Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Replacement of the glutamic acid (Glu) moiety in methotrexate (MTX) and aminopterin (AMT) by 2-amino-4-phosphonobutyric acid (APBA) and ornithine (Orn) has been found to give analogs that retain the ability to inhibit dihydrofolate reductase (DHFR) while also displaying high activity against folylpolyglutamate synthetase (FPGS). One of these compounds, the Orn analog of AMT, is the most potent FPGS inhibitor we have found to date. A model to account for the fact that side-chain analogs containing a basic and those containing an acidic terminal group can both competitively inhibit FPGS is proposed. According to this model, binding may involve interaction of an acidic terminal group on the inhibitor with a positively charged active-site residue to which the gamma-carboxyl of the folate-antifolate substrate normally binds. It may also involve the interaction of a basic terminal group on the inhibitor with a different active-site residue which is negatively rather than positively charged and to which the alpha-amino group of the incoming Glu cosubstrate must bind before an amide bond to the gamma-carboxyl of the folate-antifolate can form. The 2 oppositely charged active-site residues assumed to take part in this binding are probably situated near each other and at approximately the same distance from the pteridine-binding site. The ability of compounds to inhibit both DHFR and FPGS makes it possible in principle for such compounds to kill cells via a "self-potentiation" mechanism in which inhibition of tetrahydrofolate synthesis is complemented by interference with the subsequent conversion of tetrahydrofolates to their polyglutamate conjugates. Possible exploitation of this mechanism to overcome MTX resistance is considered.
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PMID:Synthesis and biologic activity of new side-chain-altered methotrexate and aminopterin analogs with dual inhibitory action against dihydrofolate reductase and folylpolyglutamate synthetase. 343 89

A full-length cDNA coding for a mutant dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3), cloned from a mouse fibroblast cell line grown in high concentrations of methotrexate (MTX), was microinjected into mouse embryos to produce transgenic mice. The DHFR cDNA product is 270-fold more resistant to MTX than the wild-type enzyme. Seventeen transgenic mouse lines, identified by Southern blotting of tail or spleen DNA, carried between 1 and 400 copies of the foreign gene per cell. Eight lines have thus far been tested for resistance to MTX. Control mice were treated until death; MTX was withdrawn from transgenic mice when a cumulative MTX dose uniformly fatal for controls was reached. The major site of MTX toxicity was the gastrointestinal tract, with death of controls resulting from fluid and weight loss. Transgenic animals were relatively resistant to these symptoms and tolerated significantly more MTX than control animals. These results show that genes conferring resistance to chemotherapeutic agents can, after transfer into intact organisms, produce systemic drug resistance.
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PMID:Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene. 346 29

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.
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PMID:Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants. 352 62

Dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADPH+-oxidoreductase; EC 1.5.1.3) was partially purified by affinity chromatography from three clones of the human malaria parasite Plasmodium falciparum. The three clones were representative of pyrimethamine-sensitive (clone 3D7) and pyrimethamine-resistant (clone HB3 and clone 7G8) parasites with ID50 values of 0.53 nM (3D7), 210 nM (HB3), and 540 nM (7G8), when tested in vitro against the drug. The specific activities of the partially purified DHFR differed by less than a factor of 2 between the sensitive clone 3D7 (442 +/- 39 nmol min-1 mg-1 protein) and the resistant clones HB3 (634 +/- 25 nmol min-1 mg-1 protein) and 7G8 (565 +/- 85 nmol min-1 mg-1 protein). The number of catalytic sites in partially purified DHFR from the three clones was similar and ranged from 151 to 194 pmol mg-1 protein. The Km value for NADPH was similar in all three clones (4.5-11.6 microM). The Km value for dihydrofolate was altered 13-fold comparing the sensitive clone 3D7 (3.2 +/- 0.6 microM) with the resistant clone HB3 (42.6 +/- 1.6 microM), with the Km for the resistant clone 7G8 falling in between (11.9 +/- 1.2 microM). The inhibition constants for pyrimethamine increased from 0.19 +/- 0.08 nM (3D7) to 2.0 +/- 0.3 nM (HB3) to 8.9 +/- 0.8 nM (7G8). The inhibition by pyrimethamine of the sensitive clone 3D7 was noncompetitive and competitive for the two other clones. The titration of partially purified DHFR with pyrimethamine revealed a 500-fold increase in the concentration of the drug needed to inhibit the DHFR activity by 50%, when the sensitive clone 3D7 (0.18 +/- 0.02 nM) was compared to the resistant clone 7G8 (95 +/- 16 nM). From the comparison of the specific activities and the catalytic center activities with the Km values for the substrate and the inhibition constants for pyrimethamine, both of which are altered in the resistant clones, we conclude that the molecular mechanism for pyrimethamine resistance in the three clones studied is not based on an overproduction of the DHFR but is due to a decreased affinity to antifolates by a structurally altered enzyme.
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PMID:Kinetic and molecular properties of the dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant clones of the human malaria parasite Plasmodium falciparum. 355 92

The term "high molecular weight substances" used in this paper implies mostly nucleic acids of certain molecular weight and protein, and the genetic aspects of anti-cancer chemotherapy were discussed. Studies on the mechanism of action of 5-FU have been focused on the inhibition of DNA synthesis, and it has been reported that 5-FU inhibits the growth of thymidylate synthesis deleted FM3A Thy 21 cells even in the presence of thymidine, and that the level of TTP is equal to that in control cells. On the other hand, the active metabolite of 5-FU, FdUMP, is known to bind to synthesized thymidylate and 5, 10-methylene-tetrahydrofolic acid to form a ternary complex. Recently, the cytocidal effect of 5-FU was observed in thymidylate synthetase-deficient cells in the presence of a sufficient amount of thymidine, suggesting that the cytocidal effect of 5-FU might be caused by inhibition of the RNA pathway. In this laboratory, the effect of 5-FU on polysomal patterns and the incorporation of 3H-UR into polysomes was studied in L1210 cells, but no significant difference was observed between normal and 5-FU-treated cells. Ribosomal RNA extracted from the polysomes of 5-FU treated cells appeared to contain a smaller 28S rRNA in comparison to 18S rRNA, indicating that the processing might be inhibited. Expression of mouse H-2Dd mRNA was not influenced by 5-FU at 10(-5) M up to 6 h. Methotrexate (MTX) has a chemical structure similar to folic acid, and is known to bind to DHFR (dihydrofolate reductase), and inhibit the synthesis of TMP. The cellular PRPP content is known to be increased by MTX, which inhibits purine synthesis. The level of PRPP content was found to be increased approximately 25 fold at 3 h after 10(-6) M MTX although normal bone marrow cells showed no increase even after MTX. This increased level of PRPP thus obtained in cancer cells was thought to be used for the phosphorylation of 5-FU. Clinically, sequential chemotherapy using MTX and 5-FU was employed successfully for solid tumors on the basis of the experimental evidence. In order to minimize the adverse effects of anti-cancer drugs, a technique involving the incorporation of the drug-resistant gene into normal bone marrow cells has been designed in this laboratory, and the MTX-resistant cells thus obtained are transplanted into the tumor-bearing mice.
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PMID:[The metabolism of high-molecular-weight substances in cells and the effect of anticancer drugs]. 356 94

The use and metabolism of folates by leishmanias have been studied by assessing the growth of promastigotes in defined media with different folates and the cell content of folate-metabolising enzymes. The folates present in Leishmania mexicana mexicana have been determined using HPLC. Folic acid, 5-formyltetrahydrofolate (THF) and 5-methyl-THF each supported growth of L. m. mexicana promastigotes in defined medium, whereas the parasites did not survive in the absence of folates; p-aminobenzoic acid could not replace the folate requirement. The only folate present at detectable levels in L. m. mexicana promastigotes was 5-methyl-THF. Dihydrofolate reductase (EC 1.5.1.3), methylene-THF reductase (EC 1.1.1.68), serine hydroxymethyltransferase (EC 2.1.2.1) and thymidylate synthetase (EC 2.1.1.45) were all detected in extracts of promastigotes of L. m. mexicana, L. donovani and L. major. Some of these activities were also found in extracts of amastigotes of the former two species. The enzymes of L. m. mexicana have been partially characterised. Methylene-THF reductase may be involved in the conversion in vivo of 5-methyl-THF to 5,10-methylene-THF.
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PMID:Folate utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes. 357 56

Cis-diamminediaquaplatinum(II)-ion, the biologically active form of the anticancer agent Cisplatin, reacted readily with tetrahydrofolate at pH 7 and 37 degrees C to produce a stable complex. The reaction was monitored spectrophotometrically by the change in absorbance maximum from 298 nm (tetrahydrofolate) to 275 nm (complex); occurrence of isobestic points at 282 and 327 nm indicated that a single product was formed. Purity of platinum-tetrahydrofolate, after isolation in ca. 70% yield, was established by TLC and HPLC. Elemental analysis, absorbance spectra at various pH values and nmr spectra provided evidence that the diammine platinum moiety was bridged across the N-5 and N-10 positions of tetrahydrofolate. Complexation also occurred with 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, Methotrexate and aminopterin, but not with folate or 7,8-dihydrofolate. Biological implications of these observations have been investigated. Intracellular folates in L1210 cells have been identified and quantitated via reverse phase HPLC (C18 column; tetrabutylammonium phosphate as the pairing ion) and changes in the levels of these compounds, after exposure of cells to Cisplatin, have been measured. Platinum derivatives of tetrahydrofolate or other reduced folates were not found, but there was a decrease in the level of 5,10-methenyltetrahydrofolate, accompanied by an increase in 5-formyl and 10-formyltetrahydrofolate (and perhaps tetrahydrofolate). The chemical interaction of the diaqua form of Cisplatin with Methotrexate resulted in decreased uptake of the latter by L1210 cells. The platinum complex of tetrahydrofolate was a reasonably good inhibitor (Ki = 4 microM) of L1210 dihydrofolate reductase and of the folate transport system (50% inhibition at ca. 200 microM) of L1210 cells.
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PMID:Platinum-folate compounds: synthesis, properties and biological activity. 367 4

Several fragments of the human dihydrofolate reductase gene (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate NADP+ oxidoreductase, EC 1.5.1.3) were isolated from gene-amplified KB7B cells and characterized. Recombinant plasmids containing intron sequences were constructed. Probes prepared from these plasmids were tested for dihydrofolate reductase precursor mRNA specificity via solution hybridization studies and Northern blot analysis. One probe, p0.69EH, was shown to be specific for dihydrofolate reductase RNA by its greatly enhanced level of hybridization with total RNA from dihydrofolate reductase gene-amplified versus non-amplified cells. In addition, solution hybridization studies with various classes of RNA and Northern blot analysis revealed that p0.69EH hybridizes predominantly with polyadenylated, high molecular weight, nuclear RNA species. Subsequent solution hybridization studies revealed a disproportionate 5-fluorouracil-induced increase in dihydrofolate reductase intron-containing RNA over dihydrofolate reductase mRNA. These results suggest that 5-fluorouracil incorporation into RNA may inhibit the conversion of precursor mRNA to mature mRNA.
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PMID:5-Fluorouracil augmentation of dihydrofolate reductase gene transcripts containing intervening sequences in methotrexate-resistant KB cells. 371 5

Exposure to nitrous oxide interferes selectively with the coenzyme function of vitamin B12 and causes inactivation of methionine synthetase, with subsequent impairment of folate metabolism and reduction of cellular proliferation. In a rat leukemia model (BNML) we investigated the combined administration of nitrous oxide, inactivating vitamin B12, and methotrexate (MTX), a folate antagonist inhibiting the enzyme dihydrofolate reductase. Through different mechanisms, both agents decrease the availability of tetrahydrofolate, and subsequently of other reduced folates, with increased impairment of folate-dependent synthesis of thymidylate. Effects on leukemic growth and on hematological values in rats demonstrated enhancement of the therapeutic effect of MTX by exposure to nitrous oxide. With several treatment schedules, the results of combined treatment were seen to be better than additive when compared with the effects of single agents. In particular, pretreatment of leukemic rats with nitrous oxide for 3 days before administration of MTX appeared effective. With higher doses of MTX, concomitant exposure to nitrous oxide even resulted in toxic effects. These findings were in accordance with the results of some metabolic studies performed in leukemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enhanced therapeutic effect of methotrexate in experimental rat leukemia after inactivation of cobalamin (vitamin B12) by nitrous oxide. 371 92

The self-association in aqueous solution of folic acid (FA), 7,8-dihydrofolic acid (DHFA) and 5,6,7,8-tetrahydrofolic acid (THFA) has been studied by the use of proton magnetic resonance (1H NMR) spectroscopy. At concentrations below 10 mM, all three folates exist in (monomer)2 in equilibrium dimer equilibria with association constants (Ka) equal to 400, 66 and 14 M-1 for FA, DHFA and THFA respectively. These values decreased markedly to 157, 18 and 3 M-1, for FA, DHFA and THFA respectively, in the presence of 0.8 M KCl. The high extent of dimerization of FA is believed to impede the interaction with the active site of dihydrofolate reductase (DHFR) rendering it a poor substrate. In contrast, the DHFA with a much lower Ka is a better substrate. Conditions that lower the Ka of both FA and DHFA, (i.e., 0.8M KCl) turn them into better substrates. Based on the findings of the present study, it is also predicted that dihydro MTX may be a better inhibitor of DHFR than MTX.
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PMID:Association of folate molecules as determined by proton NMR: implications on enzyme binding. 383 76


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