Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors

Mechanism of Action and Resistance Profiling

Siew Pheng Lim, Christian Guy Noble, Cheah Chen Seh, Tingjin Sherryl Soh, Abbas El Sahili, Grace Kar Yarn Chan, Julien Lescar, Rishi Arora, Timothy Benson, Shahul Nilar, Ujjini Manjunatha, Kah Fei Wan, Hongping Dong, Xuping Xie, Pei-Yong Shi, Fumiaki Yokokawa

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Original languageEnglish (US)
Article numbere1005737
JournalPLoS Pathogens
Volume12
Issue number8
DOIs
StatePublished - Aug 1 2016

Fingerprint

Viral Structural Proteins
RNA Replicase
Dengue Virus
Flavivirus
Thumb
Catalytic Domain
Genome
RNA
Replicon
X Ray Crystallography
Viral RNA
Guanosine Triphosphate
Antiviral Agents
Proteins
Cell Culture Techniques
Amino Acids

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology

Cite this

Lim, S. P., Noble, C. G., Seh, C. C., Soh, T. S., El Sahili, A., Chan, G. K. Y., ... Yokokawa, F. (2016). Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling. PLoS Pathogens, 12(8), [e1005737]. https://doi.org/10.1371/journal.ppat.1005737

Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors : Mechanism of Action and Resistance Profiling. / Lim, Siew Pheng; Noble, Christian Guy; Seh, Cheah Chen; Soh, Tingjin Sherryl; El Sahili, Abbas; Chan, Grace Kar Yarn; Lescar, Julien; Arora, Rishi; Benson, Timothy; Nilar, Shahul; Manjunatha, Ujjini; Wan, Kah Fei; Dong, Hongping; Xie, Xuping; Shi, Pei-Yong; Yokokawa, Fumiaki.

In: PLoS Pathogens, Vol. 12, No. 8, e1005737, 01.08.2016.

Research output: Contribution to journalArticle

Lim, SP, Noble, CG, Seh, CC, Soh, TS, El Sahili, A, Chan, GKY, Lescar, J, Arora, R, Benson, T, Nilar, S, Manjunatha, U, Wan, KF, Dong, H, Xie, X, Shi, P-Y & Yokokawa, F 2016, 'Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling', PLoS Pathogens, vol. 12, no. 8, e1005737. https://doi.org/10.1371/journal.ppat.1005737
Lim, Siew Pheng ; Noble, Christian Guy ; Seh, Cheah Chen ; Soh, Tingjin Sherryl ; El Sahili, Abbas ; Chan, Grace Kar Yarn ; Lescar, Julien ; Arora, Rishi ; Benson, Timothy ; Nilar, Shahul ; Manjunatha, Ujjini ; Wan, Kah Fei ; Dong, Hongping ; Xie, Xuping ; Shi, Pei-Yong ; Yokokawa, Fumiaki. / Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors : Mechanism of Action and Resistance Profiling. In: PLoS Pathogens. 2016 ; Vol. 12, No. 8.
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