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 Gabriel Birkus, Nilima Kutty, Gong-Xin He, Andrew Mulato, William Lee, Martin McDermott, and Tomas Cihlar Gilead Science, Foster City, California Received January 24, 2008; accepted April 22, 2008 ABSTRACT 9-[(R)-2-[[(S)-[[(S)-1-(Isopropoxycarbonyl)ethyl]amino] phenoxy- phosphinyl]-methoxy]propyl]adenine (GS-7340) is an isopropyl- alaninyl phenyl ester prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV; 9-[(2-phosphonometh- oxy)propyl]adenine) exhibiting potent anti-HIV activity and en- hanced ability to deliver parent TFV into peripheral blood mono- nuclear 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 en- zymes. 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 loc- alized in lysosomes and is sensitive to inhibitors of serine hydrolases. Cathepsin A, a lysosomal serine protease has re- cently 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 pro- drugs of TFV. The substrate preferences displayed by these enzymes toward TFV amidate prodrugs are nearly identical to their preferences displayed against oligopeptide substrates, in- dicating that GS-7340 and other phosphonoamidate deriva- tives of TFV should be considered peptidomimetic prodrugs of TFV. Tenofovir (TFV) is an acyclic nucleotide analog active against a variety of retroviruses including human immuno- deficiency virus (HIV) and hepatitis B virus (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 phosphoryla- tion 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 pre- viously drug-treated HIV-infected patients (for review, see Antoniou et al, 2003; Grim and Romanelli, 2003). GS-7340 (Fig. 1) is a prototype molecule representing a novel class of TFV mono-phosphonoamidate prodrugs. Un- like TDF, GS-7340 contains phenol and alanine isopropyl ester as the phosphonate masking groups (Fig. 1). Relative to Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.045526. ABBREVIATIONS: TFV, tenofovir (9-[(2-phosphonomethoxy)propyl]adenine); HIV, human immunodeficiency virus; TFVpp, TFV diphosphate; TDF, tenofovir disoproxil fumarate; GS-7340, 9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino] phenoxyphosphinyl]-methoxy]propyl]adenine; PBMC, peripheral blood mononuclear lymphocyte; DTT, dithiothreitol; BSA, bovine serum albumin; Pr3, proteinase 3; LE, leukocyte elastase; PE, pancreatic elastase; PLCE, porcine liver carboxylesterase; DFP, diisopropyl fluorophosphate; -ABA, -aminobutyric acid; E64, trans-epoxysuc- cinyl-L-leucylamido(4-guanidino)butane. 0026-895X/08/7401-92–100$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 45526/3356565 Mol Pharmacol 74:92–100, 2008 Printed in U.S.A. 92 at ASPET Journals on March 2, 2016 molpharm.aspetjournals.org Downloaded from