Critical Assessment of G Protein-Biased Agonism at the μ-Opioid Receptor

G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and one of the most common drug targets. It is now well-established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, G protein receptor kinases and βarrestins. While these signaling pathways can be activated or blocked by “balanced” agonists or antagonists, they can also be selectively activated in a “biased” response. Biased responses can be induced by biased ligands, biased receptors, or system bias, any of which can result in preferential signaling through G proteins or βarrestins. At many GPCRs, G protein- and βarrestin-mediated signaling have been shown to have distinct biochemical and physiological actions from one another and an accurate evaluation of biased signaling from pharmacology through physiology is critical for preclinical drug development. Recent structural studies have provided snapshots of GPCR-transducer complexes, which should aid in the structure-based design of novel biased therapies. Our understanding of GPCRs from two-state, on-and-off switches has evolved to that of multistate allosteric microprocessors, in which biased ligands transmit distinct structural information that is processed into distinct biological outputs. The development of biased ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.