTP also antagonized the analgesia produced by i.c.v. and intrathecal administration of [D-Pen2,D-Pen5]enkephalin (DPDPE), a highly delta selective agonist, in both analgesic tests. Differential antagonism of DPDPE, but not PL017, by the delta selective antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH in the hot-plate test indicates that PL017 and DPDPE may act at separate receptors to produce analgesia (mu and delta, respectively). In contrast, CTP did not reverse the gastrointestinal antitransit effects of intrathecal DPDPE. Schild analysis of the interactions of CTP with supraspinal mu and delta agonists in the hot-plate test indicated that although CTP antagonized PL017 in a competitive fashion (Schild slope = -1.0), the interaction of CTP with DPDPE was not competitive (Schild slope = -0.5). Naloxone, a nonselective opioid antagonist, produced competitive antagonism of both PL017 and DPDPE. In summary, we have shown that CTP is a competitive mu antagonist in vivo, in agreement with results obtained previously in vitro. In addition, CTP also blocked delta receptor-mediated analgesia, but not antitransit actions. The incongruity in delta antagonism seen in analgesic but not in the gastrointestinal assay, or in any in vitro test, might be explained best by a possible coupling of a certain population of mu and delta receptors that mediate analgesia. These findings also confirm the previously reported roles of mu, delta and kappa receptors in analgesia and in regulation of gastrointestinal function.
• Shook, J. E., Pelton, J. T., Wire, W. S., Hirning, L. D., Hruby, V. J., & Burks, T. F. (1987). Pharmacologic evaluation of a cyclic somatotatin analog with antagonist activity at Mu opioid receptors in vitro. Journal of Pharmacology and Experimental Therapeutics, 240(3), 772-777.
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  PMID: 2882015;Abstract: The cyclic somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) was evaluated for agonist and opioid antagonist actions and receptor selectivity in two bioassays: electrically stimulated guinea pig isolated ileum (GPI) and mouse isolated vas deferens (MVD). CTP (100, 300, 1000 nM) produced concentration-dependent antagonism of the mu agonist [Me-PHe3,D-Pro4]morphiceptin (PL017) in both the GPI and MVD. Schild analysis of the interactions between CTP and PL017 indicated competitive antagonism between these peptides (Schild slope GPI -0.97 ± 0.16, Schild slope MVD -1.4 ± 0.4), and also suggested that the mu receptors in the two tissues are not different (pA2 GPI 7.1 ± 0.17, pA2 MVD 6.9 ± 0.16). The effects of the delta selective agonist [D-Pen2,D-Pen5]enkephalin in the MVD were not antagonized by CTP. Likewise, in the GPI, CTP did not antagonize the kappa agonist (trans-3-4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamine (U50,488H). In comparison, naloxone antagonized both PL017 and U50,488H in the GPI, as well as [D-Pen2,D-Pen5]enkephalin and PL017 in the MVD. In the MVD, CTP also exerted weak somatostatin-like actions (35% maximal inhibition) that could not be demonstrated in somatostatin-tolerant tissues. It also showed inhibitory actions at very high concentrations (3000 and 10,000 nM) that were blocked by both naloxone and the delta antagonist N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH (ICI 174,864). ICI 174,864 antagonized [D-Pen2,D-Pen5]enkephalin in the MVD, but did not affect PL017. These results indicate that CTP is a selective mu receptor antagonist in vitro. These findings also emphasize the importance of receptor-selective antagonists such as CTP in characterizing the receptor populations in a tissue and provide an additional approach for evaluating the receptor selectivity of putative receptor-selective agonists.
• Wire, W. S., Pelton, J. T., Kazmierski, W., Hruby, V. J., Burks, T. F., & Shook, J. E. (1987). Structure-activity analysis of five constrained somatostatin like peptides with opioid antagonist properties.. Proceedings of the Western Pharmacology Society, 30, 237-241.
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  PMID: 2888128;
• Wynants, C., Sugg, E., Hruby, V. J., & Binst, G. V. (1987). Conformational study of a somatostatin analogue by high-field n.m.r. spectroscopy, in aqueous solution.. International Journal of Peptide and Protein Research, 30(4), 541-547.
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  PMID: 2892808;Abstract: The cyclic analogue of somatostatin (SRIF), D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-NH2 (CTC), exhibits good affinity for both opioid and SRIF receptor systems. Its conformational properties were examined in water by high-field proton n.m.r. spectroscopy and compared with results previously obtained with structurally related analogues SMS 201-995 and Sandoz 204-090 in the same solvent. The assignments were made using 2 D-n.m.r. methods, especially long-range connectivities between neighbouring alpha protons, and between beta and aromatic protons. The 3JNH-C alpha H and delta delta/delta T values are compatible with an equilibrium between two gamma turns involving residues 2, 3 and 4 and residues 3, 4, and 5, respectively.
• Caldwell, J. D., Hruby, V. J., Hill, P., Prange Jr., A. J., & Pedersen, C. A. (1986). Is oxytocin-induced grooming mediated by uterine-like receptors?. Neuropeptides, 8(1), 77-86.
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  PMID: 3020470;Abstract: In this study, we examined whether the mechanisms mediating the induction of grooming behavior by oxytocin (OT) is similar to mechanisms mediating the effects of OT on uterine contractility. Sprague-Dawley strain female rats were injected intracerebroventricularly (ICV) with OT or OT analogues and then were observed for grooming behaviors 25 minutes later for 30 minutes. The uterotonic analogue deamino-OT injected ICV at equimolar doses to 1 μg OT significantly elevated grooming scores although less than did OT. Other agonist analogues were not effective in inducing an increase in grooming behavior. The simultaneous ICV injection of the analogue [Pen1, Phe2, Thr4, Δ3, 4Pro7, Orn8]-OT, which blocked the effects of OT on uterine contractility, also blocked the effect of OT on grooming behavior. Injection of the same dose of this antagonist analogue did not affect the increased grooming behavior after AVP injection. Pretreatment with 5 mg/kg of the prostaglandin synthetase inhibitor indomethacin significantly inhibited OT-induced grooming. We have concluded from these data that the mechanism underlying the effect of OT on grooming is similar to its effects on uterine contractility in some respects. However, observations that the OT antagonist analogue blocked OT-but not AVP-induced grooming may suggest that more than one receptor or mechanism exists by which nonapeptides initiate excessive grooming. © 1986.
• Chan, W. Y., Hruby, V. J., Rockway, T. W., & Hlavacek, J. (1986). Design of oxytocin antagonists with prolonged action: Potential tocolytic agents for the treatment of preterm labor. Journal of Pharmacology and Experimental Therapeutics, 239(1), 84-87.
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  PMID: 3761199;Abstract: In our continuing effort to produce more potent and specific oxytocin (OT) antagonists that may have value as tocolytic agents, we have synthesized a number of new OT antagonists. Our previous studies have shown that rigid conformational structure and restricted dynamic properties are associated with antagonistic activity of the [1-penicillamine]OT ([Pen1]OT) analogs. We therefore synthesized a series of structural analogs of [Pen1,]OT; [Pen1,Thr4]-OT and [Pen1,Phe2,Thr4]OT with greater restricted conformational features. They are [Pen1,Δ3,4-Pro7]OT; [Pen1,Thr4,Δ3,4-Pro7]OT; [Pen1,Phe2,Thr4,Δ3,4-Pro7]OT; [Pen1,Orn8]OT; [Pen1,Phe2,Thr4,Δ3,4-Pro7,Orn8]OT; [Pen1,Tyr(OMethyl)2,-Thr4,Orn8]OT; [Pen1,Tyr(OEthyl)2,Thr4,Orn8]OT; [Pen1,Phe(Methyl)2,Thr4,Orn8]OT and [Pen1Phe(Ethyl)2, Thr4,Orn8]OT. As expected, all were found to be potent OT antagonists, with in vitro pA2 values ranging from 5.32 to 7.67. They were also effective OT antagonists in vivo in the term pregnant rats. Structural modifications in the above analogs produced various and interesting effects. Dehydroproline substitution for 7-proline in [Pen1]OT increased antagonistic potency, whereas in [Pen1,Thr4]OT and in [Pen1,Phe2,Thr4]OT decreased antagonistic potency. Most significantly, analogs with O-alkyl-Tyr2,Orn8 and p-alkyl-Phe2,Orn8 substitutions were found to have prolonged action both in the isolated rat uterus assays and in the term pregnant rats. Generally, substitution of the alkyl groups resulted in a reduction in anti-OT potency, and increasing the size of the alkyl substituent from a methylene group to an ethyl group diminished antagonistic potencies markedly. The 2-ethyl,8-ornithine analogs, however, retained their prolonged action. This dissociation between potency and prolonged action gives further support to our hypothesis that potency (binding and transduction) and duration of action (reversibility) may each have different molecular requirements and that OT analogs can be designed rationally based on conformational considerations for potency, specificity and long action.
• Clark, J. A., Itzhak, Y., Hruby, V. J., Yamamura, H. I., & Pasternak, G. W. (1986). [D-Pen²,D-Pen⁵]enkephalin (DPDPE): a δ-selective enkephalin with low affinity for μ₁ opiate binding sites. European Journal of Pharmacology, 128(3), 303-304.
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  PMID: 3025000;
• García-Sáinz, J., Sánchez-Sevilla, L., Pelton, J. T., Trivedi, D., & Hruby, V. J. (1986). Effects of [1-N^α-trinitrophenylhistidine, 12-homoarginine]glucagon on cyclic AMP levels and free fatty acid release in isolated rat adipocytes. BBA - Molecular Cell Research, 886(2), 310-315.
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  PMID: 3008855;Abstract: [1-Nα-Trinitrophenylhistidine, 12-homoarginine]glucagon (THG) stimulated, in a concentration-dependent fashion, lipolysis (2-fold) and cyclic AMP accumulation (50% over basal) in isolated rat adipocytes, but was much less effective than glucagon, which stimulated lipolysis 4-fold and cyclic AMP accumulation 10-15-fold. THG displaced to the right the concentration-response curves for glucagon and diminished in a concentration-dependent fashion the effects of a fixed concentration of glucagon. The data indicate that THG is a mixed agonist-antagonist (partial agonist) in isolated rat fat cells. © 1986.
• Gulya, K., Gehlert, D. R., Wamsley, J. K., Mosberg, H., Hruby, V. J., & Yamamura, H. I. (1986). Light microscopic autoradiographic localization of delta opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog. Journal of Pharmacology and Experimental Therapeutics, 238(2), 720-726.
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  PMID: 3016247;Abstract: Light microscopic autoradiography was used to visualize the neuroanatomical distribution of rat brain delta opioid receptors. Slide-mounted sections of rat brain were labeled with [3H]-[2-D-penicillamine, 5-D-penicillamine]enkephalin ([3H]DPDPE), a highly selective delta opioid agonist. Saturation isotherms of [3H]DPDPE binding to thaw-mounted brain slices gave a maximal number of binding sites of 79.9 fmol/mg of protein and an apparent dissociation constant (K(d)) of 6.3 nM. DPDPE and met-enkephalin inhibited [3H]DPDPE binding with high affinity (IC50 values of 6.3 and 13.8 nM, respectively). Putative mu opioid receptor selective ligands such as morphine sulfate, Tyr-D-Ala-Gly-NMePhe-Gyl-ol and [N-MePhe3, D-Pro4]morphiceptin (PL017) were less potent inhibitors of [3H]DPDPE binding. The rat brain areas containing the highest densities of receptors were the claustrum, basolateral amygdaloid nucleus, the caudate-putamen and nucleus accumbens, the external plexiform layer of the olfactory bulb and the olfactory tubercle. Moderate receptor density was characteristic of the hippocampal formation in which grains were seen over the molecular layer of the dentate gyrus and stratum oriens (CA1), and of the different layers of cerebral cortex. Generally, low density of binding was found over the thalamus and the septal nuclei. Low specific binding was also seen in the cerebellum, medulla oblongata and in the dorsal horn of the spinal cord. There was little specific [3H]DPDPE binding over the white matter areas.
• Gulya, K., Lui, G. K., Pelton, J. T., Kazmierski, W., Hruby, V. J., & Yamamura, H. I. (1986). H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2: a potent and selective antagonist opioid receptors.. NIDA research monograph, 75, 209-212.
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  PMID: 2893264;Abstract: H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) exhibited high affinity (IC50 = 2.80 nM) in displacing [3H]naloxone binding (nH = 0.89 +/- 0.1) and showed an exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50(naloxone) ratio of 4,840, while it displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM) in rat brain binding assays. [3H]CTOP was recently custom synthesized (spec. act.: 84 Ci/mmol) and evaluated for its in vitro binding properties towards the mu opioid receptors in rat brain membrane preparations. Association and dissociation of [3H]CTOP binding to mu opioid receptors were rapid at 25 degrees C with a kinetic Kd value of 0.67 nM. Saturation experiments gave apparent Kd value of 1.11 nM and Bmax value of 136 +/- 13 fmol/mg prot at 25 degrees C. Specific [3H]CTOP binding was inhibited by a number of different opioid and opiate ligands. Among them, putative mu opioid receptor-specific ligands, such as naloxone, naltrexone and CTOP inhibited the binding with high affinity, while delta opioid receptor-specific compounds or non-opioid drugs inhibited specific [3H]CTOP binding with low affinity or they were ineffective.
• Gulya, K., Pelton, J. T., Hruby, V. J., & Yamamura, H. I. (1986). Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors. Life Sciences, 38(24), 2221-2229.
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  PMID: 2872570;Abstract: A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [125I]CGP 23,996 (des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3,14- somatostatin), [3H]naloxone or [3H]DPDPE ([D-Pen2-D-Pen5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the syntheis of our most potent and selective mu opioid receptor compound DPheCysTyrDTrpOrnThrPenThrNH2, which we refer to as Cys2Tyr3Orn5Pen7-amide. While this octapeptide exhibited high affinity (IC50 = 2.80 nM) for an apparently single population of binding sites (nH = 0.89 ± 0.1) and exceptional selectivity for mu opioid receptos with an IC51(DPDPE)/IC50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM). Thus, Cys2Tyr3Orn5 Pen7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors. © 1986.
• Hruby, V. J. (1986). Design of conformationally constrained cyclic peptides with high delta and mu opioid receptor specificities. NIDA Research Monograph Series, No. 69, 128-147.
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  PMID: 3020412;
• Hruby, V. J., Krstenansky, J., Gysin, B., Pelton, J. T., Trivedi, D., & Mckee, R. L. (1986). Conformational considerations in the design of glucagon agonists and antagonists: Examination using synthetic analogs. Biopolymers, 25(SUPPL.), S135-S155.
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  PMID: 3022837;
• Kao, L., & Hruby, V. J. (1986). Suppression or differentiation of solvent resonance by a combination of DEFT with a two-dimensional sequence. Journal of Magnetic Resonance (1969), 70(3), 394-407.
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  Abstract: A new pulse sequence is described which combines the driven equilibrium Fourier transform (DEFT) and 2D pulse scheme, and allows simultaneous suppression of the solvent peak (H2O/D2O) in 1D and 2D experiments without perturbing the nearby solute signals. To overcome the sensitivity problem, and to allow for 2D experiments in a reasonably short time, a steady-state condition was set up by adjusting τ1 (pulse delay) and D (recycle delay) to be 2-4 times the longest spin-lattice relaxation time T1 (peptide) such that Mz (solvent) was nulled prior to the 2D pulse sequence. Longitudinal and transverse relaxation rates of the solvent were analyzed in the steady state, and a direct calculation of T1 (solvent) was performed using an equation derived in the present work. This novel pulse sequence is also extended to 2D NOE experiments with effective solvent suppression. The method is demonstrated in a variety of 1D and 2D experiments on the cyclic delta opioid receptor selective peptide {A figure is presented}. © 1986.
• Klemes, D. G., Kreutzfeld, K. L., Hadley, M. E., Cody, W. L., & Hruby, V. J. (1986). Potent and prolonged melanotropic activities of the α-MSH fragment analog, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4-9-NH₂. Biochemical and Biophysical Research Communications, 137(2), 722-728.
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  PMID: 3089218;Abstract: Ac-[Nle4, D-Phe7]-α-MSH4-9-NH2 and Ac-[Nle4]-α-MSH4-9-NH2, fragment analogs of the tridecapeptide, α-melanocyte stimulating hormone (α-MSH, α-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to α-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than α-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than α-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-α-MSH4-9-NH2 was considerably prolonged compared to α-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence. © 1986.
• Krstenansky, J. L., Trivedi, D., & Hruby, V. J. (1986). Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase. Biochemistry, 25(13), 3833-3839.
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  PMID: 3017406;Abstract: The role of the Tyr10-Ser11-Lys12-Tyr13 region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe13]glucagonamide (2), [Phe10]glucagonamide (3), [Phe10]glucagon (4), [Phe10,13]glucagon (5), [Pro11]glucagon (6), [Pro11,Gly12]glucagonamide (7), [Ala11]glucagon (8), and [Oac11-13]glucagonamide (9). These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected Nα-tert-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse-phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by α-chymotryptic peptide mapping with HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [125I]glucagon from glucagon receptors. The results of these studies indicate that while the biological "message" region of glucagon is located elsewhere, the 10-13 region has multiple roles in the glucagon-glucagon receptor interaction: (1) this region provides functional groups for direct binding interaction with the receptor, and (2) this region interacts with the receptor in such a way as to allow the "transduction message" portion of glucagon to interact and activate the receptor. © 1986 American Chemical Society.
• Krstenansky, J. L., Trivedi, D., Johnson, D., & Hruby, V. J. (1986). Conformational considerations in the design of a glucagon analogue with increased receptor binding and adenylate cyclase potencies. Journal of the American Chemical Society, 108(7), 1696-1698.
• Malek, Z. A., Kreutzfeld, K. L., Hadley, M. E., Bregman, M. D., Hruby, V. J., & Meyskens Jr., F. L. (1986). Long-term and residual melanotropin-stimulated tyrosinase activity in S91 melanoma cells is density dependent. In Vitro, 22(2), 75-81.
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  PMID: 3081484;Abstract: Cell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer culture. Continuous exposure of melanoma cells to α-melanotropin or its potent analog [Nle4, D-Phe7]-α-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins were subcultured to an initial low cell density and maintained in contact with α-MSH or [Nle4, D-Phe7]-α-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at initial densities ranging from 0.2 to 3.2 X 106 cells/flask, and exposed for 24 h to 10-7 M α-MSH, only the cultures seeded at low densities (0.2 and 0.4 X 106 cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure to melanotropin. Other flasks of various cell densities were treated with 10-7 M α-MSH or [Nle4, D-Phe7]-α-MSH for 24 h, followed by removal of the melanotropins from the culture medium. The magnitude and duration of the residual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density. These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins and express increased tyrosinase activity.
• McKee, R. L., Pelton, J. T., Trivedi, D., Johnson, D. G., Coy, D. H., Sueiras-Diaz, J., & Hruby, V. J. (1986). Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region. Biochemistry, 25(7), 1650-1656.
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  PMID: 3011069;Abstract: In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4] glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a β-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal β-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists. The partial agonism of [3-Me-His1,Arg12]-, [Phe1,Arg12]-, and [D-Ala4,Arg12]glucagon for adenylate cyclase activity in isolated liver plasma membranes observed in this study is not modulated by changes in the guanosine triphosphate (GTP) concentration. In addition, the receptor binding dose-response curve for [Phe1,Arg12]glucagon is shifted to the right in the presence of GTP to the same extent as that seen with the native hormone. Thus, the partial agonism demonstrated by these three analogues in this study is not due to a lack of modulation by GTP of the receptor binding and adenylate cyclase activities measured on liver plasma membranes. The in vivo degradation rates for glucagon and [D-Phe4]glucagon, half-lives of 5.3 and 7.5 min, respectively, were determined in this study. This slightly slower rate of degradation for [D-Phe4] glucagon is not sufficient to account for its highly potent glycogenolytic activity seen in vivo. The lack of correlation between the in vitro adenylate cyclase and the in vivo glucose release activities for these compounds is discussed. © 1986 American Chemical Society.
• Oshima, N., Kasukawa, H., Fujii, R., Wilkes, B. C., Hruby, V. J., & Hadley, M. E. (1986). Action of melanin-concentrating hormone (MCH) on teleost chromatophores. General and Comparative Endocrinology, 64(3), 381-388.
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  PMID: 3026881;Abstract: The in vitro effects of synthetic salmon melanin-concentrating hormone (MCH) on chromatophores of four teleost species were studied. In the erythrophores of the platyfish (Xiphophorus maculatus) and the swordtail (Xiphophorus helleri), and in the xanthophores and amelanotic melanophores of the medaka (Oryzias latipes), pigment aggregation took place in response to MCH even in the absence of Ca2+. In contrast to this, the leucophores of the medaka responded to MCH by the pigment dispersion but only when Ca2+ was present. The motile iridophores of the blue damselfish (Chrysiptera cyanea), which play a predominant role in coloration and its changes, were not affected by the hormone. Pharmacological studies employing various blocking agents suggest that the pigment-aggregating action of MCH is probably mediated through specific receptors possessed by the erythrophores, xanthophores, or amelanotic melanophores, while the pigment-dispersing action on the leucophores might be revealed through the receptors for melanophore-stimulating hormone (MSH). © 1986.
• Pelton, J. T., Kazmierski, W., Gulya, K., Yamamura, H. I., & Hruby, V. J. (1986). Design and synthesis of conformationally constrained somatostatin analogues with high potency and specificity for μ opioid receptors. Journal of Medicinal Chemistry, 29(11), 2370-2375.
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  PMID: 2878079;Abstract: A series of cyclic, conformationally constrained peptides related to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the μ opioid receptor. The following new peptides were prepared and tested for their μ opioid receptor potency and selectivity in rat brain binding assays: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (2, CTOP); D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (3, CTAP); D-Phe-Cys-Tyr-D-Trp-Nle-Thr-Pen-Thr-NH2 (4); D-Phe-Cys-Tyr-D-Trp-Lys-Val-Pen-Thr-NH2 (5); D-Phe-Cys-Tyr-D-Trp-Lys-Gly-Pen-Thr-NH 2(6);D-Phe-Cys-Tyr-Trp-Lys-Thr-Pen-Thr-NH2 (7); D-Tyr-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH (8); D-PhGly-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (9); and D-PhGly-Pen-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (10). The most selective peptide, 2 (CTOP), displayed both high affinity (IC50 = 3.5 nM) and exceptional selectivity (IC50 δ/IC50 μ = 4000) for μ opioid receptors. Furthermore, 2 exhibited very low affinity for somatostatin receptors in the rat brain (IC50 > 24 000 nM), with an IC50 somatostatin/IC50 μ receptor selectivity of 8750. These conformationally constrained cyclic peptides should provide new insight into the structural and conformational requirements for the μ opioid receptor and the physiological role of this receptor. © 1986 American Chemical Society.
• Shook, J. E., Pelton, J. T., Kazmierski, W., Lemcke, P. K., Wire, W. S., Hruby, V. J., & Burks, T. F. (1986). Comparison of the opioid antagonist properties of a cyclic somatostatin analog in vitro and in vivo.. NIDA research monograph, 75, 205-208.
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  PMID: 2893263;
• Smith, P. F., Krack, G., McKee, R. L., Johnson, D. G., Gandolfi, A. J., Hruby, V. J., Krumdieck, C. L., & Brendel, K. (1986). Maintenance of adult rat liver slices in dynamic organ culture. In Vitro Cellular & Developmental Biology, 22(12), 706-712.
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  PMID: 3782009;Abstract: Adult rat liver slices were maintained for 20 h in a novel organ culture system with minimal loss of viability and function. Potassium and adenosine triphosphate levels were maintained at in vivo levels, following an initial recovery period (2 to 4 h), for up to 20 h. Protein synthesis and secretion were linear for 20 and 16 h, respectively. In addition, the liver slices synthesized glycogen between 4 and 12 h in culture. Finally, the liver slices were hormonally responsive during the 20 h culture period as exemplified by glucagon-stimulated glucose production. This system provides a simple and effective method for the culture and biochemical maintenance of adult rat liver for 20 h with minimal loss of biochemical function. © 1986 Tissue Culture Association, Inc.
• Spencer, R. L., Deupree, D., Hsiao, S., Mosberg, H. I., Hruby, V., Burks, T. F., & Porreca, F. (1986). Centrally-administered opioid selective agonists inhibit drinking in the rat. Pharmacology, Biochemistry and Behavior, 25(1), 77-82.
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  PMID: 2875475;Abstract: The effects of intracerebroventricular injection of mu (morphine), kappa (dynorphin-(1-13), ethylketocyclazocine, and U50,488H), and delta ([D-Pen2,D-Pen5]enkephalin) opioid agonists on water intake of 14 hr water deprived rats was studied. All agonists caused a dose related decrease in time spent drinking, with a rank order potency of dynorphin-(1-13) > morphine > ethylketocyclazocine >[D-Pen2,D-Pen5]enkephalin = U50,488H. With the exception of morphine, all of the compounds increased the latency to begin drinking, but only at the highest doses tested. The rank order potency for this endpoint was dynorphin-(1-13) = ethylketocyclazocine > [D-Pen2,D-Pen5]enkephalin > U503, 488H. The potent inhibition of drinking following centrally-given dynorphin-(1-13), at doses that did not affect the latency to begin drinking, supports a role for endogenous dynorphin in the homeostatic control of water balance. This function may not be primarily mediated through activation of a kappa opioid receptor since dynorphin-(1-13) was 80-230 times more potent than the selective kappa agonist, U50,488H or ethylketocyclazocine. © 1986.
• Stanfield, C. F., Cody, W. L., & Hruby, V. J. (1986). Preparation of β,β-dialkyl analogues of cysteine suitable for peptide synthesis. Journal of Organic Chemistry, 51(26), 5153-5156.
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  Abstract: A general method is described for the preparation of cysteine derivatives that are substituted with one or two alkyl groups at the β-carbon. The synthesis is based on the sulfenylation of Nα-formyl-α,β-dehydro amino acid esters. The protected dehydro esters were synthesized by the condensation of ethyl isocyanoacetate with a ketone. The sulfenylation of these compounds was accomplished by refluxing with phosphorus pentasulfide to form the intermediate thiazoline, which can be hydrolyzed to the hydrochloride salt of the free sulfhydryl amino acid by heating in acid. The free sulfhydryl amino acid salt was protected as the S-p-methylbenzyl thioether, isolated as the zwitterion. The S-protected amino acids were then protected as the Nα-tert-butyloxycarbonyl derivatives and are suitable for use in solution- or solid-phase peptide synthesis. © 1986 American Chemical Society.
• Sugg, E. E., Shook, J. E., Serra, M., Korc, M., Yamamura, H. I., Burks, T. F., & Hruby, V. J. (1986). Synthesis and biological evaluation of N^α-hydroxysulfonyl-[Nle^28,31]-CCK_26-33. Life Sciences, 39(18), 1623-1629.
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  PMID: 2430160;Abstract: Two analogues of [Nle28,31]-CCK26-33 containing an N-terminal acetyl or N-terminal hydroxysulfonyl moiety were prepared and characterized. Both analogues were equipotent to native CCK26-33 in four bioassays, demonstrating that N-terminal sulfation of CCK26-33 analogues is compatible with full biological activity. © 1986.
• Sugg, E. E., Sugg, E. E., Cody, W. L., Cody, W. L., Abdel-Malek, Z., Abdel-Malek, Z., Hadley, M. E., Hadley, M. E., Hruby, V. J., & Hruby, V. J. (1986). D-isomeric replacements within the 6-9 core sequence of Ac-[Nle⁴]-α-MSH_4-11-NH₂: A topological model for the solution conformation of α-melanotropin. Biopolymers, 25(11), 2029-2042.
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  PMID: 3790700;
• Wakelam, M. J., Murphy, G. J., Hruby, V. J., & Houslay, M. D. (1986). Activation of two signal-transduction systems in hepatocytes by glucagon. Nature, 322(6083), 68-71.
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  PMID: 3018586;Abstract: The ability of glucagon to stimulate glycogen breakdown in liver played a key part in the classic identification of cyclic AMP and hormonally stimulated adenylate cyclase. But several observations indicate that glucagon can exert effects independent of elevating intracellular cAMP concentrations. These effects are probably mediated by an elevation of the intracellular concentration of free Ca2+ although the mechanism by which this occurs is unknown. We show here that glucagon, at the low concentrations found physiologically, causes both a breakdown of inositol phospholipids and the production of inositol phosphates. Indeed, we show that the glucagon analogue, (1-N-α-trinitrophenylhistidine, 12-homoarginine)glucagon (TH-glucagon), which does not activate adenylate cyclase or cause any increase in cAMP in hepatocytes yet can fully stimulate glycogenolysis, gluconeogenesis and urea synthesis, stimulates the production of inositol phosphates. This stimulation of inositol phospholipid metabolism by low concentrations of glucagon provideds a mechanism whereby glucagon can exert cAMP-independent actions on target cells. We suggest that hepatocytes possess two distinct receptors for glucagon, a GR-1 receptor coupled to stimulate inositol phospholipid breakdown and a GR-2 receptor coupled to stimulate adenylate cyclase activity.
• Wilkes, B. C., Cody, W. L., Hruby, V. J., Castrucci, A. M., & Hadley, M. E. (1986). Comparative biological activities of potent analogues of α-melanotropin. Effect of nonaromatic and para substituted aromatic amino acids at position 7. International Journal of Peptide and Protein Research, 27(6), 685-694.
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  PMID: 3489682;
• Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., & Hruby, V. J. (1986). Crystal structure analysis of deamino-oxytocin: Conformational flexibility and receptor binding. Science, 232(4750), 633-636.
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  PMID: 3008332;Abstract: Two crystal structures of deamino-oxytocin have been determined at better than 1.1 Ȧ resolution from isomorphous replacement and anomalous scattering x-ray measurements. In each of two crystal forms there are two closely related conformers with disulfide bridges of different chirality, which may be important in receptor recognition and activation.
• Burks, T. F., Hruby, V. J., Galligan, J. J., & Porreca, F. (1985). Opioids and CNS control of the gut. Japanese Journal of Smooth Muscle Research, 21(SUPPL. 1985), 101-102.
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  PMID: 3007835;
• Case, T. C., Snider, S. R., Hruby, V. J., & Rockway, T. (1985). Active and inactive L-prolyl-L-leucyl glycinamide synthetic analogs in rat models of levodopa-treated Parkinson's disease. Life Sciences, 36(26), 2531-2537.
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  PMID: 2861549;Abstract: The tripeptide, L-prolyl-L-leucyl-glycinamide (PLG) has been shown to facilitate dopaminergic mechanisms in the brain. In the present study, we evaluated the interaction of PLG and its synthetic analogs with levodopa in two animal models of Parkinson's disease. In one experiment using rats with chronic unilateral lesions of the nigrostriatal dopamine pathway, PLG and Z-PLG potentiated the contraversive rotation elicited by levodopa with carbidopa (L/C). In a second experiment using reserpinized rats, PLG, Z-PLG and cyclo-LG potentiated L/C reversal of hypokinesia. Further studies of the PLG analogs, Z-PLG and cyclo-LG as adjunctive drugs with levodopa in the treatment of parkinsonism are warranted. © 1985.
• Chaturvedi, D. N., Hruby, V. J., De, A. C., Kreutzfeld, K. L., & Hadley, M. E. (1985). Synthesis and biological evaluation of the superagonist [N(α)-chlorotriazinylaminofluorescein-Ser¹,Nle⁴,D-Phe⁷]-α-MSH. Journal of Pharmaceutical Sciences, 74(3), 237-240.
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  PMID: 2989482;Abstract: The fluorescein-labeled melanotropin [N(α)-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe7]-α-MSH, was prepared by solid-phase techniques of peptide synthesis. The biological actions of this analogue were determined in several melanocyte bioassays and were compared with the parent peptide [Nle4,D-Phe7]-α-MSH and the native hormone α-MSH. The fluorescein compound was a superpotent agonist with ~10 times more activity than α-MSH in both the frog and the lizard skin bioassays. Murine S91 melanoma cells assayed in vitro (tyrosinase bioassay) were as responsive to the fluorescein analogue as to α-MSH. The analogue exhibited ultraprolonged biological activity and the biological activities were unaffected by treatment of the analogue with α-chymotrypsin. The fluorescein-labeled melanotropin should prove useful for melanotropin receptor characterization.
• Cody, W. L., Mahoney, M., Knittel, J. J., Hruby, V. J., Maria, A., & Hadley, M. E. (1985). Cyclic melanotropins. 9. 7-D-phenylalanine analogues of the active-site sequence. Journal of Medicinal Chemistry, 28(5), 583-588.
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  PMID: 2985783;Abstract: The cyclic melanotropin Ac-Ser1-Tyr2-Ser3-Cys4-Glu 5-His6-Phe7-Arg8-Trp 9-Cys10-Lys11-Pro12-Val 13-NH2 is a highly potent agonist as determined in several melanocyte bioassays. In linear melanotropins, a D-Phe7 substitution leads to increased potency and often prolonged biological activity. In order to determine if this substitution would have the same effect in cyclic melanotropins, we have prepared a series of these analogues. The D-Phe7-substituted cyclic melanotropins Ac-[Cys4,D-Phe7,Cys10]-α-MSH 4-10-NH2 and Ac-[Cys4,D-Phe7,Cys10]-α-MSH 4-11-NH2 were both more potent than their cyclic L-Phe7-containing counterparts in either the frog or lizard skin bioassay by more than a factor of 10. Neither peptide, however, exhibited prolongation of biological activity in either assay. Substitution of D-Phe7 into the cyclic 4-12 and 4-13 sequences led to a slight or no increase in potency in both assays relative to the L-Phe7 counterparts, but the activity of the melanotropins was ultraprolonged in each assay. Ac-[Cys4,D-Phe7,Cys10]-α-MSH 4-12-NH2 was about equipotent to Ac-[Cys4,D-Phe7,Cys10]-α-MSH 4-13-NH2, again demonstrating, as with certain linear and cyclic L-Phe7-containing melanotropins, that the C-terminal amino acid valine is not required for biological activity or for superpotency. Similar to the linear D-Phe7 analogues that possessed ultraprolonged melanotropic activity, the 4-12 and 4-13 cyclic D-Phe7 analogues also displayed the phenomenon of superagonism, which is a time-dependent increase in efficacy over that produced by an equipotent concentration of the native hormone. Cyclization of certain linear melanotropins resulted in analogues with increased resistance to biological degradation by serum enzymes or purified proteolytic enzymes. Further, incorporation of a D-Phe7 into in the cyclic analogues led to melanotropins that were totally resistant to enzymatic inactivation by trypsin. © 1985 American Chemical Society.
• Darman, P. S., Landis, G. C., Smits, J. R., Hirning, L. D., Gulya, K., Yamamura, H. I., Burks, T. F., & Hruby, V. J. (1985). Conformationally restricted cyclic analogues of substance P: Insight into the receptor binding process. Biochemical and Biophysical Research Communications, 127(2), 656-662.
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  PMID: 2579658;Abstract: Three new cyclic substance P analogues were prepared to examine the possible role of a pseudocyclic turn structure for receptor recognition. In the guinea pig isolated ileum [Cy over(over(s5, C, {top square bracket}),) ys11]-SP5-11 - NH2 and [Cy over(over(s6, C, {top square bracket}),) ys11]-SP5-11 - NH2 were inactive at concentrations up to 100 μM, while [Cy over(over(s5, C, {top square bracket}),) ys6, Nle11] - SP was a weak agonist. The order of relative affinities on the rat brain radioreceptor assay was as follows: [Cy over(over(s5, C, {top square bracket}),) ys6, Nle11] - SP> [Cy over(over(s5, C, {top square bracket}),) ys11] - SP5-11 - NH2 > [Cy over(over(s6, C, {top square bracket}),) ys11] - SP5-11 - NH2. We interpret these results to indicate that a pseudocyclic structure of the 5-11 sequence may not be an important factor involved in the receptor recognition of substance P. © 1985 Academic Press, Inc.
• Gulya, K., Gehlert, D. R., Wamsley, J. K., Mosberg, H. I., Hruby, V. J., Duckles, S. P., & Yamamura, H. I. (1985). Autoradiographic localization of δ opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog. European Journal of Pharmacology, 111(2), 285-286.
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  PMID: 2990955;
• Gulya, K., Wamsley, J. K., Gehlert, D., Pelton, J. T., Duckles, S. P., Hruby, V. J., & Yamamura, H. I. (1985). Light microscopic autoradiographic localization of somatostatin receptors in the rat brain. Journal of Pharmacology and Experimental Therapeutics, 235(1), 254-258.
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  PMID: 2864434;Abstract: The binding parameters and distribution of somatostatin receptors were determined in the rat brain using in vitro light microscopic autoradiography. The proteolysis resistant somatostatin analog (des-Ala1-, Gly2-desamino-Cys3Tyr11-dicarba3,14-somatostatin; CGP 23,996) radiolabeled with 125iodine proved to be suitable for the localization of somatostatin receptors. Slide mounted tissue sections showed that 125I-CGP 23,996 bound to the somatostatin receptor with a mean K(d) value of 4.0 nM. The mean density of receptors (maximum binding) was determined to be 182 fmol/mg of protein. Both somatostatin and unlabeled CGP 23,996 displayed high-affinity binding for somatostatin receptors with IC50 values of 6 and 5 nM, respectively. The areas containing the highest densities of receptors are the basal amygdaloid nucleus, medial habenular nucleus, stratum oriens and radiatum of CA1 and CA2, and the subiculum. High receptor density can also be found in the deep layers of the cingulate cortex and in the deep layers of temporal cortex. Moderate densities occur in the caudate-putamen, the granule cell layer of the cerebellum, CA3 area of the hippocampus, the molecular layer of the dentate gyrus and in the substantia nigra. Brain areas with low specific binding include the molecular layer of the cerebellum and the corpus callosum, a white matter area.
• Hadley, M. E., Mieyr, J. H., Martin, B. E., M, A., Hruby, V. J., Sawyer, T. K., Powers, E. A., & Rao, K. (1985). [Nle⁴, D-Phe⁷]-α-MSH: A superpotent melanotropin with prolonged action on vertebrate chromatophores. Comparative Biochemistry and Physiology -- Part A: Physiology, 81(1), 1-6.
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  PMID: 2859941;Abstract: 1. 1. The in vitro and in vivo reponses of integumental chromatophores to α-MSH and a related analogue, [Nle4, D-Phe7]-α-MSH, were studied in a number of vertebrate species: the teleost, Lebistes reticulatus; the amphibians. Rana pipiens, R. catesbeiana, Xenopus laevis. Bufo alvarius, and B. cognatus; the lizard, Anolis carolinensis; the rattlesnake, Crotalus atrox. 2. 2. The α-melanotropin analogue was a superpotent agonist in the in vitro frog (R. pipiens, R. catesbeiana) and lizard (A. carolinensis) skin bioassays. 3. 3. In all species studied, the analogue exhibited ultraprolonged melanotropic activity, both in vitro and in vivo. 4. 4. This melanotropin and related analogues should prove useful in the study of numerous physiological processes, particularly when prolonged melanotropic activity is desired. © 1985.
• Hirning, L. D., Mosberg, H. I., Hurst, R., Hruby, V. J., Burks, T. F., & Porreca, F. (1985). Studies in vitro with ICI 174, 864, [D-Pen², D-Pen⁵]-enkephalin (DPDPE) and [D-Ala², NMePhe⁴, Gly-ol]-enkephalin (DAGO). Neuropeptides, 5(4-6), 383-386.
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  PMID: 2987739;Abstract: The interactions of a proposed, selective delta receptor antagonist (ICI 174, 864) and selective agonists at mu and delta receptors, [D-Ala2, NMePhe4, Gly-ol]-enkephalin (DAGO) and [D-Pen2, D-Pen5]-enkephalin (DPDPE), respectively, have been studied using the electrically-stimulated mouse isolated vas deferens (MVD) and the guinea-pig isolated ileum (GPI). Incubation of increasing concentrations of ICI 174, 864 (10, 30, 100 and 300 nM) produced a dose-related and parallel rightward displacement of the DPDPE dose-response curve in the MVD. In contrast, ICI 174, 864 (300-3000 nM) failed to affect the DAGO dose-response curve in the same tissue. Analysis of the DPDPE-ICI 174, 864 interaction in the MVD using the pA2 method revealed a Schild plot slope of -0.68 suggesting the involvement of more than one population of receptors. ICI 174, 864 (300 nM) failed to antagonize DPDPE in the GPI at doses up to 30 uM. These results suggest that (a) ICI 174, 864 acts as a selective delta antagonist in the MVD; (b) DPDPE interacts with mu receptors in the MVD but only at very high concentrations, and (c) delta receptors appear not to be of functional importance in the GPI. © 1985.
• Hruby, V. J. (1985). Design of peptide hormone and neurotransmitter analogues. Trends in Pharmacological Sciences, 6(C), 259-262.
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  Abstract: Peptides are being found to be increasingly important as hormones and neurotransmitters, and much reasearch is directed towards elucidating the physiological roles of these natural products in pain, reproduction, maintenance of blood glucose levels etc. In this review, Victor Hruby describes some of the conformational approaches that have led to significant insights into the chemical and physical properties of these compounds and so to the rational design of analogues with new and specific properties. © 1985.
• Lebl, M., Hruby, V. J., Slaninova, J., & Barth, T. (1985). Solid phase synthesis and biological activities of oxytocin carba analogues containing threonine in position 4. Collection of Czechoslovak Chemical Communications, 50(2), 418-427.
• Malek, Z. A., Kreutzfeld, K. L., Marwan, M. M., Hadley, M. E., Hruby, V. J., & Wilkes, B. C. (1985). Prolonged stimulation of S91 melanoma tyrosinase by [Nle⁴, D-Phe⁷]-substituted α-melanotropins. Cancer Research, 45(10), 4735-4740.
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  PMID: 2992767;Abstract: α-Melanotropin (α-melanocyte stimulating hormone, α-MSH) stimulates tyrosinase activity in Cloudman S91 murine melanoma cells. Three [Nle4, D-Phe7]-substituted α-melanotropin analogues, [Nle4, D-Phe7]-α-MSH, Ac[Nle4, D-Phe7]-α-MSH4-11-NH2, and Ac-[Nle4, D-Phe7]-α-MSH4-10-NH2, are at least 100-fold more effective than α-MSH in stimulating melanoma tyrosinase, the rate-limiting enzyme in melanin biosynthesis. These [Nle4, D-Phe7]-substituted melanotropin analogues induce tyrosinase activity in melanoma cells with shorter contact times than required by the native hormone, α-MSH. [Nle4, D-Phe7]-substituted melanotropins also induce a prolonged (residual) stimulation of melanoma tyrosinase. Following incubation of melanoma cells in the presence of [Nle4, D-Phe7]-α-MSH for 24 h, tyrosinase activity is maintained for up to 6 days in the absence of the melanotropin. The shorter 4-10 and 4-11 fragment analogues also exhibit residual melanotropic activity. The prolonged stimulation of tyrosinase in the absence of the analogues is maintained even though melanoma cells continue to divide about every 24 h. These results suggest that melanoma cells possess spare melanotropin receptors and that [Nle4, D-Phe7]-substituted analogues bind almost irreversibly to these receptors or to some other component of the adenylate cyclase enzyme complex responsible for enhancing tyrosinase activity and melanin production.
• Marwan, M. M., A., Z., Kreutzfeld, K. L., Hadley, M. E., Wilkes, B. C., Hruby, V. J., & Maria, A. (1985). Stimulation of S91 melanoma tyrosinase activity by superpotent α-melanotropins. Molecular and Cellular Endocrinology, 41(2-3), 171-177.
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  PMID: 3926559;Abstract: α-Melanocyte-stimulating hormone (α-MSH, α-melanotropin), [Nle4,D-Phe7]-α-MSH and related fragment analogues, Ac-[Nle4,D-Phe7]-α-MSH4-11-NH2 and Ac-[Nle4,D-Phe7]-α-MSH4-10-NH2, were studied for their ability to stimulate tyrosinase activity in Cloudman S91 mouse melanoma cells in tissue culture. All of the melanotropins stimulated tyrosinase activity in a dose-dependent manner. [Nle4,D-Phe7]-α-MSH was about 100 times more active than a-MSH as determined from the minimal effective dose (MED) required to activate the enzyme above control (basal) levels. The MED of this analogue to significantly stimulate tyrosinase activity at 24, 48 and 72 h of incubation was 10-11 M whereas the MED of α-MSH was 10-9 M at each of these times. The maximum tyrosinase activity achieved from the time of initial incubation in the presence of [Nle4,D-Phe7]-α-MSH was approximately 3-, 5- and 6-fold greater than control levels at 24, 48 and 72 h, respectively. The 2 [Nle4,D-Phe7]-substituted fragment analogues were at least as active as the tridecapeptide analogue and therefore at least 100-fold more active than a-MSH in stimulating enzyme activity. These [Nle4,D-Phe7]-substituted analogues were more active in the melanoma tyrosinase assay than in the melanoma adenylate cyclase assay or other normal melanocyte (frog and lizard skin) bioassays. © 1985.
• Oshima, N., Kasukawa, H., Fujii, R., Wilkes, B. C., Hruby, V. J., Castrucci, A. M., & Hadley, M. E. (1985). Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores. Journal of Experimental Zoology, 235(2), 175-180.
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  PMID: 4056687;Abstract: The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10-10 M. The action of this putative hormone was not blocked by α- or β-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.
• Pelton, J. T., Gulya, K., Hruby, V. J., Duckles, S. P., & Yamamura, H. I. (1985). Conformationally restricted analogs of somatostatin with high μ-opiate receptor specificity. Proceedings of the National Academy of Sciences of the United States of America, 82(1), 236-239.
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  PMID: 2857488;PMCID: PMC397007;Abstract: A series of cyclic, conformationally restricted analogs of somatostatin have been prepared and tested for their ability to inhibit the binding of [3H]naloxone and [D-Ala2, D-Leu5][3H]enkephalin to rat brain membranes. The most potent analog, D-Phe[Cys-Tyr-D-Trp-Lys-Thr-Pen]-Thr-NH2 where Pen is penicillamine in [D-Phe5,[Cys6, Tyr7, D-Trp8, Pen11]]somatostatin-(5-12)-octapeptide amide, exhibited high affinity for μ-opiate receptors (IC50 value of [3H]naloxone = 3.5 nM), being 7800 times more potent than somatostatin. The cyclic octapeptide also displayed high μ-opiate receptor selectivity with an IC50 ([D-Ala2-, D-Leu5]enkephalin)/IC50 (naloxone) ratio of 271. The high affinity and selectivity of the somatostatin analog for μ-opiate receptors may be of use in examining the physiological role(s) of the μ-opiate receptor.
• Pelton, J. T., Gulya, K., Hruby, V. J., Duckles, S., & Yamamura, H. I. (1985). Somatostatin analogs with affinity for opiate receptors in rat brain binding assay. Peptides, 6(SUPPL. 1), 159-163.
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  PMID: 2864680;Abstract: The somatostatin analogs D-Phe-Cys-D-Trp-Lys-Thr-Cys-Thr and the corresponding penicillamine compounds have been prepared and tested for their ability to displace [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin from rat brain receptors. While somatostatin and the cystine containing peptide displayed little or no preference for either receptor system, the substitution of penicillamine at position two or seven resulted in analogs that displayed opposite receptor selectivity. The substitution of tyrosine for phenylalanine at position three resulted in a large increase in opiate receptor affinity which may be related to the known requirement for a phenolic hydroxyl moiety in the rigid opiate and enkephalin systems. Conformational properties of these analogs were also examined and related to their affinity for opiate and somatostatin receptors in the rat brain. © 1985.
• Akiyama, K., Yamamura, H. I., Wilkes, B. C., Cody, W. L., Hruby, V. J., Maria, A., & Hadley, M. E. (1984). Relative stability of α-melanotropin and related analogues to rat brain homogenates. Peptides, 5(6), 1191-1195.
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  PMID: 6531272;Abstract: α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4, D-Phe7]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4, D-Phe7]-α-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4, D-Phe7]-α-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4, D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-α-MSH, Ac-[Nle4]-α-MSH4-10-NH2 and Ac-[Nle4]-α-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-α-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-α-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues. © 1984.
• Blumenstein, M., Hruby, V. J., Viswanatha, V., & Chaturvedi, D. (1984). Carbon-13 chemical shifts on oxytocin as a consequence of its interaction with neurophysins. Biochemistry, 23(10), 2153-2161.
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  PMID: 6733077;Abstract: Carbon-13 NMR was used to study the interaction of the hormone oxytocin with neurophysin (NP). Oxytocins specifically enriched to 90% 13C in the α-carbons of Leu-3 (in [3-leucine]oxytocin), Gln-4, and Leu-8 and in the carbonyl carbon of Cys-6 were synthesized, so that the effect on these positions of binding to NP could be monitored. The α-carbons of residues 3 and 4 experienced shifts of -4.2 and -1.5 ppm (negative shifts are downfield), respectively, upon binding of the hormone to NP. The carbonyl carbon of residue 6 underwent a shift of +0.7 ppm, while the α-carbon of residue 8 displayed no shift. For each enriched residue, the hormone diastereoisomer in which this residue had the D configuration was also synthesized. NMR was then used to determine the binding affinity of the various diastereoisomers to NP, as well as to measure the NMR parameters of the bound peptides. When position 3 had the D configuration, the binding affinity for NP was 10-20% that of the native hormone. For positions 4, 6, and 8, the D diastereoisomers bound with the same affinity as oxytocin. The α-carbons of D residues of positions 3 and 4 shifted by -2.5 and +0.4 ppm, respectively, the carbonyl carbon of D-Cys-6 shifted by +1.4 ppm, and the α-carbon of D-Leu-8 was unshifted on binding to NP. The shift and diastereoisomer binding data, combined with previous results involving enriched carbons and/or diastereoisomers of residues 1, 2, and 9, support the conclusion that residues 1 and 2 are most crucial for binding of oxytocin to NP, residue 3 is less important, and residues 4-9 are of only slight significance. An unequivocal interpretation of the chemical shift effects was not possible, but side chain conformation was deduced to contribute significantly. It appears that the conformations of several side chains of oxytocin bound to NP differ significantly from the predominantly averaged side chain conformations present in solution. For Cys-1, Tyr-2, and Leu-3, the change in side chain conformation is caused by direct interaction of the side chain with NP. For Gln-4, the side chain does not directly interact with NP, but its bound conformation differs from its free conformation due to changed interactions with the rest of the oxytocin molecule. The overall dissociation rate constant of oxytocin from NP was found to be 2-4 s-1, in good agreement with results from rapid kinetic experiments. Substitution of a D residue in position 3, which resulted in a decreased binding affinity for NP, also led to an increased dissociation rate constant, while the association rate constant for the D diastereoisomer was the same or somewhat higher than that of the unmodified peptide. Thus, the weaker binding of the D diastereoisomer is due to a weaker complex rather than to an unfavorable conformation of the free hormone. © 1984 American Chemical Society.
• Chaturvedi, D. N., Knittel, J. J., Hruby, V. J., Maria, A., & Hadley, M. E. (1984). Synthesis and biological actions of highly potent and prolonged acting biotin-labeled melanotropins. Journal of Medicinal Chemistry, 27(11), 1406-1410.
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  PMID: 6436488;Abstract: Biocytin derivatives of a superpotent analogue of α-melanotropin, [Nle4,D-Phe7]-α-MSH, were prepared. [Nα-Bct-Ser1,Nle4,D-Phe 7]-α-MSH and [12-Bct-Nα-dodecanoyl-Ser1,Nle 4,D-Phe7]-α-MSH were synthesized by solid-phase techniques, and the coupling of biotin and 12-aminododecanoic acid was achieved through their succinimido esters. These melanotropins possessed almost identical actions to [Nle4,D-Phe7]-α-MSH as determined by several melanocyte bioassays. Both biocytin derivatives were highly potent agonists and exhibited prolonged biological activity as determined in the frog and lizard skin bioassays. Both biotinylated peptides were at least equipotent to α-MSH in stimulating Cloudman S91 mouse melanoma tyrosinase activity. The analogues were resistant to inactivation by α-chymotrypsin. © 1984 American Chemical Society.
• Cody, W. L., Wilkes, B. C., & Hruby, V. J. (1984). Reversed-phase high-performance liquid chromatography studies of α-MSH fragments. Journal of Chromatography A, 314(C), 313-321.
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  PMID: 6526885;Abstract: α-Melanotropin (α-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Syr-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2), that is primarily known for its ability to stimulate melanosome dispersion within integumental melanocytes (F. J. H. Tilders, D. F. Swaab and T. B. van Wimersma Greidanus (Editors), Frontiers of Hormone Research, Vol. 4, Karger, Basel, 1977; J. Ramachandran, S. W. Farmer, S. Liles and C. H. Li, Biochem. Biophys. Acta, 428 (1976) 347). In our efforts to understand the relationship of structure and conformation to the biological activities of α-MSH, we have prepared a series of diastereoisomeric analogues based on the highly potent analogue Ac-[Nle4, d-Phe7]-αMSH4-11-NH2 (T. K. Sawyer, V. J. Hruby, B. C. Wilkes, M. T. Draelos, M. E. Hadley and J. Bergsneider, J. Med. Chem., 25 (1982) 1022). These analogues differed only in the amino acid substituted in the seven position, which was thought to be a critical residue for the biological activity of α-MSH. The chromatographic behavior of these analogues was examined on a C18 Vydac (16-μm) reversed-phase column with five different mobile phases. The selectivity (α) for the analogues was compared in 0.10% trifluoroacetic acid (TFA), 0.10% heptafluorobutyric acid (HFBA) and 0.25 M triethylammonium phosphate (TEAP) using either acetonitrile or methanol as the organic modifier. With only one exception all analogues substituted with a d-amino acid in the seven position were eluted prior to their l-amino acid counterparts. As expected due to the greater ionic strength, the TEAP buffer allowed the greatest selectivity for the separation of these-α-MSH analogues, but it was surprising to find that the TFA buffer had a greater influence on selectivity that the HFBA buffer with either organic modifier. The probable mechanism of retention for the α-MSH analogues in perfluoroalkanoic buffers was also investigated. In addition, the relatioship between the retention time and the hydrophobicity of the seven position substitution was examined. Although the data were somewhat limited, a lack of correlation between the hydrophobicity of the seven position residue and retention time was observed. © 1984.
• Cody, W. L., Wilkes, B. C., Muska, B. J., Hruby, V. J., Maria, A., & Hadley, M. E. (1984). Cyclic melanotropins. 5. Importance of the C-terminal tripeptide (Lys-Pro-Val). Journal of Medicinal Chemistry, 27(9), 1186-1190.
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  PMID: 6332195;Abstract: In previous work we reported that [Cys4,Cys10]-α-MSH (II) and Ac-[Cys4,Cys10]-α-MSH4-13-NH2 (III) were superpotent melanotropins. 2,3 Ac-[Cys4,Cys10]-α-MSH4-10-NH2 (VI), which constitutes the cyclic analogue of the putative active site sequence -Met4-Glu5-His6-Phe7-Arg 8-Trp9-Gly10- of α-MSH, was much less active. In the present investigation the contribution of the Lys11 and Pro12 residues of the C-terminal carboxamide tripeptide -Lys11-Pro12-Val13-NH2 to the potency of Cys4,Cys10 containing cyclic melanotropins was studied. Ac-[Cys4,Cys10]-α-MSH4-11-NH2 (V) was less potent than α-MSH in the frog and lizard skin bioassays and the mouse S-91 (Cloudman) melanoma adenylate cyclase assay but more potent than Ac-[Cys4,Cys10]-α-MSH4-10-NH2 in the three assays studied. Ac-[Cys4,Cys10]-α-MSH4-12-NH2 (IV) was considerably more potent than the cyclic 4-11 melanotropin and was, in fact, equipotent or even slightly more potent than [Cys4,Cys10]-α-MSH and Ac-[Cys4,Cys10]-α-MSH4-13-NH2 over the linear portion of the dose-response in all three bioassays. These results demonstrate that Lys11 and Pro12 but to a lesser extent Val13 of the C-terminal tripeptide sequence contributes to the potency of the cyclic melanotropins. The further substitution of a D-Phe7 for the L-Phe7 residue into the cyclic 4-12 analogue resulted in a highly potent compound Ac-[Cys4,D-Phe7,Cys10]-α-MSH 4-12-NH2 (VII) that exhibited highly prolonged biological activity. © 1984 American Chemical Society.
• Corvera, S., Huerta-Bahena, J., Pelton, J. T., Hruby, V. J., Trivedi, D., & García-Sáinz, J. (1984). Metabolic effects and cyclic AMP levels produced by glucagon, (1-N^α-trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes. BBA - Molecular Cell Research, 804(4), 434-441.
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  PMID: 6087925;Abstract: [1-Nα-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested. © 1984.
• Galligan, J. J., Mosberg, H. I., Hurst, R., Hruby, V. J., & Burks, T. F. (1984). Cerebral delta opioid receptors mediate analgesia but not the intestinal motility effects of intracerebroventricularly administered opioids. Journal of Pharmacology and Experimental Therapeutics, 229(3), 641-648.
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  PMID: 6327967;Abstract: Conformationally constrained cyclic enkephalin analogs which possess a high selectivity for the delta opioid receptor were used to determine the relative contribution of mu and delta receptors to brain-mediated changes in small intestinal propulsion and increases in hot-plate response time. Receptor preferences were determined by comparing the relative potencies of several opioid agonists in suppressing the electrically evoked contractions of the guinea-pig ileum and mouse vas deferens preparations. The ratio of IC50 values obtained in the guinea-pig ileum and the mouse vas deferens was used as an index of delta receptor selectivity. Effects on intestinal transit were determined in rats in which a silastic cannula had been implanted in the proximal duodenum and a polyethylene cannula in the right lateral cerebral ventricle (i.c.v.). Movement of a radioactive marker along the length of the small intestine after instillation into the duodenum was used to evaluate drug-induced changes in intestinal transit. The analgesic effects of i.c.v. administered opioids were determined in a second group of rats in which i.c.v. cannulas alone had been implanted. After i.c.v. administration of the agonist, the rats were placed on a 55°C hot plate and the latency to rear paw-lick was timed. Compounds which showed a preference for the mu receptor ([D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin and morphine/normorphine) were the most potent agonists at producing thermal analgesia and inhibition of small intestinal transit, whereas nonselective compounds (β-endorphin and [D-Ala2, Met5]enkephalinamide) were slightly less potent in these assays. As delta receptor selectivity increased ([D-Pen2, D-Pen5]enkephalin > [D-Pen2, L-Pen5]enkephalin > [D-Pen2, L-Cys5]enkephalin > [D-Ala2, D-Leu5]enkephalin), potency at inhibiting intestinal transit decreased markedly, whereas potency for producing thermal analgesia decreased only slightly. The analgesic effects of [D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin, [D-Pen2, L-Cys5]enkephalin, [D-Pen2, L-Pen5]enkephalin and [D-Pen2, D-Pen5]enkephalin were all antagonized by naloxone pretreatment (2.0 mg/kg). U-50,488H, a kappa selective compound, did not affect intestinal transit at any dose tested and increased hot-plate latencies only at the highest dose (125 μg). These data indicate that mu receptors exclusively mediate the intestinal motility effects of i.c.v. administered opioids, whereas both mu and delta receptors can mediate the analgesic effects in rats.
• Hirsch, M. D., O'Donohue, T. L., Wilson, R., Sawyer, T. K., Hruby, V. J., Hadley, M. E., Cody, W. L., Knittel, J. J., & Crawley, J. N. (1984). Structural and conformational modifications of α-MSH/ACTH_4-10 provide melanotropin analogues with highly potent behavioral activities. Peptides, 5(6), 1197-1201.
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  Abstract: Previous studies have identified the (4-10) heptapeptide sequence as the central core of α-MSH/ACTH peptides required for mediation of important biological activities. In the present study, the structure-activity relationships of Nle4-substituted and tsqbCys4, Cys10-bridged cyclic α-MSH analogues, which were previously shown to exhibit a wide range of melanotropic potencies from weak agonism to super potency, were examined for grooming behavioral activity in the rat following intracerebroventricular injections. The results showed that stepwise C-terminal elongation of the linear Nle4-substituted Ac-α-MSH4-10-NH2 increased grooming potencies of the peptides in a manner similar to their actions on melanocytes. The most interesting finding was the observation that cyclization of the inactive linear "central (4-10) core" of α-MSH (Ac-α-MSH4-10) to form Ac-[tsqbCys4, Cys10]-α-MSH4-10-NH2 resulted in a super potent agonist in the grooming assay. However, while cyclization of the (4-10) heptapeptide produced potent agonists on grooming behavior, the structure-activity relationships were different than the frog skin bioassay. These findings support the hypothesis that appropriate structural and confirmational modifications of α-MSH-related peptides can produce profound effects on the bioactivities of the peptides, and suggest that different structural-conformational requirements exist for α-MSH interactions with its various receptors. © 1984.
• Hruby, V. J., Krstenansky, J. L., & Cody, W. L. (1984). Chapter 30. Recent Progress in the Rational Design of Peptide Hormones and Neurotransmitters. Annual Reports in Medicinal Chemistry, 19(C), 303-312.
• Lebl, M., & Hruby, V. J. (1984). Synthesis of cyclic peptides by solid phase methodology. Tetrahedron Letters, 25(20), 2067-2068.
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  Abstract: Four carba analogues of oxytocin and a cyclic analogue of melanocyte stimulating hormone were synthesized using solid phase methodology. Purified compounds were shown to be highly biologically active. © 1984.
• Lebl, M., Cody, W. L., Wilkes, B. C., Hruby, V. J., Castrucci, A. M., & Hadley, M. E. (1984). Cyclic melanotropins. Part VII. Modified ring structures - synthesis and biological activity. International Journal of Peptide and Protein Research, 24(5), 472-479.
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  PMID: 6335135;Abstract: The highly potent cyclic analogue of α-MSH, Ac-[Cys4, Cys10]-α-MSH4-13-NH2, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin biossays were determined. It was shown that removing the terminal acetylamino group to give [Mpa4,Cys10]-α-MSH4-13-NH2 resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys4,Cys10]-α-MSH4-13-NH2 led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa4,Cys10]-α-MSH4-13-NH2, resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the ring by one methylene group to give Ac-[Hyc4,Cys10]-α-MSH4-13-NH2 led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.
• Maria, A., Hadley, M. E., & Hruby, V. J. (1984). Melanotropin bioassays: In vitro and in vivo comparisons. General and Comparative Endocrinology, 55(1), 104-111.
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  PMID: 6611282;Abstract: A reflectance method was utilized to compare the in vitro responses in three species of frogs (Rana pipiens, R. berlandieri forrei, and R. catesbeiana) and a lizard (Anolis carolinensis) to α- and β-melanotropins (α- and βp-MSH). The integumental chromatic response of the three ranid species was identical, in that α-MSH was about 2 times more potent than βp-MSH. The melanotropins were equipotent in the lizard skin bioassay. A remarkable feature of the Anolis skin assay is that skins from this lizard can be utilized repeatedly many times in one day with an extremely high degree of precision. The reflectance method was also used to determine the in vivo potencies of α-MSH and βp-MSH in the frog, R. pipiens. Surprisingly, the melanotropins were more active in the in vivo assay than in the in vitro bioassay. The darkening response of the frogs to α-MSH was reversed by 6 hr, but the response to βp-MSH persisted for more than 8 hr. When α-MSH was incubated in frog serum, the melanotropic activity was almost totally abolished by 30 min, whereas the melanotropic activity of βp-MSH was evident much longer (4 hr) in the presence of the serum. In light of the observation that the melanotropic activity of α-MSH is rapidly lost by incubation in frog serum, it is unclear why the hormone was more active as measured in vivo and why the darkening response in vivo persisted so long. © 1984.
• Maria, A., Hadley, M. E., Sawyer, T. K., & Hruby, V. J. (1984). Enzymological studies of melanotropins. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 78(3), 519-524.
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  PMID: 6332706;Abstract: 1. 1. The relative stability of natural melanotropins and related synthetic analogues to serum and purified proteolytic enzymes was studied. 2. 2. Both α- and β-MSH were rapidly inactivated by frog serum, but much more slowly by rat serum. β-MSH was more stable than α-MSH to serum inactivation. 3. 3. Both α- and β-MSH were rapidly inactivated by α-chymotrypsin and trypsin. 4. 4. The synthetic analogues, [Nle4, d-Phe7]-α-MSH and and [Cys4,Cys10]-α-MSH, were totally resistant to inactivation by frog and rat serum enzymes. 5. 5. [Nle4, d-Phe7]-α-MSH was resistant to inactivation by α-chymotrypsin and trypsin, whereas [Cys4,Cys10]-α-MSH, was partially resistant to these enzymes under similar conditions. 6. 6. Melanotropin analogues resistant to inactivation by serum enzymes may prove useful in a variety of physiological studies wherein natural melanotropins would be rapidly inactivated. © 1984.
• Porreca, F., Mosberg, H. I., Hurst, R., Hruby, V. J., & Burks, T. F. (1984). Roles of Mu, delta and kappa opioid receptors in spinal and supraspinal mediation of gastrointestinal transit effects and hot-plate analgesia in the mouse. Journal of Pharmacology and Experimental Therapeutics, 230(2), 341-348.
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  PMID: 6086883;Abstract: The opioid receptors involved in the mediation of thermal analgesia (55°C hot-plate) and inhibition of gastrointestinal transit at the spinal and supraspinal levels were studied in unanesthetized mice. Five receptor-selective compounds were evaluated for effectiveness in eliciting analgesia and inhibiting transit after both i.c.v. and intrathecal administration; these included the proposed mu agonist, [D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin (DAGO), the proposed delta agonists, [D-Pen2, L-Pen5]enkephalin (DPLPE), [D-Pen2, D-Pen5]enkephalin (DPDPE) (conformationally constrained delta selective enkephalin analogs) and [D-Thr2, Thr6, Leu5]enkephalin (DTTLE), and the proposed kappa agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate (U-50,488H), as well as the nonselective mu-acting agonist, morphine. All compound were found to produce analgesia after i.c.v. administration; the rank order of potency by the i.c.v. route was DAGO > DTTLE > morphine > DPLPE > DPDPE > U-50,488H. The analgesic effectiveness of most of these agonists given i.c.v. was evident for up to 40 min, with only DTTLE and U-50,488H having briefer time courses. Similarly, all the compounds produced analgesic responses after intrathecal administration, with the rank order of potency by this route being DTTLE > morphine > DAGO > DPLPE > DPDPE > U-50,488H, and all compounds (except U-50,488H) had durations of action of up to 20 to 40 min. These agonists also inhibited gastrointestinal transit after intrathecal administration, with a rank order of potency of DAGO > DTTLE > DPLPE > morphine > DPDPE > U-50,488H. In contrast, the most selective agonists for the delta receptor, DPLPE and DPDPE, as well as the selective kappa agonist, U-50,488H, were ineffective against transit after i.c.v. administration; the i.c.v. rank potency for inhibition of transit was DAGO > DTTLE > morphine > DPLPE, DPDPE, U-50,488H. The use of the most selective opioid agonists for the mu, delta and kappa receptors currently available along with two endpoints and sites of administration suggests that in mice 1) hot-plate analgesia at the supraspinal level is mediated by both mu and delta receptors while antitransit effects at this site are mediated exclusively by mu receptors, and 2) hot-plate analgesia at the level of the spinal cord is mediated mainly by delta receptors while inhibition of transit is mediated through delta and mu receptors.
• Vinson, G. P., Whitehouse, B. J., Bateman, A., Hruby, V. J., Sawyer, T. K., & Darman, P. S. (1984). α-MSH analogues and adrenal zona glomerulosa function. Life Sciences, 35(6), 603-610.
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  PMID: 6087070;Abstract: Recent studies on the control of adrenal zona glomerulosa function and aldosterone secretion have focussed attention on the role of MSH- like peptides. In particular, at low concentrations, α-MSH has a specific stimulatory effect on rat adrenal glomerulosa cells. The synthesis of α-MSH analogues which have potent and prolonged effects on melanocyte systems offers new methods of examining the specificity of this response. Two peptides were tested in which potential for a β-turn configuration was stabilised. These were: [Nle4, D-Phe7]-α-MSH and the cyclic [cys4, Cys10]-α-MSH. In contrast to their effects on molanocyte systems, only [cys4, Cys10]-α-MSH stimulated glomerulosa cells, and it was equipotent with a α-MSH, while [Nle4, D-Phe7]-α-MSH and shorter fragments had no effect when added alone [Nle4, D-Phe7]-α-MSH, however, augmented the response of cells already maximally stimulated with α-MSH and in this respect its actions resembled those of γ-MSH and related peptides. The augmentation produced by [Nle4, D-Phe7]-α-MSH and γ3-MSH was not additive when the two peptides were added together with α-MSH. The results suggest that the specificity of the α-MSH receptors in rat adrenal glomerulosa cells and the peptide structure-function relationships in this system are different from those described for melanocytes. © 1984.
• Wilkes, B. C., Hruby, V. J., Castrucci, A. d., Sherbrooke, W. C., & Hadley, M. E. (1984). Synthesis of a cyclic melanotropic peptide exhibiting both melanin-concentrating and -dispersing activities. Science, 224(4653), 1111-1113.
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  PMID: 6609433;Abstract: A putative melanin-concentrating hormone was synthesized. This peptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH, stimulates melanin granule aggregation within teleost melanocytes at nanomolar concentrations as does the natural purified teleost pituitary preparation. In addition, this peptide stimulates melanin granule dispersion within melanocytes of frogs and lizards. The peptide has about one six-hundredth of the activity of α-melanocyte-stimulating hormone on frog and lizard melanocytes and is a full agonist.
• Wilkes, B. C., Hruby, V. J., Sherbrooke, W. C., M., A., & Hadley, M. E. (1984). Synthesis and biological actions of melanin concentrating hormone. Biochemical and Biophysical Research Communications, 122(2), 613-619.
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  PMID: 6466330;Abstract: A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH, stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of α-melanocyte stimulating hormone (α-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied. © 1984 Academic Press, Inc.
• Wilkes, B. C., Hruby, V. J., Yamamura, H. I., Akiyama, K., Marie, A., Hadley, M. E., Andrews, J. R., & Wan, Y. -. (1984). Synthesis of tritium labeled Ac-[Nle⁴, D-Phe⁷]-α-MSH_4-11-NH₂: A superpotent melanotropin with prolonged biological activity. Life Sciences, 34(10), 977-984.
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  PMID: 6608041;Abstract: Ac-[Nle4, D-Phe7]-α-MSH4-11-NH2 an octapeptide, is a melantropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH2), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This malanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritiumlabeled congener was prepared by direct incorporation of [3H]-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors. © 1984.
• Wilkes, B. C., Sawyer, T. K., Hruby, V. J., & Hadley, M. E. (1984). Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4. International Journal of Peptide and Protein Research, 23(6), 621-629.
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  PMID: 6332085;
• Blevins, D. D., Burke, M. F., & Hruby, V. J. (1983). ROLE OF THE THIRD MOBILE PHASE SOLVENT IN BONDED PHASE CHROMATOGRAPHY.. Journal of Chromatographic Science, 21(11), 490-494.
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  Abstract: The role of the third mobile phase solvent in bonded phase chromatography is investigated on several different C//1//8 bonded phase columns. Subtle changes in selectivity are demonstrated by the addition of a third solvent to the mobile phase. The changes in selectivity can be varied dynamically by changing the percentage composition of three components in the mobile phase. For example, the use of a third component allows use of the lipophilic side chains of peptide diastereoisomers to enhance separation. In addition, the utilization of a third component allows the selective adjustment of the properties of the stationary phase so as to obtain identical selectivity for column packing materials from different sources. The observed selectivity adjustments lead to further insight in terms of the properties of the diastereoisomers as well as the active role played by the stationary phase in the separation mechanism.
• Hruby, V. J., Mosberg, H. I., Sawyer, T. K., Knittel, J. J., Rockway, T. W., Ormberg, J., Darman, P., Chan, W. Y., & Hadley, M. E. (1983). Conformational and dynamic considerations in the design of peptide hormone analogs. Biopolymers, 22(1), 517-530.
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  PMID: 6673771;Abstract: Efforts to understand the chemical-physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical-physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, the authors have prepared several semirigid [Pen 1]-oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure-activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide α-melanotropin. Some of these peptides have exceptionally prolonged in vivo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). The authors have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).
• Hruby, V. J., Rockway, T. W., Viswanatha, V., & Chan, W. Y. (1983). Pharmacological, conformational and dynamic properties of cycloleucine-2 analogues of oxytocin and [1-penicillamine]oxytoxin. International Journal of Peptide and Protein Research, 21(1), 24-34.
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  PMID: 6826279;Abstract: The solid phase syntheses of [2-cycloleucine] oxytocin and [1-penicillamine, 2-cycloleucine] oxytocin are reported. [1-Penicillamine, 2-cycloleucine] oxytocin is an oxytocin antagonist exhibiting no in vitro oxytocic activity. In the in vitro oxytocic assay, [1-penicillamine, 2-cycloleucine] oxytocin has a pA2 value of 6.70 ± 0.08 [2-Cycloleucine]-oxytocin is a full oxytocin agonist exhibiting 4.9 ± 0.5 U/mg of oxytocic activity. Neither compound possesses any measurable agonist or antagonist activity in the rate pressor assay. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T1) of the antagonist, [1-penicillamine, 2-cycloleucine] oxytocin, indicate that the antagonist exhibits similar conformational and dynamic properties as other oxytocin inhibitors previously studied. The carbon-13 nuclear magnetic resonance shift parameters and spin-lattice relaxation times (T1) of the oxytocin agonist, [2-cycloleucine] oxytocin, indicate that the agonist exhibits similar conformational and dynamic properties as oxytocin. These results are discussed in terms of the different receptor requirements for agonist and antagonist activities. It appears that there are different structural and conformational requirements at the 2-position for oxytocic agonist and antagonist activities.
• Knittel, J. J., Sawyer, T. K., Hruby, V. J., & Hadley, M. E. (1983). Structure-activity studies of highly potent cyclic [Cys⁴,Cys¹⁰]melanotropin analogues. Journal of Medicinal Chemistry, 26(2), 125-129.
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  PMID: 6600792;
• Kobobun, K., O'Donohue, T. L., Handelmann, G. E., Sawyer, T. K., Hruby, V. J., & Hadley, M. E. (1983). Behavioral effects of [4-Norleucine, 7-D-Phenylalanine]-α-melanocyte-stimulating hormone. Peptides, 4(5), 721-724.
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  PMID: 6657517;Abstract: The behavioral effects of α-melanocyte stimulating hormone (α-MSH) were compared to an α-MSH analogue that had a norleucine substituted for methionine in the four position and a D-phenylalanine substituted for L-phenylalanine in the seven position. [Nle4,D-Phe7]-α-MSH has previously been shown to be a superpotent agonist on melanocytes [17]. The present experiments indicate that [Nle4,D-Phe7]-α-MSH is equipotent to α-MSH in inducing grooming when administered intraventricularly. In contrast, the analogue has the opposite effect of α-MSH on performance of a visual discrimination task. α-MSH improves visual performance whereas [Nle4,D-Phe7]-α-MSH attenuates such performance. The contrasting activities of [Nle4,D-Phe7]-α-MSH on the physiological processes described suggest that this analogue may interact with three distinct melanotropin receptors in different ways. On melanocyte receptors the melanotropin analogue is a superagonist, on CNS melanotropin receptors involved in grooming it is equipotent to α-MSH, and on CNS receptors involved in attention, learning and memory [Nle4,D-Phe7]-α-MSH may be an antagonist of endogenous melanotropin. © 1983.
• Mosberg, H. I., Hurst, R., Hruby, V. J., Galligan, J. J., Burks, T. F., Gee, K., & Yamamura, H. I. (1983). Conformationally constrained cyclic enkephalin analogs with pronounced delta opioid receptor agonist selectivity. Life Sciences, 32(22), 2565-2569.
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  PMID: 6304440;Abstract: The enkephalin analogs, [D-Pen2, L-Cys5]- and [D-Pen2, D-Cys5]- enkephalin are cyclic compounds, conformationally constrained by virtue of their 14-membered, disulfide containing rings and by the rigidizing effect of the β,β dimethyl substituents of the penicilamine side chain. The analogs exhibit profound δ receptor specificity as assessed by their relative potencies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, exhibiting, respectively, 666 and 215 times higher potency in the latter assat system. By contrast, the receptor selectivities measured in rat brain binding assays in the absence of sodium were much more modest, the cyclic analogs being, respectively, 15.2 and 6.0 times more effective at displacing [3H] [D-Ala2, D-Leu5] enkephalin than [3H] naloxone. However, for binding assays performed in the presence of a sodium concentration equivalent to that used in the GPI and MVD assays, these binding selectivities increased to 167 and 49, respectively. © 1983.
• Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Akiyama, K., Yamamura, H. I., Galligan, J. J., & Burks, T. F. (1983). Cyclic penicillamine containing enkephalin analogs display profound delta receptor selectivities. Life Sciences, 33(SUPPL. 1), 447-450.
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  PMID: 6319901;Abstract: The cyclic, penicillamine (β, β dimethylcysteine)-containing enkephalin analogs, [D-Cys2, L-Pen5]- and [D-Cys2, D-Pen5]enkephalin and the corresponding bis-penicillamine analogs, [D-Pen2, L-Pen5]- and [D-Pen2, D-Pen5]enkephalin were synthesized and evaluated for opioid activity in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and in rat brain and neuroblastoma-glioma cell membrane binding assays. These analogs all displayed δ receptor selectivity as assessed by IC50(GPI)/IC50(MVD) ratios and by their relative potencies for displacing [3H]naloxone (NAL) vs. [3H] [D-Ala2, D-Leu5]enkephalin (DADLE) from rat brain membrane preparations. For [D-Pen2, L-Pen5]- and [D-Pen2, D-Pen5]enkephalinthe observed IC50(GPI)/IC50 (MVD) ratios (1088 and 3164) and IC50NAL/IC50DADLE ratios (371 and 175) represent a vast improvement over previously reported δ receptor selective ligands. © 1983.
• Mosberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Yamamura, H. I., Galligan, J. J., & Burks, T. F. (1983). Bis-penicillamine enkephalins possess highly improved specificity toward δ opioid receptors. Proceedings of the National Academy of Sciences of the United States of America, 80(19 I), 5871-5874.
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  PMID: 6310598;PMCID: PMC390177;Abstract: The conformationally restricted, cyclic, disulfide-containing, enkephalin analogs [2-D-penicillamine, 5-L-penicillamine]enkephalin ([D-Pen2,L-Pen5]enkephalin) and [2-D-penicillamine, 5-D-penicillamine]enkephalin ([D-Pen2,D-Pen5]enkephalin) were synthesized by solid-phase methods. Selectivities of these analogs for a single class of opioid receptor were investigated by examining relative potencies in the mouse vas deferens assay, in which the functional receptor is the δ receptor, versus the guinea pig ileum assay, in which the μ receptor is the functional receptor, and by determining their relative abilities to displace the prototypical δ receptor ligand [D-Ala2,D-Leu5]enkephalin and the prototypical μ receptor ligand naloxone from rat brain membrane preparations. Based on these comparisons [D-Pen2,L-Pen5]- and [D-Pen2,D-Pen5]enkephalin exhibited δ receptor selectivities of 1,088 and 3,164, respectively, in the bioassays, and 371 and 175, respectively, in the binding assays. Compared with the previously reported δ receptor selective analogs, [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Thr2,Leu5,Thr6]enkephalin, the bis-Pen-containing analogs provide an order of magnitude increase in δ receptor selectivity.
• Pelton, J. T., Trivedi, D., & Hruby, V. J. (1983). Re-evaluation of glucagon_1-6: The N-terminal hexapeptide of glucagon is not biologically active in the hepatic adenylate cyclase system. Life Sciences, 33(13), 1307-1314.
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  PMID: 6888179;Abstract: The N-terminal hexapeptide of glucagon and the corresponding carboxamide analog, were prepared by solid-phase synthesis and tested for biological activity in the hepatic adenylate cyclase system. Both peptides were found to be inactive, even at concentrations of 10 mM. The differences observed in the activity of our compounds compared to previous reports, is ascribed to the presence of a contaminant found in earlier preparations which activates adenylate cyclase. © 1983.
• Porreca, F., Mosberg, H. I., Hurst, R., Hruby, V. J., & Burks, T. F. (1983). A comparison of the analgesic and gastrointestinal transit effects of [D-Pen², L-Cys⁵] enkephalin after intracerebroventricular and intrathecal administration to mice. Life Sciences, 33(SUPPL. 1), 457-460.
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  PMID: 6689355;Abstract: Intrathecal (i.t.) administration of the highly delta selective peptide, [D-Pen2, L-Cys5]enkephalin (DPLCE) (1-10 μg), effectively inhibited gastrointestinal transit of an orally-given radiolabelled marker in mice. By contrast, the same doses did not affect marker transit after intracerebroventricular (i.c.v.) administration. I.c.v. or i.t. administration of the peptide effectively increased the latency to hindpaw lick using the 55° C hot-plate as the nociceptive stimulus. Maximum analgesic effects were seen with 0.3 μg given i.t. or 10 μg given i.c.v. Time-response studies showed activity for as long as 20 min after administration by either route. The differential gastrointestinal effects of DPLCE after i.c.v. and i.t. administration to mice suggest that delta receptors in the brain may mediate analgesic but not gut effects while spinal cord receptors may be less functionally selective. © 1983.
• Sawyer, T. K., Hruby, V. J., Hadley, M. E., & Engel, M. H. (1983). αmelanocyte stimulating hormone: Chemical nature and mechanism of action. Integrative and Comparative Biology, 23(3), 529-540.
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  Abstract: αMelanotropin (α-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, synthesized and secreted by the pars intermedia of the vertebrate pituitary. This peptide hormone is derived from pro-opiomelanocortin, a precursor protein which contains within its structure the sequences of other melanotropic peptides (γ- and rβ-MSH, corticotropin), and possibly other hormones. α-MSH is the physiologically relevant melanotropin secreted by the pituitary and in most vertebrates plays the essential role in adaptive color changes through its action on integumental chromatophores.The initial actions of α-MSH are mediated at the level of the melanocyte membrane and involve signal transduction from receptor to adenylate cyclase on the intracellular surface of the membrane. This results in elevated cytosolic cyclic AMP levels followed by melanosome dispersion within dermal melanocytes and melanogenesis within epidermal melanocytes. The action of α-MSH on dermal melanocytes requires calcium for transduction of signal and cyclic AMP production. Melanosome dispersion per se does not, however, require extracellular calcium. Structure-function studies of α-MSH analogues and fragments have provided important insights relative to the structural requirements of the hormone for receptor binding and transduction. Substitution of certain residues within α-MSH has led to the development of melanotropins that exhibit extraordinary potency and prolonged biological activity © 1983 by the American Society of Zoologists.
• Wilkes, B. C., Sawyer, T. K., Hruby, V. J., & Hadley, M. E. (1983). Differentiation of the structural features of melanotropins important for biological potency and prolonged activity in vitro. International Journal of Peptide and Protein Research, 22(3), 313-324.
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  PMID: 6605316;Abstract: Several α-melanotropin (α-MSH) analogues have been synthesized and tested for their melanotropin activities in order to determine the functional importance of certain amino acids near the primary active sequence of α-MSH, H-(Glu)-His-Phe-Arg-Trp-Gly-OH, on the biological activities of the hormone. In particular, we have examined the importance of the 4 and 11 positions in conjunction with the substitution of L-Phe in position 7 by D-Phe on potency amd prolonged activity of the hormone. In the frog (Rana pipiens) skin system the relative potencies were found to be: [Nle4, D-Phe7]-α-MSH (60) > α-MSH (1.0) > Ac-[NLE4, D-Phe7]-α-MSH4-11-NH2 (0.16) > Ac-[Nle4, D-Phe7]-α-MSH4-10-NH2 (0.02) ~ Ac-[D-Phe7]-α-MSH5-11-NH2 (0.01) > Ac-[Nle4}-α-MSH4-10-NH2 (0.002) = Ac-[Nle4]-α-MSH4-11-NH2 > Ac-α-MSH4-10-NH2 (0.0003) ≥ Ac-α-MSH5-11-NH2 (0.0002). On the other hand the relative potencies on the lizard (Anolis carolinensis) skin system were found to be: Ac-[Nle4, D-Phe7]-α-MSH4-10-NH2 (10) ≥ Ac-[Nle4, D-Phe7]-α-MSH4-11-NH2 (8.0) ≥ Ac-[Nle4, D-Phe7]-α-MSH (5.0) > α-MSH (1.0) = Ac-[Nle4]-α-MSH4-11-NH2 = Ac [D-Phe7]-α-MSH5-11-NH2 > Ac [Nle4]-α-MSH4-10-NH2 (0.06) > Ac-α-MSH5-11-NH2 (0.01) > Ac-α-MSH4-10-NH2 (0.004). Detailed analyses of these data suggest species-dependent differences in the stereostructural relationships of the residues in the 4, 7, and 11 positions for melanotropic potency in vitro. Particularly noteworthy is the observation that the 4-11 fragment analogue Ac-[Nle4}-α-MSH4-11-NH2 is equipotent to α-MSH in the lizard assay system, suggesting that the 1-3, 12, and 13 residues of α-MSH are not involved in the binding or transduction in this system. Examination of the ability of these α-melanotropin analogues to effect sustained biological activity (prolongation) following removal of exogenous peptide from the bioassay medium showed striking differences in the two systems. On the lizard skin assay, only [Nle4, D-Phe7]-α-MSH, Ac-[Nle4, D-Phe7]-α-MSH4-11-NH2 and Ac-[Nle4, D-Phe7]-α-MSH4-10-NH2 effect marked prolonged melanotropic activity as compared to α-MSH. In contrast, on the frog skin assay, only [Nle4, D-Phe7]-α-MSH, Ac-[Nle4, D-Phe7]-α-MSH4-11-NH2, Ac-α-MSH5-11-NH2, and Ac-[Nle4]-α-MSH4-10-NH2 exhibited significant prolonged activity. These results demonstrate that relative potency and prolongation of melanotropic activity are not directly related, but rather are the manifestation of different, species-dependent structural and topographical requirements for peptide-receptor interactions related to binding and signal transduction.
• Barfield, M., Al-Obeidi, F., Hruby, V. J., & Walter, S. R. (1982). Interproton coupling over five bonds ⁵J(H-C_α-C(O)-N-C_α-H) in the peptide moiety: The importance of specific association effects. Journal of the American Chemical Society, 104(12), 3302-3306.
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  Abstract: An experimental and theoretical study is presented of the conformational and solvent dependencies of long-range H-H coupling constants 5J(H-Cα-C(O)-N-Cα-H) in compounds which model the peptide backbone. Molecular orbital results for Fermi contact coupling in cis- and trans-N-methylacetamides do not follow a conformational dependence of the homoallylic type; negative values are predicted for most out-of-plane orientations of the Cα-H bonds. In addition, the calculated values for 5JHH′cis and 5JHH′trans are of opposite signs in the planar conformation of cyclo-(Gly-Gly) and the boat conformation of cyclo-(Gly-Tyr). However, relative sign measurements show that these two coupling constants are of the same sign in cyclo-(Gly-Tyr), and that both are positive in cyclo-(Gly-Phgly). The inclusion of five water molecules in the MO calculations for cis-N-methylacetamide and ten water molecules in association with cyclo-(Gly-Gly) led to both positive 5JHH′cis and 5JHH′trans. As a consequence, any applicability of the empirical relationship of 5J(H-Cα-C(O-N-Cα-H) to φ and ψ angles in peptides does not have any theoretical basis in the molecular oribtal theory for unhydrated amide bonds. © 1982 American Chemical Society.
• Chan, W. Y., Powell, A. M., & Hruby, V. J. (1982). Antioxytocic and antiprostaglandin-releasing effects of oxytocin antagonists in pregnant rats and pregnant human myometrial strips.. Endocrinology, 111(1), 48-54.
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  PMID: 6953012;Abstract: There is now increasing evidence that both oxytocin (OT) and prostaglandin (PG) play a role in term as well as preterm labor. OT stimulates myometrial contractions and uterine PG release. Specific OT antagonists, therefore, may be of value in the treatment of preterm labor. Recently, we have synthesized two highly potent OT antagonists, [1-penicillamine, 4-threonine]OT ([Pen1, Thr4]OT) and [1-penicillamine, 2-phenylalanine, 4-threonine]OT ([Pen1, Phe2, Thr4]OT). We have determined their antioxytocic activity in 21- to 22-day-pregnant rats and on isolated human myometrial strips obtained from term pregnant patients at caesarean section for childbirth. We also studied their effects on PG synthesis and OT-stimulated PG synthesis on uterine slices from pregnant rats. We found that the two OT antagonists were effective inhibitors of the OT responses in pregnant rats and on pregnant human myometrial strips. The two OT antagonists had no agonistic activity on PG release at a dose range that was antioxytocic. When administered together with OT, the PG-releasing action of OT was inhibited. Thus, [Pen1, Thr4]OT and [Pen1, Phe2, Thr4]OT are effective inhibitors of both the uterotonic and PG-releasing actions of OT. The potentials of OT antagonists as tocolytic agents for the treatment of preterm labor should be explored.
• Chan, W. Y., Powell, A. M., & Hruby, V. J. (1982). Effects of oxytocin antagonists on the uterotonic response and PG-releasing activity of oxytocin in pregnant rats on pregnant human myometrial strips. Biology of Reproduction, 26(Suppl. 1), No. 83.
• Hruby, V. J. (1982). Conformational restrictions of biologically active peptides via amino acid side chain groups. Life Sciences, 31(3), 189-199.
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  PMID: 6126794;Abstract: Determining the relationships between conformation and biological activity in peptide hormones and neurotransmitters is an important goal of contemporary biology. A major difficulty in these studies is the conformational flexibility of most peptides and the high dependence of the conformations on environment. The question arises whether conformations determined in solution are relevant to those important to the peptide at the membrane receptor(s). One recent approach to overcome these difficulties has been the use of conformational constraints by covalent bonding of side chain groups of residues in the peptide. In this manner linear peptides are rendered cyclic, and cyclic peptides are further conformationally constrained either by ring contractions or by other conformational constraints. Biologically active peptides specifically designed by this approach have been found to possess several useful properties including: 1) greater conformational integrity; 2) increased agonist or antagonist potency; 3) prolonged biological activity; 4) increased enzymatic stability; and 5) increased specificity for a particular receptor. Careful applications of this approach have provided important new design features for peptide structure-function studies, and new insights into peptide conformation-activity relationships for oxytocin, somatostatin, enkephalin, bradykinin, vasopressin, and other biologically active peptides. © 1982.
• Hruby, V. J. (1982). Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs. Molecular and Cellular Biochemistry, 44(1), 49-64.
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  PMID: 6283336;Abstract: Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon1-21 and glucagon1-6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon2-29 ([1-des-histidine]-glucagon), [1-Nα-carbamoylhistidine]-glucagon and [1-Nα-carbamoylhistidine, 12-Nα-carbamoyllysine]-glucagon are all partial agonists. These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine] [2-Nα-trinitrophenylserine, 12-homoarginine]-glucagon and [1-Nα-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA2 values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals. Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications. © 1982 Martinus Nijhoff/Dr W. Junk Publishers.
• Hruby, V. J., & Mosberg, H. I. (1982). Conformational and dynamic considerations in peptide structure-function studies. Peptides, 3(3), 329-336.
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  PMID: 7122270;Abstract: Most small peptide hormones and neurotransmitters are highly flexible, conformationally labile molecules in aqueous and other environments. Thus efforts to determine the relationships between conformational properties of these peptides in aqueous and other solvents and their biological activities at membrane receptors have been difficult and of limited success. One approach which may provide a more rational basis for conformation-activity relationships is the design of conformationally restricted, semi-rigid analogs of the native peptides which still possess high potency and/or antagonist properties. In addition to the increased likelihood that the conformational properties determined for these derivatives in aqueous or other solvent environments will have biological relevance, such analogs are likely to have higher specificity for particular receptors, greater in vivo stability, and perhaps even oral activity. The application of this approach to the design of highly potent oxytocin antagonists is discussed with particular emphasis on the conformational and dynamic properties which appear to differentiate agonist and antagonist analogs. The results of these studies are briefly compared with similar studies with somatostatin, angiotensin, bradykinin, α-melanotropin and enkephalin, and discussed in terms of likely further developments. © 1982.
• Hruby, V. J., Mosberg, H. I., & Viswanatha, V. (1982). Active-site studies of neurohypophyseal hormones: Comparison of oxytocin and arginine-vasopressin with analogues containing 4-D-glutamine. Journal of the American Chemical Society, 104(3), 837-841.
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  Abstract: Previous studies have indicated that oxytocin at the uterine smooth-muscle receptor and vasopressin at the mammalian antidiuretic receptor utilize different structural and conformational properties to produce their biological effects. In both cases, however, the Gln4 residue has been proposed to be of primary importance for receptor recognition (binding) but not critical for biological activity (transduction). On the basis of these considerations, it would be predicted that [D-Gln4] oxytocin and [D-Gln4,Arg8]vasopressin would be weak but full agonists at the uterine and antidiuretic receptors, respectively. We have synthesized and purified the two D-Gln4 analogues and examined their pharmacological activities in several assay systems for these hormones. In agreement with the predictions, [D-Gln4]oxytocin and [D-Gln4,Arg8]vasopressin have greatly reduced potency at the in vitro uterine and in vivo antidiuretic assay systems, respectively, and both appear to be full agonists in these assays. However, the effects are quantitatively different, with [D-Gln4] oxytocin possessing 3.3 ± 0.2 units/mg of uterotonic activity (1/170 the potency of oxytocin) and [D-Gln4,Arg8]vasopressin possessing only 0.45 ± 0.01 unit/mg (1/1100 the potency of arginine vasopressin) of antidiuretic activity. Based on carbon-13 nuclear magnetic resonance spectral data, both [D-Gln4]oxytocin and [D-Gln4,Arg8]vasopressin have very similar conformations to oxytocin and arginine-vasopressin, respectively. Interestingly [D-Gln4]oxytocin has much more reduced biological activities relative to the native hormone in the avian vasodepressor (∼0.04 unit/mg, ∼1/12000 that of oxytocin) and milk-ejecting (0.09 ± 0.02 unit/mg, ∼1/4500 that of oxytocin) assays and is a weak partial agonist in the pressor assay. [D-Gln4,Arg8]vasopressin is a weak full agonist in the pressor assay (0.260 ± 0.004 unit/mg, 1/1900 that of arginine-vasopressin) and also has weak uterotonic (∼0.11 unit/mg) and avian vasodepressor (0.10 unit/mg) activities. © 1982 American Chemical Society.
• Hruby, V. J., Mosberg, H. I., Fox, J. W., & Tu, A. T. (1982). Conformational comparisons of oxytocin agonists, partial agonists, and antagonists using laser raman and circular dichroism spectroscopy. Examination of 1-penicillamine and diastereoisomeric analogues. Journal of Biological Chemistry, 257(9), 4916-4924.
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  PMID: 7068672;Abstract: The biological activity of peptide hormones and analogues depends on the structural and conformational properties of these compounds. A comparative study of the conformational properties of diastereoisomeric analogues of oxytocin with weak agonist activities (fully active but low potency), partial agonist activity (only able to partially induce biological response), and of conformationally restricted 1-penicillamine analogues with potent antagonist activity (no intrinsic activity, but can block the hormone's activity) was made using circular dichroism and laser Raman spectroscopies. Conformational information regarding the peptide amide, disulfide, and tyrosine chromophores was obtained, and indicates differences in the hormone agonists and antagonists. The diastereoisomeric oxytocin analogues [1-hemi-D-cystine]-, [2-D-tyrosine]-, and [5-D-asparagine]-oxytocin, have spectral features consistent with overall backbone conformations similar to oxytocin itself, but with differences in side chain moieties. This suggests that the substantial decrease in potency of the diastereoisomeric oxytocin analogues is due to changes in the relative orientations of the side chains. In contrast, the 1-penicillamine analogues of the present study, [1-penicillamine, 4-threonine]- and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin, like 1-penicillamine oxytocin analogues previously examined, have different backbone and disulfide conformations than oxytocin. All the 1-penicillamine oxytocin derivatives thus far examined appear, from laser Raman and CD data, to have similar topologies. However, those of the present study seem to have more rigid conformations as evidenced by very intense amide n-π* and tyrosine π-π* CD transitions.
• Johnson, D. G., Goebel, C. U., Hruby, V. J., Bregman, M. D., & Trivedi, D. (1982). Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science, 215(4536), 1115-1116.
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  PMID: 6278587;Abstract: The glucagon analog [l-N(α)-trinitrophenylhistidine, 12-homoarginine]-glucagon (THG) was examined for its ability to lower blood glucose concentrations in rats made diabetic with streptozotocin. In vitro, THG is a potent antagonist of glucagon activation of the hepatic adenylate cyclase assay system. Intravenous bolus injections of THG caused rapid decreases (20 to 35 percent) of short duration in blood glucose. Continuous infusion of low concentrations of the inhibitor led to larger sustained decreases in blood glucose (30 to 65 percent). These studies demonstrate that a glucagon receptor antagonist can substantially reduce blood glucose levels in diabetic animals without addition of exogenous insulin.
• Mosberg, H. I., Hurst, R., Hruby, V. J., Galligan, J. J., Burks, T. F., Gee, K., & Yamamura, H. I. (1982). [D-Pen², L-cys⁵]enkephalinamide and [D-pen², D-cys⁵]enkephalinamide, conformationally constrained cyclic enkephalinamide analogs with delta receptor specificity. Biochemical and Biophysical Research Communications, 106(2), 506-512.
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  PMID: 6285922;Abstract: The conformationally constrained cyclic enkephalin analogs, [2-D-penicillamine, 5-L-cysteine]- and [2-D-penicillamine, 5-D-cysteine]enkephalinamide were synthesized and their biological activities investigated. Both analogs effectively induced thermal analgesia as measured by the in vivo hot plate test. Both analogs were effective in inhibiting muscle contractions in the guinea pig ileum and mouse vas deferens assay systems and were shown to displace both [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin from rat brain receptor preparations. The analogs exhibited a significant preference for δ-receptors over μ-receptors, an unusual finding for enkephalinamide derivatives. In addition the 5-L-cysteine containing analog was more potent than the 5-D-cysteine analog in all the in vitro assays with the exception of the guinea pig ileum system. These uncommon results are attributed to the conformational constraints imposed by the cyclization via a disulfide and by the rigidizing effect of the penicillamine. © 1982.
• Sawyer, T. K., Hruby, V. J., Darman, P. S., & Hadley, M. E. (1982). [half-Cys⁴,half-Cys¹⁰]-α-Melanocyte-stimulating hormone: A cyclic α-melanotropin exhibiting superagonist biological activity. Proceedings of the National Academy of Sciences of the United States of America, 79(6 I), 1751-1755.
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  PMID: 6281785;PMCID: PMC346058;Abstract: α-Melanocyte-stimulating hormone (α-melanotropin; α-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that reversibily darkens amphibian skins by stimulating melanosome (pigment granule) dispersion within melanophores. By using a number of in vitro melanocyte assays, we have examined the conformational requirements for α-MSH activity. Synthesis of [half-Cys4,half-Cys10]-α-MSH, a cyclic, conformationally restricted, 'isosteric' analogue of α-MSH, provided a melanotropin with a potency > 10,000 times that of the native hormone in stimulating frog (Rana pipiens) skin darkening. The cyclic analogue also showed substantially prolonged activity relative to the native hormone. [half-Cys4,half-Cys10]-α-MSH was ≃30 times more potent than α-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. By using a cell-free Cloudman S-91 mouse melanoma plasma membrane preparation, we found the cyclic analogue to be ≃3 times as potent as the native hormone in stimulating adenylate cyclase activity. These results provide insight into the conformational requirements for biological activity of α-MSH, and the comparative conformational requirements of α-MSH at a number of pigment cell receptors.
• Sawyer, T. K., Hruby, V. J., Wilkes, B. C., Draelos, M. T., Hadley, M. E., & Bergsneider, M. (1982). Comparative biological activities of highly potent active-site analogues of α-melanotropin. Journal of Medicinal Chemistry, 25(9), 1022-1027.
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  PMID: 6982339;Abstract: The heptapeptide sequence, Met-Glu-His-Phe-Arg-Trp-Gly, found in common within the primary structures of α-melanotropin (α-MSH), β-melanotropm (β-MSH), and adrenal corticotropic hormone (ACTH) may represent the active site mainly responsible for the melanotropic activities of these peptides. In α-MSH, replacement of Met by Nle at position 4 and D-Phe for its L enantiomer at position 7 produced a stereostructural analogue ([Nle4,D-Phe7]-α-MSH) with superagonist potency and extraordinarily prolonged biological activity. We have determined the extent to which these chemical modifications affect the melanotropic activity of the heptapeptide-proposed active site of α-MSH (α-MSH4-10). Although α-MSH4-10 has only about 1/100 000 the potency of α-MSH in the frog skin assay, melanotropic activity was enhanced by (1) acetylation and amidation of the N- and C-terminal residues, respectively; (2) replacement of Met by Nle; and (3) substitution of D-Phe for L-Phe. The final peptide, Ac[Nle4,D-Phe7]-α-MSH4-10-NH2, possessed one-fifth the potency of α-MSH on the frog (Rana pipiens) skin assay and was about 10 times more potent than α-MSH in the lizard (Anolis carolinensis) assay. Ac-[Nle4,D-Phe7]-α-MSH4-10-NH2 was also about 8 times more active than the native hormone in stimulating mouse melanoma adenylate cyclase. The melanotropic activity of this stereostructural heptapeptide analogue was dramatically prolonged relative to MSH in the Anolis skin assay but was less prolonged in the frog skin assay relative to the tridecapeptide analogue, [Nle4,D-Phe7]-α-MSH. Interestingly, Ac-[Tyr4]-α-MSH4-10-NH2, which was prepared to provide an analogue that might be radiolabeled, was a partial agonist in the melanoma adenylate cyclase assay. Moreover, although this compound exhibited very low potency in all assays, it exhibited exceptionally prolonged melanotropic activity in the frog skin assay. These results demonstrate that the dramatic changes in potency and prolongation of activity of the native hormone which result from substitution of Met4 by Nle4 and Phe7 by D-Phe are derived primarily, but not exclusively, from these changes within the heptapeptide active site of α-MSH. © 1982 American Chemical Society.
• Zmijewski Jr., M. J., Mikolajczak, M., Viswanatha, V., & Hruby, V. J. (1982). Biosynthesis of the antitumor antibiotic naphthyridinomycin. Journal of the American Chemical Society, 104(18), 4969-4971.
• Bool, A. M., Gray, G. H., Hadley, M. E., Heward, C. B., Hruby, V. J., Sawyer, T. K., & Yang, Y. C. (1981). Racemization effects on melanocyte-stimulating hormones and related peptides. Journal of Endocrinology, 88(1), 57-65.
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  PMID: 6970245;Abstract: Heat-alkali treatment of synthetic α- and β-melanocyte-stimulating hormones (MSH), known to cause racemization of amino acids within the peptides, results in prolongation of the darkening (melanophore dispersion) effect of these hormones on frog and lizard skins in vitro. Skins remain darkened for hours or even days if supramaximal concentrations of the racemized hormones are used. This response can be partially reversed by melatonin or noradrenaline. Heat-alkali treatment of α-MSH at either 60 or 97 °C results in a retardation of the response of the skins to the racemized peptides. In contrast, the response of frog skins to heat-alkali-treated β-MSH is immediately enhanced and potentiated. Heat-alkali treatment also prolongs and potentiates the activity of synthetic [des-acetyl]-α-MSH (in contrast to the retardation effect on the natural acetylated peptide). These data suggest a role for the N-acetyl group in the retardation phenomenon. The activity of synthetic [2-D-tyrosine]-α-MSH is much lower than that of α-MSH itself, indicating that heat-alkali treatment of the hormone may produce either potentiation or partial inactivation of the peptide, depending on the site of racemization.
• Engel, M. H., Sawyer, T. K., Hadley, M. E., & Hruby, V. J. (1981). Quantitative determination of amino acid racemization in heat-alkali-treated melanotropins: Implications for peptide hormone structure-function studies. Analytical Biochemistry, 116(2), 303-311.
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  PMID: 7316158;Abstract: The treatment of frog skins (in vitro) and frogs (in vivo) with melanotropins that have been heated briefly in aqueous alkali resulted in prolonged skin darkening. It has been postulated that this increase in melanotropic activity is related to the partial racemization of amino acid residues of the melanotropins. Quantitative determination of the extent of racemization of eight amino acids (Val, Pro, Met, Phe, Glu, Asp, Nle, Ser) present in α-melanotropin (α-MSH), [4-norleucine]-α-MSH, βporcine-melanotropin (βp-MSH), and [7-norleucine]-βp-MSH after brief heat-alkali treatment, was accomplished using a high-resolution gas chromatographic technique. Phenylalanine-7 in α-MSH and [4-norleucine]-α-MSH and phenylalanine-10 in βp-MSH and [7-norleucine]-βp-MSH were found to be partially racemized to a greater extent than expected. Other amino acid residues were also racemized to unexpected degrees. The subsequent synthesis of an α-MSH analog containing d-phenylalanine-7, [4-norleucine, 7-d-phenylalanine]-α-MSH, resulted in a highly potent melanotropin with ultralong biological activity, as determined by frog skin bioassay, stimulation of mouse melanoma cell tyrosinase activity, and activation of mouse melanoma adenylate cyclase. © 1981.
• Fox, J. A., Tu, A. T., Hruby, V. J., & Mosberg, H. I. (1981). Raman study of crystalline peptides containing β turns. Archives of Biochemistry and Biophysics, 211(2), 628-631.
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  PMID: 6171202;Abstract: The Raman spectra of crystalline H-ProLeuGlyNH2 which has a type II β turn, crystalline S-benzylCysProLeuGlyNH2 which has a type I β-turn, and crystalline gramicidin S which has two β turns and β-sheet structure in its conformation, were investigated. The amide I and amide III bands of the peptides with β turns were generally different from those which are diagnostic for α-helix and β-sheet conformations. The patterns of the amide I and amide III bands, when examined together, indicate that Raman spectra can provide diagnostic evidence for β-turn structure in peptides. © 1981.
• Hadley, M. E., Anderson, B., Heward, C. B., Sawyer, T. K., & Hruby, V. J. (1981). Calcium-dependent prolonged effects on melanophores of [4-norleucine, 7-D-phenylalanine]-α-melanotropin. Science, 213(4511), 1025-1027.
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  PMID: 6973820;Abstract: A single injection of the melanotropin analog [4-norleucine, J-D-phenylalanine]-α-melanotropin into frogs (Ratia pipiens) caused near maximum darkening of the skins of the frogs for at least 6 weeks. Injections of the natural hormone α-melanotropin or of the analog [Nle 4]-α-melanotropin also caused darkening, but this effect lasted only a few days. Morphological examination of the skins of frogs injected with [Nle4, D-Phe7]-α-melanotropin revealed that both dermal and epidermal melanophores were dispersed during the entire 6-week period. In vitro [Nle4, D-Phe7]-α-melanotropin also causes prolonged darkening of the skin of the lizard Anolis carolinensis. In the absence of the melanotropin, skins previously darkened with the analog could be lightened by removal of calcium from the incubation medium but could then be redarkened by adding calcium. The cycle could be repeated indefinitely without addition of melanotropin. These results demonstrate the role of calcium in receptor signal transduction and the prolonged biological effects of [Nle 4, D-Phe7]-α-melanotropin long after its removal from the assay medium. Copyright © 1981 AAAS.
• Hadley, M. E., Heward, C. B., Hruby, V. J., Sawyer, T. K., & Yang, Y. C. (1981). Biological actions of melanocyte-stimulating hormone.. Ciba Foundation symposium, 81, 244-262.
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  PMID: 6268380;Abstract: Melanocyte-stimulating hormone (alpha-melanotropin, MSH) may function in a number of diverse physiological roles. MSH stimulates (1) rapid translocation of melanosomes (melanin granules) in dermal melanophores to effect rapid colour change and (2) melanogenesis in normal and abnormal (melanoma) epidermal melanocytes. Both actions involve (1) initial binding of the peptide on the melanocyte membrane, (2) transduction of signal to adenylate cyclase, and (3) increased cytosolic levels of cyclic AMP. Efforts to prepare radioiodinated MSH and analogues for radioreceptor studies using melanoma membranes and intact cells reveal that conventional iodination procedures inactivate the hormone because of oxidative and iodination effects on specific structural components of the peptide. These effects can be circumvented by the use of synthetically tailored MSH analogues. Transduction of signal from receptor to adenylate cyclase requires calcium, but prostaglandin or beta-adrenoceptor stimulation of melanophores does not. The nucleotide and metal ion requirements for mouse melanoma adenylate cyclase activity have been characterized. There is both a transcriptional and translational requirement for MSH stimulation of tyrosinase activity and melanin production in melanoma cells. Melanosome translocation within melanophores is enhanced in the absence of extracellular calcium. A model for the MSH control of melanosome movements suggests a bifunctional, but compartmentalized, role for calcium in the action of MSH.
• Hruby, V. J., Agarwal, N. S., Griffen, A., Bregman, M. D., Nugent, C. A., & Brendel, K. (1981). Glucagon structure-function relationships using isolated rat hepatocytes. BBA - General Subjects, 674(3), 383-390.
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  PMID: 7236736;Abstract: The ability of glucagon and several of its semi-synthetic analogues to stimulate glucose production in isolated rat hepatocytes was measured and compared for relative potencies. The order of decreasing biological activities of glucagon in this assay was as follows: glucagon > [HArg12]-glucagon > [des-Asn28, Thr29][homoserinehydrazide27]-glucagon approx. equal to [des-His1]-glucagon > [des-Asn28, Thr29] [homoserinelactone27]-glucagon > [des-Asn28, Thr29]-[n-butylhomoserineamide27]-glucagon > glucagon1-21. Qualititatively, these results are similar to those obtained previously in the hepatic plasma membrane adenylate cylase assay. Minor exceptions were noted for the hydrazide derivative nd the partial agonist [des-His1]-glucagon, both of which were slightl y more potent relative to glucagon in the glycogenolytic assay than in the adenylate cyclase assay. The assay provides important insight into glucagon structure-function relationships. © 1981.
• Mosberg, H. I., Hruby, V. J., & Meraldi, J. (1981). Conformational study of the potent peptide hormone antagonist [1-penicillamine,2-leucine]oxytocin in aqueous solution. Biochemistry, 20(10), 2822-2828.
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  PMID: 7248250;Abstract: [1-Penicillamine,2-leucine]oxytocin is a conformationally restricted analogue of oxytoxin in which the half-cystine-1 and tyrosine-2 residues of the native hormone are replaced by half-penicillamine (β,β-dimethyl-half-cystine) and leucine, respectively. This analogue is a surprisingly potent oxytocin antagonist [Hruby, V. J., Deb, K. K., Yamamoto, D. M., Hadley, M. E., & Chan, W. Y. (1979) J. Med. Chem. 22, 7]. Extensive proton magnetic resonance experiments were performed to determine the conformational properties of this analogue in aqueous solution, and the results were compared with the previously published model for the conformation of [1-penicillamine] oxytocin. The results are consistent with a conformation similar to that of [1-penicillamine]oxytocin except that, while [1-penicillamine]oxytocin in aqueous solution possesses two 1←3 (C7) type turns involving the isoleucine-3 peptide amide proton and the half-penicillamine-1 carbonyl and the asparagine-5 peptide amide proton and the isoleucine-3 carbonyl, [1-penicillamine,2-leucine]oxytocin has only the latter 1←3 turn. This difference between the antagonists is reflected in the different φ and ψ angles in the three N-terminal residues of the two inhibitor analogues and in differences in the preferred side-chain conformations for several residues. One particular result of these conformational differences is that, whereas for [1-penicillamine]oxytocin the tyrosine-2 side chain is unable to assume the rotamer for maximal binding to the uterine receptor, [1-penicillamine,2-leucine]oxytoxin retains conformational and dynamic properties at residues two and three which are more similar to those of oxytocin. It is postulated that these conformational and dynamic properties are consistent with the stronger binding and, hence, greater antagonist activity for this penicillamine analogue relative to [1-penicillamine]oxytocin. © 1981 American Chemical Society.
• Blevins, D. D., Burke, M. F., & Hruby, V. J. (1980). Parameters affecting high performance liquid chromatographie separations of neurohypophyseal peptide hormones. Analytical Chemistry, 52(3), 420-424.
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  PMID: 7396194;Abstract: Experimental conditions and parameters involved in high performance liquid Chromatographic separations of seven neurohypophyseal hormones on several reverse phase columns were investigated. These peptides include arginine vasotocin, lysine vasopressin, arginine vasopressin, mesotocin, isotocin, oxytocin, and glumitocin. The effects of carbon chain length of the reverse phase support and organic solvent were examined. Using the appropriate solvent system and column, all of the peptides were separated from one another. Separation of the peptides was only part of the goal. The beginning of a study to understand the interaction of the peptides with the stationary phase as a function of structure was also undertaken. This study led to the conclusion that the major parameters which allow effective separation of these structurally and conformationally closely related peptides are both the eluting strength of the mobile phase and the chemical composition of the stationary phase. © 1980 American Chemical Society.
• Blevins, D. D., Burke, M. F., Hruby, V. J., & Larsen, B. R. (1980). Factors affecting the separation of arginine vasopressin peptide diastereoisomers by HPLC. Journal of Liquid Chromatography, 3(9), 1299-1318.
• Blumenstein, M., Hruby, V. J., & Viswanatha, V. (1980). The tyrosine ring of oxytocin undergoes hindered rotation when the hormone is bound to neurophysin. Biochemical and Biophysical Research Communications, 94(2), 431-437.
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  PMID: 7396908;Abstract: Tyrosine specifically enriched with 13C in the meta positions has been chemically synthesized and incorporated into oxytocin via solid phase peptide synthesis. The 13C nmr spectrum of a 1:1 mixture of the enriched hormone complexed to neurophysin was obtained. The spectrum consisted of three peaks. The two outer peaks, representing 85% of the total intensity, were of equal area, had shifts of -0.9 and +2.4 parts per million relative to the free peak, and each had a linewidth of 100 hz at 20°C, with increasing linewidths at higher temperatures. These two peaks arise from a binding mode in which tyrosine ring rotation is hindered by interaction with neurophysin. The rotation rate at 20°C is 130s-1, and at 42°C is 900s-1. The central peak occurred at the position of the resonance due to free hormone, had a temperature independent linewidth of 30-40 hz, and represented about 15% of the total intensity. We believe this peak is due to a binding mode in which tyrosine ring rotation is rapid, 104-108s-1. © 1980.
• Bregman, M. D., Sawyer, T. K., Hadley, M. E., & Hruby, V. J. (1980). Adenosine and divalent cation effects on S-91 melanoma adenylate cyclase. Archives of Biochemistry and Biophysics, 201(1), 1-7.
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  PMID: 7396490;Abstract: Adenosine inhibits S-91 melanoma membrane adenylate cyclase. This inhibition is observed with all activators tested; α-melanocyte-stimulating hormone, [norleucine4]α-melanocyte-stimulating hormone, prostaglandin-E2, and fluoride ion. This inhibition is also observed with the ribose modified adenosine analog adenine-9-β-d-arabinofuranoside. However, "R" site (stringent for the ribose moiety) analogs such as 2-chloroadenosine and 6-methyladenosine were without effect. Addition of manganous ion (Mn2+), a potent activator of adenylate cyclase, markedly enhances the inhibition by adenosine. The nucleoside specificity, Mn2+ requirement, and lack of reversal by the potent R-site antagonist, 3-isobutyl-1-methylxanthine, suggest that the S-91 melanoma membrane adenylate cyclase system contains only P-type (stringent for purine moiety) receptor sites. © 1980.
• Bregman, M. D., Trivedi, D., & Hruby, V. J. (1980). Glucagon amino groups. Evaluation of modifications leading to antagonism and agonism. Journal of Biological Chemistry, 255(24), 11725-11731.
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  PMID: 7440567;Abstract: Using native glucagon and [12-homoarginine]glucagon (analogue A), prepared in high yield and purity by new procedures, we have synthesized the following glucagon analogues by semisynthetic methods: [1-deshistidine][12-homoarginine]glucagon (analogue B); Nα-carbamoylglucagon (analogue C); Nα,Nε-dicarbamoylglucagon (analogue D); [1-Nα-carbamoylhistidine, 12-Nε-trinitrophenyllysine]glucagon (analogue II); [1-deshistidine] [2-Nα-trinitrophenylserine, 12-homoarginine]glucagon (analogue III); and [1-Nα-trinitrophenylhistidine, 12-homoarginine]glucagon (analogue IV). The introduction of hydrophylic groups at the α- and ε-amino positions of glucagon results in a reduction in potency. The α-position is also involved in biological activity. Carbamylation of the α-position results in a partial agonist (analogues C and D). The introduction of hydrophobic groups and the neutralization of the positive charge at the α- and ε-amino positions result in glucagon antagonists (analogues II, III, and IV). [1-Nα-Trinitrophenylhistidine, 12-homoarginine]glucagon (analogue IV) is the most potent inhibitor tested. Based on its competitive inhibitory action, this analogue appears to have about one-third the affinity of glucagon for the receptor site. These modifications at the ε-amino position cause an increase in the secondary structure of the peptide (as shown by circular dichroism studies) which may be related to their biological activities.
• Hruby, V. J., Mosberg, H. I., Hadley, M. E., Chan, W. Y., & Powell, A. M. (1980). Synthesis, pharmacological, conformational, and dynamic studies of the potent hormone antagonists [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin. Conformational and dynamic considerations in the design of antagonists. International Journal of Peptide and Protein Research, 16(5), 372-381.
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  PMID: 7216613;Abstract: The solid phase synthesis of [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin is reported. The two compounds have no in vitro milk ejecting activity and no in vivo or in vitro oxytocic activity, but both are potent antagonists in these three assay systems. In the in vitro oxytocic assay, '1-penicillamine, 4-threonine]- and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin have pA 2 values of 7.55 ± 0.04 and 7.67 ± 0.02, respectively, and both inhibit the uterine contractile response to oxytocin in nonpregnant and pregnant rats. [1-Penicillamine, 2-phenylalanine, 4-threonine]-oxytocin has a weak antipressor activity and at high doses, consistently caused a weak and transient fall in blood pressure in the rat. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T 1) indicate that these two new oxytocin antagonists have very similar conformation and dynamic properties to oxytocin inhibitors which have previously been examined. These results are discussed in terms of conformational and dynamic models of oxytocin antagonism at the uterus. It is suggested that conformational restrictions at the 2- and 4-positions of penicillamine-1 analogues of oxytocin are important to antagonist activity and potency.
• Hruby, V. J., Sawyer, T. K., C., Y., Bregman, M. D., Hadley, M. E., & Heward, C. B. (1980). Synthesis and structure-function studies of melanocyte stimulating hormone analogues modified in the 2 and 4(7) positions: Comparison of activities on frog skin melanophores and melanoma adenylate cyclase. Journal of Medicinal Chemistry, 23(12), 1432-1437.
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  PMID: 7452698;Abstract: The synthesis and purification of several analogues of the melanotropins with amino acid substitutions at the tyrosine-2 and methionine-4(7) positions are reported. The compounds synthesized included [4-norleucine]-α-MSH, [7-norleucine]-βp-MSH, [2-3′,5′-diiodotyrosine]-α-MSH, [2-D-tyrosine]-α-MSH, and [2-phenylalanine,4-norleucine]-α-MSH. The biological activities of these derivatives were measured and compared on normal melanocytes (frog skins) and on transformed melanocytes (mouse melanoma adenylate cyclase), over the entire dose-response range. All compounds tested were full agonists in both assay systems but varied considerably in potency. The relative potencies in the frog skin assay (α-MSH = 1.0) were as follows: [Nle7]-βp-MSH (5.2) > [Nle4]-α-MSH (2.3) > α-MSH (1.0) > [Phe2,Nle4]-α-MSH (0.80) > αp-MSH (0.55) > [I2-Tyr2]-α-MSH (0.12) > [D-Tyr2]-α-MSH (0.04). The relative potencies in the melanoma adenylate cyclase system were [Nle7]-βp-MSH (4.2) > βp-MSH (2.2) > [Nle4]-α-MSH (2.0) > α-MSH (1.0) ≈ [Phe2,Nle4]-α-MSH (0.9) > [I2-Tyr2]-α-MSH (0.40) > [D-Tyr2]-α-MSH (0.20). There appears to be some differences in structural specificity at the melanotropin receptors of the two cell systems. © 1980 American Chemical Society.
• Hruby, V. J., Viswanatha, V., & Yang, Y. C. (1980). Synthesis of S-benzyl-DL-[1-¹³C]cysteine and its incorporation into oxytocin and [8-arginine]vasopressin and related compounds by total synthesis. Separation of diastereoisomers by partition chromatography and HPLC. Journal of Labelled Compounds and Radiopharmaceuticals, 17(6), 801-812.
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  Abstract: S-Benzyl-DL-[1-13C]cysteine was prepared from Na13CN by a three step synthesis and converted to the t-butyloxycarbonyl derivative which was suitable for use in peptide synthesis. This compound was incorporated into the 1 and 6 positions of a variety of oxytocin and [8-arginine]vasopressin vasopressin derivatives and analogues via total synthesis using the solid phase method. The compounds were separated and purified by partition chromatography on Sephadex and their purity was checked by high pressure liquid chromatography. The compounds synthesized include [1-hemi-[1-13C]cystine]oxytocin, [1-hemi-D-[1-13C]cystine]oxytocin, [1-hemi-[1-13C]cysteine, 8-arginine]vasopressin, [1-hemi-D-[1-13C]cystine, 8-arginine]vasopressin, [6-hemi-[1-13C]cystine]oxytocin, [6-hemi-D-[1-13C]cystine]oxytocin, [1-hemi-D-[1-13C]cystine, 3-D-leucine]oxytocin, and [1-hemi-[1-13C]cystine, 3-D-leucine]oxytocin.
• Khan, B. A., Bregman, M. D., Nugent, C. A., Hruby, V. J., & Brendel, K. (1980). (Des-Histidine¹) (N^ε{lunate}-phenylthiocarbamoyllysine¹²)-glucagon: Effects on glycogenolysis in perfused rat liver. Biochemical and Biophysical Research Communications, 93(3), 729-736.
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  PMID: 7387671;Abstract: (Des-Histidine1) (Nε{lunate}-phenylthiocarbamoyllysine12)-glucagon, synthesized by the one-step Edman degradation procedure is a competitive inhibitor of glucagon action in the rat liver plasma membrane adenylate cyclase system. However, in the perfused rat liver, the compound did not inhibit glucagon stimulated glycogenolysis even when used at a concentration 100-fold in excess of native glucagon. Instead, it showed a weak potency, but full agonist activity, stimulating liver glycogenolysis to 100% of the level obtained by glucagon. These results are discussed in terms of the possible mechanism(s) of glucagon action. © 1980.
• Sawyer, T. K., Sanfilippo, P. J., Hruby, V. J., Engel, M. H., Heward, C. B., Burnett, J. B., & Hadley, M. E. (1980). 4-Norleucine, 7-d-phenylalanine-α-melanocyte-stimulating hormone: A highly potent α-melanotropin with ultralong biological activity. Proceedings of the National Academy of Sciences of the United States of America, 77(10 II), 5754-5758.
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  PMID: 6777774;PMCID: PMC350149;Abstract: α-Melanocyte-stimulating hormone (α-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of α-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4 (methionine) and 7 (phenylalanine) positions. [Nle 4]-α-MSH, a synthetic analogue of α-MSH, reversibly darkens frog skins and also exhibits prolonged activity after heat-alkali treatment. Synthesis of [Nle 4, D-Phe 7]-α-MSH provided an analogue with prolonged biological activity, identical to that observed with heat-alkali-treated α-MSH or [Nle 4]-α-MSH. [Nle 4, D-Phe 7]-α-MSH was resistant to enzymatic degradation by serum enzymes. In addition, this peptide exhibited dramatically increased biological activity as determined by frog skin bioassay, activation of mouse melanoma adenylate cyclase, and stimulation of mouse melanoma cell tyrosinase activity. This Nle 4, D-Phe 7 synthetic analogue of α-MSH is a very potent melanotropin, 26 times as potent as α-MSH in the adenylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged biological activity should make this analogue of α-MSH an important molecular probe for studying the melanotropin receptors of both normal and abnormal (melanoma) melanocytes.
• Viswanatha, V., & Hruby, V. J. (1980). Conversion of L-tyrosine to L-phenylalanine. Preparation of L-[3′,5′-¹³C₂]phenylalanine. Journal of Organic Chemistry, 45(10), 2010-2012.
• Wright, D. E., Agarwal, N. S., & Hruby, V. J. (1980). Synthesis of protected secretin 16-27 on a Merrifield resin. Examination of ammonolysis conditions for preparing carboxamide terminal protected peptides suitable for fragment condensation.. International Journal of Peptide and Protein Research, 15(3), 271-278.
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  PMID: 7380611;Abstract: The partially protected dodecapeptide to secretin, H-Ser(Bzl)-Ala-Arg(Tos)-Leu-Gln-Arg(Tos)-Leu-Leu-Gln-Gly-Leu-Val-NH2 (protected secretin 16-27) was prepared using a standard Merrifield resin and solid phase synthesis methods. For comparative purposes the unprotected peptide also was prepared on a benz-hydrylamine resin. Contrary to previous reports, the valine C-terminal peptide can be cleaved from the resin and the amide obtained in high yield. A variety of conditions were examined to accomplish the cleavage of the peptide from the resin in its carboxamide terminal form. The best conditions found were trans-esterification followed by ammonolysis in a mixed solvent system. A thin-layer chromatography system which clearly separates the methyl ester and carboxamide terminal secretin 16-27 was developed.
• Wright, D. E., Hruby, V. J., & Rodbell, M. (1980). Preparation and properties of glucagon analogs prepared by semi-synthesis from CNBr-glucagon. Biochimica et Biophysica Acta, 631(1), 49-58.
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  PMID: 6249392;
• Yang, Y. C., Hruby, V. J., Heward, C. B., & Hadley, M. E. (1980). Synthesis of α- and β-melanocyte stimulating hormones. International Journal of Peptide and Protein Research, 15(2), 130-138.
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  PMID: 7372404;
• Bregman, M. D., & Hruby, V. J. (1979). Synthesis and isolation of a glucagon antagonist. FEBS Letters, 101(1), 191-194.
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  PMID: 446735;
• Ebels, I., Benson, B., Bria, C. F., Richardson, D., Larsen, B. R., & Hruby, V. J. (1979). Location by paper chromatography of compensatory ovarian hypertrophy (COH) inhibiting activity in isobutanol extracts of bovine pineals. Journal of Neural Transmission, 45(1), 43-61.
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  PMID: 469521;Abstract: Bovine pineal glands were extracted according to the methods reported by Bensinger et al. (1973) and Cheesman and Fariss (1970). Isobutanol soluble COH-inhibiting activity was further separated by chromatography on Sephadex G-15 and paper chromatography. With the Bensinger method, different active pineal fractions were obtained from Sephadex G-15 columns. Certain of those fractions were further separated by paper chromatography in butanol: acetic acid: water (4:1:1) and the COH-inhibitor was localized. The pineal COH-inhibitor could also be localized by high pressure, reverse phase liquid chromatography. More COH-inhibiting activity was extracted with the Bensinger method than with aqueous and acetic acid extraction methods used earlier by us. The Cheesman extraction method for arginine vasotocin gave less regular results in our hands than the Bensinger extraction method. © 1979 Springer-Verlag.
• Heward, C. B., Yang, Y. C., Ormberg, J. F., Hadley, M. E., & Hruby, V. J. (1979). Effects of chloramine T and iodination on the biological activity of melanotropin. Hoppe-Seyler's Zeitschrift fur Physiologische Chemie, 360(12), 1851-1859.
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  PMID: 316802;
• Heward, C. B., Yang, Y. C., Sawyer, T. K., Bregman, M. D., Fuller, B. B., Hruby, V. J., & Hadley, M. E. (1979). Iodination associated inactivation of β-melanocyte stimulating hormone. Biochemical and Biophysical Research Communications, 88(1), 266-273.
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  PMID: 110327;Abstract: Contrary to other published reports, iodinated β-melanocyte stimulating hormone (β-MSH) is without biological activity as measured by frog skin bioassay, melanoma (mouse S-91) adenylate cyclase assay, or melanoma tyrosinase assay. Inactivation results in part from oxidation of the methionine residue by the chloramine T and sodium metabisulfite used in the iodination reaction. Replacement of the methionine of β-MSH with norleucine by solid phase synthesis results in an analogue which is more resistant, but not completely resistant, to inactivation. Thus, in order to obtain a biologically active radioligand for radioreceptor studies, further tailoring of the hormone and/or modification of the iodination procedure will be needed. © 1979.
• Hruby, V. J., Deb, K. K., Spatola, A. F., Upson, D. A., & Yamamoto, D. (1979). ¹³C nuclear magnetic resonance studies of the peptide hormones oxytocin, arginine vasopressin, isotocin, mesotocin, glumitocin, aspartocin, related analogues, and diastereoisomers. Use of specifically deuterated hormone derivatives for assignments and effects of structural changes on ¹³C NMR chemical shifts in peptides. Journal of the American Chemical Society, 101(1), 202-212.
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  Abstract: The 13C nuclear magnetic resonance (NMR) spectra of the naturally occurring peptide hormones oxytocin, arginine vasopressin (AVP), mesotocin, isotocin, aspartocin, and glumitocin were compared. Oxytocin derivatives specifically deuterated in the Half-Cys-1, Tyr-2, Ile-3, Half-Cys-6, Pro-7, or Gly-NH2-9 positions were used to make unequivocal assignments of most of the α and β carbon atoms, and to sort out differences in assignments previously reported. Arginine vasopressin derivatives, specifically deuterated in the Half-Cys-1, Tyr-2, Phe-3, Half-Cys-6, or Gly-N H2-9 positions, also were used for unequivocal assignments. The chemical shifts of invariant residues in these compounds were virtually unchanged from their positions in oxytocin despite the structural changes at positions 8 and/or 4, and differences in biological activities. Analogues with L-amino acid substitutions in the 1 position [(1-penicillaminc]oxytocin), 3 position [(Phe3]oxytocin), 4 position ([Leu4]oxytocin), and 2 and 4 positions ([Leu2,Leu4]oxytocin, [Ile2,Leu4]oxytocin, and [Phe2,Leu4]oxytocin), also generally showed only minor 13C NMR chemical shifts at invariant residues, though there were a few notable exceptions. An interesting observation was that except for the half-cystine residues, the Cα 13C chemical shifts of L-amino acid residues were essentially the same whatever their sequence position in the 20-membered disulfide ring moiety of these peptides. However, there were large 13C chemical shift differences (1.4-1.9 ppm) for the Cα of equivalent L-amino acids in the same molecule, when one residue was in the 20-membered ring moiety, and the other in the acyclic tripeptide moiety of the hormones. The α chemical shift of residues in the acyclic portion of the molecule were always upfield to those in the ring moiety. When D-half-cystine (positions 1 and 6) or D-tyrosine (position 2) were substituted into oxytocin or AVP, the 13C NMR spectra of the diastereoisomeric peptide hormones often showed significant chemical shift perturbations even for residues quite remote from the substitution position. The similarities and differences of 13C chemical shifts are briefly discussed in terms of the conformational and biological properties of these peptides. © 1979 American Chemical Society.
• Hruby, V. J., Deb, K. K., Yamamoto, D. M., Hadley, M. E., & Chan, W. Y. (1979). [1-Penicillamine,2-leucine]oxytocin. Synthesis and pharmacological and conformational studies of a potent peptide hormone inhibitor. Journal of Medicinal Chemistry, 22(1), 7-12.
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  PMID: 423185;Abstract: [1-Penicillamine,2-leucine]oxytocin was synthesized by the solid-phase method of peptide synthesis and purified by partition chromatography on Sephadex G-25, followed by gel filtration. The peptide was found to be a very potent competitive inhibitor of oxytocin in the oxytocic assay with a pAg of 7.14 and an inhibitor of oxytocin in the milk-ejecting assay. The compound showed no agonist activity in either of these assays, and its inhibitory activity at the uterus was of prolonged duration. The 13C nuclear magnetic resonance spectral properties and the 13C T1 (spin-lattice) relaxation times of [Pen1,Leu2]oxytocin were determined, and the results were compared with previous studies of [Pen1]oxytocin, a related competitive inhibitor, and oxytocin, the native hormone agonist. These studies indicated that the hormone inhibitors [Pen1,Leu2]oxytocin and [Pen1]oxytocin have similar conformational and dynamic properties which are different than those of the agonist, oxytocin. © 1978 American Chemical Society.
• Hruby, V. J., Upson, D. A., Yamamoto, D. M., Smith, C. W., & Walter, R. (1979). Active site studies of neurohypophyseal hormones. Comparison of oxytocin and arginine vasopressin analogues containing 2-D-tyrosine. Journal of the American Chemical Society, 101(10), 2717-2721.
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  Abstract: On the basis of differences between the proposed "biologically active" model of oxytocin at the uterine smooth muscle receptor and the proposed "biologically active" conformation of vasopressin when bound to its mammalian antidiuretic receptor, it may be anticipated that [D-Tyr2]oxytocin would exhibit markedly reduced uterotonic activity when compared to oxytocin, while [D-Tyr2]vasopressin would retain most of the antidiuretic activity of its natural congener. To test this hypothesis [D-Tyr2]oxytocin and [D-Tyr2]arginine vasopressin were prepared by the solid-phase method of peptide synthesis and evaluated pharmacologically. DL-Tyrosine was introduced into the growing peptide chain and the diastereoisomers of each of the hormones were separated by partition chromatography. The biological results of the D-Tyr-2 analogues were in agreement with the prediction derived from the "biologically active" models. [D-Tyr2]oxytocin possesses only 8.4 ± 0.3 units/mg in vitro rat uterotonic potency (cf. 546 ± 18 for oxytocin) and exhibited a reduced intrinsic activity. [D-Tyr2]arginine vasopressin retained nearly 50% (207 ± 10 units/mg) of the antidiuretic potency of arginine vasopressin (cf. 503 ± 53 units/mg) and exhibited both the same affinity and intrinsic activity in the renal medullary adenylate cyclase assay of rat as the mammalian antidiuretic hormone. © 1979 American Chemical Society.
• Johnson, R. E., Hruby, V. J., & Rupley, J. A. (1979). Thermodynamics of glucagon aggregation. Biochemistry, 18(7), 1176-1179.
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  PMID: 427106;Abstract: Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 °C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 °C: ΔG° = -7.34 kcal/mol of trimer, ΔH° = -31.2 kcal/mol, ΔS° = -80 cal/(K mol), ΔCp = -430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed. © 1979 American Chemical Society.
• Larsen, B., Fox, B. L., Burke, M., & Hruby, V. (1979). The separation of peptide mormone diastereoisomers by reverse phase high pressure liquid chromatography. Factors affecting separation of oxytocin and its diastereoisomers-structural implications.. International Journal of Peptide and Protein Research, 13(1), 12-21.
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  PMID: 33928;Abstract: Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1-hemi-D-cystine]-, (2,D-tyrosine]-, [4-D-glutamine]-, [5-D-asparagine]-, [6-hemi-D-cystine-], [7-D-proline]-, [8-D-leucine]-oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran-ammonium acetate buffer or 18% acetonitrile-ammonium acetate buffer and the μBondapak C18 support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G-25 were compared. The results are discussd in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different solution conformations of the peptides are discussed.
• Rosenblum, I. Y., Benson, B., Bria, C. F., McDonnell, D., & Hruby, V. J. (1979). Localization and chemical characterization of a partially purified bovine pineal antigonadotropin. Journal of Neural Transmission, 44(3), 197-220.
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  PMID: 571457;Abstract: An antigonadotropic substance was partially purified from aqueous extracts of bovine pineal glands by methods of gel filtration, ultrafiltration and ion exchange chromatography. Two biological tests, viz. inhibition of compensatory ovarian hypertrophy and reduction of ventral prostate weight, were used to guide the purification. The partially purified antigonadotropin was characterized chemically using techniques of UV and fluorescence spectrometry, thin layer and paper chromatography, paper electrophoresis and amino acid analysis. The results reveal a diversity of ninhydrin-positive components present in the preparations, including free and peptide-bound amino acids, as well as other unidentified components, but not including any of the commonly occurring indoles, indoleamines or catecholeamines. One peptide, oxidized glutathione, was identified in the most purified material containing the biologically active principle yet pure, synthetric glutathione has no antigonadotropic activity in the biological tests utilized. Although the chemical nature of the bovine pineal antigonadotropin remains in question it may be purified by the methods described. The activity is thought to reside in the extremely small, perhaps trace quantities of residues derived. It is believed that large scale, preparative studies will be required for structural determination. © 1979 Springer-Verlag.
• Tu, A. T., Lee, J., Deb, K. K., & Hruby, V. J. (1979). Laser raman spectroscopy and circular dichroism studies of the peptide hormones mesotocin, vasotocin, lysine vasopressin, and arginine vasopressin. Conformational analysis. Journal of Biological Chemistry, 254(9), 3272-3278.
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  PMID: 429348;
• Viswanatha, V., & Hruby, V. J. (1979). Synthesis of [3′,5′-¹³C₂]tyrosine and its use in the synthesis of specifically labeled tyrosine analogues of oxytocin and arginine-vasopressin and their 2-D-tyrosine diastereoisomers. Journal of Organic Chemistry, 44(16), 2892-2896.
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  Abstract: DL-[3′,5′-13C2]Tyrosine (91% 13C enriched) was synthesized by a ten-step synthetic scheme in an overall yield of 22% (86+% per step) using [1,3-13C2]acetone as the source of label. The enantiomers were resolved by enzymatic methods, and the labeled DL amino acid or purified enantiomers readily converted to the Nα-Boc acids suitable for peptide synthesis. Boc-DL-[3′,5′-13C2]tyrosine was used for the total synthesis of the specifically labeled peptide hormone derivatives [2-DL-[3′,5′-13C2]tyrosine]oxytocin and [2-DL-[3′,5′-13C 2]tyrosine,8-arginine]vasopressin by solid phase methods. The diastereoisomers were separated from each other by partition chromatography on Sephadex G-25 followed by gel filtration to give the following specifically labeled hormone derivatives: [2-[3′,5′-13C2]tyrosine]oxytocin, [2-D-[3′,5′-13C2]tyrosine]oxytocin, [2-[3′,5′-13C 2]tyrosine,8-arginine]vasopressin, and [2-D-[3′,5′-13C 2]tyrosine,8-arginine]vasopressin. The milk ejecting activities were determined. © 1979 American Chemical Society.
• Viswanatha, V., Larsen, B., & Hruby, V. J. (1979). Synthesis of dl-[2-¹³C]leucine and it use in the preparation of [3-dl-[2-¹³C]leucine]oxytocin and [8-dl-[2-¹³C]leucine]oxytocin. Preparative separation of diastereoisomeric peptides by partition chromatography and high pressure liquid chromatography. Tetrahedron, 35(13), 1575-1580.
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  Abstract: dl-[2-13C]Leucine was prepared by condensing the sodium salt of ethyl acetamido-[2-13C]cyanoacetate with isobutylbromide in hexamethylphosphoroustriamide followed by acid hydrolysis. N-Boc-dl-[2-13C]Leucine was prepared and incorporated into [8-dl-[2-13C]leucine]oxytocin by total synthesis. The 13C-labeled hormone derivative [8-[2-13C]leucine]oxytocin was separated from its 8-position diastereoisomer by partition chromatography. The specifically 13C-labeled peptide hormone diastereoisomeric analog [3-dl-[2-13C]leucine]oxytocin also was prepared by solid phase peptide synthesis. No suitable solvent system for partition chromatography separation of the latter diastereoisomeric peptide mixture could be found. However an excellent preparative separation of the diastereoisomers could be obtained by reverse phase high pressure liquid chromatography on a partisil 10 M9 ODS column using the solvent system 0.05 M ammonium acetate (pH 4.0), acetonitrile (81:19, v v) to give pure [3-(2-13C]leucine]oxytocin and [3-D-(2-13C]leucine]oxytocin. An excellent separation of [8[2-13C]leucine]oxytocin and the corresponding 8-D-leucine diastereoisomer derivative could also be accomplished by high pressure liquid chromatography. © 1979.
• Wyssbrod, H. R., Fischman, A. J., Live, D. H., Hruby, V. J., Agarwal, N. S., & Upson, D. A. (1979). Assignments of ¹H nuclear magnetic resonances of the cystyl, asparaginyl, and aromatic residues of arginine vasopressin in D₂O. A comparison with lysine vasopressin and oxytocin in terms of solution conformation. Journal of the American Chemical Society, 101(15), 4037-4043.
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  Abstract: The resonances of the Cα and Cβ protons of the cystyl, asparaginyl, and aromatic residues of [8-arginine]vasopressin (AVP) in D2O at pD 3.8 and 20°C were assigned in a rigorous manner by the use of isotopic isomers of AVP that contain specific replacements of protons by deuterons and by comparison of 1H NMR characteristics of AVP to those of [8-lysine]vasopressin (LVP) and oxytocin (OT). Although there is extensive overlap of resonances of Cβ protons even at 360 MHz, all of the chemical shifts of these protons and most of the couplings between them and their vicinal Cα protons could be determined, at least to a first approximation. It was concluded that the cyclic moieties (residues 1-6) of AVP, LVP, and OT possess essentially the same overall backbone conformation, and that the side-chain conformation - or rotamer populations - about the Cα-Cβ bonds of the cystyl residue (positions 1 and 6), the tyrosyl residue (position 2), and the asparaginyl residue (position 5) are similar. This study indicates that selective replacements of Cβ protons by deuterons are necessary to improve the accuracy of coupling constants extracted from 360-MHz spectra of AVP for use in conformational analysis. © 1979 American Chemical Society.
• Blumenstein, M., & Hruby, V. J. (1978). Evidence for the occurrence of a hormone conformation change when oxytocin binds to neurophysin. Federation Proceedings, 37(6), No.567.
• Ebels, I., Benson, B., Bria, C. F., McDonnell, D., Chang, S. Y., & Hruby, V. J. (1978). Location by paper chromatography of compensatory ovarian hypertrophy (COH) inhibiting activity in acetic acid extracts from bovine pineals. Journal of Neural Transmission, 42(4), 275-292.
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  PMID: 681923;Abstract: Acetic acid extracts of bovine pineals and cerebral cortex were separated on Sephadex G-25 columns. Subsequently two low molecular weight fractions, F2 and F3, were ultrafiltered through the membranes UM2 and UM05. The UM05 residues were gel filtered on Sephadex G-15 columns or chromatographed on Dowex W50-X4 columns. Fractions from these columns were tested and those which showed COH-inhibiting activity were separated by preparative paper chromatography in different solvents. The absorption spectra of those fractions were recorded and tested for COH-inhibition. By these methods, a COH-inhibitor was localized in three different solvents. Some active paper chromatography fractions were studied in high pressure, reverse phase, liquid chromatography. This latter method showed that the active fractions obtained by paper chromatography contain several orthophthalaldehyde (OPT) positive compounds. © 1978 Springer-Verlag.
• Hruby, V. J., Deb, K. K., Fox, J., Bjarnason, J., & Tu, A. T. (1978). Conformational studies of peptide hormones using laser Raman and circular dichroism spectroscopy. A comparative study of oxytocin agonists and antagonists. Journal of Biological Chemistry, 253(17), 6060-6067.
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  PMID: 681339;Abstract: A comparative study of the conformational properties of the peptide hormone agonists oxytocin and [4-glycine]oxytocin, and of the oxytocin inhibitors (antagonists) [1-penicillamine]oxytocin, and [1-penicillamine,2-leucine]oxytocin was made using laser Raman and circular dichroism spectroscopy. Conformational information regarding the disulfide, peptide amide, and tyrosine chromophores was obtained, and indicated differences for the hormone agonists and antagonists. [Gly 4]oxytocin and oxytocin have very similar Raman spectra with the v(S-S) as a singlet at 510 cm -1 and amide III bands in the range of 1260 to 1272 cm -1, while the oxytocin antagonists [Pen 1]oxytocin and [Pen 1, Leu 2]oxytocin have a singlet or doublet v(S-S) band and the amide III band at 1255 cm -1. The circular dichroism spectra of the compounds were examined over a wide range of pH values (2 to 11.5) in aqueous solution. [Gly 4]oxytocin had very similar CD spectra to those previously obtained for oxytocin. The CD spectra of [Pen 1]oxytocin and [Pen 1, Leu 2]oxytocin had important similarities to each other, and important differences with ocytocin and [Gly 4]oxytocin. Using [Pen 1, Leu 2]oxytocin which does not contain a tyrosine moiety, it was possible to assign the disulfide and amide chromophores in this compound and in [Pen 1]oxytocin. In these latter compounds the longest wavelength disulfide transition was found at 274 to 279 nm as a negative band, indicating that the disulfide C-S-S-C dihedral angle was about 100-115° with a right-handed chirality. The intensity of this band and especially that of the peptide amide transition at about 220 nm indicated that [Pen 1, Leu 2]oxytocin and [Pen 1]oxytocin were more rigid (less flexible) molecules than oxytocin and [Gly 4]oxytocin. These results are in agreement with the results of previous NMR studies on oxytocin and [Pen 1]oxytocin in aqueous solution. A conformation for the ring moiety of [Pen 1]oxytocin is suggested.
• Larsen, B., Viswanatha, V., Chang, S. Y., & Hruby, V. J. (1978). Reverse phase high pressure liquid chromatography for the separation of peptide hormone diastereoisomers. Journal of Chromatographic Science, 16(5), 207-210.
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  PMID: 670372;
• Tu, A. T., Bjarnason, J. B., & Hruby, V. J. (1978). Conformation of oxytocin studied by laser Raman spectroscopy. BBA - Protein Structure, 533(2), 530-533.
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  PMID: 647024;Abstract: The peptide backbone conformation and salient structural details of oxytocin were examined by laser Raman spectroscopy. Spectra were obtained in the solid phase, water, 2H2O, and dimethyl sulfoxide solutions. A distinct Amide I band was obtained at 1663 cm-1 for aqueous and deuterated samples and 1666 cm-1 for the solid sample. A relatively high frequency Amide III band at 1260 cm-1 was obtained. It is concluded that these Amide I and III bands arise from the "β-turn"-like conformation of oxytocin. The tyrosine side chain, according to the I850 cm-1/I830cm-1 intensity ratio, is exposed to the solvent. The S-S stretching vibration at 512 cm-1 indicates the conformation of C-C-S-S-C-C in the disulfide bridge of oxytocin in the ring is gauche-gauche-gauche. © 1978.
• Wright, D. E., Hruby, V. J., & Rodbell, M. (1978). A reassessment of structure-function relationships in glucagon. Glucagon_1-21 is a full agonist. Journal of Biological Chemistry, 253(18), 6338-6340.
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  PMID: 210180;Abstract: Glucagon1-21 has been prepared by treating native glucagon with carboxypeptidase A. Purified glucagon1-21 did not contain detectable methionine (
• Agarwal, N. S., Hruby, V. J., Katz, R., Klee, W., & Nirenberg, M. (1977). Synthesis of leucine enkephalin derivatives: Structure-function studies. Biochemical and Biophysical Research Communications, 76(1), 129-135.
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  PMID: 559493;Abstract: The solid phase synthesis of highly purified [Leu5] enkephalin and of seven derivatives including [Ala2,Leu5]-, [Ser2,Leu5]-, [Ser3,Leu5]-, [Aba2,Leu5]-, and [des-Gly2(3),Leu5]enkephalins are reported, and their morphine-like activities in neuroblastoma x glioma cell homogenates were measured. Changes at the 2, 3, and 5 positions of the enkephalin provided analogues which were all less active than [Leu5] enkephalin. The results are discussed in terms of recently suggested conformational structures for the enkephalin peptides. No melanocyte stimulating activity was observed for [Leu5] enkephalin, [Ala2,Leu5]enkephalin, or [Ser2,Leu5] enkephalin. © 1977.
• Blumenstein, M., & Hruby, V. J. (1977). Interactions of oxytocin with bovine neurophysins I and II. Use of ¹³C nuclear magnetic resonance and hormones specifically enriched with ¹³C in the glycinamide-9 and half-cystine-1 positions. Biochemistry, 16(24), 5169-5177.
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  PMID: 562672;Abstract: The specifically 13C-labeled (90% 13C-enriched) neurohypophyseal hormone derivatives [9-[2-13C]glycinamide]oxytocin (1), [1-hemi-L-[2-13C]cystine]oxytocin (2), and [1-hemi-D-[2-13C]cystine]oxytocin (3) have been synthesized by solid phase methods, and the interaction of these hormones with bovine neurophysin I and bovine neurophysin II studied using 13C nuclear magnetic resonance at 67.9 MHz and 22.6 MHz. Studies were made at a variety of hormone and protein concentrations and temperatures, but at constant pH (6.6). Under all the conditions derivatives 1 and 2 interact strongly with both neurophysins but the diastereomer 3 apparently does not. The line widths (1/πT2) of the labeled carbons of the hormones in a 0.9 to 1.0 hormone to protein mole ratio suggest considerable differences in the nature of the interaction(s) at the glycinamide-9 and half-cystine-1 residues of the hormones with the neurophysins, with the half-cystine-1 residue tightly bound with motional characteristics similar to those of the protein, while the glycinamide-9 residue of the hormones possesses additional motion. This extra motion of the glycinamide residue is equivalent to free rotation around one bond in the tripeptide tail of oxytocin. The half-cystine-1 α-carbon atom of oxytocin in the free state and when bound to neurophysin had a chemical-shift difference of about 2.7 ppm under all conditions studied, while the chemical shift for the glycinamide-9 α carbon in 1 was the same or only slightly shifted in the free hormone as compared with the complex. Of the chemical-shift difference present with 2, at most one-third can be due to a raising of the pKa of the N-terminal half-cystine residue upon binding to neurophysin. Possible reasons for the additional shift include a conformation change of the oxytocin backbone upon binding to neurophysin, or a change in the dielectric constant of the surrounding medium. Most interestingly, the glycinamide-9 α carbon of 1 is in fast exchange (>1000 s-1 at 37°C) in the hormone-neurophysin I (or neurophysin II) complex, while the half-cystine-1 α carbon in 2 is in slow exchange (
• Blumenstein, M., Hruby, V. J., Yamamoto, D., & Yang, Y. (1977). ¹³C nuclear magnetic resonance studies of the interactions of bovine neurophysins with (1-hemi-[1-¹³C]cystine)oxytocin and (1-hemi-[1-¹³C]cystine, 8-arginine)vasopressin. FEBS Letters, 81(2), 347-350.
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  PMID: 923806;
• Hadley, M. E., & Hruby, V. J. (1977). Neurohypophysial peptides and the regulation of melanophore stimulating hormone (MSH) secretion. Integrative and Comparative Biology, 17(4), 809-821.
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  Abstract: Melanophore stimulating hormone (MSH) secretion from the vertebrate pars intermedia is regulated as for other pituitary hormones, by the hypothalamus. Removal of the pituitary from hypothalamic control results in an autonomous uninhibited secretion of MSH. Thus, as for prolactin, the hypothalamus exerts a tonic inhibitory control over MSH secretion. The nature of this inhibitory mechanism is presently being debated with two general models being considered. It is suggested by some investigators that peptides of neurohypophysial hormone origin act as MSH releasing and inhibiting factors (MRF' and MIF's, respectively). In this scheme, the neurohypophysial hormones such as oxytocin would serve as prohormones which by enzymatic cleavage by hypothalamic enzymes would yield MSH releasing and/or inhibiting factors. It is suggested that the terminal tripeptide side chain is an MIF whereas the N-terminal pentapeptide sequence of oxytocin is an MRF. The data supporting this hypothesis comes from work of a few investigators that espouse this scheme. To our knowledge, the so-called MSH releasing and inhibiting factors have proven ineffective in the hands of all other investigators in regulating MSH release. © 1977 American Society of Zoologists.
• Hruby, V. J., Upson, D. A., & Agarwal, N. S. (1977). Comparative use of benzhydrylamine and chloromethylated resins in solid-phase synthesis of carboxamide terminal peptides. Synthesis of oxytocin derivatives. Journal of Organic Chemistry, 42(22), 3552-3556.
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  Abstract: Specifically deuterated derivatives of the peptide hormone oxytocin were synthesized by the solid-phase method of peptide synthesis using either the standard chloromethylated resin or the benzhydrylamine resin as the support for the syntheses, and a comparison of the overall efficiency of the syntheses on the two resins was made. [1-Hemi-DL-[β,β-2H2]cystine]oxytocin was synthesized using the standard chloromethylated resin, and the two diastereomers were separated and purified by partition chromatography and gel filtration in an overall yield of about 30%. [1-Hemi-DL-[α-2H1]cystine]oxytocin was prepared using the benzhydrylamine resin to prepare the nonapeptide resin precursor, but otherwise using essentially identical conditions as used for the synthesis on the chloromethylated resin. Again the two diastereomers were separated and purified by partition chromatography and gel filtration. The overall yield of purified diastereomers under the best conditions was about 49%. For the synthesis of the latter compounds, S-3,4-dimethylbenzyl protecting groups were used to introduce the cysteine residues. The overall yields of the peptide hormone derivatives prepared on the benzhydrylamine resin were substantially improved if HF reactions were run at lower temperatures (0°C rather than 25°C), and if the S-3,4-dimethylbenzyl rather than the S-benzyl group was used for cysteine protection. Reproducible procedures for preparing benzhydrylamine resins with amino substitution levels of 0.15-0.45 mmol of amino group/g of resin were developed.
• Meraldi, J. P., Hruby, V. J., & Brewster, A. I. (1977). Relative conformational rigidity in oxytocin and [1 penicillamine] oxytocin. A proposal for the relationship of conformational flexibility to peptide hormone agonism and antagonism. Proceedings of the National Academy of Sciences of the United States of America, 74(4), 1373-1377.
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  PMID: 266179;PMCID: PMC430763;Abstract: A comparative study of the proton and carbon 13 nuclear magnetic resonance spectral parameters of the peptide hormone oxytocin and of its competitive inhibitor [1 L penicillamine]oxytocin has been made, and the results analyzed in terms of comparative conformational and dynamic properties. The results indicate that oxytocin has a flexible conformation, while [1 L penicillamine]oxytocin has a more restricted conformation. The results provide a framework for understanding the mechanism of peptide hormone agonism and antagonism for these compounds, and an approach for understanding some features of the interaction of the hormone and related compounds with their receptor.
• Upson, D. A., & Hruby, V. J. (1977). A general method for the preparation of α-labeled amino acids. Journal of Organic Chemistry, 42(13), 2329-2330.
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  PMID: 874610;
• Yamamoto, D. M., Upson, D. A., Linn, D. K., & Hruby, V. J. (1977). Synthesis of specific deuterium labeled tyrosine and phenylalanine derivatives and their use in the total synthesis of [8-arginine]vasopressin derivatives: The separation of diastereomeric [8-arginine]vasopressin derivatives by partition chromatography. Journal of the American Chemical Society, 99(5), 1564-1570.
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  PMID: 839004;Abstract: Derivatives of tyrosine specifically deuterated at the a carbon ([α-2H1]tyrosine) and at both the α and β carbons ([α,β,β-2H3]tyrosine) and a derivative of phenylalanine specifically deuterated at the α carbon ([α-2H1]phenylalanine) have been synthesized in high yield. These labeled compounds have been resolved enzymatically, and the enantiomers and racemates have been converted to N-tert-butyloxycarbonyl derivatives. The deuterium labels were not exchanged under the conditions of the syntheses. The protected derivatives as well as specifically deuterated derivatives of S-benzylcysteine and of glycine were used to prepare specifically deuterated analogues of [8-arginine]vasopressin using solid phase peptide procedures. The use of improved synthetic procedures resulted in considerable improvements in the yields of [8-arginine]vasopressin compared with previous reports. In addition, new solvent systems for partition chromatography purification of [8-arginine]vasopressin on Sephadex were developed which allowed a facile one-step separation of diastereomers of [8-arginine]vasopressin containing a racemic amino acid at either the 1-hemicystine or the 2-tyrosine positions of the hormone. The following specifically deuterated hormone derivatives were synthesized: [9-[α,α-2H2]glycinamide,8-arginine]vasopressin (15), [1-hemi-[α-2H1]cystine,8-arginine]vasopressin (21), [1-hemi[β,β-2H2]cystine,8-arginine]vasopressin (17), [2-[α-2H1]tyrosine,8-arginine]vasopressin (19a), [2-[α,β,β-2H 3]tyrosine,8-arginine]vasopressin (20), [3-[α-2H1]phenylalanine,8-arginine]vasopressin (18a), [1-hemi-D-[α-2H1]cystine,8-arginine]vasopressin (16), [2-D-[α-2H1]tyrosine,8-arginine]vasopressin (19b), [1-hemi-D-cystine,3-[α-2H 1]phenylalanine,8-arginine]vasopressin (18b).
• Barfield, M., Hruby, V. J., & Meraldi, J. -. (1976). The dependence of geminal H-H spin-spin coupling constants on φ and ψ angles of peptides in solution. Journal of the American Chemical Society, 98(6), 1308-1314.
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  PMID: 1249364;Abstract: A theoretical study is presented of the conformational dependence of geminal H-H coupling constants in compounds which provide models for the peptide structure. Calculated results for Fermi contact coupling in N-methylacetamide, Nα-acetylglycinamide, cyclo-(-Gly-Gly-), and a three-peptide fragment having a γ turn are based on the finite perturbation theory (FPT) formulation in the semiempirical approximation of intermediate neglect of differential overlap (INDO). It is shown that the effect of the amide carbonyl is to produce a shift in the value of the geminal coupling constant to more negative values, depending on the value of the dihedral angle ψ. However, the effect of the amide nitrogen is to shift the geminal coupling constants toward more positive values depending on the dihedral angle φ. Under the combined effects of the two groups, as in Nα-acetylglycinamide, the total variation of the coupling is calculated to be 8 Hz. Agreement of calculated and experimental values is quite satisfactory in those cases in which x-ray structural data for the molecules are known. Although polarity of the solvent is known to have an effect on geminal H-H coupling, the calculated results for the three-peptide fragment having a γ turn suggests that intramolecular hydrogen bonding may not be an important factor. Based on these results, it is concluded that geminal H-H coupling constants can complement other NMR parameters as a probe of peptide structure in solution.
• Benson, B., Matthews, M. J., Hadley, M. E., Powers, S., & Hruby, V. J. (1976). Differential localization of antigonadotropic and vasotocic activities in bovine and rat pineal. Life Sciences, 19(5), 747-754.
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  PMID: 986528;Abstract: Bovine pineal glands were separated into stalk and parenchymal portions and extracted separately for both pineal antigonadotropic and neurohypophysial activities. Bioassay of these extracts localized neurohypophysial hormone activity to the stalk and antigonadotropic activity to the pineal parenchyma. Destalked rat pineals were devoid of neurohypophysial hormone activity at the concentrations employed. Whereas the injection of purified extracts containing pineal antigonadotropin reduced ventral prostate weights in mice, vasotocin was without such actions. these results fail to support pineal (parenchymal) localization of vasotocin and a reproductive role for this neurohypophysial peptide. © 1976.
• Blumenstein, M., & Hruby, V. J. (1976). Carbon-13 nuclear magnetic resonance studies of the interaction of specifically labeled (90%-¹³C) oxytocin and arginine vasopressin with neurophysins. Biochemical and Biophysical Research Communications, 68(4), 1052-1058.
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  PMID: 1267763;Abstract: Oxytocin and arginine vasopressin have been synthesized, via solid phase techniques, enriched to 90% 13C in the 2-carbon of their C-terminal glycinamide residues. In the presence of an approximately equimolar amount of bovine neurophysin II, the 13C nuclear magnetic resonance signal due to the enriched carbon in oxytocin shows a broadening which is highly dependent on both the temperature and the concentration of the neurophysin-oxytocin complex. The linewidth varies from 3 hz, observed at a protein concentration of 11mg/ml and a temperature of 37°, to 120 hz for a protein concentration of 65 mg/ml at 6°C. Similar results were obtained with arginine vasopressin. The results indicate that under conditions of low protein concentration and high temperature, the glycinamide residues of oxytocin and arginine vasopressin bound to neurophysin possess significant internal motion, while lowering the temperature and/or raising the protein-hormone concentration reduces this internal motion, probably concommitant with association of the protein-hormone complex into higher molecular weight aggregates. © 1976.
• Glasel, J. A., McKelvy, J. F., Hruby, V. J., & Spatola, A. F. (1976). Binding studies of polypeptide hormones to bovine neurophysins. Journal of Biological Chemistry, 251(10), 2929-2937.
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  PMID: 5446;Abstract: Experimental binding isotherms of [9 glycinamide 1 14C]oxytocin and [9 glycinamide 1 14C]arginine vasopressin to purified neurophysins I and II at pH = 4.4, 5.4, 6.5, 7.4, and 8.5 and 6°, 22°, and 37° in aqueous buffers are reported. For purposes of comparison, binding isotherms for [4 glycine 1 14C]oxytocin to neurophysin II and I in aqueous buffer, and [9 glycinamide 1 14C]oxytocin to neurophysin II in dimethylsulfoxide under selected conditions are also reported. A brief discussion of the interpretation of binding isotherms is entered into and apparent binding constants are derived. The results indicate that the interpretations presented in the literature up to now are much too simple. There are, in contrast, multiple binding sites of oxytocin and vasopressin to the neurophysins and large temperature dependences of the number of sites and their binding constants. We find, in fact, that at 37° the binding of neurohypophysial hormones to the supposed storage proteins is rather weak even at the pH of maximum binding.
• Hruby, V. J., Wright, D. E., Lin, M. C., & Rodbell, M. (1976). Semisynthetic glucagon derivatives for structure-function studies. Metabolism, 25(11 SUPPL.), 1323-1325.
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  PMID: 185491;
• Meraldi, J., & Hruby, V. J. (1976). Studies on the molecular association of oxytocin and related compounds in dimethyl sulfoxide [19]. Journal of the American Chemical Society, 98(20), 6408-6410.
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  PMID: 965654;
• Rosenblum, I. Y., Benson, B., & Hruby, V. J. (1976). Chemical differences between bovine pineal antigonadotropin and arginine vasotocin. Life Sciences, 18(12), 1367-1374.
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  PMID: 940419;Abstract: Studies were conducted in order to characterize chemically a partially purified antigonadotropic factor extracted from bovine pineal glands (PAG). Because several reports have appeared recently suggesting a role for arginine vasotocin (AVT) as a pineal antigonadotropin, our experiments were designed to determine the presence or absence of this nonapeptide in our material. A comparison of biologically active PAG and synthetic AVT revealed dissimilar UV absorption and fluorescence maxima, different mobilities on thin layer chromatography and paper electrophoresis, as well as different elution patterns on DEAE Sephadex ion-exchange chromatography. Amino acid analysis using 2 different methods revealed dissimilar amino acid compositions. On the basis of these and other chemical data, it is concluded that our preparations of PAG do not contain AVT. © 1976.
• Upson, D. A., & Hruby, V. J. (1976). Synthesis of specifically deuterated S-benzylcysteines and of oxytocin and related diastereomers deuterated in the half-cystine positions. Journal of Organic Chemistry, 41(8), 1353-1358.
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  PMID: 1263005;Abstract: S-Benzylcysteine derivatives specifically deuterated at the a carbon only, the β carbon only, and at both the α and β carbons have been synthesized. These labeled compounds have been enzymatically resolved and the enantiomers and reacemates have been converted to the N-tert-butyloxycarbonyl derivatives. The deuterium labels were not exchanged under the conditions of the syntheses. Condensation of the sodium salt of diethyl α-acetamidomalonate with benzyl chloromethyl sulfide followed by hydrolysis with DCl afforded S-benzyl-DL-[α-2H1]cysteine. Acetylation followed by treatment with hog renal acylase separated the stereoisomers. A Mannich reaction with [2H2]methylene diacetate, diethyl α-acetamidomalonate, and dimethylamine followed by quaternization of the amino nitrogen with methyl iodide gave diethyl α-acetamido-α-dimethylamino[2H2]methylmalonate methiodide (15). Treatment of 15 with sodium benzylmercaptide gave diethyl α-acetamido-α-benzylthio[2αH 2]methylmalonate, which was hydrolyzed with HCl to yield S-benzyl-DL-[β,β-2H2]cysteine or with DCl to afford S-benzyl-DL-[α,β,β-2H3]cysteine. These compounds were resolved as before. The preparation of S-benzyl-DL-[α,β,β-2H3]cysteine required an efficient source of ethanol-d. This deuterated solvent was prepared in quantitative yield in 2 h from tetraethoxysilane, D2O, and a catalytic amount of thionyl chloride. The protected deuterated amino acids were used in the preparation of several oxytocin analogues in which the specific deuteration appears in either the 1-hemicystine or the 6-hemicystine residues.
• Cutnell, J. D., Glasel, J. A., & Hruby, V. J. (1975). Nondipolar contributions to ¹³C relaxation in molecules. Annals of the New York Academy of Sciences, vol.248, 458-462.
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  PMID: 1054559;
• Hadley, M. E., Hruby, V. J., & Bower, S. (1975). Cellular mechanisms controlling melanophore stimulating hormone (MSH) release. General and Comparative Endocrinology, 26(1), 24-35.
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  PMID: 236974;Abstract: Melanophore stimulating hormone (MSH) release from the vertebrate pars intermedia is under an inhibitory control by the hypothalamus. Catecholamines both inhibit and stimulate MSH release from the isolated frog neurointermediate lobe or rat (and mouse) pituitary. Classical pharmacological methods using specific adrenergic receptor agonists and antagonists reveal that inhibition of MSH release by catecholamines is mediated through either alpha adrenergic receptors and/or dopamine receptors whereas stimulation of MSH release by catecholamines is mediated through beta adrenergic receptors. These results provide the physiological correlate for the morphological evidence of pars intermedia regulation by direct neuronal innervation. Acetylcholine also stimulates MSH release from the frog neurointermediate lobe and this enhanced hormone secretion is mediated through cholinergic receptors (blocked by atrophine, but not by propranolol). The possible interactions of adrenergic and cholinergic receptor mechanisms in the control of MSH release is still to be determined. Neither boiled nor acid-treated hypothalamic extracts inhibit or stimulate MSH release in vitro thus failing to implicate (by these methods) hypothalamic inhibiting or stimulating factors, if present, in the control of MSH release. Our in vitro experiments also fail to support a so-called "auto-feedback" (mass action) regulation of MSH release in either the rat, the frog, or the toad. Numerous similarities between the mechanisms involved in the control of MSH and prolactin release are apparent. © 1975.
• Hruby, V. J., & Spatola, A. F. (1975). Equilibrium dialysis studies of hormone neurophysin binding. Annals of the New York Academy of Sciences, vol.248, 451-457.
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  PMID: 235234;Abstract: From the binding studies reported here on the interaction of oxytocin and arginine vasopressin with bovine neurophysin II, a few conclusions are possible: Scatchard plots of the binding of oxytocin and arginine vasopressin to bovine neurophysin II generally are not linear over the entire binding isotherm. Thus, simple 1:1 or 1:2 binding interactions are not likely, except perhaps under a few specific conditions. The binding interactions are both temperature and pH dependent. The binding of oxytocin and of arginine vasopressin to neurophysin II is not identical.
• Lin, M. C., Wright, D. E., Hruby, V. J., & Rodbell, M. (1975). Structure-function relationships in glucagon: Properties of highly purified des-His¹-, monoiodo-, and [des-Asn²⁸,Thr²⁹](homoserine lactone²⁷)-glucagon. Biochemistry, 14(8), 1559-1563.
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  PMID: 164891;Abstract: We have compared the ability of glucagon and three highly purified derivatives of the hormone to activate hepatic adenylate cyclase (an expression of biological activity of the hormone) and to compete with [125] glucagon for binding to sites specific for glucagon in hepatic plasma membranes. Relative to that of glucagon, biological activity and affinity of [des-Asn28,Thr29](homoserine lactone27)-glucagon, prepared by CNBr treatment of glucagon, were reduced equally by 40- to 50-fold. By contrast, des-His1-glucagon, prepared by an insoluble Edman reagent and highly purified (less than 0.5% contamination with native glucagon), displayed a 15-fold decrease in affinity but a 50-fold decrease in biological activity relative to that of the native hormone. At maximal stimulating concentrations, des-His1-glucagon yielded 70% of the activity given by saturating concentrations of glucagon. Thus, des-His1-glucagon can be classified as a partial, weak agonist. Highly purified monoiodoglucagon and native glucagon displayed identical biological activity and affinity for the binding sites. Our findings suggest that the hydrophilic residues at the terminus of the carboxy region of glucagon are involved in the process of recognition at the glucagon receptor but do not participate in the sequence of events leading to activation of adenylate cyclase. The amino-terminal histidyl residue in glucagon plays an important but not obligatory role in the expression of hormone action and contributes to a significant extent in the recognition process.
• Bower, A., Hadley, M. E., & Hruby, V. J. (1974). Biogenic amines and control of melanophore stimulating hormone release. Science, 184(4132), 70-72.
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  PMID: 4544474;Abstract: Release of melanophore stimulating hormone (MSH) from the vertebrate pars intermedia is under inhibitory control by the hypothalamus. Removal of the rat pituitary or the neurointermediate lobe of the frog (Rana pipiens) to in vitro incubation medium results in rapid uninhibited release of MSH. This secretion is inhibited by norepinephrine, epinephrine, phenylephrine, and dopamine, and the inhibition is antagonized by α adrenergic receptor blocking agents. Isoproterenol stimulation of MSH secretion from isolated glands is blocked by propranolol, a β adrenergic receptor antagonist. These results implicate dopaminergic or classical α adrenergic receptors (or both) in inhibition of MSH release by catecholamines, and implicate β adrenergic receptors in stimulation of MSH release by the bioamines.
• Chan, W. Y., Hruby, V. J., & Vigneaud, V. D. (1974). Effects of magnesium ion and oxytocin inhibitors on the uterotonic activity of oxytocin and prostaglandins E2 and F2ALPHA.. Journal of Pharmacology and Experimental Therapeutics, 190(1), 77-87.
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  PMID: 4847314;
• Smith, C. W., Hruby, V. J., & Ferger, M. F. (1974). Four cyclic disulfide pentapeptides possessing the ring of isotocin and glumitocin. Journal of Medicinal Chemistry, 17(8), 873-876.
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  PMID: 4858613;Abstract: [4-Serine]tocinoic acid and [4-serine]tocinamide (the 20-membered disulfide pentapeptide and pentapeptide amide ring of isotocin and glumitocin), as well as the two deamino analogs [4-serine]deaminotocinoic acid and [4-serine]deaminotocinamide, were synthesized from four protected polypeptide precursors which had been prepared by the stepwise active ester method. All four precursors were prepared from the same intermediate Boc-Tyr(Bzl)-Ile-Ser(Bzl)-Asn-Cys(Bzl)-OBzl (1). For the preparation of the two-ring compounds containing C-terminal amides, 1 was treated with MeOH saturated with NH3 prior to condensation with either Boc-Cys(Bzl)-ONSu or β-Mpa(Bzl)-ONp. For the preparation of [4-serine]tocinoic acid and [4-serine]deaminotocinoic acid, 1 was condensed with either Z-Cys(Bzl)-ONp or β-Mpa(Bzl)-ONp. The resulting four protected precursors were then converted to the corresponding ring compounds by deprotection with Na in NH3, followed by oxidative cyclization. None of the ring compounds showed any significant oxytocic, avian vasodepressor, or rat pressor activity. All showed a slight degree of antioxytocic and antiavian vasodepressor activity but no antipressor activity. Both [4-serine]tocinoic acid and [4-serine]tocinamide showed some milk-ejecting activity.
• Spatola, A. F., Cornelius, D. A., Hruby, V. J., & Blomquist, A. T. (1974). Synthesis of oxytocin and related diastereomers deuterated in the half-cystine positions. Comparison of solid-phase and solution methods. Journal of Organic Chemistry, 39(15), 2207-2212.
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  PMID: 4859518;Abstract: Four derivatives of the neurohypophysial hormone oxytocin deuterated at the α and β positions of the two half-cystine residues have been synthesized. The substituted amino acid Boc-S-benzyl-DL-[α,β,β-2H3]cysteine was used to prepare [1-hemi-DL-[α,β,β-2H3]cystine]oxytocin and [6-hemi-DL-[α,β,β-2H3]cystine]oxytocin. The diastereomeric mixtures were separated and purified by partition chromatography and gel filtration to give [1-hemi-L-[α,β,β-2H 3]cystine]oxytocin,1ri[1-hemi-D-[α,β,β- 2H3]cystine]oxytocin, [6-hemi-L-[α,β,β-2H3]cystine]oxytocin, and [6-hemi-D-[α,β,β-2H3]cystine]oxytocin. The former two compounds were prepared by both solid-phase and solution techniques of peptide chemistry, and the two methods were compared in the synthesis of these derivatives. The solid-phase method was considerably faster and gave better overall yields, while the solution method permitted a slightly more conservative use of deuterated amino acid. It was found that much shorter deprotection and coupling times and much smaller excesses of amino acid were compatible with the solid-phase methodology.
• Brewster, A. I., & Hruby, V. J. (1973). 300 MHz nuclear magnetic resonance study of oxytocin in aqueous solution: Conformational implications. Proceedings of the National Academy of Sciences of the United States of America, 70(12), II.
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  PMID: 4521206;PMCID: PMC427333;Abstract: The 300 MHz nuclear magnetic resonance spectrum of the peptide hormone, oxytocin, in water, was obtained. Extensive nuclear magnetic resonance spindecoupling experiments, including those involving irradiation of the α proton resonances under the water peak, studies using partially deuterated hormone derivatives, and nuclear magnetic resonance studies of some precursor peptides to oxytocin, permitted us to assign most of the proton resonances of the hormone unambiguously. The J(Nα) values and temperature dependence of the chemical shift of the peptide amide and carboxamide protons of oxytocin were also obtained. These studies indicate that, in aqueous solution, oxytocin is a flexible molecule possessing several different conformations. Corresponding studies of [4 leucine] oxytocin were also made, with similar results and conclusions.
• Glasel, J. A., Heuby, V. J., McKelvy, J. F., & Spatola, A. F. (1973). Deuteron magnetic resonance studies on the microdynamical behavior of partially deuterated oxytocin with neurophysin. Journal of Molecular Biology, 79(3), 555-575.
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  PMID: 4758065;Abstract: With the use of specifically deuteron-labeled oxytocin and related molecules, deamino oxytocin and prolyl-leucyl-glycinamide, some of the intramolecular micro-mica of the free molecules and their interactions with their biological carriers, the neurophysins, have been studied. The work involves contemporary synthetic and isolation chemical techniques and computer processed nuclear magnetic resonance spectroscopy. The results show that there is considerable intramolecular motion in the "tail" of oxytocin with respect to the remainder of the molecule and that there is a rapid dynamical equilibrium between neurophysin-bound and free hormone in solution. The implications of this work on the biological action of the hormone-protein complex are discussed as well as the interpretation of high resolution nuclear magnetic resonance data in the light of these results. © 1973.
• Glasel, J. A., McKelvy, J. F., Hruby, V. J., & Spatola, A. F. (1973). Deuteron magnetic resonance studies of neurohypophysial hormones. Annals of the New York Academy of Sciences, Vol.222, 778-788.
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  PMID: 4522444;
• Hadley, M. E., Bower, A., & Hruby, V. J. (1973). Regulation of melanophore stimulating hormone (MSH) release. Yale Journal of Biology and Medicine, 46(5), 602-616.
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  PMID: 4544339;PMCID: PMC2592028;Abstract: Synthetic tocinamide and tocinoic acid, and ring structures of oxytocin, reversibly inhibit (at nanogram, or less, concentrations) the release of melanophore stimulating hormone (MSH) from the rat and hamster pituitary in vitro. These peptides are less effective (on Bufo marinus and Rana catesbeiana) or totally without effect (on Rana pipiens) on MSH release from the isolated amphibian pars intermedia. Oxytocin, lysine vasopressin, and ring structures of the vasopressins (pressinamide and pressinoic acid) are without effect of MSH release in the animals studied. The synthetic tripeptide side chain of oxytocin (L Pro L Leu Gly NH2) is devoid of in vitro MSH release inhibition in either the mammal or the frog. Crude hypothalamic extracts from either the frog or the rat 'appear' to reversibly inhibit in vitro MSH release. If, however, the extracts are heated to boiling or acid extracted, inhibition of MSH release is not observed, suggesting enzymatic or other reactions may be responsible for the loss of bioassayable MSH. The addition of MSH to crude hypothalamic extracts results in a loss of hormone activity. It is, therefore, unclear whether hypothalamic extracts provide evidence for the existence of a hypothalamic MSH release inhibiting factor (MRIF). It remains for further work to establish whether the ring structures of the neurohypophyseal hormones can be considered as possible candidates for a natural vertebrate MRIF. Both calcium and potassium ions are necessary for MSH release in vitro. Release of the hormone is inhibited by ouabain and related cardiac glycosides suggesting that a Na+-K+ pump (active transport) system is involved in MSH release. Cytochalasin B, but not colchicine (or vinblastine and vincristine) is reversibly inhibitory to hormone release and suggests that a microfilament (rather than a microtubular) system may be involved in the mechanism of acute release of MSH. However, other actions of cytochalasin B on transport mechanisms or ionic fluxes may account for its inhibition of MSH release. The relationships between an active transport system, calcium ions, and a microfilament system in the control of MSH release by hypothalamic substances, neurohypophyseal peptides, or related structures are still unclarified.
• Hruby, V. J., Muscio, F., Groginsky, C. M., Gitu, P. M., Saba, D., & Chan, W. Y. (1973). Solid-phase synthesis of [2-isoleucine,4-leucine]oxytocin and [2-phenylalanine,4-leucine]oxytocin and some of their pharmacological properties. Journal of Medicinal Chemistry, 16(6), 624-629.
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  PMID: 4733119;Abstract: [Ile2,Leu4]oxytocin and [Phe2,Leu4]oxytocin have been synthesized using the solid-phase method. In the synthesis of [Phe2,Leu4]oxytocin a benzhydrylamine resin support was used, and a diphenylmethyl carboxamide protecting group was used for the asparagine residue. The protected asparagine residue was coupled to the peptide resin with dicyclohexylcarbodiimide. The peptide was cleaved from the resin and the diphenylmethyl carboxamide and S-p-methoxybenzyl protecting groups were removed by HF(l). The disulfhydryl peptide was oxidized and purified to give the desired cyclic peptide. In the synthesis of [Ile2,-Leu4]oxytocin, tert-butyloxycarbonylasparagine was coupled to the peptide resin with dicyclohexylcarbodiimide and 1-hydroxybenzotriazole. No cyano formation was detected. [Ile2,Leu4]oxytocin and [Phe2,-Leu4]oxytocin were found to possess weak pressor activities and no antidiuretic activities. [Ile2,Leu4]-oxytocin had a mild natriuretic-diuretic activity whereas [Phe2,Leu4]oxytocin had only a weak natriuretic activity. Neither compound had a detectable oxytocic activity when assayed in the isolated uterus in a Mg2+-free van Dyke-Hastings solution. On the contrary, they inhibited the oxytocic response of oxytocin in this in vitro system. Their oxytocic inhibitory activity was found to be dependent on Ca concentration. When the Ca2+ concentration in the bathing medium was increased from 0.5 to 1.0 mM/l., the inhibitory activity of [Phe2,Leu4]oxytocin was markedly reduced while [Ile2,Leu4]oxytocin became a weak agonist.
• I., A., Hruby, V. J., Glasel, J. A., & Tonelli, A. E. (1973). Proposed conformations of oxytocin and selected analogs in dimethyl sulfoxide as deduced from proton magnetic resonance studies. Biochemistry, 12(26), 5294-5304.
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  PMID: 4760494;Abstract: The 220-MHz nmr spectra have been obtained and assigned for oxytocin, deamino-oxytocin, [4-glycine]oxytocin, [2-valine]oxytocin, [7-D-proline]oxytocin, and [1-β-mercaptopropionic acid,7-D-proline]oxytocin. Several deuterated derivatives were used for making unambiguous assignments. Conformational calculations based on measured values of the vicinal amide to α-proton coupling were used for proposing conformations for the molecules in dimethyl sulfoxide solution. Three energetically favorable conformations of oxytocin are found, one of which has a single intramolecular hydrogen bond involving the asparagine-5 backbone NH and the glutamine-4 carboxamide carbonyl. The tripeptide side chain is proposed to possess a trans-cis' junction to the ring and is folded toward the ring. In dimethyl sulfoxide oxytocin appears to possess a nonrigid conformation. Deamino-oxytocin possess a conformation similar to oxytocin. The proposed conformation for [4-glycine]oxytocin has two transannular bonds: the asparagine-5 peptide NH and carbonyl to the tyrosine-2 carbonyl and peptide NH, respectively. The conformation of [2-valine]oxytocin appears to be quite different from that of any of the other peptide analogs examined, with no intramolecular hydrogen bond. Both D-proline-7 analogs differ from oxytocin and deamino-oxytocin in the orientation of the tripeptide side chain with respect to the ring. The influence of the amino acid substitutions on the conformation is discussed.
• I., A., Hruby, V. J., Spatola, A. F., & Bovey, F. A. (1973). Carbon-13 nuclear magnetic resonance spectroscopy of oxytocin, related oligopeptides, and selected analogs. Biochemistry, 12(8), 1643-1649.
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  PMID: 4699993;Abstract: The C-13 nuclear magnetic resonance (nmr) spectra of oxytocin and several analogs in dimethyl sulfoxide solution have been studied. Assignments of all 43 carbon resonances have been made by reference to the spectra of the linear precursors and through the use of partially deuterated peptides and oxytocin derivatives. The closing of the disulfide bond yielding oxytocin from the linear nonapeptide precursor results in a number of significant chemical shift changes, particularly of the α and βcarbons, which cannot at present be interpreted in detail. Significant chemical shift differences are observed for some oxytocin analogs which may be related to conformational changes.
• Brewster, A. I., Glasel, J. A., & Hruby, V. J. (1972). Conformational studies on tocinamide and deaminotocinamide by 220 MHz nuclear magnetic resonance spectroscopy.. Proceedings of the National Academy of Sciences of the United States of America, 69(6), 1470-1474.
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  PMID: 4504362;PMCID: PMC426728;
• Hruby, V. J. (1972). A new machine for automated solid phase peptide synthesis. Analytical Chemistry, 44(2), 343-350.
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  PMID: 5059082;Abstract: A versatile, fail-safe, and simple machine for automated solid phase peptide synthesis was designed and constructed. A dual programming scheme was used. The variable operations such as kind and quantity of solvent or reagent, reaction or wash times, and other variables, are controlled by a Drum Programmer. The repetitive operations needed to get the solvent or reagent into and out of the reaction vessel are controlled by a Card Programmer. A number of failsafe devices were built into the machine to shut it off if electrical or mechanical failures occur. The operation is also monitored with a strip chart recorder which gives a permanent record of a synthesis. The machine can be operated in both automatic and manual modes.
• Hruby, V. J., Smith, C. W., Bower, S. A., & Hadley, M. E. (1972). Melanophore stimulating hormone: Release inhibition by ring structures of neurohypophysial hormones. Science, 176(4041), 1331-1332.
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  PMID: 4624585;Abstract: Tocinamide and tocinoic acid, ring structures of oxytocin, are potent inhibitors of the release of melanophore stimulating hormone from the rat and hamster pituitary in vitro. Tocinamide is effective at concentrations as low as 10-14M on the mammalian pituitary. These peptides do not affect release of the hormone on the frog (Rana pipiens) pars intermedia, but they do inhibit release in the bullfrog (Rana catesbeiana) and the toad (Bufo marinus). The specificity of the peptides on inhibition of the hormone is demonstrated by the fact that oxytocin, lysine vasopressin, and pressinoic acid and pressinamide (ring structures of the vasopressins) do not show such inhibitory activity. Hypothalamic extracts of either the frog (Rana pipiens) or the rat inhibit release of the hormone from pituitaries of either species. The inhibitory effects of tocinamide and tocinoic acid, like that of hypothalamic extracts, are reversible.
• Hruby, V. J., Smith, C. W., Linn, D. K., Ferger, M. F., & Vigneaud, V. D. (1972). The synthesis and some pharmacological properties of tocinoic acid and deaminotocinoic acid. Journal of the American Chemical Society, 94(15), 5478-5480.
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  PMID: 5040852;Abstract: Tocinoic acid, the 20-membered cyclic disulfide ring of cysteinyltyrosylisoleucylglutaminylasparaginylcysteine, which possesses the tocin ring of oxytocin, was synthesized by the stepwise p-nitrophenyl ester method. All protecting groups of the protected intermediate Z-Cys(Bzl)-Tyr-Ile-Gln-Asn-Cys(Bzl)-ONB were removed on addition of this peptide to Na in NH3. The reduced peptide was converted to purified tocinoic acid by oxidation, followed by partition chromatography and gel filtration. Deaminotocinoic acid was synthesized by similar methods. No C-terminal carboxamide derivatives could be detected. Tocinoic acid possessed 0.2-0.3 unit/mg of oxytocic activity and no detectable avian vasodepressor activity, while deaminotocinoic acid had 3.7 ± 0.3 units/mg of oxytocic activity and no detectable avian vasodepressor activity. Deaminotocinoic acid possessed a slight inhibitory effect on the avian vasodepressor activity of synthetic oxytocin.
• Walter, R., Kirchberger, M. A., & Hruby, V. J. (1972). Competitive inhibitors of neurohypophyseal hormones on adenylate cyclase from the toad urinary bladder. Experientia, 28(8), 959-960.
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  PMID: 5076336;
• Barstow, L. E., & Hruby, V. J. (1971). A simple method for the synthesis of amides. Journal of Organic Chemistry, 36(9), 1305-1306.
• Bower, S. A., Hadley, M. E., & Hruby, V. J. (1971). Comparative MSH release-inhibiting activities of tocinoic acid (the ring of oxytocin), and L-Pro-L-Leu-Gly-NH₂ (the side chain of oxytocin). Biochemical and Biophysical Research Communications, 45(5), 1185-1191.
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  PMID: 5135504;Abstract: Synthetic tocinoic acid, the ring structure of oxytocin (L-Cys-L-Tyr-L-Ile-L-Gln-L-Asn-L-Cys-OH), inhibits MSH release from the rat pituitary in vitro at less than nanogram (10-10 g/ml) levels. A similar inhibitory activity could not be demonstrated on the amphibian (Rana pipiens) pituitary. The synthetic tripeptide side chain of oxytocin (L-Pro-L-Leu-Gly-NH2) was devoid, in vitro, of MSH release inhibition in either the rat or the frog. © 1971.
• Hruby, V. J., & Chan, W. Y. (1971). [2,4-Dileucine]oxytocin. A polypeptide with natriuretic and diuretic activities, and an inhibitor of the oxytocic response of oxytocin. Journal of Medicinal Chemistry, 14(11), 1050-1051.
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  PMID: 5115201;Abstract: [2,4-Dileucine]oxytocin, an analog of oxytocin in which the tyrosine in position 2 and the glutamine in position 4 have both been formally replaced by leucine, has been synthesized from the corresponding protected nonapeptide intermediate. The analog was found to possess negligible pressor activity and no antidiuretic or oxytocic activities. However, [2,4-dileucine]oxytocin has a natriuretic activity and a weak diuretic effect. Furthermore, it was shown to be an inhibitor to oxytocin in the oxytocic in vitro assay system.
• Hruby, V. J., & Groginsky, C. M. (1971). Partition chromatography of glucagon and secretin on Sephadex. Journal of Chromatography A, 63(C), 423-428.
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  PMID: 5160851;
• Hruby, V. J., Brewster, A. I., & Glasel, J. A. (1971). NMR studies on the conformation of derivatives of the side chain of oxytocin: examples of cis-trans isomerism.. Proceedings of the National Academy of Sciences of the United States of America, 68(2), 450-453.
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  PMID: 5277099;PMCID: PMC388958;
• Hruby, V. J., Ferger, M. F., & Vigneaud, V. D. (1971). Synthesis and pharmacological properties of deaminotocinamide and a new synthesis of tocinamide. Journal of the American Chemical Society, 93(21), 5539-5542.
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  PMID: 5165679;Abstract: Deaminotocinamide (the 20-membered disulfide pentapeptide amide ring of deamino-oxytocin) and tocinamide (the corresponding disulfide pentapeptide amide ring of oxytocin) were synthesized by the stepwise p-nitrophenyl ester method using the p-nitrobenzyl ester for C-terminal carboxyl protection. p-Nitrobenzyl 5-benzyl-β-mercaptopropionyltyrosylisoleucylglutaminylasparaginyl-S- benzylcysteinate and p-nitrobenzyl N-benzyloxycarbonyl-S-benzylcysteinyltyrosylisoleucylglutaminylasparaginyl-5- benzylcysteinate were converted to the corresponding C-terminal amide compounds in liquid ammonia. The polypeptide amides were then converted to the corresponding ring compounds by treatment with sodium in liquid ammonia followed by oxidation and purification. Deaminotocinamide was found to possess 34.2 ± 3.0 units/mg of oxytocic activity, but no detectable avian vasodepressor activity. Tocinamide possessed 3.2 ± 0.2 units/mg of oxytocic activity, but no detectable avian vasodepressor activity.
• Hruby, V. J., & Ehler, K. W. (1970). O-benzyl-N-t-butyloxycarbonyl-^L-serine. Journal of Organic Chemistry, 35(5), 1690-.
• Hruby, V. J., Vigneaud, V. D., & Chan, W. Y. (1970). [2,4-Diisoleucine]-oxytocin. An analog of oxytocin with natriuretic and diuretic activities. Journal of Medicinal Chemistry, 13(2), 185-187.
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  PMID: 5418485;Abstract: [2,4-Diisoleucine]-oxytocin has been synthesized from the requisite protected nonapeptide intermediate which was prepared by the stepwise p-nitrophenyl ester method. In this analog of oxytocin, isoleucine residues replace the tyrosine and glutamine residues at the 2 and 4 positions of the hormone, and thus the isoleucine residue occurs successively at positions 2, 3, and 4. This compound possesses a very low level of avian vasodepressor, oxytocic, and pressor activities, and no antidiuretic activity. However, [2,4-diisoleucine]-oxytocin has a pronounced natriuretic activity and a mild diuretic effect.
• Takashima, H., Hruby, V. J., & Vigneaud, V. D. (1970). The synthesis of [1-deamino,4-l-leucine]-oxytocin and [1-deamino,4-l-isoleucine]-oxytocin and some of their pharmacological properties. Journal of the American Chemical Society, 92(3), 677-680.
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  PMID: 5460748;Abstract: [1-Deamino,4-L-leucine]-oxytocin and [1-deamino,4-L-isoleucine]-oxytocin have been synthesized by the solid phase method, and some of their pharmacological properties have been determined. [1-Deamino,4-L-leucine]-oxytocin was also synthesized by the p-nitrophenyl ester stepwise procedure and compared to that synthesized by the solid phase method. The two preparations were found to be identical. The oxytocic and avian vasodepressor potencies of [1-deamino,4-L-leucine]-oxytocin and [1-deamino,4-L-isoleucine]-oxytocin are two- to three-fold higher than those of [4-L-leucine]-oxytocin and [4-L-isoleucine]-oxytocin respectively. The two deamino analogs also exhibit milk-ejecting activities of approximately 150 units/mg. Neither analog possesses appreciable pressor or antidiuretic activity. A similar enhancement of oxytocic and avian vasodepressor activities had been observed when the free amino group of [4-L-valine]-oxytocin was replaced by hydrogen.
• Blomquist, A. T., Rich, D. H., Carlson, B. A., Allen, G. A., Hruby, V. J., Takashima, H., Nangeroni, L. L., Glose, P., & Vigneaud, V. D. (1969). Deuterated oxytocins: the synthesis and biological properties of a crystalline analog of deamino-oxytocin deuterated in the 5-asparagine position.. Proceedings of the National Academy of Sciences of the United States of America, 64(1), 263-266.
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  PMID: 5263009;PMCID: PMC286156;
• Hruby, V. J., & Vigneaud, V. D. (1969). Synthesis and some pharmacological activities of [2-L-valine]-oxytocin and [2-L-leucine]-oxytocin. Journal of Medicinal Chemistry, 12(5), 731-733.
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  PMID: 5818088;Abstract: [2-Valine]-oxytocin and [2-leucine]-oxytocin have been synthesized from the requisite protected nonapeptide intermediates, which were prepared by the stepwise p-nitrophenyl ester method. The analogs were isolated by partition chromatography and gel filtration on Sephadex and then tested for a number of pharmacological activities. [2-Valine]-oxytocin possesses approximately 7 units/mg of avian vasodepressor, 2 units/mg of oxytocic, 15 units/mg of milk-ejecting, 0.05 unit/mg of pressor, and less than 0.01 unit/mg of antidiuretic activities. [2-Leucine]-oxytocin has approximately 3 units/mg of avian vasodepressor, less than 0.5 unit/mg of oxytocic, about 7 units/mg of milk-ejecting, 0.05 unit/mg of pressor, and negligible antidiuretic activities. Both of these analogs are much less active than [2-isoleucine]-oxytocin. © Copyright 1989 by the American Chemical Society.
• Hruby, V. J., & Vigneaud, V. D. (1969). The detection of a Schiff base intermediate in the formation of acetone-oxytocin. Journal of the American Chemical Society, 91(13), 3624-3626.
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  Abstract: Treatment of oxytocin with 60% aqueous acetone at 0° and at a pH of approximately 5 leads to the rapid formation of a Schiff base of oxytocin which has been trapped by reduction with sodium borohydride to give [1-(N-isopropylhemi-L-cystine)]-oxytocin (N-isopropyl-oxytocin). The reduction of the Schiff base was accomplished without reduction of the disulfide bond of oxytocin. Treatment of the hormone with acetone under the same conditions for 24 hr without the addition of borohydride affords acetone-oxytocin in aproximately 50% yield. N-Isopropyl-oxytocin possesses only a trace (∼0.1 unit/mg) of oxytocic activity and does not exhibit avian vasodepressor activity. The preparation of N,N′-diisopropyl-L-cystine is also described.
• Hruby, V. J., Flouret, G., & Vigneaud, V. d. (1969). The synthesis and some of the pharmacological properties of [4-L-isoleucine]-oxytocin and [4-L-leucine]-oxytocin.. Journal of Biological Chemistry, 244(14), 3890-3894.
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  PMID: 5817165;
• Blomquist, A. T., Rich, D. H., Hruby, V. J., Nangeroni, L. L., Glose, P., & Vigneaud, V. D. (1968). Deuterated oxytocins: the synthesis and biological properties of three crystalline analogs of deamino-oxytocin deuterated in the 1-beta-mercaptopropionic acid position.. Proceedings of the National Academy of Sciences of the United States of America, 61(2), 688-692.
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  PMID: 5246000;PMCID: PMC225214;
• Chan, W. Y., Hruby, V. J., Flouret, G., & Vigneaud, V. D. (1968). 4-leucine-oxytocin: Natriuretic, diuretic, and antivasopressin polypeptide. Science, 161(3838), 280-281.
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  PMID: 5657333;Abstract: During water diuresis in anesthetized rats, 4-leucine-oxytocin increased the urine output and the rates of sodium and chloride excretion. The potassium excretion rate was only slightly increased. During vasopressin-suppressed water diuresis, 4-leucineoxytocin produced similar effects on urine and electrolyte excretions. In addition, it inhibited the vasopressin-induced free-water reabsorption, and it could reverse reabsorption to free-water clearance.
• Hruby, V. J., Yamashiro, D., & Vigneaud, V. d. (1968). The structure of acetone-oxytocin with studies on the reaction of acetone with various peptides.. Journal of the American Chemical Society, 90(25), 7106-7110.
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  PMID: 5688360;
• Blomquist, A. T., & Hruby, V. J. (1967). The preparation and properties of 1,2-bis(triphenylphosphoranyl)benzocyclobutene. Journal of the American Chemical Society, 89(19), 4996-5007.
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  Abstract: The preparation of trans-1,2-bis(triphenylphosphonio)benzocyclobutene dibromide and its conversion 1,2-bis(triphenylphosphoranyl)benzocyclobutene are described. Treatment of the dibromide salt with base followed by reaction with a benzaldehyde affords different results that depend upon the nature of the solvent. In ethanol as the major solvent, the principal products are the cis- and trans-1-benzylidenebenzocyclobutenes corresponding to the particular benzaldehyde used, together with triphenylphosphine oxide; in DMF or DMSO, the major products are the cis,cis- and/or cis,trans- and/or trans,trans-1,2-bisbenzylidenebenzocyclobutenes corresponding to the benzaldehydes used, as well as triphenylphosphine oxide. In both instances a number of minor products are obtained. The structure and stereochemistry of the major products are in accord with the chemical and physical data reported herein.
• Branda, L. A., Hruby, V. J., & Vigneaud, V. D. (1967). 2-Isoleucine-oxytocin and deamino-2-isoleucine-oxytocin: their synthesis and some of their pharmacologic activities.. Molecular Pharmacology, 3(3), 248-253.
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  PMID: 6068341;
• Hruby, V. J. (1967). Scientists and responsibility [3]. Chemical and Engineering News, 45(28), 6-7.
• Hruby, V. J. (1966). Secrecy vs. science [5]. Chemical and Engineering News, 44(21), 7-.
• Blomquist, A. T., & Hruby, V. J. (1964). 1,2-bis(triphenylphosphoranyl)benzocyclobutene [35]. Journal of the American Chemical Society, 86(22), 5041-5043.
• Johnson, A. W., Hruby, V. J., & Williams, J. L. (1964). Chemistry of ylids. X. Diphenylsulfonium alkylides - A stereoselective synthesis of epoxides. Journal of the American Chemical Society, 86(5), 918-922.
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  Abstract: The preparation and properties of diphenylsulfonium benzylide (8) and butylide (9) are described. Both ylids can be dissociated into carbenes and phenyl sulfide. The benzylide 8 reacts stereoselectively with benzaldehydes to form trans-stilbene oxides.
• Hruby, V. J., & Johnson, A. W. (1962). The decomposition of sulfur ylids to carbenes [2]. Journal of the American Chemical Society, 84(18), 3586-3587.