SARS-CoV-2 Mpro抑制剂筛选体系的构建及临床常用抗病毒中药制剂的初步筛选

Construction of Screening System for SARS-CoV-2 Mpro Inhibitors and Preliminary Screening of Antiviral Chinese Medicine Preparations

目的:利用荧光共振能量转移(Fluorescence resonance energy transfer,FRET)技术构建新冠病毒主蛋白酶(SARS-CoV-2 Mpro)中药抑制剂的体外筛选方法。方法:构建Mpro及筛选探针原核表达的工程质粒pGEX-4T-1-Mpro、pET-28a(+)-Ls-mK。使用荧光探针Ls-mK检测Mpro的生物活性及酶动力学参数,并对筛选条件进行优化。采用阳性药物GC-376及阴性对照PLpro(Papain-like protease)和TEV酶(Tobacco Etch Virus Proteinase)评价荧光探针Ls-mK的特异性及检测能力与商业化探针的差异。根据所构建的筛选体系考察五种中药制剂筛选热毒宁注射液、血必净注射液、抗病毒口服液和蒲地蓝口服液对Mpro的抑制能力。结果:本研究成功构建原核表达的工程质粒并获得纯度为90%左右的重组蛋白荧光探针Ls-mK及Mpro。荧光探针Ls-mK可成功产生FRET现象并具有良好的特异性。Mpro具有良好的生物活性。本研究所构建的筛选体系具有与商业化探针相同的检测能力,对五种中药制剂考察发现仅喜炎平注射液对Mpro具有良好的抑制活性,其IC_(50)值为(3.32±0.03)mg/mL。结论:本研究成功构建基于荧光蛋白SARS-CoV-2 Mpro的简便、灵敏和低成本的筛选体系,为新冠病毒主蛋白酶抑制剂的筛选与发现奠定了实验基础。

Objective:To construct an in vitro screening system of Chinese medicine inhibitors of SARS-CoV-2 Mpro by fluorescence resonance energy transfer(FRET)technology.Methods:The engineering plasmids pGEX-4T-1-Mpro and pET-28a(+)-Ls-mK were constructed.The biological activity and enzyme kinetic parameters of Mpro were determined by the fluorescent probe Ls-mK,and the screening conditions were optimized.The differences between fluorescent probe Ls-mK and commercial probe in specificity and detection ability were evaluated by the positive drug GC-376 and the negative control papain-like protease(PLpro)and tobacco etch virus(TEV)protease.According to the constructed screening system,the inhibitory ability of five Chinese medicine preparations against Mpro was investigated,including Reduning(热毒宁)Injection,Xuebijing(血必净)Injection,Antiviral Oral Liquid and Pudilan(蒲地蓝)Oral Liquid.Results:The engineering plasmid expressed in prokaryotes was successfully constructed,and the recombinant protein fluorescent probes Ls-mK and Mpro with a purity of about 90%were obtained.The fluorescent probe Ls-mK produced FRET phenomenon and presented good specificity.Mpro had good biological activity.The screening system constructed in this paper specifically had the same detection ability as commercial probes.It was found that only Xiyanping Injection inhibited Mpro,with an IC_(50) value of 3.32±0.03 mg/mL.Conclusion:This paper successfully constructed a simple,sensitive and low-cost screening system based on SARS-CoV-2 Mpro,which laid an experimental foundation for the screening and discovery of SARS-CoV-2 Mpro inhibitors.