Irreversible Inhibition of Thymidylate Synthase by Pyridoxine (B6) Analogues

Abstract

Three vitamin B₆ analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower K_i) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B₆ analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.     

中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关。淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原。在已完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF011L的TS基因结构、及其推定蛋白结构进行了分析。该基因全长858bp,GC含量为28．2％,编码一个长为286aa、分子量为32．7kD、等电点为7．1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS)。该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性。二级结构预测结果显示LCDV-C TS含8个a螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征。这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS。对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-C TS被单独分为一枝。进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨。     

中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关.淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原.在己完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF 011L的TS基因结构、及其推定蛋白结构进行了分析.该基因全长858bp,GC含量为28.2%,编码一个长为286aa、分子量为32.7kD、等电点为7.1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS).该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性.二级结构预测结果显示LCDV-C TS含8个α螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征.这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS.对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-CTS被单独分为一枝.进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨.     

Polymorphism of the Thymidylate Synthase Gene and Thymidylate Synthase Levels in Colon Cancer Cell Lines and Different Tissues of Colorectal Cancer Patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

Thymidylate Synthase Polymorphism and Microsatellite Instability: Association in Colorectal Cancer

5‐Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5′ and 3′untraslated region (5′–3′ UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5′UTR) is more frequently associated with MSI + and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5′UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5′ UTR) genotypes with the MSI + and MSI ?, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI +, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.     

Difference in Ki67 and Thymidylate Synthase Expression in Primary Tumour Compared with Metastatic Nodes in Breast Cancer Patients

Breast cancer is a heterogeneous disease, so therapeutic predictive biological markers need to be identified. To date an accurate evaluation of predictive markers is mainly done at the primary site; however, the main goal of adjuvant therapy for breast cancer is the control of micrometastases. The aim of this study is to assess as therapeutic and/or prognostic marker, the proliferation status of primary tumors and involved nodes as measured by Ki67 and thymidylate synthase (TS) expression, in 30 breast cancer node positive patients. TS is the main target of 5-fluorouracil (5-FU) activity, and its overexpression is one of the mechanisms of 5-FU drug resistance; however, in some studies its absence is responsible for a worse response to 5-FU. Our results show that malignant cells of involved nodes were in a post mitotic phase of the cell cycle, and show a low proliferation index and TS expression, while the primary tumours and controls, were strongly positive. On these basis we can hypothesize that these cells could be less sensitive to 5-FU. Further studies are necessary to identify other mechanisms responsible for their metastasing capability and/or for their aggressiveness.     

Radiosensitization by Thymidine Phosphorylase Inhibitor in Thymidine Phosphorylase Negative and Overexpressing Bladder Cancer Cell Lines

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p <.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ?H2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.     

DNA甲基化转移酶抑制剂与胸苷酸合成酶抑制剂体外联合用药的抗肿瘤增效作用

通过在体外分别对DNA甲基化转移酶（DNA methyhransferase,DNMT）抑制剂5-氮杂胞苷（5-azacytidine,5-aza—C）和新型胸苷酸合成酶抑制剂盐酸洛拉曲克（nolatrexed dihydrochloride．Nolatrexed）联合用药于人大肠癌细胞LoVo和人肝癌细胞Hep3B的相互作用性质的观察,探讨DNMT抑制剂和胸苷酸合成酶抑制剂联合用药的可能性．使用MTT法测定二者单独用药或联合用药的抗肿瘤活性,用抑制浓度的分数之和（sum of fractional inhibitory concentration,SFIC）值及等效剂量分析方法（isobologram）评价联合用药的作用性质．结果显示,联合用药时其SFIC值均小于或等于1,由此得到的等效剂量曲线图形表现为凹形．可见,5-aza—C和Nolatrexed体外联合用药抗肿瘤相互作用性质为明显的增效作用,DNMT抑制剂和胸苷酸合成酶抑制剂联合用药达到抗肿瘤增效作用是可行的．     

Determination of Thymidine Phosphorylase Activity in Human Blood Cells and Fibroblasts by a Nonradiochemical Assay Using Reversed-Phase High-Performance Liquid Chromatography

In this study, we demonstrated that the highest activity of thymidine phosphorylase (TP) was found in peripheral blood mononuclear (PBM) cells followed by that of thrombocytes and granulocytes whereas no activity of TP could be detected in erythrocytes. The activity of TP in leukocytes proved to be intermediate compared to the TP activity observed in PBM cells and granulocytes. The activity of TP also was readily detectable in human fibroblasts.     

Thymidylate synthetase-positive and -negative murine mammary FM3A carcinoma cells as a useful system for detecting thymidylate synthetase inhibitors

The murine mammary FM3A/O and the thymidylate (dTMP) synthetase-deficient FM3A/TS^? carcinoma cell lines can be considered as a novel and useful test system for the detection of nucleoside analogues which are directly aimed at the thymidylate synthetase. These compounds should be inhibitory for FM3A/O but not for FM3A/TS^? cells, and their inhibitory effects on FM3A/O cell growth should be readily reversed by exogenous dThd within the concentration range of 5–20 μM.     

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells

Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2′-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.     

Isolation and characterization of thymidylate synthetase mutants of Xanthomonas maltophilia

Thymidylate synthetase mutants of Xanthomonas maltophilia ATCC 13270 were isolated on a solid minimal medium containing 50 mg/l thymidine and a high concentration of trimethoprim (500 mg/l). It was found that a high concentration of trimethoprim was required to prevent background growth of the wild-type strain. The isolated mutants could grow on thymidine or dTMP at a concentration of 50 mg/l while they were unable to grow on 1000 mg/l thymine or 50 mg/l deoxyridine. Thymidylate synthetase activity was assayed in the wild-type cells and in the mutant cells but only the wild-type cells contained measurable enzyme activity.     

Intracellular thymidylate synthase inhibition by trifluorothymidine in FM3A cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5-Fluorouracil (5FU). TS activity reduced to 9% (0.1 microM TFT) and 58% (1 microM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK-) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Functional Inactivity and Mutations of p53 Differentially Affect Sensitivity to 5-Fluorouracil and Antifolate Inhibitors of Thymidylate Synthase (TS) by Altering TS Levels in Colorectal Cancer Cells

The role of p53 in altering TS expression and chemosensitivity was studied in colorectal cancer cells with wildtype, mutated, or functionally inactive p53. Cytotoxicity of TS inhibitors was studied by MTT, while PCR, Western blot, and activity assays assessed whether p53 status influenced TS expression. Lovo-175X2 cells showed increased resistance to TS inhibitors and significantly greater than wildtype expression and activity of TS. In contrast, Lovo-273X17 and Lovo-li were more sensitive to TS inhibitors and had reduced TS expression, due either to reduced TS mRNA or altered regulation of TS activity. Thus, functional inactivity and mutations of p53 differentially affect TS, potentially influencing response to TS inhibitor-based treatment.     

Selective Protection by Uridine of Growth Inhibition by 5-Fluorouracil (5FU) Mediated by 5FU Incorporation into RNA,But Not the Thymidylate Synthase Mediated Growth Inhibition by 5FU-Leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.     

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases,and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

A series of 5‐substituted analogs of 6‐aza‐2′‐deoxyuridine 5′‐monophosphate, 6‐aza‐dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin‐dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N‐atom in 6‐aza‐dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM ). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3‐(octanamido)prop‐1‐ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM ). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N‐atom furnished 6‐aza‐dTMP ( 1b ), which still was recognized as a substrate by TMPKmt.     

Protection against DNA damage-induced apoptosis by the angiogenic factor thymidine phosphorylase

Thymidine phosphorylase (TP) is involved both in pyrimidine nucleoside metabolism and in angiogenesis. TP also conferred the resistance to hypoxia-induced apoptosis of the cancer cells. In U937 cells, DNA damage-inducing agents significantly enhanced the expression of TP. Cell lines stably transfected with TP cDNA were more resistant to the DNA damage-inducing agents than the mock-transfected cells and showed augmented activity of Akt. The cytoprotective function of TP against DNA damage was independent of its enzymatic activity. The resistance to apoptosis was partially abrogated by treatment with the phosphatidyl inositol 3-kinase (PI3K) inhibitors, suggesting that the cytoprotective function of TP is mediated, at least in part, by regulation of the PI3K/Akt pathway. These findings indicate that TP expression in increased by various stress including DNA damage and that TP molecules confer resistance to DNA damage-induced apoptosis in cancer cells.     

Potent Inhibition of Macrophomate Synthase by Reaction Intermediate Analogs

Potent inhibitors for macrophomate synthase, which has recently been found to catalyze a highly unusual five-step chemical transformation, were explored. Among 11 oxalacetate analogs tested, only three analogs had moderate to relatively strong inhibitory activities (I ₅₀ 1.3-8.1 mM). On the other hand, among 35 bicyclic intermediate analogs synthesized, two diacids were found to be the most potent inhibitors (I ₅₀ 0.80, 0.84 mM) which had a much higher affinity than that of the natural substrate 2-pyrone. (-)-Enantiomers of the diacids showed 30 times stronger activity (I ₅₀ 0.34, 0.41 mM) than (+)-ones. The I ₅₀/K _m values (0.20, 0.24) showed their potent inhibitions. Competitive inhibitions were observed in two representative inhibitors.     

Ethnic Variation in the Thymidylate Synthase Enhancer Region Polymorphism among Caucasian and Asian Populations

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.     

Calmodulin Inhibitor-induced Apoptosis was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calmodulin is known to transduce Ca²⁺ signals by interacting with specific target proteins. In order to determine the role of calmodulin in regulating neuronal survival and death, we examined, whether calmodulin inhibitors induce caspase-dependent apoptotic cell death, and whether glycogen synthase kinase-3 is involved in calmodulin inhibitor-induced cell death in PC12 cells. W13, a calmodulin specific inhibitor increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation of fragmentation. Glycogen synthase kinase-3 inhibitors prevented calmodulin inhibitor-induced apoptosis. In addition, nerve growth factor and cycloheximide, a protein synthesis inhibitor, completely blocked cell death. Moreover, caspase-3 activation was accompanied by calmodulin inhibitor-induced cell death and inhibited by nerve growth factor. These results suggest that calmodulin inhibitors induce caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.