冠状病毒主蛋白酶小分子有机硒化合物抑制剂筛选

Screening of small-molecule organoselenium compounds inhibitors for the main protease of coronavirus

[目的]基于荧光共振能量转移技术(FRET)筛选抑制冠状病毒主蛋白酶(M^(pro))活性的小分子有机硒化合物。[方法]将两种冠状病毒M^(pro)蛋白在大肠杆菌(Escherichia coli)BL21(DE3)中表达并纯化;基于FRET确认M^(pro)蛋白的体外活性并对13种小分子有机硒化合物进行筛选;进一步用MTT法测定有效化合物的细胞毒性;最后利用计算机软件AutoDock 4.2.6对有效化合物和两种冠状病毒M^(pro)进行分子对接分析。[结果]成功表达纯化出具有酶活性的两种M^(pro)蛋白;筛选发现11种化合物对两种M^(pro)均具有抑制效果,其中SLL-1A-46的抑制效果最显著,其对两种M^(pro)的酶活半抑制浓度(IC_(50))分别为:23.95μmol/L(MERS-CoV)、24.97μmol/L(SARS-CoV);细胞毒性检测结果显示SLL-1A-46对三种细胞的半数毒性浓度(CC_(50))分别为75.41μmol/L(Vero E6)、39.96μmol/L(A549)和65.06μmol/L(293T);分子对接结果提示SLL-1A-46与两种M^(pro)活性位点处的结合能分别为-8.26 kcal/mol(MERS-CoV)和-8.37 kcal/mol(SARS-CoV)。[结论]初步筛选到11种抑制两种冠状病毒M^(pro)酶活性的小分子有机硒化合物,SLL-1A-46具有作为广谱冠状病毒M^(pro)抑制剂的潜力,其与两种M^(pro)的结合特点为广谱冠状病毒M^(pro)抑制剂的药物设计提供线索。

[Objective]To Screen of small molecule organic selenium compounds that inhibit the activity of coronavirus main protease(MPro)based on fluorescence resonance energy transfer technology(FRET).[Method]Two types of coronavirus M^(pro)proteins were expressed in Escherichia coli BL21(DE3)and purified.The activity of M^(pro)protein was confirmed in vitro based on FRET and 13 small molecule organic selenium compounds were screened out.Further cytotoxicity of effective compounds was determined using the MTT method.Finally,molecular docking analysis was performed on the effective compounds and three types of coronavirus M^(pro)by computer software AutoDock 4.2.6.[Result]Two types of M^(pro)proteins with enzymatic activity were successfully expressed and purified.Eleven compounds were found to have inhibitory effects on all two M^(pro),in which SLL-1A-46 exerted the most significant inhibitory effects.The enzyme activity half maximal inhibitory concentration(ICso)of SLL-1A-46 on the two M^(pro)was 23.95μmol/L for MERS-CoV,24.97μmol/L for SARS-CoV respectively.The cytotoxicity test results shown that the median cytotoxic concentration(CCso)of SLL-1A-46 on two types of cells was 75.41μmol/L for Vero E6,39.96μmol/L for A549,65.06μmol/L for 293T respectively.The molecular docking results indicated that the bonding of SLL-1A-46 in two M^(pro)active sites is-8.26 kcal/mol for MERS CoV,and-8.37 kcal/mol for SARS-CoV respectively.[Conclusion]This study preliminarily screens 1l small-molecule organoselenium compounds that inhibit the activity of coronavirus M^(pro).SLL-1A-46 has potential as a broad-spectrum coronavirus M^(pro)inhibitor.Its binding characteristics with two M^(pro)can provide clues for the drug design of broad-spectrum coronavirus M^(pro)inhibitors.