Liquid chromatography–tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals

Henneré, Gaëlle; Bécher, François; Pruvost, Alain; Goujard, Cécile; Grassi, Jacques; Bénech, Henri

doi:10.1016/s1570-0232(03)00099-0

Cited by 62 publications

(104 citation statements)

References 41 publications

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“…The methods used to quantify dNTP pools have been described (26). Briefly, P4 cells were cultivated in the presence or absence of hydroxyurea (250 M) for 18 h, trypsinized, and washed twice with 0.9% NaCl (pH 7.5) at 4°C, and a cell pellet containing 2 ϫ 10 7 cells was frozen at Ϫ70°C.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Bouchonnet

Dam²,

Mammano

et al. 2005

J Virol

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Human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) resistance mutations reduce the susceptibility of the virus to nucleoside analogues but may also impair viral DNA synthesis. To further characterize the effect of nucleoside analogue resistance mutations on the efficiency and kinetics of HIV-1 DNA synthesis and to evaluate the impact of the depletion of deoxynucleoside triphosphates (dNTP) on this process, DNA synthesis was evaluated by allowing DNA synthesis to proceed with natural HIV-1 templates and primers, either within permeabilized viral particles or in newly infected cells, and quantifying the products by real-time PCR. Three recombinant viruses derived from three pNL4-3 molecular clones expressing mutations associated with resistance to zidovudine: a clone expressing RT mutation M184V, a clone expressing mutations M41L plus T215Y (M41L؉T215Y), and clinical isolate BV34 (carrying seven resistance mutations). Following infection of P4 cells, the BV34 mutant, but not viruses expressing the M184V mutation or M41L؉T215Y, exhibited a defect in DNA synthesis. Importantly, however, for mutants carrying the M184V mutation or M41L؉T215Y mutations, a defect could be detected by using target cells in which dATP pools had been reduced by pretreatment with hydroxyurea. Based on these observations, we developed a recombinant-virus assay to assess the effects of hydroxyurea pretreatment on infectivity of viruses carrying plasma-derived RT sequences from patients with nucleoside resistance. Using this assay, we found that many, but not all, viruses carrying RT resistance mutations display an increased sensitivity to hydroxyurea, suggesting that the impact of RT resistance mutations on viral replication may be more profound in cell populations characterized by smaller dNTP pools.

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Section: Construction Of Plasmidsmentioning

confidence: 99%

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Bouchonnet

Dam²,

Mammano

et al. 2005

J Virol

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“…The fragmentor was set at 90 eV and capillary voltage 3500. (ii) An ABI API-4000 Q-trap triple quadrupole instrument was used [mass transition to a 189 fragment was monitored for each of the dNTP species, as described previously (39); N 2 gas was used for nebulization, drying, and collision and the ionization chamber temperature was 250°C]. New standard curves were created for every assay.…”

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confidence: 99%

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

Hartman

2007

Proc. Natl. Acad. Sci. U.S.A.

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Synergistically interacting gene mutations reveal buffering relationships that provide growth homeostasis through their compensation of one another. This analysis in Saccharomyces cerevisiae revealed genetic modules involved in tricarboxylic acid cycle regulation (RTG1, RTG2, RTG3), threonine biosynthesis (HOM3, HOM2, HOM6, THR1, THR4), amino acid permease trafficking (LST4, LST7), and threonine catabolism (GLY1). These modules contribute to a molecular circuit that regulates threonine metabolism and buffers deficiency in deoxyribonucleotide biosynthesis. Phenotypic, genetic, and biochemical evidence for this buffering circuit was obtained through analysis of deletion mutants, titratable alleles of ribonucleotide reductase genes, and measurements of intracellular deoxyribonucleotide pool concentrations. This circuit provides experimental evidence, in eukaryotes, for the presence of a high-flux backbone of metabolism, which was previously predicted from in silico modeling of global metabolism in bacteria. This part of the high-flux backbone appears to buffer deficiency in ribonucleotide reductase by enabling a compensatory increase in de novo purine biosynthesis that provides additional rate-limiting substrates for dNTP production and DNA synthesis. Hypotheses regarding unexpected connections between these metabolic pathways were facilitated by genome-wide but also highly quantitative phenotypic assessment of interactions. Validation of these hypotheses substantiates the added benefit of quantitative phenotyping for identifying subtleties in gene interaction networks that modulate cellular phenotypes.genetic buffering ͉ high-flux backbone of metabolism ͉ protein trafficking ͉ ribonucleotide reductase ͉ mitochondria-to-nucleus retrograde signaling pathway C ells are complex genetic systems, having evolved compensatory molecular networks that provide growth homeostasis (robustness). Conceptually, gene interactions underlie robustness by buffering environmental or genetic perturbations (1-3). Synergistic effects on the phenotype resulting from two genetic deficiencies or chemical inhibition in combination with a genetic deficiency reveal buffering relationships when the double limitation is more severe than either single limitation. Genome-wide phenotypic analysis, as possible with RNAi or use of the complete set of yeast gene deletion mutants, has enabled new approaches to investigate buffering relationships systematically (2, 4, 5). It has been shown that quantitative (strength) and qualitative (pattern) aspects of gene interaction profiles reveal how genes organize in a pathway or cellular process (4, 6). Conceptually, such sets of genes represent genetic modules that contribute buffering capacity to the cell, providing insight into how molecular circuitry is arranged to achieve robustness (7,8). Comprehensive and quantitative methods for genotypephenotype analysis are becoming available for gaining a more global and precise understanding of buffering networks (4, 6, 9). These methods permit unbiased experimental...

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“…These fractions were resuspended in Tris-methanol as described above and immediately frozen at Ϫ80°C. Intracellular triphosphorylated metabolites of AZT (AZTTP) and thymidine (dTTP) were quantified using liquid chromatography coupled with tandem mass spectrometry, as previously described for metabolites of stavudine (d4T), lamivudine (3TC), and dideoxyinosine (ddI) (5) and for natural endogenous deoxynucleotides (14). Monitoring of the selected ions, 506 to 380 and 481 to 159 for AZTTP and dTTP, respectively, was performed after electrospray ionization in the negative mode.…”

Section: Methodsmentioning

confidence: 99%

Effect of Cell Cycle Arrest on the Activity of Nucleoside Analogues against Human Immunodeficiency Virus Type 1

Wurtzer

Compain

Bénech

et al. 2005

J Virol

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Human immunodeficiency virus (HIV) reverse transcription can be notably affected by cellular activation, differentiation, and division. We hypothesized that changes in the cell cycle could also affect HIV susceptibility to nucleoside analogues, which compete with natural nucleotides for incorporation into viral DNA and inhibit viral replication through premature termination of reverse transcription. Proliferating HeLa-derived indicator cells were arrested in the S/G 2 phase with etoposide, a topoisomerase II inhibitor, or in the G 1 /S phase with aphidicolin, a polymerase ␣ inhibitor. Cell cycle arrest by both agents induced a remarkable decrease in HIV susceptibility to zidovudine (AZT). This decrease was seen both with a single-cycle infectivity assay and with a viral DNA quantitation assay, indicating that the effect of cell cycle arrest was exerted at the reverse transcription stage. The increase in the 50% inhibitory concentration (IC 50 ) seen with arrested cells was strongest for AZT (23-fold) and stavudine (21-fold) but more modest for other drugs (lamivudine, 11-fold; dideoxyinosine, 7-fold; and nevirapine, 3-fold). In drug-resistant reverse transcriptase mutants, the increase in AZT IC 50 (relative to that in dividing cells) was most prominent with a Q151M mutant and was comparable to the wild type in other drug-resistant mutants. Quantitation of intracellular pools of dTTP and AZT 5-triphosphate (AZTTP) showed that etoposide treatment induced a significant increase in intracellular dTTP and consequently a decrease in AZTTP/dTTP ratios, suggesting that the decrease in viral susceptibility to AZT was caused by reduced incorporation of the analogue into nascent viral DNA. These results emphasize the importance of cellular proliferation and deoxynucleoside triphosphate metabolism in HIV susceptibility to nucleoside analogues and underscore the need to study the activities of drugs of this class with natural target cells under physiological conditions of activation and proliferation.Nucleoside analogues, a part of most combination therapy regimens prescribed for the treatment of human immunodeficiency virus (HIV) infection, are the most widely used class of antiretroviral drugs. These compounds become active after phosphorylation into their triphosphate derivatives (15) and compete with natural endogenous deoxynucleoside triphosphates (dNTPs) for incorporation into nascent viral DNA by reverse transcriptase (RT), where they block viral DNA synthesis through a chain termination mechanism (9,23,24). The triple phosphorylation of nucleoside analogues is performed by cellular kinases that also catalyze the phosphorylation of natural endogenous deoxynucleosides (7,19,27). Although it is well established that the expression and activity of these cellular kinases are regulated by the cell cycle and by the state of activation and division of the cells (13, 29), the extent to which these parameters can affect the antiviral activity of nucleoside analogues is not known. Changes in the metabolism of nucleosides and, ...

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Liquid chromatography–tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals

Cited by 62 publications

References 41 publications

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

Effect of Cell Cycle Arrest on the Activity of Nucleoside Analogues against Human Immunodeficiency Virus Type 1

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