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

Thomas M. Smith, Siew Pheng Lim, Kimberley Yue, Scott A. Busby, Rishi Arora, Cheah Chen Seh, S. Kirk Wright, Razvan Nutiu, Pornwaratt Niyomrattanakit, Kah Fei Wan, David Beer, Pei Yong Shi, Timothy E. Benson

Research output: Contribution to journalArticle

17 Scopus citations

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 among 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. Last, 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, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.

Original languageEnglish (US)
Pages (from-to)153-163
Number of pages11
JournalJournal of Biomolecular Screening
Volume20
Issue number1
DOIs
StatePublished - Jan 30 2015
Externally publishedYes

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Keywords

  • Flaviviridae
  • LCMS
  • RNA-dependent RNA polymerase
  • RdRp
  • assay
  • dengue virus
  • inhibitor

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

Cite this

Smith, T. M., Lim, S. P., Yue, K., Busby, S. A., Arora, R., Seh, C. C., Kirk Wright, S., Nutiu, R., Niyomrattanakit, P., Wan, K. F., Beer, D., Shi, P. Y., & Benson, T. E. (2015). Identifying initiation and elongation inhibitors of dengue virus RNA polymerase in a high-throughput lead-finding campaign. Journal of Biomolecular Screening, 20(1), 153-163. https://doi.org/10.1177/1087057114551141