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)

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.
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PMID:Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase. 237 39

A new spectrophotometric method is developed and applied for the study of the inhibitory effect of triamterene, hydrochlorothiazide and their combinations on the in vitro activity of dihydrofolate reductase enzyme. The method is based on incubating the drug (0.1-1.0 microM) or a buffer control with a solution containing reduced nicotinamide adenine dinucleotide phosphate (0.5 mM), magnesium chloride (1.29 mM), and folic acid as a substrate (0.01-0.1 mM) with the dihydrofolate reductase (0.25 unit). The resulting tetrahydrofolic acid is determined by first hydrolysing it by a methanol-hydrochloric acid mixture to produce p-aminobenzoyl glutamic acid, then adding p-dimethylaminocinnamic aldehyde reagent to form a stable pink coloured product. The colour is found to develop within 5 min and is stable over 12 h, with a maximum absorption at 545 nm. A linear calibration curve is formed by using standard solutions of tetrahydrofolic acid. The presence of the studied drugs did not interfere with the determination. Lineweaver-Burk plots of the reaction kinetics, in the presence of triamterene and/or hydrochlorothiazide showed a competitive inhibition of the dihydrofolate reductase in the presence of triamterene with or without hydrochlorothiazide. A 100% inhibition is obtained by 1 microM solution of triamterene at a folic acid concentration of 0.01 mM. No measurable effect of hydrochlorothiazide at the studied concentration range is demonstrated.
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PMID:Monitoring the effect of triamterene and hydrochlorothiazide on dihydrofolate reductase activity using a new spectrophotometric method. 249 May 42

Responses of several folate-metabolizing pathways to dietary folic acid were studied in 2-week-old chicks. Oxidation of a histidine load to carbon dioxide was impaired in folate-deficient chicks. There was a curvilinear relation between oxidation and dietary folate, and maximum oxidation occurred with 2 mg supplemental folic acid/kg. Hepatic activities of glutamic acid formiminotransferase (EC 2.1.2.5) and glycine N-methyltransferase (EC 2.1.1.20) were not affected significantly (P greater than 0.05) by dietary folic acid. The activity of dihydrofolate reductase (EC 1.5.1.3) in erythrocytes was elevated in folate-deficient chicks. These studies show that the activities of two folate-dependent pathways can be used as biochemical criteria of folate status in chicks.
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PMID:Relation between some folate-dependent metabolic pathways and dietary folate content in chicks. 278 83

In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.
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PMID:Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities. 281 Mar 30

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.
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PMID:Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase. 289 42

We have used two-dimensional (2D) NMR methods to examine complexes of Lactobacillus casei dihydrofolate reductase and methotrexate (MTX) analogues having structural modifications of the benzoyl ring [the 3',5'-difluoro and 3',5'-dichloro analogues (II and III)] and also the glutamic acid moiety [the alpha- and gamma-monoamides (IV and V)]. Assignments of the 1H signals in the spectra of the various complexes were made by comparison of their 2D spectra with those of complexes containing methotrexate where we have previously assigned resonances from 32 of the 162 amino acid residues. In the complexes formed with the dihalomethotrexate analogues, the glutamic acid and pteridine ring moieties were shown to bind to the enzyme in a manner similar to that found in the methotrexate-enzyme complex. Perturbations in 1H chemical shifts of protons in Phe-49, Leu-54, and Leu-27 and the methotrexate H7 and NMe protons were observed in the different complexes and were accounted for by changes in orientation of the benzoyl ring in the various complexes (15 degrees and 25 degrees in the difluoro- and dichloromethotrexate complexes, respectively). Binding of oxidized or reduced coenzyme (NADP+ or NADPH) to the binary complexes did not result in different shifts for Leu-27, Leu-54, or Leu-19 protons, and thus, the orientation of the benzoyl ring of the methotrexate analogues is not perturbed greatly by the presence of either oxidized or reduced coenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural comparisons of complexes of methotrexate analogues with Lactobacillus casei dihydrofolate reductase by two-dimensional 1H NMR at 500 MHz. 312 5

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

Five heretofore undescribed analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized and tested as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. The meta isomer of AMT was obtained from 2,4-diamino-6-(bromomethyl)pteridine and m-(aminobenzoyl)-L-glutamic acid, while the ortho isomer was obtained via the same route by using alpha-methyl gamma-tert-butyl o-(aminobenzoyl)-L-glutamate instead of the free acid. Analogues of MTX and AMT containing a double bond in the side chain were prepared from dimethyl D,L-2-amino-4-hexenedioate and 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively. Finally, a positional isomer of MTX with the CH2CH2COOH moiety moved from the alpha-carbon to the adjacent carboxamide nitrogen was synthesized from 3-[N-(carboxymethyl)amino]propanoic acid diethyl ester and 4-amino-4-deoxy-N10-methylpteroic acid. The positional isomers of AMT were weak DHFR inhibitors and showed very little growth-inhibitory activity against L1210 murine leukemia cells or the MTX-resistant L1210/R81 mutant line in culture. The MTX and AMT analogues with the CH2CH2COOH moiety replaced by a CH2CH = CHCOOH side chain showed anti-DHFR activity similar to that of the previously described saturated compound N-(4-amino-4-deoxy-N10-methylpteroyl)-L-2-aminoadipic acid, but were less potent than the parent drugs. The MTX analogue with the CH2CH2COOH side chain displaced from C to N was weakly bound to DHFR, confirming the importance of an intact CONH moiety, and showed greatly diminished cell growth inhibitory potency relative to MTX. None of the compounds was a substrate for folylpolyglutamate synthetase (FPGS) from mouse liver. Furthermore, inhibition of folic acid polyglutamylation in vitro at equimolar 500 microM concentrations of drug and substrate was negligible. The structural changes embodied in these five novel compounds are therefore too great for binding to the FPGS active site.
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PMID:Methotrexate analogues. 31. Meta and ortho isomers of aminopterin, compounds with a double bond in the side chain, and a novel analogue modified at the alpha-carbon: chemical and in vitro biological studies. 335 53

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

Methotrexate (MTX) analogues 27a-c bearing 2, omega-diaminoalkanoic acids (ornithine and its two lower homologues) in place of glutamic acid were synthesized by routes proceeding through N2-[4-(methylamino)benzoyl]-N omega-[(1,1-dimethylethoxy)carbonyl]-2, omega-diaminoalkanoic acid ethyl esters and N2-[4-(methylamino)benzoyl]-N5-[(1,1-dimethylethoxy)carbonyl]-2, 5-diaminopentanoic acid followed by alkylation with 6-(bromomethyl)-2, 4-pteridinediamine hydrobromide. Reactions at the terminal amino group of 27-type analogues or of appropriate precursors led to other MTX derivatives whose side chains terminate in ureido, methylureido, N-methyl-N-nitrosoureido, N-(2-chloroethyl)-N-nitrosoureido, and 4-chlorobenzamido groups. Also prepared were unsymmetrically disubstituted ureido types resulting from addition of ethyl isocyanatoacetate and diethyl 2-isocyanatoglutarate to the ethyl esters of 27a,b. Of these ureido adducts (32a,b and 33a,b, respectively), only 33a was successfully hydrolyzed to the corresponding pure acid, in this instance the tricarboxylic acid 34, a pseudo-peptide analogue of the MTX metabolite MTX-gamma-Glu. Biological evaluations of the prepared compounds affirmed previous findings that the gamma-carboxyl is not required for tight binding to dihydrofolate reductase (DHFR) but is operative in the carrier-mediated transport of classical antifolates through cell membranes. High tolerance levels observed in studies against L1210 leukemia in mice suggest the reduced potency may be due not only to lower transport efficacy but also to loss of the function of intracellular gamma-polyglutamylation. The N-nitrosoureas 30 and 31 showed appreciable activity in vivo vs. L1210, but the activity did not appear to be due to antifolate action as evidenced by their poor inhibition of both L1210 DHFR and cell growth in vitro.
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PMID:Syntheses and evaluation as antifolates of MTX analogues derived from 2, omega-diaminoalkanoic acids. 402 Aug 24


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