Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs

被引:5
|
作者
Villalba, Brian [1 ,2 ]
Li, Jiawen [1 ,3 ]
Johnson, Kenneth A. [1 ]
机构
[1] Univ Texas Austin, Dept Mol Biosci, Austin, TX 78712 USA
[2] MoMa Therapeut, Cambridge, MA USA
[3] Singular Genom Syst Inc, La Jolla, CA USA
基金
美国国家卫生研究院;
关键词
hepatitis C virus (HCV); NS5B; inhibition mechanism; enzyme kinetics; RNA polymerase; viral polymerase; nucleoside; nucleotide analogue; antiviral drug; chain terminator; mericitabine; sofosbuvir; TYPE-1; REVERSE-TRANSCRIPTASE; STATE KINETIC-ANALYSIS; STRUCTURAL BASIS; NUCLEOSIDE; MECHANISM; AZT; PURIFICATION; REPLICATION; UNBLOCKING; SOFOSBUVIR;
D O I
10.1074/jbc.RA120.013422
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
NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2?-fluoro-2?C-methylcytidine-5?-triphosphate and 2?-fluoro-2?C-methyluridine-5?-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2?-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.
引用
收藏
页码:10112 / 10124
页数:13
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