论文部分内容阅读
目的:以新型冠状病毒(MERS-CoV)主蛋白酶NSP5为靶点,应用荧光共振能量转移法(FRET)建立药物筛选体系,并进行抑制剂的筛选。方法:采用原核表达系统、利用基因重组以及蛋白表达纯化技术得到目的蛋白,FRET法检测蛋白酶活性,优化反应条件以及确定酶、底物的浓度,建立药物筛选体系,对700种化合物进行了检测。结果:应用建立的药物筛选体系,筛选到抑制率较高的化合物共6种,并测得IC_(50)值,其中MDCCCL002330的IC_(50)值最低,为(0.43±0.03)μmol/L。结论:建立的药物筛选体系较为理想,适用于NSP5抑制剂的筛选,促进先导化合物的研发。
OBJECTIVE: To establish a drug screening system using fluorescence resonance energy transfer (FRET) targeting NSP5, a novel major coronavirus (MERS-CoV) protease, and to screen for inhibitors. Methods: Prokaryotic expression system was used to obtain the target protein by gene recombination and protein expression and purification. The protease activity was detected by FRET method, the reaction conditions were optimized, and the concentrations of enzyme and substrate were determined. The drug screening system was established and 700 compounds were detected. Results: A total of 6 compounds with high inhibitory rate were screened out by using established drug screening system. The IC 50 values were obtained. The IC 50 value of MDCCCL002330 was the lowest (0.43 ± 0.03) μmol / L. Conclusion: The established drug screening system is ideal for screening NSP5 inhibitors and promoting the development of lead compounds.