α-芋螺毒素[A10L]PnIA荧光探针的设计合成及活性研究  

Design,synthesis,and activity study ofα-conotoxin[A10L]PnIA fluorescent probe

作　　者：谭瑶 杨奕帅 褚召莉 长孙东亭[1] 朱晓鹏[1,2] 罗素兰 TAN Yao;YANG Yi-shuai;CHU Zhao-li;ZHANGSUN Dong-ting;ZHU Xiao-peng;LUO Su-lan(Key Laboratory of Tropical Biological Resources of Ministry of Education,School of Life and Pharmaceutical Sciences,Hainan University,Haikou 570228,China;Medical School,Guangxi University,Nanning 530004,China)

机构地区：[1]海南大学热带生物资源教育部重点实验室,海南大学生命科学与药学院,海南海口570228 [2]广西大学医学院,广西南宁530004

出　　处：《药学学报》2021年第8期2252-2259,共8页Acta Pharmaceutica Sinica

基　　金：海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC072);国家自然科学基金资助项目(31760249,81872794).

摘　　要：α7烟碱型乙酰胆碱受体(nAChR)广泛分布于中枢和外周神经系统,与多种神经系统疾病、炎症反应密切相关。α-芋螺毒素[A10L]PnIA作为靶向α7 nAChR的拮抗剂,对研究α7 nAChR相关生理、病理过程具有重要作用。利用荧光素5-羧基四甲基罗丹明标记[A10L]PnIA,体外两步法氧化折叠获得活性肽([A10L]PnIA-F)。利用非洲爪蟾卵母细胞表达体系和双电极电压钳技术检测[A10L]PnIA-F荧光肽的活性。同时,利用小鼠巨噬细胞和CCK8检测其细胞毒性。合成的[A10L]PnIA-F荧光肽,质谱鉴定其分子质量为2077.28 Da,与理论值一致;电生理测定其对鼠源α7 nAChR的半阻断剂量(IC50)为17.32 nmol·L^(-1),较[A10L]PnIA本体(IC50,13.84 nmol·L^(-1))基本保持一致;细胞毒检测结果显示,在5 nmol·L^(-1)~10μmol·L^(-1)的浓度范围内,对小鼠巨噬细胞的生长无显著抑制。结果表明α-芋螺毒素荧光探针[A10L]PnIA可以为α7 nAChR相关的神经生理、病理机制的研究提供工具。α7 nicotinic acetylcholine receptor(nAChR)is widely distributed in the central and peripheral nervous systems,and is closely related to a variety of neurological diseases and inflammation response.α-Conotoxin[A10L]PnIA,as an antagonist targetingα7 nAChR,plays an important role in studying the physiological and pathological processes involved inα7 nAChR.[A10L]PnIA was labeled with fluorescein 5-carboxytetramethylrhodamine,and the active peptide([A10L]PnIA-F)was obtained by a two-step oxidative folding procedure in vitro.The Xenopus oocyte expression system and the two-electrode voltage clamp technique were used to identify the potency of[A10L]PnIA-F fluorescent peptide,and its cytotoxicity was detected by mouse macrophages and CCK8 method.The molecular weight of[A10L]PnIA-F fluorescent peptide was identified by mass spectrometry as 2077.28 Da,which was consistent with the theoretical value.Electrophysiological determination of its halfmaximal inhibitory concentration(IC50)forα7 nAChR is 17.32 nmol·L^(-1),which is consistent with[A10L]PnIA(IC50,13.84 nmol·L^(-1)).The cytotoxicity test results showed that within the concentration range of 5 nmol·L^(-1) to 10μmol·L^(-1),there was no significant inhibition on the growth of mouse macrophages.The results showed that theα-conotoxin fluorescent probe[A10L]PnIA could provide pharmacological tools for the research ofα7 nAChRrelated neurophysiological and pathological mechanisms.

关 键 词：Α7烟碱型乙酰胆碱受体 α-芋螺毒素[A10L]PnIA 荧光探针 电生理活性 

分 类 号：R914[医药卫生—药物化学]