新型色酮小分子化合物靶向端粒全酶核心成分Dyskerin诱导胃癌细胞凋亡：设计合成及活性评价

发布时间：2018-03-16 07:36

本文选题：角化不良蛋白　切入点：诱导凋亡　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：端粒酶的上调和激活在大多数肿瘤类别中被发现并且可以诱导正常细胞的恶性转化,其在肿瘤进展中的重要作用受到广泛关注。端粒酶由端粒酶逆转录酶TERT、端粒酶RNA组分TERC和其他结合蛋白组成,他们共同参与端粒酶复合体的活化,在这些结合蛋白中的Dyskerin扮演了一个关键角色。Dyskerin的过表达已被证实在多种肿瘤进程中起到重要作用并与不良预后有关。目前的研究表明Dyskerin同时影响核糖体生物合成和端粒酶复合体的稳定。一方面Dyskerin活性的减低降低TERC水平并诱导端粒缩短;另一方面,Dyskerin功能的缺失会改变抗凋亡因子比如bcl-2和bcl-xl的信使RNA的转录和修饰。这些生物效应最终导致细胞周期阻滞和细胞凋亡。另外,还有其他相关研究指出该基因在核质穿梭、DNA损伤修复和细胞粘附中扮演重要角色。基于Dyskerin同时在细胞和机体的生理进程中起到的重要作用,靶向这些生理进程已经被探索作为新药开发的作用靶点,以期望提高选择性并优先杀死肿瘤细胞。目的:基于国内外研究和实验室经验积累,借助计算机辅助药物设计技术,设计合成靶向Dyskerin的色酮小分子化合物并进行活性评价和初步的作用机制研究。内容和方法:1、基于Dyskerin的蛋白结构分析其可能的活性位点,通过分子对接和虚拟筛选,遴选出高活性的目的蛋白小分子抑制剂的骨架结构,并通过化学合成的手段合成出一系列的目标化合物。2、对合成出的一系列化合物进行抗肿瘤活性评价,以筛选出高活性低毒性的小分子化合物。主要方法有细胞增殖实验(MTT法)、流式细胞术检测细胞凋亡(Annexin V-FITC/PI双染)、检测线粒体膜电位MPP(JC-1法)、检测细胞周期(PI单染)。3、挑选高活性的小分子化合物进行初步的作用机制研究。基于活性评价的相关结果和目标靶点的功能,通过蛋白免疫印迹实验检测其凋亡和周期阻滞相关蛋白,探寻其诱导肿瘤细胞凋亡的通路机制。4、新型小分子化合物的靶点研究。研究小分子化合物是否通过抑制设想的靶蛋白发挥作用。主要方法是检测目标蛋白及其结合蛋白的表达和调控情况并通过免疫荧光技术进行目的蛋白的定位5、TRAP-PCR-ELISA法检测小分子化合物对肿瘤细胞端粒酶活性的抑制作用。结果:借助计算机辅助药物设计技术进行结构设计,用化学合成的手段合成了一系列的小分子化合物,通过活性评价得到数个高抗肿瘤活性低毒性的色酮小分子化合物。其中化合物8的效果较好,我们挑选其进行了靶点和作用机制研究。实验结果表明,化合物8能显著降低细胞活力,并诱导胃癌细胞凋亡。其诱导胃癌细胞凋亡的机制是通过阻滞细胞周期于G2/M期、破坏线粒体功能、引起严重的DNA损伤等途径实现的。靶点研究表明,化合物8作用于胃癌细胞可以显著抑制目的蛋白Dyskerin的表达并同时抑制h TERT蛋白,导致端粒酶活性的显著下降。以上结果表明化合物8是一个高效的Dyskerin抑制剂并具有优越的体外抗肿瘤活性。结论:Dyskerin是一个很有潜力的药物作用靶点,靶向Dyskerin是抗肿瘤新药开发中的一个成功的新尝试。化合物8作为Dyskerin抑制剂是一个很有潜力的抗肿瘤新药。
[Abstract]:Telomerase upregulation and activation in most tumor types were found and can induce malignant transformation of normal cells, it plays an important role in tumor progression concern. Telomerase by telomerase reverse transcriptase TERT, telomerase RNA component TERC and other binding proteins, they participate in the activation of telomerase, in these binding proteins Dyskerin played over expression has been shown to play an important role in a variety of tumors and is associated with poor prognosis in the process of a key role in.Dyskerin. The present study shows that Dyskerin also affects the stability of ribosomal biosynthesis and telomerase complex. On the one hand to reduce the activity of Dyskerin and decrease the level of TERC induced by telomere shortening; on the other hand, the lack of the Dyskerin function will change the transcription and modification of anti apoptotic factors such as Bcl-2 and Bcl-xL messenger RNA. These biological effects. Should ultimately lead to cell cycle arrest and apoptosis. In addition, there are other related research points out that the shuttle gene in the nucleoplasm, DNA damage repair and cell adhesion plays an important role. At the same time, the important role of Dyskerin in the physiological process of cells and organisms plays based on targeting these physiological process has been explored as a target for new drugs the development, in order to improve the selectivity and preferentially kill tumor cells. Objective: Based on domestic and foreign research and laboratory experience, with the help of computer aided drug design technology, design and synthesis of small molecules targeting chromone compounds Dyskerin and study the activity evaluation and preliminary mechanism. Methods and contents: 1, protein structure analysis of Dyskerin the possible active sites based on through molecular docking and virtual screening, selection of skeleton protein inhibitors to high activity of the structure, and Chemical synthesis method of synthesis of a series of target compounds.2, anti-tumor activity evaluation of a series of compounds were synthesized and screened with small molecular compounds with high activity low toxicity. The main methods of cell proliferation assay (MTT method), cell apoptosis by flow cytometry (Annexin V-FITC/PI staining). Detection of mitochondrial membrane potential MPP (JC-1), to detect the cell cycle (PI staining).3, preliminary study on Mechanism of small molecule compounds were selected with high activity. The activity evaluation and target function based on the apoptosis and cycle arrest related protein was detected by Western blotting experiments to explore the pathway the mechanism of tumor cell apoptosis induced by.4, the research target of novel small molecule compounds. Play the role of small molecule compounds by target protein. The main method is to inhibit the idea of target protein and its detection With the expression and regulation of protein and protein localization by immunofluorescence technique 5, inhibition of TRAP-PCR-ELISA for the detection of small molecule compounds on the activity of telomerase in tumor cells. Results: with the help of computer aided drug design techniques for structural design, a series of small molecular compounds were synthesized by means of chemical synthesis, get a high number of antitumor activity and low toxicity of chromone small molecule compounds through the activity evaluation. The effect of compound 8 is better, we choose the studied targets and mechanisms. The experimental results show that the 8 compounds can significantly reduce cell viability and induce apoptosis of gastric cancer cells. The apoptosis of gastric cancer cells induced by the mechanism is through cell cycle arrest in G2/M, damage to mitochondrial function, caused by DNA damage pathway. Serious target research showed that 8 compounds on the gastric cancer The expression of cell can significantly inhibit Dyskerin protein and inhibit the H TERT protein, resulting in a significant decrease in telomerase activity. These results show that compound 8 is an efficient Dyskerin inhibitor and has superior antitumor activity in vitro. Conclusion: Dyskerin is a potential drug target, target is Dyskerin the success of a new attempt in the development of new anticancer drugs. 8 compounds as Dyskerin inhibitors is a potential anti-tumor drug.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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