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