Inhibition of Hepatitis C Virus Replicon RNA Synthesis by PSI-352938, a Cyclic Phosphate Prodrug of β-d-2′-Deoxy-2′-α-Fluoro-2′-β-C-Methylguanosine

Abstract

PSI-352938 is a novel cyclic phosphate prodrug of ␤-D-2-deoxy-2-␣-fluoro-2-␤-C-methylguanosine 5-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 M, respectively. The active 5-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. Hepatitis C virus (HCV), an important member of the Flaviviridae family of viruses, is a major health problem affecting approximately 170 million people worldwide. In the United States, where it is the leading cause of liver transplantation, nearly 2% of the U.S. population are HCV carriers (2). The current standard of care (SOC), which consists of pegylated alpha interferon (IFN-␣) and ribavirin, results in only about a 50% sustained virological response in patients infected with genotype 1 HCV, the predominant genotype in the United States and Europe (13, 18). The limited cure rate and potential side effects associated with interferon and ribavirin (10, 11) prompted the development of direct-acting antiviral agents (DAAs) in order to improve clinical efficacy and tolerability.Among the DAAs currently in clinical development, nucleoside/nucleotide analogs as inhibitors of HCV replication have the advantage of broad genotype coverage and a high barrier to resistance (14). The active 5Ј-triphosphates of nucleoside/ nucleotide analogs target the HCV NS5B RNA-dependent RNA polymerase (Pol) by serving as alternative substrate inhibitors during RNA synthesis (6). Both of the two resistance mutations identified so far, S96T and S282T, are highly conserved residues and unfit for HCV replicon replication (33). Among the nucleoside/nucleotide analogs, RG7128 (the prodrug of PSI-6130, ␤-D-2Ј-fluoro-2Ј-C-methylcytidine) is the most advanced anti-HCV nucleoside and is currently in phase IIb clinical studies. So far, results from the clinical studies showed that RG7128 is generally safe and effective in reducing the viral loads for genotype 1, 2, 3, and 4 HCV-infected patients when it is combined with SOC, with no resistance-related breakthrough (16,21,27,37).More recently, we reported results of in vitro studies characterizing PSI-7851 and PSI-7977 (the single Sp isomer of PSI-7851), phosphoramidate prodrugs of ␤-D-2Ј-fluoro-2Ј-Cmethyluridine 5Ј-monophosphate (30,45,48). While RG7128 was designed to improve the pharmacokinetic profile of PSI-6130, PSI-7851 and PSI-7977 were synthesized to bypass the nonproductive first phosphorylation step, as the corresponding nucleoside analog was incapable of being converted to the monophosphate form. In a 3-day phase I monotherapy study, a 1.95-log vira...

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

“…1. Previously we reported that replicons containing the NS5B amino acid change S96T or S282T, which confers resistance to certain nucleoside/-tide analogs, or amino acid changes (C316Y, M414T, M423T, or P495L) that confer resistance to various classes of nonnucleoside inhibitors remained fully susceptible to both PSI-352938 and PSI-353661 (4,11). In order to identify the mutation(s) that confers resistance to these compounds, selection studies were performed using replicon cells and increasing concentrations of PSI-352938 or PSI-353661.…”

“…Metabolism of PSI-352938 and PSI-353661 generates the same 5Ј-triphosphate metabolite, PSI-352666 (2Ј-F-2Ј-C-methylguanosine-triphosphate), which is similarly active against NS5B polymerases from GT 1 to 4 (11). Our previous cross-resistance studies also showed that PSI-352938 and PSI-353661 remained fully active against both the S96T and S282T replicons (4,11).…”

“…PSI-352938 and PSI-353661 inhibit HCV genotype (GT) 1b replicon replication with 50% effective concentrations (EC 50 s) of 0.13 Ϯ 0.076 M and 3.0 Ϯ 1.4 nM, respectively, and are similarly active against GT 1a and 2a replicons and infectious viruses (4,11). Metabolism of PSI-352938 and PSI-353661 generates the same 5Ј-triphosphate metabolite, PSI-352666 (2Ј-F-2Ј-C-methylguanosine-triphosphate), which is similarly active against NS5B polymerases from GT 1 to 4 (11).…”

Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus

“…It has previously been reported that PSI-6130, INX-189, and IDX-184 were less active against GT 1b S282T replicons (26,40,43). In addition, we included in these studies PSI-352938, a prodrug of 2=-F-2=-C-methylguanosine monophosphate that remains active against GT 1b replicons with the S282T change (16). Results are summarized in Table 6.…”

Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938

“…Among the compounds synthesized and tested, PSI-352938, a novel cyclic phosphate prodrug of ␤-D-2=-fluoro-2=-C-methylguanosine 5=-monophosphate with pangenotypic anti-HCV activity, showed the best profile in terms of anti-HCV activity, cytotoxicity, stability, and intracellular triphosphate production (26). Furthermore, PSI-352938 lacked cross-resistance with other modified nucleoside/nucleotide analogs targeting HCV NS5B (18), suggesting that a comple-mentary effect could be achieved when combined with other classes of nucleoside/nucleotide inhibitors. Since we previously demonstrated that the metabolism of PSI-7851, a phosphoramidate prodrug of ␤-D-2=-deoxy-2=-␣-fluoro-2=-␤-C-methyluridine-5=-monophosphate, and the metabolism of its single isomer, PSI-7977, were different in Huh 7-derived clone A replicon cells and primary human hepatocytes due to differences in the expression of certain enzymes involved in the metabolism of these compounds, we compared the metabolisms of PSI-352938 in these two cell types (24).…”