Decomposition Pathways and <i>in Vitro</i> HIV Inhibitory Effects of IsoddA Pronucleotides:  Toward a Rational Approach for Intracellular Delivery of Nucleoside 5‘-Monophosphates

Valette, Gilles; Pompon, A.; Girardet, Jean‐Luc; Cappellacci, Loredana; Franchetti, Palmarisa; Grifantini, Mario; Colla, Paolo La; Loi, A. G.; Philouze, Christian; Gosselin, Gilles; Imbach, Jean‐Louis

doi:10.1021/jm9507338

Cited by 96 publications

(88 citation statements)

References 17 publications

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“…Once the carboxyl ester is hydrolyzed, the phenol group in the phosphate moiety is released spontaneously by a nucleophilic attack of the free carboxyl group at the phosphate, resulting in the formation of the alaninyl phosphate intermediate (PSI-352707). This chemical reaction has been described previously (36). The third step in the metabolism involves deamination of PSI-352707 to release alanine to form PSI-7411, a monophosphate intermediate.…”

Section: Discussionmentioning

confidence: 91%

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Murakami¹,

Tolstykh²,

Bao³

et al. 2010

Journal of Biological Chemistry

261

308

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A phosphoramidate prodrug of 2-deoxy-2-␣-fluoro-␤-Cmethyluridine-5-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.Nucleoside analogs have long been the backbone therapy for the treatment of viral diseases such as HIV, HBV, and HSV infections (1-5). Recent studies have suggested that nucleoside analogs may be useful for treating hepatitis C virus (HCV) 3 infection (4, 6 -8). The most advanced anti-HCV nucleoside, RG7128, is a diisobutyrate nucleoside prodrug of ␤-D-2Ј-deoxy-2Ј-␣-fluoro-2Ј-␤-C-methylcytidine (PSI-6130) and is currently in phase IIb clinical studies. PSI-6130 demonstrated potent activity in the subgenomic HCV replicon assay (9); the incubation of radiolabeled PSI-6130 with either replicon cells or primary human hepatocytes resulted in the formation of the 5Ј-mono-, di-, and triphosphate metabolites of . The triphosphate metabolite (PSI-6130-TP) was shown to be a potent inhibitor of HCV NS5B RNA-directed RNA polymerase (RdRp) (11). However, incubation of replicon cells with the uridine analog, PSI-6206, resulted in no inhibition of HCV RNA production due to the inability of PSI-6206 to be phosphorylated by cellular nucleoside kinases to its monophosphate, 12). Biochemical studies showed that PSI-7411 was consecutively phosphorylated to its diphosphate, PSI-7410, by UMP-CMP kinase and its triphosphate, PSI-7409, by nucleoside diphosphate kinase (12). Inhibition studies using the replic...

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Section: Discussionmentioning

confidence: 91%

Mechanism of Activation of PSI-7851 and Its Diastereoisomer PSI-7977

Murakami¹,

Tolstykh²,

Bao³

et al. 2010

Journal of Biological Chemistry

261

308

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“…While GS-7340 and other amidate prodrugs of tenofovir successfully validated the concept of enhanced in vivo intracellular delivery of parent nucleotide, GS-9131 has been selected as a clinical development candidate based on its unique activity against HIV-1 strains resistant to approved antiretroviral nucleosides and its favorable in vivo pharmacological properties, including the ability to effectively deliver the active GS-9148 diphosphate metabolite into PBMCs (9,36). The prodrug moieties of GS-7340 and GS-9131 are structurally similar to a class of nucleoside monophosphate pro-drugs referred to as phosphoramidate pronucleotides (27,28,43,47,51). The initial step in the proposed activation mechanism for the amidate prodrugs is the hydrolysis of the carboxyester bond by an unidentified cellular hydrolase ( Fig.…”

Section: Selectively Hydrolyzed Inside Cells To Antiviral Nucleotidesmentioning

confidence: 99%

“…The initial step in the proposed activation mechanism for the amidate prodrugs is the hydrolysis of the carboxyester bond by an unidentified cellular hydrolase ( Fig. 1) (27,28,43,47,51). Hydrolysis of the ester is followed by a putative nucleophilic attack of the phosphorus by the free carboxyl group, resulting in the spontaneous elimination of phenol, producing metabolite X, which is further deaminated to the parent nucleotide analog (27,28,43,47,51).…”

Section: Selectively Hydrolyzed Inside Cells To Antiviral Nucleotidesmentioning

confidence: 99%

“…1) (27,28,43,47,51). Hydrolysis of the ester is followed by a putative nucleophilic attack of the phosphorus by the free carboxyl group, resulting in the spontaneous elimination of phenol, producing metabolite X, which is further deaminated to the parent nucleotide analog (27,28,43,47,51). The identification and thorough characterization of the enzymes involved in the intracellular activation of this class of prodrugs are essential for a more rational design of compounds with desirable pharmacological and biological properties.…”

Section: Selectively Hydrolyzed Inside Cells To Antiviral Nucleotidesmentioning

confidence: 99%

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

Birkuš

Wang

Liu

et al. 2007

Antimicrob Agents Chemother

153

162

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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]-Tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV), an acyclic nucleotide analog of dAMP, is a potent in vitro and in vivo inhibitor of human immunodeficiency virus type 1 (HIV-1) replication (2). TFV is sequentially phosphorylated in the cell by AMP kinase and nucleoside diphosphate kinase to the active species, tenofovir diphosphate (23, 39), which acts as a potent inhibitor of HIV-1 reverse transcriptase (7,49). The presence of a nonhydrolyzable phosphonic acid moiety in tenofovir circumvents an initial phosphorylation step which can be rate limiting for the activation of nucleoside analog inhibitors of HIV reverse transcriptase (3, 4).In order to increase the cellular permeability and oral bioavailability of TFV, which is a dianion at physiological pH, neutral prodrugs of TFV have been synthesized. Tenofovir disoproxil fumarate (TDF) is a bis-isopropoxycarbonyloxymethyl ester prodrug of TFV approved for the treatment of HIV. TDF is well tolerated, with infrequent development of resistance and a favorable long-term toxicity profile (2,8, 16). The oral administration of TDF results in high systemic levels of TFV (5); however, the rapid systemic degradation of TDF to TFV limits its uptake into target cells.Investigations to develop plasma-stable prodrugs which would be selectively hydrolyzed inside cells to antiviral nucleotides led to the design of GS-7340 and GS-9131{9--fluoro-1Ј-furanyladenine, respectively}, alkylalaninyl amidate phenyl ester prodrugs of TFV and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2Ј-fluoro-2Ј,3Ј-dideoxydidehydroadenosine), respectively. Both GS-7340 and GS-9131 exhibit potent in vitro anti-HIV-1 activities, favorable resistance profiles, and low cytotoxicities (9, 25). Compared to TDF, GS-7340 is significantly more stable in plasma and delivers ϳ30-fold-greater levels of active diphosphate metabolites into peripheral blood mononuclear cells (PBMCs) in vitro and in vivo (12). Compared to what was seen for TDF, oral administration of an equal dose of GS-7340 resulted in significantly higher levels of TFV accumulation both in lymphatic tissues and in PBMCs (24). While GS-7340 and other amidate prodrugs of tenofovir successfully validated the concept of enhanced in vivo intracellular delivery of parent nucleotide, GS-9131 has been selected as a clinical development candidate based on its unique activity against HIV-1 strains resistant to approved antiretroviral nucleosides and its favorable in vivo pharmacological properties, including the ability to effectively deliver the active GS-9148 diphosphate metabolite into PBMCs (9, 36). The prodrug moieties of GS-7340 and GS-9131 are structurally similar to a class of nucleoside monophosphate pro-* Corresponding author. Mailing address: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404.

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“…CatA is a lysosomal enzyme with deamidase, esterase, and carboxypeptidase activities (14)(15)(16)(17). After TAF penetrates into cells, CatA cleaves the carboxyester bond in the prodrug moiety to release a metastable metabolite, from which the phenol group is eliminated via intramolecular cyclization and hydrolysis to form TFV-Ala conjugate (18,19). Conversion of the TFV-Ala intermediate to the parent TFV occurs spontaneously due to the acidic pH within the lysosomes (20) (Fig.…”

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confidence: 99%