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δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animals. However, the role of agonist efficacy in generating different δ-receptor–mediated behaviors has not been thoroughly investigated. To this end, efficacy requirements for δ-receptor–mediated antihyperalgesia, antidepressant-like effects, and convulsions were evaluated by comparing the effects of the partial agonist BU48 and the full agonist SNC80 and changes in the potency of SNC80 after δ-receptor elimination. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. An antidepressant-like effect was evaluated in the forced swim test. Mice were observed for convulsions after treatment with SNC80 or the δ-opioid receptor partial agonist BU48. Ligand-induced G protein activation was measured by [(35)S]guanosine 5′-O-[γ-thio]triphosphate binding in mouse forebrain tissue, and δ-receptor number was measured by [(3)H]D-Pen(2,5)-enkephalin saturation binding. BU48 produced antidepressant-like effects and convulsions but antagonized SNC80-induced antihyperalgesia and G protein activation. The potency of SNC80 was shifted to the right in δ-receptor heterozygous knockout mice and naltrindole-5′-isothiocyanate–treated mice, and the magnitude of potency shift differed across assays, with the largest shift occurring in the thermal hyperalgesia assay, followed by the forced swim test and then convulsion observation. Naltrindole antagonized these SNC80-induced behaviors with similar potencies, suggesting that these effects are mediated by the same type of δ-receptor. These data suggest that δ-receptor–mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions. These findings further our understanding of the pharmacological mechanisms mediating the in vivo effects of δ-opioid receptor agonists. SIGNIFICANCE STATEMENT: δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animal models. This study evaluates pharmacological properties, specifically the role of agonist efficacy and receptor reserve, underlying these δ-receptor–mediated behaviors. These data suggest that δ-receptor–mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions.

International audience; Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than β-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Background and purpose The δ-opioid receptor is an emerging target for the management of chronic pain and depression. Biased signalling, the preferential activation of one signalling pathway over another downstream of δ-receptors, may generate better therapeutic profiles. BMS 986187 is a positive allosteric modulator of δ-receptors. Here, we ask if BMS 986187 can directly activate the receptor from an allosteric site, without an orthosteric ligand, and if a signalling bias is generated. Experimental approach We used several clonal cell lines expressing δ-receptors, to assess effects of BMS 986187 on events downstream of δ-receptors by measuring G-protein activation, β-arrestin 2 recruitment, receptor phosphorylation, loss of surface receptor expression, ERK1/ERK2 phosphorylation, and receptor desensitization. Key results BMS 986187 is a G protein biased allosteric agonist, relative to β-arrestin 2 recruitment. Despite showing direct and potent G protein activation, BMS 986187 has a low potency to recruit β-arrestin 2. This appears to reflect the inability of BMS 986187 to elicit any significant receptor phosphorylation, consistent with low receptor internalization and a slower onset of desensitization, compared with the full agonist SNC80. Conclusions and implications This is the first evidence of biased agonism mediated through direct binding to an allosteric site on an opioid receptor, without a ligand at the orthosteric site. Our data suggest that agonists targeting δ-receptors, or indeed any GPCR, through allosteric sites may be a novel way to promote signalling bias and thereby potentially produce a more specific pharmacology than can be observed by activation via the orthosteric site.

With the hope of discovering effective analgesics with fewer side effects, attention has recently shifted to allosteric modulators of the opioid receptors. In the past two years, the first chemotypes of positive or silent allosteric modulators (PAMs or SAMs, respectively) of μ- and δ-opioid receptor types have been reported in the literature. During a structure-guided lead optimization campaign with μ-PAMs BMS-986121 and BMS-986122 as starting compounds, we discovered a new chemotype that was confirmed to display μ-PAM or μ-SAM activity depending on the specific substitutions as assessed by endomorphin-1-stimulated β-arrestin2 recruitment assays in Chinese Hamster Ovary (CHO)-μ PathHunter cells. The most active μ-PAM of this series was analyzed further in competition binding and G-protein activation assays to understand its effects on ligand binding and to investigate the nature of its probe dependence.

Positive allosteric modulation of the mu-opioid receptor (MOPr), the site of action of all clinically used opioids, represents a potential approach for the management of pain. We recently reported on positive allosteric modulators of MOPr (mu-PAMs), a class A G protein coupled receptor (GPCR). This study was designed to examine the mechanism of allostery by comparing the degree to which opioid ligand structure governs modulation. To do this we examined the interaction of the mu-PAM, BMS-986122, with a chemically diverse range of MOPr orthosteric ligands. Generally, for full agonists BMS-986122 enhanced the binding affinity and potency to activate G protein with no alteration in the maximal effect. In contrast, lower efficacy agonists including morphine were insensitive to alterations in binding affinity and showed little to no change in potency to stimulate G protein. Instead, there was an increase in maximal G protein stimulation. Antagonists were unresponsive to the modulatory effects of BMS-986122. Sodium is a known endogenous allosteric modulator of MOPr and alters orthosteric agonist affinity and efficacy. The sensitivity of an orthosteric ligand to BMS-986122 was strongly correlated with its sensitivity to NaCl. In addition, BMS-986122 decreased the ability of NaCl to modulate agonist binding in an allosteric fashion. Overall, BMS-986122 displayed marked probe dependence that was based upon the efficacy of the orthosteric ligand and can be explained using the Monod-Wyman-Changeux two-state model of allostery. Furthermore, disruption of the Na(+) ion binding site may represent a common mechanism for allosteric modulation of class A GPCRs.

Opioid receptors are 7‐transmembrane domain receptors that couple to heterotrimeric G proteins. The endogenous ligands for opioid receptors are peptides which bind to the orthosteric site on the receptors. The μ‐opioid receptor is the target for opioid analgesics, while the δ‐opioid receptor has been suggested as a target for pain management, migraine and depression. Similarly, κ‐opioid receptors are involved in pain and depression and nociceptin receptors in pain and mood behaviours. However, exogenous orthosteric ligands for opioid receptors cause a myriad of on‐target side effects. Recently, selective allosteric ligands for μ‐ and δ‐opioid receptors have been described. These compounds bind to a site on the receptor distinct from the orthosteric site. Occupation of this allosteric site leads to modulation of orthosteric ligand binding affinity and/or efficacy. Allosteric modulators may be positive, negative or silent (neutral) (PAMs, NAMs or SAMs respectively). PAMs may have in vivo activity by enhancing the activity of exogenous drugs or endogenous opioid peptides. Enhancing endogenous opioid peptide activity maintains the temporal and spatial distribution of these molecules but improves, and potentially qualitatively changes, activity at their cognate receptors which could limit side effects compared with traditional opioid drugs. In this review, we describe the rationale and promise for the development of allosteric modulators for opioid receptors, the discovery of selective allosteric modulators, the identification of potential allosteric sites on opioid receptors and the mode of action of the modulators. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc

Allosteric modulators of G protein-coupled receptors (GPCRs), including opioid receptors, have been proposed as possible therapeutic agents with enhanced selectivity. BMS-986122 is a positive allosteric modulator (PAM) of the mu-opioid receptor (μ-OR). BMS-986187 is a structurally distinct PAM for the delta-opioid receptor (δ-OR) that has been reported to show 100-fold selectivity in promoting δ-OR over μ-OR agonism. Here we use ligand binding and second messenger assays to show that BMS-986187 is actually an effective PAM at μ-OR and at the kappa opioid receptor (κ-OR), but is ineffective at the nociceptin receptor (NOPR). The affinity of BMS-986187 for δ-ORs and κ-ORs is approximately 20-30 fold higher than for μ-ORs, determined using an allosteric ternary complex model. Moreover, we provide evidence, using a silent allosteric modulator as an allosteric antagonist, that BMS-986187 and BMS-986122 bind to a similar region on all three traditional opioid receptor types (μ-OR, δ-OR and κ-OR). In contrast to the dogma surrounding allosteric modulators the results indicate a possible conserved allosteric binding site across the opioid receptor family that is able to accommodate structurally diverse molecules. The findings have implications for the development of selective allosteric modulators.

Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

International audience; Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Der μ-Opioidrezeptor (μOR) ist einer der altesten pharmakologischen Angriffspunkte fur Analgesie und Sedierung. Seine Aktivierung ubertragt Informationen auf intrazellulare Effektorsysteme, die Schmerzen reduzieren, aber auch zu Toleranzentwicklung, Atemsuppression und Obstipation fuhren. Ein internationales Forscherteam um den Nobelpreistrager B. K. Kobilka stellte die Kristallstruktur des μ-OR und strukturelle Aktivierungsmechanismen dar.

As key targets for chronic pain, opioid receptors (ORs) are still at the forefront of drug discovery efforts. In the hunt for opioid analgesics that are free from adverse effects, recent high-throughput screening campaigns have focused on identifying allosteric modulators, that is molecules that bind non-conserved (allosteric) binding sites on the receptor, and modulate the potency and/or efficacy of ligands at the same (orthosteric) site as the endogenous agonist. Both positive and negative allosteric modulators (PAMs and NAMs) of μ-OR and δ-OR subtypes have recently been identified. While the recent crystal structures of ORs have revealed important details of ligand-receptor binding at the orthosteric site, both the location of allosteric sites on these receptors and the binding mode of opioid allosteric ligands are unknown. Here, we applied all-atom metadynamics to efficiently study the binding of a PAM and a NAM to the δ-OR in the presence of the orthosteric agonist SNC-80 and an explicit lipid-water environment. The dynamics of the allosteric ligands was enhanced by biasing the potential acting on the ligand-receptor distance, and a contact map reflecting the ligand-receptor interaction. The resulting energy landscapes show two deep energy minima for both the simulated PAM and NAM, which correspond to the ligands acquiring specific binding poses within two different nearby receptor pockets defined by transmembrane helices TM1, TM2, and TM7. Notably, the ligand binding poses in the putative allosteric pocket that is closest to the orthosteric ligand overlap with the allosteric site predicted by the fragment-based mapping algorithm FTMAP. In spite of most ligand-receptor interactions being the same for the simulated PAM and NAM within this pocket, there are important differences that are being tested experimentally through mutagenesis to assess their functional role.