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:2.1.1.45 (thymidylate synthase)
3,600 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro
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PMID:The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line. 1650 19

The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells.
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PMID:Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase. 1655 60

Folates play a key role in one-carbon metabolism essential for the biosynthesis of purines, thymidylate and hence DNA replication. The antifolate methotrexate has been rationally-designed nearly 60 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR) thereby achieving temporary remissions in childhood acute leukemia. Recently, the novel antifolates raltitrexed and pemetrexed that target thymidylate synthase (TS) and glycineamide ribonucleotide transformylase (GARTF) were introduced for the treatment of colorectal cancer and malignant pleural mesothelioma. (Anti)folates are divalent anions which predominantly use the reduced folate carrier (RFC) for their cellular uptake. (Anti)folates are retained intracellularly via polyglutamylation catalyzed by folylpoly-gamma-glutamate synthetase (FPGS). As the intracellular concentration of antifolates is critical for their pharmacologic activity, polyglutamylation is a key determinant of antifolate cytotoxicity. However, anticancer drug resistance phenomena pose major obstacles towards curative cancer chemotherapy. Pre-clinical and clinical studies have identified a plethora of mechanisms of antifolate-resistance; these are frequently associated with qualitative and/or quantitative alterations in influx and/or efflux transporters of (anti)folates as well as in folate-dependent enzymes. These include inactivating mutations and/or down-regulation of the RFC and various alterations in the target enzymes DHFR, TS and FPGS. Furthermore, it has been recently shown that members of the ATP-binding cassette (ABC) superfamily including multidrug resistance proteins (MRP/ABCC) and breast cancer resistance protein (BCRP/ABCG2) are low affinity, high capacity ATP-driven (anti)folate efflux transporters. This transport activity is in addition to their established facility to extrude multiple cytotoxic agents. Hence, by actively extruding antifolates, overexpressed MRPs and/or BCRP confer antifolate resistance. Moreover, down-regulation of MRPs and/or BCRP results in decreased folate efflux thereby leading to expansion of the intracellular folate pool and antifolate resistance. This chapter reviews and discusses the panoply of molecular modalities of antifolate-resistance in pre-clinical tumor cell systems in vitro and in vivo as well as in cancer patients. Currently emerging novel strategies for the overcoming of antifolate-resistance are presented. Finally, experimental evidence is provided that the identification and characterization of the molecular mechanisms of antifolate-resistance may prove instrumental in the future development of rationally-based novel antifolates and strategies that could conceivably overcome drug-resistance phenomena.
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PMID:Molecular basis of antifolate resistance. 1733 44

Methotrexate is the gold standard therapy for moderate to severe psoriasis, but there is marked interpersonal variation in its efficacy and toxicity. We hypothesized that in psoriasis patients, specific common polymorphisms in folate, pyrimidine, and purine metabolic enzymes are associated with methotrexate efficacy and/or toxicity. DNA from 203 retrospectively recruited psoriasis patients treated with methotrexate was collected and genotyped by restriction endonuclease digestion or length polymorphism assays. The reduced folate carrier (RFC) 80A allele and the thymidylate synthase (TS) 3'-untranslated region (3'-UTR) 6 bp deletion were associated with methotrexate-induced toxicity (P=0.025 and P=0.025, respectively). RFC 80A and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) 347G were associated with methotrexate discontinuation (P=0.048 and P=0.038). The TS 5'-UTR 28 bp 3R polymorphism correlated with poor clinical outcome (P=0.029), however, this was not the case when patients with palmoplantar pustular psoriasis were not included in the analysis. Stronger associations between specific polymorphisms and methotrexate-induced toxicity and discontinuation were found in a subanalysis of patients on methotrexate not receiving folic acid supplementation. We have demonstrated preliminary evidence that specific polymorphisms of enzymes involved in folate, pyrimidine, and purine metabolism could be useful in predicting clinical response to methotrexate in patients with psoriasis.
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PMID:Polymorphisms in folate, pyrimidine, and purine metabolism are associated with efficacy and toxicity of methotrexate in psoriasis. 1741 Jan 98

This study investigated associations between CpG island methylator phenotype (CIMP) colon cancer and genetic polymorphisms relevant to one-carbon metabolism and thus, potentially the provision of methyl groups and risk of colon cancer. Data from a large, population-based case-control study (916 incident colon cancer cases and 1,972 matched controls) were used. Candidate polymorphisms in methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS), transcobalamin II (TCNII), methionine synthase (MTR), reduced folate carrier (RFC), methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), dihydrofolate reductase (DHFR) and alcohol dehydrogenase 3 (ADH3) were evaluated. CIMP- or CIMP+ phenotype was based on five CpG island markers: MINT1, MINT2, MINT31, p16 and MLH1. The influence of specific dietary factors (folate, methionine, vitamin B(12) and alcohol) on these associations was also analyzed. We hypothesized that polymorphisms involved in the provision of methyl groups would be associated with CIMP+ tumors (two or more of five markers methylated), potentially modified by diet. Few associations specific to CIMP+ tumors were observed overall, which does not support the hypothesis that the provision of methyl groups is important in defining a methylator phenotype. However, our data suggest that genetic polymorphisms in MTHFR 1,298A > C, interacting with diet, may be involved in the development of highly CpG-methylated colon cancers. AC and CC genotypes in conjunction with a high-risk dietary pattern (low folate and methionine intake and high alcohol use) were associated with CIMP+ (OR = 2.1, 95% CI = 1.3-3.4 versus AA/high risk; P-interaction = 0.03). These results provide only limited support for a role of polymorphisms in one-carbon metabolism in the etiology of CIMP colon cancer.
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PMID:Genetic polymorphisms in one-carbon metabolism: associations with CpG island methylator phenotype (CIMP) in colon cancer and the modifying effects of diet. 1744 6

Antifolates that inhibit the key enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR) have found clinical utility as antitumor and antiopportunistic agents. Methotrexate {MTX, (1)} and 5-fluorouracil (5-FU) were among the first clinically useful DHFR and TS inhibitors, respectively. The development of resistance to 5-FU, its occasional unpredictable activity and toxicity resulted in the search of novel antifolates. Pemetrexed (4) and raltitrexed (5) specifically inhibit TS, and are clinically useful as antitumor agents. A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Novel antifolates have been developed that do not need to be polyglutamylated and include plevitrexed (6) and GW1843 (7). Nonclassical antifolates for antitumor and parasitic chemotherapy, such as nolatrexed (8), trimethoprim {TMP, (11)} and piritrexim {PTX, (12)}, can passively diffuse into cells and hence do not have to depend on FPGS or the reduced folate carrier (RFC). Variations in the structures of antifolates have helped delineate the structural influence on the interaction with TS, DHFR, FPGS, and RFC utilization. The differences in the active site of human and pathogen DHFR have also been exploited. The literature contains excellent reviews on the design and synthesis of antifolates prior to 1996. This two-part review discusses the design, synthesis and structural requirements for TS and DHFR inhibition and their relevance to antitumor and parasitic chemotherapy, since 1996. Monocyclic and 6-5 fused bicyclic antifolates will be discussed in Part I, while 6-6 bicyclic and tricyclic antifolates will be discussed in Part II.
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PMID:Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents: part I. 1789 13

Murine L1210 leukaemia cells expressing either the reduced folate carrier (RFC) or the membrane folate receptor (MFR) were studied in vitro and in vivo to assess the dynamics of membrane transport of two categories antifolates; folate-based inhibitors of dihydrofolate reductase (methotrexate, edatrexate, aminopterin, PT523, and PT644) and thymidylate synthase (TS) [CB3717, raltitrexed, plevitrexed (BGC9331), pemetrexed and GW1843]. The potency of in situ inhibition of TS was used as an endpoint to analyze the in vitro dynamics of RFC/MFR-membrane transport of these antifolates. Both for L1210-RFC and L1210-MFR cells, the potency of in situ TS inhibition was closely correlated with increasing affinities of these transporters for the antifolates (r = 0.64, P < 0.05 and r = -0.65, P < 0.05, respectively). Within the group of antifolates for which MFR had a low binding affinity, those that had the ability to become polyglutamylated, were more potent inhibitors of TS in situ activity than non-polyglutamatable antifolates. In vivo activity of methotrexate, edatrexate, raltitrexed and pemetrexed was assessed in L1210-RFC and L1210-MFR bearing mice that were fed either a standard or a folate-deficient chow. Dietary folate depletion significantly reduced the MTD for methotrexate (sevenfold), edatrexate (sevenfold), raltitrexed (50-fold) and pemetrexed (150-fold). Based on increased life spans, antitumor effects of methotrexate and edatrexate were markedly better in L1210-RFC bearing mice on the folate-deficient chow (ILS: 455 and 544%, respectively) than on standard chow (ILS: 213 and 263%, respectively). No therapeutic effects of methotrexate and edatrexate were observed for L1210-MFR bearing mice on either chow condition, which may be consistent with the low binding affinity for MFR. Irrespective of the folate diet status, pemetrexed and raltitrexed were inactive against both L1210-RFC and L1210-MFR bearing mice, which may be due to high circulating plasma thymidine levels. Collectively, this study underscores that modulation of dietary folate status can provide a basis within which the therapeutic effect of antifolates may be further improved.
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PMID:Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo. 1828 61

Antifolates that inhibit the key enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR) have found clinical utility as antitumor and antiopportunistic agents. Methotrexate {MTX, (1)} and 5-fluorouracil (5-FU) were among the first clinically useful DHFR and TS inhibitors, respectively. The development of resistance to 5-FU, its occasional unpredictable activity and toxicity resulted in the search of novel antifolates. Pemetrexed (4) and raltitrexed (5) are newer antifolates that specifically inhibit TS, and are clinically useful as antitumor agents. A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Recently, classical antifolates that do not need to be polyglutamylated have also been developed and include plevitrexed (6) and GW1843 (7). Nolatrexed (8), trimethoprim {TMP, (11)} and piritrexim {PTX, (12)} are nonclassical antifolates for antitumor and parasitic chemotherapy that passively diffuse into cells and hence do not have to depend on FPGS or the reduced folate carrier (RFC). Structural requirements for inhibition with antifolates have been studied extensively and novel agents that exploit key interactions in the active site of TS, DHFR, FPGS, and RFC have been proposed. This two-part review discusses the design, synthesis and structural requirements for TS and DHFR inhibition and their relevance to antitumor and parasitic chemotherapy, since 1996. Monocyclic and 6-5 fused bicyclic antifolates were discussed in Part I. The 6-6 bicyclic and tricyclic antifolates will be discussed here in Part II.
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PMID:Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents: Part II. 1828 23

Folic acid is an essential vitamin for a wide spectrum of biochemical reactions; however, unlike bacteria and plants, mammals are devoid of folate biosynthesis and thus must obtain this cofactor from exogenous sources. Therefore, folate deficiency may impair the de novo biosynthesis of purines and thymidylate and thereby disrupt DNA and RNA metabolism, homocysteine remethylation, methionine biosynthesis, and subsequent formation of S-adenosylmethionine (the universal methyl donor) which in turn may lead to altered methylation reactions. This impaired folate-dependent intracellular metabolism can lead to several key pathologies including, for example, megaloblastic anemia, homocysteinemia, cardiovascular disease, embryonic defects, in particular neural tube defects (NTDs), congenital heart defects, and possibly cancer. The current review presents and evaluates the up-to-date knowledge regarding the molecular mechanisms underlying cellular survival and/or adaptation to folate deficiency or insufficiency. These mechanisms of adaptation to folate deficiency generally associated with folate uptake, intracellular folate retention, folate-dependent metabolism, and active folate efflux specifically include: (a) Up- or downregulation of various folate-dependent enzymes like dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (b) Cellular retention of folates via polyglutamylation by the enzyme folylpoly-gamma-glutamate synthetase (FPGS), (c) Overexpression of folate influx systems including the reduced folate carrier (RFC), folate receptor (FR) as well as the proton-coupled folate transporter (PCFT), a recently identified intestinal folate influx transporter optimally functioning at the acidic microclimate of the upper intestinal epithelium, (d) Downregulation of ATP-driven folate efflux transporters of the multidrug resistance protein (MRP; ABCC) family and breast cancer resistance protein (BCRP; ABCG2) that belong to the multidrug resistance (MDR) efflux transporters of the ATP-binding cassette (ABC) superfamily. Moreover, the intricate interplay between various components of the adaptive response to folate deprivation is also discussed.
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PMID:Molecular mechanisms of adaptation to folate deficiency. 1880 93

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise-increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 microm, and established three pemetrexed-resistant lung cancer cell lines: PC6/MTA-0.4, PC6/MTA-1.6, and PC6/MTA-4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase (TS), reduced folate carrier (RFC), and folylpoly-gamma-glutamate synthetase (FPGS) genes. TS gene expression was significantly increased in PC6/MTA-1.6 and PC6/MTA-4.0 cells relative to parental cells in a pemetrexed dose-dependent manner. In contrast, the levels of RFC gene expression in PC6/MTA-0.4 cells and FPGS in PC6/MTA-1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6/MTA-4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6/MTA-4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC(50)) in 11 non-small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose-dependent, and that up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer.
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PMID:Significance of thymidylate synthase for resistance to pemetrexed in lung cancer. 1981 98


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