RESUMO
Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.
Assuntos
DCMP Desaminase/química , Nucleotídeos de Desoxicitosina/química , Inibidores Enzimáticos/química , Nucleotídeos de Timina/química , Proteínas Virais/química , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Bacteriófagos/química , Bacteriófagos/enzimologia , Cristalografia por Raios X , DCMP Desaminase/antagonistas & inibidores , DCMP Desaminase/genética , DCMP Desaminase/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Nucleotídeos de Timina/metabolismo , Tirosina/química , Tirosina/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
Assuntos
Adenocarcinoma/metabolismo , Amidas/química , Citidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes p16/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Ácidos Fosfóricos/química , Adenocarcinoma/genética , Adenocarcinoma/patologia , Citidina/síntese química , Citidina/química , Citidina/farmacologia , DCMP Desaminase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereoisomerismo , Timidilato Sintase/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution. This mutant of dCD is active, even in the absence of the allosteric regulators. The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP. The enzyme has a zinc ion-based mechanism that is similar to that of CDA. A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor. Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme. Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site. The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove. This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115. It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible. In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site.
Assuntos
Substituição de Aminoácidos/genética , Bacteriófago T4/química , Bacteriófago T4/genética , DCMP Desaminase/química , DCMP Desaminase/genética , Regulação Alostérica/genética , Arginina/genética , Sítios de Ligação/genética , Cristalização , Cristalografia por Raios X , Citidina/análogos & derivados , Citidina Desaminase/química , DCMP Desaminase/antagonistas & inibidores , Ácido Glutâmico/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nucleosídeo Desaminases/química , Estrutura Quaternária de Proteína/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Nucleosídeos de Pirimidina/química , Especificidade por Substrato/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/genética , Zinco/químicaRESUMO
Short term treatments (1-2 hrs) of human tonsillar lymphocytes by Cladribine (2-Chloro-deoxyadenosine, CdA) have suggested a new target for CdA, the inhibition of dCMP deaminase (Sasvári et al. 1994; BBRC 203, 1378). Further investigations have shown, that the dCMP-deaminase activity could be inhibited by 2-Cl-dAMP in cell free extracts of lymphocytes. The pool size of dUMP (measured by an antibody against dUMP) was also decreased in WiDr colon cancer cells by CdA. The new antimetabolite against solid tumours, Gemcitabine (2',2'-difluoro-deoxycytidine, dFdC), had similar effects on the salvage of thymidine (dThd) and deoxycytidine (dCyd) as CdA. The Ki values for 3H-dThd and 3H-dCyd incorporation into DNA were 0.16 uM and 1.0 uM dFdC, respectively. The labeling of the TTP pool increased 6-7 times, while of dCTP pool only 1.5-1.7 times, suggesting a decrease of the size of corresponding pools. Similarly to CdA, the labeling as well as the concentration of dUMP was also decreased by dFdC. Both analogues are able to increase the deoxycytidine kinase activity, necessary for their phosphorylation and therapeutic action in cells. The target(s) for the two different drugs seems to be common.
Assuntos
Cladribina/farmacologia , DCMP Desaminase/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Inibidores Enzimáticos/farmacologia , Linfócitos/metabolismo , Timidina/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular , Desoxicitidina/farmacologia , Desoxicitidina Quinase/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Fluordesoxiuridilato/metabolismo , Humanos , Técnicas In Vitro , Cinética , Linfócitos/efeitos dos fármacos , Tonsila Palatina , Timidina Quinase/metabolismo , GencitabinaRESUMO
The 2'-deoxy (2a) and 2'-ara-fluoro (3a) derivatives of zebularine [1-(beta-D-ribofuranosyl)-dihydropyrimidin-2-one, 1a] were phosphorylated in high yield to the 5'-nucleotides 2b and 3b, respectively, and characterized by HPLC, enzyme degradation, 1H, 13C and 31P NMR, and high resolution mass spectral analysis. Their inhibitory activity against partially purified MOLT-4 deoxycytidylate deaminase (dCMPD) in the presence of the allosteric effector deoxycytidine triphosphate (dCTP) and Mg+2 ion was examined. Compounds 2b and 3b inhibited dCMPD with Ki values of 2.1 x 10(-8) M and 1.2 x 10(-8) M, respectively. The parent nucleotide, zebularine monophosphate 1b was ineffective at concentrations > 100 mumol. The effect of the nucleosides, 1a-3a, as well as tetrahydrouridine (THU) and 2'-deoxy THU (dTHU), on the cellular production of DNA precursors was examined in human MOLT-4 peripheral lymphoblasts. It was shown that 1a, 2a and 3a all elevated intracellular dCTP and TTP levels in whole cells with the most powerful effect elicited by 1a. The 2'-fluoro derivative 3a was chemically phosphorylated much more cleanly and higher yield than 2a, without the formation of diphosphorylated by-products. This compound was found to be infinitely less sensitive to acid-catalyzed degradation than 2a. Since the substitution of fluorine for hydrogen had a slight potentiating effect on the dCMPD inhibitory activity while stabilizing the compound toward acid-catalyzed and enzymatic depyrimidination, compound 3b emerges as a very attractive tool for the pharmacological modulation of pyrimidine deaminase activity.
Assuntos
DCMP Desaminase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Nucleosídeos de Pirimidina/síntese química , Nucleotídeos de Pirimidina/síntese química , Nucleotídeos de Pirimidina/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citidina/análogos & derivados , DCMP Desaminase/isolamento & purificação , DCMP Desaminase/metabolismo , Desoxirribonucleotídeos/metabolismo , Estabilidade de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Cinética , Linfócitos/enzimologia , Linfócitos/metabolismo , Espectroscopia de Ressonância Magnética , Nucleosídeos de Pirimidina/isolamento & purificação , Nucleosídeos de Pirimidina/farmacologia , Nucleotídeos de Pirimidina/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Tetrahydro-dUMP, an analog of the putative transition state in aminohydrolysis of deoxycytidine monophosphate (dCMP) inhibits the allosteric enzyme deoxycytidylate aminohydrolase with high affinity. The inhibition is reversible, and its kinetics is consistent with the analog binding at the substrate site only to one and the same conformation that binds the substrate dCMP. Such kinetics is what would be expected for a transition state analog interacting in an allosteric "K system."
Assuntos
DCMP Desaminase/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Regulação Alostérica , Animais , DCMP Desaminase/antagonistas & inibidores , Nucleotídeos de Desoxiuracil/farmacologia , Cinética , Perissodáctilos , Baço/enzimologiaRESUMO
Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.
Assuntos
DCMP Desaminase/isolamento & purificação , Anticorpos/imunologia , Especificidade de Anticorpos , Cromatografia de Afinidade , Reações Cruzadas , DCMP Desaminase/antagonistas & inibidores , DCMP Desaminase/imunologia , Desoxicitidina Monofosfato/síntese química , Desoxicitidina Monofosfato/farmacologia , Escherichia coli/enzimologia , Células HeLa/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Nucleotídeos de Timina/farmacologiaRESUMO
Deoxycytidylate (dCMP) deaminase, a hexameric allosteric enzyme induced on infection of Escherichia coli by bacteriophage T4, was shown to contain two atoms of zinc per subunit by atomic absorption spectroscopy. One zinc appears to be involved in catalysis, as described for adenosine deaminase (Sharaff, A. J., Wilson, D. K., Chang, Z., and Quiocho, F. A. (1992) J. Mol. Biol. 226, 917-921) and cytidine deaminase (Yang, C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). This thesis is supported by the finding that the enzyme loses about 80% of its activity in the presence of o-phenanthroline. It has also been found that zinc is released when the enzyme is denatured in the presence of the metallochromic indicator, 4-(2-pyridylazo)resorcinol. Renaturation of the deaminase to an active form occurred in the presence but not in the absence of zinc. The second atom of zinc is proposed to be located in a region of T4-dCMP deaminase that resembles a zinc finger. This region, which has the sequence His-X3-Cys-X14-His-X3-His, would represent a zinc-binding motif that has not been described previously.
Assuntos
DCMP Desaminase/química , Fagos T/enzimologia , Zinco/análise , Sequência de Aminoácidos , Sítios de Ligação , DCMP Desaminase/antagonistas & inibidores , DCMP Desaminase/metabolismo , Metaloproteínas/química , Dados de Sequência Molecular , Fenantrolinas/farmacologia , Desnaturação Proteica , Espectrofotometria Atômica , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo , Dedos de ZincoRESUMO
Cellular metabolism studies had demonstrated previously that low cellular concentrations of 2',2'-difluorodeoxycytidine (dFdC) nucleotides are eliminated by deoxycytidylate deaminase (dCMPD), whereas dCMPD activity is inhibited at high cellular dFdC nucleotide levels (Heinemann et al., Cancer Res 52: 533-539, 1992). An assay for measuring dCMPD activity in intact human leukemia cells has now been developed to permit investigations of the interactions of dFdC nucleotides with dCMPD in intact cells in which the regulated nature of this enzyme was not disrupted. Using [14C]dCyd as the substrate, radioactivity that accumulated in dTTP was quantitated after high-pressure liquid chromotography by a radioactive flow detector. The assay was first characterized using either the dCMPD inhibitor tetrahydrodeoxyuridine (H4dUrd) which directly inhibits dCMPD, or thymidine and 5-fluoro-2'-deoxyuridine (FdUrd) which indirectly inhibit and activate dCMPD, respectively, by affecting the cellular dCTP:dTTP value. Measured by this in situ assay, there was a strong correlation between dCMPD activity and dCTP:dTTP levels. Consistent with previous studies using partially purified enzyme, incubation of cells with dFdC resulted in a concentration-dependent inhibition of dCMPD in situ. The mechanism of modulation of dCMPD by dFdC, however, was clearly different from that of thymidine and FdUrd. In addition to the effect of dFdC on cellular dCTP:dTTP, our findings also suggested an additional inhibitory mechanism, possibly a direct interaction between dCMPD and dFdC 5'-triphosphate. Thus, results obtained using this direct assay of dCMPD in intact cells support the hypothesis that dCMPD is inhibited by nucleotides of dFdC.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , DCMP Desaminase/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Leucemia Linfoide/enzimologia , Leucemia Mieloide/enzimologia , Radioisótopos de Carbono , Extratos Celulares , Cromatografia Líquida de Alta Pressão , Citidina Desaminase/metabolismo , DCMP Desaminase/metabolismo , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Nucleotídeos de Desoxicitosina/metabolismo , Humanos , Nucleotídeos de Timina/metabolismo , Células Tumorais Cultivadas , GencitabinaRESUMO
2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Citidina Trifosfato/análogos & derivados , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular , Citidina Trifosfato/síntese química , Citidina Trifosfato/metabolismo , Citidina Trifosfato/farmacologia , DCMP Desaminase/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxirribonucleotídeos/farmacologia , Humanos , Cinética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Ribonucleotídeos/isolamento & purificação , Ribonucleotídeos/metabolismo , GencitabinaRESUMO
The ratio of the steady-state kinetic Hill coefficients of two different effectors equals (under some rather weak general assumptions) the ratio in which the effectors displace each other from an enzyme. This principle can make implications of experimental allosteric enzyme kinetic data immediately apparent. We can use it to find that one molecule of the allosteric inhibitor of dCMP aminohydrolase, at moderately high effector concentrations, displaces one molecule of substrate, or one molecule of activator, whereas at very high concentrations, one molecule of inhibitor displaces two of substrate. Further use of the principle suggests that substrate, at high concentrations, binds binds to activator sites. However, ratios of substrate, activator, and inhibitor Hill coefficients are incompatible with a simple model of activation in which substrate and activator are bound to the same conformation.
Assuntos
DCMP Desaminase/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , DCMP Desaminase/antagonistas & inibidores , Nucleotídeos de Desoxicitosina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Cinética , Matemática , Perissodáctilos , Baço/enzimologia , Nucleotídeos de Timina/farmacologiaRESUMO
The effect of purine and pyrimidine deoxyribonucleosides on the activity of 5-methoxymethyl-2'-deoxycytidine (MMdCyd) against herpes simplex virus type 1 (HSV-1) was investigated. The antiviral activity of MMdCyd was decreased by deoxythymidine, deoxyuridine and deoxycytidine. Deoxyadenosine had no effect at concentrations up to 500 microM. In contrast, deoxyguanosine (dGuo) potentiated MMdCyd activity. The mean ED50 (1.5 microM) for the combination (MMdCyd plus 100 microM dGuo) was approximately 20-fold lower than that of MMdCyd (ED50 26 microM). When tetrahydrodeoxyuridine (H4dUrd, 540 microM) was added along with MMdCyd and dGuo, anti-HSV-1 activity of MMdCyd was further potentiated by 25-fold (ED50 0.06 microM). The inhibition of virus replication, as determined by the plaque reduction assay, was further confirmed by virus yield studies and by parallel observations on virus-induced cytopathogenicity. The order of decreasing effectiveness for reducing the production of infectious virus particles (virus yield) by different treatments was: MMdCyd + dGuo + H4dUrd greater than MMdCyd + DGuo greater than MMdCyd + H4dUrd greater than MMdCyd greater than dGuo + H4dUrd greater than dGuo greater than H4dUrd. The effect of dGuo and dGuo in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was determined in Vero cells infected with multiplicity of infection of 5 PFU/cell. In the presence of 100 microM dGuo, there was approximately a 3-fold, 2-fold and 12-fold increase in dCTP, dTTP and dGTP pool sizes respectively, as compared to control (untreated) cells. Treatment with H4dUrd (1.06 mM) in combination with dGuo (100 microM), resulted in an increase of the dCTP pool and a marked fall in the dTTP and dGTP pool. The possible mechanisms for potentiation of MMdCyd activity by dGuo and H4dUrd are discussed.
Assuntos
Antivirais/farmacologia , DCMP Desaminase/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Desoxiguanosina/farmacologia , Desoxirribonucleosídeos/farmacologia , Simplexvirus/efeitos dos fármacos , Tetra-Hidrouridina/análogos & derivados , Animais , Antivirais/toxicidade , Desoxicitidina/metabolismo , Desoxiguanosina/toxicidade , Simplexvirus/crescimento & desenvolvimento , Simplexvirus/metabolismo , Tetra-Hidrouridina/farmacologia , Células VeroRESUMO
The interaction between 2'-deoxycytidine (dCyd) and 1-beta-D-arabinofuranosylcytosine (ara-C), administered at pharmacologically achievable concentrations, was examined in four continuously cultured human leukemia cell lines, HL-60, KG-1, K-562, and CCRF-CEM. In three of the cell lines (HL-60, K-562, and CCRF-CEM), co-administration of 20 or 50 microM dCyd with 10 microM ara-C reduced ara-CTP formation by at least 90% and incorporation of ara-C into DNA by at least 80%. In contrast, KG-1 cells exhibited substantially smaller reductions in both ara-CTP formation and incorporation of ara-C into DNA under identical conditions. KG-1 cells were distinguished by the highest activity of the enzyme cytidine deaminase of the four lines assayed, and exhibited the smallest increments in the intracellular accumulation of both dCyd and deoxycytidine triphosphate (dCTP) in response to exogenous dCyd. Co-administration of 1 mM tetrahydrouridine (THU) or 0.5 mM deoxy-tetrahydrouridine (dTHU) had little effect on the ability of dCyd to antagonize ara-C metabolism in HL-60, KG-1 and K-562 cells. In contrast, these deaminase inhibitors substantially increased the intracellular accumulation of dCTP as well as the ability of dCyd to antagonize ara-CTP formation and incorporation of ara-C into DNA in KG-1 cells. THU and dTHU also permitted dCyd to antagonize ara-C growth inhibitory effects in KG-1 cells to the extent observed in the other leukemic cell lines. These studies suggest that the intracellular deamination of exogenous deoxycytidine may influence the degree to which this nucleoside antagonizes ara-C metabolism and toxicity in some leukemic cells. They also raise the possibility that deaminase inhibitors may be employed to modulate, and perhaps to improve, the therapeutic selectivity of pharmacologically relevant concentrations of ara-C and dCyd in the treatment of acute leukemia in man.
Assuntos
Citarabina/antagonistas & inibidores , Desoxicitidina/farmacologia , Leucemia/patologia , Tetra-Hidrouridina/análogos & derivados , Tetra-Hidrouridina/farmacologia , Arabinofuranosilcitosina Trifosfato/metabolismo , Divisão Celular/efeitos dos fármacos , Citarabina/metabolismo , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , DCMP Desaminase/antagonistas & inibidores , DCMP Desaminase/metabolismo , DNA de Neoplasias/metabolismo , Desaminação , Desoxicitidina/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Humanos , Leucemia/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Tetrahydrodeoxyuridine (dTHU) inhibits deoxycytidine deaminase and, after intracellular phosphorylation to the active 5'-monophosphate, also inhibits deoxycytidylate deaminase (dCMPD). Because in vitro studies have shown that dCMPD may regulate pyrimidine deoxynucleotide metabolism, the objective of this study was to investigate the effects of dTHU on deoxynucleotide metabolism in whole cells. Nearly complete inhibition of dCMPD, measured in intact CCRF-CEM cells by incorporation of [14C]dCyd into dTTP, occurred after a 45-min incubation with 100 microM dTHU. This was accompanied by an 8-fold dCTP pool expansion, although dATP, dTTP, dGTP, and ribonucleoside triphosphate pools were unaffected. Tetrahydrouridine, which inhibits deoxycytidine deaminase exclusively, had no effect on nucleotide pools. The dCTP pool expansion was directly proportional to the dTHU concentration (3-100 microM) and reached a maximum after 2 hr. Inhibition of ribonucleotide reductase by hydroxyurea completely prevented the dTHU-induced dCTP pool expansion, indicating that the substrate of dCMPD was derived from the ribonucleotide pool and that CDP was the predominant precursor of dCTP. dTHU-mediated inhibition of dCMPD appeared reversible. Exposure of cells to 100 microM dTHU followed by washing into fresh medium resulted in a linear decrease of the dCTP pool and an increase in the dTTP pool. The increased dCTP concentration after preincubation with dTHU was associated with an inhibition of deoxycytidine kinase, as indicated by a reduced capacity of cells to phosphorylate ara-C. dTHU is a useful new tool for investigating the role of dCMPD in the regulation of deoxynucleotide metabolism in whole cells.
Assuntos
DCMP Desaminase/antagonistas & inibidores , Nucleotídeos de Desoxicitosina/metabolismo , Desoxiuridina/análogos & derivados , Nucleotídeo Desaminases/antagonistas & inibidores , Linhagem Celular , Citarabina/metabolismo , Cistina Difosfato/metabolismo , Desoxicitidina/metabolismo , Desoxicitidina Quinase/metabolismo , Desoxiuridina/farmacologia , Hidroxiureia/farmacologia , Cinética , Ribonucleotídeo Redutases/antagonistas & inibidores , Linfócitos T/enzimologia , Tetra-Hidrouridina/farmacologia , Nucleotídeos de Timina/metabolismoRESUMO
Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained. At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP. At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.
Assuntos
DCMP Desaminase/metabolismo , Leucemia/enzimologia , Ativação Linfocitária , Linfócitos/enzimologia , Nucleotídeo Desaminases/metabolismo , Idoso , DCMP Desaminase/antagonistas & inibidores , Desoxirribonucleotídeos/farmacologia , Feminino , Humanos , Técnicas In Vitro , Leucemia/imunologia , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Fito-Hemaglutininas/farmacologiaRESUMO
Methods are described for preparing and structurally analyzing two enzymes involved in the formation of dTMP, deoxycytidylate deaminase and thymidylate synthase. In the latter case, it has been possible through the use of recombinant DNA techniques with an amplification plasmid to obtain sufficient amounts of the E. coli and T4-phage synthases to complete the entire sequence of both enzymes by employing a combination of protein and DNA sequencing methods. A comparative analysis of the L. casei and E. coli synthases has revealed a 62% conservation of sequences but an even greater homology in their hydrophobic active site regions (82%), which are primarily hydrophobic in nature. The homology between these enzymes becomes apparent by deleting a 51 amino acid segment (residues 89-139) from the L. casei synthase, which accounts for the difference in size between these enzymes. Methods for obtaining the binding sites of both substrates are described, one being the activation of the carboxyls of folate with a water soluble carbodiimide and the other, the activation of dUMP by ultraviolet light. The DNA and protein sequence of the T4-phage synthase has recently been clarified by us and is in preparation. Of great interest is the finding by Purohit and Mathews (42), based on our sequence data for the synthase, that the gene segment for the carboxyl terminal end of dihydrofolate reductase overlaps with the amino end of the gene for thymidylate synthase. The complete amino acid sequence of T2-phage deoxycytidylate deaminase has been elucidated by conventional protein sequencing methods. The binding characteristics of this enzyme for its positive allosteric effectors and substrates, as determined by equilibrium dialysis, are consistent with the cooperative nature of its kinetic responses. Consistent with these findings was the demonstration that each of the enzyme's six subunits bound an equivalent amount of substrate or allosteric modifier. Similarly the deaminase showed a marked negative change in ellipticity at 280 nm in response to increasing concentrations of dCTP, changes which could be reversed by dTTP. From the information on the enzyme's primary sequence, it should be possible to define the substrate and allosteric binding regions within the deaminase with the appropriately activated compounds. A start in this direction has been initiated by the finding that dTTP is rapidly and apparently covalently fixed to the amino terminal cyanogen bromide peptide of the enzyme in the presence of ultraviolet light.
Assuntos
DCMP Desaminase , Metiltransferases , Nucleotídeo Desaminases , Timidilato Sintase , Sequência de Aminoácidos , Sítios de Ligação , DCMP Desaminase/antagonistas & inibidores , Escherichia coli/enzimologia , Lacticaseibacillus casei/enzimologia , Metiltransferases/antagonistas & inibidores , Microscopia Eletrônica de Varredura , Nucleotídeo Desaminases/antagonistas & inibidores , Nucleotídeos , Plasmídeos , Biossíntese de Proteínas , Fagos T/enzimologia , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/genética , Raios UltravioletaAssuntos
DCMP Desaminase/antagonistas & inibidores , Nucleotídeo Desaminases/antagonistas & inibidores , Ribonucleotídeo Redutases/antagonistas & inibidores , Timidina Quinase/antagonistas & inibidores , Nucleotídeos de Timina/farmacologia , Animais , Cinética , Leucemia L1210/enzimologia , Camundongos , Relação Estrutura-AtividadeAssuntos
DCMP Desaminase/metabolismo , Nucleotídeo Desaminases/metabolismo , Baço/enzimologia , DCMP Desaminase/antagonistas & inibidores , DCMP Desaminase/isolamento & purificação , Desoxicitidina Monofosfato/metabolismo , Nucleotídeos de Desoxicitosina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Nucleotídeos de Timina/farmacologiaRESUMO
Cytidine deaminase, an enzyme found in the supernatant fluid of hepatocytes, granulocytes and tumor cells, and in plasma, degrades the antitumor agents cytosine arabinoside and 5-azacytidine. Uridine and its analogs, 3-deazauridine, 5-bromodeoxyuridine, 5-fluorodeoxyuridine and 6-azauridine, were found to competitively inhibit cytidine deaminase; the most potent inhibitor was 3-deazauridine (K(i) = 1.9 x 10(-5) M). In addition, deoxycytidylate deaminase, which degrades cytosine arabinoside monophosphate to the inactive uracil arabinoside monophosphate (K(m) = 9 x 10(-4) M), was competitively inhibited by 3-deazauridine monophosphate, as well as by the nucleotides of other uridine analogs. These results suggest that uridine analogs such as 3-deazauridine may have value in protecting cytosine arabinoside, 5-azacytidine and their monophosphate nucleotides from degration by neucleoside and nucleotide deaminases.