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

Kao, Richard Y.; To, Amanda P.C.; Ng, Louisa W.Y.; Tsui, Wayne H.W.; Lee, Terri S.W.; Tsoi, Hoi-Wah; Yuen, Kwok-Yung

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 μM range with comparable anti-SARS-CoV activity in cell-based assays.