title: Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131.
abstract: GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4'-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-2'-fluoro-1'-furanyladenine, respectively} are novel alkylalaninyl phenyl ester prodrugs of tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV) and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2'-fluoro-2', 3'-dideoxydidehydroadenosine), respectively. Both prodrugs exhibit potent antiretroviral activity against both wild-type and drug-resistant human immunodeficiency virus type 1 strains and excellent in vivo pharmacokinetic properties. In this study, the main enzymatic activity responsible for the initial step in the intracellular activation of GS-7340 and GS-9131 was isolated from human peripheral blood mononuclear cells and identified as lysosomal carboxypeptidase A (cathepsin A [CatA]; EC 3.4.16.5). Biochemical properties of the purified hydrolase (native complex and catalytic subunit molecular masses of 100 and 29 kDa, respectively; isoelectric point [pI] of 5.5) matched those of CatA. Recombinant CatA and the isolated prodrug hydrolase displayed identical susceptibilities to inhibitors and identical substrate preferences towards a panel of tenofovir phosphonoamidate prodrugs. Incubation of both enzymes with 14C-labeled GS-7340 or [3H]difluorophosphonate resulted in the covalent labeling of identical 29-kDa catalytic subunits. Finally, following a 4-h incubation with GS-7340 and GS-9131, the intracellular concentrations of prodrug metabolites detected in CatA-negative fibroblasts were approximately 7.5- and 3-fold lower, respectively, than those detected in normal control fibroblasts. Collectively, these data demonstrate the key role of CatA in the intracellular activation of nucleotide phosphonoamidate prodrugs and open new possibilities for further improvement of this important class of antiviral prodrugs.
authors.affiliation: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA. Gabriel_Birkus@gilead.com
authors.forename: Gabriel
authors.initials: G
authors.lastname: Birkus
authors.forename: Ruth
authors.initials: R
authors.lastname: Wang
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liu
authors.forename: Nilima
authors.initials: N
authors.lastname: Kutty
authors.forename: Holly
authors.initials: H
authors.lastname: MacArthur
authors.forename: Tomas
authors.initials: T
authors.lastname: Cihlar
authors.forename: Craig
authors.initials: C
authors.lastname: Gibbs
authors.forename: Swami
authors.initials: S
authors.lastname: Swaminathan
authors.forename: William
authors.initials: W
authors.lastname: Lee
authors.forename: Martin
authors.initials: M
authors.lastname: McDermott
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000409:Alanine; D044966:Anti-Retroviral Agents / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D043402:Cathepsin A / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009000:Monocytes / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D000068698:Tenofovir / Q000031:analogs & derivatives
chemical_list: D044966:Anti-Retroviral Agents; C519972:GS-9131; D011355:Prodrugs; D011994:Recombinant Proteins; D000068698:Tenofovir; D043402:Cathepsin A; C442442:tenofovir alafenamide; D000225:Adenine; D000409:Alanine