M1受体选择性激活和Gαq-PKC信号偏好的变构新位点发掘及调控机制研究

M1 muscarinic acetylcholine receptor (M1 mAChR), which is the primary subtype mediating cholinergic-hippocampal learning and memory, is one of the most potential targets for Alzheimer’s disease (AD) treatment. Positive allosteric modulators of M1 mAChR (M1-PAMs) have become a promising drug development approach for AD. However, how to optimize diverse allosteric residues and identify the favorable signalling bias of M1 receptor is still an urgent problem need to be solved. In this project, M1 mAChR crystal structure and chemical probes such as BQCA and VU0364572 are used to explore the new targetable allosteric sites and new allosteric mechanism underlying the selective modulation of Gαq-PKC signal pathway and APP metabolism. Basing on the new allosteric sites with M1 subtype-selectivity and Gαq-PKC pathway specificity, novel lead compounds of M1-PAMs are obtained following screening in our allosteric lead compounds database. The allosteric modulating effects of novel M1-PAMs are validated with our established approaches such as SPR, BRET, high-content analysis, and Aβ assays. Our results will not only help to identify the new allosteric sites and new allosteric mechanism of selective modulation of M1 mAChR by M1-PAMs, but also provide new strategies for the development of novel symptom-releasing and disease-modifying M1-PAMs for AD.

M1受体是学习记忆功能调控最关键的胆碱受体亚型和阿尔茨海默病（AD）治疗最有潜力药物靶点之一，发现新型M1受体正性变构调节剂（M1-PAM）已成为AD药物研发的新策略。然而，如何优选M1受体的多样性变构位点以及甄别有利的信号偏好仍是亟待解决的关键问题。本课题拟利用最新M1受体晶体结构和BQCA/VU0364572等化学分子探针，发掘具有靶标价值的多样性变构调节新位点；解析变构位点和受体调节特征与Gαq-PKC通路偏好性产生规律、介导AD相关APP代谢的变构调节新机制；针对兼具M1受体选择性激活和Gαq-PKC信号偏好的变构新位点进行变构低毒先导分子库的虚拟筛选，利用SPR、BRET、高内涵分析、Aβ检测等方法，探究分子探针和新型M1-PAM先导物的变构调控特性。创新成果将为发现具有靶标价值的变构调节新位点、新机制和具有认知改善和疾病修饰作用的M1-PAM新药研发提供重要的科学依据。

M胆碱受体属于G蛋白偶联受体（GPCRs），其中M1胆碱受体是学习记忆功能调控最关键的亚型及其阿尔茨海默病（AD）最有潜力的治疗靶点之一。由于内源性配体ACh结合正构位点与亚型间高度相似性，为M1高亚型选择性受体激动药研发带来巨大挑战。解析变构位点和受体调节特征，Gα通路信号偏好（signaling bias）规律，探索具信号偏向性新型M1胆碱受体正向变构调节剂（M1-PAM）具有重要科学创新价值。.为此，本课题利用M1胆碱受体晶体结构和BQCA、占诺美林等化学分子探针，采用“模块化”策略构建M1胆碱受体的M1-PAM，考察8种B-DNA-X连接体，发掘出具有重要价值的变构调节新位点（Y93、A204和L207），并基于分子动力学模拟、定点突变等方式解析关键位点D105、Y106及W157参与调控的机制，并进一步利用BRET技术构建包含36种配受体对的信号偏好评价体系，阐明M1胆碱受体激动后通过Gαq-PKC信号通路对AMPA受体及其GluA1亚基运输的调控机制，明确其对AD认知障碍的改善干预作用。.迄今变构调节剂多在筛选正位配体时偶然发现，并非由变构调节理论指导下针对变构位点筛选策略和信号偏向性考量下获得。本研究成果为后续进一步研究新型M1-PAM变构调控剂对M1胆碱受体的功能影响及其作为分子探针解析M1胆碱受体化学生物学机制提供实验依据。