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)

1. Dihydrofolate reductase was purified from Lactobacillus casei MTX/R, and studied on affinity columns containing folic acid and methotrexate. Two forms of the enzyme were interconverted by incubation with substrates. 2. Affinity columns were prepared from agarose activated with cyanogen bromide and coupled with 1,6-diaminohexane. Stable folate derivatives were covalently attached by using a carbodi-imide condensation. 3. Columns containing folic acid retarded but did not retain the enzyme. 4. Methotrexate at pH 6.0 was particularly effective for retention of the enzyme. 5. There is selective loss of one form of the enzyme during affinity chromatography in the absence of added NADPH. This loss is due to conversion into a single enzyme form on the column. 6. NADPH has a dual effect in stabilizing the enzyme and in sensitizing it to inactivation by methotrexate, particularly in the presence of glycine. 7. Protein with affinity for methotrexate, but without dihydrofolate reductase activity, may also be eluted from the columns. 8. In a single-step procedure the enzyme was purified nearly 4000-fold from mammalian skin.
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PMID:Affinity chromatography of dihydrofolate reductase. 439 20

Chain-extended analogues of methotrexate were synthesized by condensation of 4-amino-4-deoxy-N10-methylpteroic acid with esters of L-alpha-aminoadipic, L-alpha-aminopimelic, and L-alpha-aminosuberic acids, followed by ester hydrolysis with acid or base. Coupling was accomplished in up to 85% yield by the use of the peptide bond forming reagent diethyl phosphorocyanidate at room temperature. The products were found to bind bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase with an affinity comparable to methotrexate and were also equitoxic to L1210 cells in culture. Cytotoxicity increased up to 3-fold as the number of CH2 groups in the amino acid side chain was extended from two to five. The alpha-aminoadipate and alpha-aminopimelate analogues were poor substrates for carboxypeptidase G1, confirming that this enzyme has a strict requirement for a C-terminal L-glutamic acid residue. The in vivo antitumor activity of the chain-extended analogues against L1210 leukemia in mice was comparable to that of the parent drug on the qd X 9 schedule, but higher doses were required to achieve the same increase in survival. The results were consistent with findings, reported separately, that these compounds are poor substrates for folate polyglutamate synthetase and therefore would not be expected to form gamma-polyglutamates once they enter a cell. This distinctive property has potential therapeutic implications for the treatment of certain MTX-resistant tumors whose resistance may be associated with a lower than normal capacity to form gamma-polyglutamates in comparison with proliferative tissues such as intestinal mucosa or marrow.
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PMID:Methotrexate analogues. 20. Replacement of glutamate by longer-chain amino diacids: effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activity. 613 80

Methotrexate (MTX-Glu1) exerts its antitumor effects through its potent inhibition of dihydrofolate reductase (DHFR), the enzyme responsible for maintaining the cellular pool of reduced folates. Since the drug-enzyme complex (bound drug) is slowly dissociable, an excess of drug (unbound or free drug) above that required to bind all enzyme sites is required in order to compete with substrate for sites made available by enzyme-drug dissociation. We have examined the role of the polyglutamyl metabolites of MTX-Glu1 containing two to five glutamyl (MTX-Glu2-5) groups in gamma peptide linkage in maintaining an intracellular pool of free drug and in forming slowly dissociable complexes with DHFR. During 24-h incubations of ZR-75-B human breast cancer cells with 2 microM MTX-Glu1, we observed the progressive formation of derivatives with two to five glutamyl groups, which rapidly replaced the parent compound on enzyme binding sites and represented 85% of both unbound and bound intracellular drug at the end of incubation. When cells were then placed in drug-free medium, the rates of disappearance of drug and metabolites from the intracellular bound and free fractions decreased with increasing glutamyl chain length. Over 90% of both bound and free MTX-Glu1 left the cells within 1 h, greater than 90% of MTX-glu2 left within 6 h, and greater than 90% of MTX-Glu3 left the bound and free fractions within 24 h. In contrast, free MTX-Glu4 fell by only 63% and bound by only 23% after 24 h, while free MTX-Glu5 increased by 52% after 6 h in drug-free medium and bound MTX-Glu5 increased threefold after 24 h, as it replaced the other forms of drug bound to DHFR. These results suggested a rapid dissociation of MTX-GLu1 and -Glu2 from the enzyme, and a slower dissociation of the longer chain length derivatives. This conclusion was confirmed by examining the rates at which [3H]MTX-Glu1 through -Glu5 could be replaced on enzyme binding sites by a fivefold or greater excess of unlabeled MTX-Glu1. Bound [3H]MTX-Glu1 and -Glu2 had dissociation t 1/2 of 12 and 30 min, respectively, while -Glu3, -Glu4, and -Glu5 had t 1/2 of 102, 108, and 120 min. These experiments demonstrated that the longer chain polyglutamates have prolonged intracellular retention and can be dissociated less readily than MTX-Glu2 from DHFR, properties likely to make them more efficient DHFR inhibitors than the parent drug and of potential importance in extending the duration of drug action in tumor cells.
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PMID:Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase. 619 43

Computer modeling has been a valuable tool for clarifying the mechanism of action of antifolates. Some consequences of folyl and antifolyl polyglutamate synthesis can be addressed by adaptation of a network thermodynamic computer model of methotrexate action. Reversal or prevention of methotrexate cytotoxicity by 5-formyltetrahydrofolate has widely been assumed to occur through the delivery of reduced folate in substrate amounts for thymidylate synthesis, by-passing the effects of methotrexate at dihydrofolate reductase. This mechanism is inconsistent with experimental data which shows that "rescue" is a competitive phenomenon and that the transport process is incapable of delivering reduced folate at an adequate rate. Computer modeling studies are presented which predict that expansion of the total folate pool as folylpolyglutamates with "rescue" would reduce the inhibitory effect of MTX on thymidylate synthesis. Dihydrofolate polyglutamates could then accumulate to the high level needed to displace methotrexate from the small fraction of sites on dihydrofolate reductase that are sufficient to sustain tetrahydrofolate synthesis. Experimental studies with Ehrlich ascites tumor cells support this prediction. It is likely that a critical step in the protection of normal host tissues in high dose-rescue treatment regimens is the conversion of exogenously supplied 5-formyltetrahydrofolate to polyglutamyl derivatives and accumulation of total intracellular folate to higher than normal levels. Other computer simulations are presented which examine the potential significance of direct inhibition of thymidylate synthase by polyglutamyl forms of methotrexate. The model predicts that in cells with biochemical properties similar to methotrexate sensitive L1210 cells, inhibition of dihydrofolate reductase would still be the predominant site of action unless the thymidylate synthase Ki for a methotrexate polyglutamate is below about 0.1 microM. However, in methotrexate-resistant cells with elevated dihydrofolate reductase but normal membrane transport and polyglutamylation, thymidylate synthase may be the more important target enzyme.
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PMID:Predictions of a network thermodynamics computer model relating to the mechanism of methotrexate rescue by 5-formyltetrahydrofolate and to the importance of inhibition of thymidylate synthase by methotrexate-polyglutamates. 619 92

The methotrexate concentrations in the lungs or cutaneous metastases of patients with osteogenic or soft-tissue sarcoma were determined at different times after a high-dose methotrexate therapy. The levels in the metastases were 0.964 to 2.96 X 10(-7) molar six to nine days after the end of MTX infusion. They were thus 7.8 to 28 times higher than the corresponding serum levels. At the same time, an appreciable rise of dihydrofolate reductase activity was observed in the metastases. After chromatographic separation over Sephadex G15, MTX polyglutamates could be demonstrated in all tumor samples investigated so far; these amounted up to 68.3% of the total MTX. Taking into account the slower efflux of MTX polyglutamates compared to unchanged MTX, a new hypothesis for the principle of action of high-dose methotrexate therapy is discussed: the very high MTX doses lead to such high intracellular MTX concentrations even in transport-resistant tumor cells that at least part of the MTX is converted into MTX polyglutamates. Unchanged MTX flows relatively rapidly out of the cells, whereas the MTX polyglutamates only break down very slowly and thus can be cytostatically effective over a long period of time.
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PMID:Methotrexate and methotrexate polyglutamates in human sarcoma metastases after high-dose methotrexate therapy. 619 16

The uptake and tissue distribution of [3H]methotrexate [( 3H]MTX) at doses of 5 mg/kg i.p. either free or linked to anti-EL4 immunoglobulin G (AELG) or normal rabbit globulin (NRG) was studied in EL4 lymphoma-bearing C57BL/6J mice. When the uptakes of MTX-AELG, MTX-NRG, and free MTX were assayed as cell-associated 3H activity, comparison 3 hr after administration showed that uptake of MTX administered as the AELG conjugate was 2.5 times the uptake of MTX administered as the NRG conjugate and 6 times the uptake of MTX administered free. In contrast to the difference in the uptake of MTX-AELG and MTX-NRG by tumor cells, the pattern of uptake in all the other tissues studied was generally similar for the two conjugates. Conjugated MTX persisted in all tissues and serum and ascites fluid, whereas free MTX declined rapidly after reaching peak levels around 1 hr, except in EL4 cells where 45% was retained at 24 hr. The levels of intracellular MTX after administration of these three agents exceeded the intracellular dihydrofolate reductase level and correlated with the relative tumor-inhibitory effect in vivo of the agent (MTX-AELG greater than MTX-NRG greater than MTX).
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PMID:Tumor and tissue distribution of a methotrexate-anti-EL4 immunoglobulin conjugate in EL4 lymphoma-bearing mice. 646 87

Intracellular levels of DHFR can be modulated by mechanisms other than gene amplification. We found that MTX itself has an effect and the important features of this mechanism are as follows: (a) Sub-saturating doses of MTX induce intracellular DHFR activity by increasing DHFR synthesis; (b) The time-dependent effect seems quite specific for DHFR and is reversible (7); (c) Elevated DHFR synthesis is accompanied by disproportionate increases in DHFR mRNA; (d) The time scale for maximum induction is appreciably longer than the cell generation time. We suggest that part of the control involved is translational and we postulate that DHFR may regulate its own biosynthesis through feedback mechanisms. It is conceivable that the induction phenomenon could affect the clinical efficacy of MTX-therapy in some instances.
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PMID:The intracellular content of dihydrofolate reductase: possibilities for control and implications for chemotherapy. 647 40

Both the sensitivity to MTX and hCG secretion were compared in two choriocarcinoma cell lines (BeWo and HCCM-5) and study on mechanisms about MTX-resistance was described. The uptake of nucleotides and protein precursors was decreased in dose-dependent manner in both cell lines when exposed to MTX, but HCCM-5 cells showed more sensitivity to MTX. In HCCM-5 cells, both hCG and hCG-beta levels in the medium were decreased with exposure to 10(-9)M MTX, but they tended to increase when exposed to higher concentration of MTX in contrast to the inhibition of 3H-thymidine uptake. However, in BeWo cells, an inverse relationship between the incorporation of 3H-thymidine and secretion of hCG was not clearly observed. The content of dihydrofolate reductase (DHFR) in HCCM-5 cells was about one-half of that in BeWo cells. Although the peak of 3H-MTX incorporation was observed at 120 minutes in both cell lines, HCCM-5 cells incorporated about 4 times higher 3H-MTX than BeWo cells. The difference of sensitivity to MTX between those two cell lines seems to be concerned with both the decrease of MTX transport and increase of intracellular DHFR levels.
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PMID:[Characterization of two choliocarcinoma cell lines and their sensitivity to MTX]. 668 29

Cells resistant to methotrexate (L1210/R7A) and possessing an increased level of dihydrofolate reductase due to gene amplification can be detected by the technique of flow cytofluorimetry using a new fluorescent derivative of methotrexate (F-MTX) based on a putrescine linker. Comparative studies of dihydrofolate reductase enzyme and cell growth inhibition following treatment with methotrexate and F-MTX suggest that the two agents possess similar modes of action. In an artificially mixed population of cells sensitive and resistant to methotrexate it is possible, using F-MTX, to recognise and separate distinct cell subpopulations showing differential fluorescence using a fluorescence-activated cell sorter (FACS IV). The selective removal of the resistant cells within a mixed population of sensitive and resistant cells has been demonstrated for 5 X 10(-8) M vinblastine by means of flow cytometry. The effectiveness of the vinca alkaloids decreases in the order vinblastine greater than vindesine greater than vincristine, which previously was shown to be the order of effectiveness in producing collateral sensitivity.
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PMID:The use of a fluorescent methotrexate probe to monitor the effects of three vinca alkaloids on a mixed population of parental L1210 and gene-amplified methotrexate-resistant cells by flow cytometry. 673 43

The kinetics of ligand binding to dihydrofolate reductase from Lactobacillus casei (MTX/R) to form the ternary enzyme-inhibitor-coenzyme complex have been investigated by the stopped-flow fluorescence technique. The fluorescence changes observed when coenzymes or inhibitors bind to the binary complex of the enzyme with the complementary ligand occur in a single fast phase. Under pseudo-first-order conditions the reaction traces could be fitted with precision to a single-exponential decay, and apparent bimolecular rate constants in the range 2 x 10(6) to 3 x 10(7) M-1s-1 have been measured assuming a bimolecular-unimolecular model. The kinetic constants obtained suggest that prior binding of an inhibitor to the enzyme may, to a minor extent, interfere with coenzyme binding but the rates of inhibitor binding seem to be unaffected by the presence of a bound coenzyme. Dissociation rate constants appear to be less than 1 s-1 which suggests that both coenzymes and inhibitors are tightly bound in the ternary complex. An investigation of the effects of pH on the kinetics of ternary complex formation indicated the involvement of ionizable groups in ligand binding, but this shows some ligand dependence. The rates of ligand bindings to form the ternary complex are fairly high, but it is unlikely that these associations are diffusion controlled because their measured activation energies of 7.8-14.5 kcal mol-1 are higher than expected from reactions whose rates are limited by diffusion in aqeous solution.
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PMID:Kinetics of ternary complex formation between dihydrofolate reductase, coenzyme, and inhibitors. 676 38


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