Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Kicska, Greg A.; Li, Long; Hörig, Heidi; Fairchild, Craig R.; Tyler, Peter C.; Furneaux, Richard H.; Schramm, Vern L.; Kaufman, Howard L.

doi:10.1073/pnas.071050798

Cited by 180 publications

(183 citation statements)

References 25 publications

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“…1). We can minimize recycling by inclusion of the drug immucillin H, a powerful inhibitor of deoxyguanosine degradation (31), and by using HGPRT Ϫ cells. Activity Profiles of Homogeneous dCK and dGK-We prepared pure human recombinant dGK (24) and dCK (25) and compared the ability of each enzyme to phosphorylate GdR and CdR.…”

Section: Resultsmentioning

confidence: 99%

Metabolic Interrelations within Guanine Deoxynucleotide Pools for Mitochondrial and Nuclear DNA Maintenance

Leanza

Ferraro

Reichard

et al. 2008

Journal of Biological Chemistry

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Mitochondrial deoxynucleoside triphosphates are formed and regulated by a network of anabolic and catabolic enzymes present both in mitochondria and the cytosol. Genetic deficiencies for enzymes of the network cause mitochondrial DNA depletion and disease. We investigate by isotope flow experiments the interrelation between mitochondrial and cytosolic deoxynucleotide pools as well as the contributions of the individual enzymes of the network to their maintenance. To study specifically the synthesis of dGTP used for the synthesis of mitochondrial and nuclear DNA, we labeled hamster CHO cells or human fibroblasts with [ 3 H]deoxyguanosine during growth and quiescence and after inhibition with aphidicolin or hydroxyurea. At time intervals we determined the labeling of deoxyguanosine nucleotides and DNA and the turnover of dGTP from its specific radioactivity in the separated mitochondrial and cytosolic pools. In both cycling and quiescent cells, the import of deoxynucleotides formed by cytosolic ribonucleotide reductase accounted for most of the synthesis of mitochondrial dGTP, with minor contributions by cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. A dynamic isotopic equilibrium arose rapidly from the shuttling of deoxynucleotides between mitochondria and cytosol, incorporation of dGTP into DNA, and degradation of dGMP. Inhibition of DNA synthesis by aphidicolin marginally affected the equilibrium. Inhibition of DNA synthesis by blockage of ribonucleotide reduction with hydroxyurea instead disturbed the equilibrium and led to accumulation of labeled dGTP in the cytosol. The turnover of dGTP decreased, suggesting a close connection between ribonucleotide reduction and pool degradation. Mitochondrial (mt)2 deoxynucleotides are formed and regulated by a network of anabolic and catabolic enzymes located in both the cytosol and mitochondria. The enzymes are of considerable medical interest. Many drugs used in cancer and virus therapy are deoxynucleoside analogs whose activity requires phosphorylation by kinases (1) and whose efficacy and toxicity is affected by the interplay between the enzymes in the network. The enzymes may also be involved in oxidative damage by activating modified bases such as 8-oxoguanine for incorporation into RNA and DNA (2). Moreover, an increasing number of diseases with depletion of mt DNA arise from genetic deficiency of enzymes in the network (3). In some cases the genetic deficiency leads to depletion of mt deoxyribonucleoside triphosphate (dNTP) (4 -6), in other cases to overproduction (7-9). In both instances the disturbed normal balance between the four dNTPs results in disease. Early examples are the catabolic phosphorylases that degrade purine (7) or thymine nucleosides (9). Their loss causes overproduction of dGTP or dTTP, with the affected individuals suffering from immune deficiency (7,8) or mitochondrial neurogastrointestinal encephalomyopathy (9), respectively. Later discovered examples are the anabolic mt salvage enzymes deoxyguanosine kinase (dGK) (4...

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Section: Resultsmentioning

confidence: 99%

Metabolic Interrelations within Guanine Deoxynucleotide Pools for Mitochondrial and Nuclear DNA Maintenance

Leanza

Ferraro

Reichard

et al. 2008

Journal of Biological Chemistry

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“…These actions may deplete dNTPs, stall replication forks and may also result in mis-inserted deoxynucleotides because of pool imbalances. Forodesine is a transition state guanosine analog that is a potent inhibitor of purine nucleoside phosphorylase (Kicska et al, 2001), a key enzyme in the purine salvage pathway (Krenitsky, 1967). The cytotoxicity of this agent requires deoxycytidine kinase activity and the presence of deoxyguanosine (dGuo), suggesting that dGuo and not forodesine acts as a drug that needs to be phosphorylated (Kicska et al, 2001).…”

Section: Purine Nucleoside Analogsmentioning

confidence: 99%

“…Forodesine is a transition state guanosine analog that is a potent inhibitor of purine nucleoside phosphorylase (Kicska et al, 2001), a key enzyme in the purine salvage pathway (Krenitsky, 1967). The cytotoxicity of this agent requires deoxycytidine kinase activity and the presence of deoxyguanosine (dGuo), suggesting that dGuo and not forodesine acts as a drug that needs to be phosphorylated (Kicska et al, 2001). Although the exact mechanism of action of forodesine is unknown, accumulation of dGTP and deregulation of the pyrimidine deoxynucleotide pools leads to inhibition of DNA synthesis and cell death after p53 stabilization, caspase activation, changes in mitochondrial membrane potential and PARP cleavage (Kicska et al, 2001;Balakrishnan et al, 2006).…”

Section: Purine Nucleoside Analogsmentioning

confidence: 99%

“…The cytotoxicity of this agent requires deoxycytidine kinase activity and the presence of deoxyguanosine (dGuo), suggesting that dGuo and not forodesine acts as a drug that needs to be phosphorylated (Kicska et al, 2001). Although the exact mechanism of action of forodesine is unknown, accumulation of dGTP and deregulation of the pyrimidine deoxynucleotide pools leads to inhibition of DNA synthesis and cell death after p53 stabilization, caspase activation, changes in mitochondrial membrane potential and PARP cleavage (Kicska et al, 2001;Balakrishnan et al, 2006). It is postulated that this is caused by ribonucleotide reductase inhibition by increased dGTP levels (Bantia et al, 2003;Gandhi and Balakrishnan, 2007).…”

Section: Purine Nucleoside Analogsmentioning

confidence: 99%

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Nucleoside analogs: molecular mechanisms signaling cell death

2008

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Nucleoside analogs are structurally similar antimetabolites that have a broad range of action and are clinically active in both solid tumors and hematological malignancies. Many of these agents are incorporated into DNA by polymerases during normal DNA synthesis, an action that blocks further extension of the nascent strand and causes stalling of replication forks. The molecular mechanisms that sense stalled replication forks activate cell cycle checkpoints and DNA repair processes, which may contribute to drug resistance. When replication forks are not stabilized by these molecules or when subsequent DNA repair processes are overwhelmed, apoptosis is initiated either by these same DNA damage sensors or by alternative mechanisms. Recently, strategies aimed at targeting DNA damage checkpoints or DNA repair processes have demonstrated effectiveness in sensitizing cells to nucleoside analogs, thus offering a means to elude drug resistance. In addition to their DNA synthesisdirected actions many nucleoside analogs trigger apoptosis by unique mechanisms, such as causing epigenetic modifications or by direct activation of the apoptosome. A review of the cellular and molecular responses to clinically relevant agents provides an understanding of the mechanisms that cause apoptosis and may provide rationale for the development of novel therapeutic strategies.

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“…Immucillin-H (ImmH, also known as BCX-1777 or forodesine) is a non-cleavable analog of inosine and a transition state inhibitor of PNP that binds to PNP approximately 7×10 5 fold more tightly than the substrate inosine and inhibits human PNP with a K d of 56 pM 2 . ImmH has a slow rate of dissociation from the enzyme-complexes, causing potent inhibition 3, 4 .…”

Section: Introductionmentioning

confidence: 99%

Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H

Huang

Wang

et al. 2008

Leukemia Research

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Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. 3 H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas 3 H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from an ImmHresistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthineguanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.

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Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Cited by 180 publications

References 25 publications

Metabolic Interrelations within Guanine Deoxynucleotide Pools for Mitochondrial and Nuclear DNA Maintenance

Metabolic Interrelations within Guanine Deoxynucleotide Pools for Mitochondrial and Nuclear DNA Maintenance

Nucleoside analogs: molecular mechanisms signaling cell death

Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H

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