Inactivation Of Thymidylate Synthase Research Articles

Antimetabolite pemetrexed primes a favorable tumor microenvironment for immune checkpoint blockade therapy

BackgroundThe immune checkpoint blockade (ICB) targeting programmed cell death-1 (PD-1) and its ligand (PD-L1) has been proved beneficial for numerous types of cancers, including non-small-cell lung cancer (NSCLC). However, a...

Effective Timing of Surgical Resection of Colorectal Cancer Liver Metastases During Chemotherapy.

The aim of the present study was to further develop our previous study on c-Met expression in colorectal cancer and epithelial-mesenchymal transition (EMT) induced by hepatocyte growth factor (HGF), to investigate EMT in the process of liver metastases, and evaluate the effects of chemotherapy on EMT cells as a therapeutic strategy for colorectal liver metastasis. CT26 colon cancer cells were treated with 5-FU and oxaliplatin with or without HGF. The signaling pathway was evaluated by western blotting analysis, and drug resistance was evaluated by the MTT (3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide) assay. Under pretreatment with HGF for 96 h, 5 μM and 10 μM of 5-FU mediated significant growth inhibition by 72.5±3.9% and 76.2±2.4%, respectively, compared to HGF alone, and by 105.1±2.8% and 103.5±2.9%, respectively, without HGF. The expression of E2F1 was decreased significantly to 50.5±3.8% after 24 hours by HGF with a reduction of both cyclin D1 to 52.1±7.0% and E to 73.7±3.8%. Thymidylate synthase was also decreased in a time-dependent manner to 80.6±2.0% after 24 h and to 52.7±1.5% after 96 h. The presence of HGF was found to increase the 5-FU-induced death signal, JNK pathway, and inhibition of cell growth. As its mechanism, HGF was shown to decrease E2F-1 by reducing cyclin D or E by cell-cycle activation, resulting in inactivation of thymidylate synthase. The chemotherapeutic effect of 5-FU was increased in HGF- but not TGF-β-induced EMT.

Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. shRNA suppression of TS was compared with 5-fluoro-2'-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy.

Abstract 3514: Combination of 5-FU with ChoKα inhibitors as a new alternative therapy in colorectal cancer

Abstract Colorectal cancer (CRC) is the third major cause of cancer related deaths worldwide. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Despite the achieved improvements over the past years in cancer treatment, the design of novel targeted and combined therapies is still necessary. Choline kinase alpha (ChoKα), a phospholipid-related enzyme that plays a role in cell proliferation and transformation, has been found overexpressed in many different tumors, including colorectal tumors. ChoKα specific inhibitors, MN58b and TCD717, have potent antitumoral activity both in vitro and in vivo against several tumor-derived cell lines including CRC-derived tumour cells. TCD717 has recently entered clinical trials (http://clinicaltrials.gov/ct2/show/NCT01215864). We have evaluated the effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors. The sequential combination of ChoKα inhibitors with 5-FU resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. Key enzymes in the metabolic pathway of 5-FU such as Thymidylate synthase (TS) and Thymidine kinase (TK1) have been related to drug resistance, one of the major problems for the lack of success of 5-FU chemotherapy. TS is a key enzyme in the synthesis of DNA and the target enzyme of 5-FU. Several studies have demonstrated that the expression of TS mRNA or protein, predicts overall survival for colon cancer and correlates with resistance to 5-FU. Acquired resistance to 5-FU can also be caused by overproduction of TS that results from gene amplification. Regarding to 5-FU developed resistance, a salvaged pathway has been reported in which TK1 plays an important role. Thus, both TS and TK1 are directly involved in resistance to 5-FU therapy. To elucidate the mechanism of the observed synergistic effect, we examined the modulation of ChoKα inhibitors on the expression levels of TS and TK1. CRC cell lines treated with the sequential combination of ChoKα inhibitors and 5-FU show both mechanisms of TS inactivation, a formation of ternary complex due to 5-FU mechanism and a significant down-regulation of TS active due to ChoKα inhibitors, resulting in a total depletion of TS protein. We also observed that ChoKα inhibitors affects the levels of TK1, and provide the basis to support a new therapy focused on the combination of ChoKα inhibitors and 5-FU as a new approach for colorectal cancer patients. In addition, we suggest a promising role for ChoKα inhibitors as a new treatment for patients who had failed 5-FU chemotherapy or display high expression levels of TS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2011-3514

ChemInform Abstract: Mechanism-Based Inactivation of Thymidylate Synthase by 5-(3-Fluoropropyn-1-yl)-2′-deoxyuridine 5′-Phosphate.

Abstract 5-Fluoropropynyl-2′-deoxyuridine 5′-phosphate ( 3 ) was designed as a mechanism-based inactivator of thymidylate synthase (TS). The inhibitor was synthesized from 5-iodo-2′-deoxyuridine and propargyl alcohol by palladium-catalyzed coupling, followed by fluorination and selective phosphorylation. Incubation of TS with 3 , in the presence or absence of the CH 2 H 4 folate cofactor, caused rapid, irreversible inactivation of the enzyme.

Modification of Escherichia coli thymidylate synthase at tyrosine-94 by 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate.

Evidence is presented that 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate (IP-dUMP) is a mechanism-based, irreversible inactivator of Escherichia coli thymidylate synthase (TS), which covalently modifies Tyr94 at the active site of the enzyme. The inactivation of TS was time and concentration dependent and did not require the folate cofactor. Due to the rapidity of the inactivation process, accurate kinetic parameters could be determined only in the presence of saturating concentrations (1000K(M)) of the competing substrate, dUMP. Under these conditions, a K(I) of 0.36 +/- 0.09 microM and an inactivation rate constant (k(inact)) of 0.53 +/- 0.15 min(-1) were obtained from Kitz-Wilson plots. Electrospray ionization-mass spectrometry (ESI-MS) determined a 412 amu mass increase of TS after inhibition by IP-dUMP with no mass difference being detected for the TS mutants Tyr94Phe or Cys146Ala, thus indicating the importance of these residues for complex formation. The change in WT-TS mass was consistent with covalent modification by IP-dUMP, which was confirmed by proteolytic digestion of the modified protein followed by ESI-MS. By these means, a 43-residue trypsin peptide (residues 54-96), a 16-residue endoAspN peptide (residues 89-104), and an 8-residue endoAspN/endoLysC peptide (residues 89-96), each containing the IP-dUMP adduct, were observed. MS/MS analysis of the IP-dUMP-endoAspN peptide identified a modified 3-residue daughter ion, YGK (residues 94-96). A mechanistic scheme requiring the participation of Cys146 is proposed for the covalent modification of IP-dUMP by Tyr94, which, unlike an earlier proposal [Kalman, T. I., Nie, Z., and Kamat, A. (2001) Nucleosides Nucleotides Nucleic Acids 20, 869-871], does not require the release of imidazole for the activation of the inhibitor.

5-PROPYNYLPYRIMIDINE NUCLEOSIDE DERIVATIVES: RATIONALLY DESIGNED MECHANISM-BASED INACTIVATORS OF THYMIDYLATE SYNTHASE

A novel series of 5-propynyl-dUMP derivatives, with a variety of leaving groups on the side-chain, was designed as potential mechanism-based inhibitors of thymidylate synthase (TS), and synthesized from 5-iodo-2′-deoxyuridine by Pd(0)-catalyzed coupling, followed by direct phosphorylation with POCl3. All members of the series inhibited TS competitively with Ki-values of 0.015–18 μM. Analogs with fluorine or imidazole-based leaving groups caused rapid, irreversible inactivation of TS.

Reduction of 5-fluorouracil (5-FU) gastrointestinal (GI) toxicity resulting from the protection of thymidylate synthase (TS) in GI tissue by repeated simultaneous administration of potassium oxonate (Oxo) in rats.

An important cytotoxic effect of 5-fluorouracil (5-FU) is the inactivation of thymidylate synthase (TS) (EC 2.1.1.45) activity by the formation of a ternary complex consisting of covalently bound 5-fluorodeoxyuridine 5'-monophosphate (FdUMP), TS and 5,10-methylenetetrahydrofolate (CH2FH4). The gastrointestinal (GI) toxicity of 5-FU is also caused by its phosphorylation in the GI tract. Potassium oxonate (O(XO)) competitively inhibits pyrimidine phosphoribosyltransferase (EC 2.4.2.10), which converts 5-FU to 5-fluorouridine 5'-monophosphate (FUMP) in vitro. In this study the benefits of combining Oxo and tegafur (FT), which is a masked compound of 5-FU, in reducing the GI toxicity of 5-FU and in protecting the activity of TS in the normal GI tissues were evaluated. We administered orally a preparation of 1 M FT and 0.4 M 5-chloro-2,4-dihydroxypyridine (CDHP) with or without 1 M O(XO) (called S-1 and FT + CDHP, respectively) or vehicle only (control) to rats for ten consecutive days and compared the toxicity, the histopathological findings and the free TS activity in the GI tissues of the treated rats. During the experimental periods, the signs of toxicity, such as a decrease in body weight, diarrhea and death, were only observed in the rats treated with FT + CDHP. The histopathological findings in the ileum and colon samples from rats treated consecutively with S-1 on day 1, day 4, day 7 and day 10 were less frequent and more mild than in the samples from rats treated with FT + CDHP. Furthermore, the free TS activities in the ileum samples of rats given S-1 and FT + CDHP were significantly decreased compared with the activity in samples from the control rats throughout the experimental periods. The free TS activities in GI tissues of rats treated with S-1 were higher than the TS activities in tissues from rats treated with FT + CDHP daily from day 4 to day 10, although activities in S-1-treated rat were decreased to almost same low levels as in FT + CDHP-treated rats on day 1. Our results suggest that repeated simultaneous administration of Oxo and FT can effectively protect the activity of TS by decreasing FdUMP via FUMP from 5-FU in GI tissue, and may lead to a reduction in GI toxicity.

Modeling of Reaction Steps Relevant to Deoxyuridylate (dUMP) Enzymatic Methylation and Thymidylate Synthase Mechanism-Based Inhibition

Theoretical quantum mechanical ab initio Hartree-Fock calculations on molecular systems, modeling processes related to the specificity of thymidylate synthase inactivation are reported. We considered several steps of the methylation of the substrate dUMP and 4- or 5-mono- and 4,5-bisubstituted dUMP analogs, as well. The following reactions were modeled: the cysteine residue (Cys198 in the L. casei enzyme) nucleophilic attack on the substrate and the substrate C(5)-H proton abstraction. The substrate was modeled by the 1-methyluracil molecule and its structural analogs. The cysteine Cys198 residue was modeled by the methylmer-captane molecule. The substrate-enzyme binary complex was modeled by the 1-methy 1–5, 6- dihydro-6-thiomethyl-uracil (PI) molecule. The present theoretical calculations suggest that the cysteine nucleophilic attack on the substrate may result in the SH-group addition to the pyrimidine C(5)=C(6) bond in the course of a weakly exothermic reaction. The formerly presumed enolate carbanion appeared to be weakly stable or unstable and it can readily split into the thiol and pyrimidine residues. The s2-thio- (P2) and s24-dithio- (P3) substrate analogs should form stable thiolate anions after cysteine residue attachment to the C(6) position of the pyrimidine ring. Studies of the deformed PI molecule interacting with a water molecule bound to the pyrimidine C(4)=0 carbonyl residue allow a suggestion that this water molecule may be directly involved in the C(5)-H proton abstraction and may serve as a proton transmitter between the substrate and the proton acceptor residue, possibly located on the cofactor N10-nitrogen. Interaction of the pyrimidine C(4)=0 group, or its modification, with the N5,10-methylenetetrahydrofolate N(10) nitrogen atom is suggested as an additional factor influencing the inhibition process.

Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogs

N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of thymidylate synthase at an alternate high-affinity binding site.

Inactivation of thymidylate synthase at an alternate high-affinity binding site.

ChemInform Abstract: 5‐Quinone Derivatives of 2′‐Deoxyuridine 5′‐Phosphate: Inhibition and Inactivation of Thymidylate Synthase, Antitumor Cell, and Antiviral Studies.

AbstractThe iododeoxyuridine (I) is silylated and then photolytically coupled with the arenes (III), yielding the compounds (IVa) and (IVb) after desilylation.

Inactivation of thymidylate synthase by chlorine disinfectants

1. 1. The effects of two chlorine disinfectants, calcium hypochlorite (HTH) and 3-chloro-4.4-dimclhyl-2-oxazolidinonc (Agent I), on the activity of thymidylate synthase have been investigated. 2. 2. Although bolh disinfectants inactivated the enzyme, the following differences were observed: 3. 3. When the two disinfectants were used at the same total chlorine concentration, the rate and extent of inactivation were greater with Agent I than HTH. 4. 4. The substrate dUMP partially protected thyrnidylate synthase from inactivation by Agent 1, but it did not appreciably protect against inactivation by HTH. 5. 5. Large changes in the ultraviolet spectrum of the enzyme occurred when it was treated with HTH, which suggests reactions with aromatic amino acid side chains; no spectra) changes occurred when thyrnidylate synthase was treated with Agent I. 6. 6.Bocking the sulfhydryl groups of thymidylate synthase with sulfhydryl reagents prevented the irreversible inactivalion of the enzyme by Agent 1, but not by HTH.

The inactivation of thymidylate synthase by periodate

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of 10 4 − at 0°C Nearly complete inhibition resulted when the IO 4 − concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO 4 − was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by 10 4 −, with the order of effectiveness dUMP > dTMP > phosphate. Deoxyuridine, which is not a substrate, did not protect the enzyme. Titrations with dithiobis(2-nitrobenzoate) (DTNB) showed that approximately 1.5 titratable SH groups were lost when thymidylate synthase was completely inhibited by 10 4 −. Essentially no reactivation occurred when periodate-inhibited enzyme was dialyzed against buffered 2-mercaptoethanol (ME) or dithiothreitol (DTT). Enzyme that had been treated with p-hydroxymercunbenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with penodate could be completely reactivated with ME or DTT.

The inactivation of thymidylate synthase by diethyl pyrocarbonate

Thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C- methyltransferase , EC 2.1.1.45) from Lactobacillus casei was inactivated by treatment with diethyl pyrocarbonate. The inactivation was apparently due to the modification of a large proportion of the enzyme's histidine residues. Neither the substrate dUMP nor the product dTMP prevented inactivation by diethyl pyrocarbonate. The inactivated enzyme was not reactivated by treatment with hydroxylamine. These results indicate that histidine residues are involved in the maintenance of enzyme structure.

Kinetics of thymidylate synthase inhibition by disulfides

The inactivation of thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C- methyltransferase , EC 2.1.1.45) by a number of disulfides has been examined and found to be a second-order process. The apparent second-order rate constant was strongly influenced by the chemical structure of the disulfide. The data suggest that negatively charged functional groups decrease the reactivity of the disulfides and that positively charged groups enhance the reactivity. GSSG did not react with non-catalytic SH groups, since the number of SH groups of both GSSG-treated and untreated thymidylate synthase was the same. Several sulfhydryl compounds were tested for their ability to reactivate thymidylate synthase that had been inhibited by 2,2′-dithiodipyridine. Complete reactivation was obtained with either dithiothreitol or 2-mercaptoethanol. Reactivation by 2-mercaptoethanol was a second-order process.