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)

A 693-base pair gene coding for the 27,132-dalton dihydrofolate reductase (DHFR) domain of the thymidylate synthase-dihydrofolate reductase (TS-DHFR) bifunctional protein of Plasmodium falciparum was designed to have Escherichia coli codon preference and multiple unique restriction sites and was chemically synthesized. The gene was overexpressed (> 50% total cellular protein) in E. coli as insoluble inclusion bodies which could be unfolded and refolded to recover soluble enzyme activity. The refolded DHFR was purified by methotrexate-Sepharose affinity chromatography to give the homogeneous enzyme. Active site titration with methotrexate revealed that the purified protein was fully active. The purified DHFR migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 30 kDa, and gel filtration showed that the protein is a monomer. The yield of purified enzyme was about 5-6 mg/liter of bacterial culture. Kinetic properties of the purified recombinant DHFR were similar to those reported for wild type bifunctional TS-DHFR. Cassette mutagenesis of the synthetic gene was performed to give the S108N and the N51I + S108N mutants which provided DHFRs analogous to pyrimethamine-resistant mutants found in nature.
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PMID:The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants. 840 15

Triazenyl-substituted pyrimethamine derivatives 10a-s have been prepared by coupling diazotized 2,4-diamino-5-(3-amino-4-chlorophenyl)-6-ethyl pyrimidine (1c) with a series of secondary amines in aqueous sodium carbonate solution. The triazenes which are stable and poorly soluble as free bases form more soluble, but unstable, salts with alkanesulfonic acids. The lead dimethyltriazene 2,4-diamino-5[4-chloro-3-(3,3-dimethyltriazen-1-yl)phenyl]-6-et hylpyrimidine (4a) forms a crystalline ethanesulfonic acid salt (solvated with 2-propanol), which is protonated at the pyrimidine N-1 position, as determined by X-ray crystallography. The ability of these new triazenes to inhibit Pneumocystis carinii dihydrofolate reductase in vitro has been compared to that of triazene 4a. The most potent and selective compound, 2,4-diamino-5-[3-[3-[2-(acetyloxy)ethyl]-3-benzyltriazen-1-y l]-4- chlorophenyl]-6-ethylpyrimidine (14a), has an IC50 value of 0.17 microM against the microbial enzyme and potentially useful selectivity (rat liver IC50/P. carinii IC50 = 114).
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PMID:Structural studies on bioactive compounds. 28. Selective activity of triazenyl-substituted pyrimethamine derivatives against Pneumocystis carinii dihydrofolate reductase. 919 66

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimid ine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]- 6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 microM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.
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PMID:2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. 935 37

Methotrexate (MTX) treatment of rheumatoid arthritis may require increasing doses to maintain clinical efficacy. An overall plateau of clinical response is reached after only six months of treatment. To study the immunologic, biochemical and genetic effects of MTX on T cells, the Jurkat T cell line was made MTX-resistant by serial addition of methotrexate sodium into culture medium. Cells proliferated and divided successfully in MTX concentrations ranging to 15 microM. MTX resistance of Jurkat T cells in vitro was accompanied by significantly (P < 0.05) decreased expression of CD2, CD3, CD4, CD28, and CD69, IL-2 production, and MTX uptake assessed by cell association or disassociation of 3[H]-MTX or fluoresceinated MTX (FMTX), respectively. In addition, there was DHFR gene amplification and increased levels of DHFR in all resistant cell lines. Both permanent and transient phenotypic changes developed in resultant cell lines exposed to increasing concentrations of MTX in vitro. Expression of CD4 and CD25 and sensitivity to MTX returned to near-parental levels after removal of MTX from culture medium, whereas expression of CD26 and MTX uptake were significantly increased. Expression of CD2, CD3, CD69 and IL-2 production as well as the DHFR levels did not return to the parental phenotype after removal from MTX. We conclude that MTX-cultured cells express depressed levels of cell-surface markers vital for T cell function and activation. The return of enhancement of these cell-surface markers critical to T cell activation suggests a possible mechanism for the severe flares experienced by rheumatoid arthritis patients when drug treatment is discontinued.
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PMID:Long-term exposure to methotrexate induces immunophenotypic changes, decreased methotrexate uptake and increased dihydrofolate gene copy number in Jurkat T cells. 966 12

Analysis of 12 baby hamster kidney (BHK) clones in exponential growth revealed a linear relationship between cell-specific recombinant activated protein C (APC) production rates and APC mRNA levels. This correlation indicated that mRNA levels limited APC productivity. Two strategies were employed to increase APC mRNA levels and APC productivity. First, sodium butyrate was added to increase mRNA levels by two- to sixfold in five APC-producing clones to obtain up to 2.7-fold increase in APC production rate. The second strategy was to retransfect an APC-producing BHK cell line with a vector containing additional APC cDNA and a mutant DHFR. This mutant DHFR gene allowed the selection of retransfected clones in higher MTX concentrations. Over two-fold higher mRNA levels were obtained in these retransfected clones and the cell-specific APC production rate increased twofold. At the highest level of APC secretion, increases in mRNA levels did not result in higher rates of APC production. Analysis of the intracellular APC content revealed a possible saturation in the secretory pathway at high mRNA levels. The relation between mRNA level and APC secretion rate was also investigated in batch culture. The levels of total cellular RNA, APC mRNA, and beta-actin mRNA were relatively stable while cells were in the exponential growth phase, but rapidly decreased when cells reached the stationary phase. The decline of cell-specific APC mRNA levels correlated with a decline in APC secretion rates, which indicated that the mRNA levels continued to limit the rates beyond the exponential phase and into the declining growth and stationary phases of batch APC production.
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PMID:Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells. 1009 27

We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37 degrees C) by monitoring its product, tetrahydrofolate (H(4)folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchloric acid, the H(4)folate concentration was measured. Using sodium ascorbate at 20 mM, H(4)folate was stable in our assay system. The enzyme activity was also stable. The detection limit of this method was less than 1 nM of H(4)folate in the enzyme assay system, which was 1/100 lower than those for the NADPH-spectrophotometric assay, which is commonly used for analysis of DHFR activity. This value of 1 nM allowed us to control the conversion from dihydrofolate (H(2)folate) to H(4)folate less than 10% of initial substrate concentrations during assay, when we used a concentration around K(m) values reported for DHFR from various sources. The rate of reduction showed a linearity at concentrations around the K(m). The reduction rate must be evaluated exactly around the K(m), in order to obtain an accurate profile of Michaelis-Menten kinetics. This assay method has a sensitivity high enough to determine the reduction rate at H(2)folate concentrations around K(m). In addition, the assay procedure is very simple. Therefore, our method may be useful for studying DHFR.
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PMID:Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection. 1041 82

As part of a larger search for potent as well as selective inhibitors of dihydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N-[(2,4-diaminopteridin-6-yl)methyl]dibenz[b,f]azepine (4a) and the corresponding dihydrodibenz[b,f]azepine, dihydroacridine, phenoxazine, phenothiazine, carbazole, and diphenylamine analogues were synthesized from 2, 4-diamino-6-(bromomethyl)pteridine in 50-75% yield by reaction with the sodium salts of the amines in dry tetrahydrofuran at room temperature. The products were tested for the ability to inhibit DHFR from Pneumocystis carinii (pcDHFR), Toxoplasma gondii (tgDHFR), Mycobacterium avium (maDHFR), and rat liver (rlDHFR). The member of the series with the best combination of potency and species selectivity was 4a, with IC(50) values against the four enzymes of 0. 21, 0.043, 0.012, and 4.4 microM, respectively. The dihydroacridine, phenothiazine, and carbazole analogues were also potent, but nonselective. Of the compounds tested, 4a was the only one to successfully combine the potency of trimetrexate with the selectivity of trimethoprim. Molecular docking simulations using published 3D structural coordinates for the crystalline ternary complexes of pcDHFR and hDHFR suggested a possible structural interpretation for the binding selectivity of 4a and the lack of selectivity of the other compounds. According to this model, 4a is selective because of a unique propensity of the seven-membered ring in the dibenz[b,f]azepine moiety to adopt a puckered orientation that allows it to fit more comfortably into the active site of the P. carinii enzyme than into the active site of the human enzyme. Compound 4a was also evaluated for the ability to be taken up into, and retard the growth of, P. carinii and T. gondii in culture. The IC(50) of 4a against P. carinii trophozoites after 7 days of continuous drug treatment was 1.9 microM as compared with previously observed IC(50) values of >340 microM for trimethoprim and 0.27 microM for trimetrexate. In an assay involving [(3)H]uracil incorporation into the nuclear DNA of T. gondii tachyzoites as the surrogate endpoint for growth, the IC(50) of 4a after 5 h of drug exposure was 0.077 microM. The favorable combination of potency and enzyme selectivity shown by 4a suggests that this novel structure may be an interesting lead for structure-activity optimization.
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PMID:Structure-based design of selective inhibitors of dihydrofolate reductase: synthesis and antiparasitic activity of 2, 4-diaminopteridine analogues with a bridged diarylamine side chain. 1057 48

The influences of mono-, bi- and trivalent metal ions (as chloride salts) on the activity of dihydrofolate reductase (DHFR) from chicken liver have been studied to elucidate the mechanism of ion-activation of this enzyme. The results show that monovalent ions (Na+ and K+) activate DHFR at low concentration reaching a maximum activation of about 2.5 fold at 0.4-0.5 M and declining at higher concentrations. Ca2+ shows similar activation but at lower concentration, reaching a maximum at 0.1 M; activity declines with further increases in concentration. At very high concentration (> 0.4 M), Ca2+ is inhibitory. The trivalent lanthanide ions, however, show a dramatic inhibition of activity of DHFR even at very low concentration. The activity of DHFR declines to 50% of that of the control at 0.02 mM EuCl3. Intrinsic fluorescence measurements show that the ion-dependent activation in the presence of mono- and bivalent metal ions is due to the conformational changes in the protein. Energy transfer phenomenon suggests that the specific interaction of Eu3+ with Trp24 located in a loop at the active site of DHFR is responsible for the strong inhibition. The possible mechanism for the ion-inhibition is proposed and discussed.
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PMID:Lanthanide ions inhibit the activity of dihydrofolate reductase from chicken liver. 1112 90

NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways.
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PMID:Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways. 1136 92

Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.
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PMID:Operation of the CO dehydrogenase/acetyl coenzyme A pathway in both acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum. 1586 34


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