本文選題：硫氧還蛋白還原酶　切入點：熒光探針　出處：《蘭州大學》2015年博士論文　論文類型：學位論文

【摘要】：硫氧還蛋白還原酶(TrxR)是硫氧還蛋白(Trx)系統(tǒng)的重要組成部分,它在維持細胞內(nèi)氧化還原平衡中發(fā)揮著十分重要的作用。研究表明,TrxR是一個非常重要的抗腫瘤藥物作用靶點,TrxR的活性位點是其C-端的-Gly-Cys-Sec-Gly氨基酸序列。作為構(gòu)成蛋白質(zhì)的第21種氨基酸,硒半胱氨酸(Sec)殘基對維持TrxR的生物功能起著不可替代的作用。在本論文中,基于Sec的特殊化學性質(zhì),我們設計出可以選擇性識別Sec的熒光探針分子和選擇性作用于Sec殘基的小分子TrxR抑制劑,主要內(nèi)容包括以下幾章：第一章：首先對熒光探針相關(guān)知識進行了詳細的介紹,其中包括熒光探針的概念、組成及作用機理。之后介紹了生物體內(nèi)硒的主要存在形式(Sec及硒蛋白)的重要性,并對Sec探針的研究進展進行了簡單的介紹。最后對生物體內(nèi)存在的一種重要的硒蛋白-TrxR進行了介紹,著重對TrxR作為潛在的抗腫瘤藥物靶點進行了討論。第二章：通過文獻查閱,在基于硫醇探針分子識別原理的基礎(chǔ)上,設計合成了一系列潛在的Sec的熒光探針分子,經(jīng)過熒光相應測試,篩選得到熒光探針分子Sel-green,在體外對探針進一步測試后發(fā)現(xiàn)Sel-green可以選擇性識別Sec,之后利用Sel-green實現(xiàn)了對重組TrxR中的Sec的含量測定。生物實用性測試表明Sel-green可以很好的檢測細胞內(nèi)源性和外源性的Sec。Sel-green是首個可以在生理條件下選擇性識別Sec的探針分子。利用Sel-green,我們發(fā)現(xiàn)含硒化合物的細胞毒性與它們在細胞內(nèi)代謝為硒醇的能力密切相關(guān),能夠代謝為硒醇的化合物都表現(xiàn)出較高的細胞毒性。論文通過對探針構(gòu)效關(guān)系的闡述為進一步進行探針的優(yōu)化和探索硒蛋白的功能鋪平了道路。第三章：我們根據(jù)第二章構(gòu)效關(guān)系的論述內(nèi)容,對前面的探針分子進行了改進。紅光發(fā)射和近紅外發(fā)射的探針分子相比于短波長的探針分子有一系列優(yōu)點,如背景干擾小、組織穿透能力強等。本章設計合成了紅光發(fā)射和近紅外發(fā)射的熒光探針分子并對其進行了測試,得到了很好的效果。第四章：從第二章的研究內(nèi)容中發(fā)現(xiàn)淬滅基團為2,4-二硝基的熒光探針可以很好的選擇性識別Sec,也就是說Sec會與探針進行結(jié)合。TrxR的活性位點主要是其C-端的Sec殘基。所以我們推測2,4-二硝基類的熒光探針可能會抑制TrxR的活性。本章設計合成了一系列具有類似結(jié)構(gòu)的化合物,通過對TrxR活性的抑制篩選,得到了活性較好的化合物,并提出了化合物在細胞內(nèi)可能的作用機制。第五章：天然產(chǎn)物及其衍生物是藥物發(fā)現(xiàn)的重要來源。目前市場上幾乎一半的藥物是天然產(chǎn)物或其衍生物。黃腐酚(Xn)是啤酒花中存在的具有查爾酮結(jié)構(gòu)的天然產(chǎn)物,具有一定的抗腫瘤活性。本章設計合成了黃腐酚及其一系列的類似物,并對其生物活性進行了測試,通過篩選總結(jié)出這一類化合物抗腫瘤活性的基本結(jié)構(gòu)規(guī)律,并得到了活性相比于黃腐酚提高近30倍的化合物13n。通過對其作用機制的探索研究,發(fā)現(xiàn)13n是通過作用于TrxR導致活性氧的累積并誘導細胞產(chǎn)生氧化應激,最終誘導細胞凋亡來殺死癌細胞。
[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.

【學位授予單位】：蘭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R914;O657.3
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本文編號：1627636