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来缓解疼痛，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。阿片类药物引起的疼痛缓解部分是通过它们在mu-阿片受体（MOPr）上的作用发生的，MOPr激动剂也会引起欣快感、便秘、呼吸抑制和成瘾。类阿片危机是一个重大的公共卫生问题，即由于过量处方和非法使用类阿片而导致的类阿片成瘾、依赖和过量死亡比例很高。在英国，约有20万人依赖阿片类止痛药，2018年，英格兰和威尔士有2200多人死于阿片类药物，阿片类药物的过度处方每年使NHS损失超过1亿英镑。本研究探讨了配体-受体停留时间的潜力，为理解和改善阿片类药物诱导的疼痛缓解提供了一种新的方法。具体来说，该研究旨在探讨长停留时间的MOPr激动剂在突触前神经末梢比在突触后神经末梢更有效和/或导致更少的功能耐受性，而短停留时间的激动剂在突触前神经末梢比在突触后神经末梢更有效的假设。这一假设是基于以下证据：突触前mmopr能够抵抗快速脱敏；它们不是固定的，而是扩散到整个神经元膜，因此配体-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，通过活性区的效应蛋白发出信号。阿片类药物的整体功能影响取决于突触前或突触后MOPr活性的平衡，因此阿片类配体的受体停留时间可能具有功能/行为重要性。利用生物发光共振能量转移（BRET）法在转染MOPr的HEK293细胞中进行了初步实验，以表征一系列不同的阿片受体激动剂的相对效力。这是体外突触后实验。为了确定一组不同的MOPr激动剂的停留时间，将使用荧光成像板阅读器技术和纳洛酮在AtT20细胞中体外测量药物解结合率。纳洛酮只有在激动剂脱离受体后才能结合，所以纳洛酮越早减少钾外排，停留时间就越短。或者，可以使用放射性配体结合测定法。通过输精管器官浴（突触后）、LC（突触后）和VTA（多巴胺能神经元突触前、gaba能神经元突触后）脑切片电生理实验，确定不同MOPr激动剂在啮齿动物体内的相对效力。将使用单单元细胞外记录和全细胞细胞内膜片钳记录。这些MOPr激动剂的功能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/或热板停药）；呼吸抑制（全动物体积脉搏图）和成瘾的发展（条件位置偏好实验）。类似的体外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开发不良反应更少的更好的镇痛药物提供了急需的信息。

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.