Role of cathepsin A and lysosomes in the intracellular activation of novel antipapillomavirus agent GS-9191.

GS-9191, a bis-amidate prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), was designed as a topical agent for the treatment of papillomavirus-associated proliferative disorders, such as genital warts. In this study, we investigated the mechanism of conversion of GS-9191 to cPrPMEDAP. We observed that GS-9191 is hydrolyzed in the presence of the lysosomal carboxypeptidase cathepsin A (CatA) in vitro and is less efficiently metabolized in CatA-deficient fibroblasts than in control cells. In addition, knockdown of CatA by small interfering RNA (siRNA) reduced the intracellular accumulation of GS-9191 metabolites. However, intracellular CatA levels did not correlate with the susceptibility of tested cell lines to GS-9191, indicating that the CatA step is unlikely to be rate limiting for the activation of GS-9191. Further analysis showed that upon the hydrolysis of the carboxylester bond in one of the GS-9191 amidate moieties, the unmasked carboxyl group displaces L-phenylalanine 2-methylpropyl ester from the other amidate moiety. The cPrPMEDAP-L-phenylalanine conjugate (cPrPMEDAP-Phe) formed is not metabolized by Hint1 (histidine triad nucleotide binding protein 1) phosphoramidase but undergoes spontaneous degradation to cPrPMEDAP in acidic pH that can be significantly enhanced by the addition of SiHa cell extract. Pretreatment of SiHa cells with bafilomycin A or chloroquine resulted in an 8-fold increase in the intracellular concentration of cPrPMEDAP-Phe metabolite and the accumulation of GS-9191 metabolites in the lysosomal/endosomal fraction. Together, these observations indicate that the conversion of GS-9191 to cPrPMEDAP occurs in lysosomes via CatA-mediated ester cleavage, followed by the release of cPrPMEDAP, most likely through the combination of enzyme-driven and spontaneous pH-driven hydrolysis of a cPrPMEDAP-Phe intermediate.

Structural and binding analysis of pyrimidinol carboxylic acid and N-hydroxy quinazolinedione HIV-1 RNase H inhibitors.

HIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid and N-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.4 Å to 2.1 Å) crystal structures were determined with the isolated RNase H domain and reverse transcriptase (RT), which permit accurate assessment of the metal and water environment at the active site. The geometry of the metal coordination suggests that the inhibitors mimic a substrate state prior to phosphodiester catalysis. Surface plasmon resonance studies confirm metal-dependent binding to RNase H and demonstrate that the inhibitors do not bind at the polymerase active site of RT. Additional evaluation of the RNase H site reveals an open protein surface with few additional interactions to optimize active-site inhibitors.

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.

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.

Triazole derivatives as non-nucleoside inhibitors of HIV-1 reverse transcriptase--structure-activity relationships and crystallographic analysis.

A series of 3,4,5-trisubstituted 1,2,4-4H triazole derivatives was synthesized and investigated for HIV-1 reverse transcriptase inhibition. An X-ray structure with HIV-1 RT secured the binding mode and allowed the key interactions with the enzyme to be identified.

Crystal structures of HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278): implications for drug design.

Diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) have inherent flexibility, helping to maintain activity against a wide range of resistance mutations. Crystal structures were determined with wild-type and K103N HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278). These structures reveal a similar binding mode for TMC125 and TMC278, whether bound to wild-type or K103N RT. Comparison to previously published structures reveals differences in binding modes for TMC125 and differences in protein conformation for TMC278.

RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed. Binding was demonstrated via a solid state structure of the isolated p15-Ec domain of HIV-1 RT showing inhibitor and two Mn(II) ions bound to the RNase H active site.

The A62V and S68G mutations in HIV-1 reverse transcriptase partially restore the replication defect associated with the K65R mutation.

BACKGROUND: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations. METHODS: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses. RESULTS: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels. CONCLUSIONS: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.

Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131.

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.