Inhibition of Human Cytomegalovirus Protease by Monocyclic -Lactam Derivatives: Kinetic Characterization Using a Fluo
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- 作者：Pierre R. Bonneau ; Firoz Hasani ; Cé ; line Plouffe ; Eric Malenfant ; Steve R. LaPlante ; Ingrid Guse ; William W. Ogilvie ; Raymond Plante ; Walter C. Davidson ; Jerry L. Hopkins ; Maurice M. Morelock ; M
- 刊名：Journal of the American Chemical Society
- 出版年：1999
- 出版时间：April 7, 1999
- 年：1999
- 卷：121
- 期：13
- 页码：2965 - 2973
- 全文大小：149K
- 年卷期：v.121,no.13(April 7, 1999)
- ISSN：1520-5126
文摘
Recent reports have demonstrated the potential of monocyclic 
-lactam derivatives as inhibitors ofhuman cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and massspectrometry has revealed the presence of an acylenzyme intermediate suggesting that -lactams are hydrolyzedby the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic -lactamderivative for convenient kinetic characterization of this mechanism has been evaluated using 4S-(4-methylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide (1). Upon acylationof the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of thehydrolytic process. Examination of a series of progress curves by numerical analysis has provided valuableinformation on acylation and deacylation rates which relate to the IC50 values observed for -lactams. Moreimportantly the potential of compound 1 as an active site titrating agent for HCMV protease has been exploited,and a simple protocol for rapid determination of active enzyme is described. The data are consistent with theHCMV protease dimer being composed of two functional active sites. This titrating agent represents an importanttool that should significantly facilitate the characterization of this novel enzyme.
-lactam derivatives as inhibitors ofhuman cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and massspectrometry has revealed the presence of an acylenzyme intermediate suggesting that -lactams are hydrolyzedby the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic -lactamderivative for convenient kinetic characterization of this mechanism has been evaluated using 4S-(4-methylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide (1). Upon acylationof the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of thehydrolytic process. Examination of a series of progress curves by numerical analysis has provided valuableinformation on acylation and deacylation rates which relate to the IC50 values observed for -lactams. Moreimportantly the potential of compound 1 as an active site titrating agent for HCMV protease has been exploited,and a simple protocol for rapid determination of active enzyme is described. The data are consistent with theHCMV protease dimer being composed of two functional active sites. This titrating agent represents an importanttool that should significantly facilitate the characterization of this novel enzyme.