Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-d-2'-ethynyl-7-deaza-adenosine triphosphate.
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.
Latour
Derek R
DR
Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA, United States.
Jekle
Andreas
A
Javanbakht
Hassan
H
Henningsen
Robert
R
Gee
Peter
P
Lee
Ina
I
Tran
Patricia
P
Ren
Suping
S
Kutach
Alan K
AK
Harris
Seth F
SF
Wang
Sandra M
SM
Lok
Stephen J
SJ
Shaw
David
D
Li
Jim
J
Heilek
Gabrielle
G
Klumpp
Klaus
K
Swinney
David C
DC
Deval
Jerome
J
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
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases