Canonical 3'-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase.
Nucleoside chain terminators represent one of the most promising classes of antiviral drug for DNA viruses and retroviral infection; however, they have not been fully explored against RNA viral polymerases. In this report, we investigate the notion of employing canonical 3'-deoxyribonucleoside triphosphates (3'-dNTPs) as a chain terminator for hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). Using a HCV RNA transcript-dependent RNA elongating assay, we found that they inhibit NS5B RdRp with K(i) ranged from 0.7 to 23 microM. Additional structure-activity relationship studies showed that removal of 2'-hydroxyl group, elimination of ribose's 2',3'-carbon-carbon bond, or addition of 5-methyl group to a pyrimidine base is detrimental to 3'-dNTP's potency. Direct evidence was obtained that all four canonical 3'-dNTP are incorporated into elongating RNA chains and the incorporation terminates NS5B RdRp-catalyzed RNA synthesis. The K(i) values for each of 3'-dNTPs were determined in the single nucleotide incorporation experiments. The nucleoside form of 3'-dNTPs was further evaluated in a cell culture-based HCV subgenomic replicon assay. The discrepancy between the potent in vitro activity and the weak cellular activity of these chain terminators was discussed in the context of nucleoside metabolism. This proof of concept study demonstrates that canonical 3'-dNTPs can function as an effective chain terminator for HCV NS5B RdRp with cytidine as the preferred nucleoside scaffold. Our results further sheds light on the potential hurdles that need to be overcome for successful development of active nucleoside chain terminators in vivo for a viral RNA polymerase, especially the HCV NS5B RdRp.
Shim
Jaehoon
J
Drug discovery, Ribapharm Corporation, 3300 Hyland Avenue, Costa Mesa, CA 92626, USA.
Larson
Gabry
G
Lai
Vicky
V
Naim
Suhaila
S
Wu
Jim Zhen
JZ
D003562:Cytidine / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D003854:Deoxyribonucleotides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D003562:Cytidine; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase