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)

Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).
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PMID:Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs. 23 97

Interaction of several representative folate, quinazoline and pyridine nucleotide derivatives with dihydrofolate reductase from amethopterin-resistant Lactobacillus casei induces dramatic changes in its circular dichroic spectral properties. The binding of dihydrofolate induces a large extrinsic Cotton effect at 295 nm ([theta] = 113 800 deg . cm2 . dm-1). The generation of this band by dihydrofolate is strictly dependent on complex formation with a single substrate binding site and a KD = 7 . 10(-6) M. The other binary complexes examined include the enzyme . NADPH, enzyme . amethopterin, enzyme . folate, and enzyme . methasquin. All such complexes differ in spectral detail, the negative ellipticity at 330 nm being characteristic of the "folate site" complexes. The circular dichroic spectrum of the ternary complex of reductase . NADPH . methotrexate shows a positive symmetrical band centered at 360 nm ([theta] - 32 000 deg . cm2 . dm-1). Since both of the corresponding binary complexes exhibit negative bands in this region, this induced band represents a unique molecular property of the ternary complex. Chemical modification of a single tryptophan residue of the enzyme, as determined from magnetic circular dichroism spectra, results in a complete loss in the ability to bind either dihydrofolate or NADPH.
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PMID:Binary and ternary complexes of dihydrofolate reductase with substrates, coenzymes and inhibitors. Circular dichroic and magnetic circular dichroic studies. 41 36

A new method is presented for calculating a type of quantitative structure-activity relationship, given experimental data on the binding affinity of a series of ligands to a receptor site on a protein. All ligands are presumed to have known chemical structure but may be conformationally flexible, and all are presumed to bind to the same, single, fairly rigid site of the (pure) receptor protein molecule. Given the experimentally determined free energies of binding of the ligand molecules, possible binding sites are deduced in terms of geometry and the chemical character of the various parts of the site. A test of the method is given for a series of chymotrypsin inhibitors and for a series of dihydrofolate reductase inhibitors. The proposed dihydrofolate reductase site suggests that a quinazoline inhibitor may rock between two different binding nodes depending on the pK of the ring N(1).
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PMID:Distance geometry approach to rationalizing binding data. 49 May 43

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

An important unresolved issue in antifolate pharmacology is the basis for the observation that the major portion of cellular tetrahydrofolate cofactors is preserved after dihydrofolate reductase activity is abolished by antifolates despite the fact that tetrahydrofolate cofactor-dependent purine and pyrimidine biosynthesis ceases. This has been attributed to feedback inhibition of thymidylate synthase by dihydrofolate polyglutamates that accumulate in the presence of antifolates. This report combines network thermodynamic modeling and experimental observations to evaluate the effects of direct inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site with a potent lipophilic quinazoline antifolate PD130883 on folate oxidation in cells. Computer simulations predict and the data indicate that marked PD130883 suppression of thymidylate synthase only slows the rate but not the extent of tetrahydrofolate cofactor interconversion to dihydrofolate upon complete suppression of dihydrofolate reductase with trimetrexate. These observations are consistent with earlier studies from this laboratory with fluorodeoxyuridine inhibition at the deoxyuridylate binding site. Hence, the much weaker inhibition by dihydrofolate polyglutamates at the level of thymidylate synthase cannot account for the apparent preservation of tetrahydrofolate cofactor pools in cells and has virtually no pharmacologic significance under conditions in which antifolates completely suppress dihydrofolate reductase. The extent of interconversion of tetrahydrofolate cofactors to dihydrofolate is strongly influenced by residual dihydrofolate reductase catalytic activity. Exposure of cells to 0.1 microM trimetrexate results in only approximately 60% of maximum dihydrofolate levels achieved when dihydrofolate reductase activity is abolished. Network thermodynamic simulations predict, and experiments verify, that inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate site by PD130883, when dihydrofolate reductase is only partially suppressed (approximately 85%) with 0.1 microM trimetrexate, substantially decreases (31-47%) the net level of interconversion of tetrahydrofolate cofactors to dihydrofolate. Further computer simulations predict that under conditions in which residual dihydrofolate reductase activity persists within the cells (more than about 5%), feedback inhibitory effects of dihydrofolate polyglutamates as well as other weak inhibitors of thymidylate synthase can significantly limit the extent of net interconversion of tetrahydrofolate cofactors to dihydrofolate and produce an apparent "compartmentation phenomenon" in which tetrahydrofolate cofactor pools are preserved within the cell in the presence of antifolates. Residual dihydrofolate reductase activity cannot, however, account for the partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure to high trimetrexate or methotrexate levels.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effect of direct suppression of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site on the interconversion of tetrahydrofolate cofactors to dihydrofolate by antifolates. Influence of degree of dihydrofolate reductase inhibition. 182 52

5,10-Dideazatetrahydrofolate (DDATHF) is an antifolate drug, the cytotoxic effects of which can be fully reversed by hypoxanthine, suggesting that DDATHF exerts its effects by inhibiting de novo purine biosynthesis. ICI198583 is a quinazoline based inhibitor of thymidylate synthase. In this study we examine the interaction between treatment of mouse leukaemic L1210 cells with these drugs. The addition of DDATHF with ICI198583 was correlated with a decrease in ICI198583 cytotoxicity in a dose dependent manner. This protection was associated with a decrease in DNA fragmentation, and a drop in intracellular dATP pools. These results support the hypothesis that inhibitory effects on de novo purine biosynthesis by inhibitors of dihydrofolate reductase may limit cytotoxicity, and indicate that a rise in dATP pools may be an important cytotoxic signal.
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PMID:Inhibition of 2-desamino-2-methyl-10-propagyl-5,8-dideazafolic acid cytotoxicity by 5,10-dideazatetrahydrofolate in L1210 cells with decrease in DNA fragmentation and deoxyadenosine triphosphate pools. 185 63

The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.
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PMID:Synthesis and in vitro biological activity of new deaza analogues of folic acid, aminopterin, and methotrexate with an L-ornithine side chain. 201 22

The synthesis of 2'-fluoro-10-propargyl-5,8-dideazafolic acid and its 2-desamino, 2-desamino-2-hydroxymethyl, and 2-desamino-2-methoxy analogues is described. In general the synthetic route involved the coupling of diethyl N-[2-fluoro-4-(prop-2-ynylamino)benzoyl]-L-glutamate with the appropriate 6-(bromomethyl)quinazoline followed by deprotection with mild alkali. These four compounds together with the 2-desamino-2-methyl analogue were tested for their activity against L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of the L1210 cell line and of two mutant L1210 cell lines, the L1210:R7A that overproduces dihydrofolate reductase (DHFR) and the L1210:1565 that has impaired uptake of reduced folates. Compared with their non-fluorinated parent compounds, the 2'-fluoro analogues were all approximately 2-fold more potent as TS inhibitors. Similarly, they also showed improved inhibition of L1210 cell growth (1.5-5-fold), and this activity was prevented by co-incubation with thymidine. All had retained or improved activity against both the L1210:R7A and L1210:1565 cell lines.
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PMID:Quinazoline antifolate thymidylate synthase inhibitors: 2'-fluoro-N10-propargyl-5,8-dideazafolic acid and derivatives with modifications in the C2 position. 223 7

Several structurally related series of folate analogs were studied as substrates for mouse liver folylpolyglutamate synthetase (FPGS). A comparison of the kinetics of the interaction of this enzyme with folate analogs that contained the quinazoline ring in place of the pteridine ring with those of the analogous pteridines demonstrated that the quinazoline derivatives were more efficient substrates for and tighter binding inhibitors of this enzyme. A series of 2,4-diaminopyrimidine dihydrofolate reductase inhibitors were found to be substrates for FPGS; these are the first known compounds without a fused ring system analogous to the pteridine ring of the folate molecule that are substrates for FPGS. Several 5,8-dideazafolate derivatives that lack the 2-amino group had activity as substrates for FPGS equivalent to that of the corresponding 5,8-dideazafolates. When a homologous series of 5,8-dideazafolic acid analogs with hydrocarbon substituents on the 10-nitrogen were studied, these substituents were found to diminish the efficiency of utilization of these analogs as substrates for FPGS; this effect increased with increasing chain length of the hydrocarbon. It was concluded that neither the 2-amino group nor an intact pyrazine ring of folates and folate analogs are essential for the binding of folates to the active site of mouse liver FPGS but that the pyrazine ring probably serves to position other regions of the folate molecule that interact with amino acid residues in the active site. It was also inferred from these observations that the volume within the active site of FPGS above/below the pyrazine ring or near the 10-position of folate derivatives are regions of limited bulk tolerance; binding of folate analogs with substituents at these positions probably distorts the active site.
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PMID:Relative substrate activities of structurally related pteridine, quinazoline, and pyrimidine analogs for mouse liver folylpolyglutamate synthetase. 258 90

2,4-Diaminoquinazoline analogs of folate were assessed as antileishmanial agents and as dihydrofolate reductase inhibitors. Against Leishmania major in human macrophages in vitro, two compounds with tertiary amines attached directly to the quinazoline ring were remarkably active. The 50% effective doses were in the picogram per milliliter range (12 to 91 pg/ml), and the in vitro therapeutic indices were approximately 10(5). These compounds were 1,000 times more active on an absolute basis and had a 100 times more favorable therapeutic index than any compound previously tested in this model. Antileishmanial activity was not correlated with activity against Leishmania mexicana promastigote reductase, which suggests that folate utilization in general, rather than reductase activity specifically, was being inhibited.
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PMID:Antileishmanial activities of 2,4-diaminoquinazoline putative dihydrofolate reductase inhibitors. 261 Apr 96


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