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

Eisuke Murakami,Tatiana Tolstykh,Haiying Bao,Congrong Niu,Holly M Micolochick Steuer,Donghui Bao,Wonsuk Chang,Christine Espiritu,Shalini Bansal,Angela M Lam,Michael J Otto,Michael J Sofia,Phillip A Furman

doi:10.1074/jbc.m110.161802

Abstract

A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-β-C-methyluridine-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.

Highlights

Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections [1,2,3,4,5]
Materials—Recombinant human cathepsin A (CatA), carboxylesterase 1 (CES1), and carboxylesterase 2 (CES2) were purchased from R & D Systems (Minneapolis, MN); human trypsin, chymase, and neutrophil elastase were from Calbiochem; chymotrypsin was from MP Biomedicals (Solon, OH); cathepsin B, cathepsin D, cathepsin L, and lipase were from Sigma; cathepsin H, calpain 1, and caspase 1–10 were from BioVision (Mountain View, CA)
The first step in the metabolism of phosphoramidate prodrugs of nucleoside monophosphate analogs involves the hydrolysis of the carboxyl ester moiety [23, 24]

Summary

To whom correspondence may be addressed

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]. 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]. Inhibition studies using the replicase assay and purified recombinant HCV NS5B RdRp showed that PSI-7409 was a potent inhibitor of HCV RNA synthesis [10, 12]. We describe the metabolic pathway for PSI-7851 and its two diastereoisomers, PSI-7976 and PSI-7977

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION