Li Wen, Zhang Baofang, Chen Zilin
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, And Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China; State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing, 10080, China.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, And Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, China; State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing, 10080, China.
Talanta. 2021 Sep 1;232:122444. doi: 10.1016/j.talanta.2021.122444. Epub 2021 May 1.
An electrophoretically mediated microanalysis (EMMA) method for the screening of carbonic anhydrase IX inhibitors in traditional Chinese medicine (TCM) was developed. This method combines transverse diffusion of laminar flow profiles (TDLFP) and rapid polarity switching technology to achieve rapid mixing of different reactants. Different electromigration rates of different substances make it possible that incubation, separation and detection are carried out continuously in a same fused-silica capillary. In this experiment, p-nitrophenyl acetate (pNPA) was used as the substrate for the enzyme reaction, which solved the problem that capillary electrophoresis could not detect carbonate, carbon dioxide, etc., the conventional substrates of carbonic anhydrase IX. After optimizing the enzyme reaction and separation conditions, the separation of substrate and product can be finished by baseline within 4 min. The Michaelis constant of carbonic anhydrase IX was measured to be 1.2 mM. A known inhibitor acetazolamide was used to evaluate the feasibility of this method for screening carbonic anhydrase IX inhibitors, and the half-maximal inhibitory concentration (IC) was calculated to be 1.26 μM. Finally, 4 natural compounds of 15 traditional Chinese medicine standards were discovered to exhibit enzyme inhibitory activity, including polydatin, matrine, dauricine and cepharanthine, proving that the EMMA method is an effective means for screening carbonic anhydrase IX inhibitors. The results were supported by molecular docking study.
建立了一种用于筛选中药中碳酸酐酶IX抑制剂的电泳介导微分析（EMMA）方法。该方法结合层流剖面横向扩散（TDLFP）和快速极性切换技术，实现不同反应物的快速混合。不同物质的不同电迁移速率使得在同一熔融石英毛细管中连续进行孵育、分离和检测成为可能。在本实验中，以对硝基苯乙酸（pNPA）作为酶反应的底物，解决了毛细管电泳无法检测碳酸酐酶IX的常规底物碳酸、二氧化碳等的问题。优化酶反应和分离条件后，底物和产物可在4分钟内实现基线分离。测得碳酸酐酶IX的米氏常数为1.2 mM。使用已知抑制剂乙酰唑胺评估该方法筛选碳酸酐酶IX抑制剂的可行性，计算出半数抑制浓度（IC）为1.26 μM。最后，在15种中药标准品的4种天然化合物中发现具有酶抑制活性，包括白藜芦醇、苦参碱、蝙蝠葛碱和千金藤素，证明EMMA方法是筛选碳酸酐酶IX抑制剂的有效手段。分子对接研究支持了该结果。
