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)

Five methotrexate (MTX)-resistant K562 cell subclones (K562/MTX-1 approximately -5) were established and were examined for mechanisms of drug resistance. Impairment of MTX-polyglutamate formation, with membrane transport alteration, in the resistant cells was demonstrated in the previous studies (Koizumi, S. (1988) Jpn. J. Cancer Res. 79, 1230). Further analysis of sensitivity of the cells to trimetrexate (TMQ), which is not polyglutamated and does not require the reduced folate transporter, but is a potent inhibitor of human DHFR, revealed a modest decrease in sensitivity to TMQ (2.4- to 15-fold). Enzyme studies showed that the dihydrofolate reductase (DHFR) activities of these resistant subclones were very similar to that of the parent cells. The number of binding sites of these subclones for MTX calculated from Scatchard analysis was increased up to 7-fold in the K562/MTX-1 and -4 subclones and up to 3-fold in the other 3 subclones as compared to the parent cells. KD values of MTX for the DHFR in the K562/MTX-1 and -4 subclones also appeared to be altered relative to the parent cell line. Further, thymidylate synthase (TS) activity of the resistant subclones was reduced to 50% in K562/MTX-1 and -4 cells, and to 11-25% in the other subclones as compared to the parent cell line. These findings suggest that antifolate resistance in the newly established K562/MTX subclones in multifactorial with polyglutamation and transport defects accounting for the majority of resistance to MTX, and that alteration in the binding affinity of DHFR for MTX and diminished levels of TS may contribute to the 'residual' drug resistance to TMQ and have importance with respect to MTX.
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PMID:Enzyme studies of methotrexate-resistant human leukemic cell (K562) subclones. 137 15

The inhibitory effects of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline antifolate and a potent thymidylate synthase inhibitor, were evaluated in human leukemia cell lines resistant to methotrexate (MTX) and trimetrexate (TMQ). MTX-resistant MOLT-3 cell lines, MOLT-3/MTX200 and MOLT-3/MTX10,000, were cross-resistant to CB3717; however, the degree of resistance was only tenfold for both cell lines, and increased dihydrofolate reductase activity in MOLT-3/MTX10,000 had little influence on the degree of CB3717 resistance. The MOLT-3 cell line made resistant to TMQ, MOLT-3/TMQ200, was as sensitive to CB3717 as the parent line. The cell growth inhibitory effect of CB3717 on MOLT-3 was reversed by the addition of thymidine. Leucovorin also partially reversed CB3717-induced growth inhibition. Cellular uptake of MTX and 5-methyl-tetrahydrofolate was hindered by the presence of a high concentration of CB3717, whereas TMQ uptake was not influenced by CB3717. CB3717 appears to enter the cells not only through reduced folate transport system, but by other route(s). CB3717 does not share the transport pathway with TMQ. Our observations that MTX-resistant cells with increased dihydrofolate reductase are not more resistant than cells without increased enzyme activity, and that TMQ-resistant cells are not cross-resistant to CB3717, may have clinical relevance.
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PMID:N10-propargyl-5,8-dideazafolic acid (CB3717): inhibitory effects on human leukemia cell lines resistant to methotrexate or trimetrexate. 143 41

Trimetrexate (TMTX), 5,10-dideazatetrahydrofolate (DDATHF), and 10-propargyl-5,8-dideazafolate (PDDF, CB3717) are antifolates whose primary intracellular targets are dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and thymidylate synthase, respectively. Varying the medium folic acid (PteGlu) concentration over the range of 0.5 to 100 microM increasingly blocks the growth inhibitory effects of the individual antifolates in Manca human lymphoma cells, but increasingly enhances the synergistic interaction of both TMTX + DDATHF and TMTX+ PDDF combinations. Drug interactions were quantitated using the universal response surface approach, which consists of fitting a concentration-effect surface to experimental data with weighted nonlinear regression, enabling the estimation of the synergism parameter, alpha. Estimates for alpha are larger (more intense synergism) for the TMTX + DDATHF combination (alpha = 4.68 +/- 0.66 at 2 microM PteGlu; alpha = 53.6 +/- 3.7 at 40 microM PteGlu) than for the TMTX + PDDF combination (alpha = 0.690 +/- 0.25 at 2 microM PteGlu; alpha = 7.20 +/- 1.8 at 40 microM PteGlu). However, the relative increase brought about by increasing the PteGlu concentration from 2 microM to 40 microM is similar in each instance, 11- and 10-fold, respectively. These experiments suggest that the enhanced cytotoxic interaction brought about by increased PteGlu concentration with the TMTX + DDATHF combination and the TMTX + PDDF combination may share a common mechanism. The dramatic intensity of the synergism between DDATHF and TMTX at 40 microM PteGlu, along with the concept of modulation of antifolate synergism by folates, suggests future in vivo and/or clinical applications of combinations of these (or similar) compounds.
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PMID:Quantitation of folic acid enhancement of antifolate synergism. 153 66

Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipophilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance. The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression. gamma-Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads). None of the gamma-ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression. Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate. Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones. However, when present with trimetrexate, verapamil (0.3-0.6 microM) reversed the lipophilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase. This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drug-selective growth. Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates. We conclude that a gamma-radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.
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PMID:A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells. 167 47

Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.
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PMID:Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase. 182 3

Trimetrexate (TMTX), a potent inhibitor of the enzyme dihydrofolate reductase, was shown to be more active than its analogue, Methotrexate, against murine and human tumor cell lines in vitro and in vivo. We conducted two sequential phase I studies using a single bolus injection of TMTX every 14 days (Schedule A) and a weekly x 3 schedule every 4-6 weeks (Schedule B). Twenty-seven patients were treated on Schedule A with a TMTX dose range of 5 mg/m2 to 450 mg/m2 and 23 patients were treated on Schedule B with a TMTX dose range of 50 mg/m2 to 200 mg/m2. The dose limiting toxicity was myelosuppression on both schedules. The development of hematological toxicity was highly variable at different dose levels and within the same patient at a particular dose level. The nadir of blood counts was reached by Day 8 to 10 on the single dose schedule with recovery by Day 14. On Schedule B, the nadir granulocyte count occurred on Day 14 while platelet count was generally lowest by Day 20; the blood counts usually recovered 7 to 10 days after the last dose. Other common side-effects includes skin toxicity and stomatitis which were worse on the weekly schedule. Less common toxicities included mild nausea and vomiting, diarrhea, and transient deterioration in renal and hepatic functions. The occurrence of toxicity was not related to the extent of prior treatment, liver metastases, or accumulation of third space fluids. Based on our results, we recommend a starting TMTX dose for Phase II studies of 200 mg/m2 every 2 weeks or 100 mg/m2 to 125 mg/m2 on the weekly schedule.
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PMID:Phase I studies of trimetrexate using single and weekly dose schedules. 183 42

Arginine-70 of human dihydrofolate reductase (hDHFR) is a highly conserved residue which X-ray crystallographic data have shown to interact with the alpha-carboxylate of the terminal L-glutamate moiety of either folic acid or methotrexate (MTX). The rationale for this study was to introduce a conservative amino acid residue change at position 70 (Arg----Lys) which might function as a titratable group and, thus, reveal possible quantitative changes in ligand binding and kinetic parameters as a function of pH. Such a mutant enzyme (R70K) has been constructed and expressed by using site-directed mutagenesis techniques. This substitution has a dramatic effect on the binding of MTX, which displays a 22,600-fold increase in the dissociation constant (KD) at pH 7.5 compared to that of the reported wild-type enzyme value. At this pH, the KD value for dihydrofolate (FAH2) for the R70K enzyme shows only a 7-fold increase over that for the wild-type hDHFR. The pH profiles of the Michaelis and dissociation constants for FAH2 and KD values for MTX for the mutant enzyme all show a 7-8-fold increase from pH 7.5 to 8.5 as compared to its wild-type counterpart. The binding of NADPH or the nonclassical inhibitor trimetrexate (TMQ) to either the wild-type or the mutant enzyme does not show such pH-dependent characteristics. Thus, since FAH2 and MTX interact with the guanidinium side chain of arginine-70 in the wild-type hDHFR, the replacement of this residue with a lysine in the R70K mutant appears to have resulted in the introduction of a titratable group with a perturbed pKa value of ca. 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Conversion of arginine to lysine at position 70 of human dihydrofolate reductase: generation of a methotrexate-insensitive mutant enzyme. 190 50

Pneumocystis carinii was obtained in high yield from the lungs of immunosuppressed rats by rupturing mammalian host cells, washing away the soluble mammalian dihydrofolate reductase, and harvesting intact organisms in association with the mammalian plasma membranes. P. carinii dihydrofolate reductase, measured in the 100,000 x g supernatant from sonicated organisms, was obtained in yields ranging up to 62 IU per rat. The enzyme prepared in the presence of protease inhibitors was stable when frozen in liquid nitrogen. P. carinii dihydrofolate reductase differed from the mammalian enzyme in that the former was slightly inhibited by 150 mM KCl, whereas the latter was stimulated over twofold by 150 mM KCl. The standard assay for P. carinii dihydrofolate reductase contained 0.12 mM NADPH and 92 microM dihydrofolic acid. Under these conditions, the 50% inhibitory concentrations of the known inhibitors trimethoprim, trimetrexate, and pyrimethamine were 12 microM, 42 nM, and 3.8 microM, respectively. These standard compounds were also tested against dihydrofolate reductase from rat liver to allow an assessment of the selectivity of the drugs. Although it was the least potent, trimethoprim was the most selective. Pyrimethamine was more potent but was nonselective. Trimetrexate was extremely potent but was selective for mammalian dihydrofolate reductase. A series of experimental compounds was obtained from the National Cancer Institute and other sources through the Developmental Therapeutics Branch of the Division of AIDS at the National Institute of Allergy and Infectious Diseases. Among the first 87 compounds tested, 11 had 50% inhibitory concentrations below that of trimetrexate and 3 were more selective than trimethoprim. The most promising compounds in this original group were chemically related to methotrexate.
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PMID:Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs. 192 92

The metabolic disposition of trimetrexate, a nonclassical inhibitor of dihydrofolate reductase, was characterized in the rat. After iv administration of 1.2 mg/kg [14C]trimetrexate (as the glucuronate), recovery of total radioactivity in urine and feces through 144 hr was greater than 96% of dose. Trimetrexate was extensively metabolized, with only 13% of the dose excreted unchanged in urine and bile. Profiling of biliary and urinary radioactivity showed three components and unchanged drug accounted for the majority of excreted radioactivity (75% of dose). Tandem mass spectral analysis of one urinary component suggested trimetrexate had undergone N-dealkylation and oxidation to 2,4-diamino-5-methyl-6-quinazolinecarboxylic acid. Structural assignment for this metabolite was confirmed by comparison to authentic reference material. Mass spectral analysis of a second component gave a quasimolecular ion (MH)+ at m/z 532 with a key fragment ion at m/z 356 (MH-176)+, characteristic of a glucuronide conjugate. The proton NMR spectrum of this component was consistent with expectations for a glucuronide conjugate of 4'-O-desmethyl trimetrexate. Possible formation of a sulfate conjugate was explored by co-administration of unlabeled trimetrexate with [35S]sulfate to rats. A 35S-labeled component was excreted in urine, which co-eluted with the third major urinary 14C-labeled component observed in the first experiment. Mass spectrum of this component was consistent with the structure of trimetrexate-4'-O-desmethyl sulfate. In dogs, the disposition of trimetrexate was examined using stable isotope-labeled material. The dose was 10 mg/kg administered iv as a 1:1 mixture of 13C2, 15N-labeled and unlabeled trimetrexate glucuronate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Metabolic disposition of trimetrexate, a nonclassical dihydrofolate reductase inhibitor, in rat and dog. 198 48

Trimetrexate (TMTX) is an analog of methotrexate and a potent inhibitor of the enzyme dihydrofolate reductase. In this phase I study, TMTX was given intravenously to 32 patients as a constant infusion over 24 hours every 28 days. The maximum-tolerated dose of TMTX was 200 mg/m2, with myelosuppression as the dose-limiting toxicity. Other toxicities included nausea and vomiting, stomatitis, erythema and phlebitis at the site of infusion, rash and skin hyperpigmentation, and elevated serum hepatic enzymes. Two drug-related deaths occurred secondary to leukopenia and sepsis. Twenty-six patients were evaluable for antitumor response. Twenty-one patients had progressive disease, while three patients had disease stabilization. There were two partial responses observed--one in a patient with breast cancer and a second in a patient with nasopharyngeal carcinoma. TMTX pharmacokinetics were studied in 15 patients. The drug had a mean terminal half-life of 13 hours. Steady-state was not achieved during the 24-hour infusions. Only 6% of the parent compound was excreted unchanged in the urine, and CSF levels averaged less than 2% of simultaneously measured plasma levels. A dose of 150 mg/m2 is recommended for phase II trials of TMTX using this 24-hour infusion schedule.
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PMID:A phase I and pharmacokinetic study of trimetrexate using a 24-hour continuous-injection schedule. 214


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