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 transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.
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PMID:Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate. 985 20

In an effort to develop a gene-dependent enzyme/prodrug therapy (GDEPT) for tumor-specific delivery of methotrexate (MTX) we have chosen to construct mutant forms of carboxypeptidase A1 (CPA) that circumvent the requirement for trypsin-dependent activation. The basis of this strategy is that methotrexate-alpha-peptides are inefficient substrates for the reduced folate carrier (RFC) and hence cannot be internalized by cells. However, the blocking amino acid can be cleaved by CPA to liberate MTX, which is then internalized by the RFC, resulting in inhibition of dihydrofolate reductase and cytotoxicity. A battery of mutant CPAs was generated, in which the putative trypsin cleavage sites in the propeptide were mutated to the consensus recognition sequence for mammalian subtilisin-like propeptidases. These mutant forms of CPA were evaluated for expression, activation, and catalytic activity by transiently transfecting them into COS-1 cells both in the absence and in the presence of cotransfected propeptidases. CPA95 was identified as the most efficiently cleaved mutant, and further studies of this mutant indicated that the endogenously activated enzyme had kinetic parameters identical to those of the trypsin-activated wild-type protein. In addition, endogenously activated CPA95 could effectively sensitize cells to MTX-Phe in culture, decreasing the IC50 of MTX-Phe from 25- to 250-fold in squamous cell carcinoma cells expressing active CPA as compared with the parental lines.
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PMID:Toward an enzyme/prodrug strategy for cancer gene therapy: endogenous activation of carboxypeptidase A mutants by the PACE/Furin family of propeptidases. 1002 48

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.
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PMID:Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+. 1019 48

The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase. To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain. Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP). However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP. Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfr promoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter.
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PMID:Activation of the murine dihydrofolate reductase promoter by E2F1. A requirement for CBP recruitment. 1033 93

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.
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PMID:Efficient introduction of aryl bromide functionality into proteins in vivo. 1066 52

Cyromazine, an insect growth regulator, affects larval and pupal cuticles in dipterans and some other insects. The mode of action of this aminotriazine is not known yet, though it has been shown not to inhibit the synthesis of chitin and cuticular proteins. Cyromazine may, however, act on some step(s) of sclerotization of the cuticle. In the present study, we have analyzed the key enzyme for the production of sclerotization agents, phenylalanine hydroxylase (PAH), using the enzyme from Drosophila, a cyromazine-sensitive insect. PAH was studied in vitro with cyromazine and three biologically less active derivatives at concentrations ranging from 1 microM to 1 mM. None of the compounds did significantly affect PAH activity. Nor did cyromazine, fed to last instar larvae of Musca domestica, change the relative content of phenylalanine and tyrosine, or the total amount and profile of amino acids of puparial cuticles, which showed a larviform shape typical for cyromazine intoxication. Taken together, this study does not support the hypothesis that phenylalanine hydroxylase represents a target site of cyromazine. In additional studies, the conflicting results, as reported by others, on in vitro inhibition of dihydrofolate reductase (DHFR) by cyromazine were re-examined using the enzymes from larvae of the blowfly, Protophormia terraenovae, and from hen liver. There was no significant inhibition of either DHFR at 100 microM by cyromazine as well as by dicylanil, a pyrimidine analog that is biologically more active than cyromazine. In conclusion, the mode of action of cyromazine remains completely open.
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PMID:Candidate target mechanisms of the growth inhibitor cyromazine: studies of phenylalanine hydroxylase, puparial amino acids, and dihydrofolate reductase in dipteran insects. 1109 44

To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.
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PMID:Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase. 1153 21

N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithinyl]-L-phenylalanine (1), a carboxypeptidase A (CPA) cleavable prodrug was synthesized for use in an antibody directed strategy to improve the therapeutic selectivity of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (2), an extremely potent nonpoly-glutamatable DHFR inhibitor which is also highly cytotoxic. Compound 1 was shown by HPLC analysis to give a >99% yield of 2 upon incubation with bovine CPA (bCPA) for 20 min at 25 degrees C. In a spectrophotometric kinetic assay with 50 microM dihydrofolate as the competing substrate in the presence of 65 microM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a K(i) of 0.35 pM. In contrast, 1 without bCPA was a poor inhibitor of rhDHFR (K(i)>10 microM). In a 72 h growth inhibition assay against cultured CCRF-CEM human leukemic lymphoblasts, the growth inhibitory activities of 1+bCPA, 2+bCPA, and 2 alone were the same (IC(50) 1.3-1.4 nM), whereas 1 in the absence of bCPA was >100-fold less potent (IC(50) 155 nM).
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PMID:Synthesis and enzymatic activation of N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithiny]-L-phenylalanine, a candidate for antibody-directed enzyme prodrug therapy (ADEPT). 1181 34

The catalytic activity and ability to confer resistance to antifolates of Plasmodium falciparum dihydrofolate reductase (pfDHFR) through single and double mutations at Asp-54 and Phe-223 were investigated. A single Asp54Glu (D54E) mutation in the pfDHFR domain greatly decreased the catalytic activity of the enzyme and affected both the K(m) values for the substrate dihydrofolate and the K(i) values for pyrimethamine, cycloguanil and WR99210. The Phe223Ser (F223S) single mutant had unperturbed kinetics but had very poor affinity with the first two antifolates. The ability to confer high resistance to the antifolates of F223S enzyme was, however, abolished in the D54E+F223S double mutant enzyme. When D54E mutation was present together with the A16V+S108T double mutation, the effects on the K(m) values for the substrate dihydrofolate and the binding affinity of antifolates were much more pronounced. The severely impaired kinetics and poor activity observed in A16V+S108T+D54E enzyme could, however, be restored when F223S was introduced, while the binding affinities to the antifolates remained poor. The experimental findings can be explained with a model for substrate and inhibitor binding. Our data not only indicate the importance of Asp-54 of pfDHFR in catalysis and inhibitor binding, but also provide evidence that infer the potentially crucial function of the C-terminal portion of pfDHFR domain.
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PMID:Mutational analysis of Plasmodium falciparum dihydrofolate reductase: the role of aspartate 54 and phenylalanine 223 on catalytic activity and antifolate binding. 1203 52

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.
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PMID:Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin. 1270 99


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