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)

N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity. 338 30

The goals of new antifolate development are: 1) improved selectivity, 2) improved penetration into pharmacologic sanctuaries, and 3) effectiveness vs. tumors either with intrinsic or acquired resistance to methotrexate (MTX). The major target for antifolate development has been dihydrofolate reductase (DHFR), but other critical folate-dependent enzymes, i.e., thymidylate synthase, methionine synthetase, and folylpolyglutamate synthetase are also important targets for new antifolate development. The possibility that DHFR from tumor tissue differs significantly from normal tissue DHFR now seems improbable, and the ideas of the late Bill Baker to design specific inhibitors of the tumor enzyme vs. the normal tissue DHFR are unlikely to succeed. However, the experience with triazinate (Baker's antifol; TZT) indicates that transport of antifols could be exploited to provide selective toxicity, as well as to provide agents effective vs. MTX-resistant cells. This work led to a second generation of "nonclassical" folate antagonists, of which trimetrexate (JB-11; TMQ) is now in clinical trial. Uptake of TMQ is via an MTX-independent membrane system, and extremely high intracellular levels of this drug are achieved in human leukemia cells.
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PMID:Design and rationale for novel antifolates. 343 93

Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate. 346 94

Spurious methotrexate (MTX) concentrations in sera and cerebrospinal fluids from leukemia patients who were given trimethoprim (TMP) were estimated using an MTX assay kit which is based on its inhibition of dihydrofolate reductase. The interference of TMP with MTX was confirmed in the assay system. A concentration of 0.17 microgram/ml of TMP gave a value for an apparent MTX of 10 microM.
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PMID:Trimethoprim interference in methotrexate assay by an enzyme inhibition assay kit. 356 7

P388 murine leukemia cells 18.4-fold more resistant to methotrexate (MTX) than the parent, drug susceptible line, were shown to possess a 1.5-fold higher dihydrofolate reductase (EC1.5.1.3) (DHFR) activity. This is in contrast to a MTX-resistant line, obtained from adriamycin-resistant cells, which is 27.9-fold more resistant to MTX and exhibits a 22.4-fold higher DHFR activity than that of the parent. The susceptibility of the enzyme to inhibition by MTX does not markedly change with the acquired drug resistance of the cell lines studied. Thus MTX-resistant cells obtained from an adriamycin-resistant line acquired resistance due to increased activity of the target enzyme, whereas other mechanisms are responsible for the resistance of cells derived from the adriamycin-sensitive parent.
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PMID:Dihydrofolate reductase activity in adriamycin and methotrexate sensitive and resistant P388 leukemia cells. 360 90

Undifferentiated human lymphoblasts (culture LS-2) were separated according to cell size during their exponential growth phase by way of centrifugal elutriation. The cell fractions thus obtained were characterized in terms of different cell cycle stages by flow cytometric measurement of their deoxyribonucleic acid (DNA histogram), the [3H]thymidine labeling index, and by determining the rate of [3H]thymidine incorporation. In these cell fractions the activities of thymidine kinase, thymidylate synthase, DNA polymerase, dihydrofolate reductase, methionine synthase, and hexokinase were determined. The results showed that all the enzymes investigated exhibited activities in all cell fractions. With the exception of DNA polymerase, all of the enzymes exhibited the lowest level of activity in the fraction containing the highest proportion of G0 + G1 phase cells (fraction 2); the activity of thymidine kinase was particularly low. This would suggest that thymidine kinase is not active in G0 + G1 phase cells and that the activity measured in fraction 2 is perhaps attributable to contamination of this fraction by S and G2 + M phase cells.
Leukemia 1987 Mar
PMID:Relation between cell cycle stage and the activity of DNA-synthesizing enzymes in cultured human lymphoblasts: investigations on cell fractions enriched according to cell cycle stages by way of centrifugal elutriation. 366 41

Exposure to nitrous oxide interferes selectively with the coenzyme function of vitamin B12 and causes inactivation of methionine synthetase, with subsequent impairment of folate metabolism and reduction of cellular proliferation. In a rat leukemia model (BNML) we investigated the combined administration of nitrous oxide, inactivating vitamin B12, and methotrexate (MTX), a folate antagonist inhibiting the enzyme dihydrofolate reductase. Through different mechanisms, both agents decrease the availability of tetrahydrofolate, and subsequently of other reduced folates, with increased impairment of folate-dependent synthesis of thymidylate. Effects on leukemic growth and on hematological values in rats demonstrated enhancement of the therapeutic effect of MTX by exposure to nitrous oxide. With several treatment schedules, the results of combined treatment were seen to be better than additive when compared with the effects of single agents. In particular, pretreatment of leukemic rats with nitrous oxide for 3 days before administration of MTX appeared effective. With higher doses of MTX, concomitant exposure to nitrous oxide even resulted in toxic effects. These findings were in accordance with the results of some metabolic studies performed in leukemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enhanced therapeutic effect of methotrexate in experimental rat leukemia after inactivation of cobalamin (vitamin B12) by nitrous oxide. 371 92

gamma-Phosphonate analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively, by reaction with methyl D,L-2-amino-4-phosphonobutyrate followed by gentle alkaline hydrolysis. The products were compared with the corresponding D,L-homocysteic acid derivatives as inhibitors of dihydrofolate reductase and folylpolyglutamate synthetase, and as inhibitors of cell growth in culture. The gamma-phosphonates were somewhat less active than either the gamma-sulfonates or the parent drugs as inhibitors of murine dihydrofolate reductase. The MTX gamma-sulfonate and gamma-phosphonate analogues were equally inhibitory toward mouse liver folylpolyglutamate synthetase (Ki = 190 microM), but in the AMT series the gamma-phosphonate (Ki = 8.4 microM) was more potent than the gamma-sulfonate (Ki = 45 microM). The AMT analogues were consistently more inhibitory than the MTX analogues against cultured L1210 murine leukemia cells, but neither the gamma-phosphonates nor the gamma-sulfonates were as potent as their respective parent drugs. The gamma-phosphonate analogue of MTX was three times more potent than MTX against the MTX-resistant mutant line L1210/R81, but the AMT gamma-phosphonate was less potent than AMT; however, these differences were small in comparison with the level of resistance to all these compounds in the L1210/R81 line. The results suggest that N10-methyl and N10-unsubstituted compounds altered at the gamma-position do not necessarily follow identical structure-activity patterns in every test system.
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PMID:Methotrexate analogues-27. Dual inhibition of dihydrofolate reductase and folylpolyglutamate synthetase by methotrexate and aminopterin analogues with a gamma-phosphonate group in the side chain. 376 24

Methotrexate(MTX)-resistant human promyelocytic-leukaemia cells (HL-60) derived from MTX-sensitive cells have a 20-fold increase in dihydrofolate reductase (DHFR) activity as compared with the sensitive cells. This increase is not associated with a concomitant increase in DHFR protein as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunological methods using mouse anti-DHFR antibody. The rate of DHFR synthesis is similar in both cell lines. Furthermore, both the sensitive and resistant cells have similar amounts of RNA hybridizing to a DHFR complementary-DNA probe, correlating well with the lack of increase in DHFR protein. DHFR-gene dosages were similar in both types of cells. We conclude that the 20-fold increase in DHFR activity present in these MTX-resistant cells is not due to the overproduction of DHFR but due to the expression of a more active form of the enzyme.
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PMID:Increased dihydrofolate reductase activity in methotrexate-resistant human promyelocytic-leukaemia (HL-60) cells. Lack of correlation between increased activity and overproduction. 385 28

We present a general strategy for the efficient insertion of recombinant retroviral vector DNA into the mouse germ line via infection of preimplantation mouse embryos. Transgenic mice were generated that harbor a replication-competent recombinant retrovirus (delta Mo + Py M-MuLV) that lacks the Moloney murine leukemia virus (M-MuLV)-type enhancer sequence in the long terminal repeat (LTR). Instead, the LTR contains an enhancer element that permits polyoma virus F101 to grow in undifferentiated F9 embryonal carcinoma cells. Expression studies in different tissues of animals transgenic for delta Mo + Py M-MuLV indicate possibilities to target and modulate expression of retroviral recombinants in mice via their LTR enhancer sequences. In addition, 16 transgenic mice were generated that harbor proviral DNA of a defective recombinant retrovirus carrying a mutant dihydrofolate reductase gene.
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PMID:Efficient insertion of genes into the mouse germ line via retroviral vectors. 386 22


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