Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977

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.

FIGURE 1.

Metabolism of PSI-7851 in clone A and primary human hepatocytes. Clone A cells (A) or primary human hepatocytes (B) were treated with 5 μm ³H-labeled PSI-7851, and formation of the metabolites was followed up to 72 h using HPLC methodology. Cellular concentrations of PSI-7851 (○), PSI-352707 (□), PSI-7411 (△), PSI-7410 (▽), and PSI-7409 (♢) are shown.

FIGURE 2.

Expression of CES1 and CatA in HHPC and clone A cells. Western blot analysis was performed in extracts of clone A replicon cells, primary human hepatocytes, and pooled human liver cytosol using CES1-specific (A, upper panel) and CatA-specific (A, lower panel) antibodies. The same amount of total protein was loaded on the gel for the Western blot analysis. CatA (B) and CES1 (C) expression levels in human liver cytosol from 10 different single donors were examined by Western blot and compared. The expression levels were normalized against the amount of total protein loaded on the gel. Results are shown as fold-change relative to donor A.

FIGURE 3.

Effect of telaprevir and BNPP on formation of PSI-7409. Clone A (A) or primary human hepatocytes from three different donors 1–3 (B–D) were incubated with a CatA inhibitor, telaprevir, a CES1 inhibitor, BNPP, or both telaprevir and BNPP, and the formation of PSI-7409 was followed. The cellular concentrations of PSI-7409 at various concentrations of inhibitors are shown in the graphs.

FIGURE 4.

Stereospecificity. Stereoselectivity was studied by enzyme assays (A and B) and cellular metabolism assays (C and D). PSI-352707 product formation was followed by incubating PSI-7851 (□), PSI-7976 (○), or PSI-7977 (△) with human recombinant CatA (A) or CES1 (B). Time-dependent formation of PSI-7409 (active triphosphate form) in clone A cells (C) or primary human hepatocytes (HHPC) (D) was by incubating with PSI-7851 (□), PSI-7976 (○), or PSI-7977 (△). See “Experimental Procedures” for the experimental conditions.

FIGURE 5.

Enzyme activity and gene and protein expression of Hint1. A, relative Hint1 mRNA expression levels in clone A, Huh7, CEM, and primary human hepatocytes from two different donors determined by RT-PCR. B, Western blot showing expression of Hint1 protein in extracts from clone A, Huh7, CEM, and primary human hepatocytes from two different donors. The same amount of protein was loaded on the gel for the Western blot analysis.

FIGURE 6.

siRNA gene silencing. A, quantification of CatA or Hint1 mRNA expression in extracts of Huh7 cells treated with CatA siRNA, Hint1 siRNA, or only delivery media (DM). Gene expression levels relative to the DM control are shown in the graph. B, Western blot analyses of expression of CatA or Hint1 in the extract from Huh7 cells treated with CatA- or Hint1-specific siRNAs. The control experiment was done using the extract from the siRNA-untreated cells. The effect of the siRNAs on expression of GAPDH was also examined. C, amounts of PSI-352707 or total metabolites in the CatA or Hint1 knocked down cells treated with 5 μm [³H]PSI-7851 for 24 h.

FIGURE 7.

Proposed metabolic pathway for PSI-7851 and its diastereoisomers.