程序性坏死是一种新型可调控的细胞死亡方式，项目组前期发现TAK-632是一种强效程序性坏死抑制剂，但其在坏死动态发生过程中的靶标及结合位点不完全清楚，阻碍了其作用机制及结构优化的深入研究。针对现有药物靶标鉴定及位点分析方法无法适用于活细胞动态分析的局限性，本项目拟整合亲和标记与非标记靶标鉴定方法的优势及新进展，发展一种基于Click亲和色谱联合限制性酶解肽谱的活细胞内药物作用靶标及结合位点动态分析新方法，应用于TAK-632抗程序性坏死作用靶标的全面动态表征及结合位点的快速灵敏分析。在明确验证其与新靶标相互作用的基础上，阐明TAK-632抗程序性坏死的动态机制，并开展新靶标相关功能的深入研究。本方法的构建与应用对于程序性坏死发生发展机制的深入理解和新型抑制剂的靶向开发都具有重要科学意义，也可推广到其它药物与复杂疾病细胞模型，为活细胞水平药物靶标及结合位点的动态灵敏分析提供新的思路与方法。

Necroptosis is a new type of cell death that can be regulated. We found that TAK-632 is a powerful necroptosis inhibitor, but its target and binding site in various stages of necroptosis were not completely clear, which hindered the in-depth study of its mechanism of action and structural optimization. In view of the limitations of the existing drug target identification and binding site analysis methods applied in living cells, this project intends to integrate label and label-free target identification methods with the marking respective advantages and new technology progress, to develop a new method of dynamic analysis of the target and binding sites of necroptosis inhibitor in living cells based on Click affinity chromatography combined with restriction proteolysiis peptide spectrum. We apply to the comprehensive dynamic characterization the resistant target of TAK-632 in necroptosis and rapid sensitive analysis of binding sites. On the basis of clearly verifying the interaction between TAK-632 with the new target, the dynamic anti-necroptosis mechanism of TAK-632 can be elucidated, and further studies on the related functions of the new target will be carried out. The construction and application of this method have important scientific significance for the in-depth understanding of the development mechanism of necroptosis and the targeted development of new inhibitors, and can also be extended to other drugs and cell models of complex diseases, providing new ideas and methods for the dynamic sensitive analysis of drug targets and binding sites at the living cell level.