化合物8a靶向TBC1D15-GBP5信号轴抑制NLRP3炎症小体活化的机制及其对炎症性疾病的作用

Numerous studies have shown that NLRP3 inflammasome plays an important role in host response to pathogen invasion and danger signals and is involved in the development of several major human diseases, including type 2 diabetes, neurodegenerative diseases and tumor. Previous studies found that compound (+)-1, a polyketide from Daldinia eschscholzii, is biosynthetically proposed as a potent NLRP3 inflammasome activation inhibitor. We carried out structural optimization and transformation based on (+)-1 and discovered a more potent NLRP3 inflammasome inhibitor-compound 8a. We preliminarily proved TBC1D15, a GTPase activator and its role in NLRP3 inflammasome has yet unknown, to be the target of compound 8a. The project here aims to clarify the character of the 8a-TBC1D15 interaction and detailed mechanism for NLRP3 inhibition effect from the molecular, cellular and animal levels, demonstrating the role of this mechanism involved in 8a mediated anti-inflammation activity：IP, CETSA, MST, SPR, ITC et al to confirm the interaction between 8a and TBC1D15; then we’ll further clarify underlying mechanism of TBC1D15-GBP5 axis on NLRP3 inflammasome. Finally, the therapeutic effect of 8a on sepsis, colitis mice models will be explored. Results of this project will be helpful for identification new drug-target and candidate anti-inflammatory compound for NLRP3 inflammasome associated diseases.

众多研究表明，NLRP3炎症小体在机体应对病原体入侵及机体损伤信号刺激时扮演着重要的角色且与2型糖尿病、神经退行性疾病、肿瘤等人类重大疾病密切相关。前期从螳螂相关菌次级代谢产物中发现聚酮类化合物(+)-1可抑制NLRP3炎症小体活化，基于(+)-1的结构进行优化改造，得到更为高效的NLRP3炎症小体抑制剂8a。初步证实8a的靶蛋白为GTP酶激活蛋白TBC1D15，其对NLRP3炎症小体调控尚不清楚。本项目拟从分子、细胞及动物水平研究8a通过作用于TBC1D15抑制NLRP3活化的机制及对炎症的改善：采用免疫共沉淀、胞内热迁移、微量热涌动、SPR、ITC等手段验证8a与TBC1D15结合；阐明TBC1D15-GBP5信号轴对NLRP3炎症小体活化调控的机理；考察8a对败血症、溃疡性结肠炎等小鼠疾病模型的治疗效果。本项目预期为基于NLRP3炎症小体的疾病干预提供候选化合物及新的药物靶标。

NLRP3炎症小体参与了多种炎症相关疾病的发生发展，使其成为极具吸引力的药物作用靶标。前期从螳螂相关菌次级代谢产物中发现聚酮类化合物(+)-1可抑制NLRP3炎症小体活化，基于(+)-1的结构进行优化改造，得到更为高效的NLRP3炎症小体抑制剂8A。8A可选择性抑制NLRP3炎性小体激活的第二信号但不抑制第一信号。化合物8A对线粒体的稳态无影响，同时8A并不影响NLRP3的寡聚，但可以抑制ASC从细胞核向胞质的转位及其寡聚化，进而阻断NLRP3炎性小体复合物的组装，抑制caspase-1的激活。随后，我们采用细胞热迁移、微量热泳动等试验，证明了8A可与ASC结合，证实ASC是8A抑制NLRP3炎症小体活化的作用靶点。进一步，通过建立LPS诱导的小鼠败血症模型和MSU诱导的腹膜炎和痛风模型，我们考察了化合物8A的药效。化合物8A能够显著提高败血症小鼠的存活率，同时也抑制了结肠组织中的CASP1的活化。在MSU诱导的腹膜炎和痛风模型中，化合物8A给药降低了中心粒单核/巨噬细胞的浸润，并抑制了巨噬细胞中的CASP1的活化。综上所述，本项目详细探究了化合物8A的药效及作用靶点，阐明了8A靶向ASC抑制NLRP3炎症小体改善相关疾病的机制。8A作为第一个ASC的小分子抑制剂，不仅有望被用于研究ASC蛋白的结构和功能，同时其也可作为先导化合物进行药物开发。