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

Abstract

A phosphoramidate prodrug of 2-deoxy-2-␣-fluoro-␤-Cmethyluridine-5-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections (1-5). Recent studies have suggested that nucleoside analogs may be useful for treating hepatitis C virus (HCV) 3 infection (4, 6 -8). The most advanced anti-HCV nucleoside, RG7128, is a diisobutyrate nucleoside prodrug of ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylcytidine (PSI-6130) and is currently in phase IIb clinical studies. 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 . 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, 12). Biochemical studies showed that PSI-7411 was consecutively phosphorylated to its diphosphate, PSI-7410, by UMP-CMP kinase and its triphosphate, PSI-7409, by nucleoside diphosphate kinase (12). Inhibition studies using the replic...

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

“…PSI-7977, the Sp isomer, has better activity than PSI-7976 (Rp isomer) in replicon-based HCV inhibition assays (38) and is currently in phase 3 clinical trials. The 5=-triphosphate metabolite of PSI-7977, PSI-7409, inhibits HCV replication by serving as a nonobligate chain terminator (30). Cross-resistance studies have shown that PSI-7977 and PSI-7409 had reduced activity against GT 1b replicons and NS5B polymerase containing the S282T amino acid alteration, respectively (18,38).…”

Natural Product-Derived Drugs Based on β-Adrenergic Agents and Nucleosides

“…The history and potential of this compound and closely related compounds have recently been published and these papers should be consulted by interested readers. [40][41][42][43][44] It should also be mentioned that significant numbers of antiviral drugs against HIV are now delivered as preadjusted combinations of reverse transcriptase, integrase and protease inhibitors, which have the potential to reduce this disease to chronic status. An example uses emtricitabine (84) and tenofovir (82) plus the non-nucleoside reverse transcriptase inhibitor (rilpivirine; structure not shown as not a nucleoside …”

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

“…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.…”

“…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.…”