title: Metabolic activation of the anti-hepatitis C virus nucleotide prodrug PSI-352938.
abstract: PSI-352938 is a novel cyclic phosphate prodrug of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.
authors.affiliation: Pharmasset, Inc, Princeton, New Jersey, USA.
authors.forename: Congrong
authors.initials: C
authors.lastname: Niu
authors.forename: Tatiana
authors.initials: T
authors.lastname: Tolstykh
authors.forename: Haiying
authors.initials: H
authors.lastname: Bao
authors.forename: Yeojin
authors.initials: Y
authors.lastname: Park
authors.forename: Darius
authors.initials: D
authors.lastname: Babusis
authors.forename: Angela M
authors.initials: AM
authors.lastname: Lam
authors.forename: Shalini
authors.initials: S
authors.lastname: Bansal
authors.forename: Jinfa
authors.initials: J
authors.lastname: Du
authors.forename: Wonsuk
authors.initials: W
authors.lastname: Chang
authors.forename: P Ganapati
authors.initials: PG
authors.lastname: Reddy
authors.forename: Hai-Ren
authors.initials: HR
authors.lastname: Zhang
authors.forename: Joseph
authors.initials: J
authors.lastname: Woolley
authors.forename: Li-Quan
authors.initials: LQ
authors.lastname: Wang
authors.forename: Piyun B
authors.initials: PB
authors.lastname: Chao
authors.forename: Adrian S
authors.initials: AS
authors.lastname: Ray
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
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism*; D002478:Cells, Cultured; D003498:Cyclic P-Oxides / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D051528:Guanylate Kinases / Q000378:metabolism; D016174:Hepacivirus / Q000187:drug effects*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D009705:Nucleosides / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D003498:Cyclic P-Oxides; D009705:Nucleosides; C556498:PSI 352938; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009701:Nucleoside-Diphosphate Kinase; D051528:Guanylate Kinases; D010727:Phosphoric Diester Hydrolases