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)

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.
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PMID:Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies. 23 91

A number of homologous 2,4-diaminocycloalka[g]pteridines varying in ring size from 5 to 15 were prepared by (a) condensation of aminomalononitrile tosylate with alpha-oximinocycloalkanones, deoxygenation of the resulting 2-amino-3-cyanocycloalka[b]pyrazine 1-oxides, and guanidine cyclization; (b) guanidine cyclization of the above pyrazine 1-oxides to give 2,4-diaminocycloalka[g]pteridine 8-oxides, followed by deoxygenation; or (c) condensation of 2,4,5,6-tetraaminopyrimidine with a cycloalka-1,2-dione (for the cyclohepta- and cycloocta[g]pteridines only). These compounds were examined for their activity as dihydrofolate reductase inhibitors against Lactobacillus casei, rat liver, L1210, and Trypanosoma cruzi. Activity was found to depend upon ring size, with the greatest activity exhibited by the cyclododeca derivatives 31.
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PMID:Pteridines. 41. Synthesis and dihydrofolate reductase inhibitory activity of some cycloalka[g]pteridines. 41 35

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.
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PMID:Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle. 133 Oct 37

The point mutation at nucleotide 323 within the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum, which distinguishes pyrimethamine-sensitive from drug-resistant isolates, can be discriminated by the polymerase chain reaction using mutation-specific primers. The technique makes use of the principle that short oligonucleotides with a perfect match at their 3' ends, complementary to the mutation to be detected, will initiate the polymerization by Taq polymerase far more efficiently than primers with a single mismatch in this position. The Asn-108 codon was detected using a primer of 17 nucleotides with an adenosine at its 3' end, the Thr-108 codon with a 14-mer primer ending with a cytosine and the Ser-108 codon with a 16-mer containing guanidine at the critical 3' end. By selecting appropriate counterprimers, the size of the amplification products is either indicative of pyrimethamine-resistant parasites of the 7G8 type, or of pyrimethamine-sensitive parasites of the FCR-3 type or 3D7 type. The fragments obtained can be easily separated in a single lane after agarose gel electrophoresis. Coded P. falciparum DNA samples were typed unambiguously using these primers as were reconstituted parasitized blood samples stored as high salt lysates. Sensitivity, speed and specificity make this assay a realistic alternative to in vitro drug testing to monitor the resistance of P. falciparum to inhibitors of the dihydrofolate reductase.
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PMID:Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction. 218 8

Synthesis of the 10-methyl and 10-ethyl analogues of 5,10-dideazatetrahydrofolic acid (DDTHF), a potent inhibitor of glycinamide ribotide (GAR) formyltransferase, is reported. Key intermediates in the process were 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acid. Condensation of the piperidine enamines of branched 4-(p-carbomethoxyphenyl)butyraldehydes with (acetoxymethylene)malononitrile afforded 1,1-dicyano-4-piperidinobutadiene 5a,b. Subsequent reaction with alcoholic ammonium hydroxide yielded the appropriately substituted 2-amino-3-cyanopyridines 6a,b. Ring closure with guanidine gave 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acids (7a,b). Coupling with diethyl glutamate followed by ester hydrolysis afforded 10-alkyl-5,10-dideazaminopterin analogues 9a,b. Hydrolysis of the 4-amino group of 7a,b yielded the 10-alkylpteroic acids, which were coupled with diethyl glutamate, hydrogenated over PtO2, and saponified to afford 10-alkyl-5,10-dideazatetrahydrofolic acids 13a,b. Aminopterin analogues 9a,b were effective inhibitors of DHFR derived from L1210, but were less potent than methotrexate for inhibition of growth of L1210 in culture. The 10-ethyl (13b) analogue of 5,10-DDTHF was about twice as potent an inhibitor of L1210 cell growth as 5,10-DDTHF, but was only 1/7 as potent for inhibition of GAR formyltransferase. 10-Methyl analogue 13a was similar in potency to 5,10-DDTHF. All of the compounds showed moderately improved transport into L1210 cells relative to methotrexate.
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PMID:Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogues of methotrexate and tetrahydrofolic acid. 229 33

Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.
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PMID:Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid. 242 90

A series of nine 2,4-diamino-5-[6-( or 7-)quinolylmethyl]pyrimidines has been prepared by condensations of quinolinecarboxaldehydes with beta-anilinopropionitriles, followed by treatment with guanidine. All compounds has basic or methoxy substituents at the 2- or 4-positions of the quinoline ring. All of the 6-quinolylmethyl derivatives were highly inhibitory against Escherichia coli dihydrofolate reductase (DHFR), provided that an 8-substituent was present in the quinoline ring. Those compounds that had basic substituents in the 2-position of the quinoline ring were also highly specific for bacterial dihydrofolate DHFR, relative to a vertebrate counterpart. Protonation on the quinoline ring nitrogen is a possible cause of specificity.
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PMID:2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 11. Quinolylmethyl analogues with basic substituents conveying specificity. 250 33

Substitution of cysteine for proline-39 in Escherichia coli dihydrofolate reductase by oligonucleotide-directed mutagenesis positions the new cysteine adjacent to already existing cysteine-85. When the mutant protein is expressed in the E. coli cytosol, the cysteine sulfur atoms are found, by X-ray crystallographic analysis, to be in van der Waals contact but not covalently bonded to one another. In vitro oxidation by dithionitrobenzoate results in formation of a disulfide bond between residues 39 and 85 with a geometry close to that of the commonly observed left-handed spiral. Comparison of 2.0-A-refined crystal structures of the oxidized (cross-linked) and reduced (un-cross-linked) forms of the mutant enzyme shows that the conformation of the enzyme molecule was not appreciably affected by formation of the disulfide bond but that details of the molecule's thermal motion were altered. The disulfide-cross-linked enzyme is at least 1.8 kcal/mol more stable with respect to unfolding, as measured by guanidine hydrochloride denaturation, than either the wild-type or the reduced (un-cross-linked) mutant enzyme. Nevertheless, the cross-linked form is not more resistant to thermal denaturation. Moreover, the appearance of intermediates in the guanidine hydrochloride denaturation profile and urea-gradient polyacrylamide gels indicates that the folding/unfolding pathway of the disulfide-cross-linked enzyme has changed significantly.
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PMID:An engineered disulfide bond in dihydrofolate reductase. 330 20

We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase complex of P. falciparum in Escherichia coli, by constructing a gene with synthetic oligonucleotides that changed the gene's codon usages. The induced expression in an E. coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein. The product was precipitated in an inclusion body in a cell. Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl. Recombinant DHFRs with Ser or Thr at position 108 were prepared. Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108.
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PMID:Purification and characterization of dihydrofolate reductase of Plasmodium falciparum expressed by a synthetic gene in Escherichia coli. 800 23

Novel antifolates with a 6-5 fused ring system, 6,7-dihydrocyclopenta [d]pyrimidine, (3a,b and 4a,b) were synthesized on the basis of combined modification of the heterocycle and bridge regions of the folate molecule. The synthetic method involves (1) synthesis of key intermediates of tert-butyl 4-[omega-(2-substituted-3-oxocyclopentanyl) alkyl]benzoates (8a,b and 9a,b) by a carbon-carbon radical coupling of tert-butyl 4-(omega-iodoalkyl)benzoates (7a,b) with 2-substituted-2-cyclopenten-1-ones (5 and 6) utilizing tributyltin hydride, (2) cyclization of either the methyl enol-ethers derived from the 2-cyanocyclopentanones (8a,b) or the 2-(methoxycarbonyl)cyclopentanones (9a,b) themselves by treatment with guanidine which leads to 6,7-dihydrocyclopenta [d]pyrimidines with a 4-(tert-butoxycarbonyl)phenylalkyl group (11a,b and 14a,b), (3) deprotection to the corresponding carboxylic acids (12a,b and 15a,b), and (4) amidation with diethyl glutamate and deesterification. Potent dihydrofolate reductase inhibition and highly potent cell growth inhibition were found with 2,4-diaminopyrimidine-fused cyclopentene compounds containing the trimethylene (3a) or ethylene bridge (3b) but not with the corresponding 2-amino-4-hydroxy analogs (4a,b). Compounds 3a and 3b were more growth inhibitory to several tumor cell lines (P388, colon 26, colon 38, and KB) than was methotrexate, with 3a being the most potent. Both 3a and 3b gave increases in the lifespan of P388 leukemic mice comparable to that observed with MTX. Both compounds were therapeutic against colon 26 colorectal carcinoma in mice. Compound 3a was highly effective against LC-6 non-small cell lung carcinoma in nude mice.
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PMID:Synthesis and antitumor activities of novel 6-5 fused ring heterocycle antifolates: N-[4-[omega-(2-amino-4-substituted-6,7-dihydrocyclopenta [d]pyrimidin-5-yl)alkyl]benzoyl]-L-glutamic acids. 820 95


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