Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay

Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV M-pro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV M-pro in Escherichia coli, and purified the recombinant M-pro to homogeneity. The kinetic parameters of the recombinant SARS-CoV M-pro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay. Two novel small molecule inhibitors of the SARS-CoV M-pro were identified by high-throughput screening using an internally quenched fluorogenic substrate. The identified inhibitors have K-i values at low muM range with comparable anti-SARS-CoV activity in cell-based assays. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.