Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytotoxicity of trimetrexate (TMQ), a lipophilic dihydrofolate reductase inhibitor, was examined in antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate. An approximately 60-fold methotrexate (MTX)-resistant subline was developed in oxidized folate (pteroylglutamic acid: PGA) (CCRF-CEM/MTX60-PGA) from human T-cell leukemia cell line CCRF-CEM; this line exhibited impaired membrane transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX-resistant subline (CCRF-CEM/ MTX5000-PGA), which showed increased dihydrofolate reductase activity due to gene amplification in addition to further impairment of MTX transport. An approximately 140-fold MTX-resistant subline, and then a 1500-fold MTX-resistant subline were developed in reduced folate (10 nM leucovorin) (CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV); they exhibited increased dihydrofolate reductase due to gene amplification accompanied by increased intracellular drug accumulation of MTX. While CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV cells showed cross-resistance to TMQ, CCRF-CEM/MTX60-PGA and CCRF-CEM/MTX5000-PGA cells were at least as sensitive to TMQ as the parent cells. TMQ was more potent against approximately 200-fold N10-propargyl-5,8-dideazafolic-acid (CB3717)-resistant human T-cell leukemia MOLT-3 sublines developed in PGA (MOLT-3/CB3717(200)-PGA) or leucovorin (MOLT-3/CB3717(200)-LV), as compared to the parent cells; MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells were resistant to CB3717 by virtue of impaired transport, only the former possessing gene amplification of thymidylate synthase. The cytotoxicity of TMQ in both MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells was reduced by addition of leucovorin in a dose-dependent manner, suggesting intracellular folate deficiency as a cause of TMQ sensitivity. These results demonstrate that TMQ overcomes transport-impaired antifolate resistance, irrespective of gene amplification of dihydrofolate reductase or thymidylate synthase. Types of folate used during the development of antifolate resistance seem to be important in relation to the mechanism of TMQ responsiveness as well as that of antifolate resistance.
 ...
PMID:Cytotoxicity of trimetrexate against antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate. 936 39

Trimetrexate (TMTX), a potent inhibitor of the enzyme dihydrofolate reductase, is biochemically and metabolically similar to methotrexate (MTX). Fundamental differences between TMTX and MTX, however, mandate investigation of TMTX in both MTX-sensitive and MTX-resistant tumors. In a number of phase II clinical trials, the antitumor activity of single-agent TMTX has been variable, in part because of the heterogeneity of doses and schedules used and in part because of diverse patient populations. Single-agent activity has been documented in some commonly occurring tumors, including breast, non-small cell lung, and head and neck cancers. Other tumors with sensitivity to single-agent TMTX include transitional cell carcinomas of the urothelium, prostate cancer, and gastric carcinoma. In a small series of children with renal cell carcinoma, significant clinical activity was suggested. The single-agent activity of TMTX, coupled with the finding that TMTX may act as a biochemical modulator of 5-fluorouracil/leucovorin, suggests that the addition of TMTX to 5-fluorouracil/leucovorin should be investigated further. The possibility that TMTX may both exhibit single-agent activity and modulate the effectiveness of other agents makes combination therapy attractive.
 ...
PMID:Trimetrexate: experience with solid tumors. 942 24

A number of antifolate drugs, which inhibit the key enzymes in the 'thymidylate cycle', dihydrofolate reductase (DHFR) and thymidylate synthase (TS), have been developed as part of the search for analogues with superior antitumor efficacy to a 'classical' antifolate, methotrexate (MTX), and those which are active against the MTX-resistant tumor cells. Recent development of newer classes of antifolate drugs is based on the extensive understanding of the relationship between chemical structures and biological properties and of analogue interactions with target enzymes, transport proteins and folate metabolizing enzyme, folylpolyglutamate synthetase (FPGS). Tumor cells may develop resistance to an antifolate drug by virtue of, (1) amplified activity in its target enzyme, (2) impaired function of drug transport protein, e.g. reduced folate carrier (RFC), (3) induction of mutated target enzyme with low affinity for antifolate(s), and (4) defective polyglutamation of drug(s) in the cells. Recent studies have elucidated in part the molecular events involved in the resistance to antifolates. These include amplification and/or mutation of the gene encoding a target enzyme, reduced or altered gene expression of the RFC, and mutated expression of the FPGS gene. To overcome or circumvent the resistance mechanisms, new antifolates with diverse structures and different biological properties have been designed and developed for clinical use. Trimetrexate (TMQ), a lipophilic DHFR inhibitor which is not a substrate for RFC and FPGS, could overcome the MTX-resistance through impaired RFC and diminished polyglutamation, and partially through amplified DHFR. Selective inhibitors of TS with a folate structure such as raltitrexed could circumvent the resistance by virtue of DHFR overproduction, and this class of compounds which have higher substrate activities for FPGS than MTX may be of value for the treatment of myeloid leukemias in addition to lymphocytic malignancies resistant to conventional chemotherapy. Several strategies to overcome antifolate resistance by using gene therapy are currently under investigation.
 ...
PMID:Cellular and molecular mechanisms of resistance to antifolate drugs: new analogues and approaches to overcome the resistance. 947 73

The synthesis and biological activity are reported for 21 6-substituted 2,4-diaminopyrido[3,2-d]pyrimidine analogues (4-24) of piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) and as antitumor agents. Recombinant DHFR from Pneumocystis carinii (pc) and native DHFR from Toxoplasma gondii (tg) were the target enzymes tested; these organisms are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity for the pathogenic DHFR. The synthesis of S9-bridged compounds 4-6 was achieved by aryl displacement of 2,4-diamino-6-chloropyrido[3, 2-d]pyrimidine (27) with thiol nucleophiles. Oxidation of 4-6 with hydrogen peroxide in glacial acetic acid afforded the corresponding sulfone analogues 7-9. The N9-bridged compounds 10-24 were synthesized from their precursor 3-amino-6-(arylamino)-2-pyridinecarbonitriles via a thermal cyclization with chloroformamidine hydrochloride. Unlike the S9-bridged compounds, the arylamino side chains of the N9-bridged analogues were introduced prior to the formation of the 2, 4-diaminopyrido[3,2-d]pyrimidine nucleus. A reversed two-atom-bridged analogue (25) was also synthesized using a synthetic strategy similar to that utilized for compounds 10-24. The IC50 values of these compounds against pcDHFR ranged from 0.0023 x 10(-6) M for 2,4-diamino-6-(N-methyl-3',4'-dimethoxyanilino)pyrido[3, 2-d]pyrimidine (21), which was the most potent, to 90.4 x 10(-6) M for 2,4-diamino-6-(4'-methoxyanilino)pyrido[3,2-d]pyrimidine (12), which was the least potent. The three S9-bridged compounds tested were more potent than the corresponding sulfone-bridged compounds for all three DHFRs. N9-Methylation increased the potency by as much as 17 000-fold (compounds 15 and 21). None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was again 21 with an IC50 value of 0.00088 x 10(-6) M and the least potent was 12 with an IC50 of 2.8 x 10(-6) M. N9-Methylation afforded an increase in potency of up to 770-fold (compound 15 NH vs 21 N-CH3) compared to the corresponding N9-H analogue. In contrast to pcDHFR, several analogues had a greater selectivity ratio for tgDHFR compared to trimetrexate (TMQ) or PTX, most notably 2, 4-diamino-6-[(3',4'- dimethoxyphenyl)thio]pyrido[3,2-d]pyrimidine (4), 2,4-diamino-6-[(2'-methoxyphenyl)sulfonyl]pyrido[3, 2-d]pyrimidine (7), and 2,4-diamino-6-(2', 5'-dimethoxyanilino)pyrido[3,2-d]pyrimidine (14) which combined relatively high potency at 10(-7)-10(-8) M along with selectivity ratios of 3.97, 6.67, and 4.93, respectively. Several analogues synthesized had better selectivity ratios than TMQ or PTX for both pcDHFR and tgDHFR, and the potencies of the N9-methylated compounds were comparable to or greater than that of TMQ or PTX. Selected compounds were evaluated as inhibitors of the growth of a variety of tumor cells in culture. The N9-CH3 analogues were, in general, highly potent with GI50 values in the nanomolar range. The N9-H and S9 analogues were less potent with GI50 values in the millimolar to micromolar range.
 ...
PMID:6-Substituted 2,4-diaminopyrido[3,2-d]pyrimidine analogues of piritrexim as inhibitors of dihydrofolate reductase from rat liver, Pneumocystis carinii, and Toxoplasma gondii and as antitumor agents. 980 92

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]++ +thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 23 or 40 microM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, alpha, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 23 microM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 microM PteGlu, the intensity of the combined action in all cell lines was increased However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.
 ...
PMID:Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin -6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034): comparison across sensitive and resistant human tumor cell lines. 995 21

Various gene alterations are involved in the drug resistance of leukemia cells. To understand the mechanism that underlies the emergence of cells with such gene alterations in human leukemia, we performed clonal analysis of the gene expression of mutant dihydrofolate reductase (DHFR) and mdr1 in trimetrexate-resistant human leukemia MOLT-3 cells. Trimetrexate-resistant (70- and 60-fold) sublines were developed in the presence or absence of an exogenous supply of thymidine (MOLT-3/TMQ70/Th+, MOLT-3/TMQ60/Th-, respectively). Ten clonal lines were isolated by methyl cellulose cloning from each of the 2 trimetrexate-resistant MOLT-3 sublines. All the clonal lines from the 2 sublines expressed mutated DHFR mRNA, with a base change (T --> C) at the second position of codon 31, as well as the wild-type mRNA, in accordance with cross-resistance to methotrexate. On the other hand, mdr1 mRNA expression was demonstrated by reverse-transcription polymerase chain reaction only in clonal lines from MOLT-3/TMQ70/Th+ cells. mdr1 mRNA expression in clonal lines from MOLT-3/TMQ70/Th+ cells and subclonal lines subsequently obtained from the 3 clonal lines with different mdr1 mRNA expression levels was heterogeneous, and its high expression levels were correlated with acquisition of the multidrug resistance (MDR) phenotype. Polymerase chain reaction-based assay for separate microsatellites, mfd27 and mfd41, demonstrated genomic instability among clonal and subclonal lines of MOLT-3. The clonal analysis of polymorphic microsatellites also suggested that emergence of MDR in trimetrexate-resistant MOLT-3 cells in thymidine was not only heterogeneous but also progressively expanding among clones. Genomic instability may play a role in the establishment and clonal evolution of drug resistance in leukemia cells.
 ...
PMID:Microsatellite instability and clonal heterogeneity of MDR1 messenger RNA expression in trimetrexate-resistant human leukemia MOLT-3 cells developed in thymidine. 1036 Aug 22

Folates have been co-administered with some antifolates to diminish host toxicity; however, the extent to which this will reduce antitumor activity is not known. To further clarify this issue, studies were undertaken to characterize and quantitate the impact of alterations in intracellular folate levels on the activities of a variety of antifolates in L1210 murine leukemia cells. Intracellular folate cofactor levels increased almost in proportion to the increase in extracellular 5-formyltetrahydrofolate (5-CHO-THF) over a concentration range that encompassed physiological levels of 5-methyltetrahydrofolate. This resulted in a spectrum of increases in the ic50 values of antifolates upon continuous exposure to drugs [Lometrexol (DDATHF) (70x) > trimetrexate (TMQ) (30x), multitargeted antifolate, LY231514 (ALIMTA) (30x) > Raltitrexed, Tomudex (ZD1694) (10x), 6R-2',5'-thienyl-5,10-dideazatetrahydrofolic acid (LY309887) (10x) > methotrexate (MTX) (6x) > (2S)-2-[o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido]-4-(tetrazol-5-yl) butyric acid (ZD9331) (3x), N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) (3x)]. Upon a 4-hr pulse exposure to drug, the ic50 values for DDATHF and ALIMTA were increased > 180- and 5-fold, respectively, with only a 2.5-fold increase in the extracellular 5-CHO-THF level within the physiological range. The reductions in drug sensitivities could be attributed to decreases in accumulation of polyglutamate derivatives of ALIMTA and DDATHF. Hence, in these studies, natural folates diminished the activity of agents that undergo polyglutamation by suppression of the formation of these active congeners at the level of folylpolyglutamate synthetase. For inhibitors of dihydrofolate reductase, the suppressive effect of endogenous folates appears to be due to competition between the antifolate and dihydrofolate at the level of the target enzyme. These data should be carefully considered in the design of regimens with antifolates, which incorporate co-administration of folates.
 ...
PMID:Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells. 1127 72

The results of the crystal structure determination of human dihydrofolate reductase (hDHFR) as a binary complex with the potent N9-C10 reversed-bridge antifolate inhibitor 2,4-diamino-6-[N-(3',4',5'-trimethoxybenzyl)-N-methylamino]pyrido[2,3-d]pyrimidine (1) are reported for two independent polymorphic rhombohedral R3 lattices [R3(1) and R3(2)]. Data from these two crystal forms were refined to 1.90 A resolution for complex R3(1), with R = 0.186 for 9689 data, and to 1.80 A resolution for complex R3(2), with R = 0.194 for 13 305 data. Changes in the loop geometry between the two structures reflects contact differences in the packing environments in the two R3 lattices. The largest changes (between 0.5 and 1.7 A) are observed for the loop regions encompassing residues 16-25, 40-48, 81-89, 99-108, 143-148 and 161-169. Comparison of the intermolecular contacts of these loops reveals that the R3(2) lattice is more tightly packed, as reflected in its smaller V(M) value and smaller solvent content. The conformation of inhibitor (1) is similar in both structures and the N9-C10 bridge geometry is more similar to that observed for the normal C9-N10 bridge of trimetrexate (TMQ) than to the other N9-C10 reversed-bridge antifolates previously reported. The effect of the N9-C10 reversed-bridge geometry is to distort the bridge from coplanarity with the pyrido[2,3-d]pyrimidine ring system and to twist the C10 methylene conformation towards a gauche conformation. This also influences the conformation of the methoxybenzyl ring, moving it away from a trans position and placing the 5'-methoxy group deeper within the hydrophobic pocket made by Leu60, Pro61 and Asn64 of the hDHFR active site.
 ...
PMID:Analysis of two polymorphic forms of a pyrido[2,3-d]pyrimidine N9-C10 reversed-bridge antifolate binary complex with human dihydrofolate reductase. 1265 84

In order to examine the intracellular locus of the folic acid (PteGlu)-enhanced synergies of trimetrexate (TMQ) plus the thymidylate synthase (TS) inhibitor, raltitrexed (RTX), and TMQ plus the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, AG2034, comprehensive protection studies with thymidine (dThd) and hypoxanthine (HX) were conducted in a 96-well plate cell growth inhibition (sulforhodamine B) assay. Current modeling techniques were extended to characterize these protection patterns involving multiple-agent interaction. Wild-type human ileocecal HCT-8 cells and DW2, a subline deficient in folylpoly-gamma-glutamate synthetase (FPGS) were individually treated for 96 h with TMQ, AG2034 and a 1:1 mixture of TMQ:AG2034 or with TMQ, RTX, and a 1:1 mixture of TMQ:RTX in the presence of PteGlu (2.3 or 40 micro M) and the protection agents (10 micro M dThd and/or 100 micro M HX). Drug treatments were randomly assigned to wells. Both isobols and 3-dimensional concentration-effect surfaces were used to assess the nature and the intensity of drug interactions. The structural Hill model was fitted to data with weighted non-linear regression for most cases. A so-called 'double Hill' model was sometimes more appropriate when a plateau in the middle of the concentration-effect curve was found. In HCT-8 and DW2 cells at 2.3 and 40 micro M PteGlu, inhibition of DHFR by TMQ induced antithymidylate and antipurine effects; AG2034 and RTX selectively inhibited de novo purine or thymidine synthesis, respectively. dThd protection increased the PteGlu-enhancement of the TMQ + AG2034 synergy, whereas HX protection increased the PteGlu-enhancement of the TMQ + RTX synergy. The PteGlu-enhanced synergies of TMQ + AG2034 and TMQ + RTX occur primarily through inhibition of purine synthesis and inhibition of thymidylate synthesis, respectively. These results further substantiate the hypothesis that the nonpolyglutamylatable DHFR inhibitor, TMQ, acts as a modulator by decreasing the protection by PteGlu of cells against the polyglutamylatable AG2034 and RTX.
 ...
PMID:Thymidine and hypoxanthine protection patterns of the folic acid-enhanced synergies for combinations of trimetrexate plus a polyglutamylatable inhibitor of purine or thymidylate synthesis against human ileocecal HCT-8 cells. 1285 89

Structural data are reported for the first example of the potent antifolate inhibitor 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxy-N-methylanilino)methyl]pyrido[2,3-d]pyrimidine (1) in complex with human dihydrofolate reductase (hDHFR) and NADPH. Small differences in crystallization conditions resulted in the growth of two different forms of a binary complex. The structure determination of an additional crystal of a ternary complex of hDHFR with NADPH and (1) grown under similar conditions is also reported. Diffraction data were collected to 2.1 A resolution for an R3 lattice from a hDHFR ternary complex with NADPH and (1) and to 2.2 A resolution from a binary complex. Data were also collected to 2.1 A resolution from a binary complex with hDHFR and (1) in the first example of a tetragonal P4(3)2(1)2 lattice. Comparison of the intermolecular contacts among these structures reveals differences in the backbone conformation (1.9-3.2 A) for flexible loop regions (residues 40-46, 77-83 and 103-107) that reflect differences in the packing environment between the rhombohedral and tetragonal space groups. Analysis of the packing environments shows that the tetragonal lattice is more tightly packed, as reflected in its smaller V(M) value and lower solvent content. The conformation of the inhibitor (1) is similar in all structures and is also similar to that observed for TMQ, the parent quinazoline compound. The activity profile for this series of 5-deaza N-substituted non-classical trimethoxybenzyl antifolates shows that the N10-CH(3) substituted (1) has the greatest potency and selectivity for Toxoplasma gondii DHFR (tgDHFR) compared with its N-H or N-CHO analogs. Models of the tgDHFR active site indicate preferential contacts with (1) that are not present in either the human or Pneumocystis carinii DHFR structures. Differences in the acidic residue (Glu30 versus Asp for tgDHFR) affect the precise positioning of the diaminopyridopyrimidine ring, while changes in other residues, particularly at positions 60 and 64 (Leu versus Met and Asn versus Phe), involve interactions with the trimethoxybenzyl substituents.
 ...
PMID:Analysis of three crystal structure determinations of a 5-methyl-6-N-methylanilino pyridopyrimidine antifolate complex with human dihydrofolate reductase. 1292 91


<< Previous 1 2 3 4 5 6 7 8 Next >>