Developing chemical probes that target specific inflammasomes

 描述（由申请人提供）：炎性小体是涉及针对感染和自身免疫性/自身炎性疾病的免疫防御的关键先天免疫信号传导平台。尽管它们具有重要的生理和病理功能，但对炎性小体特异性化学探针的迫切需求尚未得到满足，并且没有报道的炎性小体抑制剂直接与炎性小体受体/传感器蛋白结合。这不仅阻碍了炎性小体领域中的机制研究的进展，而且阻碍了针对炎性病症（例如系统性红斑狼疮（SLE）、银肩病、cryopyrin相关周期性综合征（CAPS）、痛风、2型糖尿病、阿尔茨海默病和动脉粥样硬化）的特异性和有效治疗剂的开发。本实验室为我们理解AIM 2和IFI 16炎性小体的结构和功能做出了重要贡献：我们建立了AIM 2和IFI 16识别序列非依赖性dsDNA的结构基础，表征了调节AIM 2受体的自抑制机制，阐明了炎性小体受体和信号适配器的pyrin，CARD和TIR结构域介导的信号传导机制。我们将利用我们的专业知识和经验，并测试的假设，结合炎性受体/传感器或适配器在关键域接口的化学化合物将稳定其结构的不同构象状态，从而调节其功能。为此，我们将探索一种双管齐下的方法来鉴定AIM 2和NLRP 3炎性小体特异性的化学探针。在目标1中，我们将对特异性结合AIM 2、NLRP 3或ASC的化学探针进行基于结构的虚拟筛选。我们预计，结合和稳定的分子内结构域-结构域界面和AIM 2和NLRP 3的自抑制结构的小分子可能有助于抑制它们的激活。另一方面，使用竞争性抑制ASC丝形成的化合物破坏ASC PYD寡聚化界面也可抑制炎性小体活化。在目标2中，我们将进行三个独立的体外试验，使我们能够筛选和验证结合和调节ASC聚合和斑点形成的炎性小体特异性化合物。这些测定包括使用纯化的AIM 2、NLRP 3和ASC蛋白的热荧光测定和ASC聚合测定，以及使用稳定表达荧光天蓝色标记的ASC蛋白的骨髓来源的巨噬细胞系的ASC斑点形成测定。我们预计，将鉴定和验证通过ASC特异性调节AIM 2炎性体、NLRP 3炎性体或两者活性的化学探针。重要的是，该项目的成功将满足对高质量和新型化学工具的未满足需求，以探测特定炎性小体的功能，并刺激针对炎症性疾病的特异性和有效治疗方法的开发。

 DESCRIPTION (provided by applicant): The inflammasomes are crucial innate immune signaling platforms implicated in immune defense against infections and autoimmune/autoinflammatory disorders. Despite their important physiological and pathological functions, the critical need for inflammasome-specific chemical probes has not been met and none of the reported inflammasome inhibitors directly engage the inflammasome receptor/sensor proteins. This not only impedes the progress of mechanistic studies in the inflammasome field, but also hampers the development of specific and potent therapeutics for inflammatory disorders, such as systemic lupus erythematosus (SLE), psoriasis, cryopyrin-associated periodic syndromes (CAPS), gout, type 2 diabetes, Alzheimer's disease, and atherosclerosis. My lab has made major contributions to our understanding of the structure and function of the AIM2 and IFI16 inflammasomes: we established the structural basis of sequence-independent dsDNA recognition by AIM2 and IFI16, characterized the autoinhibition mechanism that regulates the AIM2 receptor, elucidated the signaling mechanisms mediated by the pyrin, CARD and TIR domains from the inflammasome receptors and signaling adapters. We will leverage our expertise and experience and test the hypothesis that chemical compounds that bind the inflammasome receptors/sensors or adapter at critical domain interfaces will stabilize different conformational states of their structures and thus modulate ther functions. To this end, we will explore a two-pronged approach to identify chemical probes specific for the AIM2 and NLRP3 inflammasomes. In aim 1, we will perform structure-based virtual screening for chemical probes that specifically bind AIM2, NLRP3 or ASC. We anticipate that small molecules that bind and stabilize the intramolecular domain-domain interfaces and the autoinhibited structures of AIM2 and NLRP3 may serve to suppress their activation. On the other hand, disruption of the ASC PYD oligomerization interface using chemical compounds that competitively inhibit the ASC filament formation may also inhibit inflammasome activation. In aim 2, we will carry out three independent in vitro assays that allow us to screen and validate inflammasome-specific chemical compounds that bind and regulate the ASC polymerization and speck formation. These assays include the thermofluor assay and the ASC polymerization assay using purified AIM2, NLRP3 and ASC proteins, and the ASC speck formation assay using a bone marrow-derived macrophage cell line stably expressing fluorescent cerulean-tagged ASC protein. We anticipate that chemical probes that specifically modulate the activities of either the AIM2 inflammasome, the NLRP3 inflammasome, or both through ASC, will be identified and validated. Importantly, the success of this project will fulfill unmet needs for hig quality and novel chemical tools to probe the function of specific inflammasomes, as well as to stimulate the development of specific and potent therapeutics against inflammatory disorders.