Inhibition of Hepatitis C Virus RNA Replication by 2′-Modified Nucleoside Analogs

Abstract

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. Hepatitis C virus (HCV)1 infection is the leading cause of sporadic, post-transfusion, non-A non-B hepatitis (1, 2). One hundred seventy million people worldwide are thought to be infected with hepatitis C virus of which an estimated 4 million reside in the United States (3). Approximately 80% of infected individuals progress to chronic infection. Long term chronic HCV infection can lead to liver cirrhosis and to hepatocellular carcinoma (4 -6). Currently, the recommended therapy is treatment with a combination of interferon ␣2b and ribavirin, which results in a sustained viral response in 40% of patients (7,8). Investigational therapies using a combination of pegylated interferon and ribavirin have lead to an sustained viral response in 54% of patients, but the response rate (42%) of patients harboring HCV genotype 1 is lower (9, 10). Consequently, additional therapies for HCV infection are needed.Antiviral chemotherapies based on administration of analogs of deoxynucleosides have been widely successful as treatment for HIV, herpes virus, and hepatitis B infection (11,12). Intracellular phosphorylation of the nucleoside analog to the triphosphate creates the active form of the inhibitor that then serves as a substrate for the viral polymerase. Generally, incorporation of the nucleotide analog at the 3Ј-end of the replicating viral DNA causes termination of DNA synthesis, owing to the lack of the 3Ј-hydroxyl required for extension. These successes suggest that an investigation of ribonucleoside analogs as inhibitors of HCV replication would be worthwhile.The HCV NS5B protein, the RNA-dependent polymerase responsible for the synthesis of the viral RNA geno...

The non‐immunosuppressive cyclosporin DEBIO‐025 is a potent inhibitor of hepatitis C virus replication in vitro†

“…The preparation of DEBIO-025 was based on the synthetic strategy for D-MeAla 3 -EtXaa 4 -cyclosporin analogs described previously. 23 CsA was purchased from Fluka Chemie GmbH (9471 Buchs, Switzerland), 2Ј-C-methyl-Cytidine, 24 25 and VX-950 [26][27][28] were synthesized by standard methods.…”

Design, Synthesis, and Antiviral Activity of Adenosine 5‘-Phosphonate Analogues as Chain Terminators against Hepatitis C Virus

“…This result is consistent with the report in which addition of a 2′-methyl group to ATP makes the resulting triphosphate a potent HCV inhibitor. 16 Interestingly, the antiviral activity of compounds 3 and 4 does not differ much despite their different sugar moieties. This conclusion indicates that the ribose bridge oxygen is not critical for cellular activity.…”

“…Although they possess the 3′-hydroxyl group for elongation, the conformation distortion relayed from the phosphonate bond could prevent them from further chain elongation. Nucleoside analogues that retain a 3′-hydroxyl have been reported as a chain terminator, such as 2′-methyl ATP 16 for HCV NS5B RdRp, and arabinosyladenine triphosphate 27 and arabinofuranosylcytidine triphosphate 28 for DNA polymerase . Nevertheless, there is a slight possibility that a lower concentration of trinucleotides formed in the reaction makes any chain elongation difficult to detect under the described assay conditions.…”

“…It has been reported that addition of a 2′-methyl group onto ATP yields a potent chain terminator for HCV NS5B RdRp. 16 Hence, an adenosine phosphonate with the 2′-methyl addition was also conceived (Figure 2, compound 5). Since the ionic character of a phosphonic acid would present an obstacle for cellular permeability, an S-acyl-2-thioethyl (SATE) prodrug (5b) for compound 5 was also prepared.…”

Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

“…Various small molecule HCV RdRp inhibitors such as nucleoside analogues (16,17) and non-nucleoside inhibitors (NNI) (15, 18 -22) were synthesized and reported to be efficient NS5B inhibitors. After conversion to nucleoside triphosphate by cell host machinery, nucleoside analogue competes with natural NTP at the catalytic site of RdRp and terminates the elongation on incorporation.…”