选择性作用于硒半胱氨酸的荧光探针和抗肿瘤药物的设计合成及其生物测试

发布时间：2018-03-18 02:33

  本文选题：硫氧还蛋白还原酶　切入点：荧光探针　出处：《兰州大学》2015年博士论文　论文类型：学位论文

【摘要】：硫氧还蛋白还原酶(TrxR)是硫氧还蛋白(Trx)系统的重要组成部分,它在维持细胞内氧化还原平衡中发挥着十分重要的作用。研究表明,TrxR是一个非常重要的抗肿瘤药物作用靶点,TrxR的活性位点是其C-端的-Gly-Cys-Sec-Gly氨基酸序列。作为构成蛋白质的第21种氨基酸,硒半胱氨酸(Sec)残基对维持TrxR的生物功能起着不可替代的作用。在本论文中,基于Sec的特殊化学性质,我们设计出可以选择性识别Sec的荧光探针分子和选择性作用于Sec残基的小分子TrxR抑制剂,主要内容包括以下几章：第一章：首先对荧光探针相关知识进行了详细的介绍,其中包括荧光探针的概念、组成及作用机理。之后介绍了生物体内硒的主要存在形式(Sec及硒蛋白)的重要性,并对Sec探针的研究进展进行了简单的介绍。最后对生物体内存在的一种重要的硒蛋白-TrxR进行了介绍,着重对TrxR作为潜在的抗肿瘤药物靶点进行了讨论。第二章：通过文献查阅,在基于硫醇探针分子识别原理的基础上,设计合成了一系列潜在的Sec的荧光探针分子,经过荧光相应测试,筛选得到荧光探针分子Sel-green,在体外对探针进一步测试后发现Sel-green可以选择性识别Sec,之后利用Sel-green实现了对重组TrxR中的Sec的含量测定。生物实用性测试表明Sel-green可以很好的检测细胞内源性和外源性的Sec。Sel-green是首个可以在生理条件下选择性识别Sec的探针分子。利用Sel-green,我们发现含硒化合物的细胞毒性与它们在细胞内代谢为硒醇的能力密切相关,能够代谢为硒醇的化合物都表现出较高的细胞毒性。论文通过对探针构效关系的阐述为进一步进行探针的优化和探索硒蛋白的功能铺平了道路。第三章：我们根据第二章构效关系的论述内容,对前面的探针分子进行了改进。红光发射和近红外发射的探针分子相比于短波长的探针分子有一系列优点,如背景干扰小、组织穿透能力强等。本章设计合成了红光发射和近红外发射的荧光探针分子并对其进行了测试,得到了很好的效果。第四章：从第二章的研究内容中发现淬灭基团为2,4-二硝基的荧光探针可以很好的选择性识别Sec,也就是说Sec会与探针进行结合。TrxR的活性位点主要是其C-端的Sec残基。所以我们推测2,4-二硝基类的荧光探针可能会抑制TrxR的活性。本章设计合成了一系列具有类似结构的化合物,通过对TrxR活性的抑制筛选,得到了活性较好的化合物,并提出了化合物在细胞内可能的作用机制。第五章：天然产物及其衍生物是药物发现的重要来源。目前市场上几乎一半的药物是天然产物或其衍生物。黄腐酚(Xn)是啤酒花中存在的具有查尔酮结构的天然产物,具有一定的抗肿瘤活性。本章设计合成了黄腐酚及其一系列的类似物,并对其生物活性进行了测试,通过筛选总结出这一类化合物抗肿瘤活性的基本结构规律,并得到了活性相比于黄腐酚提高近30倍的化合物13n。通过对其作用机制的探索研究,发现13n是通过作用于TrxR导致活性氧的累积并诱导细胞产生氧化应激,最终诱导细胞凋亡来杀死癌细胞。
[Abstract]:Thioredoxin reductase (TrxR) is thioredoxin (Trx) is an important part of the system, it is in the maintenance of intracellular oxidation plays an important role in reducing balance. The results show that TrxR is a very important target of anti-cancer drugs, the active site of TrxR is -Gly-Cys-Sec-Gly amino acid sequence the end of the C-. As a twenty-first amino acids protein, selenium cysteine (Sec) residues in biological function to maintain TrxR plays an irreplaceable role. In this paper, the special chemical properties based on Sec, we design a fluorescent probe molecule and selective action can choose the identification of small molecule Sec TrxR inhibitors for Sec residues, the main contents include the following chapters: the first chapter: firstly, the fluorescence probe related knowledge are introduced in detail, including the concept of fluorescent probe, composition and mechanism. After the introduction of the The main form of selenium in organisms (Sec and selenoproteins) the importance and research progress of the Sec probe were introduced. Finally, a kind of important selenoprotein -TrxR in organism were introduced, focusing on TrxR as potential anticancer drug targets are discussed. The second chapter: through the literature consult, based on thiol probe principle of molecular recognition, fluorescent probe molecules of a series of potential Sec were designed and synthesized through the corresponding fluorescence test, screened fluorescent probe molecule Sel-green in vitro to probe into one step after the test found that Sel-green can selectively recognize Sec, after the realization of the use of Sel-green content of recombinant TrxR the determination of Sec. The biological practical tests show that Sel-green can be a very good Sec.Sel-green detection of cell endogenous and exogenous is first selected under physiological conditions The probe molecule selective recognition of Sec. Using Sel-green, we found that the cell toxicity and their intracellular metabolic capacity for alcohol is closely related to the selenium selenium compounds, alcohol compounds capable of selenium metabolism showed higher cytotoxicity. The structure-activity relationship of the probe this paves the way for further probe the optimization and exploration of selenoprotein function. In the third chapter, we discuss the content: according to the structure-activity relationship of the second chapter, on the front of the probe molecules were improved. Molecular probe probe molecules red emission and NIR emission compared to the short wavelength has a series of advantages, such as low background noise, strong penetration capability. In this chapter, the design and synthesis of fluorescent probe molecules red emission and NIR emission and the test, obtained very good results. The fourth chapter: from the quenching of the second chapter. Destroy the groups as the fluorescent probe 2,4- two nitro selective recognition of Sec good, that is to say Sec binds to the active site of.TrxR and probes are mainly Sec residues in the C- terminal. So we speculated that the fluorescent probe 2,4- two nitro compounds could inhibit the activity of TrxR. A series of compounds with similar structures the synthesis of this chapter design, by inhibiting the activity of TrxR screening compounds were obtained, and put forward the mechanism of compound may in the cells. The fifth chapter: natural products and their derivatives is an important source of drug discovery. The drug on the market, almost half of the natural products or their derivatives of xanthohumol. (Xn) is a chalcone natural product hops exist, has antitumor activity. Analogues of xanthohumol and synthesized a series of the design, and its biological activity Tested by screening, summed up the basic structure of the antitumor activity of these compounds, and has been compared to the activity of xanthohumol increased nearly 30 times the compound 13n. on the research of its mechanism, found that 13N is accumulated by acting on TrxR in reactive oxygen and induce oxidative stress. The final induction of apoptosis to kill cancer cells.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R914;O657.3
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本文编号：1627636