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 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

Serine hydroxymethyltransferase and the trifunctional enzyme C1-tetrahydrofolate synthase have been purified to near homogeneity from L1210 cells. Kinetic constants (Km and kcat) have been determined for both folate and non-folate substrates. The effect of increasing glutamate chain length on affinity and catalytic efficiency were determined for the four activities. The studies show that the structural and catalytic properties of the two L1210 enzymes are very similar to the corresponding enzymes purified from rabbit liver. Antibodies to both rabbit serine hydroxymethyltransferase and C1-tetrahydrofolate synthase cross-react with the corresponding L1210 enzymes. The intracellular concentration of active sites of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in L1210 cells are both 9 microM. The combined concentration of these two enzymes exceeds the previously reported concentration of 10 microM for total intracellular folates. A network thermodynamic computer model of one carbon metabolism (Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. (1989) J. Biol. Chem. 264, 17016-17023) suggests that complete inhibition of cytosolic serine hydroxymethyltransferase would neither significantly decrease the rates of biosynthesis of purines and thymidylate nor significantly alter the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate with subsequent inhibition of dihydrofolate reductase.
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PMID:Purification and properties of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from L1210 cells. 237 84

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

The thermodynamic parameters of the binding of antifolate drugs to bovine liver dihydrofolate reductase (EC 1.5.1.3., 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase) have been measured with a flow microcalorimetric method. These parameters are greatly influenced by the structure of the inhibitor and/or by the presence of NADPH and above all by temperature. For all the compounds studied, binding at 37 degrees C is driven by favourable enthalpy variations, whereas entropy variations are unfavourable. At 10 degrees C, reactions are both enthalpically and entropically driven. These effects can be explained by a partial thermal denaturation of dihydrofolate reductase at 37 degrees C, which is restructured by NADPH and/or the antifolate. The refolding induced by the antifolate trimetrexate may explain its high association constant in the binary system (without NADPH), and the weaker cooperative effect of NADPH in the ternary system, as compared to methotrexate. In contrast, the poor affinity of trimethoprim for mammalian dihydrofolate reductase in binary and ternary systems at 37 degrees C is the result of a weaker stabilizing effect of this compound as regards temperature increase. Heat capacity variation linked to the complex formation reaction showed that this conformational transition is more pronounced between 25 and 37 degrees C than between 10 and 25 degrees C. Thus, the ability of the inhibitors to give to dihydrofolate reductase a more stable thermal behaviour at 37 degrees C is determinant in their binding.
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PMID:Comparative thermodynamic study of the interaction of some antifolates with dihydrofolate reductase. 240 Jul 75

Previous studies from this laboratory have shown that the addition of leucovorin to tumor cells dissociates methotrexate, but not methotrexate polyglutamates, from dihydrofolate reductase (L. H. Matherly, D. W. Fry, and I. D. Goldman, Cancer Res., 43: 2694-2699, 1983). To further assess the importance of these interactions to leucovorin rescue, antifolate growth inhibition toward L1210 cells in the presence of leucovorin was correlated with the metabolism of (6S)-5-formyl tetrahydrofolate to dihydrofolate as a measure of dihydrofolate reductase activity. Growth inhibition (greater than 95%) by methotrexate (5-10 microM) following its intracellular polyglutamylation during a 3-h preexposure, or by continuous treatment with high levels of the lipophilic antifolate, trimetrexate (1 microM), was only slightly diminished by 10 microM leucovorin (15-25%). High-pressure liquid chromatographic analyses of the derivatives formed from radiolabeled (6S)-5-formyl tetrahydrofolate under these conditions showed an incomplete conversion to dihydrofolate and metabolism to predominantly 10-formyl tetrahydrofolate. Neither of the antifolates interfered appreciably with the metabolism of the folate derivatives to polyglutamates. Growth inhibition in the presence of leucovorin correlated with the accumulation of dihydrofolate (1.5-2.2 nmol) from radiolabeled (6S)-5-formyl tetrahydrofolate, reflecting continued suppression of dihydrofolate reductase activity at these drug concentrations. With lower equitoxic levels of the trimetrexate (7.5 nM), the provision of leucovorin allowed for a restoration of cell growth to a level greater than 90% of control. Under these conditions, control levels of dihydrofolate (0.2 nmol) were formed from radiolabeled cofactor, consistent with sustained dihydrofolate reductase activity. These findings support a role for the activation of dihydrofolate reductase as an important component of the reversal of the effects of diaminoantifolates by leucovorin, presumably by a competitive displacement of drug from the enzyme. Since no displacement occurs in cells which have accumulated methotrexate polyglutamates, or in the presence of high levels of trimetrexate, it appears that the concentration of unbound drug within cells is a significant determinant of the extent of this competitive binding interaction. From these considerations, the high levels of methotrexate polyglutamates that accumulate in sensitive tumors relative to bone marrow and gastrointestinal cells would appear to represent an important factor for the selectivity of leucovorin rescue in vivo.
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PMID:Antifolate polyglutamylation and competitive drug displacement at dihydrofolate reductase as important elements in leucovorin rescue in L1210 cells. 241 28

Recent studies have clarified the critical role that polyglutamylation plays in methotrexate (MTX) action. Polyglutamate derivatives of MTX bind to dihydrofolate reductase (DHFR) with affinities comparable to the monoglutamate, but their retention in cells results in a sustained block in tetrahydrofolate (FH4) synthesis. One important element in the selectivity of MTX action is the preferential buildup and retention of these polyglutamyl forms in susceptible tumor cells as compared to host cells of the bone marrow or gastrointestinal mucosa. This selectivity in the accumulation of MTX polyglutamyl forms has now been further shown to play an important role in the selectivity of leucovorin rescue and may provide a unique new approach to nucleoside protection as well. This paper reviews the current understanding of the biochemical basis for leucovorin rescue and its selectivity. Important elements in leucovorin rescue are reactivation of DHFR with depression of cellular dihydrofolate (FH2) and provision of folate substrate to circumvent the block in FH4 synthesis. Selectivity of leucovorin rescue may be attributed to direct inhibition by MTX polyglutamyl forms, as well as FH2 polyglutamates that accumulate in their presence, at the levels of thymidylate synthase and transformylation during purine nucleotide biosynthesis. The presence of cellular MTX polyglutamates impairs reactivation of endogenous DHFR activity by leucovorin metabolites, and the resultant maintenance of high cellular levels of cellular FH2 and the polyglutamyl derivations of MTX impair the utilization of added FH4 in susceptible tumor cells. This paper also develops the concept of "early" nucleoside protection in antifolate therapy. In this approach, nucleosides are administered simultaneously with a pulse of MTX to provide early host protection from the cytotoxic effects of modest doses of MTX. Cessation of protection occurs at a time when extracellular and intracellular monoglutamate has fallen to low levels, and the polyglutamyl forms of the drug are present in susceptible tumors but not in host tissues of the gut and bone marrow. Data are presented to demonstrate that increased doses of MTX can be administered in normal and tumor-bearing animal systems as well as in humans by this technique.
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PMID:Biochemical factors in the selectivity of leucovorin rescue: selective inhibition of leucovorin reactivation of dihydrofolate reductase and leucovorin utilization in purine and pyrimidine biosynthesis by methotrexate and dihydrofolate polyglutamates. 244 54

This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 microM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [3H]deoxyuridine incorporation into DNA or [14C]formate incorporation into purines. When the interval of exposure to 100 microM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (approximately 50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 microM 7-OH MTX on cell growth were reversed completely by 10 microM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.
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PMID:Further studies on the pharmacologic effects of the 7-hydroxy catabolite of methotrexate in the L1210 murine leukemia cell. 246 76

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

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.
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PMID:Atypical transient state kinetics of recombinant human dihydrofolate reductase produced by hysteretic behavior. Comparison with dihydrofolate reductases from other sources. 249 21

Serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified from a pyrimethamine sensitive strain of Plasmodium chabaudi. Km values of 2.91 and 1.08 mM were determined for tetrahydrofolate and serine, respectively. The effects of pH, of temperature and of some potential inhibitors were determined. The enzyme was also partially purified from a pyrimethamine-resistant strain of P. chabaudi and subjected to the same regime. No differences between the enzymes from the two sources could be detected. It would appear that the changes in properties in the enzymes dihydrofolate reductase and thymidylate synthetase associated with the development of drug resistance in P. chabaudi were not reflected in any obvious alterations in serine hydroxymethyltransferase.
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PMID:Serine hydroxymethyltransferase from pyrimethamine-sensitive and -resistant strains of Plasmodium chabaudi. 249 46


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