The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture.
Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.
Klumpp
Klaus
K
Roche Palo Alto LLC, CA 94304, USA. klaus.klumpp@roche.com
Lévêque
Vincent
V
Le Pogam
Sophie
S
Ma
Han
H
Jiang
Wen-Rong
WR
Kang
Hyunsoon
H
Granycome
Caroline
C
Singer
Margaret
M
Laxton
Carl
C
Hang
Julie Qi
JQ
Sarma
Keshab
K
Smith
David B
DB
Heindl
Dieter
D
Hobbs
Chris J
CJ
Merrett
John H
JH
Symons
Julian
J
Cammack
Nick
N
Martin
Joseph A
JA
Devos
Rene
R
Nájera
Isabel
I
D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D003562:Cytidine; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C508950:4'-azidocytidine