Mechanistic Study of HCV Polymerase Inhibitors at Individual Steps of the Polymerization Reaction

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• 作者：Yaya Liu ; Wen W. Jiang ; John Pratt ; Todd Rockway ; Kevin Harris ; Sudthida Vasavanonda ; Rakesh Tripathi ; Ron Pithawalla ; Warren M. Kati
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：September 26, 2006
• 年：2006
• 卷：45
• 期：38
• 页码：11312 - 11323
• 全文大小：792K
• 年卷期：v.45,no.38(September 26, 2006)
• ISSN：1520-4995

文摘

Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporationand the individual steps employed by this enzyme during a catalytic cycle. In this paper, we appliedvarious biochemical tools and examined the mechanism of polymerase catalysis. We found that formationof a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiationof primer strand synthesis. Various groups have reported several classes of small molecule inhibitors ofhepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitorsis not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms ofinhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitivelybind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation stepsof polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect tothe active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiationpocket in the active site and inhibit only the initiation step of de novo RNA polymerization. Thebenzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNAsynthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiationpocket using inhibitor-inhibitor cross competition studies. This information will be very important indesigning combination therapies using two small molecule drugs to treat hepatitis C virus.