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．     

组蛋白脱乙酰酶抑制剂的抗癌作用

组蛋白乙酰转移酶和组蛋白脱乙酰酶分别催化组蛋白的乙酰化和脱乙酰基反应,调节组蛋白的乙酰化水平,从而调控基因表达。这些过程与恶性肿瘤的发生具有密切的关系。组蛋白脱乙酰酶抑制剂通过增加细胞内组蛋白的乙酰化程度,调节多种基因的表达水平,抑制肿瘤细胞的增殖、诱导细胞分化和凋亡。该文从抑制细胞增殖、诱导细胞分化、诱导细胞凋亡和抗血管形成等4个方面介绍组蛋白脱乙酰酶抑制剂的抗癌机制,并简要介绍它们的分类。     

四核苷酸Ap Gp Ap Gp的合成

本文报道了酵母tRNA~(ala)分子中第42—45核苷酸片段——四核苷酸AGAGp的合成。首先我们化学合成了N,2′,5′-全乙酰化腺苷-3′-磷酸N,N-二甲氨基甲叉鸟苷-3′-磷酸的二环己基码啉胍盐,经DCC缩合、脱保护得到Ap Gp,然后通过氯甲酸乙酯环化形成ApG>p,再经RNaseN_1酶促合成为四核苷酸AGAGp。     

胸苷酸合成酶mRNA亲和多肽的鉴定及其对翻译过程的抑制作用

胸苷酸合成酶(thymidylate synthase, TS)是催化生物体内胸苷酸合成所必需的酶, 多年来一直作为肿瘤化疗的靶点. 研究表明, TS是一种RNA结合蛋白, 可以与其自身mRNA的2个位点结合, 使mRNA翻译受阻. 本文以mRNA体外展示技术进行了由大容量多肽库(>10¹³)中筛选胸苷酸合成酶mRNA亲和多肽的研究, 对随机库进行了12轮循环的选择及扩增. 结果表明, 与初始库相比, 经选择循环之后, 碱性氨基酸及芳香族的苯丙氨酸含量明显增加, 它们在TS RNA与蛋白质的相互作用中扮演着重要角色. 按其理化特性对每一随机位点的氨基酸进行分类, 并与初始库比较, 发现位点1, 12, 17和18具有明显的带正电荷的特性, 表明碱性侧链参与了与RNA的结合. 二级结构预测表明, 随着筛选的进行, 与TS mRNA 亲和的多肽显示出明显的螺旋倾向, 而且形成螺旋的区域富含碱性氨基酸. 凝胶迁移及体外翻译实验证实, 选择循环之后的多肽能够与TS mRNA高度亲和, 并能抑制TS mRNA的翻译. 本研究表明mRNA体外展示方法得到的亲和多肽可以用作新的TS RNA的翻译抑制剂, 并有可能成为一类新型的抗肿瘤药物.     

流式细胞术检测组蛋白乙酰化水平方法的建立与应用

组蛋白去乙酰化酶抑制剂是近年来出现的一类新的抗肿瘤药物,在艾滋病等其他疾病中同样也受到关注。但是在基础和临床研究中,目前还缺乏统一可靠的组蛋白乙酰化水平的检测手段。本文利用全血和外周血单个核细胞,通过一系列的对比实验,比较了不同样品处理温度(冰上和室温)、破膜方法(细胞内因子染色破膜和核内因子染色破膜)、抗体剂量(抗体滴定)和抗体孵育时间(时间梯度)等实验条件对流式细胞术检测的影响,最终建立了一套基于流式细胞术的组蛋白乙酰化水平检测手段。同时,将优化后的流式细胞检测技术应用于西达本胺(目前国内唯一上市的组蛋白去乙酰化酶抑制剂)的体外实验和临床试验,结果均证明本文建立的组蛋白乙酰化流式细胞检测方法可以作为基础和临床研究中一个可靠、快速、便捷的检测手段。     

信号传导拮抗物对大豆细胞植保素和异黄酮积累的影响

磷酸酶的抑制剂花萼海绵诱癌素A、芫菁素和冈田酸都能诱导大豆植保素(大豆素)的积累。相反,激酶的抑制剂K252a几乎完全阻止它们诱导大豆素的合成。与磷酸酶抑制剂相比较,酵母细胞壁激发子(YE)有利于诱导黄苷元、染料木苷等异黄酮中间体的积累,而磷酸酶的抑制剂有利于大豆素的合成。YE和芫菁素还呈现出协同诱导大豆素积累的效果。通过磷脂酶A2的抑制剂与阻止线粒体ATP合酶活性的化合物的分别处理,发现茉莉酸信号传导途径是参与调控大豆植保素合成的重要途径之一,而植保素的合成需要线粒体提供能量。     

PARP酶抑制剂未引起整合的外源LacZ基因的丢失

使用聚ADP核糖聚合酶（PARP）NAD位点抑制剂苯甲酞胺（BA）研究了降低PARP酶活性对外源LacZ基因整合稳定性的影响．利用DNA体外重组技术将LacZ基因全序列插入到真核表达载体pSV2neo的HindⅢ位点,构建了一个具有真核细胞neo基因筛选标记和LacZ基因的真核表达重组体pSV2neo-beta-gal．将该重组体导入HeLa细胞,经G418筛选获得了能稳定表达β-半乳糖苷酶的HeLa转化细胞系HeLa-beta-gal．使用PARP酶抑制剂苯甲酸胺处理细胞5周,随后进行细胞基因组Southern杂交分析与细胞内容物β-半乳苷酶的活性检测．结果表明,经BA处理的HeLa-beta-gal细胞β-半乳糖苷酶的活性及杂交带密度与未经BA处理的HeLa-beta-gal细胞相比未有明显区别．结合以前的结果可以认为,PARP酶抑制剂BA并不能导致所有外源基因的丢失,且使用PARP酶抑制剂BA引起外源基因的丢失具有选择性．     

小麦蓝矮病植原体胸苷酸激酶基因(tmk)的分离、原核表达及酶活性分析

[目的]克隆、表达小麦蓝矮病(WBD)植原体胸苷酸激酶基因(tmk),并分析酶活性,进一步研究胸苷酸激酶在植原体感染宿主及繁殖过程中的功能和作用机理,更好地防治植原体病害.[方法]PCR方法扩增tmk基因并进行序列分析,连接pET30a( )表达载体后原核表达,经Ni-NTA柱层析纯化后进行酶催化活性分析.[结果]首次从小麦蓝矮病(WBD)植原体基因组中分离出胸苷酸激酶基因(tmk),该基因包含tmk-1和tmk-2两种,大小分别为630 bp和624 bp,其编码的氨基酸序列均包含3个与结合NTP/NMP相关的保守功能区.表达的融合蛋白TMK-1活性极低,酶活仅16.4 U/mg,而 TMK-2酶活高达112.41 U/mg,且其最适催化条件为32℃、pH 7.3、1.5 mmol/L Mg2 和 1 mmol/L ATP.[结论]分析了胸苷酸激酶活性中心的一级结构序列及其催化活性随条件变化而改变的性质,为深入研究小麦蓝矮病植原体胸苷酸激酶在侵染寄主及其在宿主体内增殖的转录性质奠定基础.     

斑马鱼胸苷酸合成酶与自身mRNA的相互作用

斑马鱼(zebrafish,Danio rerio)是生物学领域中公认的研究脊椎类生物的模式生物.胸苷酸合成酶(thymidylate synthase,TS)是DNA从头合成的限速酶,多年来一直作为肿瘤化疗的重要靶酶.前期的研究表明,人和大肠杆菌中TS能与自身的mRNA结合,在翻译水平上具有反馈抑制自调控现象.斑马鱼作为药物模型的研究已成为热点研究领域,为了探讨斑马鱼的胸苷酸合成酶的调控规律,以及与人TS的相关性,利用原核表达,纯化获得高均一性斑马鱼TS蛋白,采用凝胶迁移研究了TS和其mRNA的体外结合,采用免疫共沉淀:RT-PCR技术研究了它们在体内的相互作用,实验结果表明,斑马鱼的TS在体内外均与自身的mRNA存在特异性的相互作用.研究说明,斑马鱼和人的TS具有高度生物学过程相关性,为构建斑马鱼抗肿瘤药理模型提供了理论基础.     

脱乙酰壳多糖处理增加人参细胞皂苷的累积和皂苷合成关键酶基因的转录

脱乙酰壳多糖处理可以诱导人参细胞产生H2 O2 ,增加人参皂苷的累积 ,提高鲨烯合酶 (squalenesynthase,GSS)与鲨烯环氧酶 (squaleneepoxidase,GSE)基因的转录水平。质膜NADPH氧化酶的抑制剂DPI,H2 O2 的淬灭剂DMTU与DHC可以抑制脱乙酰壳多糖的这些效应 ,暗示脱乙酰壳多糖可以活化质膜NADPH氧化酶而产生H2 O2 ,H2 O2 进而作为第二信使诱导gss与gse基因转录以及皂苷的合成。质膜钙通道抑制剂LaCl3与内质网钙通道抑制剂RR ,以及蛋白激酶抑制剂K2 5 2a都能削弱脱乙酰壳多糖促进皂苷积累和gss、gse转录的效应 ,说明胞内Ca2 浓度的升高与蛋白质磷酸化都参与了脱乙酰壳多糖诱导的gss、gse的转录以及皂苷的合成     

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.     

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Interferon effects upon fluorouracil metabolism by HL-60 cells

In order to better understand the synergistic antiproliferative effects of interferon in combination with fluorouracil (FUra), we studied effects of alpha 2-interferon upon FUra induced inhibition of thymidylate synthase of HL-60 cells. The 50% inhibitory dose for FUra decreased from approximately 75 microM to 10 microM following interferon treatment, as measured by whole cell activity assays. Enhanced FUra inhibition of cytosolic [3H] - FdUMP binding of interferon treated cells was also noted. FdUMP accumulation following FUra treatment increased over 10 fold in interferon treated cells, but dUMP did not increase. These results suggest that interferon can sensitize cells to FUra inhibition of thymidylate synthase by enhancing accumulation of FdUMP.     

Building a common feature hypothesis for thymidylate synthase inhibition

A set of 21 highly flexible competitive inhibitors of thymidylate synthase (TS; EC 2.1.1.45) covering a wide activity range (IC50 = 6 nM-100 microM) has been investigated by three-dimensional quantitative structure-activity relationship (3D-QSAR). CATALYST was used to generate three-dimensional hypotheses to study the common interaction features among a set of thymidylate synthase inhibitor. The verification of the hypothesis was achieved by using the molecules outside the training set.     

Different Activities of 5-Hydroxy-dUMP and 5-Hydroxymethyl-dUMP in Thymidylate Synthase-Catalyzed Reaction in View of Molecular Modeling and Structural Studies

In order to explain different activities shown by 5-hydroxy-dUMP (substrate) and its close analogue 5-hydroxymethyl-dUMP (slow-binding inhibitor) in the reaction catalyzed by thymidylate synthase, studies have been undertaken involving (i) ab initio RHF simulations, (ii) comparative analysis of crystallographic structures available from CSD, and (iii) QSAR analysis of experimental results describing thymidylate synthase interaction with various 5-substituted dUMP analogues. Assuming substrate activity of 5-hydroxy-dUMP to be associated with proton release from the C(5) hydroxyl in the enzyme-catalyzed reaction, acidities of 5-hydroxy and 5-hydroxymethyl substituents in dUMP molecule were compared. The results indicate the 5-hydroxyl deprotonation to be easier and supported by resonance electronic effect, pointing to a probable mechanism of different activities of the two dUMP analogues in thymidylate synthase reaction. The possibility is discussed that 5-mercapto-dUMP and 5-hydroseleno-dUMP, previously assumed to be inhibitors, could be also substrates for thymidylate synthase, as the 5-mercaptyl and 5-hydroselenidyl appear to be deprotonated even more easily than the 5-hydroxyl. Copyright 2000 Academic Press.     

Characteristics of thymidylate synthase purified from a human colon adenocarcinoma

Thymidylate synthase has been purified greater than 4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH2-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH2-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu1-7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlu1 and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folypolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 microM) or CH2-H4PteGlu (4.3 microM) were detected when polyglutamates of [6R]CH2-H4PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGlu5. In addition, CH2-H4PteGlu4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH2-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH2-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.     

Sulfamide antifolates inhibiting thymidylate synthase: synthesis,enzyme inhibition and cytotoxicity

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.