Josef Vagner

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• Gillman, S., Schiff, H., Pederson, W., Sandoval, E. C., Rivera, B., Dussor, G., Price, T. J., Vágner, J., DeFea, K., & Boitano, S. (2023).

  A humanized protease-activated receptor-2 mouse as a model for allergic asthma

  . Physiology, 38(S1). doi:10.1152/physiol.2023.38.s1.5734467
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  Asthma is a complex disease characterized by airway hyperresponsiveness (AHR) inflammation, and mucus overproduction, and has differential presentation in males and females. There exists strong demand to improve upon current asthma treatments by targeting upstream signaling pathways, including the G-protein coupled receptor protease-activated receptor-2 (PAR2). We have recently shown effectiveness of full (C391) and ß-arrestin biased (C781) PAR2 antagonism in limiting allergen-induced asthma indicators in mouse models (C57Bl/6 and Balb/C). To better test PAR2 antagonists as asthma drugs, we have developed a transgenic mouse model that expresses human PAR2 without mouse PAR2 expression in a C57Bl/6 background (htgPAR2). The htgPAR2 mice have heightened responses to asthma allergens [house dust mite (HDM) or Alternaria alternata] following acute exposure. Limited sex differences were observed in the HDM challenge model. However, when challenged with A. alternata there was significantly higher AHR in the male mice and significantly higher inflammatory and mucus responses in the female mice. The full and ß-arrestin biased PAR2 antagonists also displayed differential effects with C781 providing broader control of allergen-induced indicators (AHR, inflammation and mucus overproduction). We conclude that the htgPAR2 mouse model is a promising tool for preclinical in vivo screening of potential asthma treatments targeting PAR2. Grants from the National Institute of Health (NS098826, AI140257, HL16024, HL152942) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
• Kume, M., Ahmad, A., DeFea, K., Vágner, J., Dussor, G., Boitano, S., & Price, T. J. (2023).

  Protease-Activated Receptor 2 (PAR2) expressed in sensory neurons contributes to signs of pain and neuropathy in paclitaxel treated mice

  . The Journal of Pain, 24(11), 1980-1993. doi:10.1101/2023.02.12.528175
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  ABSTRACT Background and Purpose Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a common, dose-limiting side effect of cancer therapy. Protease-activated receptor 2 (PAR2) is implicated in a variety of pathologies, including CIPN. In this study, we demonstrate the role of PAR2 expressed in sensory neurons in a paclitaxel (PTX)-induced model of CIPN in mice. Experimental Approach CIPN was induced in both PAR2 knockout/WT mice and mice with PAR2 ablated in sensory neurons via the intraperitoneal injection of paclitaxel. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. We then examined immunohistochemical staining of dorsal root ganglion (DRG) and hind paw skin samples from CIPN mice to measure satellite cell gliosis and intra-epidermal nerve fiber (IENF) density. Pharmacological reversal of CIPN pain was tested with the PAR2 antagonist C781 Key Results Mechanical allodynia caused by paclitaxel treatment was alleviated in PAR2 knockout mice of both sexes. In the PAR2 sensory neuronal conditional knockout (cKO) mice, both mechanical allodynia and facial grimacing were attenuated in mice of both sexes. In the dorsal root ganglion of the paclitaxel-treated PAR2 cKO mice, satellite glial cell activation was reduced compared to control mice. IENF density analysis of the skin showed that the paclitaxel-treated control mice have a reduction in nerve fiber density while the PAR2 cKO mice had a comparable skin innervation as the vehicle-treated animals. Similar results were seen with satellite cell gliosis in the DRG where gliosis induced by PTX was absent in PAR cKO mice. Finally, C781 was able to transiently reverse established PTX-evoked mechanical allodynia. Conclusions and Implications Our work demonstrates that PAR2 expressed in sensory neurons plays a key role in paclitaxel-induced mechanical allodynia, spontaneous pain and signs of neuropathy, suggesting PAR2 as a possible therapeutic target in multiple aspects of paclitaxel CIPN.
• Kume, M., Ahmad, A., Shiers, S., Burton, M. D., DeFea, K., Vágner, J., Dussor, G., Boitano, S., & Price, T. J. (2023).

  C781, a β-Arrestin Biased Antagonist at Protease-Activated Receptor-2 (PAR2), Displays in vivo Efficacy Against Protease-Induced Pain in Mice

  . J Pain, 24(4), 605-616. doi:10.1016/j.jpain.2022.11.006
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  Given the limited options and often harmful side effects of current analgesics and the suffering caused by the opioid crisis, new classes of pain therapeutics are needed. Protease-activated receptors (PARs), particularly PAR2, are implicated in a variety of pathologies, including pain. Since the discovery of the role of PAR2 in pain, development of potent and specific antagonists has been slow. In this study, we describe the in vivo characterization of a novel small molecule/peptidomimetic hybrid compound, C781, as a β-arrestin-biased PAR2 antagonist. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. Pharmacokinetic studies were done to assess pharmacokinetic/pharmacodynamic relationship in vivo. We used both prevention and reversal paradigms with protease treatment to determine whether C781 could attenuate protease-evoked pain. C781 effectively prevented and reversed mechanical and spontaneous nociceptive behaviors in response to small molecule PAR2 agonists, mast cell activators, and neutrophil elastase. The ED50 of C781 (intraperitoneal dosing) for inhibition of PAR2 agonist (20.9 ng 2-AT)-evoked nociception was 6.3 mg/kg. C781 was not efficacious in the carrageenan inflammation model. Pharmacokinetic studies indicated limited long-term systemic bioavailability for C781 suggesting that optimizing pharmacokinetic properties could improve in vivo efficacy. Our work demonstrates in vivo efficacy of a biased PAR2 antagonist that selectively inhibits β-arrestin/MAPK signaling downstream of PAR2. Given the importance of this signaling pathway in PAR2-evoked nociception, C781 exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development. PERSPECTIVE: Our work provides evidence that PAR2 antagonists that only block certain aspects of signaling by the receptor can be effective for blocking protease-evoked pain in mice. This is important because it creates a rationale for developing safer PAR2-targeting approaches for pain treatment.
• Mason, B. N., Hassler, S., DeFea, K., Boitano, S., Vágner, J., Price, T. J., & Dussor, G. (2023).

  PAR2 activation in the dura causes acute behavioral responses and priming to glyceryl trinitrate in a mouse migraine model

  . The Journal of Headache and Pain, 24(42). doi:10.1186/s10194-023-01574-5
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  Migraine is a severely debilitating disorder that affects millions of people worldwide. Studies have indicated that activation of protease-activated receptor-2 (PAR2) in the dura mater causes headache responses in preclinical models. It is also well known that vasodilators such as nitric oxide (NO) donors can trigger migraine attacks in migraine patients but not controls. In the current study we examined whether activation of PAR2 in the dura causes priming to the NO donor glyceryl trinitrate (GTN).A preclinical behavioral model of migraine was used where stimuli (PAR2 agonists: 2at-LIGRL-NH2 (2AT) or neutrophil elastase (NE); and IL-6) were applied to the mouse dura through an injection made at the intersection of the lamdoidal and sagittal sutures on the skull. Following dural injection, periorbital von Frey thresholds and facial grimace responses were measured until their return to baseline. GTN was then given by intraperitoneal injection and periorbital hypersensitivity and facial grimace responses observed until they returned to baseline.We found that application of the selective PAR2 agonist 2at-LIGRL-NH2 (2AT) onto the dura causes headache-related behavioral responses in WT but not PAR2-/- mice with no differences between sexes. Additionally, dural PAR2 activation with 2AT caused priming to GTN (1 mg/kg) at 14 days after primary dural stimulation. PAR2-/- mice showed no priming to GTN. We also tested behavioral responses to the endogenous protease neutrophil elastase, which can cleave and activate PAR2. Dural neutrophil elastase caused both acute responses and priming to GTN in WT but not PAR2-/- mice. Finally, we show that dural IL-6 causes acute responses and priming to GTN that is identical in WT and PAR2-/- mice, indicating that IL-6 does not act through PAR2 in this model.These results indicate that PAR2 activation in the meninges can cause acute headache behavioral responses and priming to an NO donor, and support further exploration of PAR2 as a novel therapeutic target for migraine.
• Schiff, H., Gillman, S., Pederson, W., Dussor, G., Price, T., Vagner, J., DeFea, K., & Boitano, S. (2023).

  Beta-arrestin-biased protease-activated receptor-2 antagonists preferentially limit allergen-induced responses in vivo.

  . Physiology, 38(S1). doi:10.1152/physiol.2023.38.s1.5734466
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  Biased signaling in G protein-coupled receptors (GPCRs) has emerged as a target for drug development. Protease-activated receptor-2 (PAR2) is a GPCR present in the airway epithelium with biased signaling that has been shown to trigger both detrimental effects [airway hyperresponsiveness (AHR), inflammation, mucus overproduction] and beneficial effects (bronchorelaxation) associated with allergen-induced asthma. These dual effects have been shown to be dictated by the two primary signaling pathways downstream of PAR2 activation: Gαq/Ca 2+ signaling is associated with bronchorelaxation, and β-arrestin/MAPK signaling is associated with AHR, inflammation and mucus overproduction. We have developed full (C391 which antagonizes Gαq/Ca 2+ and β-arrestin/MAPK signaling) and biased (C781 which antagonizes β-arrestin/MAPK signaling) antagonists of PAR2 and tested their efficacy in acute allergen-induced asthma mouse models. Both full and β-arrestin/MAPK signaling compounds attenuated AHR and inflammation in mouse models, and to a lesser extent, mucus overproduction. In a human bronchial model, the full PAR2 antagonist C391 prevented PAR2- dependent bronchial relaxation while the biased PAR2 antagonist C781 did not. Antagonism by C781 and other biased PAR2 antagonists can fine tune and effectively improve safety profiles for drug development in asthma treatment. Grants from the National Institute of Health (NS098826, AI140257, HL16024, HL152942) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
• Vagner, J. (2022). Alternaria Alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression. Biochemical and Biophysical Research Communication, 591, 13-19. doi:10.1016/j.bbrc.2021.12.090
• Boitano, S., DeFea, K. A., Vagner, J., Price, T. J., Kiela, P. R., Khanna, R., Moutal, A., Schiff, H. V., Rivas, C. M., Boitano, S., DeFea, K. A., Vagner, J., Price, T. J., Kiela, P. R., Khanna, R., Moutal, A., Schiff, H. V., & Rivas, C. M. (2021). Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.. Biophysical and Biochemical Research Communications, 591, 13-19. doi:10.1016/j.bbrc.2021.12.090
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  Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to initiate the physiological response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiological responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.
• Mwirigi, J., Kume, M., Hassler, S. N., Ahmad, A., Ray, P. R., Jiang, C., Chamessian, A., Mseeh, N., Ludwig, B. P., Rivera, B. D., Nieman, M. T., Van de Ven, T., Ji, R. R., Dussor, G., Boitano, S., Vagner, J., & Price, T. J. (2021). A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist. The journal of pain.
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  The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3 mice. We characterized other nociceptive phenotypes in PAR3 mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3 mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. Perspective: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.
• Hassler, S. N., Kume, M., Mwirigi, J. M., Ahmad, A., Shiers, S., Wangzhou, A., Ray, P. R., Belugin, S. N., Naik, D. K., Burton, M. D., Vagner, J., Boitano, S., Akopian, A. N., Dussor, G., & Price, T. J. (2020). The cellular basis of protease-activated receptor 2-evoked mechanical and affective pain. JCI insight, 5(11).
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  Protease-activated receptor 2 (PAR2) has long been implicated in inflammatory and visceral pain, but the cellular basis of PAR2-evoked pain has not been delineated. Although PAR2-evoked pain has been attributed to sensory neuron expression, RNA-sequencing experiments show ambiguous F2rl1 mRNA detection. Moreover, many pharmacological tools for PAR2 are nonspecific, acting also on the Mas-related GPCR family (Mrg) that are highly enriched in sensory neurons. We sought to clarify the cellular basis of PAR2-evoked pain. We developed a PAR2-conditional knockout mouse and specifically deleted PAR2 in all sensory neurons using the PirtCre mouse line. Our behavioral findings show that PAR2 agonist-evoked mechanical hyperalgesia and facial grimacing, but not thermal hyperalgesia, are dependent on PAR2 expression in sensory neurons that project to the hind paw in male and female mice. F2rl1 mRNA is expressed in a discrete population (~4%) of mostly small-diameter sensory neurons that coexpress the Nppb and IL31ra genes. This cell population has been implicated in itch, but our work shows that PAR2 activation in these cells causes clear pain-related behaviors from the skin. Our findings show that a discrete population of DRG sensory neurons mediate PAR2-evoked pain.
• Burt, J. M., Ek Vitorin, J. F., Vagner, J., Solan, J. L., Lampe, P. D., Boitano, S. A., Cotter, M. L., Burt, J. M., Ek Vitorin, J. F., Vagner, J., Solan, J. L., Lampe, P. D., Boitano, S. A., & Cotter, M. L. (2019). The lipidated connexin mimetic peptide SRPTEKT-Hdc is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform. Am J Physiol Cell Physiol, 317(4), C825-C842.
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 microM, 1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl (Hdc) lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-Hdc) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca(2+)-wave propagation (IC50 64.8 pM) and HCh-mediated dye uptake (IC50 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT-Hdc inhibition of Ca(2+)-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca(2+)-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT-Hdc potently inhibited GJCh-mediated Ca(2+)-wave propagation (IC50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca(2+)-wave propagation, dye coupling, and dye uptake were largely unaffected (IC50 12.3 muM) by SRPTEKT-Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT-Hdc is a potent inhibitor of physiological Ca(2+)-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.
• Cotter, M. L., Boitano, S., Lampe, P. D., Solan, J. L., Vagner, J., Ek-Vitorin, J. F., & Burt, J. M. (2019). The lipidated connexin mimetic peptide SRPTEKT- is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform. American journal of physiology. Cell physiology, 317(4), C825-C842.
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 µM, 1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl () lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca-wave propagation (IC 64.8 pM) and HCh-mediated dye uptake (IC 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT- inhibition of Ca-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT- potently inhibited GJCh-mediated Ca-wave propagation (IC 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca-wave propagation, dye coupling, and dye uptake were largely unaffected (IC 12.3 μM) by SRPTEKT- in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT- is a potent inhibitor of physiological Ca-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.
• Hart, N. J., Weber, C., Papas, K. K., Limesand, S. W., Vagner, J., & Lynch, R. M. (2019). Multivalent activation of GLP-1 and sulfonylurea receptors modulates β-cell second-messenger signaling and insulin secretion. American journal of physiology. Cell physiology, 316(1), C48-C56.
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  Linking two pharmacophores that bind different cell surface receptors into a single molecule can enhance cell-targeting specificity to cells that express the complementary receptor pair. In this report, we developed and tested a synthetic multivalent ligand consisting of glucagon-like peptide-1 (GLP-1) linked to glibenclamide (Glb) (GLP-1/Glb) for signaling efficacy in β-cells. Expression of receptors for these ligands, as a combination, is relatively specific to the β-cell in the pancreas. The multivalent GLP-1/Glb increased both intracellular cAMP and Ca, although Ca responses were significantly depressed compared with the monomeric Glb. Moreover, GLP-1/Glb increased glucose-stimulated insulin secretion in a dose-dependent manner. However, unlike the combined monomers, GLP-1/Glb did not augment insulin secretion at nonstimulatory glucose concentrations in INS 832/13 β-cells or human islets of Langerhans. These data suggest that linking two binding elements, such as GLP-1 and Glb, into a single bivalent ligand can provide a unique functional agent targeted to β-cells.
• Hassler, S. N., Ahmad, F. B., Burgos-Vega, C. C., Boitano, S., Vagner, J., Price, T. J., & Dussor, G. (2019). Protease activated receptor 2 (PAR2) activation causes migraine-like pain behaviors in mice. Cephalalgia : an international journal of headache, 39(1), 111-122.
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  Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice.
• Hassler, S., Ahmad, F. B., Burgos-Vega, C. C., Boitano, S. A., Vagner, J., Price, T. J., & Dussor, G. (2019). Protease activated receptor 2 (PAR2) activation casues migraine-like pain behaviors in mice. Cephalalgia, 39(1), 111-122. doi:10.1177/0333102418779548
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  BACKGROUND: Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice. METHODS: Ratiometric Ca(++) imaging was performed on primary trigeminal and dural cell cultures after application of 2at-LIGRL-NH2, a specific protease activated receptor 2 agonist. Cutaneous hypersensitivity and facial grimace was measured in wild-type and protease activated receptor 2(-/-) mice after dural application of 2at-LIGRL-NH2 or compound 48-80, a mast cell degranulator. Behavioral experiments were also conducted in mice after dural application of 2at-LIGRL-NH2 (2AT) in the presence of either C391, a selective protease activated receptor 2 antagonist, or sumatriptan. RESULTS: 2at-LIGRL-NH2 evoked Ca(2+) signaling in mouse trigeminal neurons, dural fibroblasts and in meningeal afferents. Dural application of 2at-LIGRL-NH2 or 48-80 caused dose-dependent grimace behavior and mechanical allodynia that were attenuated by either local or systemic application of C391 as well as in protease activated receptor 2(-/-) mice. Nociceptive behavior after dural injection of 2at-LIGRL-NH2 was also attenuated by sumatriptan. CONCLUSIONS: Functional protease activated receptor 2 receptors are expressed on both dural afferents and fibroblasts and activation of dural protease activated receptor 2 produces migraine-like behavioral responses. Protease activated receptor 2 may link resident immune cells to meningeal nociceptor activation, driving migraine-like pain and implicating protease activated receptor 2 as a therapeutic target for migraine in humans.
• Cotter, M. L., Boitano, S., Vagner, J., & Burt, J. M. (2018). Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca-wave propagation. American journal of physiology. Cell physiology, 315(2), C141-C154.
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  Connexin (Cx) mimetic peptides (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) reversibly inhibit hemichannel (HCh) and gap junction channel (GJCh) function in a concentration- and time-dependent manner (HCh: ~5 µM, 1 h). We hypothesized that addition of a hexadecyl tail to SRPTEKT (SRPTEKT- Hdc) would improve its ability to concentrate in the plasma membrane and consequently increase its inhibitory efficacy. We show that SRPTEKT- Hdc inhibited intercellular Ca-wave propagation in Cx43-expressing MDCK and rabbit tracheal epithelial cells in a time (61-75 min)- and concentration (IC: 66 pM)-dependent manner, a concentration efficacy five orders of magnitude lower than observed for the nonlipidated Gap27. HCh-mediated dye uptake was inhibited by SRPTEKT- Hdc with similar efficacy. Following peptide washout, HCh-mediated dye uptake was restored to control levels, whereas Ca-wave propagation was only partially restored. Scrambled and reverse sequence lipidated peptides had no detectable inhibitory effect on Ca-wave propagation or dye uptake. Cx43 expression was unchanged by SRPTEKT- Hdc incubation; however, Triton-insoluble Cx43 was reduced by SRPTEKT- Hdc exposure and reversed following washout. In summary, our results show that SRPTEKT- Hdc blocked HCh function and intercellular Ca signaling at concentrations that minimally affected dye coupling. Selective inhibition of intercellular Ca signaling, likely indicative of channel conformation-specific SRPTEKT- Hdc binding, could contribute significantly to the protective effects of these mimetic peptides in settings of injury. Our data also demonstrate that lipidation represents a paradigm for development of highly potent, efficacious, and selective mimetic peptide inhibitors of hemichannel and gap junction channel-mediated signaling.
• Cromey, B., McDaniel, A., Matsunaga, T., Vagner, J., Kieu, K. Q., & Banerjee, B. (2018). Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging. Journal of biomedical optics, 23(4), 1-8.
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  Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery.
• Karolak, A., Estrella, V. C., Huynh, A. S., Chen, T., Vagner, J., Morse, D. L., & Rejniak, K. A. (2018). Targeting Ligand Specificity Linked to Tumor Tissue Topological Heterogeneity via Single-Cell Micro-Pharmacological Modeling. Scientific reports, 8(1), 3638.
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  Targeted therapy has held promise to be a successful anticancer treatment due to its specificity towards tumor cells that express the target receptors. However, not all targeting drugs used in the clinic are equally effective in tumor eradication. To examine which biochemical and biophysical properties of targeted agents are pivotal for their effective distribution inside the tumor and their efficient cellular uptake, we combine mathematical micro-pharmacological modeling with in vivo imaging of targeted human xenograft tumors in SCID mice. The mathematical model calibrated to experimental data was used to explore properties of the targeting ligand (diffusion and affinity) and ligand release schemes (rates and concentrations) with a goal to identify the properties of cells and ligands that enable high receptor saturation. By accounting for heterogeneities typical of in vivo tumors, our model was able to identify cell- and tissue-level barriers to efficient drug uptake. This work provides a base for utilizing experimentally measurable properties of a ligand-targeted agent and patient-specific attributes of the tumor tissue to support the development of novel targeted imaging agents and for improvement in their delivery to individual tumor cells.
• Moy, J. K., Khoutorsky, A., Asiedu, M. N., Black, B. J., Kuhn, J. L., Barragán-Iglesias, P., Megat, S., Burton, M. D., Burgos-Vega, C. C., Melemedjian, O. K., Boitano, S., Vagner, J., Gkogkas, C. G., Pancrazio, J. J., Mogil, J. S., Dussor, G., Sonenberg, N., & Price, T. J. (2017). The MNK-eIF4E Signaling Axis Contributes to Injury-Induced Nociceptive Plasticity and the Development of Chronic Pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 37(31), 7481-7499.
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  Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation ( ). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from mice. These effects were recapitulated in mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in and mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain. Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5' cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain.
• Price, T. J., Sonenberg, N., Dussor, G. G., Mogil, J. S., Pancrazio, J. J., Gkogkas, C. G., Vagner, J., Boitano, S., Melemedjian, O. K., Burgos-Vega, C. C., Burton, M. D., Megat, S., Barragan-Iglesias, P., Kuhn, J. L., Black, B. J., Asiedu, M. N., Khoutorsky, A. A., & Moy, J. K. (2017). The MNK - eIF4E signaling axis controls injury-induced nociceptive plasticity and the transition to chronic pain. Journal of Neuroscience, 37(31), 7481-7499. doi:10.1523/JNEUROSCI.0220-17.2017
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  Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4E(S209A) ). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca(2+) imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4E(S209A) mice. These effects were recapitulated in Mnk1/2(-/-) mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4E(S209A) and Mnk1/2(-/-) mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5' cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain.
• Cohen, A. S., Patek, R., Enkemann, S. A., Johnson, J. O., Chen, T., Toloza, E., Vagner, J., & Morse, D. L. (2016). Delta-Opioid Receptor (δOR) Targeted Near-Infrared Fluorescent Agent for Imaging of Lung Cancer: Synthesis and Evaluation In Vitro and In Vivo. Bioconjugate chemistry, 27(2), 427-38.
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  In the United States, lung cancer is the leading cause of cancer death and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta-opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR-targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high and low endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR-specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer.
• Harpel, K., Baker, R. D., Amirsolaimani, B., Mehravar, S., Vagner, J., Matsunaga, T. O., Banerjee, B., & Kieu, K. (2016). Imaging of targeted lipid microbubbles to detect cancer cells using third harmonic generation microscopy. Biomedical optics express, 7(7), 2849-60.
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  The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained and ultrasound gel may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate, label-free imaging of tissues and cells with high resolution and contrast. We have recently determined a novel application of MPM to be used for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.
• Huynh, A. S., Estrella, V., Stark, V. E., Cohen, A. S., Chen, T., Casagni, T. J., Josan, J. S., Lloyd, M. C., Johnson, J., Kim, J., Hruby, V. J., Vagner, J., & Morse, D. L. (2016). Tumor Targeting and Pharmacokinetics of a Near-Infrared Fluorescent-Labeled δ-Opioid Receptor Antagonist Agent, Dmt-Tic-Cy5. Molecular pharmaceutics, 13(2), 534-44.
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  Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.
• Kim, J. V., Megat, S., Moy, J. K., Asiedu, M. N., Mejia, G. L., Vagner, J., & Price, T. J. (2016). Neuroligin 2 regulates spinal GABAergic plasticity in hyperalgesic priming, a model of the transition from acute to chronic pain. Pain, 157(6), 1314-1324.
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  Plasticity in inhibitory receptors, neurotransmission, and networks is an important mechanism for nociceptive signal amplification in the spinal dorsal horn. We studied potential changes in GABAergic pharmacology and its underlying mechanisms in hyperalgesic priming, a model of the transition from acute to chronic pain. We find that while GABAA agonists and positive allosteric modulators reduce mechanical hypersensitivity to an acute insult, they fail to do so during the maintenance phase of hyperalgesic priming. In contrast, GABAA antagonism promotes antinociception and a reduction in facial grimacing after the transition to a chronic pain state. During the maintenance phase of hyperalgesic priming, we observed increased neuroligin (nlgn) 2 expression in the spinal dorsal horn. This protein increase was associated with an increase in nlgn2A splice variant mRNA, which promotes inhibitory synaptogenesis. Disruption of nlgn2 function with the peptide inhibitor, neurolide 2, produced mechanical hypersensitivity in naive mice but reversed hyperalgesic priming in mice previously exposed to brain-derived neurotrophic factor. Neurolide 2 treatment also reverses the change in polarity in GABAergic pharmacology observed in the maintenance of hyperalgesic priming. We propose that increased nlgn2 expression is associated with hyperalgesic priming where it promotes dysregulation of inhibitory networks. Our observations reveal new mechanisms involved in the spinal maintenance of a pain plasticity and further suggest that disinhibitory mechanisms are central features of neuroplasticity in the spinal dorsal horn.
• Weterings, E., Gallegos, A. C., Dominick, L. N., Cooke, L. S., Bartels, T. N., Vagner, J., Matsunaga, T. O., & Mahadevan, D. (2016). A novel small molecule inhibitor of the DNA repair protein Ku70/80. DNA repair, 43, 98-106.
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  Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.
• Weterings, E., Mahadevan, D., Vagner, J., & Matsunaga, T. O. (2016). A Novel Small Molecule Inhibitor of the DNA Repair Protein Ku70/80. DNA Repair, 43, 98-106.
• Boitano, S., Hoffman, J., Flynn, A. N., Asiedu, M. N., Tillu, D. V., Zhang, Z., Sherwood, C. L., Rivas, C. M., DeFea, K. A., Vagner, J., & Price, T. J. (2015). The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo. British journal of pharmacology, 172(18), 4535-4545.
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  Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described.
• Burt, J. M., Vagner, J., Boitano, S. A., & Maura, C. L. (2017). Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca2+-wave propagation. American Journal of Physiology: Cell Physiology, 315(2), C141-C154. doi:10.1152/ajpcell.00156.2017
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap 27: SRPTEKTIFII, and Gap36: KRDPCHQVDCFLSRPTEK) have been used as reversible, Cx-specific inhibitors of gap junction intercellular communication. Typically, these peptides require relatively high concentrations (i.e., 100–200 μM with IC50s ~20–30 μM) to achieve inhibition. We hypothesized that lipidation of Cx mimetic peptides would improve their ability to concentrate in the plasma membrane and thus more effectively interfere with intercellular signaling. A hexadecyl (Hdc) lipid tail was added to the conserved SRPTEKT peptide sequence of Gap27 (SRPTEKT-Hdc) and its efficacy in inhibiting mechanically-induced intercellular Ca2+-wave propagation in confluent cultures of both Madin-Darby canine kidney epithelial cells expressing Cx43 (MDCK43) and primary-cultured rabbit tracheal epithelial cells (RTECs) assessed. SRPTEKT-Hdc reversibly inhibited in a time- and concentration- dependent manner intercellular Ca2+-wave propagation in both cell types with an IC50 in the picomolar range (i.e., 31.8–138.9 pM), five orders of magnitude lower than the non-lipidated Gap27 peptide. SRPTEKT-Hdc was less effective at inhibiting dye coupling. Scrambled and reverse sequence lipidated peptides had no detectable inhibitory effect on Ca2+-wave propagation. Immunoblot analysis revealed no change in Cx43 expression, but a small reduction in plaque-associated Cx43 following SRPTEKT-Hdc incubation. Our results suggest SRPTEKT-Hdc potently inhibits, in a channel conformation-specific manner, intercellular IP3 signaling at concentrations that minimally affect dye (electrical) coupling. In summary, lipidation of mimetic peptides represents a paradigm for development of highly potent, efficacious and selective inhibitors of gap junction-mediated intercellular signaling.
• Lipovka, Y., Chen, H., Vagner, J., Price, T. J., Tsao, T. S., & Konhilas, J. P. (2015). Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase. Bioscience reports, 35(5).
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  Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα.
• Steyn, L. V., Ananthakrishnan, K., Anderson, M. J., Patek, R., Kelly, A., Vagner, J., Lynch, R. M., & Limesand, S. W. (2015). A Synthetic Heterobivalent Ligand Composed of Glucagon-Like Peptide 1 and Yohimbine Specifically Targets β Cells Within the Pancreas. Molecular imaging and biology, 17(4), 461-70.
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  β Cell specificity for a heterobivalent ligand composed of glucagon-like peptide-1 (GLP-1) linked to yohimbine (GLP-1/Yhb) was evaluated to determine its utility as a noninvasive imaging agent.
• Tillu, D. V., Hassler, S. N., Burgos-Vega, C. C., Quinn, T. L., Sorge, R. E., Dussor, G., Boitano, S., Vagner, J., & Price, T. J. (2015). Protease-activated receptor 2 activation is sufficient to induce the transition to a chronic pain state. Pain, 156(5), 859-867.
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  Protease-activated receptor type 2 (PAR2) is known to play an important role in inflammatory, visceral, and cancer-evoked pain based on studies using PAR2 knockout (PAR2(-/-)) mice. We have tested the hypothesis that specific activation of PAR2 is sufficient to induce a chronic pain state through extracellular signal-regulated kinase (ERK) signaling to protein synthesis machinery. We have further tested whether the maintenance of this chronic pain state involves a brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (trkB)/atypical protein kinase C (aPKC) signaling axis. We observed that intraplantar injection of the novel highly specific PAR2 agonist, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at), evokes a long-lasting acute mechanical hypersensitivity (median effective dose ∼12 pmoles), facial grimacing, and causes robust hyperalgesic priming as revealed by a subsequent mechanical hypersensitivity and facial grimacing to prostaglandin E2 (PGE2) injection. The promechanical hypersensitivity effect of 2-at is completely absent in PAR2(-/-) mice as is hyperalgesic priming. Intraplantar injection of the upstream ERK inhibitor, U0126, and the eukaryotic initiation factor (eIF) 4F complex inhibitor, 4EGI-1, prevented the development of acute mechanical hypersensitivity and hyperalgesic priming after 2-at injection. Systemic injection of the trkB antagonist ANA-12 similarly inhibited PAR2-mediated mechanical hypersensitivity, grimacing, and hyperalgesic priming. Inhibition of aPKC (intrathecal delivery of ZIP) or trkB (systemic administration of ANA-12) after the resolution of 2-at-induced mechanical hypersensitivity reversed the maintenance of hyperalgesic priming. Hence, PAR2 activation is sufficient to induce neuronal plasticity leading to a chronic pain state, the maintenance of which is dependent on a BDNF/trkB/aPKC signaling axis.
• Boitano, S., Hoffman, J., Tillu, D. V., Asiedu, M. N., Zhang, Z., Sherwood, C. L., Wang, Y., Dong, X., Price, T. J., & Vagner, J. (2014). Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering. PloS one, 9(6), e99140.
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  Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.
• Elshan, N. G., Patek, R., Vagner, J., & Mash, E. A. (2014). Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Analytical biochemistry, 464, 24-9.
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  Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination as well as the success of metal ion removal on attempted purification. We compared the use of Empore chelating disks and Chelex 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore chelating disks were found to give much higher recoveries of the probes under the conditions used. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.
• Hart, N. J., Chung, W. J., Weber, C., Ananthakrishnan, K., Anderson, M., Patek, R., Zhang, Z., Limesand, S. W., Vagner, J., & Lynch, R. M. (2014). Hetero-bivalent GLP-1/glibenclamide for targeting pancreatic β-cells. Chembiochem : a European journal of chemical biology, 15(1), 135-45.
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  G protein-coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell-specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin-secreting pancreatic β-cells are the sulfonylurea-1 (SUR1) and the glucagon-like peptide-1 (GLP-1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP-1 (7-36 GLP-1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β-cells, by using fluorescence microscopy or time-resolved saturation and competition binding assays, respectively. Once the ligand binds to β-cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium-labelled GLP-1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high-affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β-cell targeting in vivo.
• Sherwood, C. L., Daines, M. O., Price, T. J., Vagner, J., & Boitano, S. (2014). A highly potent agonist to protease-activated receptor-2 reveals apical activation of the airway epithelium resulting in Ca2+-regulated ion conductance. American journal of physiology. Cell physiology, 307(8), C718-26.
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  The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca(2+)-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2 (-/-) mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca(2+) concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca(2+)-activated Cl(-) and large-conductance K(+) channels that were blocked with a Ca(2+)-activated Cl(-) channel inhibitor and clotrimazole, respectively. Stimulation of Cl(-) efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents.
• Alleti, R., Vagner, J., Dehigaspitiya, D. C., Moberg, V. E., Elshan, N. G., Tafreshi, N. K., Brabez, N., Weber, C. S., Lynch, R. M., Hruby, V. J., Gillies, R. J., Morse, D. L., & Mash, E. A. (2013). Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors. Bioorganic & medicinal chemistry, 21(17), 5029-38.
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  Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.
• Barkey, N. M., Preihs, C., Cornnell, H. H., Martinez, G., Carie, A., Vagner, J., Xu, L., Lloyd, M. C., Lynch, V. M., Hruby, V. J., Sessler, J. L., Sill, K. N., Gillies, R. J., & Morse, D. L. (2013). Development and in vivo quantitative magnetic resonance imaging of polymer micelles targeted to the melanocortin 1 receptor. Journal of medicinal chemistry, 56(16), 6330-8.
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  Recent emphasis has focused on the development of rationally designed polymer-based micelle carriers for drug delivery. The current work tests the hypothesis that target specificity can be enhanced by micelles with cancer-specific ligands. In particular, we describe the synthesis and characterization of a new gadolinium texaphyrin (Gd-Tx) complex encapsulated in an IVECT micellar system, stabilized through Fe(III) cross-linking and targeted with multiple copies of a specific ligand for the melanocortin 1 receptor (MC1R), which has been evaluated as a cell-surface marker for melanoma. On the basis of comparative MRI experiments, we have been able to demonstrate that these Gd-Tx micelles are able to target MC1R-expressing xenograft tumors in vitro and in vivo more effectively than various control systems, including untargeted or un-cross-linked Gd-Tx micelles. Taken in concert, the findings reported herein support the conclusion that appropriately designed micelles are able to deliver contrast agent payloads to tumors expressing the MC1R.
• Tafreshi, N. K., Silva, A., Estrella, V. C., McCardle, T. W., Chen, T., Jeune-Smith, Y., Lloyd, M. C., Enkemann, S. A., Smalley, K. S., Sondak, V. K., Vagner, J., & Morse, D. L. (2013). In vivo and in silico pharmacokinetics and biodistribution of a melanocortin receptor 1 targeted agent in preclinical models of melanoma. Molecular pharmaceutics, 10(8), 3175-85.
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  The melanocortin 1 receptor (MC1R) is overexpressed in most melanoma metastases, making it a promising target for imaging of melanomas. In this study, the expression of MC1R in a large fraction of patients with melanoma was confirmed using mRNA and tissue microarray. Here, we have characterized the in vivo tumor and tissue distribution and pharmacokinetics (PK) of uptake and clearance of a MC1R specific peptidomimetic ligand conjugated to a near-infrared fluorescent dye. We propose an interdisciplinary framework to bridge the different time and space scales of ligand-tumor-host interactions: intravital fluorescence microscopy to quantify probe internalization at the cellular level, a xenograft tumor model for whole body pharmacokinetics, and a computational pharmacokinetic model for integration and interpretation of experimental data. Administration of the probe into mice bearing tumors with high and low MC1R expression demonstrated normalized image intensities that correlated with expression levels (p < 0.05). The biodistribution study showed high kidney uptake as early as 30 min postinjection. The PK computational model predicted the presence of receptors in the kidneys with a lower affinity, but at higher numbers than in the tumors. As the mouse kidney is known to express the MC5R, this hypothesis was confirmed by both coinjection of a ligand with higher MC5R affinity compared to MC1R and by injection of lower probe concentrations (e.g., 1 nmol/kg), both leading to decreased kidney accumulation of the MC1R ligand. In addition, through this interdisciplinary approach we could predict the rates of ligand accumulation and clearance into and from organs and tumors, and the amount of injected ligand required to have maximum specific retention in tumors. These predictions have potential to aid in the translation of a targeted agent from lab to the clinic. In conclusion, the characterized MC1R-specific probe has excellent potential for in vivo detection of melanoma metastases. The process of cell-surface marker validation, targeted imaging probe development, and in vitro, in vivo, and in silico characterization described in this study can be generally applied to preclinical development of targeted agents.
• Josan, J. S., R, C., Yoo, B., Lynch, R. M., Pagel, M. D., Vagner, J., & Hruby, V. J. (2011). Fluorescent and lanthanide labeling for ligand screens, assays, and imaging.. Methods in molecular biology (Clifton, N.J.), 716, 89-126.
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  PMID: 21318902;PMCID: PMC3365840;Abstract: The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening -studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.
• Vagner, J., Boitano, S., Flynn, A. N., Schulz, S. M., Hoffman, J., Price, T. J., & Vagner, J. -. (2011). Potent agonists of the protease activated receptor 2 (PAR2). Journal of medicinal chemistry, 54(5).
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  Novel peptidomimetic pharmacophores to PAR(2) were designed based on the known activating peptide SLIGRL-NH(2). A set of 15 analogues was evaluated with a model cell line (16HBE14o-) that highly expresses PAR(2). Cells exposed to the PAR(2) activating peptide with N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH(2)) initiated increases in intracellular calcium concentration ([Ca(2+)](i) EC(50) = 0.84 μM) and in vitro physiological responses as measured by the xCELLigence real time cell analyzer (RTCA EC(50) = 138 nM). We discovered two selective PAR(2) agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca(2+) EC(50) = 1.77 μM, RTCA EC(50) = 142 nM) and compound 2 (6-aminonicotinyl; Ca(2+) EC(50) = 2.60 μM, RTCA EC(50) = 311 nM). Unlike the previously described agonist, these novel agonists are devoid of the metabolically unstable 2-furoyl modification and thus provide potential advantages for PAR(2) peptide design for in vitro and in vivo studies. The novel compounds described herein also serve as a starting point for structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists and antagonists of PAR(2).
• Vagner, J., Hruby, V. J., & Vagner, J. -. (2006). High throughput synthesis of peptides and peptidomimetics. Chimica oggi, 24(4).
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  Peptide synthesis has been developed into one of the most efficient synthetic procedures in organic chemistry. The problems of orthogonal functional group protection and amide bond formation without racemization have been developed in a number of ingenious strategies. Optimization, in particular, has been achieved in stepwise solid phase synthesis. This in turn made possible the development of combinatorial synthesis allowing the synthesis of millions of peptide compounds of high purity in a few days. A variety of methodologies and strategies have been developed and continue to be developed to determine structures and to evaluate peptides and peptidomimetics. The development of methods for solid phase synthesis of a variety of organic and inorganic structures using similar strategies as in peptide synthesis are being vigorously pursued. However, existing instrumentation and technology is not sufficient to cover current demands for peptides, and thus new approaches and technologies for cost-effective synthesis of peptide arrays are needed.

Proceedings Publications

• Weterings, E., Mahadevan, D., Matsunaga, T. O., & Vagner, J. (2016, April 2016). A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80.. In 107th Annual Meeting of the American Association for Cancer Research,.
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  Meeting abstract and poster presentation