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Antimicrobial Agents and Chemotherapy, February 2007, p. 543-550, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00968-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cathepsin A Is the Major Hydrolase Catalyzing the Intracellular Hydrolysis of the Antiretroviral Nucleotide Phosphonoamidate Prodrugs GS-7340 and GS-9131{triangledown}

Gabriel Birkus,* Ruth Wang, Xiaohong Liu, Nilima Kutty, Holly MacArthur, Tomas Cihlar, Craig Gibbs, Swami Swaminathan, William Lee, and Martin McDermott

Gilead Science, 333 Lakeside Drive, Foster City, California 94404

Received 3 August 2006/ Returned for modification 20 September 2006/ Accepted 22 November 2006

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.

* Corresponding author. Mailing address: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404. Phone: (650) 522-5944. Fax: (650) 522-5890. E-mail: Gabriel_Birkus{at}gilead.com.

{triangledown} Published ahead of print on 4 December 2006.

Antimicrobial Agents and Chemotherapy, February 2007, p. 543-550, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00968-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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