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)

Deoxynucleoside kinases catalyze the 5'-phosphorylation of 2'-deoxyribonucleosides with nucleoside triphosphates as phosphate donors. One of the cellular kinases, deoxycytidine kinase (dCK), has been shown to phosphorylate several L-nucleosides that are efficient antiviral agents. In this study we investigated the potentials of stereoisomers of the natural deoxyribonucleoside to serve as substrates for the recombinant cellular deoxynucleoside kinases. The cytosolic thymidine kinase exhibited a strict selectivity and phosphorylated only beta-D-Thd, while the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK) as well as dCK all had broad substrate specificities. TK2 phosphorylated Thd and dCyd stereoisomers in the order: beta-D- > or = beta-L- >> alpha-D- > or = alpha-L-isomer. dCK activated both enantiomers of beta-dCyd, beta-dGuo, and beta-dAdo with similar efficiencies, and alpha-D-dCyd also served as a substrate. dGK phosphorylated the beta-dGuo enantiomers with no preference for the ribose configuration; alpha-L-dGuo was also phosphorylated, and beta-L-dAdo and beta-L-dCyd were substrates but showed reduced efficiencies. The anomers of the 2',3'-dideoxy-D-nucleosides (ddNs) were tested, and TK2 and dCK retained their low selectivities. Unexpectedly, alpha-dideoxycytidine (ddC) was a 3-fold better substrate for dCK than beta-ddC. Similarly, alpha-dideoxythymidine (ddT) was a better substrate for TK2 than beta-ddT. dGK did not accept any D-ddNs. Thus, TK2, dCK, and dGK, similar to herpes simplex virus type 1 thymidine kinase (HSV-1 TK), showed relaxed stereoselectivities, and these results substantiate the functional similarities within this enzyme family. Docking simulations with the Thd isomers and the active site of HSV-1 TK showed that the viral enzyme may in some respects serve as a model for studying the substrate specificities of the cellular enzymes.
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PMID:Stereoisomeric selectivity of human deoxyribonucleoside kinases. 1060 35

Preclinical aspects of a potent anti-hepatitis B virus (HBV) L-nucleoside, 1-(2-fluoro-5-methyl-beta-L-arabino-furanosyl)uracil (L-FMAU) are described. L-FMAU was prepared from L-ribose derivatives via either L-xylose or L-arabinose. L-FMAU shows potent antiviral activity against hepatitis B virus (EC50 5.0 microM in H1 cells) with high selectivity in vitro. L-FMAU is not incorporated into mitochondrial DNA and no significant lactic acid production was observed in vitro. L-FMAU is phosphorylated by thymidine kinase as well as deoxycytidine kinase, ultimately to the triphosphate, which inhibits HBV DNA polymerase as the mechanism of antiviral action. Preliminary in vivo toxological studies suggest no apparent toxicity for 30 days at 50 mg/kg/day in mice and for 3 months in woodchucks (10 mg/kg/day). L-FMAU also has respectable bioavailability in rats. L-FMAU shows potent anti-HBV activity in vivo against woodchuck hepatitis virus in chronically infected woodchucks and there is no significant virus rebound after cessation of the drug treatment.
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PMID:Preclinical investigation of L-FMAU as an anti-hepatitis B virus agent. 1072 61

Mitochondrial thymidine kinase (TK2) phosphorylates pyrimidine nucleosides to monophosphates and is expressed constitutively through the cell cycle in all cells. Because of the overlap of its substrate specificity with that of the cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), it has been difficult to determine the role of TK2 in activating nucleosides used in chemotherapy. In this report, we described the construction of a recombinant Escherichia coli strain which could be used to test if TK2 activity is limiting for the toxicity of nucleosides. Enzymes of bacterial origin which are involved in thymidine and deoxyuridine anabolism and catabolism were eliminated, and the cDNA for human TK2 was introduced. In the crude extract of the engineered E. coli, the level of thymidine kinase was, after induction of TK2 expression, several hundred fold higher than in the control strain. Several pharmacologically interesting nucleoside analogues, including 3'-azidothymidine, 2',3'-didehydro-2',3'-dideoxythymidine, and 2', 3'-dideoxy-beta-L-3'-thiacytidine, were tested for their effects on the growth of this recombinant strain. For a comparison, the phosphorylation of these compounds was determined with purified recombinant TK1, TK2, and dCK. A correlation was observed between the phosphorylation of several of these compounds by TK2 and their effects on bacterial growth. These results demonstrate that activation of growth-inhibiting pyrimidine nucleosides can be catalyzed by TK2, and together with recombinant E. coli strains expressing other cellular nucleoside kinases, this whole-cell bacterial system may serve as a tool to predict the efficacy and side effects of chemotherapeutic nucleosides.
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PMID:Expression of human mitochondrial thymidine kinase in Escherichia coli: correlation between the enzymatic activity of pyrimidine nucleoside analogues and their inhibitory effect on bacterial growth. 1079 56

The deoxynucleoside kinase reaction is often rate-limiting in the anabolism of pharmacologically active anti-cancer nucleosides. The levels of thymidine kinase (TK), deoxycytidine kinase, deoxyguanosine kinase (dGK), and thymidylate kinase were determined in leukocyte extracts from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). The extracts from AML patients showed significantly higher TK activity than the ones from CLL patients. There were no differences in the levels of the other three kinases. In the case of dGK, the determinations were carried out with both an immunoblotting assay and selective enzyme activity measurements.
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PMID:Deoxynucleoside anabolic enzyme levels in acute myelocytic leukemia and chronic lymphocytic leukemia cells. 1127 69

OBJECTIVES: In vitro activities of thymidine kinase (TK, EC 2.7.1.21), adenosine kinase (AK, EC 2.7.1.20) and deoxycytidine kinase (dCK, EC 2.7.1.74) enzymes involved in the salvage pathway of DNA precursor synthesis, in homogenates of the rat liver and kidney, were examined. Type I iodothyronine-5'-deiodinase (5'D-I) is the main enzyme responsible for peripheral metabolism of thyroid hormones. This occurs especially in the liver, kidney and muscle. The activity of 5'D-I is inhibited bypropylthiouracil (PTU), an antithyroid drug. METHODS: The liver and kidney were collected from rats pretreated in vivo with either a 0.1% solution of PTU in drinking water for 2 weeks or injected with levothyroxine (L-T(4), 50 &mgr;g/kg BW, daily) for 2 weeks. The enzyme activities were measured by ascending chromatography and expressed asthe amounts of radioactive reaction products of the phosphorylation of dThd (for TK), ofdAdo (for AK and dCK) and of dGuo (for dCK). RESULTS: In liver homogenates, PTU-pretreatment decreased the activities of the three enzymes when compared to control values and those of L-T(4)-treated animals; also L-T(4) injections decreased the AK and dCK activities in the liver homogenates. PTU-pretreatment increased TK activity and the rate of dGuo phosphorylation in kidney homogenates, when compared to controls and to the L-T(4)-pretreated animals. Conversely, both PTU- and L-T(4)-pretreatment reduced the rate of dAdo phosphorylation in kidney homogenates. CONCLUSION: Changes in the activities of examined enzymes which participate inpyrimidine orpurine metabolism of the salvage pathway of DNA synthesis in the liver afterPTU-pretreatment (as shown herein) are similar to the changes of the 5'D-I activity after PTU-treatment (as reported by others). Thus, the observations suggest a role of the salvage pathway of DNA synthesis in the peripheral metabolism of thyroid hormones.
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PMID:Pyrimidine and purine salvage deoxyribonucleoside metabolism in hepatic and renal homogenates from rats pretreated with propylthiouracil or L-thyroxine. 1145 32

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.
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PMID:Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB. 1170 77

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.
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PMID:Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides. 1173 47

After pulsed low dose rate irradiation the activities of deoxycytidine kinase and thymidine kinase 1 and 2 were increased 1.5-2-fold 6 h after treatment. Twenty-four hours after treatment the activities of these enzymes had returned to control levels. We presume that the increase of enzyme activities is part of an adaptive response to irradiation and that this increase could be an explanation for the increased survival in the initial part of the SW-1573 cell survival curve. The observation that not only S-phase specific thymidine kinase 1 but also mitochondrial thymidine kinase 2 increases, implies that both these enzymes play a role in an adaptive response of cells to irradiation.
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PMID:Enhanced levels of deoxycytidine kinase and thymidine kinase 1 and 2 after pulsed low dose rate irradiation as an adaptive response to radiation. 1174 72

The biological evaluation of mononucleotide prodrugs (pronucleotides) of various nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), zalcitabine (ddC) and lamivudine (3TC) was reported in human T-lymphoid MOLT-4/8 cells which were grown continuously for more than 1 year in a medium containing cytarabine (Ara-C). In this cell line, expression of deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) was decreased in comparison to parental cells (3.8 and 2.9-fold, respectively). The lower mRNA level of TK1 correlated significantly with lower enzyme activity, whereas no dCK activity was detectable. In Ara-C-resistant cells, anti-HIV-1 effects of ddC, 3TC and AZT were more than 100-fold lower compared with parental cells. In contrast, the corresponding mononucleoside phosphotriesters bearing S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protection retained anti-HIV-1 activity due to their ability to bypass the first monophosphorylation step catalyzed by dCK or TK1. The results demonstrate that in vitro selection of T-lymphoid cells in the presence of Ara-C results in cross-resistance to deoxycytidine (ddC, 3TC) and thymidine (AZT) analogs and that these cellular resistance mechanisms can be bypassed by the use of bis(SATE) pronucleotides.
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PMID:S-acyl-2-thioethyl (SATE) pronucleotides are potent inhibitors of HIV-1 replication in T-lymphoid cells cross-resistant to deoxycytidine and thymidine analogs. 1175 Sep 40

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.
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PMID:Cytotoxic activity of 2',2'-difluorodeoxycytidine, 5-aza-2'-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene. 1205 82


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