Structural Basis of Inhibition Specificities of 3C and 3C-like Proteases by Zinc-coordinating and Peptidomimetic Compounds

doi:10.1074/jbc.M807947200

Structural Basis of Inhibition Specificities of 3C and 3C-like Proteases by Zinc-coordinating and Peptidomimetic Compounds*

^‡Structural Biology Program, Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan, ^§Institute of Biological Chemistry, ^¶National Core Facility of High-Throughput Protein Crystallography, and ^∥Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan, ^**Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan, ^‡‡Department of Medical Biotechnology and Laboratory Science, Chang Gung University, and ^§§Clinical Virology Laboratory, Department of Clinical Pathology, Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan, and ^¶¶TaiGen Biotechnology, Taipei 114, Taiwan

2 To whom correspondence may be addressed. Tel.: 886-2-2785-5696, ext. 6070; Fax: 886-2-2788-9759; E-mail: phliang{at}gate.sinica.edu.tw. ³ To whom correspondence may be addressed. Tel.: 886-2-2788-1981; Fax: 886-2-2788-2043; E-mail: ahjwang{at}gate.sinica.edu.tw.

Abstract

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3C^pro) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CL^pro) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CL^pro. As shown in the present study, some of these compounds were also found to be active against 3C^pro of CV strain B3 (CVB3). Several crystal structures of 3C^pro from CVB3 and 3CL^pro from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His⁴⁰-Cys¹⁴⁷ catalytic dyad of CVB3 3C^pro. The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Footnotes

↵4 The abbreviations used are: CV, coxsackievirus; 3C^pro, 3C protease; RV, rhinovirus; 3CL^pro, 3CL protease; CoV, coronavirus; SARS, severe acute respiratory syndrome; EPDTC, N-ethyl-N-phenyldithiocarbamate; Dabcyl, 4-(4-dimethylaminophenylazo)benzoic acid; Edans, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MPD, methyl-2,4-pentanediol; HPLC, high pressure liquid chromatography; DMSO, dimethyl sulfoxide; PEG, polyethylene glycol; RT, reverse transcription.
↵* This work was supported by grants from Academia Sinica and the National Science Council, Taiwan (NSC-95-3112-B-001-015-Y to A. H.-J. W.) for the National Core Facility of High-Throughput Protein Crystallography at Academia Sinica, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

The atomic coordinates and structure factors (codes 2ZTZ, 2ZU1, 2ZTY, 2ZTX, 2ZU2, 2ZU3, 2ZU4, and 2ZU5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
↵1 Both authors contributed equally to this work.
- Received October 16, 2008.
- Revision received December 26, 2008.
The American Society for Biochemistry and Molecular Biology, Inc.