Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA, United States.
Antiviral Res. 2010 Aug;87(2):213-22. doi: 10.1016/j.antiviral.2010.05.003. Epub 2010 May 12.
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 EC(50) around or below 1microM. 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+/-0.016microM. 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.
登革热病毒（DENV）是黄病毒科的一种新兴病原体，目前尚无疫苗或抗病毒治疗方法，对全球公共卫生构成严重威胁。从理论上讲，小分子有几种可能的方式来抑制 DENV 的复制周期。在这里，我们表明，核苷类似物β-d-2'-乙炔基-7-脱氮腺苷以约 1μM 或以下的 EC（50）抑制所有 4 种血清型的代表性菌株。在酶促测定中，我们使用膜相关的天然复制酶复合物以及来自每种 DENV 血清型的重组 RNA 聚合酶，提供了证据表明β-d-2'-乙炔基-7-脱氮腺苷三磷酸（2'E-7D-ATP）通过与天然核苷酸底物竞争，以与天然核苷酸底物竞争的方式靶向聚合酶活性位点，表观 K（i）为 0.060+/-0.016μM。在单核苷酸掺入实验中，2'E-7D-ATP 的催化效率比天然 ATP 低 10 倍，并且掺入的核苷酸类似物会立即导致链终止。生物信息学和定点诱变的组合表明，2'E-7D-ATP 在所有血清型中均具有同等效力，因为登革热病毒的核苷酸结合位点残基是保守的。总体而言，β-d-2'-乙炔基-7-脱氮腺苷为开发登革热病毒聚合酶抑制剂提供了有希望的支架。
