Activation of 9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) and Other Tenofovir Phosphonoamidate Prodrugs by Human Proteases

Abstract

9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine (GS-7340) is an isopropylalaninyl phenyl ester prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV; 9-[(2-phosphonomethoxy)propyl]adenine) exhibiting potent anti-HIV activity and enhanced ability to deliver parent TFV into peripheral blood mononuclear cells (PBMCs) and other lymphatic tissues in vivo. The present study focuses on the intracellular metabolism of GS-7340 and its activation by a variety of cellular hydrolytic enzymes. Incubation of human PBMCs in the presence of GS-7340 indicates that the prodrug is hydrolyzed slightly faster to an intermediate TFV-alanine conjugate (TFV-Ala) in quiescent PBMCs compared with activated cells (0.21 versus 0.16 pmol/min/10 6 cells). In contrast, the conversion of TFV-Ala to TFV and subsequent phosphorylation to TFV-diphosphate occur more rapidly in activated PBMCs. The activity of GS-7340 hydrolase producing TFV-Ala in PBMCs is primarily localized in lysosomes and is sensitive to inhibitors of serine hydrolases. Cathepsin A, a lysosomal serine protease has recently been identified as the primary enzyme activating GS-7340 in human PBMCs. Results from the present study indicate that in addition to cathepsin A, a variety of serine and cysteine proteases cleave GS-7340 and other phosphonoamidate prodrugs of TFV. The substrate preferences displayed by these enzymes toward TFV amidate prodrugs are nearly identical to their preferences displayed against oligopeptide substrates, indicating that GS-7340 and other phosphonoamidate derivatives of TFV should be considered peptidomimetic prodrugs of TFV. (De Clercq, 2003). TFV contains catabolically stable phosphonate moiety; therefore, unlike with nucleoside analogs, its intracellular activation requires only two phosphorylation steps to yield its active form, TFV diphosphate (TFVpp). The phosphorylation of TFV is catalyzed by AMP kinase and nucleoside diphosphate kinase (Robbins et al., 1995). TFVpp is a potent competitive inhibitor of HIV reverse transcriptase, acting as an obligatory DNA chain terminator (Suo and Johnson, 1998). However, the presence of two negative charges on the TFV molecule limits its cellular permeability and precludes oral administration. To overcome these limitations, various TFV prodrugs containing lipophilic groups masking the charged phosphonate moiety have been designed. Among these, tenofovir disoproxil fumarate (TDF; Viread) has been approved for the treatment of HIV infection. Because of its favorable resistance profile and long-term tolerability, TDF therapy is broadly used in treatment of both naive and previously drug-treated HIV-infected patients (for review, see Antoniou et al, 2003;Grim and Romanelli, 2003). Tenofovir (TFV) is an acyclic nucleotide analog active against a variety of retroviruses including human immunodeficiency virus (HIV) and hepatitis B virusGS-7340 (Fig. 1) is a prototype molecule representing a novel class of TFV mono-phosphonoamidate prodru...

Reduction Sensitive Lipid Conjugates of Tenofovir: Synthesis, Stability, and Antiviral Activity

“…TAF is an isopropylalaninyl phenyl ester that requires two disparate enzymes for prodrug release: carboxyesterase and cathepsin A. 21 The latter enzyme, cathepsin A, is a serine protease localized almost exclusively to lysosomal endosomes and ensures selective intracellular delivery of TFV. Similarly, 1 also relies on the catalytic activity of an intracellular hydrolase, phospholipase C and/or sphingomylenase, to liberate TFV within the cytosol.…”

Prodrugs of Phosphonates and Phosphates: Crossing the Membrane Barrier

“…Whether diamidate (e.g. 70 ), or aryl amidate ( 71 ), these compounds appear to be released through an initial enzymatic hydrolysis of the amino acid carboxyl ester [159]. In the case of diamidates and aryloxy amino acid amidates this initial step is catalyzed by carboxypeptidase Y (cathepsin A) or carboxylesterase I [160], to afford the corresponding carboxylate ( 72 or 73 ).…”

Practical considerations for developing nucleoside reverse transcriptase inhibitors

“…The stability of GS-7340 is about 200 fold greater than TDF, so that levels are detectable in the circulation. GS-7340 is converted by lysosomal protease cathepsin A (CatA) in PBM cells to an alanine metabolite of TFV (TFV-Ala), which then degrades to TFV under acidic pH in lysosomes [94]. PK studies in beagle dogs demonstrated a rapid plasma clearance with 34-fold greater 24 hr TFV-DP exposures in PBM cells compared to TDF, and five to 15-fold greater levels of GS-7340 metabolite was observed in lymphatic tissues compared with TDF.…”