John M Streicher

Interests

Research

I am interested in the molecular mechanisms underlying signal transduction, with a focus on GPCRs and the opioid receptors. By understanding which cascades and transduction mechanisms lead to which aspects of the overall response, both good and bad, we can design drugs which activate only the cascades we want, potentially improving the therapeutic index of new analgesic drugs. In my lab we investigate both sides of this approach, describing new signal transduction mechanisms, and screening experimental drugs for development and eventual human use.

Courses

Scholarly Contributions

Chapters

• Lei, W., Duron, D. I., Stine, C., & Streicher, J. M. (2019). Role of Heat Shock Protein 90 in Regulating Downstream Signal Transduction Cascades. In Heat Shock Proteins. Springer, Cham. doi:10.1007/978-3-030-03952-3_9
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  Heat shock protein 90 (Hsp90) regulates a broad swathe of proteins critical for normal and pathological cell function. One major class of regulated proteins are signal transduction molecules, such as the Mitogen Activated Protein Kinases (MAPK), G Protein Coupled Receptor (GPCR) regulatory kinases, and similar. Hsp90 regulates these signaling proteins by promoting proper folding and protein stability, however, Hsp90 also regulates signaling activation and association/targeting of mature proteins during the course of acute signal transduction. As these signaling proteins are ubiquitously expressed in most cells and are downstream of numerous different receptor systems, Hsp90 regulation of signaling proteins is strongly and broadly impactful. In this chapter, we will discuss the main themes of signaling protein regulation by Hsp90, and highlight several crucial signaling protein families. We will discuss the impact of Hsp90 on signaling downstream of multiple receptor systems, and subsequent effects on physiology and pathophysiology. We will also suggest means to manipulate these regulatory relationships to improve clinical therapy, and future directions for the field of Hsp90 signaling regulation.

Journals/Publications

• Banik, R. K., Sia, T., Ibrahim, M. M., Sivanesan, E., Uhelski, M., Pena, A., Streicher, J. M., & Simone, D. A. (2023). Increases in local skin temperature correlate with spontaneous foot lifting and heat hyperalgesia in both incisional inflammatory models of pain. Pain reports, 8(5), e1097.
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  This study investigated if a localized increase in skin temperature in rat models of incisional and inflammatory pain correlates with the intensity of spontaneous and evoked pain behaviors.
• Barber, K. R., Vizcarra, V. S., Zilch, A., Majuta, L., Diezel, C. C., Culver, O. P., Hughes, B. W., Taniguchi, M., Streicher, J. M., Vanderah, T. W., & Riegel, A. C. (2023). The Role of Ryanodine Receptor 2 in Drug-Associated Learning. bioRxiv : the preprint server for biology.
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  Type-2 ryanodine receptor (RyR2) ion channels facilitate the release of Ca from stores and serve an important function in neuroplasticity. The role for RyR2 in hippocampal-dependent learning and memory is well established and chronic hyperphosphorylation of RyR2 (RyR2P) is associated with pathological calcium leakage and cognitive disorders, including Alzheimer's disease. By comparison, little is known about the role of RyR2 in the ventral medial prefrontal cortex (vmPFC) circuitry important for working memory, decision making, and reward seeking. Here, we evaluated the basal expression and localization of RyR2 and RyR2P in the vmPFC. Next, we employed an operant model of sucrose, cocaine, or morphine self-administration (SA) followed by a (reward-free) recall test, to reengage vmPFC neurons and reactivate reward-seeking and re-evaluated the expression and localization of RyR2 and RyR2P in vmPFC. Under basal conditions, RyR2 was expressed in pyramidal cells but not regularly detected in PV/SST interneurons. On the contrary, RyR2P was rarely observed in PFC somata and was restricted to a different subcompartment of the same neuron - the apical dendrites of layer-5 pyramidal cells. Chronic SA of drug (cocaine or morphine) and nondrug (sucrose) rewards produced comparable increases in RyR2 protein expression. However, recalling either drug reward impaired the usual localization of RyR2P in dendrites and markedly increased its expression in somata immunoreactive for Fos, a marker of highly activated neurons. These effects could not be explained by chronic stress or drug withdrawal and instead appeared to require a recall experience associated with prior drug SA. In addition to showing the differential distribution of RyR2/RyR2P and affirming the general role of vmPFC in reward learning, this study provides information on the propensity of addictive drugs to redistribute RyR2P ion channels in a neuronal population engaged in drug-seeking. Hence, focusing on the early impact of addictive drugs on RyR2 function may serve as a promising approach to finding a treatment for substance use disorders.
• Gabriel, K. A., & Streicher, J. M. (2023). HSP90 inhibition in the mouse spinal cord enhances opioid signaling by suppressing an AMPK-mediated negative feedback loop. Science signaling, 16(780), eade2438.
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  Opioids and other agonists of the μ-opioid receptor are effective at managing acute pain, but their chronic use can lead to tolerance that limits their efficacy. We previously reported that inhibiting the chaperone protein HSP90 in the spinal cords of mice promotes the antinociceptive effects of opioids in a manner that involved increased activation of the kinase ERK. Here, we found that the underlying mechanism involves the relief of a negative feedback loop mediated by the kinase AMPK. Intrathecal treatment of male and female mice with the HSP90 inhibitor 17-AAG decreased the abundance of the β1 subunit of AMPK in the spinal cord. The antinociceptive effects of 17-AAG with morphine were suppressed by intrathecal administration of AMPK activators and enhanced by an AMPK inhibitor. Opioid treatment increased the abundance of phosphorylated AMPK in the dorsal horn of the spinal cord, where it colocalized with a neuronal marker and the neuropeptide CGRP. Knocking down AMPK in CGRP-positive neurons enhanced the antinociceptive effects of morphine and demonstrated that AMPK mediated the signal transduction between HSP90 inhibition and ERK activation. These data suggest that AMPK mediates an opioid-induced negative feedback loop in CGRP neurons of the spinal cord and that this loop can be disabled by HSP90 inhibition to enhance the efficacy of opioids.
• Khanna, R., Streicher, J., Moutal, A., Duran, P., Loya‐López, S., Ran, D., Tang, C., Calderon‐Rivera, A., Gomez, K., Stratton, H. J., Huang, S., Xu, Y., Wijeratne, E. M., Perez‐Miller, S., Shan, Z., Cai, S., Gabrielsen, A. T., Dorame, A., Masterson, K. A., , Alsbiei, O., et al. (2023). The natural product argentatin C attenuates postoperative pain via inhibition of voltage‐gated sodium and T‐type voltage‐gated calcium channels. British Journal of Pharmacology. doi:10.1111/bph.15974
• Ranaldi, R., Timken, P., Parasram, D., Ali, T., Zhang, S., Moukha-Chafiq, O., Augelli-Szafran, C., & Streicher, J. M. (2023). The D3 receptor antagonist SR 21502 reduces cue-induced reinstatement of methamphetamine-seeking in rats. Neuroscience letters, 806, 137237.
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  There is as of yet no FDA-approved medication for methamphetamine use disorder. Although dopamine D3 receptor antagonists have been shown to be useful in reducing methamphetamine seeking in animal models their translation to the clinic has been hindered because currently tested compounds can produce dangerously high blood pressure. Thus, it is important to continue to explore other classes of D3 antagonists. We report here the effects of SR 21502, a selective D3 receptor antagonist, on cue-induced reinstatement (i.e., relapse) of methamphetamine-seeking in rats. In Experiment 1, rats were trained to self-administer methamphetamine under a fixed ratio schedule of reinforcement followed by extinction of the response. Then, animals were tested with one of several doses of SR 21502 on cue-induced reinstatement of responding. SR 21502 significantly reduced cue-induced reinstatement of methamphetamine-seeking. In Experiment 2, animals were trained to lever press for food under a PR schedule and tested with the lowest dose of SR 21502 that caused a significant reduction in Experiment 1. These animals responded on average 8 times more than the vehicle-treated rats in Experiment 1, eliminating the possibility that SR 21502-treated rats in Experiment 1 responded less because they were incapacitated. In summary, these data suggest that SR 21502 may selectively inhibit methamphetamine-seeking and may constitute a promising pharmacotherapeutic agent for methamphetamine or other drug use disorders.
• Schwarz, A. M., Keresztes, A., Bui, T., Hecksel, R. J., Peña, A., Lent, B., Gao, Z. G., Gamez-Rivera, M., Seekins, C. A., Chou, K., Appel, T. L., Jacobson, K. A., Al-Obeidi, F. A., & Streicher, J. M. (2023). Terpenes from Induce Antinociception in Mouse Chronic Neuropathic Pain via Activation of Spinal Cord Adenosine A Receptors. bioRxiv : the preprint server for biology.
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  Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including . A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, β-pinene, α-humulene, and β-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the efficacy and mechanism of these terpenes in relieving chronic pain. We first tested for antinociceptive efficacy by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal efficacy to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs. either alone. We then used the adenosine A receptor (AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the AR to identify this receptor as the mechanism for terpene antinociception in CIPN. cAMP and binding studies and modeling studies further suggested that the terpenes act as AR agonists. Together these studies identify terpenes as potential therapeutics for chronic neuropathic pain, and identify a receptor mechanism in the spinal cord for this activity.
• Stefanucci, A., Minosi, P., Pieretti, S., Tanguturi, P., Molnar, G., Scioli, G., Marinaccio, L., Della Valle, A., Streicher, J. M., & Mollica, A. (2023). Design of Analgesic Trivalent Peptides with Low Withdrawal Symptoms: Probing the Antinociceptive Profile of Novel Linear and Cyclic Peptides as Opioid Pan Ligands. ACS chemical neuroscience, 14(3), 506-515.
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  The discovery of efficacious and safe analgesics with reduced side effects is the foremost challenge in the pain field. In this work, we report the and evaluation of linear and cyclic analogues of biphalin with the aim to complete the series of structural modifications previously applied in the development of opioid peptides incorporating a xylene bridge. Replacement of Tyr by Dmt (2,5-dimethyltyrosine) in the linear biphalin analogue and cyclic analogue resulted in two new compounds (namely, and ) endowed with improved KOR/MOR/DOR binding affinity. Both compounds showed a strong antinociceptive profile in models of nociception, allodynia, and hyperalgesia via the tail flick, hot plate, and formalin tests after intracerebroventricular and subcutaneous administration. One of these ligands, , was also tested in tolerance and dependence studies, exhibiting very little withdrawal symptoms.
• Streicher, J. M., Stefanucci, A., Minosi, P., Pieretti, S., Tanguturi, P., Molnar, G., Scioli, G., Marinaccio, L., Della Valle, A., & Mollica, A. (2023). Design of Analgesic Trivalent Peptides with Low Withdrawal Symptoms: Probing the Antinociceptive Profile of Novel Linear and Cyclic Peptides as Opioid Pan Ligands. ACS Chemical Neuroscience. doi:10.1021/acschemneuro.3c00005
• Szabó, L. Z., Tanguturi, P., Goodman, H. J., Sprőber, S., Liu, C., Al-Obeidi, F., Bartlett, M. J., Falk, T., Kumirov, V. K., Heien, M. L., Streicher, J. M., Polt, R., Szabó, L. Z., Tanguturi, P., Goodman, H. J., Sprőber, S., Liu, C., Al-Obeidi, F., Bartlett, M. J., , Falk, T., et al. (2023). Structure-Based Design of Glycosylated Oxytocin Analogues with Improved Selectivity and Antinociceptive Activity. ACS medicinal chemistry letters, 14(2), 163-170.
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  Acute and chronic pain is often treated with opioids despite the negative side effects of constipation, physical dependence, respiratory depression, and overdose. The misuse of opioid analgesics has given rise to the opioid crisis/epidemic, and alternate nonaddictive analgesics are urgently needed. Oxytocin, a pituitary hormone, is an alternative to the small molecule treatments available and has been used as an analgesic as well as for the treatment and prevention of opioid use disorder (OUD). Clinical implementation is limited by its poor pharmacokinetic profile, a result of the labile disulfide bond between two cysteine residues in the native sequence. Stable brain penetrant oxytocin analogues have been synthesized by replacement of the disulfide bond with a stable lactam and glycosidation of the C-terminus. These analogues show exquisite selectivity for the oxytocin receptor and potent antinociception in mice following peripheral (i.v.) administration, supporting further study of their clinical potential.
• Tanguturi, P., & Streicher, J. M. (2023). The role of opioid receptors in modulating Alzheimer's Disease. Frontiers in pharmacology, 14, 1056402.
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  Alzheimer's disease (AD) is a complex neurological disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles. Long term investigation of AD pathogenesis suggests that β-site amyloid precursor protein [APP] cleaving enzyme 1 (BACE1) and γ-secretase enzymes promote the amyloidogenic pathway and produce toxic Aβ peptides that are predisposed to aggregate in the brain. Hence, the targeted inhibition of BACE1/γ-secretase expression and function is a promising approach for AD therapy. Several reports have suggested that the opioid family of G-protein coupled receptors modulate the etiology of AD progression. It has also been found that changes in the signaling pathways of opioid receptors increased the expression of BACE1 and γ-secretase, and is strongly correlated with abnormal production of Aβ and pathogenesis of AD. Thus, the opioid receptor family is a promising candidate for targeted drug development to treat AD. In this review, we outline the involvement and mechanisms of opioid receptor signaling modulation in Alzheimer's Disease progression.
• Apostol, C. R., Bernard, K., Tanguturi, P., Molnar, G., Bartlett, M. J., Szabò, L., Liu, C., Ortiz, J. B., Saber, M., Giordano, K. R., Green, T. R., Melvin, J., Morrison, H. W., Madhavan, L., Rowe, R. K., Streicher, J. M., Heien, M. L., Falk, T., & Polt, R. (2022). Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism. Frontiers in drug discovery, 1.
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  There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP's poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues ( and ) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson's disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.
• Duran, P., Loya-López, S., Ran, D., Tang, C., Calderon-Rivera, A., Gomez, K., Stratton, H. J., Huang, S., Xu, Y. M., Wijeratne, E. M., Perez-Miller, S., Shan, Z., Cai, S., Gabrielsen, A. T., Dorame, A., Masterson, K. A., Alsbiei, O., Madura, C. L., Luo, G., , Moutal, A., et al. (2022). The natural product Argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels. British journal of pharmacology.
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  Postoperative pain occurs in as many as 70% of the over 230 million surgeries performed annually worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is of utmost importance to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel and biologically active compounds for development of safe analgesics. Hence, in this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels which have important roles in nociceptive sensory processing.
• Fullerton, E. F., Karom, M. C., Streicher, J. M., Young, L. J., & Murphy, A. Z. (2022). Age-Induced Changes in μ-Opioid Receptor Signaling in the Midbrain Periaqueductal Gray of Male and Female Rats. The Journal of neuroscience : the official journal of the Society for Neuroscience, 42(32), 6232-42.
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  Opioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. The present study investigated the impact of advanced age and biological sex on opioid signaling in the ventrolateral periaqueductal gray (vlPAG) in the presence of chronic inflammatory pain. Assays measuring μ-opioid receptor (MOR) radioligand binding, GTPγS binding, receptor phosphorylation, cAMP inhibition, and regulator of G-protein signaling (RGS) protein expression were performed on vlPAG tissue from adult (2-3 months) and aged (16-18 months) male and female rats. Persistent inflammatory pain was induced by intraplantar injection of complete Freund's adjuvant (CFA). Adult males exhibited the highest MOR binding potential (BP) and highest G-protein activation (activation efficiency ratio) in comparison to aged males and females (adult and aged). No impact of advanced age or sex on MOR phosphorylation state was observed. DAMGO-induced cAMP inhibition was highest in the vlPAG of adult males compared with aged males and females (adult and aged). vlPAG levels of RGS4 and RGS9-2, critical for terminating G-protein signaling, were assessed using RNAscope. Adult rats (both males and females) exhibited lower levels of vlPAG RGS4 and RGS9-2 mRNA expression compared with aged males and females. The observed age-related reductions in vlPAG MOR BP, G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in RGS4 and RGS9-2 vlPAG expression, provide potential mechanisms whereby the potency of opioids is decreased in the aged population.Opioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. In the present study, we observed age-related reductions in ventrolateral periaqueductal gray (vlPAG) μ-opioid receptor (MOR) binding potential (BP), G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in regulator of G-protein signaling (RGS)4 and RGS9-2 vlPAG expression, providing potential mechanisms whereby the potency of opioids is decreased in the aged population. These coordinated decreases in opioid receptor signaling may explain the previously reported reduced potency of opioids to produce pain relief in females and aged rats.
• Keresztes, A., Olson, K., Nguyen, P., Lopez-Pier, M. A., Hecksel, R., Barker, N. K., Liu, Z., Hruby, V., Konhilas, J., Langlais, P. R., & Streicher, J. M. (2022). Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling. Pain, 163(1), 146-158.
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  The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.
• Milnes, T. M., Vanderah, T. W., Streicher, J. M., Langlais, P. R., Cottier, K. E., Almuslim, M., Palomino, S. M., Vivek, A., & Wahl, J. R. (2022). Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome. Biology of sex differences.
• Palomino, S. M., Levine, A. A., Wahl, J., Liktor-Busa, E., Streicher, J. M., & Largent-Milnes, T. M. (2022). Inhibition of HSP90 Preserves Blood-Brain Barrier Integrity after Cortical Spreading Depression. Pharmaceutics, 14(8).
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  Cortical spreading depression (CSD) is a pathophysiological mechanism underlying headache disorders, including migraine. Blood-brain barrier (BBB) permeability is increased during CSD. Recent papers have suggested that heat shock proteins (HSP) contribute to the integrity of the blood-brain barrier. In this study, the possible role of HSP90 in CSD-associated blood-brain barrier leak at the endothelial cell was investigated using an in vitro model, for the blood-endothelial barrier (BEB), and an in vivo model with an intact BBB. We measured barrier integrity using trans endothelial electric resistance (TEER) across a monolayer of rodent brain endothelial cells (bEnd.3), a sucrose uptake assay, and in situ brain perfusion using female Sprague Dawley rats. CSD was induced by application of 60 mM KCl for 5 min in in vitro experiments or cortical injection of KCl (1 M, 0.5 µL) through a dural cannula in vivo. HSP90 was selectively blocked by 17-AAG. Our data showed that preincubation with 17-AAG (1 µM) prevented the reduction of TEER values caused by the KCl pulse on the monolayer of bEnd.3 cells. The elevated uptake of C-sucrose across the same endothelial monolayer induced by the KCl pulse was significantly reduced after preincubation with HSP90 inhibitor. Pre-exposure to 17-AAG significantly mitigated the transient BBB leak after CSD induced by cortical KCl injection as determined by in situ brain perfusion in female rats. Our results demonstrated that inhibition of HSP90 with the selective agent 17-AAG reduced CSD-associated BEB/BBB paracellular leak. Overall, this novel observation supports HSP90 inhibition mitigates KCl-induced BBB permeability and suggests the development of new therapeutic approaches targeting HSP90 in headache disorders.
• Polt, R. L., Falk, T., Heien, M. L., Streicher, J. M., Rowe, R. K., Madhavan, L., Morrison, H. W., Melvin, J., Green, T. R., Giordano, K. R., Saber, M., Ortiz, J. B., Liu, C., Szabò, L., Bartlett, M. J., Molnar, G., Tanguturi, P., Bernard, K., & Apostol, C. R. (2022). Design and Synthesis of Novel Brain Penetrant Glycopeptide Analogues of PACAP with Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism.. Frontiers in drug discovery, Vol. 1:(818003). doi:https://doi.org/10.3389/fddsv.2021.818003
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  There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP’s poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson’s disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.
• Stefanucci, A., Della Valle, A., Scioli, G., Marinaccio, L., Pieretti, S., Minosi, P., Szucs, E., Benyhe, S., Masci, D., Tanguturi, P., Chou, K., Barlow, D., Houseknecht, K., Streicher, J. M., & Mollica, A. (2022). Discovery of κ Opioid Receptor (KOR)-Selective d-Tetrapeptides with Improved Antinociceptive Effect after Peripheral Administration. ACS medicinal chemistry letters, 13(11), 1707-1714.
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  Peripherally active tetrapeptides as selective κ opioid receptor (KOR) agonists have been prepared in good overall yields and high purity following solid-phase peptide synthesis via Fmoc protection strategy. Structural modifications at the first and second position of the FF(d-Nle)R-NH () were contemplated with aromatic side chains containing d-amino acids, such as (d)-F-Phe, (d)-F-Phe, (d)-F-Phe, which led to highly selective and efficacious KOR agonists endowed with strong antinociceptive activity following intravenous (i.v.) and subcutaneous (s.c.) administration in the tail flick and formalin tests. These results suggest potential clinical applications in the treatment of neuropathic and inflammatory pain.
• Streicher, J. M., Langlais, P. R., Konhilas, J. P., Hruby, V., Liu, Z., Barker, N. K., Hecksel, R., Lopez-Pier, M. A., Nguyen, P., Olson, K., & Keresztes, A. (2022). Antagonism of the Mu-Delta Opioid Receptor Heterodimer Enhances Opioid Anti-Nociception by Activating Src and CaMKII Signaling. Pain.
• Streicher, J. M., Varga, B. R., & Majumdar, S. (2022). Strategies towards safer opioid analgesics—A review of old and upcoming targets. British Journal of Pharmacology. doi:10.1111/bph.15760
• Tanguturi, P., Pathak, V., Zhang, S., Moukha-Chafiq, O., Augelli-Szafran, C. E., & Streicher, J. M. (2022). Correction: Tanguturi et al. Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists. 2021, , 6693. Molecules (Basel, Switzerland), 27(6).
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  There was an error in the original publication [...].
• Wahl, J. R., Vivek, A., Palomino, S. M., Almuslim, M., Cottier, K. E., Langlais, P. R., Streicher, J. M., Vanderah, T. W., Liktor-Busa, E., & Largent-Milnes, T. M. (2022). Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome. Pharmaceutics, 14(7).
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  Pathologies of the blood-brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood-brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic "checkpoint" of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation.
• Apostol, C. R., Tanguturi, P., Szabò, L. Z., Varela, D., Gilmartin, T., Streicher, J. M., & Polt, R. (2021). Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP. Molecules (Basel, Switzerland), 26(16).
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  The search for efficacious treatment of neurodegenerative and progressive neuroinflammatory diseases continues, as current therapies are unable to halt or reverse disease progression. PACAP represents one potential therapeutic that provides neuroprotection effects on neurons, and also modulates inflammatory responses and circulation within the brain. However, PACAP is a relatively long peptide hormone that is not trivial to synthesize. Based on previous observations that the shortened isoform PACAP is capable of inducing neuroprotection in vitro, we were inspired to synthesize shortened glycopeptide analogues of PACAP. Herein, we report the synthesis and in vitro characterization of glycosylated PACAP analogues that interact strongly with the PAC1 and VPAC1 receptors, while showing reduced activity at the VPAC2 receptor.
• LaVigne, J. E., Hecksel, R., Keresztes, A., & Streicher, J. M. (2021). Cannabis sativa terpenes are cannabimimetic and selectively enhance cannabinoid activity. Scientific reports, 11(1), 8232.
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  Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an "entourage effect" whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral effects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our findings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage effect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.
• Lee, Y. S., Remesic, M., Ramos-Colon, C., Wu, Z., LaVigne, J., Molnar, G., Tymecka, D., Misicka, A., Streicher, J. M., Hruby, V. J., & Porreca, F. (2021). Multifunctional Enkephalin Analogs with a New Biological Profile: MOR/DOR Agonism and KOR Antagonism. Biomedicines, 9(6).
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  In our previous studies, we developed a series of mixed MOR/DOR agonists that are enkephalin-like tetrapeptide analogs with an N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety at the C-terminus. Further SAR study on the analogs, initiated by the findings from off-target screening, resulted in the discovery of (, Dmt-DNle-Gly-Phe(-Cl)-Ppp), a multifunctional ligand with MOR/DOR agonist and KOR antagonist activity (GTPγS assay: IC = 52 nM, I = 122% cf. IC = 59 nM, I = 100% for naloxone) with nanomolar range of binding affinity ( = 1.3 nM cf. = 2.4 nM for salvinorin A). Based on its unique biological profile, is considered to possess high therapeutic potential for the treatment of chronic pain by modulating pathological KOR activation while retaining analgesic efficacy attributed to its MOR/DOR agonist activity.
• Liktor-Busa, E., Keresztes, A., LaVigne, J., Streicher, J. M., & Largent-Milnes, T. M. (2021). Analgesic Potential of Terpenes Derived from. Pharmacological reviews, 73(4), 98-126.
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  Pain prevalence among adults in the United States has increased 25% over the past two decades, resulting in high health-care costs and impacts to patient quality of life. In the last 30 years, our understanding of pain circuits and (intra)cellular mechanisms has grown exponentially, but this understanding has not yet resulted in improved therapies. Options for pain management are limited. Many analgesics have poor efficacy and are accompanied by severe side effects such as addiction, resulting in a devastating opioid abuse and overdose epidemic. These problems have encouraged scientists to identify novel molecular targets and develop alternative pain therapeutics. Increasing preclinical and clinical evidence suggests that cannabis has several beneficial pharmacological activities, including pain relief. contains more than 500 chemical compounds, with two principle phytocannabinoids, Δ-tetrahydrocannabinol (Δ-THC) and cannabidiol (CBD). Beyond phytocannabinoids, more than 150 terpenes have been identified in different cannabis chemovars. Although the predominant cannabinoids, Δ-THC and CBD, are thought to be the primary medicinal compounds, terpenes including the monoterpenes -myrcene, -pinene, limonene, and linalool, as well as the sesquiterpenes -caryophyllene and -humulene may contribute to many pharmacological properties of cannabis, including anti-inflammatory and antinociceptive effects. The aim of this review is to summarize our current knowledge about terpene compounds in cannabis and to analyze the available scientific evidence for a role of cannabis-derived terpenes in modern pain management. SIGNIFICANCE STATEMENT: Decades of research have improved our knowledge of cannabis polypharmacy and contributing phytochemicals, including terpenes. Reform of the legal status for cannabis possession and increased availability (medicinal and recreational) have resulted in cannabis use to combat the increasing prevalence of pain and may help to address the opioid crisis. Better understanding of the pharmacological effects of cannabis and its active components, including terpenes, may assist in identifying new therapeutic approaches and optimizing the use of cannabis and/or terpenes as analgesic agents.
• Morgan, M. M., Peecher, D. L., & Streicher, J. M. (2021). Use of home cage wheel running to assess the behavioural effects of administering a mu/delta opioid receptor heterodimer antagonist for spontaneous morphine withdrawal in the rat. Behavioural brain research, 397, 112953.
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  Opioid abuse is a major health problem. The objective of the present study was to evaluate the potentially disruptive side effects and therapeutic potential of a novel antagonist (D24M) of the mu-/delta-opioid receptor (MOR/DOR) heterodimer in male rats. Administration of high doses of D24M (1 & 10 nmol) into the lateral ventricle did not disrupt home cage wheel running. Repeated twice daily administration of increasing doses of morphine (5-20 mg/kg) over 5 days depressed wheel running and induced antinociceptive tolerance measured with the hot plate test. Administration of D24M had no effect on morphine tolerance, but tended to prolong morphine antinociception in non-tolerant rats. Spontaneous morphine withdrawal was evident as a decrease in body weight, a reduction in wheel running and an increase in sleep during the normally active dark phase of the circadian cycle, and an increase in wheel running and wakefulness in the normally inactive light phase. Administration of D24M during the dark phase on the third day of withdrawal had no effect on wheel running. These data provide additional evidence for the clinical relevance of home cage wheel running as a method to assess spontaneous opioid withdrawal in rats. These data also demonstrate that blocking the MOR/DOR heterodimer does not produce disruptive side effects or block the antinociceptive effects of morphine. Although administration of D24M had no effect on morphine withdrawal, additional studies are needed to evaluate withdrawal to continuous morphine administration and other opioids in rats with persistent pain.
• Streicher, J. M., Langlais, P. R., Konhilas, J. P., Hruby, V., Liu, Z., Barker, N. K., Hecksel, R., Lopez-Pier, M. A., Nguyen, P., Olson, K., & Keresztes, A. (2021). Antagonism of the Mu-Delta Opioid Receptor Heterodimer Enhances Opioid Anti-Nociception by Activating Src and CaMKII Signaling. Pain.
• Streicher, J. M., Langlais, P., & Campbell, C. (2021). Inhibition of Heat shock protein 90 in the Spinal Cord Enhances Opioid Anti-Nociception by Disabling an Inhibitory GABA Circuit. The Journal of Pain, 22(5), 597-598. doi:10.1016/j.jpain.2021.03.080
• Streicher, J. M., Lavigne, J. E., Keresztes, A., & Hecksel, R. (2021). Cannabis sativa terpenes are cannabimimetic and selectively enhance cannabinoid activity.. Scientific reports, 11(1), 8232. doi:10.1038/s41598-021-87740-8
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  Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an "entourage effect" whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral effects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our findings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage effect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.
• Tanguturi, P., Pathak, V., Zhang, S., Moukha-Chafiq, O., Augelli-Szafran, C. E., & Streicher, J. M. (2021). Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists. Molecules (Basel, Switzerland), 26(21).
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  The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, have been developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45127/SRI-45128 as inverse agonists. Then, these compounds were evaluated in vitro for their binding affinity by radioligand binding and functional activity by S-GTPγS coupling and cAMP accumulation in cells expressing the human DOR. All three compounds demonstrated high binding affinity and selectivity at the DOR, and all three displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45127/SRI-45128). Together, these results demonstrate that we have designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.
• Varga, B. R., Streicher, J. M., & Majumdar, S. (2021). Strategies towards safer opioid analgesics-A review of old and upcoming targets. British journal of pharmacology.
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  Opioids continue to be of use for the treatment of pain. Most clinically used analgesics target the μ opioid receptor whose activation results in adverse effects like respiratory depression, addiction and abuse liability. Various approaches have been used by the field to separate receptor-mediated analgesic actions from adverse effects. These include biased agonism, opioids targeting multiple receptors, allosteric modulators, heteromers and splice variants of the μ receptor. This review will focus on the current status of the field and some upcoming targets of interest that may lead to a safer next generation of analgesics.
• Wu, Z., Tymecka, D., Streicher, J. M., Remesic, M., Ramos-colon, C., Porreca, F., Molnar, G., Misicka, A., Lee, Y. S., Lavigne, J., & Hruby, V. J. (2021). Multifunctional Enkephalin Analogs with a New Biological Profile: MOR/DOR Agonism and KOR Antagonism.. Biomedicines, 9(6). doi:10.3390/biomedicines9060625
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  In our previous studies, we developed a series of mixed MOR/DOR agonists that are enkephalin-like tetrapeptide analogs with an N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety at the C-terminus. Further SAR study on the analogs, initiated by the findings from off-target screening, resulted in the discovery of LYS744 (6, Dmt-DNle-Gly-Phe(p-Cl)-Ppp), a multifunctional ligand with MOR/DOR agonist and KOR antagonist activity (GTPγS assay: IC50 = 52 nM, Imax = 122% cf. IC50 = 59 nM, Imax = 100% for naloxone) with nanomolar range of binding affinity (Ki = 1.3 nM cf. Ki = 2.4 nM for salvinorin A). Based on its unique biological profile, 6 is considered to possess high therapeutic potential for the treatment of chronic pain by modulating pathological KOR activation while retaining analgesic efficacy attributed to its MOR/DOR agonist activity.
• Cai, S., Tuohy, P., Ma, C., Kitamura, N., Gomez, K., Zhou, Y., Ran, D., Bellampalli, S. S., Yu, J., Luo, S., Dorame, A., Yen Ngan Pham, N., Molnar, G., Streicher, J. M., Patek, M., Perez-Miller, S., Moutal, A., Wang, J., & Khanna, R. (2020). A modulator of the low-voltage-activated T-type calcium channel that reverses HIV glycoprotein 120-, paclitaxel-, and spinal nerve ligation-induced peripheral neuropathies. Pain, 161(11), 2551-2570.
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  The voltage-gated calcium channels CaV3.1-3.3 constitute the T-type subfamily, whose dysfunctions are associated with epilepsy, psychiatric disorders, and chronic pain. The unique properties of low-voltage-activation, faster inactivation, and slower deactivation of these channels support their role in modulation of cellular excitability and low-threshold firing. Thus, selective T-type calcium channel antagonists are highly sought after. Here, we explored Ugi-azide multicomponent reaction products to identify compounds targeting T-type calcium channel. Of the 46 compounds tested, an analog of benzimidazolonepiperidine-5bk (1-{1-[(R)-{1-[(1S)-1-phenylethyl]-1H-1,2,3,4-tetrazol-5-yl}(thiophen-3-yl)methyl]piperidin-4-yl}-2,3-dihydro-1H-1,3-benzodiazol-2-one) modulated depolarization-induced calcium influx in rat sensory neurons. Modulation of T-type calcium channels by 5bk was further confirmed in whole-cell patch clamp assays in dorsal root ganglion (DRG) neurons, where pharmacological isolation of T-type currents led to a time- and concentration-dependent regulation with a low micromolar IC50. Lack of an acute effect of 5bk argues against a direct action on T-type channels. Genetic knockdown revealed CaV3.2 to be the isoform preferentially modulated by 5bk. High voltage-gated calcium, as well as tetrodotoxin-sensitive and -resistant sodium, channels were unaffected by 5bk. 5bk inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, 5bk did not bind human mu, delta, or kappa opioid receptors. 5bk reversed mechanical allodynia in rat models of HIV-associated neuropathy, chemotherapy-induced peripheral neuropathy, and spinal nerve ligation-induced neuropathy, without effects on locomotion or anxiety. Thus, 5bk represents a novel T-type modulator that could be used to develop nonaddictive pain therapeutics.
• Duron, D. I., Hanak, F., & Streicher, J. M. (2020). Daily intermittent fasting in mice enhances morphine-induced antinociception while mitigating reward, tolerance, and constipation. Pain, 161(10), 2353-2363.
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  The opioid epidemic has plagued the United States with high levels of abuse and poor quality of life for chronic pain patients requiring continuous use of opioids. New drug discovery efforts have been implemented to mitigate this epidemic; however, new medications are still limited by low efficacy and/or high side effect and abuse potential. Intermittent fasting (IF) has recently been shown to improve a variety of pathological states, including stroke and neuroinflammation. Numerous animal and human studies have shown the benefits of IF in these disease states, but not in pain and opioid treatment. We thus subjected male and female CD-1 mice to 18-hour fasting intervals followed by 6-hour feed periods with standard chow for 1 week. Mice that underwent this diet displayed an enhanced antinociceptive response to morphine both in efficacy and duration using thermal tail-flick and postoperative paw incision pain models. While showing enhanced antinociception, IF mice also demonstrated no morphine reward and reduced tolerance and constipation. Seeking a mechanism for these improvements, we found that the mu-opioid receptor showed enhanced efficacy and reduced tolerance in the spinal cord and periaqueductal gray, respectively, from IF mice using a S-GTPγS coupling assay. These improvements in receptor function were not due to changes in mu-opioid receptor protein expression. These data suggest that a daily IF diet may improve the therapeutic index of acute and chronic opioid therapies for pain patients in the clinic, providing a novel tool to improve patient therapy and reduce potential abuse.
• Duron, D. I., Lei, W., Barker, N. K., Stine, C., Mishra, S., Blagg, B. S., Langlais, P. R., & Streicher, J. M. (2020). Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway. Science signaling, 13(630).
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  Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating μ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.
• Faouzi, A., Uprety, R., Gomes, I., Massaly, N., Keresztes, A. I., Le Rouzic, V., Gupta, A., Zhang, T., Yoon, H. J., Ansonoff, M., Allaoa, A., Pan, Y. X., Pintar, J., Morón, J. A., Streicher, J. M., Devi, L. A., & Majumdar, S. (2020). Synthesis and Pharmacology of a Novel μ-δ Opioid Receptor Heteromer-Selective Agonist Based on the Carfentanyl Template. Journal of medicinal chemistry, 63(22), 13618-13637.
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  In this work, we studied a series of carfentanyl amide-based opioid derivatives targeting the mu opioid receptor (μOR) and the delta opioid receptor (δOR) heteromer as a credible novel target in pain management therapy. We identified a lead compound named that exhibits high G-protein activity at μ-δ heteromers compared to the homomeric δOR or μOR and low β-arrestin2 recruitment activity at all three. Furthermore, exhibits distinct signaling profile, as compared to the previously identified agonist targeting μ-δ heteromers, CYM51010. Pharmacological characterization of supports the utility of this compound as a molecule that could be developed as an antinociceptive agent similar to morphine in rodents. characterization reveals that maintains untoward side effects such as respiratory depression and reward behavior; together, these results suggest that optimization of is necessary for the development of therapeutics that suppress the classical side effects associated with conventional clinical opioids.
• LaVigne, J., Keresztes, A., Chiem, D., & Streicher, J. M. (2020). The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor. Pharmacological reports : PR, 72(2), 465-471.
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  Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles.
• Langlais, P., Streicher, J. M., Langlais, P. R., Gabriel, K., Duron, D. I., & Barker, N. (2020). Does DUSP15 promote activation of ERK MAPK signaling after Hsp90 inhibition in the spinal cord to promote opioid anti‐nociception?. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05649
• Langlais, P., Streicher, J. M., Langlais, P. R., Keresztes, A., Hurtado, K., & Barker, N. K. (2020). Does the Mu‐Delta Opioid Receptor Heterodimer Repress Akt Kinase to Reduce Opioid Anti‐Nociception?. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.06535
• Langlais, P., Streicher, J. M., Sieffert, M. M., Langlais, P. R., Keresztes, A., & Barker, N. K. (2020). Investigation of the Signaling Mechanisms of the Mu‐Delta Opioid Receptor Heterodimer. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05218
• Largent-Milnes, T. M., Canals, M., & Streicher, J. M. (2020). Editorial: Novel Molecular Targets for the Treatment of Pain. Frontiers in molecular neuroscience, 13, 625714.
• Mishra, S. J., Streicher, J. M., Mishra, S., Duron, D. I., Blagg, B. S., & Bejarano, P. (2020). Identification of the Hsp90 Isoforms and Co-Chaperones that Repress Opioid Anti-Nociception in the Spinal Cord. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05462
• Stefanucci, A., Dimmito, M. P., Molnar, G., Streicher, J. M., Novellino, E., Zengin, G., & Mollica, A. (2020). Developing Cyclic Opioid Analogues: Fluorescently Labeled Bioconjugates of Biphalin. ACS medicinal chemistry letters, 11(5), 720-726.
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  The development of bioconjugates is of pivotal importance in medicinal chemistry due to their potential applications as therapeutic agents to improve the targeting of specific diseases, decrease toxicity, or control drug release. In this work we achieved the synthesis and characterization of three novel opioid peptides fluorescently labeled, analogues of cyclic biphalin derivatives, namely , , and . Among them, compound , containing a dansyl-maleimide motif, exhibited an excellent binding affinity and functional potency for the δ-opioid receptor (DOR). also demonstrated a strong fluorescence emission spectrum ranging from 300 to 700 nm. These features could be highly desirable for medical and biological applications needed for targeting the DOR, including imaging, and as a lead for the design of fluorescent probes.
• Stine, C., Coleman, D. L., Flohrschutz, A. T., Thompson, A. L., Mishra, S., Blagg, B. S., Largent-Milnes, T. M., Lei, W., & Streicher, J. M. (2020). Heat shock protein 90 inhibitors block the antinociceptive effects of opioids in mouse chemotherapy-induced neuropathy and cancer bone pain models. Pain, 161(8), 1798-1807.
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  Heat shock protein 90 (Hsp90) is a ubiquitous signal transduction regulator, and Hsp90 inhibitors are in clinical development as cancer therapeutics. However, there have been very few studies on the impact of Hsp90 inhibitors on pain or analgesia, a serious concern for cancer patients. We previously found that Hsp90 inhibitors injected into the brain block opioid-induced antinociception in tail flick, paw incision, and HIV neuropathy pain. This study extended from that initial work to test the cancer-related clinical impact of Hsp90 inhibitors on opioid antinociception in cancer-induced bone pain in female BALB/c mice and chemotherapy-induced peripheral neuropathy in male and female CD-1 mice. Mice were treated with Hsp90 inhibitors (17-AAG, KU-32) by the intracerebroventricular, intrathecal, or intraperitoneal routes, and after 24 hours, pain behaviors were evaluated after analgesic drug treatment. Heat shock protein 90 inhibition in the brain or systemically completely blocked morphine and oxymorphone antinociception in chemotherapy-induced peripheral neuropathy; this effect was partly mediated by decreased ERK and JNK MAPK activation and by increased protein translation, was not altered by chronic treatment, and Hsp90 inhibition had no effect on gabapentin antinociception. We also found that the Hsp90 isoform Hsp90α and the cochaperone Cdc37 were responsible for the observed changes in opioid antinociception. By contrast, Hsp90 inhibition in the spinal cord or systemically partially reduced opioid antinociception in cancer-induced bone pain. These results demonstrate that Hsp90 inhibitors block opioid antinociception in cancer-related pain, suggesting that Hsp90 inhibitors for cancer therapy could decrease opioid treatment efficacy.
• Streicher, J. M., & Duron, D. I. (2020). Daily Intermittent Fasting in Mice Enhances Morphine Induced Anti-Nociception while Mitigating Multiple Side Effects. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.04700
• Streicher, J. M., Langlais, P. R., Blagg, B. S., Mishra, S., Stine, C., Barker, N. K., Lei, W., & Duron, D. I. (2020). Inhibition of spinal cord Hsp90 enhances morphine anti-nociception by activating an ERK/RSK pathway.. Science Signaling.
• Streicher, J. M., Lavigne, J., & Hecksel, R. (2020). In Defense of the “Entourage Effect”: Terpenes Found in Cannabis sativa Activate the Cannabinoid Receptor 1 In Vitro. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.04020
• Streicher, J. M., Stefanucci, A., Pieretti, S., Novellino, E., Mollica, A., Macedonio, G., Lei, W., Houseknecht, K. L., Dimmito, M. P., Ciarlo, L., & Barlow, D. (2020). Potent, Efficacious, and Stable Cyclic Opioid Peptides with Long Lasting Antinociceptive Effect after Peripheral Administration.. Journal of medicinal chemistry, 63(5), 2673-2687. doi:10.1021/acs.jmedchem.9b01963
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  Four novel fluorinated cyclic analogues of biphalin with excellent to modest binding affinity for μ-, δ-, and κ-receptors were synthesized. The cyclic peptides have a combination of piperazine or hydrazine linker with or without a xylene bridge. Among the ligands, MACE3 demonstrated a better activity than biphalin after intravenous administration, and its corresponding analogue incorporating the hydrazine linker (MACE2) was able to induce longer lasting analgesia following subcutaneous administration. An analogue of MACE2 containing 2,6-dimethyl-l-tyrosine (MACE4) showed the best potency and in vivo antinociceptive activity of this series.
• Vekariya, R. H., Lei, W., Ray, A., Saini, S. K., Zhang, S., Molnar, G., Barlow, D., Karlage, K. L., Bilsky, E. J., Houseknecht, K. L., Largent-Milnes, T. M., Streicher, J. M., & Ananthan, S. (2020). Synthesis and Structure-Activity Relationships of 5'-Aryl-14-alkoxypyridomorphinans: Identification of a μ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects. Journal of medicinal chemistry, 63(14), 7663-7694.
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  We previously identified a pyridomorphinan (, SRI-22138) possessing a 4-chlorophenyl substituent at the 5'-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed μ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5'- and 14-positions of this molecule gave insights into the structure-activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.
• Vekariya, R. H., Streicher, J. M., Lei, W., & Ananthan, S. (2020). A Novel Mu-Delta Opioid Agonist Demonstrates Enhanced Efficacy With Reduced Tolerance and Dependence in Mouse Neuropathic Pain Models.. The journal of pain, 21(1-2), 146-160. doi:10.1016/j.jpain.2019.05.017
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  Numerous studies have demonstrated a physiological interaction between the mu opioid receptor (MOR) and delta opioid receptor (DOR) systems. A few studies have shown that dual MOR-DOR agonists could be beneficial, with reduced tolerance and addiction liability, but are nearly untested in chronic pain models, particularly neuropathic pain. In this study, we tested the MOR-DOR agonist SRI-22141 in mice in the clinically relevant models of HIV Neuropathy and Chemotherapy-Induced Peripheral Neuropathy (CIPN). SRI-22141 was more potent than morphine in the tail flick pain test and had equal or enhanced efficacy versus morphine in both neuropathic pain models, with significantly reduced tolerance. SRI-22141 also produced no jumping behavior during naloxone-precipitated withdrawal in CIPN or naïve mice, suggesting that SRI-22141 produces little to no dependence. SRI-22141 also reduced tumor necrosis factor-α and cyclooxygenase-2 in CIPN in the spinal cord, suggesting an anti-inflammatory mechanism of action. The DOR-selective antagonist naltrindole strongly reduced CIPN efficacy and anti-inflammatory activity in the spinal cord, without affecting tail flick antinociception, suggesting the importance of DOR activity in these models. Overall, these results provide compelling evidence that MOR-DOR agonists could have strong efficacy with reduced side effects and an anti-inflammatory mechanism in the treatment of neuropathic pain. PERSPECTIVE: This study demonstrates that a MOR-DOR dual agonist given chronically in chronic neuropathic pain models has enhanced efficacy with strongly reduced tolerance and dependence, with a further anti-inflammatory effect in the spinal cord. This suggests that MOR-DOR dual agonists could be effective treatments for neuropathic pain with reduced side effects.
• Bellampalli, S. S., Ji, Y., Moutal, A., Cai, S., Wijeratne, E. M., Gandini, M. A., Yu, J., Chefdeville, A., Dorame, A., Chew, L. A., Madura, C. L., Luo, S., Molnar, G., Khanna, M., Streicher, J. M., Zamponi, G. W., Gunatilaka, A. A., & Khanna, R. (2019). Betulinic acid, derived from the desert lavender Hyptis emoryi, attenuates paclitaxel-, HIV-, and nerve injury-associated peripheral sensory neuropathy via block of N- and T-type calcium channels. Pain, 160(1), 117-135.
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  The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.
• Khanna, R., Yu, J., Yang, X., Moutal, A., Chefdeville, A., Gokhale, V., Shuja, Z., Chew, L. A., Bellampalli, S. S., Luo, S., François-Moutal, L., Serafini, M. J., Ha, T., Perez-Miller, S., Park, K. D., Patwardhan, A. M., Streicher, J. M., Colecraft, H. M., & Khanna, M. (2019). Targeting the CaVα-CaVβ interaction yields an antagonist of the N-type CaV2.2 channel with broad antinociceptive efficacy. Pain, 160(7), 1644-1661.
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  Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, β, γ and α2δ subunits. The β subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVβ's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the β subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVβ and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVβ function and ultimately be developed as a nonopioid therapeutic for chronic pain.
• Lei, W., Duron, D. I., Stine, C., Mishra, S., Blagg, B. S., & Streicher, J. M. (2019). The Alpha Isoform of Heat Shock Protein 90 and the Co-chaperones p23 and Cdc37 Promote Opioid Anti-nociception in the Brain. Frontiers in molecular neuroscience, 12, 294.
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  Opioid activation of the mu opioid receptor (MOR) promotes signaling cascades that evoke both analgesic responses to pain and side effects like addiction and dependence. Manipulation of these cascades, such as by biased agonism, has great promise to improve opioid therapy. However, the signaling cascades of the MOR are in general poorly understood, providing few targets for drug development. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel and crucial regulator of opioid anti-nociception in the brain by promoting ERK MAPK activation. In this study, we sought to identify the molecular isoforms and co-chaperones by which Hsp90 carried out this role, which could provide specific targets for future clinical intervention. We used novel selective small molecule inhibitors as well as CRISPR/Cas9 gene editing constructs delivered by the intracerebroventricular () route to the brains of adult CD-1 mice to target Hsp90 isoforms (Hsp90α/β, Grp94) and co-chaperones (p23, Cdc37, Aha1). We found that inhibition of the isoform Hsp90α fully blocked morphine anti-nociception in a model of post-surgical paw incision pain, while blocking ERK and JNK MAPK activation, suggesting Hsp90α as the main regulator of opioid response in the brain. We further found that inhibition of the co-chaperones p23 and Cdc37 blocked morphine anti-nociception, suggesting that these co-chaperones assist Hsp90α in promoting opioid anti-nociception. Lastly, we used cycloheximide treatment in the brain to demonstrate that rapid protein translation within 30 min of opioid treatment is required for Hsp90 regulation of opioid response. Together these studies provide insight into the molecular mechanisms by which Hsp90 promotes opioid anti-nociception. These findings thus both improve our basic science knowledge of MOR signal transduction and could provide future targets for clinical intervention to improve opioid therapy.
• Lei, W., Vekariya, R. H., Ananthan, S., & Streicher, J. M. (2019). A Novel Mu-Delta Opioid Agonist Demonstrates Enhanced Efficacy With Reduced Tolerance and Dependence in Mouse Neuropathic Pain Models. The journal of pain : official journal of the American Pain Society.
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  Numerous studies have demonstrated a physiological interaction between the mu opioid receptor (MOR) and delta opioid receptor (DOR) systems. A few studies have shown that dual MOR-DOR agonists could be beneficial, with reduced tolerance and addiction liability, but are nearly untested in chronic pain models, particularly neuropathic pain. In this study, we tested the MOR-DOR agonist SRI-22141 in mice in the clinically relevant models of HIV Neuropathy and Chemotherapy-Induced Peripheral Neuropathy (CIPN). SRI-22141 was more potent than morphine in the tail flick pain test and had equal or enhanced efficacy versus morphine in both neuropathic pain models, with significantly reduced tolerance. SRI-22141 also produced no jumping behavior during naloxone-precipitated withdrawal in CIPN or naïve mice, suggesting that SRI-22141 produces little to no dependence. SRI-22141 also reduced tumor necrosis factor-α and cyclooxygenase-2 in CIPN in the spinal cord, suggesting an anti-inflammatory mechanism of action. The DOR-selective antagonist naltrindole strongly reduced CIPN efficacy and anti-inflammatory activity in the spinal cord, without affecting tail flick antinociception, suggesting the importance of DOR activity in these models. Overall, these results provide compelling evidence that MOR-DOR agonists could have strong efficacy with reduced side effects and an anti-inflammatory mechanism in the treatment of neuropathic pain. PERSPECTIVE: This study demonstrates that a MOR-DOR dual agonist given chronically in chronic neuropathic pain models has enhanced efficacy with strongly reduced tolerance and dependence, with a further anti-inflammatory effect in the spinal cord. This suggests that MOR-DOR dual agonists could be effective treatments for neuropathic pain with reduced side effects.
• Olson, K. M., Duron, D. I., Womer, D., Fell, R., & Streicher, J. M. (2019). Comprehensive molecular pharmacology screening reveals potential new receptor interactions for clinically relevant opioids. PloS one, 14(6), e0217371.
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  Most clinically used opioids are thought to induce analgesia through activation of the mu opioid receptor (MOR). However, disparities have been observed between the efficacy of opioids in activating the MOR in vitro and in inducing analgesia in vivo. In addition, some clinically used opioids do not produce cross-tolerance with each other, and desensitization produced in vitro does not match tolerance produced in vivo. These disparities suggest that some opioids could be acting through other targets in vivo, but this has not been comprehensively tested. We thus screened 9 clinically relevant opioids (buprenorphine, hydrocodone, hydromorphone, morphine, O-desmethyl-tramadol, oxycodone, oxymorphone, tapentadol, tramadol) against 9 pain-related receptor targets (MOR, delta opioid receptor [DOR], kappa opioid receptor [KOR], nociceptin receptor [NOP], cannabinoid receptor type 1 [CB1], sigma-1 receptor [σ1R], and the monoamine transporters [NET/SERT/DAT]) expressed in cells using radioligand binding and functional activity assays. We found several novel interactions, including monoamine transporter activation by buprenorphine and σ1R binding by hydrocodone and tapentadol. Tail flick anti-nociception experiments with CD-1 mice demonstrated that the monoamine transporter inhibitor duloxetine selectively promoted buprenorphine anti-nociception while producing no effects by itself or in combination with the most MOR-selective drug oxymorphone, providing evidence that these novel interactions could be relevant in vivo. Our findings provide a comprehensive picture of the receptor interaction profiles of clinically relevant opioids, which has not previously been performed. Our findings also suggest novel receptor interactions for future investigation that could explain some of the disparities observed between opioid performance in vitro and in vivo.
• Shan, Z., Cai, S., Yu, J., Zhang, Z., Vallecillo, T. G., Serafini, M. J., Thomas, A. M., Pham, N. Y., Bellampalli, S. S., Moutal, A., Zhou, Y., Xu, G. B., Xu, Y. M., Luo, S., Patek, M., Streicher, J. M., Gunatilaka, A. A., & Khanna, R. (2019). Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Physalin F via Block of CaV2.3 (R-Type) and CaV2.2 (N-Type) Voltage-Gated Calcium Channels. ACS chemical neuroscience, 10(6), 2939-2955.
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  No universally efficacious therapy exists for chronic pain, a disease affecting one-fifth of the global population. An overreliance on the prescription of opioids for chronic pain despite their poor ability to improve function has led to a national opioid crisis. In 2018, the NIH launched a Helping to End Addiction Long-term plan to spur discovery and validation of novel targets and mechanisms to develop alternative nonaddictive treatment options. Phytochemicals with medicinal properties have long been used for various treatments worldwide. The natural product physalin F, isolated from the Physalis acutifolia (family: Solanaceae) herb, demonstrated antinociceptive effects in models of inflammatory pain, consistent with earlier reports of its anti-inflammatory and immunomodulatory activities. However, the target of action of physalin F remained unknown. Here, using whole-cell and slice electrophysiology, competition binding assays, and experimental models of neuropathic pain, we uncovered a molecular target for physalin F's antinociceptive actions. We found that physalin F (i) blocks CaV2.3 (R-type) and CaV2.2 (N-type) voltage-gated calcium channels in dorsal root ganglion (DRG) neurons, (ii) does not affect CaV3 (T-type) voltage-gated calcium channels or voltage-gated sodium or potassium channels, (iii) does not bind G-protein coupled opioid receptors, (iv) inhibits the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in spinal cord slices, and (v) reverses tactile hypersensitivity in models of paclitaxel-induced peripheral neuropathy and spinal nerve ligation. Identifying CaV2.2 as a molecular target of physalin F may spur its use as a tool for mechanistic studies and position it as a structural template for future synthetic compounds.
• Stefanucci, A., Dimmito, M. P., Macedonio, G., Ciarlo, L., Pieretti, S., Novellino, E., Lei, W., Barlow, D., Houseknecht, K. L., Streicher, J. M., & Mollica, A. (2019). Potent, Efficacious, and Stable Cyclic Opioid Peptides with Long Lasting Antinociceptive Effect after Peripheral Administration. Journal of medicinal chemistry.
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  Four novel fluorinated cyclic analogues of biphalin with excellent to modest binding affinity for μ-, δ-, and κ-receptors were synthesized. The cyclic peptides have a combination of piperazine or hydrazine linker with or without a xylene bridge. Among the ligands, demonstrated a better activity than biphalin after intravenous administration, and its corresponding analogue incorporating the hydrazine linker () was able to induce longer lasting analgesia following subcutaneous administration. An analogue of containing 2,6-dimethyl-l-tyrosine () showed the best potency and antinociceptive activity of this series.
• Stefanucci, A., Lei, W., Pieretti, S., Dimmito, M. P., Luisi, G., Novellino, E., Nowakowski, M., Koźmiński, W., Mirzaie, S., Zengin, G., Streicher, J. M., & Mollica, A. (2019). Novel Cyclic Biphalin Analogues by Ruthenium-Catalyzed Ring Closing Metathesis: and Biological Profile. ACS medicinal chemistry letters, 10(4), 450-456.
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  In this work we report the application of the ring-closing metathesis (RCM) to the preparation of two cyclic olefin-bridged analogues of biphalin (Tyr-d-Ala-Gly-Phe-NH-NH ← Phe ← Gly ← d-Ala ← Tyr), using the second generation Grubbs' catalyst. The resulting - and -cyclic isomers were identified, fully characterized, and tested at μ (ΜΟR), δ (DOR), and κ (KOR) opioid receptors and for antinociceptive activity. Both were shown to be full agonists at MOR and potential partial antagonists at DOR, with low potency KOR agonism. They also share a strong antinociceptive effect after intracerebroventricular (i.c.v.) and intravenous (i.v.) administration, higher than that of the cyclic biphalin analogues containing a disulfide bridge between the side chains of two d-Cys or d-Pen residues, previously described by our group.
• Stefanucci, A., Lei, W., Pieretti, S., Novellino, E., Dimmito, M. P., Marzoli, F., Streicher, J. M., & Mollica, A. (2019). On resin click-chemistry-mediated synthesis of novel enkephalin analogues with potent anti-nociceptive activity. Scientific reports, 9(1), 5771.
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  Here, we report the chemical synthesis of two DPDPE analogues 7a (NOVA1) and 7b (NOVA2). This entailed the solid-phase synthesis of two enkephalin precursor chains followed by a Cu-catalyzed azide-alkyne cycloaddition, with the aim of improving in vivo analgesic efficacy versus DPDPE. NOVA2 showed good affinity and selectivity for the μ-opioid receptor (K of 59.2 nM, EC of 12.9 nM, E of 87.3%), and long lasting anti-nociceptive effects in mice when compared to DPDPE.
• Streicher, J. M. (2019). The Role of Heat Shock Proteins in Regulating Receptor Signal Transduction. Molecular pharmacology.
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  Heat shock proteins (Hsp) are a class of stress-inducible proteins that mainly act as molecular protein chaperones. This chaperone activity is diverse, including assisting in nascent protein folding, and regulating client protein location and translocation within the cell. The main proteins within this family, particularly Hsp70 and Hsp90, also have a highly diverse and numerous set of protein clients, which when combined with the high expression levels of these proteins (2%-6% of total protein content), establishes these molecules as "central regulators" of cell protein physiology. Among these client proteins, Hsps regulate numerous signal transduction and receptor regulatory kinases, and indeed directly regulate some receptors themselves. This makes the Hsps, particularly Hsp90, also central regulators of signal transduction machinery, with important impacts on endogenous and drug ligand responses. Among these roles, Hsp90 in particular acts to maintain mature signaling kinases in a metastable conformation permissive for signaling activation. In this review, we will focus on the roles of the Hsps, with a special focus on Hsp90, in regulating receptor signaling and subsequent physiological responses. We will also explore potential means to manipulate Hsp function to improve receptor-targeted therapies. Overall, Hsps are important regulators of receptor signaling that are receiving increasing interest and exploration, particularly as Hsp90 inhibitors progress towards clinical approval for the treatment of cancer. Understanding the complex interplay of Hsp regulation of receptor signaling may provide important avenues to improve patient treatment.
• Streicher, J. M. (2019). The role of heat shock protein 90 in regulating pain, opioid signaling, and opioid antinociception. Vitamins and hormones, 111, 91-103.
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  Heat shock protein 90 (Hsp90) is one of the central signal transduction regulators of the cell. Via client interactions with hundreds of proteins, including receptors, receptor regulatory kinases, and downstream signaling regulators, Hsp90 has a crucial and wide-ranging impact on signaling in response to numerous drugs with impacts on resultant physiology and behavior. Despite this importance, however, Hsp90 has barely been studied in the context of pain and the opioid receptor system, leaving open the possibility that Hsp90 could be manipulated to improve pain therapeutic outcomes, a current area of massive medical need. In this review, we will highlight the known roles of Hsp90 in directly regulating the initiation and maintenance of the pain state. We will also explore how Hsp90 regulates signaling and antinociceptive responses to opioid analgesic drugs, with a special emphasis on ERK MAPK signaling. Understanding this new and growing area will improve our understanding of how Hsp90 regulates signaling and physiology, and also may provide new ways to treat pain, and perhaps reduce the severe impact of the ongoing opioid addiction and overdose crisis.
• Yu, J., Gunatilaka, A. A., Yu, J., Wijeratne, E. M., Streicher, J. M., Shan, Z., Moutal, A., Luo, S., Li, W., Khanna, R., Khanna, M., Ji, Y., Gunatilaka, A. A., Dorame, A., Cai, S., & Bellampalli, S. S. (2019). (-)-Hardwickiic Acid and Hautriwaic Acid Induce Antinociception via Blockade of Tetrodotoxin-Sensitive Voltage-Dependent Sodium Channels.. ACS chemical neuroscience, 10(3), 1716-1728. doi:10.1021/acschemneuro.8b00617
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  For an affliction that debilitates an estimated 50 million adults in the United States, the current chronic pain management approaches are inadequate. The Centers for Disease Control and Prevention have called for a minimization in opioid prescription and use for chronic pain conditions, and thus, it is imperative to discover alternative non-opioid based strategies. For the realization of this call, a library of natural products was screened in search of pharmacological inhibitors of both voltage-gated calcium channels and voltage-gated sodium channels, which are excellent targets due to their well-established roles in nociceptive pathways. We discovered (-)-hardwickiic acid ((-)-HDA) and hautriwaic acid (HTA) isolated from plants, Croton californicus and Eremocarpus setigerus, respectively, inhibited tetrodotoxin-sensitive sodium, but not calcium or potassium, channels in small diameter, presumptively nociceptive, dorsal root ganglion (DRG) neurons. Failure to inhibit spontaneous postsynaptic excitatory currents indicated a preferential targeting of voltage-gated sodium channels over voltage-gated calcium channels by these extracts. Neither compound was a ligand at opioid receptors. Finally, we identified the potential of both (-)-HDA and HTA to reverse chronic pain behavior in preclinical rat models of HIV-sensory neuropathy, and for (-)-HDA specifically, in chemotherapy-induced peripheral neuropathy. Our results illustrate the therapeutic potential for (-)-HDA and HTA for chronic pain management and could represent a scaffold, that, if optimized by structure-activity relationship studies, may yield novel specific sodium channel antagonists for pain relief.
• Cai, S., Bellampalli, S. S., Yu, J., Li, W., Ji, Y., Wijeratne, E. M., Dorame, A., Luo, S., Shan, Z., Khanna, M., Moutal, A., Streicher, J. M., Gunatilaka, A. A., & Khanna, R. (2018). (-)-Hardwickiic acid and Hautriwaic acid induce antinociception via blockade of tetrodotoxin-sensitive voltage-dependent sodium channels. ACS chemical neuroscience.
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  For an affliction that debilitates an estimated 50 million of U.S. adults, the current chronic pain management approaches are inadequate. The Centers for Disease Control and Prevention have called for a minimization in opioid prescription and use for chronic pain conditions, and thus, it is imperative to discover alternative non-opioid based strategies. For the realization of this call, a library of natural products was screened in search of pharmacological inhibitors of both voltage-gated calcium channels and voltage-gated sodium channels, excellent targets due to their well-established roles in nociceptive pathways. We discovered (-)-hardwickiic acid ((-)-HDA) and hautriwaic acid (HTA) isolated from plants, Croton californicus and Eremocarpus setigerus, respectively, inhibited tetrodotoxin-sensitive sodium, but not calcium or potassium, channels in small diameter, presumptively nociceptive, dorsal root ganglion (DRG) neurons. Failure to inhibit spontaneous post-synaptic excitatory currents indicated a preferential targeting of voltage-gated sodium channels over voltage-gated calcium channels by these extracts. Neither compound was a ligand at opioid receptors. Finally, we identified the potential of both (-)-HDA and HTA to reverse chronic pain behavior in preclinical rat models of HIV-sensory neuropathy, and for (-)-HDA specifically, in chemotherapy-induced peripheral neuropathy. Our results illustrate the therapeutic potential for (-)-HDA and HTA for chronic pain management and could represent a scaffold, that, if optimized by structure-activity relationship studies, may yield novel specific sodium channel antagonists for pain relief.
• Edwards, K. A., Havelin, J. J., Mcintosh, M. I., Ciccone, H. A., Pangilinan, K., Imbert, I., Largent-Milnes, T. M., King, T., Vanderah, T. W., & Streicher, J. M. (2018). A Kappa Opioid Receptor Agonist Blocks Bone Cancer Pain Without Altering Bone Loss, Tumor Size, or Cancer Cell Proliferation in a Mouse Model of Cancer-Induced Bone Pain. The journal of pain : official journal of the American Pain Society, 19(6), 612-625.
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  Breast cancer metastasizes to bone, diminishing quality of life of patients because of pain, fracture, and limited mobility. Cancer-induced bone pain (CIBP) is characterized as moderate to severe ongoing pain, primarily managed by mu opioid agonists such as fentanyl. However, opioids are limited by escalating doses and serious side effects. One alternative may be kappa opioid receptor (KOR) agonists. There are few studies examining KOR efficacy on CIBP, whereas KOR agonists are efficacious in peripheral and inflammatory pain. We thus examined the effects of the KOR agonist U50,488 given twice daily across 7 days to block CIBP, tumor-induced bone loss, and tumor burden. U50,488 dose-dependently blocked tumor-induced spontaneous flinching and impaired limb use, without changing tactile hypersensitivity, and was fully reversed by the KOR antagonist nor-binaltorphimine. U50,488 treatment was higher in efficacy and duration of action at later time points. U50,488 blocked this pain without altering tumor-induced bone loss or tumor growth. Follow-up studies in human cancer cell lines confirmed that KOR agonists do not affect cancer cell proliferation. These studies suggest that KOR agonists could be a new target for cancer pain management that does not induce cancer cell proliferation or alter bone loss.
• Olson, K. M., Keresztes, A., Tashiro, J. K., Daconta, L. V., Hruby, V. J., & Streicher, J. M. (2018). Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the Mu-Delta Opioid Receptor Heterodimer that Reduces Morphine Withdrawal in Mice. Journal of medicinal chemistry.
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  A major limitation in the study of the mu-delta opioid receptor heterodimer (MDOR) is that few selective pharmacological tools exist and no heteromer-selective antagonists. We thus designed a series of variable-length (15-41 atoms) bivalent linked peptides with selective but moderate/low-affinity pharmacophores for the mu and delta opioid receptors. We observed a U-shaped MDOR potency/affinity profile in vitro, with the 24-atom spacer length (D24M) producing the highest MDOR potency/affinity (
• Shah, N., Kumar, S., Zaman, N., Pan, C. C., Bloodworth, J. C., Lei, W., Streicher, J. M., Hempel, N., Mythreye, K., & Lee, N. Y. (2018). TAK1 activation of alpha-TAT1 and microtubule hyperacetylation control AKT signaling and cell growth. Nature communications, 9(1), 1696.
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  Acetylation of microtubules (MT) confers mechanical stability necessary for numerous functions including cell cycle and intracellular transport. Although αTAT1 is a major MT acetyltransferase, how this enzyme is regulated remains much less clear. Here we report TGF-β-activated kinase 1 (TAK1) as a key activator of αTAT1. TAK1 directly interacts with and phosphorylates αTAT1 at Ser237 to critically enhance its catalytic activity, as mutating this site to alanine abrogates, whereas a phosphomimetic induces MT hyperacetylation across cell types. Using a custom phospho-αTAT1-Ser237 antibody, we screen various mouse tissues to discover that brain contains some of the highest TAK1-dependent αTAT1 activity, which, accordingly, is diminished rapidly upon intra-cerebral injection of a TAK1 inhibitor. Lastly, we show that TAK1 selectively inhibits AKT to suppress mitogenic and metabolism-related pathways through MT-based mechanisms in culture and in vivo. Collectively, our findings support a fundamental new role for TGF-β signaling in MT-related functions and disease.
• Streicher, J. M., & Bilsky, E. J. (2018). Peripherally Acting μ-Opioid Receptor Antagonists for the Treatment of Opioid-Related Side Effects: Mechanism of Action and Clinical Implications.. Journal of pharmacy practice, 31(6), 658-669. doi:10.1177/0897190017732263
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  Opioid receptors are distributed throughout the central and peripheral nervous systems and on many nonneuronal cells. Therefore, opioid administration induces effects beyond analgesia. In the enteric nervous system (ENS), stimulation of µ-opioid receptors triggers several inhibitory responses that can culminate in opioid-induced bowel dysfunction (OBD) and its most common side effect, opioid-induced constipation (OIC). OIC negatively affects patients' quality of life (QOL), ability to work, and pain management. Although laxatives are a common first-line OIC therapy, most have limited efficacy and do not directly antagonize opioid effects on the ENS. Peripherally acting µ-opioid receptor antagonists (PAMORAs) with limited ability to cross the blood-brain barrier have been developed. The PAMORAs approved by the U S Food and Drug Administration for OIC are subcutaneous and oral methylnaltrexone, oral naloxegol, and oral naldemedine. Although questions of cost-effectiveness and relative efficacy versus laxatives remain, PAMORAs can mitigate OIC and improve patient QOL. PAMORAS may also have applications beyond OIC, including reducing the increased cardiac risk or potential tumorigenic effects of opioids. This review discusses the burden of OIC and OBD, reviews the mechanism of action of new OIC therapies, and highlights other potential opioid-related side effects mediated by peripheral opioid receptors in the context of new OIC therapies.
• Streicher, J. M., Stine, C., Lei, W., Duron, D., & Blagg, B. (2018). Heat shock protein 90 promotes opioid anti-nociception in the brain and represses opioid anti-nociception in the spinal cord through the differential regulation of ERK MAPK signaling. The Journal of Pain, 19(3), S100-S101. doi:10.1016/j.jpain.2017.12.227
• Keresztes, A., & Streicher, J. M. (2017). Synergistic interaction of the cannabinoid and death receptor systems - a potential target for future cancer therapies?. FEBS letters, 591(20), 3235-3251.
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  Cannabinoid receptors have been shown to interact with other receptors, including tumor necrosis factor receptor superfamily (TNFRS) members, to induce cancer cell death. When cannabinoids and death-inducing ligands (including TNF-related apoptosis-inducing ligand) are administered together, they have been shown to synergize and demonstrate enhanced antitumor activity in vitro. Certain cannabinoid ligands have been shown to sensitize cancer cells and synergistically interact with members of the TNFRS, thus suggesting that the combination of cannabinoids with death receptor (DR) ligands induces additive or synergistic tumor cell death. This review summarizes recent findings on the interaction of the cannabinoid and DR systems and suggests possible clinical co-application of cannabinoids and DR ligands in the treatment of various malignancies.
• Lei, W., Mullen, N., McCarthy, S., Brann, C., Richard, P., Cormier, J., Edwards, K., Bilsky, E. J., & Streicher, J. M. (2017). Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain. The Journal of biological chemistry, 292(25), 10414-10428.
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  Recent advances in developing opioid treatments for pain with reduced side effects have focused on the signaling cascades of the μ-opioid receptor (MOR). However, few such signaling targets have been identified for exploitation. To address this need, we explored the role of heat-shock protein 90 (Hsp90) in opioid-induced MOR signaling and pain, which has only been studied in four previous articles. First, in four cell models of MOR signaling, we found that Hsp90 inhibition for 24 h with the inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) had different effects on protein expression and opioid signaling in each line, suggesting that cell models may not be reliable for predicting pharmacology with this protein. We thus developed an in vivo model using CD-1 mice with an intracerebroventricular injection of 17-AAG for 24 h. We found that Hsp90 inhibition strongly blocked morphine-induced anti-nociception in models of post-surgical and HIV neuropathic pain but only slightly blocked anti-nociception in a naive tail-flick model, while enhancing morphine-induced precipitated withdrawal. Seeking a mechanism for these changes, we found that Hsp90 inhibition blocks ERK MAPK activation in the periaqueductal gray and caudal brain stem. We tested these signaling changes by inhibiting ERK in the above-mentioned pain models and found that ERK inhibition could account for all of the changes in anti-nociception induced by Hsp90 inhibition. Taken together, these findings suggest that Hsp90 promotes opioid-induced anti-nociception by an ERK mechanism in mouse brain and that Hsp90 could be a future target for improving the therapeutic index of opioid drugs.
• Olson, K. M., Lei, W., Keresztes, A., LaVigne, J., & Streicher, J. M. (2017). Novel Molecular Strategies and Targets for Opioid Drug Discovery for the Treatment of Chronic Pain. The Yale journal of biology and medicine, 90(1), 97-110.
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  Opioid drugs like morphine and fentanyl are the gold standard for treating moderate to severe acute and chronic pain. However, opioid drug use can be limited by serious side effects, including constipation, tolerance, respiratory suppression, and addiction. For more than 100 years, we have tried to develop opioids that decrease or eliminate these liabilities, with little success. Recent advances in understanding opioid receptor signal transduction have suggested new possibilities to activate the opioid receptors to cause analgesia, while reducing or eliminating unwanted side effects. These new approaches include designing functionally selective ligands, which activate desired signaling cascades while avoiding signaling cascades that are thought to provoke side effects. It may also be possible to directly modulate downstream signaling through the use of selective activators and inhibitors. Separate from downstream signal transduction, it has also been found that when the opioid system is stimulated, various negative feedback systems are upregulated to compensate, which can drive side effects. This has led to the development of multi-functional molecules that simultaneously activate the opioid receptor while blocking various negative feedback receptor systems including cholecystokinin and neurokinin-1. Other novel approaches include targeting heterodimers of the opioid and other receptor systems which may drive side effects, and making endogenous opioid peptides druggable, which may also reduce opioid mediated side effects. Taken together, these advances in our molecular understanding provide a path forward to break the barrier in producing an opioid with reduced or eliminated side effects, especially addiction, which may provide relief for millions of patients.
• Porreca, F., Roberts, E., Navratilova, E., Streicher, J. M., Yue, X., LaVigne, J., Xie, J. Y., De Felice, M., Kopruszinski, C. M., Eyde, N., Remeniuk, B., Hernandez, P., Goshima, N., Ossipov, M., King, T., Dodick, D., & Rosen, H. (2017). Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention. Cephalalgia, 37(8), 780-794. doi:10.1177/0333102417702120
• Starnowska, J., Costante, R., Guillemyn, K., Popiolek-Barczyk, K., Chung, N. N., Lemieux, C., Keresztes, A., Van Duppen, J., Mollica, A., Streicher, J., Vanden Broeck, J., Schiller, P. W., Tourwé, D., Mika, J., Ballet, S., & Przewlocka, B. (2017). Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice. ACS chemical neuroscience, 8(10), 2315-2324.
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  The lower efficacy of opioids in neuropathic pain may be due to the increased activity of pronociceptive systems such as substance P. We present evidence to support this hypothesis in this work from the spinal cord in a neuropathic pain model in mice. Biochemical analysis confirmed the elevated mRNA and protein level of pronociceptive substance P, the major endogenous ligand of the neurokinin-1 (NK1) receptor, in the lumbar spinal cord of chronic constriction injury (CCI)-mice. To improve opioid efficacy in neuropathic pain, novel compounds containing opioid agonist and neurokinin 1 (NK1) receptor antagonist pharmacophores were designed. Structure-activity studies were performed on opioid agonist/NK1 receptor antagonist hybrid peptides by modification of the C-terminal amide substituents. All compounds were evaluated for their affinity and in vitro activity at the mu opioid (MOP) and delta opioid (DOP) receptors, and for their affinity and antagonist activity at the NK1 receptor. On the basis of their in vitro profiles, the analgesic properties of two new bifunctional hybrids were evaluated in naive and CCI-mice, representing models for acute and neuropathic pain, respectively. The compounds were administered to the spinal cord by lumbar puncture. In naive mice, the single pharmacophore opioid parent compounds provided better analgesic results, as compared to the hybrids (max 70% MPE), raising the acute pain threshold close to 100% MPE. On the other hand, the opioid parents gave poor analgesic effects under neuropathic pain conditions, while the best hybrid delivered robust (close to 100% MPE) and long lasting alleviation of both tactile and thermal hypersensitivity. The results presented emphasize the potential of opioid/NK1 hybrids in view of analgesia under nerve injury conditions.
• Stefanucci, A., Lei, W., Hruby, V. J., Macedonio, G., Luisi, G., Carradori, S., Streicher, J. M., & Mollica, A. (2017). Fluorescent-labeled bioconjugates of the opioid peptides biphalin and DPDPE incorporating fluorescein-maleimide linkers. Future medicinal chemistry, 9(9), 859-869.
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  The conjugation of fluorescent labels to opioid peptides is an extremely challenging task, which needs to be overcome to create new classes of probes for biological assays.
• Streicher, J. M., & Bilsky, E. J. (2017). Peripherally Acting µ-Opioid Receptor Antagonists for the Treatment of Opioid-Related Side Effects: Mechanism of Action and Clinical Implications. Journal of pharmacy practice, 897190017732263.
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  Opioid receptors are distributed throughout the central and peripheral nervous systems and on many nonneuronal cells. Therefore, opioid administration induces effects beyond analgesia. In the enteric nervous system (ENS), stimulation of µ-opioid receptors triggers several inhibitory responses that can culminate in opioid-induced bowel dysfunction (OBD) and its most common side effect, opioid-induced constipation (OIC). OIC negatively affects patients' quality of life (QOL), ability to work, and pain management. Although laxatives are a common first-line OIC therapy, most have limited efficacy and do not directly antagonize opioid effects on the ENS. Peripherally acting µ-opioid receptor antagonists (PAMORAs) with limited ability to cross the blood-brain barrier have been developed. The PAMORAs approved by the U S Food and Drug Administration for OIC are subcutaneous and oral methylnaltrexone, oral naloxegol, and oral naldemedine. Although questions of cost-effectiveness and relative efficacy versus laxatives remain, PAMORAs can mitigate OIC and improve patient QOL. PAMORAS may also have applications beyond OIC, including reducing the increased cardiac risk or potential tumorigenic effects of opioids. This review discusses the burden of OIC and OBD, reviews the mechanism of action of new OIC therapies, and highlights other potential opioid-related side effects mediated by peripheral opioid receptors in the context of new OIC therapies.
• Streicher, J. M., & Keresztes, A. (2017). Synergistic interaction of the cannabinoid and death receptor systems - a potential target for future cancer therapies?. FEBS Letters, 591(20), 3235-3251. doi:10.1002/1873-3468.12863
• Xie, J. Y., De Felice, M., Kopruszinski, C. M., Eyde, N., LaVigne, J., Remeniuk, B., Hernandez, P., Yue, X., Goshima, N., Ossipov, M., King, T., Streicher, J. M., Navratilova, E., Dodick, D., Rosen, H., Roberts, E., & Porreca, F. (2017). Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention. Cephalalgia : an international journal of headache, 37(8), 780-794.
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  Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.
• Ramos-Colon, C. N., Lee, Y. S., Remesic, M., Hall, S. M., LaVigne, J., Davis, P., Sandweiss, A. J., McIntosh, M. I., Hanson, J., Largent-Milnes, T. M., Vanderah, T. W., Streicher, J., Porreca, F., & Hruby, V. J. (2016). Structure-Activity Relationships of [des-Arg(7)]Dynorphin A Analogues at the κ Opioid Receptor. Journal of medicinal chemistry, 59(22), 10291-10298.
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  Dynorphin A (Dyn A) is an endogenous ligand for the opioid receptors with preference for the κ opioid receptor (KOR), and its structure-activity relationship (SAR) has been extensively studied at the KOR to develop selective potent agonists and antagonists. Numerous SAR studies have revealed that the Arg(7) residue is essential for KOR activity. In contrast, our systematic SAR studies on [des-Arg(7)]Dyn A analogues found that Arg(7) is not a key residue and even deletion of the residue does not affect biological activities at the KOR. In addition, it was also found that [des-Arg(7)]Dyn A(1-9)-NH2 is a minimum pharmacophore and its modification at the N-terminus leads to selective KOR antagonists. A lead ligand, 14, with high affinity and antagonist activity showed improved metabolic stability and could block antinociceptive effects of a KOR selective agonist, FE200665, in vivo, indicating high potential to treat KOR mediated disorders such as stress-induced relapse.
• Streicher, J. M., Pangilinan, K., Lavigne, J., Edwards, K., & Bogusz, J. (2016). (285) Phosphatidylethanolamine-binding protein regulates Mu opioid receptor induced βarrestin2 recruitment and downstream signaling. The Journal of Pain, 17(4), S47. doi:10.1016/j.jpain.2016.01.191
• Zhang, Y., Williams, D. A., Zaidi, S. A., Yuan, Y., Braithwaite, A., Bilsky, E. J., Dewey, W. L., Akbarali, H. I., Streicher, J. M., & Selley, D. E. (2016). 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(4'-pyridylcarboxamido)morphinan (NAP) Modulating the Mu Opioid Receptor in a Biased Fashion. ACS chemical neuroscience.
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  Mounting evidence has suggested that G protein-coupled receptors can be stabilized in multiple conformations in response to distinct ligands, which exert discrete functions through selective activation of various downstream signaling events. In accordance with this concept, we report biased signaling of one C6-heterocyclic substituted naltrexamine derivative, namely, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(4'-pyridylcarboxamido)morphinan (NAP) at the mu opioid receptor (MOR). NAP acted as a low efficacy MOR partial agonist in the G protein-mediated [(35)S]GTPγS binding assay, whereas it did not significantly induce calcium flux or β-arrestin2 recruitment. In contrast, it potently blocked MOR full agonist-induced β-arrestin2 recruitment and translocation. Additionally, NAP dose-dependently antagonized MOR full agonist-induced intracellular calcium flux and β-arrestin2 recruitment. Further results in an isolated organ bath preparation confirmed that NAP reversed the morphine-induced reduction in colon motility. Ligand docking and dynamics simulation studies of NAP at the MOR provided more supporting evidence for biased signaling of NAP at an atomic level. Due to the fact that NAP is MOR selective and preferentially distributed peripherally upon systemic administration while β-arrestin2 is reportedly required for impairment of intestinal motility by morphine, biased antagonism of β-arrestin2 recruitment by NAP further supports its utility as a treatment for opioid-induced constipation.
• Edwards, K. A., Havelin, J. J., Mcintosh, M. I., Ciccone, H. A., Pangilinan, K., Imbert, I., Largent-Milnes, T. M., King, T., Vanderah, T. W., & Streicher, J. M. (2017). A Kappa Opioid Receptor Agonist Blocks Bone Cancer Pain Without Altering Bone Loss, Tumor Size, or Cancer Cell Proliferation in a Mouse Model of Cancer-Induced Bone Pain. Journal of Pain.
• Frankowski, K. J., Slauson, S. R., Lovell, K. M., Phillips, A. M., Streicher, J. M., Zhou, L., Whipple, D. A., Schoenen, F. J., Prisinzano, T. E., Bohn, L. M., & Aubé, J. (2015). Potency enhancement of the κ-opioid receptor antagonist probe ML140 through sulfonamide constraint utilizing a tetrahydroisoquinoline motif. Bioorganic & medicinal chemistry, 23(14), 3948-56.
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  Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined with additional structure-activity relationship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.
• Stevenson, G. W., Luginbuhl, A., Dunbar, C., LaVigne, J., Dutra, J., Atherton, P., Bell, B., Cone, K., Giuvelis, D., Polt, R., Streicher, J. M., & Bilsky, E. J. (2015). The mixed-action delta/mu opioid agonist MMP-2200 does not produce conditioned place preference but does maintain drug self-administration in rats, and induces in vitro markers of tolerance and dependence. Pharmacology, biochemistry, and behavior, 132, 49-55.
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  Previous work in our laboratories provides preclinical evidence that mixed-action delta/mu receptor glycopeptides have equivalent efficacy for treating pain with reduced side effect profiles compared to widely used mu agonist analgesics such as morphine. This study evaluated the rewarding and reinforcing effects of a lead candidate, mixed-action delta/mu agonist MMP-2200, using a conditioned place preference assay as well as a drug self-administration procedure in rats. In place conditioning studies, rats underwent a 2-week conditioning protocol and were then tested for chamber preference. Rats receiving MMP-2200, at previously determined analgesic doses, could not distinguish between the drug and saline-paired chamber, whereas rats receiving the opioid agonist morphine showed a strong preference for the morphine-paired chamber. In self-administration studies, rats were trained to respond for the high efficacy mu opioid receptor agonist fentanyl on an FR5 schedule of reinforcement. Following complete dose-response determinations for fentanyl, a range of doses of MMP-2200 as well as morphine were tested. Relative to the mu agonist morphine, MMP-2200 maintained a significantly lower number of drug infusions. To begin investigating potential molecular mechanisms for the reduced side effect profile of MMP-2200, we also examined βarrestin2 (βarr2) recruitment and chronic MMP-2200 induced cAMP tolerance and super-activation at the human delta and mu receptors in vitro. MMP-2200 efficaciously recruited βarr2 to both receptors, and induced cAMP tolerance and super-activation equivalent to or greater than morphine at both receptors. The in vivo findings suggest that MMP-2200 may be less reinforcing than morphine but may have some abuse potential. The reduced side effect profile cannot be explained by reduced βarr2 recruitment or reduced cAMP tolerance and superactivation at the monomeric receptors in vitro.
• Streicher, J. M., Pangilinan, K., Lavigne, J., & Edwards, K. (2015). Phosphatidylethanolamine-Binding Protein Regulates Mu Opioid Receptor Induced βarrestin2 Recruitment and Downstream Signaling. The FASEB Journal, 29.
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  The signaling regulators and pathways of the mu-opioid receptor (MOR) are being studied to develop functionally selective MOR ligands. Such novel analgesic drugs may alleviate pain without the side...
• Lavigne, J., Edwards, K., Bilsky, E. J., Porreca, F., Ananthan, S., Roberts, E., Roberts, E., Hruby, V. J., Hruby, V. J., Streicher, J. M., Streicher, J. M., Roberts, E., & Porreca, F. (2014). The development of bifunctional ligands as novel therapeutics for chronic pain (1061.5). The FASEB Journal, 28. doi:10.1096/fasebj.28.1_supplement.1061.5
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  Opioid drugs can be efficacious in the treatment of chronic pain, but are plagued by the development of serious side effects such as tolerance and dependence that worsen with chronic use. Recent in...
• Schmid, C. L., Streicher, J. M., Meltzer, H. Y., & Bohn, L. M. (2014). Clozapine acts as an agonist at serotonin 2A receptors to counter MK-801-induced behaviors through a βarrestin2-independent activation of Akt. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 39(8), 1902-13.
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  The G protein-coupled serotonin 2A receptor (5-HT2AR) is a prominent target for atypical antipsychotic drugs, such as clozapine. Although clozapine is known to inhibit 5-HT2AR signaling through G protein-dependent mechanisms, it differs from classic GPCR antagonists, in that it also induces 5-HT2AR internalization and activates Akt signaling via a 5-HT2AR-mediated event. In this regard, clozapine may also be considered a functionally selective agonist. The cognate neurotransmitter at the 5-HT2AR, serotonin, also induces 5-HT2AR internalization and Akt phosphorylation. Serotonin promotes interactions with the scaffolding and regulatory protein, βarrestin2, which results in the recruitment and activation of Akt. These interactions prove to be critical for serotonin-induced, 5-HT2AR-mediated behavioral responses in mice. Herein, we sought to determine whether clozapine also utilizes βarrestin2-mediated mechanisms to induce 5-HT2AR signaling, and whether this interaction contributes to its behavioral effects in mice. We demonstrate that unlike serotonin, clozapine-mediated 5-HT2AR internalization and Akt phosphorylation is independent of receptor interactions with βarrestin2. Moreover, clozapine-mediated suppression of MK-801 and phencyclidine (PCP)-induced hyperlocomotion is βarrestin2 independent, although it is dependent upon Akt. These results demonstrate that pharmacologically oppositional ligands, serotonin and clozapine, utilize differential mechanisms to achieve the same 5-HT2AR-meadiated downstream events: Akt phosphorylation and receptor internalization. Although βarrestin2 has no effect on clozapine's actions in vivo, Akt phosphorylation is required for clozapine's efficacy in blocking MK-801- and PCP-induced models of schizophrenic behaviors in mice.
• Streicher, J. M., Mullen, N., Moses-fynn, E., Lavigne, J., & Edwards, K. (2014). Identification of novel signaling regulators of the mu opioid receptor (1066.1). The FASEB Journal, 28.
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  Recent evidence has suggested that specific downstream signaling pathways of the mu opioid receptor (MOR) carry out specific aspects of the behavioral response, such as antinociception or side effe...
• Zhang, Y., Braithwaite, A., Yuan, Y., Streicher, J. M., & Bilsky, E. J. (2014). Behavioral and cellular pharmacology characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) as a mu opioid receptor selective ligand. European journal of pharmacology, 736, 124-30.
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  Mu opioid receptor (MOR) selective antagonists and partial agonists have been used for the treatment of opioid abuse and addiction. Our recent efforts on the identification of MOR antagonists have provided several novel leads displaying interesting pharmacological profiles. Among them, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan (NAQ) showed sub-nanomolar binding affinity to the MOR with significant selectivity over the delta opioid receptor (DOR) and the kappa opioid receptor (KOR). Its central nervous system penetration capacity together with marginal agonism in the MOR-GTPγS binding assay made it a very interesting molecule for developing novel opioid abuse and addiction therapeutic agents. Therefore, further pharmacological characterization was conducted to fully understand its biological profile. At the molecular and cellular level, NAQ not only induced no translocation of β-arrestin2 to the MOR, but also efficaciously antagonized the effect of DAMGO in MOR-βarr2eGFP-U2OS cells in the β-arrestin2 recruitment assay. At the in vivo level, NAQ displayed a potent inhibition of the analgesic effect of morphine in the tail-flick assay (ID50=1.19 mg/kg). NAQ (10 mg/kg) also significantly decreased the hyper-locomotion induced by acute morphine without inducing any vertical jumps. Meanwhile NAQ precipitated lesser withdrawal symptoms in morphine dependent mice than naloxone. In conclusion, NAQ may represent a new chemical entity for opioid abuse and addiction treatment.
• Schmid, C. L., Streicher, J. M., Bohn, L. M., Bohn, L. M., Streicher, J. M., & Bohn, L. M. (2013). The atypical antipsychotic clozapine induces 5-HT2ARmediated signaling and behavioral events in a beta-arrestin2- independent but Akt-dependent manner. The FASEB Journal, 27.
• Schmid, C. L., Streicher, J. M., Groer, C. E., Munro, T. A., Zhou, L., & Bohn, L. M. (2013). Functional selectivity of 6'-guanidinonaltrindole (6'-GNTI) at κ-opioid receptors in striatal neurons. The Journal of biological chemistry, 288(31), 22387-98.
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  There is considerable evidence to suggest that drug actions at the κ-opioid receptor (KOR) may represent a means to control pain perception and modulate reward thresholds. As a G protein-coupled receptor (GPCR), the activation of KOR promotes Gαi/o protein coupling and the recruitment of β-arrestins. It has become increasingly evident that GPCRs can transduce signals that originate independently via G protein pathways and β-arrestin pathways; the ligand-dependent bifurcation of such signaling is referred to as "functional selectivity" or "signaling bias." Recently, a KOR agonist, 6'-guanidinonaltrindole (6'-GNTI), was shown to display bias toward the activation of G protein-mediated signaling over β-arrestin2 recruitment. Therefore, we investigated whether such ligand bias was preserved in striatal neurons. Although the reference KOR agonist U69,593 induces the phosphorylation of ERK1/2 and Akt, 6'-GNTI only activates the Akt pathway in striatal neurons. Using pharmacological tools and β-arrestin2 knock-out mice, we show that KOR-mediated ERK1/2 phosphorylation in striatal neurons requires β-arrestin2, whereas Akt activation depends upon G protein signaling. These findings reveal a point of KOR signal bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indicator when developing biased agonists at the KOR.
• Zhou, L., Lovell, K. M., Frankowski, K. J., Slauson, S. R., Phillips, A. M., Streicher, J. M., Stahl, E., Schmid, C. L., Hodder, P., Madoux, F., Cameron, M. D., Prisinzano, T. E., Aubé, J., & Bohn, L. M. (2013). Development of functionally selective, small molecule agonists at kappa opioid receptors. The Journal of biological chemistry, 288(51), 36703-16.
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  The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.
• Béguin, C., Potuzak, J., Xu, W., Liu-Chen, L., Streicher, J. M., Groer, C. E., Bohn, L. M., Carlezon, W. A., & Cohen, B. M. (2012). Differential signaling properties at the kappa opioid receptor of 12-epi-salvinorin A and its analogues. Bioorganic & medicinal chemistry letters, 22(2), 1023-6.
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  The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12-epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as β-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the β-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the β-arrestin mediated signaling pathway activated through KOPR.
• Frankowski, K. J., Hedrick, M. P., Gosalia, P., Li, K., Shi, S., Whipple, D., Ghosh, P., Prisinzano, T. E., Schoenen, F. J., Su, Y., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Milan, L., Heynen-Genel, S., Mangravita-Novo, A., Vicchiarelli, M., Smith, L. H., , Streicher, J. M., et al. (2012). Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy. ACS chemical neuroscience, 3(3), 221-236.
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  Herein we present the outcome of a high throughput screening (HTS) campaign-based strategy for the rapid identification and optimization of selective and general chemotypes for both kappa (κ) opioid receptor (KOR) activation and inhibition. In this program, we have developed potent antagonists (IC(50) < 120 nM) or agonists of high binding affinity (K(i) < 3 nM). In contrast to many important KOR ligands, the compounds presented here are highly modular, readily synthesized and, in most cases, achiral. The four new chemotypes hold promise for further development into chemical tools for studying the KOR or as potential therapeutic lead candidates.
• Hedrick, M., Gosalia, P., Li, K., Frankowski, K., Shi, S., Prisinzano, T., Schoenen, F., Aube, J., Su, Y., Stonich, D., Vasile, S., Sergienko, E., Gray, W., Hariharan, S., Milan, L., Heynen-Genel, S., Vicchiarelli, M., Mangravita-Novo, A., Streicher, J. M., , Smith, L., et al. (2011). Antagonist for the Kappa Opioid Receptor. NIH Molecular Libraries Program.
• Streicher, J. M., Groer, C. E., Munro, T. A., Beguin, C., Cohen, B. M., Bohn, L. M., Bohn, L. M., Streicher, J. M., & Bohn, L. M. (2011). 6'-Guanidinonaltrindole (6'-GNTI) is a potent and functionally unique kappa opioid agonist that displays bias against beta-arrestin recruitment and receptor internalization. The FASEB Journal, 25.
• Tarselli, M. A., Raehal, K. M., Brasher, A. K., Streicher, J. M., Groer, C. E., Cameron, M. D., Bohn, L. M., & Micalizio, G. C. (2011). Synthesis of conolidine, a potent non-opioid analgesic for tonic and persistent pain. Nature chemistry, 3(6), 449-53.
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  Management of chronic pain continues to represent an area of great unmet biomedical need. Although opioid analgesics are typically embraced as the mainstay of pharmaceutical interventions in this area, they suffer from substantial liabilities that include addiction and tolerance, as well as depression of breathing, nausea and chronic constipation. Because of their suboptimal therapeutic profile, the search for non-opioid analgesics to replace these well-established therapeutics is an important pursuit. Conolidine is a rare C5-nor stemmadenine natural product recently isolated from the stem bark of Tabernaemontana divaricata (a tropical flowering plant used in traditional Chinese, Ayurvedic and Thai medicine). Although structurally related alkaloids have been described as opioid analgesics, no therapeutically relevant properties of conolidine have previously been reported. Here, we describe the first de novo synthetic pathway to this exceptionally rare C5-nor stemmadenine natural product, the first asymmetric synthesis of any member of this natural product class, and the discovery that (±)-, (+)- and (-)-conolidine are potent and efficacious non-opioid analgesics in an in vivo model of tonic and persistent pain.
• Drews, O., Tsukamoto, O., Liem, D., Streicher, J., Wang, Y., & Ping, P. (2010). Differential regulation of proteasome function in isoproterenol-induced cardiac hypertrophy. Circulation research, 107(9), 1094-101.
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  Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation.
• Papanicolaou, K. N., Streicher, J. M., Ishikawa, T., Herschman, H., Wang, Y., & Walsh, K. (2010). Preserved heart function and maintained response to cardiac stresses in a genetic model of cardiomyocyte-targeted deficiency of cyclooxygenase-2. Journal of molecular and cellular cardiology, 49(2), 196-209.
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  Cyclooxygenase-1 and -2 are rate-limiting enzymes in the formation of a wide array of bioactive lipid mediators collectively known as prostanoids (prostaglandins, prostacyclins, and thromboxanes). Evidence from clinical trials shows that selective inhibition of the second isoenzyme (cyclooxygenase-2, or Cox-2) is associated with increased risk for serious cardiovascular events and findings from animal-based studies have suggested protective roles of Cox-2 for the heart. To further characterize the function of Cox-2 in the heart, mice with loxP sites flanking exons 4 and 5 of Cox-2 were rendered knockout specifically in cardiac myocytes (Cox-2 CKO mice) via cre-mediated recombination. Baseline cardiac performance of CKO mice remained unchanged and closely resembled that of control mice. Furthermore, myocardial infarct size induced after in vivo ischemia/reperfusion (I/R) injury was comparable between CKO and control mice. In addition, cardiac hypertrophy and function four weeks after transverse aortic constriction (TAC) was found to be similar between the two groups. Assessment of Cox-2 expression in purified adult cardiac cells isolated after I/R and TAC suggests that the dominant source of Cox-2 is found in the non-myocyte fraction. In conclusion, our animal-based analyses together with the cell-based observations portray a limited role of cardiomyocyte-produced Cox-2 at baseline and in the context of ischemic or hemodynamic challenge.
• Streicher, J. M., Kamei, K., Ishikawa, T., Herschman, H., & Wang, Y. (2010). Compensatory hypertrophy induced by ventricular cardiomyocyte-specific COX-2 expression in mice. Journal of molecular and cellular cardiology, 49(1), 88-94.
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  Cyclooxygenase-2 (COX-2) is an important mediator of inflammation in stress and disease states. Recent attention has focused on the role of COX-2 in human heart failure and diseases owing to the finding that highly specific COX-2 inhibitors (i.e., Vioxx) increased the risk of myocardial infarction and stroke in chronic users. However, the specific impact of COX-2 expression in the intact heart remains to be determined. We report here the development of a transgenic mouse model, using a loxP-Cre approach, which displays robust COX-2 overexpression and subsequent prostaglandin synthesis specifically in ventricular myocytes. Histological, functional, and molecular analyses showed that ventricular myocyte specific COX-2 overexpression led to cardiac hypertrophy and fetal gene marker activation, but with preserved cardiac function. Therefore, specific induction of COX-2 and prostaglandin in vivo is sufficient to induce compensated hypertrophy and molecular remodeling.
• Streicher, J. M., Ren, S., Herschman, H., & Wang, Y. (2010). MAPK-activated protein kinase-2 in cardiac hypertrophy and cyclooxygenase-2 regulation in heart. Circulation research, 106(8), 1434-43.
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  Activation of p38 mitogen-activated protein kinase (MAPK) has a significant impact on cardiac gene expression, contractility, extracellular matrix remodeling, and inflammatory response in heart. The p38 kinase pathway also has a controversial role in cardiac hypertrophy. MAPK-activated protein kinase-2 (MK2) is a well-established p38 downstream kinase, yet its contribution to p38-mediated pathological response in heart has not been investigated.
• Streicher, J. M., & Wang, Y. (2008). The role of COX-2 in heart pathology. Cardiovascular & hematological agents in medicinal chemistry, 6(1), 69-79.
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  Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins, and an important anti-inflammation drug target. Recent focus has been placed on the role of COX-2 in heart function and pathology, due to the finding that specific COX-2 inhibitors significantly increased the risk of heart disease in chronic users. However, the exact role of COX-2 in cardiac physiology and disease remains controversial due to the conflicting data reported. Roughly equal numbers of reports have shown either a detrimental role or a protective role for COX-2 in heart in experimental models. Here we attempt to provide a background on the more general roles of COX-2 in pathophysiology, as well as molecular mechanisms employed to control COX-2 expression. This background provides a basis for better understanding the functional role of COX-2 in human heart pathologies, based on the results of COX-2 pharmacological inhibitor studies in humans as well as COX-2 expression in human heart disease. Furthermore, we will explore the experimental evidence implicating different intracellular molecular signaling cascades that regulate COX-2 expression in cardiomyocytes. All of this data permits a more mechanistic understanding of the published studies using pharmacological inhibitors of COX-2 in experimental models of heart pathology. Lastly, we will examine the use of genetic manipulation of COX-2 in mice as one of the future avenues in an attempt to resolve the role of COX-2 in cardiac physiology and pathology.
• Wang, Y., Streicher, J. M., Ren, S., Pu, H., & Gao, J. (2007). The Role of MK2 in p38 MAPK Induced Cardiac COX-2 Regulation and Heart Failure. Journal of Cardiac Failure, 13(6), S84. doi:10.1016/j.cardfail.2007.06.335
• Bethea, C. L., Streicher, J. M., Mirkes, S. J., Sanchez, R. L., Reddy, A. P., & Cameron, J. L. (2005). Serotonin-related gene expression in female monkeys with individual sensitivity to stress. Neuroscience, 132(1), 151-66.
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  Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. In this study, the expression of four genes pivotal to serotonin neural function was assessed in monkeys previously categorized as highly stress resistant (n=3; normal menstrual cyclicity through two stress cycles), medium stress resistant (n=5; ovulatory in the first stress cycle but anovulatory in the second stress cycle), or low stress resistant (i.e. stress-sensitive; n=4; anovulatory as soon as stress is initiated). In situ hybridization and quantitative image analysis was used to measure mRNAs coding for SERT (serotonin transporter), 5HT1A autoreceptor, MAO-A and MAO-B (monoamine oxidases) at six levels of the dorsal raphe nucleus (DRN). Optical density (OD) and positive pixel area were measured with NIH Image software. In addition, serotonin neurons were immunostained and counted at three levels of the DRN. Finally, each animal was genotyped for the serotonin transporter long polymorphic region (5HTTLPR). Stress sensitive animals had lower expression of SERT mRNA in the caudal region of the DRN (P
• Bethea, C. L., Streicher, J. M., Coleman, K., Pau, F. K., Moessner, R., & Cameron, J. L. (2004). Anxious behavior and fenfluramine-induced prolactin secretion in young rhesus macaques with different alleles of the serotonin reuptake transporter polymorphism (5HTTLPR). Behavior genetics, 34(3), 295-307.
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  Anxiety is a normal aspect of human personality, which can manifest in a variety of disorders and other negative traits. The primary treatment for anxiety is the class of drugs known as the selective serotonin reuptake inhibitors (SSRIs), which bind to the serotonin reuptake transporter. The upstream region of the gene that codes for this transporter contains a polymorphism that is an insertion/deletion event that in turn, produces long (l) and short (s) alleles in the population. This particular polymorphism in the serotonin transporter, the 5HTTLPR (serotonin transporter linked polymorphic region), is thought to be involved in the genesis of anxious traits and disorders. Most studies with human subjects have examined adult behavior, which may derive from diverse experiential and environmental backgrounds, as well as genetic differences. To better isolate the effect of genetics, we genotyped 128 infant and juvenile monkeys for the 5HTTLPR and tested for behavioral response in four testing paradigms designed to elicit fearful-anxious behaviors: a free play, remote-controlled car, human intruder, and novel fruit test. The s/s monkeys were found to be behaviorally inhibited in the free play test, engaged in more fear behaviors in the remote-controlled car test, and threatened more in the stare portion of the human intruder test, even though a small number of monkeys were assessed. There was no difference between genotypes of either sex in the prolactin response to fenfluramine. These data indicate greater anxiety in the s/s monkeys for distinct facets of anxious behavior, which are independent of a global neurohormonal challenge test. These neurobehavioral data support recent neuroimaging findings in humans indicating the importance of the 5HTTLPR for amygdala-dependent anxious behavior.
• Huan, J., Streicher, J. M., Bleyle, L. A., & Koop, D. R. (2004). Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells. Toxicology and applied pharmacology, 199(3), 332-43.
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  The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.
• Bethea, C. L., Lu, N. Z., Reddy, A., Shlaes, T., Streicher, J. M., & Whittemore, S. R. (2003). Characterization of reproductive steroid receptors and response to estrogen in a rat serotonergic cell line. Journal of neuroscience methods, 127(1), 31-41.
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  Study of the cellular and molecular consequences of steroid hormone action in the serotonin neural system will provide new avenues for pharmacotherapeutic intervention in mental illness related to reproductive function. However, it is difficult to probe intracellular mechanisms with whole animal models. We sought the steroid receptor compliment and estrogen response of two rat serotonin cell lines in order to determine if they could be of future assistance in this matter. Immunohistochemistry with a panel of antibodies, RT-PCR and a serotonin ELISA were utilized to characterize the RN46A-V1 cells (herein called RN46A), and the subclone RN46A-B14 (herein called B14) that is stably transfected with brain derived neurotrophic factor (BDNF). RN46A and B14 cells express estrogen receptor beta (ERbeta), androgen receptors (AR) and nuclear factor kappa B (NFkappaB) but not estrogen receptor alpha (ERalpha) or progestin receptors (PR). RT-PCR confirmed the presence of ERbeta and the absence of ERalpha and PR in both cell lines. B14 cells contain more immunodetectable BDNF and serotonin than the RN46A parent line. In addition, immunofluorescence for the serotonin reuptake transporter (SERT) was observed in the cell body region of undifferentiated B14 cells. After differentiation at a nonpermissive temperature, SERT immunostaining was observed in both the cell body region and along the extent of the axons. Serotonin content as determined by ELISA was higher in B14 than RN46A cells. Estrogen (0.1 and 1.0 nM) stimulated serotonin in the B14 cells in serum free medium. In summary, the RN46A cells and the B14 subclone contain the same compliment of nuclear steroid receptors as rat raphe serotonin neurons and thus may provide a convenient in vitro model for study of intracellular mechanisms of action of steroid hormones in the context of a serotonin neuron.
• Bethea, C. L., Lu, N. Z., Gundlah, C., & Streicher, J. M. (2002). Diverse actions of ovarian steroids in the serotonin neural system. Frontiers in neuroendocrinology, 23(1), 41-100.
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  All of the serotonin-producing neurons of the mammalian brain are located in 10 nuclei in the mid- and hindbrain regions. The cells of the rostal nuclei project to almost every area of the forebrain and regulate diverse neural processes from higher order functions in the prefrontal cortex such as integrative cognition and memory, to limbic system control of arousal and mood, to diencephalic functions such as pituitary hormone secretion, satiety, and sexual behavior. The more caudal serotonin neurons project to the spinal cord and interact with numerous autonomic and sensory systems. All of these neural functions are sensitive to the presence or absence of the ovarian hormones, estrogen and progesterone. We have shown that serotonin neurons in nonhuman primates contain estrogen receptor beta and progestin receptors. Thus, they are targets for ovarian steroids which in turn modify gene expression. Any change in serotoninergic neural function could be manifested by a change in any of the projection target systems and in this manner, serotonin neurons integrate steroid hormone information and partially transduce their action in the CNS. This article reviews the work conducted in this laboratory on the actions of estrogens and progestins in the serotonin neural system of nonhuman primates. Comparisons to results obtained in other laboratory animal models are made when available and limited clinical data are referenced. The ability of estrogens and progestins to alter the function of the serotonin neural system at various levels provides a cellular mechanism whereby ovarian hormones can impact cognition, mood or arousal, hormone secretion, pain, and other neural circuits.

Proceedings Publications

• Streicher, J. M., Porreca, F., Olson, K. M., Lavigne, J., & Hruby, V. J. (2015). Differential Potencies for Endogenous Dynorphins Indicate Functional Selectivity at the Delta Opioid Receptor. In Peptides 2015, Proceedings of the 24th American Peptide Symposium, 223-224.
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  Opioid receptors (ORs) are G-protein Coupled Receptors (GPCRs), which mediate analgesia, tolerance, withdrawal, GI transit. Classically, ORs couple inhibitory Gi/o proteins and recruit arrestin – a multifaceted scaffold molecule implicated in opioid mediated effects including tolerance, constipation, dysphoria and naseua [1,2]. Upon activation -arrestin and Gi/o induce downstream signaling responses such as reduced cAMP levels. Recent drug discovery efforts identified several functionally selective exogenous opiates which prefer certain signaling pathways at a given receptor – such as G stimulation – to others – such as -arrestin recruitment and generate desired pharmacological properties [3,4]. Noting that most of the 20+ endogenous opioid peptides are nonselective and some opiates display functional selectivity, two important points emerge. First, endogenous and exogenous ligands, such as those used during studies, do not necessarily generate the same effects. Second, two different endogenous opioid peptides may differentially activate a given receptor. Dynorphin A (DynA) and Dynorphin B (DynB) are considered OR agonists, despite binding to the OR at 1.29nM and 3.39 nM [1], respectively. The Dynorphins start with the 5 amino acid Leuenkephalin (Leu-Enk) sequence – YGGFL – traditionally considered a OR agonist followed by distinct C-terminal sequences. Thus, we ask: Do Dynorphin A (1-17), Dynorphin B (1-13) and Leuenkephalin induce functionally selective signaling at the OR?

Poster Presentations

• Bernard, K., Bernard, K., Madhavan, L., Lujan, A., Lujan, A., Falk, T., Corenblum, M. J., Corenblum, M. J., Polt, R. L., Saez, J. L., Saez, J. L., Streicher, J. M., Bartlett, M. J., Bartlett, M. J., Szabò, L. Z., Tanguturi, P., Tanguturi, P., Apostol, C. R., Apostol, C. R., , Apostol, C. R., et al. (2022, Spring). Multimodal effects of systemic PACAP glycopeptide delivery in rodent models of Parkinson’s disease.. 16th International Conference on Alzheimer’s and Parkinson’s Diseases and related neurological disorders, AD/PD™.
• Bernard, K., Lujan, A., Corenblum, M. J., Saez, J. L., Bartlett, M. J., Tanguturi, P., Apostol, C. R., Szabò, L. Z., Streicher, J. M., Polt, R. L., Falk, T., & Madhavan, L. (2022, Spring). Multimodal effects of systemic PACAP glycopeptide delivery in rodent models of Parkinson’s disease.. 16th International Conference on Alzheimer’s and Parkinson’s Diseases and related neurological disorders, AD/PD™.
• Falk, T., Falk, T., Heien, M. L., Heien, M. L., Polt, R. L., Polt, R. L., Streicher, J. M., Streicher, J. M., Madhavan, L., Madhavan, L., Sherman, S. J., Sherman, S. J., Szabo, L., Szabo, L., Apostol, C., Apostol, C., Molnar, G., Molnar, G., Liu, C., , Liu, C., et al. (2021, Spring). Evaluation of a neuroprotective PACAP glycopeptide as systemically delivered CNS active drug to treat motor and cognitive symptoms in two rodent models of Parkinson’s disease.. 15th International Conference on Alzheimer’s and Parkinson’s Diseases and related neurological disorders, AD/PD™.
• Falk, T., Heien, M. L., Polt, R. L., Streicher, J. M., Madhavan, L., Sherman, S. J., Szabo, L., Apostol, C., Molnar, G., Liu, C., Bartlett, M. J., & Bernard, K. (2021, January). Evaluation of a neuroprotective PACAP glycopeptide as systemically delivered CNS active drug to treat Parkinson’s disease.. Society for Neuroscience Global Connectome Conference.
• Falk, T., Heien, M. L., Polt, R. L., Streicher, J. M., Madhavan, L., Sherman, S. J., Szabò, L., Apostol, C. R., Molnar, G., Liu, C., Bartlett, M. J., Morrison, H. W., Lujan, A., & Bernard, K. (2021, Fall). Evaluation of a systemically delivered PACAP glycopeptide as a neuroprotective agent in 2 rodent models of Parkinson’s Disease.. Society for Neuroscience Meeting.
• Falk, T., Polt, R. L., Heien, M. L., Streicher, J. M., Ronaldson, P. T., Szabo, L., Molnar, G., Bartlett, M. J., Liu, C., Apostol, C., & Bernard, K. (2020, Summer). Preclinical evaluation of glycosylated PACAP Hormones to treat Parkinson’s disease and Stroke.. Arizona Alzheimer’s Consortium Annual Abstracts, 2020.
• Falk, T., Polt, R. L., Heien, M. L., Streicher, J. M., Ronaldson, P. T., Szabo, L., Molnar, G., Bartlett, M. J., Liu, C., Apostol, C., & Bernard, K. (2020, Summer). Preclinical evaluation of glycosylated PACAP Hormones to treat Parkinson’s disease and Stroke.. Arizona Alzheimer’s Consortium Annual Abstracts.
• Polt, R. L., Heien, M. L., Falk, T., Streicher, J. M., Ronaldson, P. T., Rowe, R., Szabo, L., Molnar, G., Bernard, K., Bartlett, M. J., Liu, C., & Apostol, C. (2020, Summer). Glycosylated PACAP Hormones as Potential Therapy for Parkinsonism, Stroke and Traumatic Brain Injury.. American Chemical Society National Meeting.
• Sieffert, M. M., Keresztes, A., Barker, N. K., Langlais, P. R., & Streicher, J. M. (2020, April/Spring). Investigation of the Signaling Mechanisms of the Mu-Delta Opioid Receptor Heterodimer. Experimental Biology.
• Streicher, J. M., Langlais, P. R., Barker, N. K., Duron, D., & Gabriel, K. (2020, April/Spring). Does DUSP15 promote activation of ERK MAPK signaling after Hsp90 inhibition in the spinal cord to promote opioid anti-nociception?. Experimental Biology.
• Streicher, J. M., Langlais, P. R., Barker, N. K., Keresztes, A., & Hurtado, K. (2020, April/Spring). Does the Mu-Delta Opioid Receptor Heterodimer Repress Akt Kinase to Reduce Opioid Anti-Nociception?. Experimental Biology.
• Streicher, J. M., Hruby, V., Langlais, P. R., Liu, Z., Barker, N. K., Hguyen, P., Olson, K., & Keresztes, A. (2019, July/Summer). The Mu-Delta Opioid Receptor Heterodimer Acts as a Negative Feedback Brake to Reduce Opioid Anti-Nociception by Repression of CaMKII and Src Signaling. International Narcotics Research Conference - 50TH ANNIVERSARY MEETING.
• Streicher, J. M., Hruby, V., Langlais, P. R., Liu, Z., Barker, N. K., Hguyen, P., Olson, K., & Keresztes, A. (2019, May/Summer). Mu-Delta Opioid Receptor Heterodimer Antagonists: Novel Ligands that Enhance Opioid Analgesia while Reducing Opioid Withdrawal. 2019 NIH Pain Consortium Symposium - Pain Across the Lifespan.
• Polt, R. L., Polt, R. L., Szabo, L., Szabo, L., Smith, C. L., Smith, C. L., Liu, C., Liu, C., Molnar, G., Molnar, G., Heien, M. L., Heien, M. L., Bartlett, M. J., Bartlett, M. J., Apostol, C. R., Apostol, C. R., Hay, M., Hay, M., Falk, T., , Falk, T., et al. (2018, Fall). Glycopeptides as Systemically Delivered CNS Active Drugs from Endogenous Peptide Hormones. Society for Neuroscience.
• Polt, R. L., Streicher, J. M., Heien, M. L., Falk, T., Molnar, G., Bartlett, M. J., Szabo, L., Liu, C., & Apostol, C. C. (2018, Fall). Design and Synthesis of Brain Penetrant Glycopeptide Analogues of Pituitary Adenylate Cyclase Activating Peptide (PACAP) for the Treatment of Parkinson’s Disease. Peptide Therapeutic Symposium at the Salk Institute in San Diego.
• Polt, R. L., Szabo, L., Smith, C. L., Liu, C., Molnar, G., Heien, M. L., Bartlett, M. J., Apostol, C. R., Hay, M., Falk, T., & Streicher, J. M. (2018, Fall). Glycopeptides as Systemically Delivered CNS Active Drugs from Endogenous Peptide Hormones. Society for Neuroscience Meeting.
• Streicher, J. M., Langlais, P. R., Barker, N. K., Keresztes, A., & Sieffert, M. M. (2020, April/Spring). Investigation of the Signaling Mechanisms of the Mu-Delta Opioid Receptor Heterodimer. Experimental Biology.