Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.1.1.45 (thymidylate synthase)
 3,600 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.
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PMID:Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line. 1266 51

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

The alpha-isoform of the glycosylphosphatidylinositol cell membrane tethered folate receptor (alpha-FR) is overexpressed in some carcinomas (notably ovarian carcinomas) relative to normal tissues. The nonpolyglutamatable folate-based thymidylate synthase (TS) inhibitor, CB300638 (TS K(i) = 0.24 nM) displayed an IC(50) of 0.0028 microM for the inhibition of the growth of human A431-FBP cells transfected with the alpha-FR. In contrast, the IC(50) for the neotransfected A431 cells was 0.81 microM (300-fold higher). Similarly, this compound inhibited the growth of human KB cells that constitutively overexpress the alpha-FR with an IC(50) of 0.0036 microM. These data were derived from cells grown in a physiological concentration of folate (20 nM R,S-leucovorin). Incubation of KB cells with a 1 microM excess of folic acid (FA), to selectively block uptake via the alpha-FR, increased the CB300638 IC(50) to 0.39 microM. The relatively low potency of CB300638 under these conditions, or in cell lines not expressing the alpha-FR, is ascribed to its low affinity for the ubiquitously expressed folate transporter, the reduced-folate carrier (K(i) for inhibition of [(3)H]methotrexate transport >100 microM). The high potency of CB300638 in alpha-FR-overexpressing cell lines is attributable to high affinity of the alpha-FR (53% of FA) and efficient endosomal trafficking mediated by the alpha-FR. Sixteen-h exposure to CB300638 inhibited the rate of (3)H(2)O release from 5-[(3)H]dUrd (in situ TS assay) in A431, A431-FBP, and KB cells with IC(50) values of 0.1 microM, 0.005 microM, and 0.002 microM, respectively. The coaddition of 1 micro M FA increased the IC(50)s for A431-FBP and KB cells to approximately 0.1 microM consistent with alpha-FR-mediated transport of CB300638. In conclusion, alpha-FR-mediated uptake of CB300638 leads to TS and growth inhibition that is highly selective for alpha-FR overexpressing tumor cell lines. The low expression of the alpha-FR in normal tissues, particularly those sensitive to TS inhibitors, together with the low affinity of CB300638 for the reduced-folate carrier, suggests that the compound may have potential as an antitumor agent with a high therapeutic index.
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PMID:Selective delivery of CB300638, a cyclopenta[g]quinazoline-based thymidylate synthase inhibitor into human tumor cell lines overexpressing the alpha-isoform of the folate receptor. 1283 49

Pemetrexed (ALIMTA, MTA) is a novel thymidylate synthase (TS) inhibitor and has shown activity against colon cancer, mesothelioma and nonsmall-cell lung cancer. We induced resistance to Pemetrexed in the human colon cancer cell line WiDr by using a continuous exposure to stepwise increasing Pemetrexed concentrations (up to 20 microM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 microM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low folate conditions. Mechanisms of resistance to Pemetrexed were determined at the level of TS, folylpolyglutamate synthetase (FPGS) and reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor Thymitaq (6- and 42-fold, respectively) but not to 5-fluorouracil. The ratios of TS mRNA:beta actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold (P=0.01) and 18-fold (P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 microM dUMP increased from 30 pmol/hr/10(6) cells in WiDr cells to 2201 and 7663 pmol/hr/10(6) cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained Pemetrexed resistance. Altogether, these results indicate that resistance to Pemetrexed in the colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (mRNA and protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.
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PMID:Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression. 1290 42

Most antifolate drugs are efficiently transported by the reduced-folate carrier (RFC). However, several also bind with high affinity to the alpha-isoform of the folate receptor (alpha-FR) and there is evidence to suggest that this transport mechanism may contribute to their activity when the receptor is highly overexpressed or when the extracellular folate concentration is very low. In particular, the presence of the alpha-FR on tumour cell lines sensitises them to brief exposures to ZD9331. Nevertheless, it is the ubiquitous expression of the RFC in normal tissues that reduces patient tolerability to antifolate drugs. The overexpression of the alpha-FR in some epithelial tumours and its restricted distribution in normal tissues suggests an opportunity for the development of antifolates specifically targeted at alpha-FR overexpressing tumours. Potent cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) have been discovered with high and low affinity for the alpha-FR and RFC, respectively. This class of agent is represented by CB300638 (TS Ki=0.24 nM) that displays high potency (IC50 approximately 3 nM) for A431-FBP cells (transfected with the alpha-FR) and KB cells (constitutive overexpression). Importantly, this activity is approximately 300-fold higher than for alpha-FR negative cell lines such as A431. In mice bearing the KB tumour xenograft we have demonstrated localisation of CB300638 to tumour and, more importantly, specific inhibition of TS in tumour and not in normal tissues. Data supports the clinical development of this class of agent with the prediction that toxicity would be reduced compared with conventional antifolate drugs. There are a number of challenges to this development posed by the uniqueness of the compounds and these are discussed.
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PMID:Antifolates targeted specifically to the folate receptor. 1509 10

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 micromol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC(50), in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.
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PMID:Loss of reduced folate carrier function and folate depletion result in enhanced pemetrexed inhibition of purine synthesis. 1570 1

Folate metabolism supports the synthesis of nucleotides as well as the transfer of methyl groups. Polymorphisms in folate-metabolizing enzymes have been shown to affect risk of colorectal neoplasia and other malignancies. Using data from a population-based incident case-control study (1,600 cases and 1,962 controls), we investigated associations between genetic variants in the reduced folate carrier (RFC), thymidylate synthase (TS), methionine synthase (MTR), and 5,10-methylenetetrahydrofolate reductase (MTHFR) and colon cancer risk. The TS enhancer region (TSER) variant was associated with a reduced risk among men [2rpt/2rpt versus 3rpt/3rpt wild-type; odds ratio (OR), 0.7; 95% confidence interval, 0.6-0.98] but not women. When combined genotypes for both TS polymorphisms (TSER and 3'-untranslated region 1494delTTAAAG) were evaluated, ORs for variant genotypes were generally below 1.0, with statistically significantly reduced risks among women. Neither MTR D919G nor RFC 80G>A polymorphisms were associated with altered colon cancer risk. Because folate metabolism is characterized by interrelated reactions, we evaluated gene-gene interactions. Genotypes resulting in reduced MTHFR activity in conjunction with low TS expression were associated with a reduced risk of colon cancer. When dietary intakes were taken into account, individuals with at least one variant TSER allele (3rpt/2rpt or 2rpt/2rpt) were at reduced risk in the presence of a low folate intake. This study supports findings from adenoma studies indicating that purine synthesis may be a relevant biological mechanism linking folate metabolism to colon cancer risk. A pathway-based approach to data analysis is needed to help discern the independent and combined effects of dietary intakes and genetic variability in folate metabolism.
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PMID:Polymorphisms in the reduced folate carrier, thymidylate synthase, or methionine synthase and risk of colon cancer. 1628 71

BGC 945 is a cyclopenta[g]quinazoline-based, thymidylate synthase inhibitor specifically transported into alpha-folate receptor (alpha-FR)-overexpressing tumors. Affinity of BGC 945 for the alpha-FR is 70% of the high-affinity ligand folic acid. In contrast to conventional antifolates, BGC 945 has low affinity for the widely expressed reduced-folate carrier (RFC). The K(i) for isolated thymidylate synthase is 1.2 nmol/L and the IC(50) for inhibition of the growth of alpha-FR-negative mouse L1210 or human A431 cells is approximately 7 micromol/L. In contrast, BGC 945 is highly potent in a range of alpha-FR-overexpressing human tumor cell lines (IC(50) approximately 1-300 nmol/L). Pharmacokinetic variables measured following i.v. injection of 100 mg/kg BGC 945 to KB tumor-bearing mice showed rapid plasma clearance (0.021 L/h) and tissue distribution. The terminal half-lives in plasma, liver, kidney, spleen, and tumor were 2, 0.6, 5, 21, and 28 hours, respectively. Tumor BGC 945 concentration at 24 hours was approximately 1 nmol/g tissue, at least 10-fold higher than that in plasma or normal tissues. Inhibition of thymidylate synthase in tissues leads to increased incorporation of 5-[(125)I]-iodo-2'-deoxyuridine ([(125)I]dUrd) into DNA. Forty-eight hours after injection of 100 mg/kg 6RS-BGC 945 ([(125)I]dUrd injected at 24 hours), tumor was the only tissue with incorporation above control level (6-fold). The RFC-mediated thymidylate synthase inhibitor plevitrexed also increased uptake of [(125)I]dUrd in tumor (10-fold) but, in contrast, also caused increased incorporation in other normal tissues such as spleen and small bowel (4.5- and 4.6-fold, respectively). These data suggest that BGC 945 selectively inhibits thymidylate synthase in alpha-FR-overexpressing tumors and should cause minimal toxicity to humans at therapeutic doses.
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PMID:BGC 945, a novel tumor-selective thymidylate synthase inhibitor targeted to alpha-folate receptor-overexpressing tumors. 1635 84

Genetic instability, including chromosomal imbalance, is important in the pathogenesis of lymphoproliferative disorders such as non-Hodgkin lymphoma (NHL). DNA synthesis and methylation, which are closely linked to folate metabolism and transport, may be affected by polymorphisms in genes involved in these pathways. Folate metabolism polymorphisms have been linked to acute lymphoblastic leukemia and colorectal cancer. To evaluate whether genetic variation in folate metabolism and transport may have a role in determining the risk of developing NHL, we analyzed several polymorphisms using DNA obtained as part of a large U.K. population-based case-control study of lymphoma. Polymorphisms studied include methylenetetrahydrofolate reductase (MTHFR) 677 C >T and 1298 A >C, methionine synthase (MTR) 2756 A>G, serine hydroxymethyltransferase (SHMT1) 1420 C >T, thymidylate synthase (TYMS) 1494del6 and 28-bp repeat, and reduced folate carrier (RFC) 80 G >A. Increased risks for NHL [odds ratio (OR), 1.48; 95% confidence intervals (CI), 1.12-1.97], and marginal zone lymphoma (OR, 3.38; 95% CI, 1.30-8.82) were associated with the TYMS 2R/3R variant. Marginal increased risks were also observed for diffuse large B cell lymphoma with the TYMS homozygous 6 bp deletion (OR, 1.61; 95% CI, 0.99-2.60) and for follicular lymphoma with RFC 80AA (OR, 1.44; 95% CI, 0.94-2.22) and TYMS 28-bp repeat 2R/3R (OR, 1.45; 95% CI, 0.96-2.2). We observed no association between NHL and haplotypes for MTHFR or TYMS. These findings are somewhat inconsistent with those of others, but may reflect differences in circulating folate levels between study populations. Thus, further investigations are warranted in larger series with dietary information to determine the roles that genetics and folic acid status play in the etiology of lymphoma.
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PMID:Risk of non-Hodgkin lymphoma associated with polymorphisms in folate-metabolizing genes. 1636 25


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