Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs.
The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.
Carroll
Steven S
SS
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. steve_carroll@merck.com
Tomassini
Joanne E
JE
Bosserman
Michele
M
Getty
Krista
K
Stahlhut
Mark W
MW
Eldrup
Anne B
AB
Bhat
Balkrishen
B
Hall
Dawn
D
Simcoe
Amy L
AL
LaFemina
Robert
R
Rutkowski
Carrie A
CA
Wolanski
Bohdan
B
Yang
Zhucheng
Z
Migliaccio
Giovanni
G
De Francesco
Raffaele
R
Kuo
Lawrence C
LC
MacCoss
Malcolm
M
Olsen
David B
DB
D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D002478:Cells, Cultured; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005782:Gels; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D000241:Adenosine; D000255:Adenosine Triphosphate; D003562:Cytidine; D003570:Cytidine Triphosphate; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D005782:Gels; D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C052203:2'-O-methylcytidine; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase