Investigating ligand-receptor dwell time as a form of spatio-temporal bias at the mu-opioid receptor

Abstract

Opioid drugs, such as morphine, are clinically important due to the pain-relieving effects they induce via activation of the µ-opioid receptor (MOPr). As well as analgesia, opioids also cause various adverse effects that limit their clinical usefulness including respiratory depression, constipation and tolerance. In order to develop better opioid analgesics that avoid these adverse effects, greater breadth and depth of understanding about opioid signalling via MOPr is needed. One possible advance is with “biased” agonists, whereby different agonists can induce different signalling profiles. Whilst the pursuit of biased MOPr agonists that preferentially activate G proteins over arrestins has yielded little success, other forms of biased agonism offer promising new avenues of research. The work in this thesis sought to investigate a novel form of spatio-temporal bias at MOPr. Specifically, it explores the hypothesis that MOPr agonists that have longer dwell time (i.e. bind to the receptor for longer) induce responses more strongly at presynaptic MOPrs compared with at post-synaptic MOPrs. Using fluorescent imaging plate reader (FLIPR) membrane potential dye experiments in AtT20 cells, the relative dwell times of a selection of MOPr agonists including the under-studied compounds BU168 and BU186, were determined. Etorphine, BU168, carfentanil and BU186 were characterised as MOPr agonists with long dwell times, relative to DAMGO and morphine. The classical signalling bias of BU168 and BU186 was then assessed, alongside morphine and etorphine, using bioluminescence resonance energy transfer (BRET) assays in HEK293T cells and no bias was found for any of the agonists relative to DAMGO. To assess spatial bias, the efficacies and potencies at presynaptic and post-synaptic MOPrs were compared by testing agonists in mouse vas deferens and FLIPR assays, respectively. Etorphine, BU168, carfentanil and BU186 all showed significant preference for presynaptic MOPr signalling, relative to that of DAMGO. Using these agonists with long dwell times – as well as others identified as having equal dwell times to DAMGO – positive correlation was found in support of the hypothesis that dwell time drives presynaptic spatial bias. Finally, antinociceptive tail withdrawal assays in mice found that etorphine, BU168 and BU186 were all more potent than morphine, providing preliminary evidence that agonists with long dwell times might be better analgesics. In the future, more comprehensive assessments of the in vivo profiles of MOPr agonists with long dwell times are needed to test whether presynaptic bias confers a wider therapeutic window. The findings in this thesis also precipitate a wider discussion about the mechanisms by which long dwell time and/or presynaptic bias may arise. Overall, this thesis provides correlative evidence for spatio-temporal bias at MOPr that could be useful therapeutically or as a tool for investigating the mechanisms underpinning opioid pharmacology further.

Date of Award	17 Mar 2024
Original language	English
Awarding Institution	University of Bath
Supervisor	Chris Bailey (Supervisor), Sue Wonnacott (Supervisor) & Eamonn Kelly (Supervisor)