本文選題：細(xì)胞壞死 + 小分子抑制劑��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：細(xì)胞壞死被證明是細(xì)胞凋亡、細(xì)胞自噬之外另一類程序性細(xì)胞死亡的一種。對(duì)細(xì)胞壞死信號(hào)通路中RIPK1、RIPK3和MLKL的鑒定及功能的研究,讓細(xì)胞壞死信號(hào)的具體轉(zhuǎn)導(dǎo)和調(diào)控分子機(jī)制被逐步揭示。雖然MLKL被證明是細(xì)胞壞死的最終執(zhí)行者,但是對(duì)于MLKL如何轉(zhuǎn)移到細(xì)胞膜并造成細(xì)胞膜的通透性改變,還有待進(jìn)一步研究。程序性細(xì)胞壞死在很多疾病中都有非常重要的作用,因此抗壞死藥物開發(fā)也是目前的一個(gè)重要研究方向。我們?cè)噲D通過本次抗細(xì)胞壞死小分子抑制劑的篩選,一方面結(jié)合化學(xué)生物學(xué)的研究方法對(duì)小分子抑制劑的靶標(biāo)進(jìn)行鑒定,從而對(duì)細(xì)胞壞死的分子機(jī)理作進(jìn)一步深入探索;另一方面由于目前已知的抗壞死小分子在作為藥物開發(fā)前體還存在多方面的限制因素,新型苗頭化合物的發(fā)現(xiàn)有希望為新的抗壞死藥物設(shè)計(jì)提供思路。以TNF-α、Smacmimetic和Z-VAD.fmk誘導(dǎo)程序性細(xì)胞壞死的篩選方法,我們對(duì)含31萬化合物的小分子庫(kù)進(jìn)行抗細(xì)胞壞死活性篩選,最終得到約180個(gè)活性苗頭化合物。結(jié)合三維結(jié)構(gòu)分類、不同細(xì)胞死亡活性比較及細(xì)胞壞死關(guān)鍵標(biāo)志物檢測(cè)等方法,我們對(duì)苗頭化合物在細(xì)胞壞死信號(hào)通路中的作用節(jié)點(diǎn)及方式進(jìn)行了初步分析和分類,得到類Nec-1、類NSA及新型抗壞死分子等多種類別化合物。在mRIPK3聚合誘導(dǎo)細(xì)胞壞死、Thermal shift assay等其他篩選方法中我們還得到一類通過抑制RIPK3激酶活性的抗壞死活性化合物,根據(jù)篩選目的,最終我們對(duì)C236N12和S37J14兩個(gè)苗頭化合物進(jìn)行了抗壞死分子機(jī)理的深入研究。通過生物化學(xué)和化學(xué)生物學(xué)等方法,最終確定這兩個(gè)小分子都以MLKL作為作用靶標(biāo)蛋白,通過共價(jià)結(jié)合MLKL的Cys86位點(diǎn)對(duì)MLKL進(jìn)行修飾,阻斷其形成多聚體及向膜結(jié)構(gòu)的轉(zhuǎn)移,從而起到抑制細(xì)胞壞死的作用。在研究的過程中,開發(fā)出了通過誘導(dǎo)RIPK3或MLKL形成聚合體,而不依賴上游信號(hào)轉(zhuǎn)導(dǎo)過程,直接激活細(xì)胞壞死的壞死誘導(dǎo)系統(tǒng)。該系統(tǒng)對(duì)于抗壞死小分子藥物的篩選、活性分子作用位點(diǎn)判斷及細(xì)胞壞死信號(hào)激活機(jī)制的研究都有非常重要的作用。通過對(duì)C236N12的構(gòu)效關(guān)系研究和結(jié)構(gòu)優(yōu)化,最終我們將其抗細(xì)胞壞死活性EC50優(yōu)化到約為2 nM,有望開發(fā)為高特異性的抗壞死前體藥物。
[Abstract]:Cell necrosis has been shown to be a form of apoptosis, a programmed cell death in addition to autophagy. The identification and function of RIPK1, RIPK3 and MLKL in the cell necrosis signal pathway were studied, so that the specific transduction and regulation molecular mechanism of cell necrosis signal were revealed step by step. Although MLKL has been proved to be the ultimate executor of cell necrosis, it remains to be further studied how MLKL can transfer to the cell membrane and change the permeability of the cell membrane. Programmed cell necrosis plays an important role in many diseases, so the development of antinecrotic drugs is also an important research direction. On the one hand, we try to identify the target of small molecular inhibitor by the method of chemical biology, so as to further explore the molecular mechanism of cell necrosis. On the other hand, because the known anti-necrotic small molecules are still limited by many factors as drug development precursors, the discovery of new seedling compounds may provide ideas for the design of new anti-necrotic drugs. Using the screening method of TNF- 偽 Smacmimetic and Z-VAD.fmk to induce programmed cell necrosis, we screened the small molecular library containing 310000 compounds for anti-necrosis activity, and finally obtained about 180 active seedling compounds. Based on the three dimensional structural classification, the comparison of cell death activity and the detection of key markers of cell necrosis, we preliminarily analyzed and classified the action nodes and patterns of seedling compounds in cell necrosis signal pathway. Nec-1-like, NSA-like and new anti-necrotic molecules were obtained. In other screening methods, such as mRIPK3 polymerization induced cell necrosis and thermal shift assay, we have also obtained a class of anti-necrotic compounds by inhibiting the activity of RIPK3 kinase. Finally, we studied the molecular mechanism of anti-necrosis of two seedling compounds, C236N12 and S37J14. By means of biochemical and chemical biological methods, the two small molecules were determined to be MLKL as target proteins, and MLKL was modified by covalent binding to the Cys86 site of MLKL to block the formation of polymers and the transfer to membrane structure. Thus play a role in inhibiting cell necrosis. In the course of the study, a necrotizing induction system was developed to directly activate cell necrosis by inducing RIPK3 or MLKL to form aggregates without relying on upstream signal transduction processes. This system plays an important role in the screening of anti-necrotic small molecule drugs, the judgement of active molecular action sites and the study of activation mechanism of cell necrosis signal. By studying the structure-activity relationship of C236N12 and optimizing its structure, the EC50 of C236N12 was optimized to about 2 nm, which is expected to be a highly specific antinecrotic precursor drug.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R91

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