α-芋螺毒素LvIA与α3β2乙酰胆碱受体相互作用分子机理研究

..α-Conotoxins (α-CTxs) are effective antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs) with a high selectivity, which are from venom of cone snails in tropical ocean with the realistic medicine potential. The nAChR subtypes are important in physiology of both the central and peripheral nervous system, which are key targets of drugs to treat many difficult miscellaneous diseases, such as pain, addiction, Parkinson's disease, breast and lung carcinoma etc. So far, the composition, structure and roles of nAChR subtypes remain to be elucidated. We discovered a unique and analgesic α-conotoxin LvIA from Hainan Conus lividus, which is the first ligand reported to show highest selectivity for α3β2 vs. α6β2 nAChRs until now. Previous conotoxins targeting α3β2 block α6β2* nAChR too, which are poorly selective between α3β2 vs. α6β2* nAChRs. In mammal nerve system, especially in dopaminergic regions, α6β2* nAChR accompanies α3β2 expression simultaneously. Generally, α3β2 nAChR expression overlaps with that of α6β2* nAChR. Structure of α3β2 nAChR is too similar with α6β2* nAChR to differentiate by far. However their functions are distinct different between each other. This multidisciplinary research project would focus on interaction mechanism of binding site and mode of action between α-conotoxin LvIA and its receptor α3β2 nAChR by mutations of both toxin and receptor, as well as corresponding bioassay. The results would be valuable for developing α-conotoxin LvIA as potent innovative medication of nAChRs-related diseases, and research tool of neuroscience.

..来自热带海洋生物芋螺毒液中的α-芋螺毒素，是烟碱乙酰胆碱受体各种亚型选择性最好的特异阻断剂，具有巨大的新药研发价值。乙酰胆碱受体介导众多中枢和外周神经系统的生理功能，是筛选诊断和治疗一大类重要疾病药物的关键靶点，如疼痛、成瘾、帕金森病、癌症等。至今对该受体各种亚型的精细结构和功能知之甚少。我们发现了一个独特的α-芋螺毒素LvIA，具有很强的镇痛活性，是迄今对α3β2和α6β2* 这两个极其相似亚型区分度最好的配体化合物，国际上之前发现的α3β2阻断剂，都同时阻断α6β2*亚型。在哺乳动物神经系统中，尤其是多巴胺神经元区域，α3β2和α6β2*两个亚型总是伴随表达，它们的结构极其相似，极难区分，却有着迥然不同的药理学功能。本项目拟通过毒素和受体的双方面突变，检测相应的功能变化，解析毒素受体相互作用位点、以及作用方式的分子机制，为LvIA应用于神经科学研究和创新药物研发提供研究基础。..

本项目围绕α-芋螺毒素LvIA与α3β2乙酰胆碱受体相互作用的分子机理，进行了系统深入的研究，取得了很好的研究成果。2019-2022年项目执行期间，按计划书要求顺利开展了各项研究工作。主要包括α-芋螺毒素LvIA及其受体复合物的同源模型构建，毒素受体分子对接分析，LvIA丙氨酸扫描点突变体设计与人工合成；开展α3β2乙酰胆碱受体（nAChR）突变型设计与制备，对受体突变型的功能进行检测，对难以表达的困难突变型受体进行条件优化；开展LvIA点突变肽与受体突变型的结合活性研究，以及有意义突变体对其他nAChR受体相似亚型的结合活性检测；在此基础上重构LvIA及其受体复合物的同源模型，设计并人工合成LvIA多点突变体，研究LvIA多点突变体与α3β2 nAChR受体突变型的结合活性，发现了LvIA优化突变体，检测了对其他nAChR受体亚型的结合活性和选择性；大量合成LvIA优化突变体，在动物模型上评价了镇痛活性；综合分析了毒素受体双边突变的靶点结合活性，阐明了α-芋螺毒素LvIA及其优化突变体与α3β2 nAChR受体相互作用的分子机理。. 以通讯作者在《Journal of Medicinal Chemistry》、《Marine Drugs》、《Toxins》、《European Journal of Pharmacology》等期刊上，发表该项目第一标注资助的SCI论文15 篇。已培养研究生10名，其中博士毕业生4名、硕士毕业生6名。远远超额完成了既定研究目标和人才培养任务。这些研究结果对哺乳动物脑功能研究、α3β2受体的结构与功能及其相关疾病发病机理研究和治疗新药研发，具有非常重要的科学意义。