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)

Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.
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PMID:Amplification of protein expression in a cell free system. 128 16

The effects of methotrexate (inhibiting dihydrofolate reductase) and nitrous oxide (inactivating methionine synthase) on intracellular folate coenzyme levels of leukemic cells were studied. Blast cells from 10 cases of acute myeloid leukemia (AML) and 5 cases of acute lymphoid leukemia (ALL) were incubated with 5 x 10(-8) M [3H] 5-formyltetrahydrofolate (5-formylTHF) for 18 h to label intracellular folate pools, which were subsequently quantitated by high performance liquid chromatography (HPLC). In AML, 5-methylTHF made up 53% of the total folate pool followed by 10-formylTHF (26%), 5-formylTHF (10%), THF (9%) and DHF (1%). Cells from ALL differed from AML (p less than 0.05) with respect to 10-formylTHF (17%) and DHF (10%). Exposure to nitrous oxide (8 h) caused an equal decrease of 10-formylTHF and 5-formylTHF in both AML (30%) and ALL (45%), whereas 5-methylTHF increased (130%). Methotrexate (4 h, 10(-6) M) caused an accumulation of DHF and a decrease of 5-methylTHF in both AML (32%) and ALL (12%). A specific reduction of the 10-formylTHF (50%) and 5-formylTHF (25%) pools was noticed in ALL. Exposure to nitrous oxide prior to methotrexate treatment aggravated the reduction of 10-formylTHF and 5-formylTHF presumably by impaired replenishment from the 5-methylTHF pool. In conclusion, this study demonstrates a significant difference in folate coenzyme distribution between cells from AML and ALL. Moreover it is shown that nitrous oxide and methotrexate treatment of leukemic cells cause an accumulation of 5-methylTHF and DHF respectively at the expense of other folate forms. The presence of substantial amounts of DHF in cells from ALL together with the specific reduction of 10-formylTHF (necessary for purine synthesis) during MTX treatment may in part explain the efficacy of methotrexate in the treatment of ALL.
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PMID:Effect of nitrous oxide and methotrexate on folate coenzyme pools of blast cells from leukemia patients. 201 7

The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.
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PMID:Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid. 229 24

5-Fluorouracil (5-FU)-resistant L1210 cell line (L1210/5-FU-1) was established in this laboratory, and maintained by serial passage in the peritoneal cavities of BDF1 mice. This and another 5-FU-resistant cell line (L1210/5-FU-2) showed approximately 50-fold increase in resistance to 5-FU, i.e., IC50 of 5-FU determined for wild type L1210 cells was 3 x 10(-7) M, whereas those for 5-FU-resistant lines, L1210/5-FU-1 and L1210/5-FU-2 were 1.65 x 10(-5) M and 1.35 x 10(-5) M, respectively. The incorporation of 3H-5-FU into L1210/5-FU-1 cells was about 57% of that observed in wild type L1210 cells. Northern blot analysis of DHFR mRNA obtained from 5-FU-sensitive and -resistant cell lines revealed four distinct bands of 1.6 kb, 1.2 kb, 1.0 kb and 0.75 kb in length. Although all these bands showed higher density in autoradiography in 5-FU-resistant lines than in wild type, no extra band was observed. Southern blot analysis of DHFR DNA, digested with the restriction enzymes, EcoRI, BamHI or HindIII, revealed no rearrangement. However, all the fragments were expanded, showing that DHFR gene increased in 5-FU-resistant cells. The karyotype analysis carried out for L1210/5-FU-1 showed abnormal banding region in a part of chromosome X, and this chromosomal aberration was considered to be the reflection of the amplification of DHFR gene. Many investigators have reported that thymidylate synthetase (TS), a target enzyme for 5-FU, increased in 5-FU-resistant cells and that the increase of TS was responsible for the drug resistance to 5-FU. The increase both in DHFR mRNA and DHFR DNA suggested the increase in DHFR and also in N5, N10 methylenetetrahydrofolate (methylene THF), a coenzyme of TS. The increase of methylene THF, together with the increase of TS, might result in the resistance of the cells to 5-FU.
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PMID:The changes in the levels of dihydrofolate reductase mRNA and its gene dosage in 5-fluorouracil-resistant L1210 cells. 239 54

Folic acid metabolism in eukaryotic cells consists of a network of enzymatic reactions in which 1-carbon (C1) units at three different oxidation states are 1) interconverted while linked to the 5- and/or 10-positions of tetrahydrofolate, or 2) added to, or taken from, tetrahydrofolate. Particularly important in the latter category are reactions involving C1-tetrahydrofolate adducts in the synthesis of inosinate, thymidylate, serine, and methionine. Tetrahydrofolate, a central component of the network, can be generated from: 1) folate, via the NADPH-dependent dihydrofolate reductase; 2) 5-methyltetrahydrofolate via the methyl B12-dependent methionine synthetase; or 3) 5-formyltetrahydrofolate via a sequence of reactions beginning with the ATP-dependent isomerization to 5,10-methenyltetrahydrofolate or via transfer of the formyl group to glutamate. Because of the close relationship of folic acid metabolism to cell replication, folate-dependent enzymes provide excellent targets for cancer chemotherapy. This potential has not yet been realized, however, except for dihydrofolate reductase and thymidylate synthetase, which are strongly inhibited by the anti-cancer agents methotrexate (MTX) and FUra. The following enzymes are particularly attractive as targets for future exploitation in chemotherapy: 1) the two transformylases involved in purine nucleotide synthesis, 2) serine hydroxymethyltransferase, 3) methionine synthetase, and 4) methylenetetrahydrofolate dehydrogenase. Suggestions are also made for the development of new agents based upon a strategy of enzyme-targeted chemotherapy.
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PMID:Folic acid metabolism and its disruption by pharmacologic agents. 312 3

The title compounds were prepared in extensions of a general synthetic approach used earlier to prepare 5-alkyl-5-deaza analogues of classical antifolates. Wittig condensation of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (2a) and its 5-methyl analogue 2b with [4-(methoxycarbonyl)benzylidene] triphenylphosphorane gave 9,10-ethenyl precursors 3a and 3b. Hydrogenation (DMF, ambient, 5% Pd/C) of the 9,10-ethenyl group of 3b followed by ester hydrolysis led to 4-[2-(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)ethyl]ben zoi c acid (5), which was converted to 5-methyl-5,10-dideazaaminopterin (6) via coupling with dimethyl L-glutamate (mixed-anhydride method using i-BuOCOCl) followed by ester hydrolysis. Standard hydrolytic deamination of 6 gave 5-methyl-5,10-dideazafolic acid (7). Intermediates 3a and 3b were converted through concomitant deamination and ester hydrolysis to 8a and 8b. Peptide coupling of 8a,b (using (EtO)2POCN) with diesters of L-glutamic acid gave intermediate esters 9a and 9b. Hydrogenation of both the 9,10 double bond and the pyrido ring of 9a and 9b (MeOH-0.1 N HCl, 3.5 atm, Pt) was followed by ester hydrolysis to give 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (11a) and the 5-methyl analogue 11b. Biological evaluation of 6, 7, 11a, and 11b for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for growth inhibition and transport characteristics toward L1210 cells revealed 6 to be less potent than methotrexate in the inhibition of DHFR and cell growth. Compounds 6, 11a, and 11b were transported into cells more efficiently than methotrexate. Growth inhibition IC50 values for 11a and 11b were 57 and 490 nM, respectively; the value for 11a is in good agreement with that previously reported (20-50 nM). In tests against other folate-utilizing enzymes, 11a and 11b were found to be inhibitors of glycinamide ribonucleotide formyltransferase (GAR formyltransferase) from one bacterial (Lactobacillus casei) and two mammalian (Manca and L1210) sources with 11a being decidedly more inhibitory than 11b. Neither 11a nor 11b inhibited aminoimidazolecarboxamide ribonucleotide formyltransferase. These results support reported evidence that 11a owes its observed antitumor activity to interference with the purine de novo pathway with the site of action being GAR formyltransferase.
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PMID:Synthesis and antifolate activity of 5-methyl-5,10-dideaza analogues of aminopterin and folic acid and an alternative synthesis of 5,10-dideazatetrahydrofolic acid, a potent inhibitor of glycinamide ribonucleotide formyltransferase. 318 24

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

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

The kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been investigated by using progress curve, initial velocity, product inhibition, and dead-end inhibition studies as well as isotope effects. The results indicate that the reaction conforms to a random mechanism involving two dead-end complexes, viz., enzyme-DHF-THF and enzyme-NADP-DHF. At higher concentrations, DHF causes substrate inhibition by combining at the NADPH binding site on the enzyme. The steady-state velocity data can be analyzed adequately on the basis that rapid-equilibrium conditions apply. However, this can be only an approximate description of the reaction since the isotope effects observed with NADPD demonstrate clearly that catalysis cannot be rate limiting at pH 7.4. The choice of conditions for analysis of progress-curve data is discussed in the Appendix.
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PMID:Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli. 675 19

Previously, 8-deazafolic acid (17) was shown to be a potent inhibitor of the folate-dependent bacteria, Streptococcus faecium (ATCC 8043) and Lactobacillus casei (ATCC 7469), and to have activity against lymphoid leukemia L1210 in mice. To examine the 5,6,7,8-tetrahydro derivatives, a new synthesis of 17 was developed from 8-deaza-2,4-dichloro-6-methylpteridine. Treatment of the latter with aqueous base gave the corresponding pteridin-4(3H)-one, which was aminated with ammonia to give 8-deaza-6-methylpterin (9). Bromination of 9 gave mainly 8-deaza-6-(tribromomethyl)pterin, which on reaction with p-aminobenzoyl-L-glutamic acid resulted in the formation of the 9-oxo derivative of 17. In contrast, bromination of the 2-acetyl derivative of 9 gave mainly the corresponding 6-(bromomethyl)pterin, which was converted to 17 in 23% yield (from 9). Hydrogenation of 17 at atmospheric pressure and room temperature was unsuccessful either in a basic medium or formic acid. In trifluoroacetic acid, overreduction occurred to give a mixture containing 8-deaza-5,6,7,8-tetrahydro-6-methylpterin and the 5,6,7,8-tetrahydro derivative of 17. The latter was characterized by conversion to the methenyl analogue 21, which was also prepared by hydrogenation of the 10-formyl derivative of 17. Treatment of 21 with hydroxide gave 8-deaza-10-formyl-5,6,7,8-tetrahydrofolic acid. Compound 21 showed cytotoxicity to cultured H.Ep.-2 cells and was tested as an inhibitor of bovine dihydrofolic reductase. Lineweaver-Burk analysis indicated inhibition competitive with dihydrofolate.
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PMID:New synthesis of N-[4-[[(2-amino-4(3H)-oxopyrido[3,2-d]pyrimidin-6-yl)methyl]amino]benzoyl]-L-glutamic acid (8-deazafolic acid) and the preparation of some 5,6,7,8-tetrahydro derivatives. 694 61


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