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对SARS冠状病毒主蛋白酶(SARS-Co V M~(pro))进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,t对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M~(pro)的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC_(50))分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC_(50)值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。
Heterologous recombination and purification of the SARS-Co VM pro (SARS-Co VM pro pro), using it as an in vitro drug screening model based on fluorescence resonance energy transfer (FRET) The inhibitory activity and mechanism of the selected compounds on the inhibition of SARS-CoV M ~ (pro) were investigated from the kinetic point of view. The results showed that five compounds with inhibitory rate> 80% and quenching rate <20% were P-1-08, P-1-19, P-2-24, (IC 50) were 0.69 ± 0.05μmol / L, 1.19 ± 0.41μmol / L, 0.14 ± 0.01μmol / L, 1.36 ± 0.07μmol / L, 0.36 ± 0.03μmol / L. The inhibitory effect of compounds P-1-08, P-1-19, P-2-24 and P-2-54 on the major protease of SARS coronavirus was irreversible, and the inhibitory effect of compound P-2-28 was reversible . According to the Lineweaver-Burk plot and the Dixon plot, compound P-2-28 was found to competitively inhibit the major protease of SARS coronavirus with a Ki constant of 0.81 μmol / L. The inhibitory effect of P-2-28 was further confirmed by competitive inhibition of substrate concentration, IC 50 and Ki values. The discovery of this inhibitor laid the foundation for the study of the major protease inhibitor of SARS coronavirus and provided the lead compound for the anti-SARS virus drug development.