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Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high throughput lead finding campaign

Smith, Thomas, Lim, Siew Pheng, Yue, Qing, Busby, Scott, Arora, Rishi, Seh, Cheah Chen, Wright, Kirk, Nutiu, Razvan, Wan, Kah Fei, Beer, David John, Shi, Pei-Yong, Benson, Timothy and Niyomrattanakit, Pornwaratt (2015) Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high throughput lead finding campaign. Journal of Biomolecular Screening, 20 (1). pp. 153-163.


Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of Dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved amongst the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a “closed” to “open” conformation to accommodate the viral RNA template. Inhibitors that lock the “closed” or block the “open” conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template, but is label-free, and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a “closed” conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Lastly, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium-to high throughput, robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

Item Type: Article
Keywords: Flaviviridae; Dengue Virus; RNA-dependent RNA polymerase; RdRp; LCMS screening, assay, inhibitor
Date Deposited: 13 Oct 2015 13:12
Last Modified: 13 Oct 2015 13:12


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