title: Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-d-2'-ethynyl-7-deaza-adenosine triphosphate.
abstract: Dengue virus (DENV), an emerging pathogen from the Flaviviridae family with neither vaccine nor antiviral treatment available, causes a serious worldwide public health threat. In theory, there are several ways by which small molecules could inhibit the replication cycle of DENV. Here, we show that the nucleoside analogue beta-d-2'-ethynyl-7-deaza-adenosine inhibits representative strains of all four serotypes of DENV with an EC(50) around or below 1microM. Using membrane-associated native replicase complex as well as recombinant RNA polymerase from each DENV serotype in enzymatic assays, we provide evidence that beta-d-2'-ethynyl-7-deaza-adenosine triphosphate (2'E-7D-ATP) targets viral replication at the polymerase active site by competing with the natural nucleotide substrate with an apparent K(i) of 0.060+/-0.016microM. In single-nucleotide incorporation experiments, the catalytic efficiency of 2'E-7D-ATP is 10-fold lower than for natural ATP, and the incorporated nucleotide analogue causes immediate chain termination. A combination of bioinformatics and site-directed mutagenesis demonstrates that 2'E-7D-ATP is equipotent across all serotypes because the nucleotide binding site residues are conserved in dengue virus. Overall, beta-d-2'-ethynyl-7-deaza-adenosine provides a promising scaffold for the development of inhibitors of dengue virus polymerase.
authors.affiliation: Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA, United States.
authors.forename: Derek R
authors.initials: DR
authors.lastname: Latour
authors.forename: Andreas
authors.initials: A
authors.lastname: Jekle
authors.forename: Hassan
authors.initials: H
authors.lastname: Javanbakht
authors.forename: Robert
authors.initials: R
authors.lastname: Henningsen
authors.forename: Peter
authors.initials: P
authors.lastname: Gee
authors.forename: Ina
authors.initials: I
authors.lastname: Lee
authors.forename: Patricia
authors.initials: P
authors.lastname: Tran
authors.forename: Suping
authors.initials: S
authors.lastname: Ren
authors.forename: Alan K
authors.initials: AK
authors.lastname: Kutach
authors.forename: Seth F
authors.initials: SF
authors.lastname: Harris
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Wang
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lok
authors.forename: David
authors.initials: D
authors.lastname: Shaw
authors.forename: Jim
authors.initials: J
authors.lastname: Li
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Heilek
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
authors.forename: David C
authors.initials: DC
authors.lastname: Swinney
authors.forename: Jerome
authors.initials: J
authors.lastname: Deval
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D019295:Computational Biology; D017124:Conserved Sequence; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D003716:Dengue Virus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases