title: Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.
abstract: The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Xu
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Susan P
authors.initials: SP
authors.lastname: Colby-Germinario
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Said
authors.initials: S
authors.lastname: Hassounah
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Quashie
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Yingshan
authors.initials: Y
authors.lastname: Han
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Maureen
authors.initials: M
authors.lastname: Oliveira
authors.affiliation: Champlain Exploration Pharma, Inc., Montreal, Quebec, Canada.
authors.forename: Brent R
authors.initials: BR
authors.lastname: Stranix
authors.affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada Department of Medicine, McGill University, Montreal, Quebec, Canada Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada mark.wainberg@mcgill.ca.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wainberg
mesh_terms: D000330:Aedes; D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D002614:Chelating Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D006224:Cricetinae; D003716:Dengue Virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004847:Epithelial Cells / Q000187:drug effects / Q000821:virology; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D062105:Molecular Docking Simulation; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010847:Picolines / Q000138:chemical synthesis / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000138:chemical synthesis / Q000494:pharmacology*; D013450:Sulfones / Q000138:chemical synthesis / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D002614:Chelating Agents; C000611450:DMB220; C471213:His-His-His-His-His-His; D006877:Hydroxamic Acids; C582223:NS5 protein, dengue virus; D009842:Oligopeptides; D010847:Picolines; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D013450:Sulfones; D017361:Viral Nonstructural Proteins; D006639:Histidine; D012324:RNA-Dependent RNA Polymerase