Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

Gao, Wen Yi; Johns, David G.; Chokekuchai, S; Mitsuya, Hiroaki

doi:10.1073/pnas.92.18.8333

Cited by 64 publications

(40 citation statements)

References 23 publications

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“…At the conceptual level, understanding how an inhibitor that should have identical effects on all four activities of rNDP reductase achieves apparent selective inhibition of just one of those activities in vivo relates to our long-standing interest in using in vitro enzymology to learn how enzymes behave within their natural environment, the living cell. At the practical level, hydroxyurea has been used for some time as an anticancer agent, which targets ribonucleotide reductase, and HU has shown activity as an antiviral agent in HIV-infected cells (6,19,20). In these more recent studies, HU displayed a synergistic effect when applied together with 2Ј,3Ј-dideoxyinosine, but only additive effects in combination with dideoxycytidine or dideoxyuridine.…”

Section: Discussionmentioning

confidence: 99%

Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase

Hendricks

Mathews

1998

Journal of Biological Chemistry

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Hydroxyurea inhibits DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools, suggesting that the activities of rNDP reductase are differentially sensitive to hydroxyurea. In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyurea addition. Also, we followed enzyme activity directly, by using a recently developed assay that allows simultaneous monitoring of the four activities, in the presence of substrates and effectors at concentrations that approximate the intracellular environment. We found the primary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effectors accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions where the enzymatic reaction is proceeding, further accelerated radical decay. Alternative models are discussed, to account for selective depletion of purine nucleotide pools by hydroxyurea. Hydroxyurea (HU)1 inhibits DNA replication in cells that use the class I form of ribonucleotide reductase (RNR) by inactivating the tyrosyl radical required for enzyme activity (1). This inhibition is also observed in viruses such as vaccinia, which encodes a class I RNR that is closely related in structure and regulation to the mammalian cell RNRs. HU inhibits the de novo synthesis of deoxyribonucleotides, thereby starving the host cell and viral replication complexes for precursors. One might expect the inhibition of RNR by a drug like HU to result in an equivalent decrease in the rates of formation of all four products of the enzyme. However, when our laboratory measured the triphosphate forms of these products, the 2Ј-deoxyribonucleoside 5Ј-triphosphates (dNTPs) in vaccinia virus-infected cells, we found that hydroxyurea treatment had different effects upon the four dNTP pools; dATP was the most severely depleted, dCTP and dGTP were depleted to intermediate levels, and dTTP actually accumulated to some 2-fold over control values (2). Similar results had earlier been noted with uninfected mammalian cells (3-5). In vaccinia virus-infected cells, addition of deoxyadenosine to the culture medium, along with an adenosine deaminase inhibitor, reversed the inhibition of virus growth by HU (2). Also, labeling studies that estimated RNR flux rates in vivo confirmed that ADP reduction was the most severely affected of the four RNR activities. These observations suggested that the reduction of ADP is a specific biological target of hydroxyurea in vivo. This finding, in turn, has spurred attempts to ...

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Section: Discussionmentioning

confidence: 99%

Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase

Hendricks

Mathews

1998

Journal of Biological Chemistry

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“…2a). HU has been shown to preferentially deplete dATP pools in mammalian cells (25,26), and there was a tendency for purine (particularly dATP) pools to fluctuate acutely whenever threonine metabolism and RNR activity were perturbed in combination (Fig. 4 a and b).…”

Section: Functional Interactions Between Dntp and Threonine Metabolismmentioning

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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“…The effect of HU on HIV replication might be mediated by two molecular mechanisms: (i) depletion of intracellular levels of dATP, resulting in a favorable shift of the purine analogue (ddI) triphosphate/dATP ratio; or (ii) enhancement of the mammalian pyrimidine kinase activities in salvage pathways, resulting in increased phosphorylation of the anti-HIV pyrimidine analogues (zidorudine [AZT], 3TC, and ddC). Several in vitro and in vivo studies have demonstrated the benefit of adding HU to anti-HIV therapeutic regimens that contain ddI, adefovir, d4T, AZT, 3TC and/or ddC (5,6,12,18,20). HU might also inhibit HIV replication by decreasing T-cell proliferation.…”

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confidence: 99%

In Vitro Hydroxyurea Decreases Th1 Cell-Mediated Immunity

Weinberg

2001

Clin Diagn Lab Immunol

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Hydroxyurea (HU) is used in the treatment of hematologic disorders and is sometimes added to antiretroviral combination therapy to potentiate human immunodeficiency virus (HIV) suppression. However, HU has toxic effects on rapidly dividing cells, including the effectors of the immune response. To determine whether HU affects specific T-cell responses, we measured lymphocyte proliferation and cytokine production in response to microbial antigen and mitogen stimulation in the presence of added HU (10 to 1,000 M). HU treatment of peripheral blood mononuclear cells obtained from HIV-infected patients and uninfected controls decreased lymphocyte proliferation and gamma interferon production compared with untreated cells. Interleukin-2 (IL-2) and IL-10 production was not affected by HU. The HU-mediated decrease of lymphocyte proliferation was similar in peripheral blood mononuclear cells from HIV-infected patients and from uninfected controls. The inhibitory effect of HU required continuous exposure to the drug and could be reverted by washing the drug out of the culture environment. These findings suggest that HU-containing therapeutic regimens might decrease Th1-cell-mediated immune responses in vivo.

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Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

Abstract: We and other groups have recently reported

Cited by 64 publications

References 23 publications

Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase

Differential Effects of Hydroxyurea upon Deoxyribonucleoside Triphosphate Pools, Analyzed with Vaccinia Virus Ribonucleotide Reductase

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

In Vitro Hydroxyurea Decreases Th1 Cell-Mediated Immunity

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