Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5′-triphosphate in comparison to cellular DNA polymerases α and β

doi:10.1016/0006-291X(87)91078-3

Biochemical and Biophysical Research Communications

Volume 148, Issue 1, 14 October 1987, Pages 78-85

https://doi.org/10.1016/0006-291X(87)91078-3 Get rights and content

Abstract

The sugar-modified dTTP analogues 2′,3′-didehydro-2′,3′-dideoxythymidine 5′-triphosphate (ddeTTP), 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP), 3′-fluorothymidine 5′-triphosphate (FdTTP), and 3′-azidothymidine 5′-triphosphate (N₃dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the ID_so values of the compounds for the HIV-RT (ranging from 0.03 μM for ddeTTP to 0.1 μM for ddTTP) and the cellular DNA polymerase α (> 200 μM). DNA polymerase β is partially affected by N₃dTTP (ID_so=31 μM) and by the other analogues (ID_so=1–2.2 μM). FdTTP has proved as effective as N₃dTTP (ID_so=0.05 μM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K₁ values (about 0.05 μM).

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Cited by (94)

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Some aspects of the mechanism of biological action of FLT have been studied including its phosphorylation in a cell-free system and in various cell lines, incorporation into DNA in a cell-free system and in vitro termination of DNA synthesis as well as antiproliferative activity towards human cell lines (Langen et al., 1972; Chidgeavadze et al., 1985; Matthes et al., 1988). FLT 5′-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (RT) (Cheng et al., 1987; Matthes et al., 1987). In addition, the development of HIV mutants resistant to FLT is slower than of mutants resistant to other RT inhibitors.
Various thiated analogues of thymine 2′,3′-dideoxy-3′-fluoronucleoside (FLT) and their 5′-monophosphates and 5′-triphosphates were prepared with the use of modified multistep procedures. The thiated analogues of FLT and FLTMP were evaluated against the wild type and drug- and multidrug-resistant strains of HIV-1, using the replicative phenotyping format of the deCIPhR assay, and showed potent inhibition of drug-resistant HIV-1 strains at low cytotoxicity. Additionally, inhibition of recombinant drug resistant forms of reverse transcriptase from single and multiple HIV-1 mutants by the synthesized 5′-triphosphates was investigated. The strongest inhibition was observed for K103N and Δ67 mutants and the most potent anti-HIV-1 activity against drug resistant strains and the lowest cytotoxicity was exerted by S⁴FLTMP and FLTMP which may be regarded as potential anti-HIV/AIDS agents.
Partial selective inhibition of HIV-1 reverse transcriptase and human DNA polymerases γ and β by thiated 3'-fluorothymidine analogue 5'-triphosphates
2010, Antiviral Research
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It is worth to notice, that besides examined in this work host polymerases, other cellular polymerases also exposed to triphosphate forms of tested thymidine analogues are potential targets for cytotoxicity. However, Matthes et al. demonstrated poor sensitivity of polymerase α for both 4-SFLTTP and FLTTP with IC50 values of 140 μM and above 200 μM, respectively (Matthes et al., 1987, 1989). Additionally, many other inhibition studies showed that polymerase α was less sensitive to other thymidine nucleotide analogues than polymerase γ and mostly similarly or even less sensitive to such compounds than polymerase β (Faraj et al., 2000; Hart et al., 1992; Martin et al., 1994; Matthes et al., 1987).
3′-Deoxy-3′-fluorothymidine (FLT, alovudine^®) belongs to the most potent agents inhibiting HIV-1 replication. Its 5′-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase γ, by nucleoside analogue 5′-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5′-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases γ (pol γ) and β (pol β) was investigated. It was shown that 3′-deoxy-3′-fluoro-4-thiothymidine 5′-triphosphate (4-SFLTTP) and 3′-deoxy-3′-fluoro-2-thiothymidine 5′-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with $K_{i}^{app}$ values of 0.091 and 0.022 μM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol γ with $K_{i}^{app}$ of 0.174 μM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with $K_{i}^{app}$ of 0.055 μM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol β, with $K_{i}^{app}$ values of 16.84 and 4.04 μM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3′-fluorothymidine 5′-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol γ than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989, Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT.
Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.
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2010, Tetrahedron
Citation Excerpt :
Especially, their corresponding 5′-triphosphates acted as competitive inhibitors of some DNA polymerases by incorporating into a growing DNA chain and terminating it at a site that was strictly complementary to the corresponding template bases.128 Some 5′-triphosphates of 3′-fluoro-modified nucleoside analogues have been shown to be very strong inhibitors of HIV and/or HBV replication at the cellular level.129–132 However, the synthesis and investigation of the biological activity of 3′-deoxy-3′-fluoro nucleoside analogues have received relatively little attention.
Fluorinated nucleosides: Synthesis and biological implication
2008, Journal of Fluorine Chemistry
A number of important pharmaceuticals have been discovered and developed based on fluorinated analogs of biologically active nucleosides. The introduction of fluorine into a nucleoside structure at an appropriate position has modulated and/or improved the pharmacological properties of a molecule. The present review deals with the synthetic methodology, structural and biological implication of carbohydrate-modified fluoronucleosides.
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Development and optimization of anti-HIV nucleoside analogs and prodrugs: A review of their cellular pharmacology, structure-activity relationships and pharmacokinetics
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Significant improvements in antiviral therapy have been realized over the past 10 years. Numerous nucleoside analogs, as well as prodrugs of active compounds, have been synthesized and tested for anti-HIV activity. In addition to the five nucleoside analogs currently used clinically for the treatment of HIV infection, a broad spectrum of anti-HIV nucleoside analogs (including 2′,3′-dideoxynucleoside analogs, oxathiolanyl 2′,3′-dideoxynucleoside analogs, dioxolanyl 2′,3′-dideoxynucleoside analogs, carbocyclic 2′,3′-dideoxynucleoside analogs and acyclic nucleoside analogs) and their prodrugs (including ester prodrugs, phospholipid prodrugs, dihydropyridine prodrugs, pronucleotides and dinucleotide analogs), targeted at HIV reverse transcriptase, are reviewed with focus on structure-activity relationships, cellular pharmacology and pharmacokinetics. Several of these anti-viral agents show promise in the treatment of AIDS.

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^☆: Presented in part at FEBS advanced course: “Antiviral Drug Development: A Multidisciplinary Approach”, I1 Chiocco, Italy, May 10–23, 1987.

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Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5′-triphosphate in comparison to cellular DNA polymerases α and β☆

Abstract

Lancet

Virology

J. Biol. Chem

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Nature