Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:024500 (thymidylate synthase)
 2,970 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship between the antitumor activity and the inhibition of thymidylate synthase after oral administration of 5-FU, FT-207 or UFT was examined. The inhibition of tumor growth on sarcoma-180 after oral administration of 5-FU (20 mg/kg), FT-207 (120 mg/kg) or UFT (30 mg/kg) was 40%, 50% or 70%, respectively. It was found that the inhibition of thymidylate synthase in sarcoma-180 tumor tissue after single oral administration of 5-FU (20 mg/kg), FT-207 (30 or 120 mg/kg) or UFT (30 mg/kg) were about 45%, 20%, 50% or 65%, respectively, but the activities of other enzymes involved in DNA synthesis were not almost inhibited. Thymidylate synthase was inhibited more severely by oral administration of UFT (30 mg/kg). The activities of other enzymes were not inhibited even by continuous oral administration of these drugs for 6 or 11 days. The extent of inhibition of thymidylate synthase seemed to be parallel to that of inhibition of tumor growth. These results suggest that the inhibition of thymidylate synthase by FdUMP converted from 5-FU, FT-207 or UFT plays a major role in their antitumor actions.
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PMID:[Studies on the mechanism of antitumor activity of 5-FU and its derivatives--relationship between the inhibition of tumor growth and the inhibition of thymidylate synthetase in vivo]. 642 1

A series of 2-amino-4-hydroxy-quinazolines was synthesized and evaluated as inhibitors of colon adenocarcinoma and the folate-dependent enzymes, thymidylate synthase and dihydrofolate reductase. Of the quinazolines tested, 5,8-dideazaisopteroylglutamate, (IAHQ), when administered at 85 mg/kg on days 2 and 10 after tumor implantation delayed the growth of colon tumor No. 38, and resulted in 6 of 20 tumor-free animals at 90 days. In contrast, methotrexate had no effect on the growth of colon tumor No. 38 at maximally tolerated doses. IAHQ was also active against human colon adenocarcinoma cells (HCT-8) in tissue culture, requiring a concentration of 5 X 10(-7) M to inhibit cell growth 50% after 72 hours continuous exposure. Since IAHQ was an effective substrate for folylpolyglutamate synthetase, we examined the effects of IAHQ and its possible tri-gamma-glutamyl metabolite, 5,8-dideazaisoPteGLu3, on thymidylate synthase and dihydrofolate reductase. Neither IAHQ nor 5,8-dideazaisoPteGlu3 stimulated significant binding of 5-fluorodeoxyuridylate to thymidylate synthase. This was consistent with the observation that IAHQ antagonized the killing of HCT-8 cells by 5-fluorouracil. 5,8 DideazaisoPteGlu3 bound more tightly to thymidylate synthase than dihydrofolate reductase as indicated by Kis of 0.09 and 0.7 microM when deoxyuridylate and dihydropteroylglutamate, respectively, were the variable substrates. Inhibition studies also revealed that binding of IAHQ and 5,8-dideazaisoPteGlu3 to thymidylate synthase is promoted and not antagonized by deoxyuridylate. The data suggests that the biochemical basis for the antitumor effects of IAHQ is the intracellular conversion of IAHQ to poly-gamma-glutamyl metabolites, which inhibit thymidylate synthase via formation of an inhibitor-deoxyuridylate-enzyme complex.
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PMID:Effects of 5,8-dideazaisopteroylglutamate and its possible tri-gamma-glutamyl metabolite (5,8-dideazaisoPteGlu3) on colon adenocarcinoma, and the folate dependent enzymes thymidylate synthase and dihydrofolate reductase. 668 93

Reported antifolate activity against leukemia L1210 by N-[14-[[(2-amino-4-hydroxy-6-quinazolinyl)methyl]-propargylamino]benzoyl]]-L-glu tamic acid through potent inhibition of thymidylate synthase (EC 2.1.1.45) prompted us to include the propargyl group in a study of the effect on folate metabolism and membrane transport of replacing the 10-methyl group of methotrexate with other groups. Along with the propyl (8a) and octyl (8b) homologues of methotrexate, the propargyl compound 8c was prepared for evaluation. Syntheses of 8a,b were achieved by a standard multistep sequence involving preparation of the side-chain precursors via tosylated intermediates and then their alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. The side-chain precursor to 8c was prepared by direct alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with propargyl bromide and was separated from unchanged amine and dipropargyl coproduct by a combination of methods, including dry-column chromatography and recrystallization. Subsequent steps leading to 8c were like those used to prepare 8a,b. Biological evaluations of the three compounds consisted of studies of their effects on enzyme inhibition [(dihydrofolate reductase (EC 1.5.1.3) and thymidylate synthase)], L1210 cell growth inhibition, cellular membrane transport with various murine cell types (L210, S180, Ehrlich, and epithelial), in vivo (mice) activity vs. L1210 leukemia and S180 ascites, and plasma clearance in mice. The in vivo results vs. S180 ascites offered evidence that 8c might have a better therapeutic index against this tumor than methotrexate, but no other result from either of these compounds suggested significant superiority over methotrexate.
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PMID:10-Propargylaminopterin and alkyl homologues of methotrexate as inhibitors of folate metabolism. 710 7

Classical antifolate inhibitors of thymidylate synthase (TS) often require the reduced folate uptake system in order to exert their antitumor effects. In addition, these analogues are polyglutamylated via the enzyme folylpoly-gamma-glutamate synthetase (FPGS), which prevents analogue efflux from the cell and usually increases their inhibitory potency against TS. Impaired function of the reduced folate uptake system and that of FPGS are potential sources of resistance to such antifolates. We designed and synthesized a classical 6-5 ring-fused analogue N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3- d]pyrimidin-5-yl)thio]-benzoyl]-L-glutamic acid (5) and a nonclassical 6-5 ring-fused analogue 2-amino-6-methyl-5-(pyridin-4-ylthio)-3,4-dihydro-4-oxo-7H-pyrrolo [2,3- d]pyrimidine (6) as TS inhibitors and antitumor agents. The syntheses of analogues 5 and 6 were achieved via the oxidative addition of the sodium salt of ethyl 4-mercaptobenzoate or 4-mercaptopyridine to 2-(pivaloylamino)-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyri midine (17) in the presence of iodine. For the synthesis of 5 the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 5 was a potent inhibitor of human and bacterial TS with IC50 values of 42 and 21 nM, respectively. Compound 6 was 10-fold less potent than 5 against human TS but more than 4700-fold less potent than 5 against Lactobacillus casei TS. The classical analogue 5 was neither a substrate nor an inhibitor of human FPGS derived from CCRF-CEM cells. Compound 5 was cytotoxic to CCRF-CEM and FaDu tumor cell lines as well as to an FPGS-deficient subline of CCRF-CEM. Thymidine protection studies established that TS was the primary target of 5.
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PMID:5-Arylthio-substituted 2-amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine antifolates as thymidylate synthase inhibitors and antitumor agents. 747 77

Pre-operative chemotherapy with concomitant use of UFT and CDDP (UFT: oral administration of 400 mg/day for 2 weeks till one day before operation, CDDP, one intravenous drip of 40 mg/m2 one week before operation) was used for 24 untreated cases of advanced stomach cancer diagnosed as resectable pre-operatively, and the histological antitumor effect analyzed in dissected preparation and the thymidylate synthase inhibition rate (TSIR: %) in tumor tissue were examined. The average administration dose of CDDP was 61.1 mg/body, and the average total administration dose of UFT was 5.0 g/body. The histological antitumor effect was grade 0 in 8 cases (33.3%), grade 1a in 10 cases (41.7%), grade 1b in 5 cases (20.8%), and grade 2 in 1 case (4.2%). TSIR in tumor tissue was under 10% in 2 cases (9.1%); over 10% and under 20% in 4 cases (18.2 %); over 20% and under 30% in 6 cases (27.3%); over 30% and under 40% in 5 cases (22.7%); over 40% and under 50% in 3 cases (13.6%); over 50 % in 2 cases (9.1%), and not measurable in 2 cases, with the average of 29.0%. The correlation was observed between histological anti-tumor effect and TSIR in tumor tissue (p < 0.05). These results suggest the possibility that the anti-tumor effect can be estimated at the in vivo level from measurement of TSIR in tumor tissue.
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PMID:[UFT/CDDP preoperative chemotherapy for progressive gastric cancer--histological antitumor effects and thymidylate synthase inhibition rate]. 748 25

We previously reported (Matherly et al., J Biol Chem 267: 23253-23260, 1992) that impaired methotrexate transport in a drug-resistant CCRF-CEM variant (CEM/MTX) involved the synthesis of a structurally altered isoform of the "classical" carrier for methotrexate and related derivatives. Although CEM/MTX cells were highly resistant (162- to 300-fold) to assorted antifolate substrates for the classical transporter, including methotrexate, aminopterin, 10-ethyl-10-deazaaminopterin, ICI D1694, and 1843U89, they were only 3.6-fold resistant to (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF). These divergent antifolate sensitivities were not associated with appreciable differences in the levels of dihydrofolate reductase, thymidylate synthase, and 5'-phosphoribosylglycinamide (GAR) transformylase, or the expression of a high affinity membrane folate binding protein receptor in either line. The initial rate of [14C]DDATHF influx was increased 2.9-fold over that for [3H]methotrexate in parental cells (at 2 microM). Whereas [14C]DDATHF initial uptake was, likewise, increased over [3H]methotrexate in CEM/MTX cells (5.3-fold), influx of both compounds was impaired substantially (95-97%). For the parent, influx of [14C]DDATHF was inhibited by substrates for the classical transporter including unlabeled DDATHF, methotrexate, (6R,S)-5-formyl tetrahydrofolate, 10-ethyl-10-deazaaminopterin, ICI D1694, 1843U89, and folic acid. The synthesis of a modified transporter in CEM/MTX cells was accompanied by significant changes in the binding of all these transport substrates. In spite of its impaired transport, [14C]DDATHF (at 2 microM), unlike methotrexate, continued to accumulate in CEM/MTX cells, eventually reaching 62% of the parental drug levels after 4 hr. At this time, 53% (parent) and 71% (CEM/MTX) of the intracellular radioactivity from [14C]DDATHF was identified as polyglutamates. DDATHF polyglutamates in CEM/MTX cells after 4 hr reached 90% of the levels measured in parental cells. While significant levels of methotrexate polyglutamates were detected in the parental line, methotrexate polyglutamylation was negligible in intact CEM/MTX cells. The specific activity of folylpolyglutamate synthetase was measured in cell-free extracts from parental and CEM/MTX cells using aminopterin, methotrexate, and DDATHF as substrates; in each case, CEM/MTX cells showed 2-fold higher enzyme activity than parental cells. These data show that even for tumor cells with severely impaired antifolate transport, the extensive conversion of DDATHF to polyglutamyl forms required for GAR transformylase inhibition preserves high levels of antitumor activity.
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PMID:Determinants of the disparate antitumor activities of (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate and methotrexate toward human lymphoblastic leukemia cells, characterized by severely impaired antifolate membrane transport. 750 26

Mice bearing subcutaneously implanted EMT6 mammary adenocarcinoma were treated with leucovorin or folic acid by continuous subcutaneous infusion or bolus intraperitoneal injection. (6R)-5,10-Methylenetetrahydrofolate pools in cytosolic extracts of the tumor, marrow, and gut were measured by analysis of the ternary complex with thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) and 5-fluoro-2'-deoxyuridylate, and the polyglutamate distribution in the (6R)-5,10-methylenetetrahydrofolate pool was analyzed by native gel electrophoresis. Bolus intraperitoneal administration of either leucovorin or folic acid caused dose-dependent expansion of the (6R)-5,10-methylenetetrahydrofolate pool in the tumor, but not in the marrow or gut. For example, the AUC (0-5 hr) in the tumor increased from a baseline value of 8.2 to 20 nmol/mg protein.hr after a bolus dose of 1.5 mmol/kg of leucovorin or folic acid, whereas the increase in marrow and gut was 2- to 4-fold lower. Continuous subcutaneous infusion at the same total dosage over 3 days gave AUC (0-96 hr) values of 134 nmol/mg protein.hr for controls as compared with 347 nmol/mg protein.hr for the leucovorin group and 254 nmol/mg protein.hr for the folic acid group. In contrast to bolus treatment, the increase in (6R)-5,10-methylenetetrahydrofolate in the marrow and small intestine with both leucovorin and folic acid infusion was similar to the increase in the tumor. Thus, intraperitoneal bolus injection was tumor selective, but subcutaneous continuous infusion was not. Longer-chain polyglutamates of (6R)-5,10-methylenetetrahydrofolate in the tumor after bolus treatment with 0.375 and 0.75 mmol/kg of leucovorin or folic acid increased relative to controls. At higher doses of 1.5 and 2.25 mmol/kg, an increase was observed only in the mono/diglutamate fraction. In marrow, on the other hand, the mono/diglutamate fraction, but not the longer-chain polyglutamates, increased at all doses. In the constant infusion regimen, longer-chain polyglutamates increased in all three tissues, though in gut and marrow the mono/diglutamate fraction increased more than in tumor. Leucovorin and folic acid were converted to (6R)-5,10-methylenetetrahydrofolate more efficiently but less selectively during a 3-day subcutaneous infusion than after an intraperitoneal bolus. Longer-chain polyglutamates were selectively increased in tumor by both regimens of leucovorin administration.
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PMID:Leucovorin and folic acid regimens for selective expansion of murine 5,10-methylenetetrahydrofolate pools. 753 77

Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease. We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-L-glutamic acid), a drug currently in phase III clinical trial. The degree of resistance was > 20,000 for the human lymphoblastoid cell line W1L2:R and approximately 14 for the ovarian carcinoma cell line CH1:R. In both cases resistance was associated with increased TS activity. The W1L2:R cell line had an approximately 100-fold increase in TS gene copy number and mRNA levels and a 500- to 1000-fold increase in enzyme levels determined using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern and Western blotting. The CH1:R cell line had an approximately 2- to 2.5-fold increase in TS gene copy number, mRNA and protein levels. In both cell lines the fold resistance determined was significantly higher than the fold increase in target enzyme DNA, mRNA or protein levels. Small changes in TS levels may therefore translate to clinically significant alterations in drug sensitivity.
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PMID:Molecular characterisation of two cell lines selected for resistance to the folate-based thymidylate synthase inhibitor, ZD1694. 753 19

Previous work in our laboratory showed that UFT (mixed compound of tegafur and uracil, molar ratio 1:4, respectively) caused the prolonged reduction of dTTP in L1210 leukemia cells in comparison with 5-fluorouracil (5-FU). The purpose of this study was to assess the effect of UFT on cell cycle distribution and thymidylate synthase activity of a leukemia cell line as compared with 5-FU. UFT and 5-FU were orally given to BDF1 mice bearing L1210 ascites tumor on day 3 after the tumor inoculation. Cell cycle distribution patterns at 24 hr after the drug administration showed a higher percentage of S phase in tumor cells treated with UFT than in those treated with 5-FU. Until 6 hr after the oral administration of the drugs, UFT inhibited the incorporation of [3H] deoxyuridine into DNA more long than 5-FU did. These results indicated that UFT has longer and stronger inhibitory effects on DNA replication than 5-FU in vivo under the employed experimental conditions (i.e., low and single doses of these fluorinated pyrimidines).
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PMID:Effects of UFT (mixed compound of tegafur and uracil) on cell kinetics and inhibition of thymidylate synthase in L1210 ascites tumor. 755 12

Variation of the bridge linking the heterocyclic ring and p-aminobenzoyl-L-glutamate portions of our previously described classical 2,4-diaminofuro[2,3-d]pyrimidines 1 and 2 are reported as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and as antitumor agents. Specifically -CH2CH2- and -CH2NHCH2- bridged analogues, N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) ethyl]benzoyl]-L-glutamic acid (3) and N-[4-[[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl) methyl]amino]methyl]benzoyl]-L-glutamic acid (4), respectively, were synthesized. Compound 3 was obtained via a Wittig reaction of the tributylphosphonium salt of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (5) and methyl 4-formylbenzoate (6) followed by reduction and coupling with the diethyl ester of L-glutamic acid. Compound 4 was synthesized by the nucleophilic displacement of 5 with diethyl N-[4-(aminomethyl)benzoyl]-L-glutamate (15) and saponification. Both analogues were evaluated in vitro as inhibitors of DHFRs from (recombinant) human, human CCRF-CEM cells, and Lactobacillus casei. Compound 3 showed moderate activity (IC50 10(-6)-10(-7) M). Compound 4 was essentially inactive (IC50 10(-5) M, CCRF-CEM). The compounds were also evaluated against TS from (recombinant) human and L. casei and were of low activity (IC50 10(-5) M). The three-atom-bridged analogue 4 was somewhat more inhibitory to human TS than methotrexate (MTX). Compound 3 inhibited the growth of tumor cells in culture (IC50 10(-7) M) while 4 showed a low level of growth inhibitory activity. The inhibition of the growth of leukemia CCRF-CEM cells by both compounds parallels their inhibition of CCRF-CEM DHFR. Analogue 3 was a good substrate for human folylpolyglutamate synthetase (FPGS) derived from CCRF-CEM cells (Km 8.5 microM). Further evaluation of the growth inhibitory activity of 3 against the MTX-resistant subline of CCRF-CEM cells (R30dm) with decreased FPGS indicated that poly-gamma-glutamylation was important for its action. Protection studies with 3 in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin [(6R,S-5-formyltetrahydrofolate] or by a combination of thymidine and hypoxanthine, suggesting an antifolate effect directed at DHFR.
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PMID:Effect of bridge region variation on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines. 756 10


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