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 (original) (raw)
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Antimicrobial Agents and Chemotherapy, 2008
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Cellular phosphorylation of anti-HIV nucleosides
Journal of Biological …, 1996
Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho-and triphospho-derivatives of azidothymidine ...
Journal of Medicinal Chemistry, 1997
The decomposition pathways in peripheral blood mononuclear cells (PBMCs) and the in vitro anti-HIV-1 activity of the structurally similar 3′-azido-3′-deoxythymidine (AZT) phosphoramidates 1-6 and 3′-fluoro-3′-deoxythymidine (FLT) phosphoramidates 7-10 are reported. The AZT phosphoramidates exhibited no cytotoxicity toward CEM cells at concentrations as high as 100 µM, whereas the FLT phosphoramidates 9 and 10 had CC 50 values of 95.6 and 35.1 µM, respectively. All 10 compounds exhibited no cytotoxicity toward PBMCs at concentrations as high as 100 µM and were effective at inhibiting viral replication. In particular, the AZT phosphomonoester amidate 4 displayed comparable antiviral acitivity to the parent nucleoside analog AZT. Mechanistic studies on the amino acid carbomethoxy ester phosphomonoester amidates revealed that their decomposition pathway differs from that of amino acid carbomethoxy ester aryl phosphodiester amidates of nucleotide prodrugs. AZT phosphomonoester amidates are internalized by lymphocytes to the same extent as AZT by a nonsaturable process. In lymphocytes, the amino acid carbomethoxy ester phosphomonoester amidates of AZT are not significantly metabolized to either AZT or the mono-, di-, or triphosphate of AZT. The amount of active anabolite, AZT-5′-triphosphate, formed in PBMCs incubated with the AZT phosphomonoester amidates 3 and 4 was 2-and 3-fold less than that observed after treatment with AZT, respectively. In contrast, FLT phosphomonoester amidates are rapidly converted to FLT-5′-monophosphate by a process that is antagonized by the corresponding AZT derivative 4. These results suggest that the metabolism of aromatic amino acid carbomethoxy ester phosphomonoester amidate nucleotide prodrugs by PBMCs does not require prior conversion to the corresponding carboxylic acid before proceeding to P-N bond cleavage.
Antiviral Research, 1995
Recently the ribonucleotide reductase (RR) inhibitors, specifically hydroxyurea, have gained attention for their in vitro activity against HIV These compounds act upon cellular enzymes and would be less likely to produce resistant retroviral mutants than direct antiviral agents. We evaluated hydroxyurea and the other RR inhibitors 3,4-dihydroxybenzohydroxamic acid (Didox) and 3,4,5trihydroxybenzamidoxime (Trimidox) in the RMuLV model. Splenomegaly a marker for RMul3/infection, was reduced or completely inhibited by both Didox (500, 250, and 125 mg/kg, qd) and Trimidox (250, 125, and 62.5 mg/kg, qd) in a dosedependent manner while hydroxyurea reduced splenomegaly to control levels at all doses (1000, 500, 300, and 100 mg/kg, qd). Preliminary evidence suggests that viremia was not reduced by these drugs, except by the lowest concentration of Trimidox, and at the highest level of hydroxyurea; this hydroxyurea concentration showed considerable toxicity This discordance of two of the main markers of infection in RMuLV (splenomegaly and viremia) following treatment is unexpected and experiments directed at providing an explanation are ongoing. It may not be necessary for these compounds to inhibit virus replication as monotherapies since they have synergistic activity in vitro with nucleoside analogue antiviral drugs. Reduction of competing pools of naturally occurring deoxynucleotides by RR inhibitors probably accounts for this synergy Further studies of the RR inhibitors, including combination studies with nucleoside analogues, are in progress.
Journal of Biological Chemistry, 1998
ATP hydrolyzing activity of a mutant ␣ 3  3 ␥ subcomplex of F 0 F 1 -ATP synthase (⌬NC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E., Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221). This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F 0 F 1 -ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, ⌬NC F 0 F 1 -ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by ⌬NC F 0 F 1 -ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by ⌬NC F 0 F 1 -ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.
Antimicrobial Agents and Chemotherapy, 2003
Ribavirin is an approved broad-spectrum antiviral drug. A liver-targeting prodrug of ribavirin, viramidine, is in clinical trial in an attempt to provide a better therapeutic index. The conversion of viramidine to ribavirin, and of ribavirin to an inactive metabolite through adenosine deaminase, is reported. Kinetic analysis indicates that adenosine deaminase is likely involved in activation of viramidine in vivo, and the process is highly pH sensitive. The differential activities of two consecutive deamination reactions are kinetically studied and interpreted based on adenosine deaminase structural information. A comprehensive understanding of the viramidine and ribavirin deamination mechanism should help in designing better nucleoside therapeutics in the future.
Mini Reviews in Medicinal Chemistry, 2004
Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required for the pharmacological activity of these compounds in the chemotherapeutic treatment of viral infections and cancer. Human thymidylate kinase (TMPK), apart from its essential physiological role in the biosynthesis of TTP, is also required for the activation of thymidine analogs, such as the clinically used anti-HIV prodrugs AZT and d4T. This enzyme is rate determining in the three-step cascade of AZT phosphorylation. Our structural work on human, yeast and E. coli TMPKs, in conjunction with sequence homology analyses and biochemical data, has demonstrated that three loops are crucial for the function of this enzyme: the first is the highly conserved P-loop motif, which binds and positions the phosphoryl groups of ATP, the second critical loop contains the DR(Y/H) motif that supplies a catalytic arginine and is also important for the binding and positioning of the magnesium ion complexed to ATP, and the third loop is the so-called Lid-region that is a flexible stretch which closes on ATP when it binds. Modifications of the sugar moieties of nucleoside monophosphates are shown to exert drastic effects on the enzyme's conformation and, thus, reduced activity. Our structural work on several TMPKs has formed the basis for generating mutants of human TMPK that are about 100 times more efficient in phosphorylating AZTMP. These enzyme variants could potentially be introduced into HIV-targeted cells in order to significantly improve AZT's antiviral activity.
Biochemistry, 2016
We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PR S17), selected to represent high resistance by machine learning on genotype-phenotype data. Three of five mutations in PR S17 correlating with major drug resistance, M46L, G48V and V82S, and five of eleven natural variations, differ from two clinically derived extreme mutants, PR20 and PR22 bearing 19 and 22 mutations, respectively. PR S17 , which forms a stable dimer (<10 nM), is ~10-and 2-fold less efficient in processing the Gag polyprotein relative to the wild-type and PR20, respectively, but maintains the same cleavage order. Isolation of a model precursor of PR S17 flanked by the 56 amino acid transframe region (TFP-p6pol) at its Nterminus, which is impossible when expressing an analogous PR20 precursor, allowed systematic comparison of inhibition of TFP-p6pol-PR S17 and mature PR S17. Resistance of PR S17 to 8 protease inhibitors (PIs) relative to PR ranges from 1.5 to 5 orders of magnitude increase in K i from 0.01 to 8.4 μM. Amprenavir, darunavir, atazanavir and lopinavir, the most effective of the 8 PIs, inhibit precursor autoprocessing at the p6pol/PR site with IC 50 ranging from ~7.5 to 60 μM. Thus this process, crucial for stable dimer formation, shows ~200 to 800-fold weaker inhibition than the mature PR S17. TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC 50 of 15 μM); amprenavir shows a 2-fold increase in IC 50 (~15 μM), and atazanavir and lopinavir show increased IC 50 of >42 μM and >70 μM, respectively.
Chemistry - A European Journal, 2011
Nucleoside phosphoramidates (NPs) are a class of nucleotide analogues that have been developed as potential antiviral/antitumoral prodrugs. Recently we have shown that some amino acid nucleoside phosphoramidates (aaNPs) can act as substrate for viral polymerase like HIV-1 RT. Here we report the synthesis and hydrolysis study of a series of new aaNPs containing either natural or modified nucleobase in order to define the basis for their differential reactivity. Aqueous stability, kinetics and hydrolysis pathways were studied by NMR in different solution pD (5-7) and temperature. It was observed that kinetics and mechanism (P-N and/or P-O bond cleavage) of hydrolysis largely depend on the nature of nucleobase and amino acid moiety. Aspartyl NPs were found to be more reactive than Gly or β-Ala NPs. For aspartyl NPs, the order of reactivity of nucleobase was 1-deazaadenine > 7deazaadenine > adenine > thymine ≥ 3deazaadenine. Notably, neutral aqueous solution of Asp-1-deaza-dAMP degraded spontaneously even at 4 °C via exclusive P-O bond hydrolysis (50fold reactivity difference of Asp-1deaza-dAMP vs. Asp-3-deaza-dAMP at pD 5, 70 °C). Conformational studies by NMR and molecular modelling suggest the involvement of protonated N3 atom of adenine and 1/7deazaadenine in the intramolecular catalysis of the hydrolysis reaction via rare syn conformation.