Opioids remain unsurpassed as our most effective treatment for severe pain despite their propensity to induce serious on-target adverse effects. Both the therapeutic and adverse effects of opioids are mediated through their activation of the μ opioid receptor (MOPr), a G protein-coupled receptor (GPCR). The disclosure of biased signalling at GPCRs has heralded the development of a new generation of refined GPCR-targeting drugs. G protein-biased agonists at MOPr have been developed in the anticipation that they will be effective analgesics, devoid of the adverse-effects of traditional, balanced opioids. Additionally, G protein-biased MOPr agonists also have the potential to induce less receptor desensitization than balanced opioids due to their low coupling to traditional pathways of receptor regulation (arrestin and GRK). This implies that tolerance to their effects may develop slower and to a lesser extent than in the case of traditional opioids, making them clinically beneficial. As such, we sought to characterise the receptor desensitization and regulation induced by G protein-biased MOPr agonists. Firstly, we characterised the biased signalling profiles of a series of putatively G protein-biased agonists at MOPr in recombinant systems using BRET assays of G protein coupling and arrestin recruitment. These assays demonstrated that the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased agonist at MOPr with a similar intrinsic activity for G protein signalling as morphine. In contrast, we demonstrate that the reportedly G protein-biased agonist PZM21 is in fact a non-biased lower efficacy agonist. We then investigated the receptor desensitization induced by previously characterised MOPr agonists in rat locus coeruleus (LC) neurones using patch clamp electrophysiology. It was hypothesised that G protein-biased agonists would induce less rapid MOPr desensitization due to their weak coupling to arrestin and GRK pathways. Intriguingly, the G protein-biased agonist Compound 1 conversely induced substantial receptor desensitization in LC neurones, to a greater degree than the balanced opioid morphine. Using pharmacological tools, it was determined that Compound 1-induced desensitization in LC neurones was in fact GRK-dependent, but PKC-independent. MOPr phosphorylation and internalization was studied in recombinant systems to outline the potential mechanisms underlying the implied GRK-dependent, arrestin-independent mechanism of Compound 1-induced desensitization. In these assays, Compound 1 induced minimal MOPr phosphorylation and internalization, in line with its G protein-biased profile. Together, the work within this thesis characterised Compound 1 as a novel G protein-biased agonist at MOPr, which intriguingly induces substantial receptor desensitization through GRK. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitization and the subsequent development of tolerance.
|Date of Award||26 May 2021|
|Supervisor||Chris Bailey (Supervisor), Sue Wonnacott (Supervisor) & Eamonn Kelly (Supervisor)|
- G protein-coupled receptor
- Biased agonism
- Receptor desensitization
- mu opioid receptor
- G protein-coupled receptor kinases