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.

FIG. 1.

GS-7340 and GS-9131 metabolic activation pathway. Met X, metabolite X.

FIG. 2.

Purification of prodrug hydrolase. (A) Anion-exchange chromatography. PBMC extract from 15 × 10⁹ cells (75 to 85 ml) was diluted 1:10 (vol/vol) with Q15 buffer A and loaded onto an anion-exchange column. Bound protein was eluted and assayed for both GS-7340 and GS-9131 hydrolase activity as described in the text. (B) Hydrophobic interaction chromatography. The Q15 pool was processed as described in the text and loaded onto a butyl Sepharose HIC column. Bound protein was eluted and assayed for prodrug hydrolase activity. Symbols: •, GS-7340 hydrolase activity; ○, GS-9131 hydrolase activity; ---, salt concentration gradient. c, concentration.

FIG. 3.

SDS-PAGE resolution and identification of proteins in the ConA pool. Proteins present in the ConA pool were concentrated to 60 μl and resolved using SDS-PAGE (4 to 20% acrylamide gradient gel, NUPAGE; Invitrogen, Inc., Carlsbad, CA). Lanes: 1, ConA beads after elution; 2, ConA beads eluted with 8 M urea; 3, concentrated ConA pool (10 μl). Protein bands in the 10-μl lane were visualized using silver staining. Adjacent areas of the gel in an unstained lane (50 μl aliquot loaded) corresponding to a stained protein band were excised and identified by MS-MS sequencing of tryptic peptides generated by digestion. The resultant peptides were purified by passage through a C₁₈ ZipTip column, analyzed by positive ESI-MS, and sequenced using MS-MS fragmentation. Proteins were identified from the generated sequences by BLAST analysis of the NCBI nonredundant protein/peptide database (1). Proteins indicated to the right: 1, fibrinogen; 2, N-acetylglucosamidase; 3, acid α-glucosidase; 4, sulfamidase; 5, fibrinogen; 6, α-fucosidase; 7, iduronate sulfatase; 8, concanavalin A; 9, CatA; 10, cathepsin Z/X.

FIG. 4.

Labeling of prodrug hydrolase and recombinant CatA with ¹⁴C-labeled GS-7340 and [³H]DFP. Labeling of prodrug hydrolase, recombinant CatA, and concanavalin A with ¹⁴C-labeled GS-7340 and [³H]DFP was performed with both native (A) and denatured (B) enzymes as described in the text. Proteins were analyzed by SDS-PAGE and visualized by exposure to X-ray film. Incubation mixtures contained 1.0 mM ¹⁴C-labeled GS-7340 and prodrug hydrolase (lanes 1 and 7), recombinant CatA (lanes 2 and 8) or concanavalin A (lanes 5 and 11); 25 μM [³H]DFP or prodrug hydrolase (lanes 3 and 9), and recombinant CatA (lanes 4 and 10) or concanavalin A (lanes 6 and 12). (C) Aliquots of recombinant CatA (50 ng; lane 2) and 1 μg each of the Q-Source (lane 3), HIC (lane 4), and ConA (lane 5) pools were resolved using SDS-PAGE, transferred to a PVDF membrane, incubated with human cathepsin-A antibody, and visualized with a fluorescently labeled secondary antibody. M, molecular mass marker.

FIG. 5.

Metabolism of GS-7340 and GS-9131 in CatA+/− fibroblasts. Fibroblasts deficient in CatA (CatA−) (GM05076 and GM02438) and control fibroblasts (Cat A+) (GM00409, GM5400, GM05757B, and HEL) were cultivated and incubated with ¹⁴C-labeled GS7340 or ¹⁴C-labeled GS9131 (10 μM; 0.521 μCi/ml) for 30 to 240 min. At selected time points, medium was removed and cells were processed as described in the text. Associated ¹⁴C-labeled metabolites were extracted from the cells and expressed as picomoles/10⁶ cells. Cell volume was used to calculate metabolite concentration (μM).