Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977.
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.
Murakami
Eisuke
E
Pharmasset, Inc, Princeton, New Jersey 08540, USA. emurakami@pharmasset.com
Tolstykh
Tatiana
T
Bao
Haiying
H
Niu
Congrong
C
Steuer
Holly M Micolochick
HM
Bao
Donghui
D
Chang
Wonsuk
W
Espiritu
Christine
C
Bansal
Shalini
S
Lam
Angela M
AM
Otto
Michael J
MJ
Sofia
Michael J
MJ
Furman
Phillip A
PA
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
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
Chem Res Toxicol. 2005 Sep;18(9):1371-7
16167828
Drug Metab Pharmacokinet. 2006 Jun;21(3):173-85
16858120
Antimicrob Agents Chemother. 2010 Jan;54(1):305-11
19841155
Mol Pharmacol. 2007 Sep;72(3):686-94
17537833
Curr Opin Pharmacol. 2008 Oct;8(5):522-31
18835365
Antimicrob Agents Chemother. 2008 Feb;52(2):458-64
17999967
Hepatology. 2007 Sep;46(3):640-8
17879366
J Virol. 2003 Oct;77(19):10689-94
12970456
J Biol Chem. 2005 Apr 15;280(15):15356-61
15703176
Future Med Chem. 2009 Nov;1(8):1429-52
21426058
Antivir Chem Chemother. 1998 Nov;9(6):473-9
9865385
Rev Med Virol. 2008 Jan-Feb;18(1):19-34
17966115
J Pharmacol Exp Ther. 2004 Aug;310(2):469-76
15082749
Biochemistry. 2002 Jul 23;41(29):9003-14
12119013
Mol Pharm. 2007 Mar-Apr;4(2):208-17
17217311
Antimicrob Agents Chemother. 2007 Feb;51(2):503-9
17101674
Biochem Pharmacol. 2009 Jan 15;77(2):238-47
18983829
Nat Rev Drug Discov. 2007 Dec;6(12):1001-18
18049474
J Med Chem. 1996 May 10;39(10):1981-90
8642557
Antivir Chem Chemother. 2006;17(2):79-87
17042329
J Biol Chem. 2007 Oct 12;282(41):29812-20
17698842
Curr Opin Pharmacol. 2004 Oct;4(5):453-64
15351349
Toxicol Appl Pharmacol. 2007 May 15;221(1):1-12
17442360
J Mol Biol. 2007 Nov 2;373(4):978-89
17870088
Infect Disord Drug Targets. 2006 Mar;6(1):17-29
16787301
Biochim Biophys Acta. 1978 May 11;524(1):85-93
656450
Cell Mol Life Sci. 1999 Dec;56(11-12):894-907
11212324
Antivir Chem Chemother. 1998 Mar;9(2):109-15
9875382
J Med Chem. 2009 Sep 10;52(17):5520-30
19645484
J Submicrosc Cytol Pathol. 1989 Apr;21(2):275-9
2752360
Cell Mol Life Sci. 2006 Jan;63(2):163-86
16389458
J Biol Chem. 2007 May 18;282(20):15137-47
17337452
Herpes. 2001 Mar;8(1):12-6
11867011
Antimicrob Agents Chemother. 2007 Feb;51(2):543-50
17145787
Science. 2000 Dec 8;290(5498):1972-4
11110665
Drug Metab Dispos. 2006 Oct;34(10):1734-41
16837570