• YAKHAK HOEJI
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• v.46 no.6
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• pp.416-425
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• 2002

A comparative study to validate the reliability of a fully automated docking program, FlexiDock, was carried out to predict the binding modes of DHFR-inhibitor complex. The inhibitors were extracted from the crystallographically determined DHFR-NADP$^{+}$(H)-inhibitor ternary complexes of human, Escherichia coli and Candida albicans and then docked back into the remaining DHFR-NADP$^{+}$(H) binary complexes using FlexiDock. The resulting conformations and orientations were compared to the original crystal complex structures for reproducibility. Then, folate, the substrate, and known inhibitors such as methotrexate, piritrexim and trimethoprim were docked into the wild-type human DHFR and their binding modes were compared with X-ray crystallographic or other modeling data. The root mean square deviations (RMSDs) for ligands ranged from 1.14 to 1.57$\AA$, and the protein backbone RMSDs from 0.94 to 1.26$\AA$. FlexiDock reproduced the orientations and binding modes of all seven ligands in good agreement with the crystal structures. It proved to be a reliable and efficient program in studying binding modes of DHFR-inhibitor complexes of different species, and the information obtained from this work may provide additional insight into the design of new agents with improved activity.ity.

• Bulletin of the Korean Chemical Society
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• v.32 no.7
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• pp.2433-2442
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• 2011

The three dimensional quantitative structure activity relationship (3D QSAR) models were developed using Comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA) and docking studies. The fit of Quinazolinone antifolates inside the active site of modeled bovine dihydrofolate reductase (DHFR) was assessed. Both ligand based (LB) and receptor based (RB) QSAR models were generated, these models showed good internal and external statistical reliability that is evident from the $q^2_{loo}$, $r^2_{ncv}$ and $r^2_{pred}$. The identified key features enabled us to design new Quinazolinone analogues as DHFR inhibitors. This study is a building bridge between docking studies of homology modeled bovine DHFR protein as well as ligand and target based 3D QSAR techniques of CoMFA and CoMSIA approaches.

• Parasites, Hosts and Diseases
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• v.28 no.2
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• pp.79-84
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• 1990

Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro·dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses do novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reject the specific proliferation of Texoplasma. It can be exploited to characterize the elects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis. Key words: Toxoplasma gondii, uracil salvage, dihydrofolate reductase, thymidylate synthase TMP biosynthesis.

• Korean Journal of Veterinary Research
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• v.48 no.3
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• pp.243-250
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• 2008

A calmodulin-dihydrofolate reductase (DHFR) sandwich fusion protein was generated by insertion of calmodulin into the $\beta$-bulge region of DHFR to observe the effects of structurally constraining the calmodulin structure. The calcium binding properties of the sandwich protein were almost identical to calmodulin. Similar to calmodulin ($10.7 {\mu}M$), the sandwich protein bound four equivalents of calcium, with half saturation ($K_{0.5}$) observed at a [$Ca^{2+}$] of $8{\mu}M$. However, nicotinamide adenine dinucleotide (NAD) kinase activation property of the sandwich protein was lower than that of calmodulin. The sandwich protein activated NAD kinase, but to only half of the level obtained with calmodulin. The K 0.5 for both calmodulin and the sandwich protein were approximately the same (1-2 nM). Methylation analyses of the sandwich protein show that insertion of calmodulin into DHFR results in a large decrease in methylation. The $V_{max}$ observed with the sandwich protein (95 nmole/min/ml) was only 22% of the value observed with calmodulin (436 nmol/min/ml) in the presence of calcium. Addition of trimethoprim to the reaction significantly inhibited the observed methylation rate. Overall, the data suggest that the insertion of calmodulin into the DHFR structure has little effect on calcium binding by the individual lobes of calmodulin, but may constrain the lobes in a manner that results in altered interaction with the calmodulin-dependent proteins, and severely perturbed the methyltransferase recognition site.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3461-3464
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• 2012

Methotrexate (MTX) is an important drug for the treatment of childhood acute lymphoblastic leukemia (ALL). However, related toxicity occurs in many organs which may cause interruption of treatment, morbidity, and mortality. Single nucleotide polymorphisms (SNPs) of dihydrofolate reductase (DHFR) and gamma glutamyl hydrolase (GGH) are known to alter their enzymatic activity and thus affect the metabolism of MTX and influence the effectiveness. Therefore, we hypothesized that genetic variations of DHFR and GGH genes may influence the risk of toxicity after high dose MTX. The study population comprised of 105 children with ALL who were treated according to the modified St Jude Total XV protocol. The patients received 2.5 or $5g/m^2$ of MTX for 5 doses during the consolidation phase. Genotyping of DHFR 829C>T and GGH-401C>T was performed using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The GGH-401CT and TT genotypes were associated with increased risk of leukopenia and thrombocytopenia after high dose MTX (OR 2.97, 95%CI; 1.24-7.13 and OR 4.02, 95%CI; 1.58-10.26). DHFR 829C>T was not associated with toxicity. In conclusion, the GGH-401CT and TT genotypes were found to increase the risk of severe leukopenia and thrombocytopenia after exposure to high dose MTX for childhood ALL therapy.

• Archives of Pharmacal Research
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• v.25 no.6
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• pp.807-816
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• 2002

Dihydrofolate reductases (DHFR) of human, Candida albicans and E. coli were docked with their original ligands of X-ray crystal complex using QXP (Quick eXPlore), a docking program. Conditions to reproduce the crystal structures within the root mean square deviation (rmsd) of 2.00 $\AA$ were established. Applying these conditions, binding modes and species-specificities of a novel antibacterial compound, $N^4-(2-acetoxyethoxymethyl)-2-acetylpyridine$ thiosemicarbazone (MTSC), were studied. As the results, the docking program reproduced the crystal structures with average rmsd of six ligands as 0.91 $\AA$ ranging from 0.49 to 1.45 $\AA$. The interactions including the numbers of hydrogen bonds and hydrophobic interactions were the same as the crystal structures and superposition of the crystal and docked structures almost coincided with each other. For AATSC, the results demonstrated that it could bind to either the substrate or coenzyme sites of DHFR in all three species with different degrees of affinity. It confirms the experimentally determined kinetic behavior of uncompetitive inhibition against either the inhibitor or the coenzyme. The docked MTSC overlapped well with the original ligands and major interactions were consistent with the ones in the crystal complexes. The information generated from this work should be useful for future development of antibacterial and antifungal agents.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.994-1004
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• 2005

The dihydrofolate reductase (dhfr) promoter contains cis-acting element for the transcription factors Spl and E2F. Transcription of dhfr gene shows maximal activity during the Gl/S phase of cell cycle. The member of the Spl transcriptional factor family can act as both negative and positive regulators of gene expression. There was a report that Spl-Rb and E2F4-pl30 complexes cooperate to establish stable repression of dhfr gene expression in CHOC400 cells. Here, we examined the role of HDAC in dhfr, cyclin E, and cyclin A gene regulation using the histone deacetylation inhibitor, trichostatin A (TSA) in U2OS and C33A cells, a Rb-positive human osteosarcoma cell line, and a Rb-negative cervical carcinoma cell line, respectively. When the dhfr promoter constructs were applied in U2OS cells, TSA markedly stimulated over 14-fold of dhfr promoter activity through dhfr-Spl sites by the deletion of an E2F element. In contrast, the deletion of dhfr-Spl binding sites completely abolished promoter stimulation by TSA. The dhfr promoter activity including dhfr-Spl sites increased only 2-fold in C33A cells. Promoter activity containing only dhfr-E2F site did not have much effect by the treatment of TSA in both U2OS and C33A cells. On the other hand, treatment with TSA induced significantly mRNA expression of dhfr and cyclin E, whereas levels of cyclin A decreased in U2OS cells, but had no effect in C33A cells. These results indicate that TSA have contradictory effect, activation of dhfr and cyclin E genes on Gl phase, and down-regulation of cyclin A on G2 phase through transcriptional regulation in U2OS cells.

• Parasites, Hosts and Diseases
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• v.49 no.2
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• pp.125-131
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• 2011

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.57-63
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• 1996

The biliary excretion kinetics of the active folate derivatives were examined after an intravenous injection of methotrexate at doses of 0.3 and 10mg/kg to clarify the mechanism of the acute decrease in the plasma folate by the dihydrofolate reductase inhibitors. Even at a higher dose than used in the clinical therapy, methotrexate did not cause any acute depletion of folate denvatives in the excreted bile. Therefore, the decrease in the plasma folate appeared not to be related with the biliary excretion process of folates. A factor responsible for the plasma folate depletion by DHFR inhibitors may be due to the malabsorption of folate derivatives excreted into the small intestine.

• Journal of Nutrition and Health
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• v.49 no.2
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• pp.72-79
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• 2016

Purpose: The initiation of mandatory folic acid fortification using pteroylmonoglutamic acid (PteGlu) has reduced the rate of congenital malformations. However, it also appears to be responsible for several adverse effects, including increased cancer incidence. This may be related to physicho-chemical characteristics of PteGlu. This study examines the potential effect of high concentrations of PteGlu on a population subjected to mandatory folic acid fortification using an in vitro model. Methods: Caco-2 (colorectal cancer) and MCF7 (breast cancer) cell lines were cultured at 6 different PteGlu concentrations (0, 0.1, 1, 50, 250, and $500{\mu}g/ml$) for 6 days. Cell growth was determined using thiazolyl blue tetrazolium bromide assay. The genotype of dihydrofolate reductase 19bp deletion/insertion (DHFR 19-del) was also scored in cell lines using a restriction fragment length polymorphism technique to examine whether genetic variations may factor in cell proliferation. Results: PteGlu exhibited differential growth promoting properties between cell lines. Caco-2 cells did not show a significant growth difference at low concentrations compared to control, however, at higher concentrations, the growth showed a contrasting trend in the early experimental period, while MCF7 showed enhanced cell growth at all concentrations. The DHFR 19-del genotype differed in the two cell lines. Conclusions: Altered response to PteGlu by Caco-2 and MCF7 may reflect a tissue specific disease aetiology or genotype specific differential enzyme activity, for example by DHFR, to critical levels of PteGlu. As folic acid fortification is a blanket intervention, and DHFR and other enzyme activities vary between individuals, PteGlu intake may have an as yet undefined effect on health. These findings may be relevant when considering mandatory folic acid fortification for disease prevention.