The mechanism of action of beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine involves a second metabolic pathway leading to beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate, a potent inhibitor of the hepatitis C virus RNA-dependent RNA polymerase

Abstract

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.

FIG. 1.

Chemical structures of PSI-6130, RO2433, and PSI-7672.

FIG. 2.

HPLC chromatograms of extracts of clone A replicon cells incubated for 48 h with PSI-6130 in the absence of zebularine (top panel) and in the presence of 10 μM (middle panel) and 50 μM (bottom panel) zebularine. Peaks were identified from the chromatograms of the standards. The PSI-6130-MP and RO2433-MP peaks were close to the background levels, with retention times of 8.57 min and 11.83 min, respectively.

FIG. 3.

Proposed metabolic pathway for PSI-6130. The kinetic values for the phosphorylation of PSI-6130, PSI-6130-MP, and PSI-6130-DP are from previously published data (19).