Inhibition of dengue virus polymerase by blocking of the RNA tunnel

Pornwaratt Niyomrattanakit, Yen Liang Chen, Hongping Dong, Zheng Yin, Min Qing, J. Frasier Glickman, Kai Lin, Dieter Mueller, Hans Voshol, Joanne Y.H. Lim, Shahul Nilar, Thomas H. Keller, Pei Yong Shi

Research output: Contribution to journalArticle

77 Scopus citations

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)20]. Chemical modification of the initial "hit" improved the compound potency to an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC50 of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

Original languageEnglish (US)
Pages (from-to)5678-5686
Number of pages9
JournalJournal of virology
Volume84
Issue number11
DOIs
StatePublished - Jun 1 2010
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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  • Cite this

    Niyomrattanakit, P., Chen, Y. L., Dong, H., Yin, Z., Qing, M., Glickman, J. F., Lin, K., Mueller, D., Voshol, H., Lim, J. Y. H., Nilar, S., Keller, T. H., & Shi, P. Y. (2010). Inhibition of dengue virus polymerase by blocking of the RNA tunnel. Journal of virology, 84(11), 5678-5686. https://doi.org/10.1128/JVI.02451-09