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

Opioids have been used to relieve pain and abused for their psychotropic effects for centuries and remain the most
effective treatment for significant pain. Opioid-induced pain relief occurs in part through their action at the mu-opioid
receptor (MOPr) and MOPr agonists also induce euphoria, constipation, depression of breathing and addiction. The
opioid crisis, whereby high rates of opioid addiction, dependence and overdose deaths due to over-prescription and
illicit use of opioids, is a major public health concern. In Britain around 200,000 people are dependent on opioid
painkillers, in 2018 more than 2,200 people in England and Wales died from opiates and over-prescription of opioids
costs the NHS over £100m per year.
This research investigates the potential for ligand-receptor dwell time to offer a new approach to understanding and
improving opioid-induced pain relief. Specifically, it aims to explore the hypothesis that MOPr agonists with long dwell
times will be more potent and/or cause less functional tolerance at presynaptic nerve terminals than at postsynaptic
nerve terminals, whereas short dwell time agonists will be less potent at presynaptic nerve terminals than at
postsynaptic. The hypothesis is based on evidence that presynaptic MOPrs are: resistant to rapid desensitisation;
and not immobilised, rather they diffuse throughout the neuronal membrane, so ligand-receptor complexes may form
at the nerve terminal and/or extrasynaptically then laterally diffuse to the nerve terminal to signal via effector proteins
at the active zone. The overall functional effects of opioids differ depending on the balance of presynaptic or
postsynaptic MOPr activity, and the receptor dwell time of an opioid ligand may therefore have functional/behavioural
importance.
Preliminary experiments using Bioluminescence Resonance Energy Transfer (BRET) assays in HEK293 cells
transfected with MOPr have been carried out, to characterise a battery of different opioid agonists in terms of relative
potencies. These are an in vitro post-synaptic assay.
To determine the dwell-time of a battery of different MOPr agonists, the rate of drug-unbinding will be measured in
vitro in AtT20 cells using Fluorescent Imaging Plate Reader technology and naloxone. Naloxone can only bind when
the agonist has come off the receptor, so the sooner naloxone reduces potassium efflux the shorter the dwell time.
Alternatively, radio-ligand binding assays may be used.
Relative potency of different MOPr agonists will be determined ex vivo in rodents using vas deferens organ bath
(postsynaptic) and LC (postsynaptic) and VTA (presynaptic in dopaminergic neurons, postsynaptic in GABAergic
neurons) brain slice electrophysiology experiments. Single-unit extracellular recordings and whole-cell intracellular
patch-clamp recordings will be used.
Functional behavioural effects of these MOPr agonists will also be tested in vivo in rodents, e.g. analgesic effects (tail
flick and/or hot plate withdrawal); respiratory depression (whole-animal plethysmography) and development of
addiction (conditioned place preference experiments). Similar ex vivo and in vivo experiments may also be used
alongside repeated opioid exposure protocols, to investigate how ligand-receptor dwell-time affects opioid tolerance.
Findings from this research may inform the urgently needed development of better analgesic drugs with fewer
adverse effects.