Screening And Research For Inhibitors Of SARS Coronavirus Main Protease

SARS coronavirus is a single strand RNA coronavirus with the largest genome can cause severe acute respiratory syndrome(SARS).As the major proteolytic enzymes of SARS virus,SARS coronavirus main protease(SARS-CoV MPr0)plays the key role in viral replication and pathogenicity and it has highly conservative in the same genus coronavirus,it is an ideal target for anti-SARS virus drugs.Therefore,The discovery and development of its inhibitor or drug not only has important theoretical significance.but also contains significant practical value.The issue using the SARS coronavirus main protease as a model with the high-throughput drug screening techniques,researched and developed the treatments of SARS for SARS coronavirus main protease inhibitors.First heterologous recombinant expression and purification of SARS-CoV MPr0,then as a target,detected the protease activity through the change of fluorescence intensity with the using of drug screening model in vitro.Then evaluated the inhibition activity in vitro of 275 kinds of compounds from the small molecules fragment library and protease inhibitor library.We obtained 5 compounds which inhibition rate>80%,quenching rate<20%by screening.They are P-1-08,P-1-19,P-2-24,P-2-28 and P-2-54.Their half effective inhibitoryconcentration(IC50)were:0.69±0.05?mol/L,1.19±0.4 1?mol/L,0.14±0.01?mol/L,1.36±0.07?mol/L and 0.36±0.03?mol/L,respectively.Then discussed their inhibition mechanism to SARS-CoV Mpro through kinetics and docking.The results found that P-1-08,P-1-19,P-2-24 and P-2-54 can bind to SARS-CoV Mpro active site Cys-145 thought a covalent bond,they are irreversible inhibition,P-2-28 combined with SARS-CoV Mpro by the way of forming hydrogen bonds with Asn-142,Gly-143 and Gln-189,is reversible inhibition.According to Lineweaver-Burk and Dixon diagram found that P-2-28 was competitive inhibition for SARS-CoV Mpro.the inhibition constant K1 was 0.81?mol/L.Through study the relationship of substrate concentration,IC50 and Ki.further validated P-2-28 was competitive inhibition.The found of these compounds lay a foundation of SARS-CoV Mpro inhibitors,and provided a lead compound for the development of anti-SARS virus drug.