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.
Receptor heteromers often display distinct pharmacological and functional properties compared to the individual receptor constituents. In this study, we compared the properties of the DOP-KOP heteromer agonist, 6'-guanidinonaltrindole (6'-GNTI), with agonists for DOP ([D-Pen2,5]enkephalin [DPDPE]) and KOP (U50488) in peripheral sensory neurons in culture and in vivo. In primary cultures, all three agonists inhibited PGE 2 -stimulated cAMP accumulation as well as activated extracellular signal-regulated kinase ½ (ERK) with similar efficacy. ERK activation by U50488 was Gi-protein mediated but that by DPDPE or 6'-GNTI was Gi-protein independent (i.e., pertussis toxin insensitive). Brief pretreatment with DPDPE or U50488 resulted in loss of cAMP signaling, however, no desensitization occurred with 6'-GNTI pretreatment. In vivo, following intraplantar injection, all three agonists reduced thermal nociception. The dose-response curves for DPDPE and 6'-GNTI were monotonic whereas the curve for U50488 was an inverted U-shape. Inhibition of ERK blocked the downward phase and shifted the curve for U50488 to the right. Following intraplantar injection of carrageenan, antinociceptive responses to either DPDPE or U50488 were transient but could be prolonged with inhibitors of 12/15-lipoxgenases (LOX). By contrast, responsiveness to 6'-GNTI remained for a prolonged time in the absence of LOX inhibitors. Further, pretreatment with the 12/15-LOX metabolites, 12-and 15hydroxyeicosatetraenoic acid, abolished responses to U50488 and DPDPE but had no effect on 6'-GNTI-mediated responses either in cultures or in vivo. Overall, these results suggest that DOP-KOP heteromers exhibit unique signaling and functional regulation in peripheral sensory neurons and may be a promising therapeutic target for the treatment of pain.
The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR localize to the brain and share overlapping signal transduction facets that contribute to their roles in cognition, mood, learning, and memory. Achieving selective targeting of these receptors is challenged by the similarity in their 5-HT orthosteric binding pockets. A fragment-based discovery approach was employed to design and synthesize novel oleamide analogues as selective 5-HT2CR or dual 5-HT2CR/5-HT2AR positive allosteric modulators (PAMs). Compound 13 (JPC0323) exhibited on-target properties, acceptable plasma exposure and brain penetration, as well as negligible displacement to orthosteric sites of ∼50 GPCRs and transporters. Furthermore, compound 13 suppressed novelty-induced locomotor activity in a 5-HT2CR-dependent manner, suggesting 5-HT2CR PAM, but not 5-HT2AR, activity at the level of the whole organism at the employed doses of 13. We discovered new selective 5-HT2CR PAMs and first-in-class 5-HT2CR/5-HT2AR dual PAMs that broaden the pharmacological toolbox to explore the biology of these vital receptors.
The occurrence of opioid overdose is reaching epidemic proportions; an estimated 115 Americans die daily from an opioid overdose. The current pharmacological treatment relies exclusively on naloxone (Narcan), a competitive antagonist at mu opioid receptors, that can reverse the respiratory depression and sedation produced by opioid agonists like heroin and fentanyl. However, naloxone's effectiveness is limited by its short half‐life. By contrast, methocinnamox (MCAM) has been shown to be a long‐acting mu opioid receptor antagonist and as such, may be more effective for reversing opioid overdose. However, rigorous characterization of the pharmacological properties of MCAM has not yet been done. Here we characterized the antagonist properties of MCAM using HEK cell lines that stably express either human mu, delta or kappa opioid receptors along with a bioluminescent cAMP sensor. Opioid receptor agonist concentration response curves were obtained in response to either DAMGO (mu agonist), DPDPE (delta agonist) or U50488 (kappa agonist) for inhibition of forskolin‐stimulated cellular cAMP levels.We first compared MCAM to that of naloxone at mu receptors. Incubation with the mu receptor agonist DAMGO inhibited forskolin‐stimulated cAMP levels by 40% ± 2 % with an EC50 of 10 nM. Pretreatment with MCAM (10 nM, 10 × Ki) for 15 min shifted the concentration response curve (CRC) to DAMGO to the right 1000‐fold and suppressed the maximal response by 50%. The magnitude of MCAM's antagonist effect increased following a 2h pretreatment. Pretreatment with naloxone (NLX, 100 nM, 10 × Ki) for 15 min also antagonized DAMGO‐mediated inhibition of stimulated cAMP levels. However, in contrast to MCAM, the shift in the DAMGO CRC by NLX was fully surmountable and there was no further antagonist effect with 2h of NLX pretreatment. Furthermore, MCAM‐mediated antagonism following a 2h pretreatment was unchanged after a rigorous washout period whereas antagonism produced by NLX was fully reversible. At delta opioid receptors, pretreatment with MCAM (20 nM, 10 × Ki at delta) for either 15 min or 2h shifted the DPDPE CRC ~10‐fold to the right with no change in the maximal response. Similarly, the CRC for the kappa agonist, U50488, was shifted ~10‐fold to the right with no change in the maximal response following pretreatment for either 15 min or 2h with MCAM (50 nM, 10 × Ki at kappa). Furthermore, antagonism produced by MCAM was fully reversible at both delta‐ and kappa opioid receptors.The time‐dependent nature of antagonism, the depression of the maximal mu agonist response, the lack of reversibility following wash‐out, and the lack of long‐term antagonist effects at either delta or kappa opioid receptors suggests that MCAM is a selective, long acting (perhaps irreversible) mu opioid receptor antagonist.Support or Funding InformationSupported by the ARTT Center of Excellence, UT Health San Antonio.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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