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dc.titlePotent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling
dc.contributor.authorLim S.P.
dc.contributor.authorNoble C.G.
dc.contributor.authorSeh C.C.
dc.contributor.authorSoh T.S.
dc.contributor.authorEl Sahili A.
dc.contributor.authorChan G.K.Y.
dc.contributor.authorLescar J.
dc.contributor.authorArora R.
dc.contributor.authorBenson T.
dc.contributor.authorNilar S.
dc.contributor.authorManjunatha U.
dc.contributor.authorWan K.F.
dc.contributor.authorDong H.
dc.contributor.authorXie X.
dc.contributor.authorShi P.-Y.
dc.contributor.authorYokokawa F.
dc.identifier.citationLim 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.
dc.description.abstractFlaviviruses 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.
dc.rightsAttribution 4.0 International
dc.sourceUnpaywall 20191101
dc.subjectnonstructural protein 5
dc.subjectRNA directed RNA polymerase
dc.subjectantivirus agent
dc.subjectNS5 protein, dengue virus
dc.subjectnucleic acid synthesis inhibitor
dc.subjectRNA directed RNA polymerase
dc.subjectviral protein
dc.subjectantiviral therapy
dc.subjectbinding affinity
dc.subjectbiochemical analysis
dc.subjectcontrolled study
dc.subjectDengue virus
dc.subjectenzyme activity
dc.subjectenzyme assay
dc.subjectenzyme inhibition assay
dc.subjectgene expression
dc.subjectluciferase assay
dc.subjectprotein expression
dc.subjectreverse genetics
dc.subjectvirus morphology
dc.subjectvirus replication
dc.subjectX ray crystallography
dc.subjectZika virus
dc.subjectA-549 cell line
dc.subjectantagonists and inhibitors
dc.subjectDengue virus
dc.subjectdrug design
dc.subjectdrug effects
dc.subjectprotein domain
dc.subjectsurface plasmon resonance
dc.subjectA549 Cells
dc.subjectAntiviral Agents
dc.subjectCrystallography, X-Ray
dc.subjectDengue Virus
dc.subjectDrug Design
dc.subjectNucleic Acid Synthesis Inhibitors
dc.subjectProtein Domains
dc.subjectRNA Replicase
dc.subjectSurface Plasmon Resonance
dc.subjectViral Nonstructural Proteins
dc.contributor.departmentDEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL)
dc.contributor.departmentDUKE-NUS MEDICAL SCHOOL
dc.contributor.departmentDEPT OF BIOLOGICAL SCIENCES
dc.contributor.departmentDEPT OF MICROBIOLOGY & IMMUNOLOGY
dc.description.sourcetitlePLoS Pathogens
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