Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate.
SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.
Kuo
Chih-Jung
CJ
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, ROC.
Chi
Ya-Hui
YH
Hsu
John T-A
JT
Liang
Po-Huang
PH
D000595:Amino Acid Sequence; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D009282:Naphthalenesulfonates / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000378:metabolism
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D010450:Endopeptidases