Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay |
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Authors: | 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 |
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Affiliation: | Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, China. rytkao@hkucc.hku.hk |
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Abstract: | Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity. The kinetic parameters of the recombinant SARS-CoV Mpro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay. Two novel small molecule inhibitors of the SARS-CoV Mpro were identified by high-throughput screening using an internally quenched fluorogenic substrate. The identified inhibitors have Ki values at low microM range with comparable anti-SARS-CoV activity in cell-based assays. |
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