乙酰胆碱酯酶抑制剂微量筛选模型的比较研究

目的为新药发现阶段乙酰胆碱酯酶(acetylcholinest-erase,AChE)抑制剂的筛选提供实用的微量筛选方法。方法分别以电鳗AChE、大鼠大脑匀浆、蛇毒AChE为酶源,以96孔板为载体,在生理pH值和温度的条件下,用正交试验法研究酶浓度、底物浓度、反应时间、显色剂浓度对酶反应速率的影响,确定酶反应最佳条件。在研究样品溶媒DMSO对酶反应的影响和SDS终止酶反应的效果后,最终确定筛选模型运行条件并对492种贵州民族药植物提取物进行筛选研究。结果采用上述3种酶源均成功构建了微量化的AChE抑制剂筛选模型。筛选结果表明,电鳗AChE有较高的筛选灵敏度,而蛇毒AChE的筛选结果则与大鼠大脑匀浆的筛选结果有较好的一致性。结论以上述3种具有代表性的酶源构建的AChE抑制剂微量筛选模型均具备了简便、快速、可靠、经济、灵活的优点。其中电鳗AChE最适合用于新药发现阶段粗提物样品的筛选,而高纯度的蛇毒AChE更适合用于从化合物库中大规模筛选AChE抑制剂。

Comparative study on microassays for screening acetylcholinesterase inhibitors

Aim To provide practical microassays for screening acetylcholinesterase (AChE) inhibitors in drug discovery.Methods The optimal conditions of assaying the activity of AChEs from electric eel,rat brain homogenate and cobra venom were determined in 96-well plates under physiological pH value and temperature by orthogonal matrix method.The concentrations of AChE,substrate and DTNB,and reaction time were optimized.After the effects of sample solvent (DMSO) used in the assay and stopping reagent on enzyme activity were assessed,the assay conditions were finally selected,and 492 kinds of extracts from Guizhou ethno-drugs were screened.Results Practical microassays for screening AChE inhibitors were successfully constituted by using AChEs mentioned above.The data analysis of screening results revealed that electric eel AChE possessed a high sentivity to inhibitors,and cobra venom AChE shared high similarity with rat brain homogenate in positive results.Conclusion Microassays constituted in this work possessed advantages of being easy,rapid,reliable,cost saving and flexible.AChE from electric eel was especially suitable for screening AChE inhibitors from extracts,and AChE from cobra venom was more suitable to be used in screening AChE inhibitors from large numbers of compounds.