title: Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay.
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.
authors.affiliation: Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, China. rytkao@hkucc.hku.hk
authors.forename: Richard Y
authors.initials: RY
authors.lastname: Kao
authors.forename: Amanda P C
authors.initials: AP
authors.lastname: To
authors.forename: Louisa W Y
authors.initials: LW
authors.lastname: Ng
authors.forename: Wayne H W
authors.initials: WH
authors.lastname: Tsui
authors.forename: Terri S W
authors.initials: TS
authors.lastname: Lee
authors.forename: Hoi-Wah
authors.initials: HW
authors.lastname: Tsoi
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases