药学学报, 2021, 56(8): 2252-2259
引用本文:
谭瑶, 杨奕帅, 褚召莉, 长孙东亭, 朱晓鹏, 罗素兰. α-芋螺毒素[A10L]PnIA荧光探针的设计合成及活性研究[J]. 药学学报, 2021, 56(8): 2252-2259.
TAN Yao, YANG Yi-shuai, CHU Zhao-li, ZHANGSUN Dong-ting, ZHU Xiao-peng, LUO Su-lan. Design, synthesis, and activity study of α-conotoxin[A10L]PnIA fluorescent probe[J]. Acta Pharmaceutica Sinica, 2021, 56(8): 2252-2259.

α-芋螺毒素[A10L]PnIA荧光探针的设计合成及活性研究
谭瑶1, 杨奕帅1, 褚召莉1, 长孙东亭1, 朱晓鹏1,2*, 罗素兰1,2*
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
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2 077.28 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-1~10 μmol·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。
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     
Design, synthesis, and activity study of α-conotoxin[A10L]PnIA fluorescent probe
TAN Yao1, YANG Yi-shuai1, CHU Zhao-li1, ZHANGSUN Dong-ting1, ZHU Xiao-peng1,2*, LUO Su-lan1,2*
1. Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China;
2. Medical School, Guangxi University, Nanning 530004, China
Abstract:
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 targeting α7 nAChR, plays an important role in studying the physiological and pathological processes involved in α7 nAChR.[A10L]PnIA was labeled with fluorescein 5-carboxytetramethylrho-damine, 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 2 077.28 Da, which was consistent with the theoretical value. Electrophysiological determination of its halfmaximal inhibitory concentration (IC50) for α7 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 10 μmol·L-1, there was no significant inhibition on the growth of mouse macrophages. The results showed that the α-conotoxin fluorescent probe[A10L]PnIA could provide pharmacological tools for the research of α7 nAChRrelated neurophysiological and pathological mechanisms.
Key words:    α7 nicotinic acetylcholine receptor    α-conotoxin[A10L]PnIA    fluorescent probe    electrophysio-logical activity   
收稿日期: 2021-04-12
DOI: 10.16438/j.0513-4870.2021-0528
基金项目: 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).
通讯作者: 朱晓鹏,Tel:86-898-66289538,E-mail:biozxp@163.com;罗素兰,Tel:86-898-66289538,E-mail:luosulan2003@163.com
Email: biozxp@163.com;luosulan2003@163.com
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