基于比色法原理的新冠病毒主蛋白酶小分子抑制剂高通量筛选模型的优化与应用  

Discovery of a novel SARS-CoV-2 main protease inhibitor by a simple and optimized colorimetric screening assay

作　　者：闫干干 闫浩浩 刘志成 戚海燕 刘晓丽 刘晓平[1] 张晶[2] 陈云雨 YAN Gan-gan;YAN Hao-hao;LIU Zhi-cheng;QI Hai-yan;LIU Xiao-li;LIU Xiao-ping;ZHANG Jing;CHEN Yun-yu(Institute for Drug Screening and Evaluation,Wannan Medical College,Wuhu 241002,China;Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]皖南医学院,药物筛选与评价研究所,安徽芜湖241002 [2]中国医学科学院、北京协和医学院医药生物技术研究所,北京100050

出　　处：《药学学报》2022年第10期3019-3026,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81370087,81703546);安徽省自然科学基金资助项目(1808085QH265);安徽省高等学校自然科学研究项目(KJ2019ZD30,KJ2021A0839,YJS20210549);中国医学科学院医学与健康科技创新工程资助项目(2021-I2M-1-054);皖南医学院青年骨干人才资助项目(wyqnyx202104)。

摘　　要：本研究优化并建立了新冠病毒主蛋白酶(main protease,M^(pro))小分子抑制剂比色法高通量筛选模型,以期快速筛选天然产物来源的新型苗头化合物。基于比色法原理,以TSAVLQ-pNA(para-nitroanilide)作为M^(pro)水解底物,通过优化pNA底物浓度、M^(pro)工作浓度、最佳反应时间、二甲基亚砜耐受浓度等影响因素,建立M^(pro)小分子抑制剂比色法高通量筛选模型并用于天然产物化合物库的快速筛选。通过一系列反应条件优化,选择0.4μmol·L^(-1) M^(pro)和100μmol·L^(-1) pNA底物,成功地建立了Z'因子值为0.9的比色法高通量筛选模型。通过对天然产物化合物库进行高通量筛选,证实了白果新酸(ginkgolic acid C13:0)在体外对M^(pro)酶活性具有良好的竞争性抑制作用。本研究成功建立了新冠病毒M^(pro)小分子抑制剂比色法高通量筛选模型,为抗新冠病毒药物先导化合物的筛选与发现奠定了实验基础。For rapid discovery of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)main protease(M^(pro))inhibitors from a natural product library,a novel colorimetric screening assay was developed.According to the colorimetric principle,the synthetic peptide TSAVLQ-para-nitroanilide(pNA)was used as the M^(pro) hydrolysis substrate.Subsequently,the working concentration of pNA substrate,M^(pro) working concentration,hydrolysis time and DMSO tolerance were optimized for the development of a simple and robust colorimetric screening assay.Through these systematic optimizations,we selected 0.4μmol·L^(-1) M^(pro) and 100μmol·L^(-1) pNA substrate as the optimal working concentrations in this colorimetric screening assay,and a high Z'factor of 0.9 was achieved.Using this screening assay,natural product ginkgolic acid C13:0(GA13:0)was identified as a novel competitive M^(pro) inhibitor in vitro.Taken together,we have successfully developed a simple and optimized colorimetric screening assay,which will be vital for the discovery of novel SARS-CoV-2 M^(pro) inhibitors.

关 键 词：新冠病毒 主蛋白酶抑制剂 比色法 荧光共振能量转移 荧光偏振 白果新酸 

分 类 号：R965[医药卫生—药理学]