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)

Transfer of drug resistance genes to hematopoietic cells is being studied as a means to protect against the myelosuppression associated with cancer chemotherapy and as a strategy for the in vivo selection and amplification of genetically modified cells. The goal of this study was to test if retroviral-mediated gene transfer of a dihydrofolate reductase (DHFR) variant (L22Y) could be used for in vivo selection of transduced myeloid cells and to determine what proportion of transduced cells was required for protection from myelosuppression. Based on previous work suggesting that selection with antifolates may also require inhibition of nucleoside transport mechanisms, mice transplanted with DHFR-transduced bone marrow cells were treated with trimetrexate and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine riboside phosphate. In vivo selection of transduced myeloid progenitors was seen in the bone marrow and in circulating mature peripheral blood cells following drug treatment. These results show that the novel combination of the L22Y-DHFR cDNA, trimetrexate and nitrobenzylmercaptopurine riboside phosphate can be used to select for transduced myeloid cells, and that this approach warrants further study in large animal models. A bicistronic vector containing a human CD24 reporter gene was used to determine the number of modified cells needed for chemoprotection. Partial protection from neutropenia was seen when greater than 10% of myeloid cells expressed the vector, and high levels of protection were obtained when the proportion exceeded 30%. These results suggest that gene transfer may be useful for myeloprotection in certain pediatric cancers, but that more efficient gene transfer will be required to apply this approach to adult cancer patients.
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PMID:Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells. 1101 66

The oxygenation, the growth rate and the metastatic potential of a solid tumor depend on its vascularization and, in particular, on angiogenesis; a therapeutic approach affecting angiogenesis has been suggested as an alternative to conventional ones. Especially the study of the metabolism in the cells of the vessel wall should be a useful prerequisite for this approach. In this connection, an enzyme histochemical study was performed to characterize the blood vessels in a solid tumor (Ehrlich carcinoma). The following enzymes were considered: (a) alkaline phosphatase, involved in the transcellular phosphate transport and in the response to inflammatory and growth promoting factors; (b) dihydrofolate reductase, involved in the metabolism of tetrahydrofolate (for the synthesis of nucleic acids and the metabolism of serine and glycine); (c) purine nucleoside phosphorylase, involved in the degradation of purines and, in particular, of extracellular ATP and ADP; (d) xanthine oxidoreductase, engaged in the same degradation path and leading to the formation of urate, a strong antioxidant. Various patterns of enzyme activities were observed in the vessel wall. In particular, thin linear capillaries (presumed to be host capillaries penetrating the tumor) were identified for the intense positivity of alkaline phosphatase, dihydrofolate reductase and purine nucleoside phosphorilase; tortuous capillaries with variable diameters (presumed to be induced by angiogenesis from the host vessels) were negative for the alkaline phosphatase and expressed an heterogeneous pattern for the dihydrofolate reductase. All the data suggest a different vessel behaviour concerning the response to cytokines and to inflammatory stimuli.
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PMID:Enzyme histochemical studies on tumor blood vessels. 1132 3

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.
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PMID:Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells. 1132 83

Currently, low levels of stable gene transfer into hematopoietic tissues of large animals and humans continues to limit the clinical application of gene therapy. One strategy for overcoming this problem is to selectively expand, in vivo, the population of successfully gene-modified cells. Recent work has shown that nucleoside transport inhibition in combination with antifolates can be used to select in vivo for hematopoietic stem cells expressing drug-resistant dihydrofolate reductase (DHFR). In this study we investigated whether trimetrexate (TMTX) and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose phosphate (NBMPR-P) can be used to select for tyr22-variant DHFR expressing transgenic hematopoietic cells in a murine bone marrow transplant model. Our results indicate that 40 mg/kg TMTX and 20 mg/kg NBMPR-P can be used in combination to expand transgene-positive progenitor cells 3- to 4-fold immediately following drug administration. In addition, long-term progenitor populations were expanded 2- to 3-fold in primary recipients, to approximately 5 months following drug administration. Secondary transplants conducted with marrow from primary recipients 5 months following drug administration revealed a statistically significant selective expansion of transgene-positive cells in the spleens and peripheral blood of these animals. No such expansion was observed in groups of mice treated with TMTX alone or NBMPR-P alone. We conclude that TMTX + NBMPR-P can be used to selectively expand transgenic tyr22-variant DHFR expressing murine hematopoietic stem cells in vivo.
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PMID:In vivo selection of antifolate-resistant transgenic hematopoietic stem cells in a murine bone marrow transplant model. 1175 96

Diseases caused by pathogenic trypanosomatids cause great suffering throughout the developing world. New drugs for these diseases are urgently needed. Recent technological advances have permitted the identification and validation of numerous drug targets in these organisms. However, efforts to develop inhibitors of these targets, that may then be taken forward for development into new drugs, have been comparatively scarce. In this review we discuss the design, synthesis and evaluation of inhibitors of two drug targets in trypanosomatids, 6-phosphogluconate dehydrogenase, the third enzyme of the pentose phosphate pathway, and dihydrofolate reductase, a key enzyme involved in DNA synthesis. Enzyme inhibitors can only be useful as drugs if they can enter cells and bind to their targets. Therefore we also discuss approaches to designing molecules that can specifically cross the plasma membrane of African trypanosomes via unusual nutrient transporters.
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PMID:Perspectives for new drugs against trypanosomiasis and leishmaniasis. 1196 68

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20,000, and 40,000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 degrees C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX-PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX-PEG40000 conjugate for 6 days at 37 degrees C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX-PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC(50) values of the tested compounds increased with the size of the drug-polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC(50) value ranging from approximately 0.01 to 0.05 microM), while the IC(50) values of the polymer conjugates were higher (IC(50) value for MTX-PEG750, 2000 and 5000: approximately 0.6-3 microM; for MTX-PEG10000 and 20000: approximately 2-7 microM; and for MTX-PEG40000: > 6 microM). Subsequently, MTX-PEG5000, MTX-PEG20000, and MTX-PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX-HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX-PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX-PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX-PEG20000 at a dose of 40 mg/kg showed antitumor efficacy comparable to MTX, but MTX-PEG40000 at a dose of 20 mg/kg was superior to MTX and demonstrated antitumor activity of the same order as MTX-HSA (20 mg/kg).
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PMID:Polyethylene glycol conjugates of methotrexate varying in their molecular weight from MW 750 to MW 40000: synthesis, characterization, and structure-activity relationships in vitro and in vivo. 1212 Nov 33

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.
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PMID:NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity. 1450 65

An improved microscale synthesis of 2-tritiated isopropanol ([2-3H]iPrOH) and R-tritiated reduced beta-nicotinamide adenine dinucleotide 2(')-phosphate (R-[4-3H]NADPH) is presented. The current procedure offers high yield, high purity, and small-quantity synthesis and is shorter than previous procedures. [2-3H]iPrOH was prepared by reduction of acetone with [3H]NaBH(4) under reflux conditions. [2-3H]iPrOH was used to reduce NADP(+) to R-[4-3H]NADPH with alcohol dehydrogenase from Thermoanaerobium brockii at 40 degrees C. This equilibrium reaction was drawn toward products by trapping the acetone with an excess of semicarbazide. This improvement enables a better control of the reaction time, as the enzymatic reduction became rate determining. Greater than 75% of the product was identified as reduced cofactor by reverse-phase (RP) HPLC. Longer reaction led to decomposition of the product and lower yield. Purification was carried out by semipreparative RP HPLC followed by lyophilization and yielded a compound of high purity that was preserved at -80 degrees C. Applications of the new procedure to a wide variety of specific radioactivities ranging from carrier-free to a few Ci/mmol are discussed. The intriguing formation of radioactive NADP(+) by-product (the major product in some reported procedures), was also studied and minimized in the procedure described below. Finally, the usage of the labeled cofactor is demonstrated with the enzymes dihydrofolate reductase and thymidylate synthase.
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PMID:Microscale synthesis of 2-tritiated isopropanol and 4R-tritiated reduced nicotinamide adenine dinucleotide phosphate. 1459 25

We have studied the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) and the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH); the substrate is 5-protonated 7,8-dihydrofolate, and the product is tetrahydrofolate. The potential energy surface is modeled by a combined quantum mechanical-molecular mechanical (QM/MM) method employing Austin model 1 (AM1) and a simple valence bond potential for 69 QM atoms and employing the CHARMM22 and TIP3P molecular mechanics force fields for the other 21 399 atoms; the QM and MM regions are joined by two boundary atoms treated by the generalized hybrid orbital (GHO) method. All simulations are carried out using periodic boundary conditions at neutral pH and 298 K. In stage 1, a reaction coordinate is defined as the difference between the breaking and forming bond distances to the hydride ion, and a quasithermodynamic free energy profile is calculated along this reaction coordinate. This calculation includes quantization effects on bound vibrations but not on the reaction coordinate, and it is used to locate the variational transition state that defines a transition state ensemble. Then, the key interactions at the reactant, variational transition state, and product are analyzed in terms of both bond distances and electrostatic energies. The results of both analyses support the conclusion derived from previous mutational studies that the M20 loop of DHFR makes an important contribution to the electrostatic stabilization of the hydride transfer transition state. Third, transmission coefficients (including recrossing factors and multidimensional tunneling) are calculated and averaged over the transition state ensemble. These averaged transmission coefficients, combined with the quasithermodynamic free energy profile determined in stage 1, allow us to calculate rate constants, phenomenological free energies of activation, and primary and secondary kinetic isotope effects. A primary kinetic isotope effect (KIE) of 2.8 has been obtained, in good agreement with the experimentally determined value of 3.0 and with the value 3.2 calculated previously. The primary KIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary KIE, with a value of 1.13, is almost entirely due to dynamical effects on the reaction coordinate, especially tunneling.
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PMID:Reaction-path energetics and kinetics of the hydride transfer reaction catalyzed by dihydrofolate reductase. 1462 3

Nicotinamide-containing cofactors are ubiquitous in biological systems. Consequently, numerous assays have been developed to study such systems that involve a variety of derivatives and isotopically labeled forms of these cofactors. Often the nicotinamide ring is labeled at the C-4 position which is directly involved in the hydride transfer chemistry catalyzed by nicotinamide-dependent enzymes. A label remote from the reactive center is often also required to follow the course of a reaction or the location of the cofactor. Since the necessary labeling pattern can be as unique as the designed experiment, these cofactors need to be synthesized, analyzed, and, most preferably, preserved. The micro-scale preservation of reduced nicotinamides has long been a challenge due to the inherent lability of the reduced cofactors. Furthermore, it has been found that the reduced 2'-phosphorylated cofactor is even less stable (i.e., reduced nicotinamide adenine dinucleotide phosphate (NADPH) is more labile than reduced nicotinamide adenine dinucleotide). Presented here are methods that were established to purify nicotinamide cofactors via reverse-phase high-performance liquid chromatography (HPLC) and, most importantly, to stabilize NADPH under optimal conditions for long-term storage. Additionally, an analytical HPLC method which achieves 7-min resolution between oxidized and reduced cofactors was developed. This method also results in over 4-min resolution of these nicotinamide cofactors from various derivatives of folic acid. This analysis affords a new analytical assay for the dihydrofolate reductase-catalyzed reaction and several dehydrogenases involved in folic acid metabolism.
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PMID:Purification, analysis, and preservation of reduced nicotinamide adenine dinucleotide 2'-phosphate. 1470 76


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