Critical Assessment of G Protein-Biased Agonism at the μ-Opioid Receptor
Authors: Alexander Gillis, Andrea Kliewer, Eamonn Kelly, Graeme Henderson, Macdonald J. Christie, Stefan Schulz, Meritxell Canals
Journal: Trends in Pharmacological Sciences (2020)

G protein-biased agonists of the µ-opioid receptor have been hypothesized to be an improved class of opioid analgesicsEarly studies in the β-arrestin2-knockout mouse that suggested a separation between the signaling mediating analgesia versus side effects have not been reproduced, and a 'G protein-biased' mutant MOPr mouse does not support the original proposal.There is now evidence for a G protein-dependent signal mediating deleterious opioid effects.The previously observed G protein bias of many recently developed MOPr agonists has been confounded by assay amplification. Such ligands may in fact be unbiased, with low intrinsic efficacy.Current evidence does not support the proposal that G protein-biased agonism at the MOPr will provide substantially improved therapeutic profiles.Low intrinsic efficacy represents an alternative mechanism by which novel opioids may display wider therapeutic windows. G protein-biased agonists of the μ-opioid receptor (MOPr) have been proposed as an improved class of opioid analgesics. Recent studies have been unable to reproduce the original experiments in the β-arrestin2-knockout mouse that led to this proposal, and alternative genetic models do not support the G protein-biased MOPr agonist hypothesis. Furthermore, assessment of putatively biased ligands has been confounded by several factors, including assay amplification. As such, the extent to which current lead compounds represent mechanistically novel, extremely G protein-biased agonists is in question, as is the underlying assumption that β-arrestin2 mediates deleterious opioid effects. Addressing these current challenges represents a pressing issue to successfully advance drug development at this receptor and improve upon current opioid analgesics. G protein-biased agonists of the μ-opioid receptor (MOPr) have been proposed as an improved class of opioid analgesics. Recent studies have been unable to reproduce the original experiments in the β-arrestin2-knockout mouse that led to this proposal, and alternative genetic models do not support the G protein-biased MOPr agonist hypothesis. Furthermore, assessment of putatively biased ligands has been confounded by several factors, including assay amplification. As such, the extent to which current lead compounds represent mechanistically novel, extremely G protein-biased agonists is in question, as is the underlying assumption that β-arrestin2 mediates deleterious opioid effects. Addressing these current challenges represents a pressing issue to successfully advance drug development at this receptor and improve upon current opioid analgesics. G protein-biased agonists (see Glossary) of the μ-opioid receptor (MOPr) have been widely proposed to be a novel, substantially improved class of analgesics [1.Siuda E.R. et al.Biased mu-opioid receptor ligands: a promising new generation of pain therapeutics.Curr. Opin. Pharmacol. 2017; 32: 77-84Crossref PubMed Scopus (99) Google Scholar,2.Smith J.S. et al.Biased signalling: from simple switches to allosteric microprocessors.Nat. Rev. Drug Discov. 2018; 17: 243-260Crossref PubMed Scopus (327) Google Scholar]. The prototypical such agonist, oliceridine (TRV130), has proceeded to Phase III clinical trials [3.Viscusi E.R. et al.APOLLO-1: a randomized placebo and active-controlled phase III study investigating oliceridine (TRV130), a G protein-biased ligand at the µ-opioid receptor, for management of moderate-to-severe acute pain following bunionectomy.J. Pain Res. 2019; 12: 927-943Crossref PubMed Scopus (72) Google Scholar], and was recently approved in the USA for use in acute pain. Existing, clinically approved opioid analgesics, such as morphine, oxycodone, and fentanyl, are MOPr agonists that provide pain relief that is unmatched by other drug classes. Current opioids have an array of adverse effects, including respiratory depression, constipation, and euphoria, as well as inducing tolerance and dependence over time. These important limitations of opioid analgesics have all been proposed to be addressed by G protein-biased MOPr agonists (Box 1). However, recent results have brought into question the hypothesis that underpins the proposed mechanism of action of this anticipated new drug class, that β-arrestin2 mediates deleterious opioid effects. In addition, there is evidence that challenges both the extent of the G protein bias of lead compounds and the extent to which such compounds are likely to represent improved analgesics.Box 1The G Protein-Biased MOPr Agonism HypothesisG protein-biased MOPr agonists maintain the eponymous G protein signal, established to mediate analgesia, while minimizing recruitment of β-arrestin to the receptor. MOPr/β-arrestin interactions are proposed to mediate, or to positively facilitate, deleterious opioid effects, including respiratory depression, constipation, tolerance, and physical dependence. Therefore, MOPr agonists, which are G protein biased are hypothesized to be dramatically improved, less addictive analgesics that induce lesser respiratory depression and constipation (Figure I). However, the initial β-arrestin2-knockout results [10.Raehal K.M. et al.Morphine side effects in beta-arrestin 2 knockout mice.J. Pharmacol. Exp. Ther. 2005; 314: 1195-1201Crossref PubMed Scopus (413) Google Scholar,38.Bohn L.M. et al.Enhanced morphine analgesia in mice lacking beta-arrestin 2.Science. 1999; 286: 2495-2498Crossref PubMed Scopus (767) Google Scholar,40.Bohn L.M. et al.Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence.Nature. 2000; 408: 720-723Crossref PubMed Scopus (695) Google Scholar] have not been repeated in later experiments [22.Kliewer A. et al.Morphine-induced respiratory depression is independent of β-arrestin2 signalling.Br. J. Pharmacol. 2020; 177: 2923-2931Crossref PubMed Scopus (93) Google Scholar,26.Azevedo Neto J. et al.Biased versus partial agonism in the search for safer opioid analgesics.Molecules. 2020; 25: E3870Crossref PubMed Scopus (27) Google Scholar,42.Koblish M. et al.TRV0109101, a G protein-biased agonist of the µ-opioid receptor, does not promote opioid-induced mechanical allodynia following chronic administration.J. Pharmacol. Exp. Ther. 2017; 362: 254-262Crossref PubMed Scopus (26) Google Scholar], while studies of mice expressing a phosphorylation-deficient mutant MOPr that does not recruit β-arrestins also do not support the G protein-biased agonist hypothesis [23.Kliewer A. et al.Phosphorylation-deficient G-protein-biased mu-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects.Nat. Commun. 2019; 10: 367Crossref PubMed Scopus (128) Google Scholar]. Furthermore, substantial evidence has accumulated for G protein mechanisms mediating respiratory depression and constipation, as well as analgesia.The profile of apparently G protein-biased (see main text), minimally β-arrestin-recruiting lead compounds is not significantly improved over established opioids, such as morphine [76.Conibear A.E. Kelly E. A biased view of mu-opioid receptors?.Mol. Pharmacol. 2019; 96: 542-549Crossref PubMed Scopus (54) Google Scholar]. The prototypical G protein-biased MOPr agonist, oliceridine (but see [51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar,53.Vasudevan L. et al.Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform.Biochem. Pharmacol. 2020; 177113910Crossref PubMed Scopus (25) Google Scholar]), was initially reported in rodent studies to have an improved separation in potency between antinociceptive effects and both respiratory depression and constipation [6.DeWire S.M. et al.A G protein-biased ligand at the mu-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine.J. Pharmacol. Exp. Ther. 2013; 344: 708-717Crossref PubMed Scopus (407) Google Scholar]. Clinical trial results of this drug candidate have been mixed, with persistent respiratory depression and constipation, but a marginally improved therapeutic window observed in some measures compared with morphine [3.Viscusi E.R. et al.APOLLO-1: a randomized placebo and active-controlled phase III study investigating oliceridine (TRV130), a G protein-biased ligand at the µ-opioid receptor, for management of moderate-to-severe acute pain following bunionectomy.J. Pain Res. 2019; 12: 927-943Crossref PubMed Scopus (72) Google Scholar,77.Soergel D.G. et al.Biased agonism of the mu-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers.Pain. 2014; 155: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar,78.Ayad S. et al.Evaluating the incidence of opioid-induced respiratory depression associated with oliceridine and morphine as measured by the frequency and average cumulative duration of dosing interruption in patients treated for acute postoperative pain.Clin. Drug Investig. 2020; 40: 755-764Crossref PubMed Scopus (15) Google Scholar]. In regard to abuse-related effects, volunteers rated oliceridine similarly to morphine in a drug effects questionnaire [77.Soergel D.G. et al.Biased agonism of the mu-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers.Pain. 2014; 155: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar], and, in rodents, both the self-administration of, and the facilitation of, intracranial self-stimulation induced by oliceridine was similar to other opioids [79.Altarifi A.A. et al.Effects of acute and repeated treatment with the biased mu opioid receptor agonist TRV130 (oliceridine) on measures of antinociception, gastrointestinal function, and abuse liability in rodents.J. Psychopharmacol. 2017; 31: 730-739Crossref PubMed Scopus (107) Google Scholar,80.Zamarripa C.A. et al.The G-protein biased mu-opioid agonist, TRV130, produces reinforcing and antinociceptive effects that are comparable to oxycodone in rats.Drug Alcohol Depend. 2018; 192: 158-162Crossref PubMed Scopus (51) Google Scholar]. Additionally, oliceridine generalized to fentanyl in a drug-discrimination procedure, similarly to other MOPr agonists [69.Schwienteck K.L. et al.Effectiveness comparisons of G-protein biased and unbiased mu opioid receptor ligands in warm water tail-withdrawal and drug discrimination in male and female rats.Neuropharmacology. 2019; 150: 200-209Crossref PubMed Scopus (21) Google Scholar]. A second putatively G protein-biased compound, PZM21 (but see [48.Hill R. et al.The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.Br. J. Pharmacol. 2018; 175: 2653-2661Crossref PubMed Scopus (107) Google Scholar,51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar]), was initially reported not to induce respiratory depression, constipation, or to be rewarding in a conditioned-place preference test in mice [8.Manglik A. et al.Structure-based discovery of opioid analgesics with reduced side effects.Nature. 2016; 537: 185-190Crossref PubMed Scopus (544) Google Scholar]. However, several later studies found substantial respiratory depression to be induced by PZM21 [48.Hill R. et al.The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.Br. J. Pharmacol. 2018; 175: 2653-2661Crossref PubMed Scopus (107) Google Scholar,51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar], albeit with some variation in potency between experimenters, while the observation of minimal conditioned-place preference has been reproduced [81.Kudla L. et al.Functional characterization of a novel opioid, PZM21, and its effects on the behavioural responses to morphine.Br. J. Pharmacol. 2019; 176: 4434-4445Crossref PubMed Scopus (17) Google Scholar]. A recent examination of the reinforcing effects of PZM21 in non-human primates showed similar abuse liability to oxycodone [82.Ding H. et al.Antinociceptive, reinforcing, and pruritic effects of the G-protein signalling-biased mu opioid receptor agonist PZM21 in non-human primates.Br. J. Anaesth. 2020; 125: 596-604Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar], and antinociceptive tolerance comparable to morphine develops following prolonged treatment [48.Hill R. et al.The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.Br. J. Pharmacol. 2018; 175: 2653-2661Crossref PubMed Scopus (107) Google Scholar].Recently, the G protein-bias factor of a family of newly developed compounds was correlated to the separation in potency between antinociception and respiratory depression [7.Schmid C.L. et al.Bias factor and therapeutic window correlate to predict safer opioid analgesics.Cell. 2017; 171: 1165-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar]. Most significantly, SR17018 was reported not to induce respiratory depression [7.Schmid C.L. et al.Bias factor and therapeutic window correlate to predict safer opioid analgesics.Cell. 2017; 171: 1165-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar], although again later studies have shown decreases in respiratory rate at minimally antinociceptive doses [51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar]. Studies of this ligand are significantly hampered by extremely poor solubility [46.Grim T.W. et al.A G protein signaling-biased agonist at the mu-opioid receptor reverses morphine tolerance while preventing morphine withdrawal.Neuropsychopharmacology. 2020; 45: 416-425Crossref PubMed Scopus (34) Google Scholar,51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar], and a recent study reported high, DAMGO-like arrestin recruitment by SR17018 [58.Gutman E.S. et al.G-protein biased opioid agonists: 3-hydroxy-N-phenethyl-5-phenylmorphans with three-carbon chain substituents at C9.RSC Med. Chem. 2020; 11: 896-904Crossref Google Scholar]. G protein-biased MOPr agonists maintain the eponymous G protein signal, established to mediate analgesia, while minimizing recruitment of β-arrestin to the receptor. MOPr/β-arrestin interactions are proposed to mediate, or to positively facilitate, deleterious opioid effects, including respiratory depression, constipation, tolerance, and physical dependence. Therefore, MOPr agonists, which are G protein biased are hypothesized to be dramatically improved, less addictive analgesics that induce lesser respiratory depression and constipation (Figure I). However, the initial β-arrestin2-knockout results [10.Raehal K.M. et al.Morphine side effects in beta-arrestin 2 knockout mice.J. Pharmacol. Exp. Ther. 2005; 314: 1195-1201Crossref PubMed Scopus (413) Google Scholar,38.Bohn L.M. et al.Enhanced morphine analgesia in mice lacking beta-arrestin 2.Science. 1999; 286: 2495-2498Crossref PubMed Scopus (767) Google Scholar,40.Bohn L.M. et al.Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence.Nature. 2000; 408: 720-723Crossref PubMed Scopus (695) Google Scholar] have not been repeated in later experiments [22.Kliewer A. et al.Morphine-induced respiratory depression is independent of β-arrestin2 signalling.Br. J. Pharmacol. 2020; 177: 2923-2931Crossref PubMed Scopus (93) Google Scholar,26.Azevedo Neto J. et al.Biased versus partial agonism in the search for safer opioid analgesics.Molecules. 2020; 25: E3870Crossref PubMed Scopus (27) Google Scholar,42.Koblish M. et al.TRV0109101, a G protein-biased agonist of the µ-opioid receptor, does not promote opioid-induced mechanical allodynia following chronic administration.J. Pharmacol. Exp. Ther. 2017; 362: 254-262Crossref PubMed Scopus (26) Google Scholar], while studies of mice expressing a phosphorylation-deficient mutant MOPr that does not recruit β-arrestins also do not support the G protein-biased agonist hypothesis [23.Kliewer A. et al.Phosphorylation-deficient G-protein-biased mu-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects.Nat. Commun. 2019; 10: 367Crossref PubMed Scopus (128) Google Scholar]. Furthermore, substantial evidence has accumulated for G protein mechanisms mediating respiratory depression and constipation, as well as analgesia. The profile of apparently G protein-biased (see main text), minimally β-arrestin-recruiting lead compounds is not significantly improved over established opioids, such as morphine [76.Conibear A.E. Kelly E. A biased view of mu-opioid receptors?.Mol. Pharmacol. 2019; 96: 542-549Crossref PubMed Scopus (54) Google Scholar]. The prototypical G protein-biased MOPr agonist, oliceridine (but see [51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar,53.Vasudevan L. et al.Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform.Biochem. Pharmacol. 2020; 177113910Crossref PubMed Scopus (25) Google Scholar]), was initially reported in rodent studies to have an improved separation in potency between antinociceptive effects and both respiratory depression and constipation [6.DeWire S.M. et al.A G protein-biased ligand at the mu-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine.J. Pharmacol. Exp. Ther. 2013; 344: 708-717Crossref PubMed Scopus (407) Google Scholar]. Clinical trial results of this drug candidate have been mixed, with persistent respiratory depression and constipation, but a marginally improved therapeutic window observed in some measures compared with morphine [3.Viscusi E.R. et al.APOLLO-1: a randomized placebo and active-controlled phase III study investigating oliceridine (TRV130), a G protein-biased ligand at the µ-opioid receptor, for management of moderate-to-severe acute pain following bunionectomy.J. Pain Res. 2019; 12: 927-943Crossref PubMed Scopus (72) Google Scholar,77.Soergel D.G. et al.Biased agonism of the mu-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers.Pain. 2014; 155: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar,78.Ayad S. et al.Evaluating the incidence of opioid-induced respiratory depression associated with oliceridine and morphine as measured by the frequency and average cumulative duration of dosing interruption in patients treated for acute postoperative pain.Clin. Drug Investig. 2020; 40: 755-764Crossref PubMed Scopus (15) Google Scholar]. In regard to abuse-related effects, volunteers rated oliceridine similarly to morphine in a drug effects questionnaire [77.Soergel D.G. et al.Biased agonism of the mu-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers.Pain. 2014; 155: 1829-1835Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar], and, in rodents, both the self-administration of, and the facilitation of, intracranial self-stimulation induced by oliceridine was similar to other opioids [79.Altarifi A.A. et al.Effects of acute and repeated treatment with the biased mu opioid receptor agonist TRV130 (oliceridine) on measures of antinociception, gastrointestinal function, and abuse liability in rodents.J. Psychopharmacol. 2017; 31: 730-739Crossref PubMed Scopus (107) Google Scholar,80.Zamarripa C.A. et al.The G-protein biased mu-opioid agonist, TRV130, produces reinforcing and antinociceptive effects that are comparable to oxycodone in rats.Drug Alcohol Depend. 2018; 192: 158-162Crossref PubMed Scopus (51) Google Scholar]. Additionally, oliceridine generalized to fentanyl in a drug-discrimination procedure, similarly to other MOPr agonists [69.Schwienteck K.L. et al.Effectiveness comparisons of G-protein biased and unbiased mu opioid receptor ligands in warm water tail-withdrawal and drug discrimination in male and female rats.Neuropharmacology. 2019; 150: 200-209Crossref PubMed Scopus (21) Google Scholar]. A second putatively G protein-biased compound, PZM21 (but see [48.Hill R. et al.The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.Br. J. Pharmacol. 2018; 175: 2653-2661Crossref PubMed Scopus (107) Google Scholar,51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar]), was initially reported not to induce respiratory depression, constipation, or to be rewarding in a conditioned-place preference test in mice [8.Manglik A. et al.Structure-based discovery of opioid analgesics with reduced side effects.Nature. 2016; 537: 185-190Crossref PubMed Scopus (544) Google Scholar]. However, several later studies found substantial respiratory depression to be induced by PZM21 [48.Hill R. et al.The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.Br. J. Pharmacol. 2018; 175: 2653-2661Crossref PubMed Scopus (107) Google Scholar,51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar], albeit with some variation in potency between experimenters, while the observation of minimal conditioned-place preference has been reproduced [81.Kudla L. et al.Functional characterization of a novel opioid, PZM21, and its effects on the behavioural responses to morphine.Br. J. Pharmacol. 2019; 176: 4434-4445Crossref PubMed Scopus (17) Google Scholar]. A recent examination of the reinforcing effects of PZM21 in non-human primates showed similar abuse liability to oxycodone [82.Ding H. et al.Antinociceptive, reinforcing, and pruritic effects of the G-protein signalling-biased mu opioid receptor agonist PZM21 in non-human primates.Br. J. Anaesth. 2020; 125: 596-604Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar], and antinociceptive tolerance comparable to morphine develops following prolonged treatment [48.Hill R. et al.The novel mu-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.Br. J. Pharmacol. 2018; 175: 2653-2661Crossref PubMed Scopus (107) Google Scholar]. Recently, the G protein-bias factor of a family of newly developed compounds was correlated to the separation in potency between antinociception and respiratory depression [7.Schmid C.L. et al.Bias factor and therapeutic window correlate to predict safer opioid analgesics.Cell. 2017; 171: 1165-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar]. Most significantly, SR17018 was reported not to induce respiratory depression [7.Schmid C.L. et al.Bias factor and therapeutic window correlate to predict safer opioid analgesics.Cell. 2017; 171: 1165-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar], although again later studies have shown decreases in respiratory rate at minimally antinociceptive doses [51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar]. Studies of this ligand are significantly hampered by extremely poor solubility [46.Grim T.W. et al.A G protein signaling-biased agonist at the mu-opioid receptor reverses morphine tolerance while preventing morphine withdrawal.Neuropsychopharmacology. 2020; 45: 416-425Crossref PubMed Scopus (34) Google Scholar,51.Gillis A. et al.Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.Sci. Signal. 2020; 13eaaz3140Crossref PubMed Scopus (109) Google Scholar], and a recent study reported high, DAMGO-like arrestin recruitment by SR17018 [58.Gutman E.S. et al.G-protein biased opioid agonists: 3-hydroxy-N-phenethyl-5-phenylmorphans with three-carbon chain substituents at C9.RSC Med. Chem. 2020; 11: 896-904Crossref Google Scholar]. The target of opioid analgesics, MOPr, is a G protein coupled receptor (GPCR) that signals predominantly through activation of the Gαi/o and βγ proteins. MOPr activation alters neuronal function through well-established G protein signaling mechanisms, including postsynaptic activation of G protein coupled inwardly rectifying potassium channels (GIRK), causing hyperpolarization and inhibition of neurons [4.Williams J.T. et al.Enkephalin opens potassium channels on mammalian central neurones.Nature. 1982; 299: 74-77Crossref PubMed Scopus (186) Google Scholar]. Presynaptic inhibition of neurotransmission also occurs through G protein signaling of MOPr, predominantly via the inhibition of voltage-gated calcium channels (VGCC) [5.Schroeder J.E. et al.Activation of mu opioid receptors inhibits transient high- and low-threshold Ca2+ currents, but spares a sustained current.Neuron. 1991; 6: 13-20Abstract Full Text PDF PubMed Scopus (178) Google Scholar]. G protein signaling of the MOPr can be negatively regulated via a system of intracellular C-terminal phosphorylation by various kinases, and β-arrestin binding common to most GPCRs. In addition to negative regulation of G protein signaling, the recruitment of β-arrestin to MOPr has been proposed to transduce a G protein-independent signal [1.Siuda E.R. et al.Biased mu-opioid receptor ligands: a promising new generation of pain therapeutics.Curr. Opin. Pharmacol. 2017; 32: 77-84Crossref PubMed Scopus (99) Google Scholar,2.Smith J.S. et al.Biased signalling: from simple switches to allosteric microprocessors.Nat. Rev. Drug Discov. 2018; 17: 243-260Crossref PubMed Scopus (327) Google Scholar]. To date, however, the nature of this putative signal has not been described and it remains unclear how β-arrestin signaling mediates discrete physiological effects. Central to the proposed benefit of putatively G protein-biased MOPr agonists [6.DeWire S.M. et al.A G protein-biased ligand at the mu-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine.J. Pharmacol. Exp. Ther. 2013; 344: 708-717Crossref PubMed Scopus (407) Google Scholar, 7.Schmid C.L. et al.Bias factor and therapeutic window correlate to predict safer opioid analgesics.Cell. 2017; 171: 1165-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 8.Manglik A. et al.Structure-based discovery of opioid analgesics with reduced side effects.Nature. 2016; 537: 185-190Crossref PubMed Scopus (544) Google Scholar, 9.Varadi A. et al.Mitragynine/corynantheidine pseudoindoxyls as opioid analgesics with mu agonism and delta antagonism, which do not recruit beta-arrestin-2.J. Med. Chem. 2016; 59: 8381-8397Crossref PubMed Scopus (158) Google Scholar] is the hypothesis that MOPr/β-arrestin interactions mediate opioid-induced respiratory depression and constipation [1.Siuda E.R. et al.Biased mu-opioid receptor ligands: a promising new generation of pain therapeutics.Curr. Opin. Pharmacol. 2017; 32: 77-84Crossref PubMed Scopus (99) Google Scholar], based on results obtained using the β-arrestin2-knockout mouse [10.Raehal K.M. et al.Morphine side effects in beta-arrestin 2 knockout mice.J. Pharmacol. Exp. Ther. 2005; 314: 1195-1201Crossref PubMed Scopus (413) Google Scholar]. Opioid-induced respiratory depression is the major cause of overdose death, and multiple genetic knockout studies have shown that it results from activation of MOPr rather than δ- or κ-opioid receptor subtypes (DOPr and KOPr) or the opioid-related nociceptin/orphanin FQ receptor (NOPr) [7.Schmid C.L. et al.Bias factor and therapeutic window correlate to predict safer opioid analgesics.Cell. 2017; 171: 1165-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,11.Matthes H.W. et al.Activity of the delta-opioid receptor is partially reduced, whereas activity of the kappa-receptor is maintained in mice lacking the mu-receptor.J. Neurosci. 1998; 18: 7285-7295Crossref PubMed Google Scholar,12.Hill R. et al.Fentanyl depression of respiration: comparison with heroin and morphine.Br. J. Pharmacol. 2020; 177: 254-266Crossref PubMed Scopus (43) Google Scholar], as does inhibition of gastrointestinal transit [13.Kieffer B.L. Gaveriaux-Ruff C. Exploring the opioid system by gene knockout.Prog. Neurobiol. 2002; 66: 285-306Crossref PubMed Scopus (498) Google Scholar]. Expression of MOPr is abundant throughout the respiratory network of the brainstem [14.Montandon G. Slutsky A.S. Solving the opioid crisis: respiratory depression by opioids as cr