Biased agonism at G protein–coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus β-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as β-arrestin recruitment. At the μ-opioid receptor (MOR), G protein–biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in β-arrestin–mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and β-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

Background and Purpose: GPCRs can signal through both G proteins and β-arrestin2.For the μ-opioid receptor, early experimental evidence from a single study suggested that G protein signalling mediates analgesia, whereas β-arrestin2 signalling mediates respiratory depression and constipation. Consequently, for more than a decade, much research effort has been focused on developing biased μ-opioid agonists that preferentially target G protein signalling over β-arrestin signalling, as it was believed that such drugs would be analgesics devoid of respiratory depressant activity. However, the prototypical compounds that have been developed based on this concept have so far failed in clinical and preclinical development.Experimental Approach: The present study was set up to re-examine opioid-induced respiratory depression in β-arrestin2 knockout mice. To this end, a consortium was formed consisting of three different laboratories located in different countries to evaluate independently opioid-induced respiratory depression.Key Results: Our consensus results unequivocally demonstrate that the prototypical μ-opioid agonist morphine (3.75-100 mgÁkg −1 s.c. or 3-30 mgÁkg −1 i.p.) as well as the potent opioid fentanyl (0.05-0.35 mgÁkg −1 s.c.) do indeed induce respiratory depression and constipation in β-arrestin2 knockout mice in a dose-dependent manner indistinguishable from that observed in wild-type mice.Conclusion and Implications: Our findings do not support the original suggestion that β-arrestin2 signalling plays a key role in opioid-induced respiratory depression and call into question the concept of developing G protein-biased μ-opioid receptor agonists as a strategy for the development of safer opioid analgesic drugs.Abbreviation: βarr2−/−, β-arrestin2 knockout.

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 proteinbiased 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. Current View of MOPr Biased SignalingG protein-biased agonists (see Glossary) of the μ-opioid receptor (MOPr) have been widely proposed to be a novel, substantially improved class of analgesics [1,2]. The prototypical such agonist, oliceridine (TRV130), has proceeded to Phase III clinical trials [3], 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.

Evidence from several novel opioid agonists and knockout animals suggests that improved opioid therapeutic window, notably for analgesia versus respiratory depression, is a result of ligand bias downstream of activation of the m-opioid receptor (MOR) toward G protein signaling and away from other pathways, such as arrestin recruitment. Here, we argue that published claims of opioid bias based on application of the operational model of agonism are frequently confounded by failure to consider the assumptions of the model. These include failure to account for intrinsic efficacy and ceiling effects in different pathways, distortions introduced by analysis of amplified (G protein) versus linear (arrestin) signaling mechanisms, and nonequilibrium effects in a dynamic signaling cascade. We show on both theoretical and experimental grounds that reduced intrinsic efficacy that is unbiased across different downstream pathways, when analyzed without due considerations, does produce apparent but erroneous MOR ligand bias toward G protein signaling, and the weaker the G protein partial agonism is the greater the apparent bias. Experimentally, such apparently G protein-biased opioids have been shown to exhibit low intrinsic efficacy for G protein signaling when ceiling effects are properly accounted for. Nevertheless, such agonists do display an improved therapeutic window for analgesia versus respiratory depression. Reduced intrinsic efficacy for G proteins rather than any supposed G protein bias provides a more plausible, sufficient explanation for the improved safety. Moreover, genetic models of G protein-biased opioid receptors and replication of previous knockout experiments suggest that reduced or abolished arrestin recruitment does not improve therapeutic window for MORinduced analgesia versus respiratory depression. SIGNIFICANCE STATEMENT Efforts to improve safety of m-opioid analgesics have focused on agonists that show signaling bias for the G protein pathway versus other signaling pathways. This review provides theoretical and experimental evidence showing that failure to consider the assumptions of the operational model can lead to large distortions and overestimation of actual bias. We show that low intrinsic efficacy is a major determinant of these distortions, and pursuit of appropriately reduced intrinsic efficacy should guide development of safer opioids.