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:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neisseria gonorrhoeae dihydrofolate reductase undergoes a time-dependent, irreversible inactivation by 2,4-diamino-5-[3,5-dimethoxy-4-(p-bromoacetamidophenoxy)benzyl] pyrimidine. The kinetics of inactivation are consistent with the reversible formation of an enzyme-inhibitor complex followed by covalent binding to the enzyme. The reversible component is competitive with dihydrofolate and has an inhibitor binding constant of 10 nM. Irreversible inactivation proceeds as a pseudo first-order process with a minimum inactivation half-time of 20 min and a Ki of 28 nM. Using radiolabeled inhibitor, it was shown that approximately 1 mol of ligand was covalently bound to the enzyme/mol of methotrexate binding site when the enzyme was completely inhibited. Radiolabeled inhibitor remained associated with the enzyme following denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide cleavage of the 14C-labeled enzyme-inhibitor complex yielded only one radioactive polypeptide, and sequence determinations showed that His-25 was modified by covalent attachment of the inhibitor. When dihydrofolate reductases from Lactobacillus casei, Streptococcus faecium, Escherichia coli, SR-1 rodent lymphoma, and chicken liver were tested with the affinity label, only the L. casei enzyme showed a time-dependent increase in inhibition. These data, along with comparisons of known amino acid sequences and x-ray crystal structures, were used to make predictions concerning the three-dimensional conformation of the gonococcal enzyme.
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PMID:Species-specific irreversible inhibition of Neisseria gonorrhoeae dihydrofolate reductase by a substituted 2,4-diamino-5-benzylpyrimidine. 643 42

Levels of mRNA for the enzyme dihydrofolate reductase (EC 1.5.1.3) were determined in growth-stimulated 3T6 cells which contained wild-type dosage of the gene coding for this enzyme. As in the case of methotrexate-resistant cells having highly amplified levels of genes for dihydrofolate reductase, an increase in dihydrofolate reductase mRNA by a factor of 2-4 can be determined when cells enter the S phase. This increase is inhibited by sodium butyrate (which inhibits growth-stimulated 3T6 cells in mid G1 phase) but not by hydroxyurea (which inhibits in early S phase). We conclude that with the available methods it is possible to study the regulation of S phase-specific enzymes after growth stimulation at the level of the mRNA, even if gene amplification is not possible or desirable.
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PMID:Increased levels of dihydrofolate reductase mRNA can be measured in normal, growth-stimulated mouse fibroblasts. 668 60

The chemical synthesis of 11-oxahomoaminopterin (1) has been carried out using procedures which were also found to be applicable to the synthesis of 11-oxahomofolic acid (2). Reaction of 1-bromo-4-[p-(caarbomethoxy)phenoxy]-2-butanone (10) with sodium azide gave 1-azido-4-[p-(carbomethoxy)phenoxy]-2-butanone (11). Protection of the carbonyl group of 11 as the ethylene ketal and subsequent base hydrolysis of the product gave 1-azido-4-(p-carboxyphenoxy)-2-butanone ketal (13). The glutamate conjugate 14 was prepared from 13 by the isobutyl chloroformate method and was hydrogenated to diethyl N-[(alpha-amino-2-oxo-4-butanoyl)-p-anisoyl]-L-glutamate ketal (15). Reaction of 15 with 6-chloro-2,4-diamino-5-nitropyrimidine (16) and 2-amino-6-chloro-4-hydroxy-5-nitropyrimidine (17) and deprotection of the corresponding products gave the intermediates 18 and 19, which were elaborated to 1 and 2 using a series of steps involving deprotection, dithionite reduction, cyclization, oxidation, and hydrolysis. Although 11-oxahomoaminopterin showed antifolate activity against two folate-requiring microorganisms and inhibited Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound 1 was also tested for its ability to be transported via the methotrexate transport system using the L-1210 and Ehrlich tumor cell lines, and these results are compared with those of related analogues. The growth inhibitory activity of 1 in the L-1210 cell lines in culture was found to be 15 times weaker than that of methotrexate.
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PMID:Folate analogues altered in the C9-N10 bridge region. 18. Synthesis and antitumor evaluation of 11-oxahomoaminopterin and related compounds. 679 26

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.
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PMID:Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase. 716 15

We have studied a line of 3T6 mouse embryo fibroblasts grown in progressively increasing concentrations of methotrexate. Resistance of cells to low concentrations of the inhibitor (less than 5 X 10(-5) M) is attributed to selective multiplication of the genes coding for dihydrofolate reductase and the resulting elevation of enzyme content. Cells isolated at a higher methotrexate concentration (4 X 10(-4) M) contain high levels of a dihydrofolate reductase with a reduced affinity for methotrexate. The altered dihydrofolate reductase exhibits a 270-fold reduction in binding affinity for methotrexate as measured by equilibrium dialysis (Kd = 5.4 X 10(-8) M versus 2 X 10(-10) M for the wild type enzyme). While binding to NADPH is unchanged, the Km for dihydrofolate is increased 3-fold over wild type enzyme and the turnover number for the reduction of dihydrofolate to tetrahydrofolate is decreased 20-fold. The altered dihydrofolate reductase shows a broader and more predominant acidic peak in its pH profile for this reaction. The molecular weights of the altered and wild type enzymes are identical as determined by sodium dodecyl sulfate-gel electrophoresis, but 2-dimensional electrophoresis reveals a significant basic shift in the migration of the altered enzyme. Studies with various folic acid analogs suggest that modifications involving the para-aminobenzoyl moiety of the inhibitor molecules are associated with the most dramatic differential binding between the altered and wild type dihydrofolate reductases.
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PMID:Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. 728 96

A comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins synthesized by antifolate-sensitive (DC-3F) and -resistant (DC-3F/A3 and DC-3F/MQ19) Chinese hamster lung fibroblast cells has shown that an apparent molecular weight difference exists between the dihydrofolate reductases overproduced by the resistant sublines. Translation in a rabbit reticulocyte lysate system of polysomal and cytoplasmic mRNAs from the three sublines has shown that the overproduction of dihydrofolate reductase by resistant cells is accompanied by increased levels of dihydrofolate reductase-specific mRNA and that the different molecular weight forms of dihydrofolate reductase are encoded by their respective mRNAs.
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PMID:Antifolate-resistant chinese hamster cells. Evidence from independently derived sublines for the overproduction of two dihydrofolate reductases encoded by different mRNAs. 735 12

Methotrexate has been approved for the treatment of refractory rheumatoid arthritis by several regulatory agencies, including the Food and Drug Administration. The tendency is now to prescribe it at earlier stages of the disease. Methotrexate is a well known antifolate. Its exact mechanism of action in rheumatoid arthritis remains uncertain. The polyglutamated derivatives of methotrexate are potent inhibitors of various enzymes, including dihydrofolate reductase and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase. Inhibitory effects on cytokines, particularly interleukin-1, and on arachidonic acid metabolism, as well as effects on proteolytic enzymes, have been reported. Some of them may be linked to the antifolate properties of methotrexate. Overall, the drug appears to act in rheumatoid arthritis as an anti-inflammatory agent with subtle immunomodulating properties. Direct inhibitory effects on rapidly proliferating cells in the synovium have also been suggested. Methotrexate is usually given orally. Marked interindividual variation in its bioavailability has been found. Food intake has no significant effect on the pharmacokinetics of oral methotrexate. Methotrexate undergoes significant metabolism. The functionally important metabolites are the polyglutamated derivatives of methotrexate, which are selectively retained in the cells. Less than 10% of a dose of methotrexate is oxidised to 7-hydroxy-methotrexate, irrespective of the route of administration. This metabolite is extensively (91 to 93%) bound to plasma proteins, in contrast to the parent drug (35 to 50% bound). Methotrexate is mainly excreted by the kidneys. It undergoes tubular secretion and may thereby compete with various organic acid compounds. Early placebo-controlled trials demonstrated that weekly low dosage methotrexate produced early symptomatic improvement in most rheumatoid arthritis patients. Two meta-analyses showed that methotrexate is among the most efficacious of slow-acting antirheumatic agents, together with parenteral gold (sodium aurothiomalate), penicillamine and sulfasalazine. Furthermore, in the short term context of clinical trials, methotrexate has one of the best efficacy/toxicity ratios. There is little evidence that methotrexate, or any available slow-acting antirheumatic agent, is a true disease-modifying drug. However, the probability that a patient will continue methotrexate therapy over time appears quite favourable compared with any other slow-acting antirheumatic drug. Combination therapy with slow-acting drugs has been advised for the management of rheumatoid arthritis, but the evidence currently available does not support general use of combination therapy including methotrexate. Almost all investigations indicated that toxic effects, rather than lack of response, were the major reason for discontinuing methotrexate therapy.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Methotrexate in rheumatoid arthritis. An update. 751 Jun 20

Thymidylate synthetase (TS) is the only enzyme that catalyzes the formation of thymidine nucleotides in Angiostrongylus cantonensis. A fraction enriched in TS was obtained from the gravid nematode by gel filtration and affinity chromatography using methotrexate-agarose. TS, which was well separated from dihydrofolate reductase, has a relative molecular mass of 66 kDa. By electrophoresis in sodium dodecyl sulphate gel, a major protein band corresponding to 31 kDa was observed. This band was shown to be TS by comparing the electrophoretic mobility with an enzyme preparation bound with [6-3H]5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). Therefore, the enzyme is composed of two identical or very similar subunits. Velocity studies and product inhibition patterns revealed that the TS reaction undergoes a sequential mechanism in which 2'-deoxyuridine 5'-monophosphate (dUMP) is the first substrate added to the active site and thymidine 5'-monophosphate is the last product released. The apparent Km values for dUMP and 5,10-methylenetetrahydrofolate are 10 and 185 microM, respectively. FdUMP and trimethoprim inhibited the parasite TS competitively with dUMP and the Ki values of 23.5 nM and 852 microM, respectively. Methotrexate was a noncompetitive inhibitor of TS. At 0.2 mM 5,10-methylenetetrafolate, 1 mM methotrexate inhibited the activity by 74%.
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PMID:Angiostrongylus cantonensis: characterization of thymidylate synthetase. 800 63

The toxicology and safety pharmacology of brodimoprim (Ro 10-5970), a new dihydrofolate reductase inhibitor for antimicrobial chemotherapy, are reported. The toxicity is compared to trimethoprim and other dihydrofolate reductase inhibitors of the trimethoprim type. In safety pharmacological tests brodimoprim showed no significant activity in the cardiovascular, autonomic neuroeffector or central nervous systems. Effects on urine volume and sodium and potassium excretion were not noted. The acute toxicity of brodimoprim after oral administration is low. In repeated dose studies in rats, oral doses up to 50 mg/kg/day were generally well tolerated. In the baboon no toxic effects were seen even at repeated doses up to 150 mg/kg/day; on the other hand the dog was found to be particularly sensitive to treatment as 20 mg/kg/day were poorly tolerated. Target organ systems included central nervous system, liver, red blood cell parameters and thyroid gland. Doses up to 100 mg/kg/day were not teratogenic or embryotoxic in rabbits. However, treatment with 100 mg/kg/day in reproductive toxicity studies in rats induced effects on fetal/litter weight, survival of offspring and litter size and increased the incidence of skeletal variations and malformed offspring. No mutagenicity was found in the tests performed with brodimoprim. In comparison brodimoprim shows the typical toxicity known for trimethoprim and analogs. In pyrimethamine toxic effects occurred at considerably lower doses than in the other compared compounds.
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PMID:Preclinical toxicology and safety pharmacology of brodimoprim in comparison to trimethoprim and analogs. 819 31

A series of eight previously undescribed 2,4-diaminothieno[2,3-d]pyrimidine analogues of the potent dihydrofolate reductase (DHFR) inhibitors trimetrexate (TMQ) and piritrexim (PTX) were synthesized as potential drugs against Pneumocystis carinii and Toxoplasma gondii, which are major causes of severe opportunistic infections in AIDS patients. 2,4-Diamino-5-methyl-6-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 3,4,5-trimethoxy or 2,5-dimethoxy substitution in the aryl/aralkyl moiety and 2,4-diamino-5-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 2,5-dimethoxy substitution in the aryl/aralkyl moiety were obtained by reaction of the corresponding 2-amino-3-cyanothiophenes with chloroformamidine hydrochloride. The aryl group in the 5,6-disubstituted analogues was either attached directly to the hetero ring or was separated from it by one or two carbons, whereas the aryl group in the 5-monosubstituted analogues was separated from the hetero ring by two or three carbons. 2-Amino-3-cyano-5-methyl-6-(aryl/alkyl)thiophene intermediates for the preparation of the 5,6-disubstituted analogues were prepared from omega-aryl-2-alkylidene-malononitriles and sulfur in the presence of a secondary amine, and 2-amino-3-cyano-4-(aryl/aralkyl)thiophene intermediates for the preparation of the 5-monosubstituted analogues were obtained from omega-aryl-1-chloro-2-alkylidenemalononitriles and sodium hydrosulfide. Synthetic routes to the heretofore unknown ylidenemalononitriles, and the ketone precursors thereof, were developed. The final products were tested in vitro as inhibitors of DHFR from Pneumocystis carinii, Toxoplasma gondii, rat liver, beef liver, and Lactobacillus casei. A selected number of previously known 2,4-diaminothieno[2,3-d]pyrimidines lacking the 3,4,5-trimethoxyphenyl and 2,5-dimethoxyphenyl substitution pattern of TMQ and PTX, respectively, were also tested for comparison. None of the compounds was as potent as TMQ or PTX, and while some of them showed some selectivity in their binding to Pneumocystis carinii and Toxoplasma gondii versus rat liver DHFR, this effect was not deemed large enough to warrant further preclinical evaluation.
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PMID:2,4-Diaminothieno[2,3-d]pyrimidine analogues of trimetrexate and piritrexim as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. 823 96


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