摘要
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, 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 recombinant expression and purification of SARS coronavirus main protease（SARS-CoV M^pro）,Then utilized it as the target,used drug screening model in vitro based on fluorescence resonance energy transfer（FRET）,evaluated inhibition activity of 96 compounds from the protease inhibitor focused library,and explored the inhibition of compounds to SARS-CoV M^pro from the perspective of kinetics.The result show that 5 compounds are obtained of which inhibition rate 80%,quenching rate 20% by screened,they are P-1-08,P-1-19,P-2-24,P-2-28 and P-2-54.Their half effective inhibitory concentration（IC50）are 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,respectively.The inhibition of P-1-08,P-1-19,P-2-24 and P-2-54 to SARS-CoV M^pro is irreversible.P-2-28 is reversible inhibition,according to Dixon diagram and Lineweaver-Burk curve found it is competitive inhibition for SARS-CoV M^pro,the inhibition constant Ki is 0.81μmol/L.Through the study about relationship of substrate concentration,IC50 and Ki,validated P-2-28 was competitive inhibition.The discovery of this inhibitor set up the foundation of resource to SARS-CoV M^pro inhibitors,and provided a lead compound for the development of antiSARS virus drug.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2016年第4期35-42,共8页
China Biotechnology
基金
天津市科技支撑计划重点项目(10ZCKFSY08800
10ZCKFSY08700)资助项目
