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)

5-Deaza-10-propargylfolic acid (4), an analogue of the thymidylate synthase (TS) inhibitor 10-propargyl-5,8-dideazafolic acid (PDDF, 1), was prepared via alkylation of diethyl N-[4-(propargylamino)benzoyl]-L-glutamate (7) by 2-amino-6-(bromomethyl)-4(3H)-pyrido[2,3-d]pyrimidinone (15). Bromomethyl intermediate 15 was prepared from the corresponding hydroxymethyl precursor 14 by treatment with 48% HBr. Hydroxymethyl compound 14 was obtained by deamination of reported 2,4-diaminopyrido[2,3-d]pyrimidine-6-methanol (12a) in refluxing 1 N NaOH. Both 12a and its 5-methyl-substituted analogue 12b were converted to versatile 6-bromomethyl intermediates 13a and 13b from which important antifolates may be readily derived. Alkylation of 7 by 13a,b led to 10-propargyl-5-deazaaminopterin (5) and 5-methyl-10-propargyl-5-deazaaminopterin (6). As an inhibitor of TS from H35F/F cells, 4 gave an IC50 value showing it to be approximately 6-fold less inhibitory than PDDF (90 nM for 4 vs 14 nM for PDDF). In in vitro studies, IC50 (microM) values obtained for 4 vs L1210 and S180 of 1.50 and 2.35, respectively, were similar to those obtained for PDDF (2.61 and 1.97). Against HL60 cells, 4 was about 7-fold more cytotoxic than PDDF (IC50 values 0.72 and 5.29 microM). Inclusion of thymidine did not establish TS as the site of cytotoxic action for either 4 or PDDF in the cell lines used. In in vivo tests against L1210 in mice, 4 failed to show therapeutic effect. The 2,4-diamino compounds 5 and 6 were as potent inhibitors of DHFR from L1210 cells as MTX and 7- and 35-fold, respectively, more inhibitory than MTX toward L1210 cell growth. In mediated influx into L1210 cells, 5 and 6 were transported 2.7- and 8.5-fold, respectively, more readily than MTX. Against the EO771 mammary adenocarcinoma in mice, 6 produced greater antitumor effect than MTX. A dose of 36 mg/kg per day for 5 days caused no toxic deaths while the average tumor volume among 10 mice was reduced to 8-9% of that of the control, and 20% of the test animals were rendered tumor free.
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PMID:Synthesis and antifolate evaluation of the 10-propargyl derivatives of 5-deazafolic acid, 5-deazaaminopterin, and 5-methyl-5-deazaaminopterin. 173 51

Two species of DHFR were identified in wild-type L1210 murine leukemia cells by analysis of the kinetics of the binding of MTX and dissociation of the MTX-enzyme complex at pH 5.0 and pH 7.2. The two forms of DHFR were also distinguished by immunoinhibition of the binding of MTX and the catalytic reduction of FH2 to FH4 using an antiserum raised to the purified high affinity form of DHFR. The Ka for the binding of MTX by the low affinity form of the enzyme is 4.5 x 10(7) M-1, substantially lower than the reported Ka for the binding of this drug by the high affinity enzyme. The low affinity form of the enzyme catalyzed the reduction of FH2 to FH4 at a rate slower than the high affinity form of DHFR.
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PMID:Evidence for kinetic and immunologic heterogeneity of dihydrofolate reductase in L1210 leukemia cells. 178 10

Transient neurologic dysfunction associated with high-dose methotrexate and citrovorum factor rescue (MTX-CF) has been previously reported. At the biochemical level, there are at least two important pathways in central nervous system metabolism which might be disturbed by MTX: MTX may deplete the cell of the de novo synthesis of purine nucleotides and thymidylate through its action on dihydrofolate reductase (DHFR), and also inhibit dihydropteridine reductase (DHPR), an enzyme maintaining the cofactor of phenylalanine-hydroxylase in its active tetrahydrogenated form (tetrahydrobiopterin), and hence interfere with the supply of the neurotransmitters derived from tyrosine and tryptophan. We describe such a neurologic disease in a patient with acute lymphoblastic leukemia (ALL) receiving chemotherapy. Significant increase in cerebrospinal fluid biopterins supports the hypothesis of an inhibition of dihydropteridine reductase by MTX, and provides additional suggestions in terms of etiology, diagnosis and treatment.
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PMID:[Early neurotoxicity of high-dose of methotrexate and tetrahydrobiopterin deficiency]. 179 49

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate. 190 81

Two-dimensional 1H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severely broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. At 303 K, NOESY spectra with mixing times of 100 ms did not show interconversion between these isomers. However, exchange cross-peaks were observed in a 700-ms NOESY spectrum at 323 K which demonstrated that these isomers are interconverting slowly on the NMR time scale. Many of the side chains with clearly doubled resonances were located in the beta-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.
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PMID:Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli. 199 78

The effects of methotrexate (inhibiting dihydrofolate reductase) and nitrous oxide (inactivating methionine synthase) on intracellular folate coenzyme levels of leukemic cells were studied. Blast cells from 10 cases of acute myeloid leukemia (AML) and 5 cases of acute lymphoid leukemia (ALL) were incubated with 5 x 10(-8) M [3H] 5-formyltetrahydrofolate (5-formylTHF) for 18 h to label intracellular folate pools, which were subsequently quantitated by high performance liquid chromatography (HPLC). In AML, 5-methylTHF made up 53% of the total folate pool followed by 10-formylTHF (26%), 5-formylTHF (10%), THF (9%) and DHF (1%). Cells from ALL differed from AML (p less than 0.05) with respect to 10-formylTHF (17%) and DHF (10%). Exposure to nitrous oxide (8 h) caused an equal decrease of 10-formylTHF and 5-formylTHF in both AML (30%) and ALL (45%), whereas 5-methylTHF increased (130%). Methotrexate (4 h, 10(-6) M) caused an accumulation of DHF and a decrease of 5-methylTHF in both AML (32%) and ALL (12%). A specific reduction of the 10-formylTHF (50%) and 5-formylTHF (25%) pools was noticed in ALL. Exposure to nitrous oxide prior to methotrexate treatment aggravated the reduction of 10-formylTHF and 5-formylTHF presumably by impaired replenishment from the 5-methylTHF pool. In conclusion, this study demonstrates a significant difference in folate coenzyme distribution between cells from AML and ALL. Moreover it is shown that nitrous oxide and methotrexate treatment of leukemic cells cause an accumulation of 5-methylTHF and DHF respectively at the expense of other folate forms. The presence of substantial amounts of DHF in cells from ALL together with the specific reduction of 10-formylTHF (necessary for purine synthesis) during MTX treatment may in part explain the efficacy of methotrexate in the treatment of ALL.
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PMID:Effect of nitrous oxide and methotrexate on folate coenzyme pools of blast cells from leukemia patients. 201 7

Fluorescein-methotrexate (F-MTX) has been synthesized by an improved procedure and separated via chromatography on DEAE-Trisacryl into the alpha- and gamma-isomers. Purity of each isomer was verified by TLC, HPLC, and absorbance spectra. Identity of the alpha- and gamma-isomers was established by the following biological criteria: the gamma-isomer inhibited dihydrofolate reductase and was hydrolyzed by carboxypeptidase G2 (at the pteroate-glutamate linkage). The alpha-isomer, conversely, was unreactive in both systems, which is consistent with the specificity of these enzymes. Based upon these results, the gamma-isomer was selected for covalent labeling of proteins. Fluorescent bands were observed when the 22 kDa human dihydrofolate reductase and the 18 kDa folate transporter from Lactobacillus casei were treated with gamma-F-MTX (activated by N-hydroxysuccinimide) and subjected to SDS-PAGE. The probe was also useful for visualizing in situ the micromolar folate transport protein in L1210 cells.
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PMID:Visualization of folate transport proteins by covalent labeling with fluorescein methotrexate. 211 51

Acquired resistance of the L1210 leukemia in mice developed with less rapidity during therapy with edatrexate (10-ethyl-10-deazaaminopterin, EDX) than with MTX. Since this was explained only partially by the somewhat greater antitumor activity of EDX, this result may also reflect a difference in biochemical phenotypes selected in each case. Among 20 sublines selected for resistance to MTX, a reduction in influx, an elevation of DHFR, and a reduction of DHFR inhibition by MTX were all delineated. Among 14 sublines selected for resistance to EDX, both a reduction in influx and an elevation in level of DHFR were also commonly found. In addition, however, 7 of 14 EDX-resistant sublines exhibited a reduction in the level of folylpolyglutamate synthetase (FPGS) activity. Clonal derivatives of these 7 EDX-resistant cell lines exhibited 2- to 28-fold reductions in FPGS activity and a commensurate reduction in [3H]-MTX polyglutamate formation in situ following exposure to [3H]-MTX during growth in mice. An analysis of the kinetics and relative substrate preferences for FPGS from variant and parental L1210 cells revealed that the various changes in FPGS activity were at the level of the Vmax rather than Km. These results derived from an in vivo tumor model provide further evidence for a role of FPGS as a determinant of cytotoxicity and acquired resistance to classical folate analogs. They also provide evidence in the same pharmacologic model for a manifestation of resistance to 4-aminofolates in vivo that involves all of the alterations of its primary target, transport, and metabolism that have ever been associated with acquired resistance in cell culture systems.
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PMID:Preferential selection during therapy in vivo by edatrexate compared to methotrexate of resistant L1210 cell variants with decreased folylpolyglutamate synthetase activity. 220 78

A simple, highly sensitive radioassay was developed for the activity of a newly discovered inhibitor of dihydrofolate reductase (DHFR), aditoprim. The procedure is based on the inhibition of binding of [3H]-methotrexate ([3H]MTX) with bacterial dihydrofolate reductase by the antifolate, aditoprim. The analytic sensitivity using this binding inhibition method was less than 5 ng in plasma. The procedure developed requires no extraction of the drug from the plasma. The variation of simultaneous duplicate determinations was 6.3 per cent, whereas the variability of plasma samples assayed on different days was less than 11 per cent. The assay developed was applied to study the pharmacokinetics of aditoprim in the goat. In comparison with trimethoprim (TMP), the new inhibitor of DHFR, aditoprim, had a longer half-life and a larger volume of distribution, suggesting enhanced and prolonged antibacterial activity of aditoprim over TMP.
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PMID:Pharmacokinetics of aditoprim in goats using a radioassay. 220 3

As DHFR is the main molecular target of MTX, a widely used anticancer drug, its level in human tissues is likely to be one of the factors determining tissue sensitivity towards this drug. Forty-one biopsies were analyzed for their DHFR activity by a convenient spectrofluorometric assay developed in our laboratory; this sensitive method proved to be suitable for measurements in very small human samples. Statistical analysis of the results showed that (i) DHFR activity is not an index of tumorogenicity, at least in the cases studied, (ii) tumorous extracts contain modulators of DHFR activity.
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PMID:Activity measurements in human tissues of the methotrexate molecular target: a novel fluorometric assay. 226 53


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