Mechanism of Activation of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

Abstract

) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K m of 81 M and a k cat of 0.007 s ؊1 , but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K i ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 M. Similar results were obtained with 2-C-methyladenosine triphosphate (K i ‫؍‬ 1.5 M) and 2-C-methylcytidine triphosphate (K i ‫؍‬ 1.6 M). NS5B with the S282T mutation, which is known to confer resistance to 2-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.Hepatitis C virus (HCV) is an RNA virus which possesses a single-stranded positive-sense RNA as the viral genome. This viral RNA plays important roles during viral replication, as it serves as an mRNA for viral protein synthesis, a template for RNA replication, and a nascent RNA genome for a newly formed virus (17). HCV NS5B RNA-dependent RNA polymerase is a key enzyme in viral RNA replication. This enzyme, which does not require a primer for initiation of RNA synthesis, catalyzes de novo RNA synthesis (8, 11). Nucleoside analogs have been used to treat viral infections, such as herpes simplex virus, human immunodeficiency virus, and hepatitis B virus infections (5,6,21). These drugs are designed to inhibit viral polymerases by a process called chain termination, in which DNA synthesis is quenched by incorporating the triphosphate forms of these drugs, which lack the 3Ј-hydroxyl group on the sugar moiety. In order for nucleoside analogs to inhibit a viral polymerase, they must be transported into the cell and converted to the active 5Ј-triphosphate form by cellular kinases. 2Ј-C-Methylnucleosides have been investigated as anti-HCV agents targeting HCV NS5B RNA polymerase (2, 20). 2Ј-C-Methyladenosine (2Ј-C-Me-A) and 2Ј-C-methylguanosine (2Ј-C-Me-G) showed potent anti-HCV activities in a cell-based replicon assay, and their triphosphate forms inhibited replicase and NS5B RNA polymerase in vitro (20). In addition, 2Ј-C-Me-A exhibited significant activity against HCV in a cell culture system which involves complete HCV replication and which produces infectious HCV (16). A resistant replicon has been selected by passage of HCV in the...

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

“…PSI-6130 demonstrated potent activity in the subgenomic HCV replicon assay (9); the incubation of radiolabeled PSI-6130 with either replicon cells or primary human hepatocytes resulted in the formation of the 5Ј-mono-, di-, and triphosphate metabolites of PSI-6130 (10 -12). The triphosphate metabolite (PSI-6130-TP) was shown to be a potent inhibitor of HCV NS5B RNA-directed RNA polymerase (RdRp) (11). However, incubation of replicon cells with the uridine analog, PSI-6206, resulted in no inhibition of HCV RNA production due to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases to its monophosphate, PSI-7411 (10,12).…”

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

“…Recently, ␤-D-2Ј-deoxy-2Ј-fluoro-2Ј-C-methylcytidine (PSI-6130) has been identified as a potent and selective inhibitor of HCV replication in the subgenomic replicon system with little or no cytotoxicity in various human cell lines or bone marrow precursor cells (16). The corresponding triphosphate of PSI-6130 is an inhibitor of HCV NS5B competitive with natural CTP (17). Conversion to the active 5Ј-triphosphate form by cellular kinases is an important part of the mechanism of action for nucleoside analogs.…”

Combinations of 2'-C-Methylcytidine Analogues with Interferon-α2b and Triple Combination with Ribavirin in the Hepatitis C Virus Replicon System

“…Although valopicitabine was recently evaluated in clinical trials and the combination of valopicitabine with IFN-a (plus RBV) has led to successful viral RNA suppression in a number of HCV-infected individuals, this drug was found to have gastrointestinal toxicity leading to its discontinuation in its present form [6]. Another novel cytidine analogue, β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130, 2′-F-C-MeC has demonstrated potent and specific in vitro anti-HCV activity with no apparent cytotoxicity [7,8]. Recently, in a 14 day Phase I monotherapy study, a new prodrug of 2′-F-C-MeC named R7128 was shown to be highly potent at lowering levels of HCV RNA, with a 2.7-log reduction in the viral load of individuals infected with HCV genotype 1 who had failed prior IFN therapy [9,10].…”