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Title: Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling
Authors: Lim S.P. 
Noble C.G.
Seh C.C.
Soh T.S.
El Sahili A.
Chan G.K.Y. 
Lescar J.
Arora R.
Benson T.
Nilar S.
Manjunatha U. 
Wan K.F.
Dong H.
Xie X.
Shi P.-Y. 
Yokokawa F.
Keywords: nonstructural protein 5
RNA directed RNA polymerase
antivirus agent
NS5 protein, dengue virus
nucleic acid synthesis inhibitor
RNA directed RNA polymerase
viral protein
antiviral therapy
binding affinity
biochemical analysis
controlled study
Dengue virus
enzyme activity
enzyme assay
enzyme inhibition assay
gene expression
luciferase assay
protein expression
reverse genetics
virus morphology
virus replication
X ray crystallography
Zika virus
A-549 cell line
antagonists and inhibitors
Dengue virus
drug design
drug effects
protein domain
surface plasmon resonance
A549 Cells
Antiviral Agents
Crystallography, X-Ray
Dengue Virus
Drug Design
Nucleic Acid Synthesis Inhibitors
Protein Domains
RNA Replicase
Surface Plasmon Resonance
Viral Nonstructural Proteins
Issue Date: 2016
Citation: Lim S.P., Noble C.G., Seh C.C., Soh T.S., El Sahili A., Chan G.K.Y., Lescar J., Arora R., Benson T., Nilar S., Manjunatha U., Wan K.F., 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 12 (8) : e1005737. ScholarBank@NUS Repository.
Rights: Attribution 4.0 International
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 ?M 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. ? 2016 Lim et al.
Source Title: PLoS Pathogens
ISSN: 15537366
DOI: 10.1371/journal.ppat.1005737
Rights: Attribution 4.0 International
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