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 semicarbazones, thiosemicarbazones and acetyl-hydrazones of phthalimide, o-benzosulfimide, naphthalimide and diphenimide demonstrated potent cytotoxicity against murine and human leukemia cell growth and cultured cell growth from human solid tumors. The major site of inhibition in L1210 leukemia cells was DNA synthesis after 60 min incubated with the agents at 25, 50 and 100 microM. De novo synthesis of purines at the regulatory enzyme sites of PRPP amidotransferase and IMP dehydrogenase were the major targets of the agent. Thymidylate synthetase, dihydrofolate reductase and ribonucleoside reductase activities were inhibited by the agents in a manner which would contribute to the overall reduction of DNA synthesis and cell death. d(NTP) pools were significantly reduced and the evidence suggests that the agents interacted with DNA affording DNA strand scission which would interfere with both template utilization by the polymerases and also ultimately reduce nucleic acid synthesis.
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PMID:Cytotoxicity of imides-N-alkyl semicarbazones, thiosemicarbazones, acetylhydrazones and related derivatives. 775 77

Thymidylate synthetase (TS) is the only enzyme that catalyzes the formation of thymidine nucleotides in Angiostrongylus cantonensis. A fraction enriched in TS was obtained from the gravid nematode by gel filtration and affinity chromatography using methotrexate-agarose. TS, which was well separated from dihydrofolate reductase, has a relative molecular mass of 66 kDa. By electrophoresis in sodium dodecyl sulphate gel, a major protein band corresponding to 31 kDa was observed. This band was shown to be TS by comparing the electrophoretic mobility with an enzyme preparation bound with [6-3H]5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). Therefore, the enzyme is composed of two identical or very similar subunits. Velocity studies and product inhibition patterns revealed that the TS reaction undergoes a sequential mechanism in which 2'-deoxyuridine 5'-monophosphate (dUMP) is the first substrate added to the active site and thymidine 5'-monophosphate is the last product released. The apparent Km values for dUMP and 5,10-methylenetetrahydrofolate are 10 and 185 microM, respectively. FdUMP and trimethoprim inhibited the parasite TS competitively with dUMP and the Ki values of 23.5 nM and 852 microM, respectively. Methotrexate was a noncompetitive inhibitor of TS. At 0.2 mM 5,10-methylenetetrafolate, 1 mM methotrexate inhibited the activity by 74%.
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PMID:Angiostrongylus cantonensis: characterization of thymidylate synthetase. 800 63

Trimetrexate is a powerful inhibitor of the dihydrofolate reductase of Pneumocystis carinii. AIDS patients (n = 215) with moderate to severe P. carinii pneumonia were enrolled in a double-blind study of trimetrexate plus leucovorin versus trimethoprim-sulfamethoxazole (TMP-SMZ) for 21 days. By study day 10, study therapy failed because of lack of efficacy in 16% of patients assigned to TMP-SMZ and 27% assigned to trimetrexate (P = .064), and the PAO2-PaO2 improved significantly faster with TMP-SMZ. By study day 21, failure rates were 20% with TMP-SMZ and 38% with trimetrexate (P = .008), with respective mortality rates of 12% and 20% (P = .088). By study day 49, the difference in mortality (16% vs. 31%) was significant (P = .028). The cumulative incidence of serious and treatment-terminating adverse events including hematologic toxicities was less with trimetrexate (P < .001). Thus, trimetrexate plus leucovorin was effective, albeit inferior to TMP-SMZ, for moderately severe P. carinii pneumonia but was better tolerated than TMP-SMZ.
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PMID:Trimetrexate with leucovorin versus trimethoprim-sulfamethoxazole for moderate to severe episodes of Pneumocystis carinii pneumonia in patients with AIDS: a prospective, controlled multicenter investigation of the AIDS Clinical Trials Group Protocol 029/031. 801 93

Transfer of shigella R-plasmids in vivo has seldom been demonstrated. Strains of Shigella dysenteriae type 1 and Shigella flexneri type 5b were isolated from a Bulgarian traveller who visited Vietnam and developed dysentery, which was treated with trimethoprim/sulfamethoxazole (TMP/SMZ) for a short time. Both species of shigellae are unusual in Bulgaria where strains of S. sonnei predominate. Both shigella strains were multiresistant to the same antimicrobial agents. Each strain contained a 48-kilobase plasmid that conferred the entire resistance phenotype to a susceptible Escherichia coli. Restriction endonuclease patterns of plasmid DNA from the respective strains were identical. Transmissible plasmids of the same resistance phenotypes and restriction patterns were isolated from the patient's colonic E. coli. Transconjugants hybridized to a dihydrofolate reductase type I-DNA probe. These studies support the hypothesis that R-plasmid transfer may occur between non-pathogenic, faecal strains and pathogenic shigellae, a process that may have been facilitated by inadequate treatment with TMP/SMZ at the onset of the illness.
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PMID:In vivo R-plasmid transfer in a patient with a mixed infection of shigella dysentery. 814 99

The basis for the high affinity and selectivity of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine, TMP] and several close structural analogues is reviewed. Methoxy group substitution on the benzyl group of 2,4-diaminobenzylpyrimidine markedly affects both Escherichia coli dihydrofolate reductase (DHFR) Ki values and in vitro antibacterial activity. TMP is several hundred-fold more potent than the unsubstituted benzylpyrimidine, and the monomethoxy and dimethoxy analogues are of intermediate activity. However, equilibrium dissociation constants determined in the absence of cofactor (NADPH) show that the binding of these diaminobenzylpyrimidines in the enzyme-inhibitor binary complex is considerably weaker and does not vary among the compounds. Thus, the TMP binding affinity of E. coli DHFR is increased by NADPH in the ternary complex, and this increased affinity (cooperativity) varies with methoxy group substitution. In contrast, mouse DHFR has a weaker binding affinity for diaminobenzylpyrimidines, and none of the analogues show strong NADPH cooperative effects. The difference in the magnitude of NADPH/TMP cooperativity between bacterial and mammalian DHFR is an important factor in selectivity. The E. coli enzyme binds TMP more avidly in binary complex, and an additional selectivity factor of 30-fold arises from differences in cooperativity. Although the X-ray crystal structures of bacterial and vertebrate DHFR have been studied extensively, no single hypothesis convincingly explains the molecular basis of TMP selectivity. However, information on the three-dimensional structure of the enzyme has been used to rationally design novel, high-affinity inhibitors.
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PMID:Basis of selectivity of antibacterial diaminopyrimidines. 819 30

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg83b27/vgBG heterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.
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PMID:The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis. 823 10

The vestigial (vg) gene of Drosophila melanogaster encodes a nuclear protein which plays a key role in wing formation but is also involved in other developmental processes. We have previously shown that depletion of the dTMP pool by aminopterin, an inhibitor of the enzyme dihydrofolate reductase, or by fluorodeoxyuridine, an inhibitor of thymidylate synthetase, induces nicks in the wings of wild-type flies and a strong vg phenotype in vgBG/+ flies and also in individuals heterozygous for a deficiency of the vg locus (vgB/+). Furthermore, specific alterations of the vg locus, caused by intronic insertions, are associated with resistance to these drugs. In this paper, we show that: (1) depletion of the dTMP pool by aminopterin leads to a decrease in the amount of vg transcripts; (2) insertion of the retrotransposon 412 in the vgBG mutant, which is resistant to aminopterin, leads to the formation of a truncated transcript that is prematurely terminated in the long terminal repeat of this transposable element; and (3) aminopterin also affects the level of this truncated transcript. These results indicate that alterations of the wing by inhibitors of dTMP synthesis are caused by an effect of these drugs on levels of vg transcripts; the resistance to such agents observed for the vgBG strain is not due to a qualitatively different effect of this drug on the vg transcript but, rather, is related to the expression of a modified Vg protein encoded by a truncated transcript. These results are compatible with a role for vestigial in modulating cell proliferation.
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PMID:Vestigial gene expression in Drosophila melanogaster is modulated by the dTMP pool. 862 52

We have expressed catalytically active Toxoplasma gondii dihydrofolate-thymidylate synthase (DHFR-TS) and the individual TS and DHFR domains in Escherichia coli using the T7 promoter of pET-15b. DHFR-TS constituted approximately 10% of the total soluble cell protein and was purified using methotrexate-Sepharose chromatography to yield 10 mg of homogeneous DHFR-TS per liter of culture. The DHFR domain was recovered as insoluble inclusion bodies which could be unfolded and refolded to recover soluble, active enzyme. The TS domain was overexpressed as a soluble protein by growing the cells at 24 degrees C; this is the first report of the expression of an active TS domain from a bifunctional enzyme. The kcat and K(m) values for DHFR-TS are similar to those of other previously characterized protozoan DHFRs and TSs. The antimicrobial antifolates, TMP and Pyr, inhibit DHFR activity of the bifunctional protein in accord with their effects in crude enzyme preparations and in vivo systems. Kinetic parameters and Ki values for TMP and Pyr with the isolated DHFR domain were identical to the values for DHFR in the bifunctional enzyme. Evidence of kinetic channeling of the dihydrofolate product of TS to the DHFR domain in the bifunctional enzyme was obtained by kinetic and inhibition studies. Properties such as yield, stability, and activities of the recombinant T. gondii DHFR-TS provide clear advantages over other bifunctional DHFR-TSs as a model for future studies.
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PMID:Heterologous expression and characterization of the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of Toxoplasma gondii. 863 82

Classical and nonclassical isosteric C8-N9 bridged analogues of the multitargeted antifolate LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and as antitumor and antiopportunistic infection agents. The syntheses of the analogues were accomplished by reductive amination of the appropriate anilines with 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (28) followed by saponification of the ethyl esters, for the classical analogue 6. The N9-methyl analogues were obtained from the N9-H precursors by reductive methylation. In general, the nonclassical compounds 7-17 were similar in potency to TMP against Toxoplasma gondii DHFR, with selectivity ratios greater than 38 and 21 for 11 and 16, respectively. These compounds were poor inhibitors of Pneumocystis carinii DHFR and rat liver DHFR. The nonclassical analogues were also inactive against TS. The classical analogue 6 was a marginal inhibitor of isolated human TS (IC50 = 46 microM) and of human DHFR (IC50 = 10 microM), however, it was a potent inhibitor of the growth of two human head and neck squamous cell carcinoma cell lines and of CCRF-CEM human lymphoblastic leukemia cells in culture and was similar to LY231514 against ZR-75-1 human breast carcinoma cell line. Evaluation of 6 against MTX-resistant sublines indicated that DHFR is not the major target of 6. Metabolite protection studies of the growth inhibitory activity of 6 suggest that TS is a major target of this drug and that polyglutamyl forms of 6 may serve as the intracellular TS inhibitors. These studies also suggest that 6 has a site of action in addition to sites in the folate pathway.
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PMID:Synthesis, antifolate, and antitumor activities of classical and nonclassical 2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines. 1138 44

Novel 2,4-diaminopyrimidines bearing N,N-disubstituted aminomethyl residues at the 5-position were designed as dihydrofolate reductase (DHFR) inhibitors. These compounds were obtained by treatment of 1-[(2,4-diamino-5-pyrimidinyl)methyl]pyridinium bromide with secondary amines in a polar solvent and in the presence of triethylamine at room temperature. The procedure was found to be very efficient and suitable for application in high-throughput synthesis. In addition, we found that high-throughput screening for enzymatic and in vitro antibacterial activity could be performed on crude reaction mixtures, thus avoiding any purification step. Over 1200 proprietary secondary amines were selected for high-throughput synthesis, based on structural and diversity-related criteria, and the resulting products were submitted to high-throughput screening. A greater number of hits, and significantly more active compounds, were obtained through structure-based library design than through diversity-based library design. Different classes of inhibitors of DHFR were identified in this way, including compounds derived from di-, tri-, and tetracyclic amines. In general, these products showed high activity against the enzymes derived from both TMP-sensitive and TMP-resistant Streptococcus pneumoniae. Some compounds possessed appreciable selectivity for the bacterial over the human enzyme, whereas other compounds were not at all selective. In most cases, active enzyme inhibitors also displayed antibacterial activity.
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PMID:Novel dihydrofolate reductase inhibitors. Structure-based versus diversity-based library design and high-throughput synthesis and screening. 1277 35


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