Neuroinflammation and the NLRP3 inflammasome, the regulator of the maturation and production of IL-1ß, have been indicated a critical role in the pathogenesis of Alzheimer's disease (AD). Therefore, it would be of significant importance to develop novel NLRP3 inflammasome inhibitors with well defined mode of action (MOA), which will complement ongoing molecular and genetic studies, help further unravel the roles of NLRP3 inflammasome in the pathogenesis of AD, and ultimately facilitate the development of novel NLRP3 inflammasome inhibitors as effective AD treatments. Recently, we have developed a small molecule and selective NLRP3 inflammasome inhibitor. We have demonstrated that this inhibitor can cross the BBB and exhibit in vivo activities in AD mouse model. Herein, we plan to identify the protein target of this lead compound within the NLRP3 inflammasome complex and define the MOA for this novel chemotype. Two specific aims are proposed to accomplish our goals. In aim 1, chemical probes that can label interacting proteins are designed based on the chemical structure of lead compound. In aim 2, rationally designed chemical probes will be employed to define the MOA of this lead as NLRP3 inflammasome inhibitor. The goal of this aim is to identify the protein components within the NLRP3 inflammasome platform that interact with this lead compound. We have developed a progressive and tiered plan to accomplish our goals: 1) Four chemical probes will be designed and synthesized; 2) inhibitory potency and selectivity on the production of IL-1ß will be assessed in macrophage cells for the designed chemical probes; 3) two complementary assay systems will be employed to identify the binding partners; 4) proteomic analysis will be conducted to elucidate the protein identity.