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

几个世纪以来，阿片类药物一直被用于缓解疼痛，并因其精神作用而被滥用，并且仍然是治疗严重疼痛的最有效方法。阿片样物质诱导的疼痛缓解部分通过其对μ-阿片样物质受体（MOPr）的作用而发生，并且MOPr激动剂还诱导欣快、便秘、呼吸抑制和成瘾。阿片类药物危机是一个重大的公共卫生问题，由于过量处方和非法使用阿片类药物，阿片类药物成瘾、依赖和过量死亡率很高。在英国，大约有20万人依赖阿片类止痛药，2018年，英格兰和威尔士有2,200多人死于阿片类药物，过度处方阿片类药物每年花费NHS超过1亿英镑。这项研究调查了配体-受体停留时间的潜力，为理解和改善阿片类药物引起的疼痛缓解提供了一种新的方法。具体而言，其目的是探索这样的假设，即具有长停留时间的MOPr激动剂在突触前神经末梢处比在突触后神经末梢处更有效和/或引起更少的功能耐受，而短停留时间激动剂在突触前神经末梢处比在突触后神经末梢处更不有效。这一假说是基于证据表明，突触前MOPrs是：抵抗快速脱敏;而不是固定的，而是它们扩散整个神经元膜，所以配体-受体复合物可能形成神经末梢和/或突触外，然后横向扩散到神经末梢，通过效应蛋白在活性区发出信号。阿片类药物的总体功能效应取决于突触前或突触后MOPr活性的平衡，因此阿片配体的受体停留时间可能具有功能/行为重要性。为了确定一组不同MOPr激动剂的停留时间，将使用荧光成像板读取器技术和纳洛酮在AtT 20细胞中体外测量药物解结合的速率。纳洛酮仅在激动剂脱离受体时才能结合，因此纳洛酮越早减少钾流出，停留时间越短。或者，可以使用放射性配体结合试验。不同MOPr激动剂的相对效力将使用输精管器官浴在啮齿动物中离体测定（突触后）和LC（突触后）和VTA（多巴胺能神经元突触前，GABA能神经元突触后）脑片电生理实验。这些MOPr激动剂的功能性行为效应也将在啮齿动物体内进行测试，例如镇痛效应（甩尾和/或热板戒断）;呼吸抑制（全动物体积描记法）和成瘾的发展（条件性位置偏爱实验）。类似的离体和体内实验也可以与重复的阿片类药物暴露方案一起使用，以研究配体-受体停留时间如何影响阿片类药物耐受性。这项研究的发现可能为急需开发更好的、副作用更少的镇痛药物提供信息。

Opioids have been used to relieve pain and abused for their psychotropic effects for centuries and remain the mosteffective treatment for significant pain. Opioid-induced pain relief occurs in part through their action at the mu-opioidreceptor (MOPr) and MOPr agonists also induce euphoria, constipation, depression of breathing and addiction. Theopioid crisis, whereby high rates of opioid addiction, dependence and overdose deaths due to over-prescription andillicit use of opioids, is a major public health concern. In Britain around 200,000 people are dependent on opioidpainkillers, in 2018 more than 2,200 people in England and Wales died from opiates and over-prescription of opioidscosts the NHS over £100m per year.This research investigates the potential for ligand-receptor dwell time to offer a new approach to understanding andimproving opioid-induced pain relief. Specifically, it aims to explore the hypothesis that MOPr agonists with long dwelltimes will be more potent and/or cause less functional tolerance at presynaptic nerve terminals than at postsynapticnerve terminals, whereas short dwell time agonists will be less potent at presynaptic nerve terminals than atpostsynaptic. 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 format the nerve terminal and/or extrasynaptically then laterally diffuse to the nerve terminal to signal via effector proteinsat the active zone. The overall functional effects of opioids differ depending on the balance of presynaptic orpostsynaptic MOPr activity, and the receptor dwell time of an opioid ligand may therefore have functional/behaviouralimportance.Preliminary experiments using Bioluminescence Resonance Energy Transfer (BRET) assays in HEK293 cellstransfected with MOPr have been carried out, to characterise a battery of different opioid agonists in terms of relativepotencies. 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 invitro in AtT20 cells using Fluorescent Imaging Plate Reader technology and naloxone. Naloxone can only bind whenthe 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 GABAergicneurons) brain slice electrophysiology experiments. Single-unit extracellular recordings and whole-cell intracellularpatch-clamp recordings will be used.Functional behavioural effects of these MOPr agonists will also be tested in vivo in rodents, e.g. analgesic effects (tailflick and/or hot plate withdrawal); respiratory depression (whole-animal plethysmography) and development ofaddiction (conditioned place preference experiments). Similar ex vivo and in vivo experiments may also be usedalongside 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 feweradverse effects.