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)

Pneumocystis carinii and Toxoplasma gondii are opportunistic pathogens of immunosuppressed patients that are susceptible to therapy with inhibitors of dihydrofolate reductase (DHFR). The DHFR of these two organisms was characterized to facilitate the identification of more selective inhibitors. Similar to all reported protozoa, T. gondii has a bifunctional enzyme, of 120,000 Da, that possesses both DHFR and thymidylate synthase (TS) activity. Unexpectedly, P. carinii DHFR activity was present on a small molecule, of 26,000 Da. T. gondii DHFR and TS activity coeluted during affinity chromatography using a methotrexate-Sepharose column, whereas P. carinii DHFR and TS activity could be separated by affinity chromatography using the same column. P. carinii DHFR could be easily distinguished from rat DHFR, which is similar in size, by the differences in Km for dihydrofolate (P. carinii, 17.6 +/- 3.9 microM; rat, 4.0 +/- 2.2 microM). Since all protozoa reported have a large molecular weight, bifunctional DHFR, these studies support the classification of P. carinii as a fungus. These studies also provide a basis for the development of more effective therapeutic agents for these pathogens.
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PMID:Characterization of dihydrofolate reductase of Pneumocystis carinii and Toxoplasma gondii. 235 16

The synthesis and biological activity of two series of nonclassical thymidylate synthase (TS) inhibitors are described. The first is a series of 10-propargyl-5,8-dideazafolic acid derivatives (10a-j) and the second is a series of the analogous 2-desamino derivatives (13a-c,k), both bearing a more lipophilic substituent on the phenyl ring than the CO-glutamate of classical antifolates. Compounds 10a-j were prepared in a straightforward manner, generally by treatment of N-[6-(bromomethyl)-3,4-dihydro-4-oxo-2-quinazolinyl]-2,2-dimethylprop anamide (6) with various phenyl-substituted N-propargylanilines (8), followed by deprotection. Compounds 13a-c,k were prepared similarly from [6-(bromomethyl)-4-oxo-3(4H)-quinazolinyl] methyl 2,2-dimethylpropanoate (11). The compounds were tested for inhibition of purified L1210 TS and for inhibition of L1210 cell growth in vitro. Several of these nonclassical analogues approached the TS inhibitory potency of 10-propargyl-5,8-dideazafolic acid (1, CB3717), a glutamate-containing TS inhibitor. 2-Amino target compounds 10a-j were generally potent inhibitors of L1210 TS, with IC50s within the range of 0.51-11.5 microM, compared to 0.05 microM for 1. The order of potency for phenyl substitution at the 4-position in this series was the following: COCF3 greater than or equal to NO2 greater than or equal to CONH2 greater than or equal to COCH3 greater than SO2NMe2 greater than CN much greater than OCF3 greater than or equal to F. The 2-desamino target compounds 13a-c,k also exhibited significant, although diminished, TS inhibition. Both series were growth inhibitory to cells in tissue culture and this inhibition could be reversed by thymidine alone, indicating that the primary target was TS. None of the compounds was a potent inhibitor of dihydrofolate reductase. These studies indicate that the presence of the glutamate moiety in folate analogues is not an absolute requirement for potent inhibition of TS.
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PMID:Potent inhibition of thymidylate synthase by two series of nonclassical quinazolines. 236 85

The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.
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PMID:Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase. 236 90

We examined the in vitro activity of 2-desamino-5,8-dideazafolate and 2-desamino-N10-propargyl-5,8-dideazafolate (desamino-CB3717), the more water-soluble 2-desamino analogues of 5,8-dideazafolate and N10-propargyl-5,8-dideazafolic acid (CB3717). We report Ki values for the inhibition of L1210 thymidylate synthase (TS) of 2 and 0.027 microM for 2-desamino-5,8-dideazafolate and desamino-CB3717, respectively, indicating a 30- and 10-fold loss in TS-inhibitory activity compared with the corresponding 2-NH2 compounds. The synthetic tri- and tetrapolyglutamate derivatives of desamino-CB3717 were 66- and 101-fold more potent than the monoglutamate form as inhibitors of TS. Both desamino compounds were more potent as inhibitors of L1210 and W1L2 cell growth than were their 2-amino counterparts. 2-Desamino-5,8-dideazafolate retains quite good activity against both the TS-overproducing W1L2:C1 line and the L1210 cell line grown in the presence of thymidine, suggesting that a secondary locus of action may be involved. This other target is a folate-dependent enzyme as evidenced by the protection of the inhibition of cell growth by the addition of hypoxanthine or folinic acid together with thymidine. The methotrexate-resistant, dihydrofolate reductase-overproducing L1210:R7A cell line is cross-resistant to 2-desamino-5,8-dideazafolate, which suggests that dihydrofolate reductase is the other target. An L1210 subline (1565) unable to transport reduced folates is 10-fold resistant to desamino-CB3717 and 2-desamino-5,8-dideazafolate but is not cross-resistant to CB3717 or 5,8-dideazafolate. The removal of the 2-amino function of CB3717 did not affect folylpolyglutamate synthetase substrate activity (CB3717 Km = 48 microM, desamino-CB3717 Km = 40 microM). However, both 5,8-dideazafolate and its desamino analogue were about 10-fold better substrates for folylpolyglutamate synthase than were the N10-propargyl compounds, and this may contribute to their good growth-inhibitory properties. In vivo, desamino-CB3717 cured approximately 75% of mice bearing the L1210:ICR tumor at doses of 50 mg/kg daily for 5 days and above (maximum tolerated dose greater than 1000 mg/kg daily for 5 days).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activity of the thymidylate synthase inhibitor 2-desamino-N10-propargyl-5,8-dideazafolic acid and related compounds in murine (L1210) and human (W1L2) systems in vitro and in L1210 in vivo. 238 29

The nucleotide sequence of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene in pyrimethamine-resistant (PyrR) mutants of Plasmodium falciparum selected in vitro was examined to determine if specific mutations in DHFR were associated with drug resistance. We analysed the sequence of genomic DNA from strain FCR3, from eight previously isolated PyrR parasites derived from FCR3, and from strain Honduras-1. We found that: (1) five PyrR FCR3 mutants, FCR3-D4-D8, had an identical nucleotide change and a novel single amino acid change (Phe to Ser) at amino acid 223 of DHFR; (2) our originally reported nucleotide sequence of the DHFR-TS gene was of the PyrR strain Honduras-1, and was not of FCR3; (3) three PyrR mutants, FCR3-D1, D2, and D3, thought to have been derived from the FCR3 strain, were in fact isolates of Honduras-1. We also examined the chromosomal DNA of PyrR mutants by pulsed-field gradient gel (PFG) electrophoresis. The PyrR mutants FCR3-D1, D2, and D3 had several chromosome size polymorphisms compared to FCR3. In two of the PyrR FCR3 mutants, FCR3-D7 and D8, the chromosome 4-size DNA of FCR3 that the DHFR-TS probe normally hybridised to was not observed. Instead, in FCR3-D7, a chromosome larger than the chromosome 4-size DNA was observed to hybridise to the DHFR-TS probe. In FCR3-D8, two chromosomes that hybridised to the DHFR-TS probe were found. One of them was larger than FCR-3 chromosome 4-size DNA, and the other was smaller than FCR3 chromosome 1-size DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dihydrofolate reductase mutations and chromosomal changes associated with pyrimethamine resistance of Plasmodium falciparum. 240 91

Leishmania tropica promastigotes selected for resistance to the dihydrofolate reductase inhibitor, methotrexate, or the thymidylate synthase inhibitor, 5,8-dideaza-10-propargyl folate, overproduce a bifunctional thymidylate synthase-dihydrofolate reductase and possess a 30-kilobase region of amplified DNA. Five fragments, resulting from BglII digestion of this amplified DNA, were cloned into vectors and utilized as probes to examine mRNA in these organisms. Four mRNA species which hybridize to the amplified DNA sequences were found in both resistant and wild-type Leishmania, but were about 40-fold more abundant in the drug-resistant cells. Three of the four mRNAs are transcribed from the same strand of DNA, are clustered, and appear to have partial overlapping sequences. The thymidylate synthase-dihydrofolate reductase gene was localized to a specific region of the amplified unit of DNA by hybridization with mouse cDNA containing thymidylate synthase sequences and with a synthetic oligonucleotide 41 nucleotides in length, prepared on the basis of the partial amino acid sequence of the Leishmania enzyme. Furthermore, mRNA hybrid-selected using a plasmid containing sequences of the putative gene was shown to direct in vitro synthesis of the bifunctional protein.
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PMID:DNA amplification in antifolate-resistant Leishmania. The thymidylate synthase-dihydrofolate reductase gene and abundant mRNAs. 240 75

We have characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with less than 0.1 microM MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 microM) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 microM) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 microM drug. Two cell lines with resistance to 10 microM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 microM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.
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PMID:Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate. 241 16

Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.
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PMID:Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid. 242 90

The cytogenetic study of colorectal carcinomas is consistent with the following sequence in the tumor evolution: rearrangement of chromosome 17 with loss of 17p and often gain of 17q, loss of chromosome 18, frequent del(5q), frequent del(1p) correlated with the gain of an early replicating X. At least one gene directly involved in nucleotide synthesis, especially in the de novo pathways for thymidine is located on each of these chromosomes or chromosomes segments. A model established on the gene dosage effect, which likely results of these chromosome imbalances, may be proposed: (1) increase of thymidine kinase activity (chromosome 17q) and thus of the salvage pathway of thymidine synthesis (2) decrease of thymidine de novo pathways by decreased of thymidylate synthase (chromosome 18) and of dihydrofolate reductase (chromosome 5q) and thus accumulation of 2'-deoxyuridine-5'-P, which saves 2'-deoxycytidine 5'-P (3) decrease of cytidylate (or uridylate) kinase (chromosome 1p) and thus accumulation of 2-deoxycytidine-5-PP and of uridine-5-P (UMP) decreasing the metabolisation of orotidine-5'-P, precursor of 2-deoxycytidine-5-PP, which (4) saves -D-5-ribosyl-PP (PRPP) or even conversion of orotidine-5'-P in PRPP. The later is the immediate precursor of nucleotides in their major salvage pathways synthesis: PRPP + base----nucleotide + PPi. This reaction which would be much activated needs hypoxanthine phosphorybosyl transferase (HPRT). Its gene is carried by chromosome X which is here duplicated in its active early replicating form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of increased salvage pathways of nucleotide synthesis by dosage effect due to chromosome imbalances may be fundamental in carcinogenesis: the example of colorectal carcinoma. 242 58

A series of "stretched" methotrexate (MTX) analogues containing up to five 4-aminobutyryl (Gab) spacers between the 4-amino-4-deoxy-N10-methylpteroyl (MeAPA) moiety and the glutamate (Glu) side chain was prepared. Interest in these compounds stemmed from their relationship to MTX gamma-polyglutamates, from which they differ only in lacking "internal" alpha-carboxyl groups. The ability of the MeAPA-Gabn-Glu derivatives to inhibit dihydrofolate reductase (DHFR) and thymidylate synthase (TS) in vitro and to inhibit the growth of tumor cells in culture was evaluated. The IC50 for DHFR inhibition increased progressively from 0.082 to 0.84 microM as the number of Gab spacers was varied from one to five. At the same time the introduction of Gab spacers was found to produce substantial TS inhibition (Ki 0.1-0.4 microM) similar to that reported for MTX polyglutamates. Despite the activity of the MeAPA-Gabn-Glu derivatives as combined inhibitors of TS and DHFR, there was a steep loss of cell growth inhibitory potency as the number of Gab spacers was increased. This most likely reflects low cell uptake and the fact that when n greater than 1 there is almost total abolition of substrate activity for folylpolyglutamate synthetase, which had previously been observed with n = 1.
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PMID:Methotrexate analogues. 29. Effect of gamma-aminobutyric acid spacers between the pteroyl and glutamate moieties on enzyme binding and cell growth inhibition. 242 79


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