Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-D-2′-ethynyl-7-deaza-adenosine triphosphate

被引：35

作者：

Latour, Derek R. ^{[1
]}

Jekle, Andreas ^{[1
]}

Javanbakht, Hassan ^{[1
]}

Henningsen, Robert ^{[1
]}

Gee, Peter ^{[1
]}

Lee, Ina ^{[1
]}

Tran, Patricia ^{[1
]}

Ren, Suping ^{[1
]}

Kutach, Alan K. ^{[1
]}

Harris, Seth F. ^{[1
]}

Wang, Sandra M. ^{[1
]}

Lok, Stephen J. ^{[1
]}

Shaw, David ^{[1
]}

Li, Jim ^{[1
]}

Heilek, Gabrielle ^{[1
]}

Klumpp, Klaus ^{[1
]}

Swinney, David C. ^{[1
]}

Deval, Jerome ^{[1
]}

机构：

[1] Roche Palo Alto LLC, Palo Alto, CA USA

来源：

ANTIVIRAL RESEARCH | 2010年 / 87卷 / 02期

关键词：

Dengue virus; NS5; polymerase; Nucleotide; Chain terminator; Single-nucleotide incorporation; Steady-state kinetics; HEPATITIS-C VIRUS; ANTIVIRAL NUCLEOSIDE ANALOGS; CRYSTAL-STRUCTURE; NONSTRUCTURAL PROTEIN-5; NUCLEAR-LOCALIZATION; DRUG DISCOVERY; REPLICATION; INITIATION; RESISTANCE; DESIGN;

D O I：

10.1016/j.antiviral.2010.05.003

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

Dengue virus (DENV), an emerging pathogen from the Flaviviridae family with neither vaccine nor antiviral treatment available, causes a serious worldwide public health threat. In theory, there are several ways by which small molecules could inhibit the replication cycle of DENV. Here, we show that the nucleoside analogue beta-D-2'-ethynyl-7-deaza-adenosine inhibits representative strains of all four serotypes of DENV with an EC50 around or below 1 M. Using membrane-associated native replicase complex as well as recombinant RNA polymerase from each DENV serotype in enzymatic assays, we provide evidence that beta-D-2'-ethynyl-7-deaza-adenosine triphosphate (2'E-7D-ATP) targets viral replication at the polymerase active site by competing with the natural nucleotide substrate with an apparent K-i of 0.060 +/- 0.016 mu M. In single-nucleotide incorporation experiments, the catalytic efficiency of 2'E-7D-ATP is 10-fold lower than for natural ATP, and the incorporated nucleotide analogue causes immediate chain termination. A combination of bioinformatics and site-directed mutagenesis demonstrates that 2'E-7D-ATP is equipotent across all serotypes because the nucleotide binding site residues are conserved in dengue virus. Overall, beta-D-2'-ethynyl-7-deaza-adenosine provides a promising scaffold for the development of inhibitors of dengue virus polymerase. (C) 2010 Elsevier B.V. All rights reserved.

引用

页码：213 / 222

页数：10

共 40 条

[1] De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase [J].