國家衛生研究院 NHRI:Item 3990099045/1153
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    题名: Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate
    作者: Kuo, CJ;Chi, YH;Hsu, JTA;Liang, PH
    贡献者: Division of Biotechnology and Pharmaceutical Research
    摘要: 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 K-d of the dimer was 100 muM. 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 (k(cat) = 1.9 s(-1) and K-m = 17 muM) 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. (C) 2004 Elsevier Inc. All rights reserved.
    关键词: Biochemistry & Molecular Biology;Biophysics
    日期: 2004-06-11
    關聯: Biochemical and Biophysical Research Communications. 2004 Jun;318(4):862-867.
    Link to: http://dx.doi.org/10.1016/j.bbrc.2004.04.098
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0006-291X&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000221776400010
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=2442435903
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