Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay

被引：50

作者：

Kao, RY ^{[1
]}

To, APC ^{[1
]}

Ng, LWY ^{[1
]}

Tsui, WHW ^{[1
]}

Lee, TSW ^{[1
]}

Tsoi, HW ^{[1
]}

Yuen, KY ^{[1
]}

机构：

[1] Univ Hong Kong, Dept Microbiol, Hong Kong, Hong Kong, Peoples R China

来源：

FEBS LETTERS | 2004年 / 576卷 / 03期

关键词：

severe acute respiratory syndrome; Coronavirus; 3C-like protease; main protease; fluorogenic substrate; small molecule inhibitor;

D O I：

10.1016/j.febslet.2004.09.026

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 muM range with comparable anti-SARS-CoV activity in cell-based assays. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

引用

页码：325 / 330

页数：6

共 22 条

[1] Coronavirus main proteinase (3CLpro) structure:: Basis for design of anti-SARS drugs [J].

Anand, K ;

Ziebuhr, J ;

Wadhwani, P ;

Mesters, JR ;

Hilgenfeld, R .

SCIENCE, 2003, 300 (5626) :1763-1767

[2] Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro [J].

Bacha, U ;

Barrila, J ;

Velazquez-Campoy, A ;

Leavitt, SA ;

Freire, E .

BIOCHEMISTRY, 2004, 43 (17) :4906-4912

[3] Identification of a novel coronavirus in patients with severe acute respiratory syndrome [J].

Drosten, C ;

Günther, S ;

Preiser, W ;

van der Werf, S ;

Brodt, HR ;

Becker, S ;

Rabenau, H ;

Panning, M ;

Kolesnikova, L ;

Fouchier, RAM ;

Berger, A ;

Burguière, AM ;

Cinatl, J ;

Eickmann, M ;

Escriou, N ;

Grywna, K ;

Kramme, S ;

Manuguerra, JC ;

Müller, S ;

Rickerts, V ;

Stürmer, M ;

Vieth, S ;

Klenk, HD ;

Osterhaus, ADME ;

Schmitz, H ;

Doerr, HW .

NEW ENGLAND JOURNAL OF MEDICINE, 2003, 348 (20) :1967-1976

[4] Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase [J].

Fan, KQ ;

Wei, P ;

Feng, Q ;

Chen, SD ;

Huang, CK ;

Ma, L ;

Lai, B ;

Pei, JF ;

Liu, Y ;

Chen, JG ;

Lai, LH .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2004, 279 (03) :1637-1642

[5] A CONTINUOUS FLUORESCENCE-BASED ASSAY OF HUMAN CYTOMEGALOVIRUS PROTEASE USING A PEPTIDE SUBSTRATE [J].

HOLSKIN, BP ;

BUKHTIYAROVA, M ;

DUNN, BM ;

BAUR, P ;

DECHASTONAY, J ;

PENNINGTON, MW .

ANALYTICAL BIOCHEMISTRY, 1995, 227 (01) :148-155

[6] 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism [J].

Huang, CK ;

Wei, P ;

Fan, KQ ;

Liu, Y ;

Lai, LH .

BIOCHEMISTRY, 2004, 43 (15) :4568-4574

[7] Identifying inhibitors of the SARS coronavirus proteinase [J].

Jenwitheesuk, E ;

Samudrala, R .

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2003, 13 (22) :3989-3992

[8] Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides [J].

Kati, WM ;

Sham, HL ;

McCall, JO ;

Montgomery, DA ;

Wang, GT ;

Rosenbrook, W ;

Miesbauer, L ;

Buko, A ;

Norbeck, DW .

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1999, 362 (02) :363-375

[9] A novel coronavirus associated with severe acute respiratory syndrome [J].

Ksiazek, TG ;

Erdman, D ;

Goldsmith, CS ;

Zaki, SR ;

Peret, T ;

Emery, S ;

Tong, SX ;

Urbani, C ;

Comer, JA ;

Lim, W ;

Rollin, PE ;

Dowell, SF ;

Ling, AE ;

Humphrey, CD ;

Shieh, WJ ;

Guarner, J ;

Paddock, CD ;

Rota, P ;

Fields, B ;

DeRisi, J ;

Yang, JY ;

Cox, N ;

Hughes, JM ;

LeDuc, JW ;

Bellini, WJ ;

Anderson, LJ .

NEW ENGLAND JOURNAL OF MEDICINE, 2003, 348 (20) :1953-1966

[10] Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate [J].

Kuo, CJ ;

Chi, YH ;

Hsu, JTA ;

Liang, PH .

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2004, 318 (04) :862-867

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