抗细胞坏死小分子化合物的筛选及抗坏死活性的分子机理研究

发布时间：2018-07-02 21:11

本文选题：细胞坏死 + 小分子抑制剂　；参考：《北京协和医学院》2017年博士论文

【摘要】：细胞坏死被证明是细胞凋亡、细胞自噬之外另一类程序性细胞死亡的一种。对细胞坏死信号通路中RIPK1、RIPK3和MLKL的鉴定及功能的研究,让细胞坏死信号的具体转导和调控分子机制被逐步揭示。虽然MLKL被证明是细胞坏死的最终执行者,但是对于MLKL如何转移到细胞膜并造成细胞膜的通透性改变,还有待进一步研究。程序性细胞坏死在很多疾病中都有非常重要的作用,因此抗坏死药物开发也是目前的一个重要研究方向。我们试图通过本次抗细胞坏死小分子抑制剂的筛选,一方面结合化学生物学的研究方法对小分子抑制剂的靶标进行鉴定,从而对细胞坏死的分子机理作进一步深入探索;另一方面由于目前已知的抗坏死小分子在作为药物开发前体还存在多方面的限制因素,新型苗头化合物的发现有希望为新的抗坏死药物设计提供思路。以TNF-α、Smacmimetic和Z-VAD.fmk诱导程序性细胞坏死的筛选方法,我们对含31万化合物的小分子库进行抗细胞坏死活性筛选,最终得到约180个活性苗头化合物。结合三维结构分类、不同细胞死亡活性比较及细胞坏死关键标志物检测等方法,我们对苗头化合物在细胞坏死信号通路中的作用节点及方式进行了初步分析和分类,得到类Nec-1、类NSA及新型抗坏死分子等多种类别化合物。在mRIPK3聚合诱导细胞坏死、Thermal shift assay等其他筛选方法中我们还得到一类通过抑制RIPK3激酶活性的抗坏死活性化合物,根据筛选目的,最终我们对C236N12和S37J14两个苗头化合物进行了抗坏死分子机理的深入研究。通过生物化学和化学生物学等方法,最终确定这两个小分子都以MLKL作为作用靶标蛋白,通过共价结合MLKL的Cys86位点对MLKL进行修饰,阻断其形成多聚体及向膜结构的转移,从而起到抑制细胞坏死的作用。在研究的过程中,开发出了通过诱导RIPK3或MLKL形成聚合体,而不依赖上游信号转导过程,直接激活细胞坏死的坏死诱导系统。该系统对于抗坏死小分子药物的筛选、活性分子作用位点判断及细胞坏死信号激活机制的研究都有非常重要的作用。通过对C236N12的构效关系研究和结构优化,最终我们将其抗细胞坏死活性EC50优化到约为2 nM,有望开发为高特异性的抗坏死前体药物。
[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- 伪 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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R91

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