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 point mutation at nucleotide 323 within the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum, which distinguishes pyrimethamine-sensitive from drug-resistant isolates, can be discriminated by the polymerase chain reaction using mutation-specific primers. The technique makes use of the principle that short oligonucleotides with a perfect match at their 3' ends, complementary to the mutation to be detected, will initiate the polymerization by Taq polymerase far more efficiently than primers with a single mismatch in this position. The Asn-108 codon was detected using a primer of 17 nucleotides with an adenosine at its 3' end, the Thr-108 codon with a 14-mer primer ending with a cytosine and the Ser-108 codon with a 16-mer containing guanidine at the critical 3' end. By selecting appropriate counterprimers, the size of the amplification products is either indicative of pyrimethamine-resistant parasites of the 7G8 type, or of pyrimethamine-sensitive parasites of the FCR-3 type or 3D7 type. The fragments obtained can be easily separated in a single lane after agarose gel electrophoresis. Coded P. falciparum DNA samples were typed unambiguously using these primers as were reconstituted parasitized blood samples stored as high salt lysates. Sensitivity, speed and specificity make this assay a realistic alternative to in vitro drug testing to monitor the resistance of P. falciparum to inhibitors of the dihydrofolate reductase.
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PMID:Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction. 218 8

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.
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PMID:Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene. 250 70

The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 A away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to greater than 45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 microM for H45Q (similar to wild type) but from 8 to 5000 microM for R44L.
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PMID:Effects of distal point-site mutations on the binding and catalysis of dihydrofolate reductase from Escherichia coli. 267 72

Dihydrofolate reductase with a molecular weight of 22,000 has been purified by salt precipitation and methotrexate-Sepharose affinity chromatography from mouse livers having a mean weight of 2.4 g each. When the same purification procedure was followed using livers with a mean weight of 1.4 g or less, a protein with a molecular weight of 27,500 co-purified with dihydrofolate reductase. This 27.5 kDa species was recovered with dihydrofolate reductase following a second passage through the affinity column and it reacted by Western immunoblotting with a monospecific polyclonal antiserum raised to the purified 22 kDa enzyme. The two proteins could not be separated in a native state to compare their functional activity, but the 27.5 kDa protein appeared to have catalytic reductase activity when assayed directly on the polyacrylamide gel following non-denaturing electrophoresis. The catalytic activity of the mixture of the purified proteins was stoichiometrically inhibited by a molar equivalent of methotrexate.
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PMID:Age-dependent expression of a novel protein in mouse liver immunologically and functionally homologous with dihydrofolate reductase. 280 23

A series of 2,4-diamino-5-aryl-6-ethylpyrimidines embracing basic substituents in the 5-aryl ring was synthesized and evaluated for inhibitory activity against rat liver dihydrofolate reductase (DHFR). Maximal enzyme inhibition was observed for compounds bearing a benzylamino (19) or N-alkylbenzylamino substituent (29 and 30) in the 4-position of the phenyl ring and a nitro group in the 3-position, the corresponding 3-amino, 3-azido, or unsubstituted analogues proving only weakly active or inactive as DHFR inhibitors. Selected compounds were also screened in vivo against a methotrexate-resistant tumor, the M5076 murine reticulosarcoma, and antitumor activity in general paralleled activity against DHFR, the (3,4-dichlorobenzyl)amino analogue 26 proving the least toxic compound to exhibit significant antitumor activity. The X-ray crystal structure of the ethanesulfonic acid salt of the N-methylbenzylamino compound 29 has been determined to facilitate future molecular modeling studies in this new series of DHFR inhibitors.
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PMID:Structural studies on bioactive compounds. 8. Synthesis, crystal structure, and biological properties of a new series of 2,4-diamino-5-aryl-6-ethylpyrimidine dihydrofolate reductase inhibitors with in vivo activity against a methotrexate-resistant tumor cell line. 281 Mar 35

The chromatin structure of the promoter region of the human dihydrofolate reductase gene was determined using a variety of nucleases including DNase I, micrococcal nuclease, several restriction endonucleases, exonuclease III, and Bal31. Two separate regions from -670 to -340 (the distal hypersensitive region) and from -170 to +150 (the proximal hypersensitive region) were shown to be essentially free of proteins as indicated by their accessibility to both endo- and exonucleases. Within the proximal hypersensitive region, one protein appears to be bound at the start site for transcription. A 170-base pair fragment between the two hypersensitive regions was highly resistant to all nucleases tested. Multiple barriers against exonuclease digestion and resistance to dissociation by high salt concentrations suggest that more than one protein is tightly bound to this region. The upstream sequence from -670 and the downstream sequence from +150 were shown to be packaged into nucleosomes. The selective accessibility of certain sites to micrococcal nuclease cutting indicates that the initial nucleosomes are phased upstream from the distal hypersensitive region. There appears to be a protein bound between the phased nucleosomes and the upstream boundary of the distal hypersensitive region. These results suggest that the normal nucleosome array is interrupted by about 900 base pairs of nucleosome-free DNA to which several nuclear proteins bind in a DNA sequence-specific manner.
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PMID:Chromatin structure of the human dihydrofolate reductase gene promoter. Multiple protein-binding sites. 300 70

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.
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PMID:Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. 324 60

The interaction of dihydrofolate reductase (DHFR) from Escherichia coli with drugs such as methotrexate (MTX) and 2,4-diamino-6,7-dimethylpteridine (DAM) has been studied by means of site-directed mutagenesis, fluorescence spectroscopy, and steady-state as well as transient kinetics. A strictly conserved residue at the dihydrofolate binding site of DHFR, phenylalanine-31, has been replaced with tyrosine or valine to ascertain the importance for binding of this hydrophobic amino acid, which interacts with both the pteridine ring and the p-aminobenzoyl moiety. The first mutation (Phe-31----Tyr) has a minimal effect on the binding of the classical inhibitor, DAM. On the other hand, the second mutation (Phe-31----Val) has increased the dissociation constant of DAM from the DHFR.NADPH.DAM ternary complex over 150-fold (greater than 3 kcal/mol). The dissociation constant of DAM from the (Val31-DHFR).DAM binary complex was too large to be measured fluorometrically. More importantly, these mutations have decreased the overall tight binding of MTX, from 100- to 140-fold (corresponding to a loss of binding energy of 2.2-2.4 kcal/mol) for the Tyr-31 and Val-31 mutants, respectively. These results indicate that hydrophobic interactions between MTX and DHFR are at least as important as formation of the MTX.DHFR salt bridge in the tight binding of MTX.
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PMID:Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase. 327 76

The importance of the ionic interaction due to the formation of the salt bridge between the Asp-27 and the pteridine ring in Escherichia coli dihydrofolate reductase-methotrexate complex has been studied by using the free-energy perturbation method. The calculation suggests that the ion-pair contribution to the binding energy is insignificant, as the enzyme surroundings do not stabilize the salt bridge to the extent of the desolvation of the charged groups. The activation barrier for the proton exchange between the pteridine ring and the Asp-27 is calculated to be 20.1 kcal/mol (1 cal = 4.184 J) by using the coordinate-coupled perturbation method, implying that this may be a channel to the proton exchange from the pteridine ring to the solvent. The Gibbs-energy difference of binding between the Asn-27 and Ser-27 is calculated to be 3.2 kcal/mol and is mainly due to the electrostatic interactions.
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PMID:Probing the salt bridge in the dihydrofolate reductase-methotrexate complex by using the coordinate-coupled free-energy perturbation method. 338 Jul 91

Two properties of the aromatic azido group have been exploited in the design of a novel dihydrofolate reductase inhibitor--lipophilicity and biotransformability. 2,4-Diamino-5-(3-azido-4-chlorophenyl)-6-ethylpyrimidine (MZP, 4), which can be prepared in three high-yielding synthetic steps from the antimalarial drug pyrimethamine (1), has a pKa of 7.19, a log P of 2.81 and inhibitory activity against the P388, L1210, B16 and M5076 tumours in vivo. The ethanesulphonic acid salt of MZP (MZPES, 6) is a potent inhibitor of rat liver dihydrofolate reductase (Ki 1.60 nM) and L1210 cells in vitro. Injections of MZPES in water (10 mg ml-1) at pH 4.1 can be sterilised by filtration and are stable provided they are protected from light.
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PMID:The aromatic azido group in anti-cancer drug design: application in the development of novel lipophilic dihydrofolate reductase inhibitors. 344 94


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