Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid). In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate. Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition. E. coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate. B. bacteriovourus grew at a normal rate on these depleted E. coli cells but with somewhat reduced cell yield. Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E. coli somewhat more than it inhibited growth on normal E. coli, but the effects were small compared with inhibition of axenic growth of the mutant. Total bdellovibrio DNA after growth on the depleted E. coli in the presence or absence of methotrexate exceeded the initial quanity of E. coli DNA present. Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA. The data are consistent with the prediction that intraperiplasmic growth of B. bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers. The data also indicate that B. bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth. The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B. bacteriovorus.
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PMID:Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus. 109 May 93

The de novo pathway of thymidylate synthesis (i.e., methylation of dUMP to dTMP) is directly folate dependent and indirectly vitamin B12 (cobalamins) dependent. In deficiency of these vitamins, this pathway is impaired, and exogenous deoxyuridine (dU) fails to suppress adequately in vitro incorporation of [3H]thymidine (3H-TdR) into DNA via the salvage pathway (i.e., abnormal dU suppression). This abnormality is corrected by the addition of folate compounds (analogues) and/or vitamin B12 depending on the nature of the underlying deficiency. We studied the effects of addition of PteGlu, 5-methyl THF (5-CH3-FH4), 5-formyl-THF (5-CHO-FH4), and hydroxy-cobalamin (OH-cbl) on 3H-TdR incorporation into DNA and thymidine kinase activity (salvage pathway), and on [3H]deoxyuridine (3H-dU) incorporation and dU suppression values (de novo pathway) in cultures of normal and megaloblastic bone marrows. The results showed that 3H-TdR incorporation into DNA and the salvage enzyme, thymidine kinase, activity were greater and 3H-dU incorporation into DNA less in megaloblastic cells as compared with normal cells. The addition of folates significantly reduced 3H-TdR incorporation and thymidine kinase activity and enhanced 3H-dU incorporation in folate and vitamin B12-deficient cells except that 5-CH3-FH4 had no effect on vitamin B12-deficient cells. None of these additives had any significant effect on normal cells. This study also showed that the addition of the deficient vitamin(s) to the "control tubes" in the dU suppression test is inappropriate, as these vitamins may at least partially correct the defect in cellular DNA synthesis caused by the deficiencies of these vitamins and may mask these deficiencies in the results of the in vitro correction of the dU suppression abnormalities in mild cases of megaloblastic anemia.
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PMID:In vitro DNA synthesis by megaloblastic bone marrow: effect of folates and cobalamins on thymidine incorporation and de novo thymidylate synthesis. 270 38

Inhibition of dihydrofolate reductase by the folate analog, methotrexate (MTX) results in a depletion of tetrahydrofolate dependent one carbon transfer reactions in amino acid and nucleic acid biosynthesis. When human cells (either HeLa or normal skin fibroblasts) are exposed to MTX in a defined medium containing dialyzed fetal calf serum, essential and non-essential amino acids, and purine source, the thymidylate pools alone are depleted. Under these conditions exposure to 10(-6) M MTX induces mitochondrial mutagenesis, measured as an increase in the frequency of chloramphenicol resistant (CAPR) colonies, without altering the rate of nuclear mutation monitored by determining the frequency of 6-thioguanine resistance (TGr). The occurrence of CAPR mutations is time, and MTX concentration dependent and the frequency of CAPR can be decreased quantitatively by adding thymidine to the culture medium. This mitochondrial specific mutagenesis can also be achieved using the thymidylate synthetase inhibitor, 5-fluorodeoxyuridine further implicating thymidylate pools as the mediator of this effect. During the course of exposure to 10(-6) M MTX the thymidine kinase deficient HeLa BU25 cell line exhibits a progressive depletion and degradation of mitochondrial DNA suggesting that the mutagenesis and DNA degradation represent portions of a progressive process. The basis for the selective sensitivity of the mitochondrial genome to thymidylate depletion mutagenesis may be the consequence of its differences from the nuclear genome in mechanisms of DNA replication or repair.
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PMID:Induction of mitochondrial mutations in human cells by methotrexate. 399 29

Growth inhibition by methotrexate (MTX) of a cultured mouse mammary carcinoma FM3A line and its mutants deficient in thymidine kinase and thymidylate synthetase was studied under a variety of conditions. In medium containing 10 microM thymidine, the thymidylate synthetase-deficient mutants were slightly more resistant to MTX than the wild-type line and the thymidine kinase-deficient mutant. The addition of both 10 microM thymidine and 50 microM hypoxanthine to the medium completely eliminated the inhibition by MTX of the wild-type and thymidylate synthetase-deficient mutants but had no effect on inhibition of the thymidine kinase-deficient mutant. Thus, addition of either thymidine or purine alone was not sufficient to protect FM3A cells against the growth inhibitory effect of MTX. In contrast, addition of a small amount of 5-methyltetrahydrofolate to medium containing 10 microM thymidine caused an increase of several orders of magnitude in the resistance of thymidylate synthetase-deficient mutants to MTX but did not affect that of the wild-type line. Wild-type cells became almost as resistant to MTX as did the mutant cells by addition of 1 microM 5-fluorodeoxyuridine as well as a small amount of 5-methyltetrahydrofolate with thymidine. These results show directly that thymidylate synthetase is essential in determining the cytotoxicity of MTX by modulating the intracellular tetrahydrofolate pool.
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PMID:Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FM3A cells. 645 98

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.
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PMID:Biochemical and cellular pharmacology of 1843U89, a novel benzoquinazoline inhibitor of thymidylate synthase. 842 62

Cervical carcinoma is an AIDS-defining illness. The expression of folate receptors (FRs) in cervical carcinoma (HeLa-IU1) cells was modulated by stable transduction of FR cDNA encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). Although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) FCS, [methyl-3H]thymidine incorporation into DNA was significantly increased in sense cells in 10% serum; therefore, the basis for this discrepancy was investigated. The activity of thymidine kinase (TK) was subsequently directly correlated with the extent of FR expression in single cell-derived clones of transduced cells. This elevated TK activity was not a result of recruitment of the salvage pathway based on the presence of adequate dTTP pools, normal thymidylate synthase (TS) activity, persistence of increased thymidine incorporation despite the exogenous provision of excess 5,10-methylene-tetrahydrofolate, and documentation of adequate folates in sense cells. The increase in TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as demonstrated by augmented phosphorylation of 3'-azido-3'-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT. Conversely, sense cells were highly resistant to methotrexate, but this was reversed by the addition of AZT. The direct correlation of FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression.
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PMID:Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity. 1002 88

A novel strategy was developed for the synthesis of N(7)-purine acyclic nucleosides 9 and 14. The key step involved the reaction between [2-(p-methoxyphenyloxy)ethoxy]methyl chloride and N(9)-tritylated nucleobases 6 or 11 followed by concomitant self-detritylation. N(7)-Guanine acyclic nucleoside 9 exhibited antiviral activity, but was phosphorylated by both HSV and Vero cell thymidine kinases. Thus, it showed more potent cellular toxicity than acyclovir (2). N(7)-Adenine acyclic nucleoside 14 was found to be an excellent antiviral agent as well as a good inhibitor of calf mucosal adenosine deaminase. This inhibitory property allows for a greater expression of antiviral activity of antiviral agents, such as N(9)-adenine acyclic nucleoside 1 and ara-A (3). Compound 14 was phosphorylated neither by herpes simplex virus (HSV) thymidine kinase nor by Vero cell thymidine kinase, yet it enhanced the rate constant for the monophosphorylation of acyclovir (2) by HSV thymidine kinase. Consequently, the combination of acyclovir (2) and 14 exhibited greater antiviral activity than acyclovir alone. 7-[2-(Phosphonomethoxy)ethyl]adenine (20) was also synthesized. The key step involved the reaction of 9-(2-cyanoethyl)adenine (15) with methyl iodoacetate in the presence of lithium 2,2,6,6-tetramethylpiperidine in THF. Unlike 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, 4), the N(7)-isomer 20 was not phosphorylated effectively by 5-phosphoribosyl 1-pyrophosphate synthetase (PRPP synthetase). Thus, it did not exhibit pronounced antiviral activity. Dinucleotide 5'-monophosphate 24 and its butenolide ester 25 were also synthesized. Compound 24 showed substrate activity toward PRPP synthetase and exhibited notable activity against DNA viruses. The antiviral activity of the ester derivative 25 was found to be higher than that of the parent molecule 24. Dinucleotide 5'-monophosphate 24 is susceptible to degradation by snake venom and spleen phosphodiesterases. However, its respective butenolide ester derivative 25 was completely resistant to snake venom and spleen enzymes. Butenolide ester derivatives 28 and 29 were also synthesized and exhibited notable anti-DNA virus and anti-retrovirus activity in vitro. Compounds 2, 4, 9, 14, 20, 24, 25, and 28 were also evaluated for their inhibitory effect on HSV-1-induced mortality in NMRI mice. N(7)-adenine acyclic nucleoside 14 [LD(50) (intraperitoneal, ip) 950 mg/kg], nucleotide-containing butenolide 25 [LD(50) (ip) 675 mg/kg], and butenolide 28 [LD(50) (ip) 710 mg/kg] were found to be potent anti-HSV-1 agents in vivo. In addition, butenolide 28 efficiently decreased tumor formation induced by Moloney murine sarcoma virus (MSV) in NMRI mice while significantly increasing the survival time of MSV-infected mice.
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PMID:Design, synthesis, and biological evaluation of novel nucleoside and nucleotide analogues as agents against DNA viruses and/or retroviruses. 1160 36

Pyrimidine antagonists, for example, 5-fluorouracil (5-FU), cytarabine (ara-C) and gemcitabine (dFdC), are widely used in chemotherapy regimes for colorectal, breast, head and neck, non-small-cell lung cancer, pancreatic cancer and leukaemias. Extensive metabolism is a prerequisite for conversion of these pyrimidine prodrugs into active compounds. Interindividual variation in the activity of metabolising enzymes can affect the extent of prodrug activation and, as a result, act on the efficacy of chemotherapy treatment. Genetic factors at least partly explain interindividual variation in antitumour efficacy and toxicity of pyrimidine antagonists. In this review, proteins relevant for the efficacy and toxicity of pyrimidine antagonists will be summarised. In addition, the role of germline polymorphisms, tumour-specific somatic mutations and protein expression levels in the metabolic pathways and clinical pharmacology of these drugs are described. Germline polymorphisms of uridine monophosphate kinase (UMPK), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and methylene tetrahydrofolate reductase (MTHFR) and gene expression levels of OPRT, UMPK, TS, DPD, uridine phosphorylase, uridine kinase, thymidine phosphorylase, thymidine kinase, deoxyuridine triphosphate nucleotide hydrolase are discussed in relation to 5-FU efficacy. Cytidine deaminase (CDD) and 5'-nucleotidase (5NT) gene polymorphisms and CDD, 5NT, deoxycytidine kinase and MRP5 gene expression levels and their potential relation to dFdC and ara-C cytotoxicity are reviewed.
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PMID:Genetic factors influencing pyrimidine-antagonist chemotherapy. 1604 92

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