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

Abstract

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