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)

N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates. Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated. Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.
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PMID:The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine. 1073 74

The potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) and six of its B-ring (5-deaza, 8-deaza, and 5,8-dideaza) analogues were compared in terms of their ability to: (a) inhibit the growth of CCRF-CEM human leukemic lymphoblasts, and (b) utilize the reduced folate carrier (RFC) in these cells as measured in a competition assay of [(3)H]methotrexate ([(3)H]MTX) influx. The IC(50) values of the hemiphthaloylornithine derivatives against CCRF-CEM cells after 72 hr of drug exposure varied from 0.64 to 1.3 nM as compared with 14 nM for MTX and 4.4 nM for aminopterin (AMT). The K(i) values of these compounds in the [(3)H]MTX influx assay were in the 0.3 to 0.7 microM range as compared with a K(i) of 5.4 microM for AMT and a K(t) of 7.1 microM for MTX. As a group, the affinities of these compounds for the RFC were approximately 10-fold greater than those of their respective glutamate analogues. These results indicate that, in addition to their previously reported tight binding to dihydrofolate reductase, a property contributing to the high potency of PT523 and its B-ring analogs as inhibitors of tumor cell growth is their strong affinity for the RFC.
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PMID:Efficient utilization of the reduced folate carrier in CCRF-CEM human leukemic lymphoblasts by the potent antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine (PT523) and its B-ring analogues. 1080 43

Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26-A, C26-10, C26-G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5-fluorouracil (5-FU) alone or in combination with leucovorin (LV) (2-5-fold), to the thymidylate synthase (TS) inhibitors, AG337 (2-7-fold), ZD1694 (3-49-fold), ZD9331 (3-40-fold), LY231514 (2-21-fold) or GW1843U89 (4-29-fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2-50-fold) compared with their HF variants cultured in standard medium containing up to 8 microM folic acid. LV could only increase sensitivity to 5-FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2-7-fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7-fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3-fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5-FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5-FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models.
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PMID:Folate depletion increases sensitivity of solid tumor cell lines to 5-fluorouracil and antifolates. 1095 84

The relationship between loss of functional p53 and human reduced folate carrier (hRFC) levels and function was examined in REH lymphoblastic leukemia cells, which express wild type p53, and in p53-null K562 cells (K562(pTet-on/p53)) engineered to express wild type p53 under control of a tetracycline-inducible promoter. Activation of p53 in REH cells by treatment with daunorubicin was accompanied by decreased ( approximately 5-fold) levels of hRFC transcripts and methotrexate transport. Treatment of K562(pTet-on/p53) cells with doxycycline resulted in a dose-dependent expression of p53 protein and transcripts, increased p21 protein, decreased dihydrofolate reductase, and G(1) arrest with decreased numbers of cells in S-phase. p53 induction was accompanied by up to 3-fold decreases in hRFC transcripts transcribed from the upstream hRFC-B promoter and similar losses of hRFC protein and methotrexate uptake capacity. Expression of p15 in an analogous inducible system in K562 cells resulted in a nearly identical decrease of S-phase cells and dihydrofolate reductase without effects on hRFC levels or activity. When the hRFC-B promoter was expressed as full-length and basal promoter-luciferase reporter constructs in K562(pTet-on/p53) cells, induction of p53 with doxycycline resulted in a 3-fold loss of promoter activity, which was reversed by cotransfection with a trans-dominant-negative p53. These studies show that wild type p53 acts as a repressor of hRFC gene expression, via a mechanism that is independent of its effects on cell cycle progression.
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PMID:Repression of human reduced folate carrier gene expression by wild type p53. 1110 43

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.
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PMID:mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method. 1118 7

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.
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PMID:Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis. 1184 83

Antimetabolites are active chemotherapeutic agents for many solid tumor and hematologic malignancies. Folate antagonists, purine analogues, and pyrimidine analogues are the three main categories of antimetabolites. Methotrexate, the most studied folate antagonist, is effective in many malignancies. Methotrexate inhibits dihydrofolate reductase, which leads to accumulation of polyglutamated folates, causing further inhibition of thymidylate synthase and glycinamide ribonucleotide formyltransferase. Subsequently, the lack of reduced folate substrates impairs synthesis of purine nucleotides, thymidylate, and certain amino acids, which can lead to cell death. However, methotrexate resistance develops through several mechanisms, including decreased folate carrier-mediated membrane transport, dihydrofolate reductase gene amplification, specific transcription-translational modifications, and downregulation of intracellular methotrexate polyglutamation. Antifolate drug development has focused on agents designed to overcome different aspects of methotrexate resistance. This article reviews the enzymatic targets for antifolates, describes the known mechanisms of antifolate resistance, and summarizes the current development of novel antifolate agents. Discussed specifically are trimetrexate, edatrexate, raltitrexed, pemetrexed, ZD9331, lometrexol, LY309887, GW1843, OSI-7904(L), and nolatrexed, all of which have unique clinical pharmacology and are in various stages of development. The toxicity of antifolates has been sporadic and difficult to predict clinically. Supplementation with folic acid and vitamin B(12) has been shown to reduce the toxicity of pemetrexed without affecting efficacy and has increased the therapeutic index for this novel agent.
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PMID:Novel antifolate drugs. 1258 28

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.
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PMID:Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity. 1273 54

Previous studies have shown that decreased expression of the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR) are associated with intrinsic and acquired methotrexate resistance, respectively, in osteosarcoma (OS). It has also been shown in colorectal cancer that E2F-1 expression correlates with thymidylate synthase (TS) and, to a lesser extent, DHFR expression. To begin to investigate the regulation of DHFR and RFC expression in OS samples, mRNA expression of E2F-1 and E2F-4 were measured in OS tumor samples and related to DHFR, RFC, and TS mRNA expression. Using fluorescent quantitative real-time PCR, 112 human OS patient samples were investigated for potential E2F-1/E2F-4:DHFR, E2F-1/E2F-4:RFC, and E2F-1/E2F-4:TS correlations. The expression ranges for each gene are as follows: DHFR, 0.02-33.13 (median = 0.20); RFC, 0.02-229.13 (median = 1.91); TS, 0.01-9.99 (median = 0.15); E2F-1, 0.05-69.07 (median = 0.52); and E2F-4, 0.24-52.35 (median = 1.45). Spearman correlation coefficients (r(s)) for E2F-1:DHFR, E2F-1:RFC, E2F-1:TS, E2F-4:DHFR, E2F-4:RFC, and E2F-4:TS were calculated to be 0.53, 0.63, 0.60, 0.41, 0.58, and 0.33, respectively (P < 0.001). On the basis of this data, moderate correlations exist between E2F-1/E2F-4 and DHFR, RFC, and TS. These results suggest E2F-1/E2F-4 may play a role in the regulation of RFC expression, which has not been reported previously. The E2F transcription factors are also related to DHFR and TS expression in OS samples, suggesting a possible involvement in methotrexate resistance. Although E2F mRNA levels correlate with DHFR, RFC, and TS mRNA expression, additional experiments are necessary to determine the direct effects of these transcription factors and identify other proteins that may influence this relationship.
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PMID:mRNA expression levels of E2F transcription factors correlate with dihydrofolate reductase, reduced folate carrier, and thymidylate synthase mRNA expression in osteosarcoma. 1281 32

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.
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PMID:Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques. 1458 36


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