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)

Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.
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PMID:Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase. 251 77

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.
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PMID:The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase. 252 27

Folate analogs that inhibit dihydrofolate reductase result in only partial interconversion of tetrahydrofolate cofactors to dihydrofolate with preservation of the major portion of reduced cellular folate cofactors in L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of dihydrofolate polyglutamates that accumulate in the presence of antifolates block thymidylate synthase to prevent depletion of reduced folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in purine and pyrimidine biosynthesis in L1210 murine leukemia cells exposed to antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM trimetrexate there is an almost instantaneous inhibition of [3H] deoxyuridine or [14C]formate incorporation into nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total folate pool. Pretreatment of cells with fluorodeoxyuridine, to inhibit thymidylate synthase by about 95% followed by trimetrexate only slows the rate of folate interconversion (t1/2 approximately 25 min) but not the final dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of thymidylate synthase by 97, 98, and 99% should increase the half-time of dihydrofolate rise after trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal thymidylate synthase activity. The data reflect the high degree of catalytic activity of thymidylate synthase relative to tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this enzyme that is necessary to slow the rate of folate interconversions after addition of antifolates. The model predicts, and the data demonstrate, that virtually any residual thymidylate synthase activity will permit the interconversion of all tetrahydrofolate cofactors available for oxidation to dihydrofolate when dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of tetrahydrofolate-dependent purine and pyrimidine biosynthesis after antifolates in these cells can be accounted for solely on the basis of tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of thymidylate synthase by dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after antifolates, can account for the rapid but partial interconversion of reduced folate cofactors to dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates. Experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamylates in antifolate action in cells. 252 54

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from the monogenetic kinetoplastid protozoan, Crithidia fasciculata, was isolated and characterized. The gene is located on a single chromosome of approximately one megabase, and shows significant sequence similarity to other eukaryotic and prokaryotic DHFR and TS genes. There is a single low-abundance polyadenylated DHFR-TS transcript of approximately 3100 nt. One major miniexon splice site was identified by primer extension analysis. The 5' flanking region of the gene is divergently transcribed and shows strong similarities to a consensus DHFR promoter as well as to other eukaryotic 'housekeeping' gene promoter regions. A sequence downstream of the DHFR promoter consensus region is complementary to the 3' end of the C. fasciculata miniexon-derived RNA. This suggests a means by which the two separately transcribed RNAs may be juxtaposed for trans-splicing. In the 3' flanking region of the DHFR-TS gene, there is a sequence that is present in all of the chromosomes from this species and also from Leishmania tarentolae.
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PMID:Structure, genomic organization and transcription of the bifunctional dihydrofolate reductase-thymidylate synthase gene from Crithidia fasciculata. 254 Apr 36

Treatment of H35 hepatoma cells with the lipid soluble dihydrofolate reductase inhibitors metoprine and trimetrexate cause a nearly 10-fold increase in the toxicity of the antipyrimidine folate analogue PDDF and the antipurine folate analogue DDATHF. Evaluation of these interactions by the combination index developed by Chou (17-20) yields results conforming to synergistic interactions. The capacity of PDDF to inhibit thymidylate synthase in intact cells as measured by tritium release from [5-3H]deoxyuridine was increased by approximately the same amount by preincubation with the dihydrofolate reductase inhibitors. The primary effect of the reductase inhibitors in causing greater activity may be a reduction in cellular folates which can cause 5,10-CH2H4PteGlun to decrease and cellular PDDF (polyglutamates) to increase. These conditions would favor inhibition of thymidylate synthase by PDDF by promoting formation of the stable, inhibited PDDF (polyglutamates)-thymidylate synthase-dUMP complex (12).
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PMID:The enhancement of the activity of 10-propargyl-5,8-dideazafolate and 5,10-dideazatetrahydrofolate by inhibitors of dihydrofolate reductase. 262 72

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.
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PMID:Isolation and expression of the Pneumocystis carinii thymidylate synthase gene. 267 92

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.
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PMID:Analysis of T4 bacteriophage deletion mutants that lack td and frd genes. 267 2

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.
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PMID:Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum. 267 19

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinii DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinii DHFR and, in fact, is a more potent inhibitor of human DHFR.
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PMID:Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene. 268 53

The classic inhibitor of dihydrofolate reductase (DHFR), methotrexate (MTX), has been shown to be an effective inducer of the differentiation of HL-60 promyelocytic leukemia cells (Bodner A.J. et al.; J. Natl. Cancer Inst. 67:1025-1030; 1981). We have obtained evidence that induction of the differentiation of these cells by MTX, as well as by other folic acid antagonists, is the result of the effects of these agents on purine and thymine nucleotide biosynthesis. Thymidine (10 microM) completely blocked both the cytotoxicity and induction of differentiation produced by the specific inhibitor of thymidylate synthase (TS), N10-propargyl-5,8-dideazafolic acid (CB-3717). Thymidine also blocked the acute cytotoxicity caused by MTX and trimetrexate (TMQ); the induction of differentiation and the loss of proliferative capacity, however, were only partially prevented by thymidine. Hypoxanthine (100 microM), which completely restored antifolate-depleted purine nucleotide levels, had no effect on either the cytotoxicity or the induction of maturation produced by these agents. The growth inhibitory effects and the induction of differentiation caused by dideazatetrahydrofolic acid (DDATHF), which acts on de novo purine nucleotide biosynthesis rather than on DHFR or TS, was completely prevented by hypoxanthine. Hypoxanthine also completely prevented the inhibition of cellular replication and induction of differentiation by MTX and TMQ when combined with thymidine. The findings suggest that the depletion of intracellular thymine nucleotide levels by the antifolates, MTX, TMQ, and CB-3717 is the primary event involved in the maturation of HL-60 leukemia cells produced by these agents and that maturation occurs concomitantly with a high level of cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of the induction of the differentiation of HL-60 leukemia cells by antifolates. 270 Sep 13


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