title: 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.
authors.affiliation: Pharmasset, Inc, Princeton, New Jersey 08540, USA. emurakami@pharmasset.com
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
authors.forename: Tatiana
authors.initials: T
authors.lastname: Tolstykh
authors.forename: Haiying
authors.initials: H
authors.lastname: Bao
authors.forename: Congrong
authors.initials: C
authors.lastname: Niu
authors.forename: Holly M Micolochick
authors.initials: HM
authors.lastname: Steuer
authors.forename: Donghui
authors.initials: D
authors.lastname: Bao
authors.forename: Wonsuk
authors.initials: W
authors.lastname: Chang
authors.forename: Christine
authors.initials: C
authors.lastname: Espiritu
authors.forename: Shalini
authors.initials: S
authors.lastname: Bansal
authors.forename: Angela M
authors.initials: AM
authors.lastname: Lam
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Otto
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sofia
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Furman
mesh_terms: D000998:Antiviral Agents / Q000493:pharmacokinetics* / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D043402:Cathepsin A / Q000378:metabolism; D002460:Cell Line; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D006868:Hydrolysis; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D011355:Prodrugs / Q000493:pharmacokinetics* / Q000494:pharmacology; D012367:RNA, Viral / Q000378:metabolism; D000069474:Sofosbuvir; D013237:Stereoisomerism; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects* / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002265:Carboxylic Ester Hydrolases; D043402:Cathepsin A; D009703:Nucleoside-Phosphate Kinase; D011355:Prodrugs; D012367:RNA, Viral; D000069474:Sofosbuvir; D014542:Uridine Monophosphate; D017361:Viral Nonstructural Proteins; C019979:cytidylate kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C474609:CES1 protein, human; C468629:CTSA protein, human
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