Abstract ID 130118Poster Board 294GPR52 is an orphan G protein-coupled receptor (GPCR) that is a promising target for neuropsychiatric disorders including schizophrenia and substance abuse disorders. GPR52 is a constitutively active receptor that couples to Gs/olf G proteins to stimulate adenylyl cyclase and increase intracellular cAMP. As with most understudied GPCRs, there is a paucity of structural diversity among known GPR52 ligands. To expand the chemical space of GPR52 ligands, we utilized a proprietary ligand based-pharmacophore model and virtual screening method to identify new potential compounds for GPR52. Greater than 500k synthesis-ready compounds were screened in silico for their structural similarity to the GPR52 agonist c17. This screening provided a compound priority list of 400 predicted GPR52 ligands. These priority compounds were obtained and subsequently screened at 10 μM using a Glosensor cAMP assay in HEK293 cells expressing human GPR52. The primary screen showed 21 hit compounds activated GPR52 cAMP signaling at > 40% of the maximal response to the positive control agonist 4-(3-(3-fluoro-5-(trifluoromethyl) benzyl)-5-methyl-1 H-1,2,4-triazol-1-yl)-2-methylbenzamide (FTBMT). Subsequent re-testing confirmed activities, and the five most active compounds were evaluated in concentration responses to determine potency (EC50) and efficacy (Emax). All five compounds exhibited low micromolar potency (ranging from 1.6 to 4.0 μM) and were partial agonists when compared to FTBMT. Two promising validated compounds included 2-B3 (N-(5-{[3-(trifluoromethyl)phenyl]methyl}-1,3-thiazol-2-yl)cyclopropanecarboxamide) and 5-E1 (ethyl 2-benzyl-4-methyl-1,3-thiazole-5-carboxylate). Compound 2-B3 showed lower partial agonist activity (Emax = 39 ± 2%) with a potency (pEC50) of 5.80 ± 0.07 [1.6 μM]. Compound 5-E1 showed moderate partial agonist activity (Emax = 71 ± 2%) and potency (pEC50) of 5.47 ± 0.02 [3.4 μM]. 2-B3 was docked into the previously solved GPR52 crystal structure (PBD 6LI0) using AMDock program. 2-B3 docked into a similar binding site as the agonist c17, with potential hydrogen bonding at the ECL2 amino acid D188. Structural derivatives of agonist 2-B3 are being actively screened to define structure activity relationships important for GPR52 potency and efficacy. Taken together, this virtual screening with pharmacological validation provides a viable method for the discovery of ligands for orphan GPCRs. Compounds 2-B3 and 5-E1 are promising GPR52 agonist ligands amenable to further chemical optimization to expand ligand diversity for GPR52.This work was supported in part by grant# T32DA007287 from the National Institutes of Health and the UTMB Center for Addiction Sciences and Therapeutics.

Orphan GPR52 is emerging as a promising neurotherapeutic target. Optimization of previously reported lead 4a employing an iterative drug design strategy led to identification of a series of unique GPR52 agonists, such as 10a (PW0677), 15b (PW0729) and 24f (PW0866), with improved potency and efficacy. Intriguingly, compounds 10a and 24f showed greater bias for G protein/cAMP signaling and induced significantly less in vitro desensitization than parent compound 4a, indicating that reducing GPR52 β-arrestin activity with biased agonism results in sustained GPR52 activation. Further exploration of compounds 15b and 24f indicated improved potency and efficacy, excellent target selectivity, but limited brain exposure warranting further optimization. These balanced and biased GPR52 agonists provide important pharmacological tools to study GPR52 activation, signaling bias, and therapeutic potential for neuropsychiatric and neurological diseases.

Abstract ID 93671Poster Board 549G protein-coupled receptors (GPCRs) are the largest family of eukaryotic membrane receptors, mediate diverse physiological functions, and are targets for ∼30% of marketed drugs, including opioids used to treat pain. Despite considerable resource allocation for more than a decade, the opioid epidemic continues to grow, with >80,000 American lives lost per year due to opioid overdose. The kappa opioid receptor (KOR) is highly expressed in the nervous system and is a promising therapeutic target for pain. Activation of KOR in peripheral sensory neurons produces profound antinociceptive responses, and peripherally-restricted KOR agonists avoid the addictive and deadly adverse effects of centrally-targeted opioids. However, development of peripherally-restricted KOR agonists has been limited by unidentified regulatory pathways that reduce effectiveness and/or induce hyperalgesia.A single injection of norbinaltorphimine (norBNI) can result in weeks-long inhibition of KOR via an unknown mechanism. This long-term antagonism is not through sustained receptor occupancy, but requires norBNI-mediated KOR activation and subsequent c-Jun N-terminal kinase (JNK) signaling. We have shown that this long-term antagonism requires protein synthesis, as this effect is blocked by protein translation inhibitors. Here we identify that the scaffolding protein 14-3-3γ is upregulated in response to norBNI-mediated JNK activation. Knockdown of 14-3-3γ by siRNA eliminated norBNI’s long-term antagonism of KOR-mediated antinociception in the rat hind paw and KOR-mediated cAMP signaling in primary cultures of sensory neurons. Thus, 14-3-3γ is a critical mediator necessary for the long-term KOR antagonism by norBNI. Notably, not all KOR-mediated signaling pathways were disrupted by norBNI pretreatment, as KOR-mediated activation of extracellular signal regulated kinase (ERK) was maintained. Further, overexpression of 14-3-3γ in CHO cells also prevented KOR-mediated cAMP signaling, and these effects appeared selective to KOR as other Gi coupled receptors were not affected.Next, we sought to understand the mechanism of 14-3-3γ inhibition of KOR. The family of 14-3-3 proteins are ubiquitously expressed, highly conserved scaffolds that can modulate signaling complexes through protein-protein interactions with a large and diverse set of interacting partners. Thus, we considered that KOR and 14-3-3γ may form a complex in cells that can bias KOR signaling. KOR and 14-3-3γ co-immunoprecipitate in transfected CHO and HEK293T cells, suggesting the formation of a KOR-14-3-3γ complex. This idea is supported by recent literature identifying 14-3-3 proteins as regulators of certain G protein-coupled receptors. Molecular modeling suggests that 14-3-3γ may interact directly with KOR in a similar manner as beta-arrestins, via the KOR phosphorylated C-terminus or intracellular loops. Ongoing studies are investigating the molecular underpinnings of this interaction, which may be widely applicable to the GPCR family as ∼90% of GPCRs have predicted 14-3-3 binding motifs.This work was supported by the National Institutes of Health grant [R01 DA038645] to WC and KB; RJ was supported in part by the Translational Science Training program at UT Health San Antonio (TST128233); And MW was supported by the NIDA T32 Postdoctoral Training Program in Drug Abuse Research: Behavior & Neurobiology [T32DA031115] and a National Research Service Award [F32DA056212].

Naloxone (NLX) is a competitive antagonist at the mu opioid receptor and is currently the only pharmacological intervention available to treat an opioid overdose. Unfortunately, NLX is limited by its surmountability and its rapid metabolism/clearance can lead to a reemergence of narcotic effects (renarcotization). Methocinnamox (MCAM) is a long‐acting antagonist at mu receptors both in vivo and in vitro. In addition to its orthosteric antagonism, we have found that MCAM differentially modulates orthosteric properties (affinity and/or efficacy) of mu agonists via an allosteric mechanism (Zamora, Smith, et al. (2021). Pharmacology Research & Perspectives. https://doi.org/10.1002/prp2.887). To further explore the interactions between MCAM and the human mu opioid receptor, we compared the rate by which MCAM and NLX reverse cAMP inhibition by the mu receptor agonists DAMGO and fentanyl. Treatment with either DAMGO or fentanyl (~ 30 x EC50) inhibited forskolin (FSK)‐stimulated intracellular cAMP levels. When administered 20 min after agonist, NLX (400 nM; ~100 x Ki) reversed DAMGO‐ and fentanyl‐mediated inhibition of cAMP production at a similar rate (25.4% ± 2.5 min‐1 vs 26.8% ± 5.5 min‐1, respectively). Since reversal by NLX was driven mostly by dissociation of the agonists, the similar rate of reversal by NLX indicate that DAMGO and fentanyl dissociate from the receptor at similar rates. Reversal by MCAM (100 nM; ~ 100 x Ki) however was ligand‐dependent. The rate of reversal of DAMGO‐mediated inhibition by MCAM was 11.7% ± 1.3 min‐1, whereas reversal of fentanyl was slower (4.1% ± 0.9 min‐1; P<0.001). The ligand‐dependence of MCAM’s reversal kinetics on DAMGO vs fentanyl’s inhibition of cAMP production is consistent with the reported allosteric action of MCAM, and may be explained by modulation of ligand dissociation by MCAM. Together, these data suggest in addition to its orthosteric antagonism, MCAM may allosterically modulate the binding of mu agonists.

Long-term inhibition of kappa opioid receptor (KOR) signaling in peripheral pain-sensing neurons is a potential obstacle for development of peripherally-restricted KOR agonists that produce analgesia. Such a long-term inhibitory mechanism is invoked from activation of c-Jun N-terminal kinase (JNK) that follows a single injection of the KOR antagonist norbinaltorphimine (norBNI). This effect requires protein synthesis of an unknown mediator in peripheral pain-sensing neurons. Using 2D difference gel electrophoresis with tandem mass spectrometry, we have identified that the scaffolding protein 14-3-3γ is upregulated in peripheral sensory neurons following activation of JNK with norBNI. Knockdown of 14-3-3γ by siRNA eliminates the long-term reduction in KOR-mediated cAMP signaling by norBNI in peripheral sensory neurons in culture. Similarly, knockdown of 14-3-3γ in the rat hind paw abolished the norBNI-mediated long-term reduction in peripheral KOR-mediated antinociception. Further, overexpression of 14-3-3γ in KOR expressing CHO cells prevented KOR-mediated inhibition of cAMP signaling. These long-term effects are selective for KOR as heterologous regulation of other receptor systems was not observed. These data suggest that 14-3-3γ is both necessary and sufficient for the long-term inhibition of KOR by norBNI in peripheral sensory neurons.

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4-5 months) and aged (26-27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Abstract Opioid overdose is a leading cause of death in the United States. The only treatment available currently is the competitive antagonist, naloxone (Narcan®). Although naloxone is very effective and has saved many lives, as a competitive antagonist it has limitations. Due to the short half‐life of naloxone, renarcotization can occur if the ingested opioid agonist remains in the body longer. Moreover, because antagonism by naloxone is surmountable, renarcotization can also occur in the presence of naloxone if a relatively larger dose of opioid agonist is taken. In such circumstances, a long‐lasting, non‐surmountable antagonist would offer an improvement in overdose treatment. Methocinnamox (MCAM) has been reported to have a long duration of antagonist action at mu opioid receptors in vivo. In HEK cells expressing the human mu opioid receptor, MCAM antagonism of mu agonist‐inhibition of cAMP production was time‐dependent, non‐surmountable and non‐reversible, consistent with (pseudo)‐irreversible binding. In vivo, MCAM injected locally into the rat hindpaw antagonized mu agonist‐mediated inhibition of thermal allodynia for up to 96 h. By contrast, antagonism by MCAM of delta or kappa agonists in HEK cells and in vivo was consistent with simple competitive antagonism. Surprisingly, MCAM also shifted the concentration‐response curves of mu agonists in HEK cells in the absence of receptor reserve in a ligand‐dependent manner. The shift in the [D‐Ala2,N‐MePhe4,Gly‐ol5]‐enkephalin (DAMGO) concentration‐response curve by MCAM was insensitive to naloxone, suggesting that in addition to (pseudo)‐irreversible orthosteric antagonism, MCAM acts allosterically to alter the affinity and/or intrinsic efficacy of mu agonists.

Opioid overdose is a leading cause of death in the US. Naloxone (NLX), a mu opioid receptor (MOR) competitive antagonist, is the only treatment currently available for reversing opioid overdose. Unfortunately, NLX’s effects are limited by a short duration of action and ease of surmountability by opioid agonists. Methocinnamox (MCAM) is a novel MOR antagonist that has a long duration of action in vivo and holds promise as a better treatment for overdose and perhaps opioid use disorder. In this study, we compared the antagonist properties of MCAM to that of NLX and beta‐funaltrexamine (β‐FNA; an irreversible MOR antagonist). In HEK cells that express human MOR, DAMGO inhibited forskolin‐stimulated cAMP levels in a concentration dependent manner with an EC50 of 10 nM and a maximal inhibition of 40%. Pretreatment (15 min or 2 hrs) with NLX (100 nM, 10 x Ki) shifted the DAMGO concentration response curve (CRC) to the right, 100‐fold, in a fully surmountable manner that was independent of pretreatment time and fully reversed following NLX washout. Pretreatment with β‐FNA (10 nM) reduced the DAMGO maximal response in a time‐dependent, non‐surmountable and irreversible manner with no effect on the potency of DAMGO. By contrast to both β‐FNA and NLX, pretreatment with MCAM (10 nM) reduced the maximal response in a time‐dependent, non‐surmountable, non‐washable, and irreversible manner and shifted the DAMGO CRC to the right 1000‐fold. We hypothesized that this rightward shift in the DAMGO CRC may be due to allosteric properties of MCAM at MOR. A hallmark of allosterism is ligand dependence, so we next tested effects of MCAM, β‐FNA and NLX on a different mu opioid agonist, fentanyl. As expected, pretreatment with β‐FNA reduced the maximal response to fentanyl in a non‐surmountable manner without affecting potency, whereas NLX pretreatment shifted the CRC of fentanyl to the right (decreased the potency) in a manner that was fully surmountable and independent of pretreatment time. By contrast, pretreatment with MCAM reduced the maximal response to fentanyl in a non‐surmountable manner and shifted the CRC to the left (increased potency). Altogether, these data suggest that MCAM may be an irreversible orthosteric antagonist and an allosteric modulator at MOR.Support or Funding InformationSupported by NIH/NIDA RO1 grant DA048214

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Chemotherapy‐induced peripheral neuropathy (CIPN) is a prevalent adverse effect of anti‐cancer chemotherapeutics like paclitaxel (PTX), a microtubule targeting agent known to induce severe and persistent neuropathic pain. The development of CIPN not only diminishes the quality of life of cancer patients, but also downgrades their cancer prognosis as dose reduction or cessation of their anti‐cancer regimen are the only clinical interventions currently available for CIPN. Thus, the development of a pharmacological intervention that limits the neuropathic side‐effects of anti‐cancer chemotherapy is urgently needed. Here we tested the neuroprotective effects of the aminopropyl carbazole, P7C3‐A20 (A20), and several A20 derivatives in a rat model of paclitaxel‐induced peripheral neuropathy. All experiments were done by investigators blinded to the drug treatments. Racemic A20 (2.2, 6.6, 20 mg/kg, i.p., q.d.), or vehicle, was administered daily to male Sprague‐Dawley rats over a 14‐day period beginning 2 days before PTX treatment (11.7 mg/kg/day, i.p.; every other day for 3 injections). A20 dose‐dependently attenuated PTX‐induced mechanical allodynia in rats. Evaluation of intraepidermal nerve fiber (IENF) density in rat paw biopsies using immunohistochemical staining revealed that treatment with PTX produced a profound loss in IENF density that was prevented by treatment with A20. Further, statistical analysis indicated strong correlations between IENF density, a diagnostic marker of CIPN in humans, and nociceptive threshold to mechanical stimuli. Enantiomer specific mixtures of A20 (20 mg/kg, i.p., q.d.), racemic A20 (20 mg/kg, i.p., q.d.), or vehicle were then administered utilizing the same behavioral paradigm and PTX dosing schedule as above. Consistent with our previous data, racemic A20 attenuated PTX‐induced mechanical allodynia and the S‐enantiomer was identified as the active component of the racemic mixture. We have also tested the neuroprotective efficacy of four novel A20 derivatives coded as compounds A through D. The A20 derivatives were administered daily at 20 mg/kg by i.p. injection and mechanical allodynia was assayed as described above. One compound, Drug C, provided robust protection against PTX‐induced mechanical allodynia, whereas the compounds A, B, and D demonstrated limited neuroprotective efficacy. Taken together, A20 and perhaps its derivatives are an exciting new class of pharmacological agents that may have clinical utility for alleviating peripheral neuropathies caused by various anti‐cancer chemotherapeutics.Support or Funding InformationSupported in part by Calico Life Sciences and Proneurotech.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chemotherapy-induced peripheral neuropathy (CIPN) arises from collateral damage to peripheral afferent sensory neurons by anticancer pharmacotherapy, leading to debilitating neuropathic pain. No effective treatment for CIPN exists, short of dose-reduction which worsens cancer prognosis. Here, we report that stimulation of nicotinamide phosphoribosyltransferase (NAMPT) produced robust neuroprotection in an aggressive CIPN model utilizing the frontline anticancer drug, paclitaxel (PTX). Daily treatment of rats with the first-in-class NAMPT stimulator, P7C3-A20, prevented behavioral and histologic indicators of peripheral neuropathy, stimulated tissue NAD recovery, improved general health, and abolished attrition produced by a near maximum-tolerated dose of PTX. Inhibition of NAMPT blocked P7C3-A20-mediated neuroprotection, whereas supplementation with the NAMPT substrate, nicotinamide, potentiated a subthreshold dose of P7C3-A20 to full efficacy. Importantly, P7C3-A20 blocked PTX-induced allodynia in tumored mice without reducing antitumoral efficacy. These findings identify enhancement of NAMPT activity as a promising new therapeutic strategy to protect against anticancer drug-induced peripheral neurotoxicity.