Article
Understanding the reaction mechanism of SARS 3C-like proteinase for structural based drug design against SARS
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Authors
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Luhua Lai
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Ying Liu
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Keqiang Fan
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Changkang Huang
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Ping Wei
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Zhenming Liu
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Shiyong Liu
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Hao Chen
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Jianfeng Pei
- State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry, Peking University, Beijing, China
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Jianguo Chen
- College of Life Science, Peking University, Beijing, China
| Published: | May 26, 2004 |
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Outline
Text
The 3C-like proteinase of SARS coronavirus has been proposed to be a key target for structural based drug design against SARS. In order to understand the substrate specificity, enzyme catalytic mechanism and to develop efficient inhibitors, we have cloned, expressed and purified SARS 3C-like proteinase. Analytic gel filtration and ultracentrifuge studies confirmed that the enzyme presents in solution as a dynamic mixture of monomer and dimer. Enzyme activity studies reveal that the enzyme dimer is the major active form for catalytic reaction and the dimer interface can serve as a novel site for inhibitor design. Compounds designed to bind with the interface were shown to be active inhibitors against this enzyme.
We also studied the substrate specificity and enzyme mechanism. SARS 3C-like proteinase can cut all the 11 peptides corresponding to the 11 cleavage sites on the virus poly-protein with different efficiency, which is closely related to the secondary structures of the substrate peptides in addition to sequence selectivity. A continuous colorimetric assay for SARS 3C-like proteinase was developed to study the enzyme mechanism and for inhibitor screen. The pKa values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and can not be explained by a thiolate-imidazolium ion-pair model. The structure flexibility of SARS 3C-like proteinase was studied by molecular dynamics simulations and several conformers were applied in structural based inhibitor design. Compounds from virtual database screen were confirmed to be active in the enzyme and cell based assays.
References
- 1.
- Fan KQ, Wei P, Feng Q, Chen SD, Huang CK, Ma L, Lai B, Pei JF, Liu Y, Chen JG, Lai LH. Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase. Journal of Biological Chemistry 2004; 279(3):1637-1642
- 2.
- Huang CK, Wei P, Fan KQ, Liu Y, Lai LH. 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism. Biochemistry, in press
