HIGH-THROUGHPUT ASSAYS TO IDENTIFY INHIBITORS OF CARD-CARD INTERACTIONS

描述（由申请人提供）：先天免疫反应对于病原体和无菌损伤的稳态炎症反应的启动是重要的。它们还启动适应性免疫系统的激活，以长期保护人体免受病原体的侵害。然而，炎症本身可能是病理性的。近年来，许多炎症综合征被追溯到先天免疫系统中淋巴结样受体（NLR）分支的不适当激活。在某些情况下，这些源于病原体启动激活，在其他情况下是无菌损伤，还有一些是由于受体本身的遗传错误。到目前为止，还没有小分子能够抑制NLR的激活或信号传导。在本提案中，我们描述了开发检测的计划，该检测将允许高通量筛选NLR启动信号的抑制剂。NLR发起的信号依赖于与下游适配器的交互。许多这些相互作用是通过所谓的CARD结构域发生的，它介导受体-受体相互作用。CARD结构域在结构和功能上都得到了广泛的研究。这些研究表明它们是稳定、紧凑、保守的结构。方便的是，结构研究表明，apaf1 - card与Procaspase 9-CARD复合物的蛋白链的N和C末端都是溶剂可接近的，并且相当接近，这表明这些末端可以被修饰而不损失相互作用的能力。在以前的工作中，我们已经开发了一个基于b-内酰胺酶（Bla）的蛋白质片段互补系统。Bla可以分裂成两个片段，它们不会自发重组形成活性Bla，但当这些片段与其他两个相互作用的蛋白质融合后靠近时，这些片段会重组形成活性Bla。在这项工作中，我们建议将Bla片段融合到不同的相互作用CARD结构域，如Apaf1和Procaspase 9 CARD结构域，在同一细胞中表达这两种嵌合蛋白，并测试Bla的功能。我们的初步结果表明，Apaf1-CARD和Procaspase 9-CARD确实会发生这种情况。然后，我们将开发表达嵌合体的稳定细胞系，并使用它们在小分子文库中识别相互作用的抑制剂，这些小分子文库要么是市售的，要么是通过NIH分子文库筛选中心网络获得的。抑制剂最初的特点是它们抑制CARD- CARD相互作用的能力，然后它们抑制各自NLR启动的信号传导的能力。

DESCRIPTION (provided by applicant): Innate immune responses are important for initiation of homeostatic inflammatory responses to pathogens as well as sterile injury. They also initiate activation of the adaptive immune system for long term protection against pathogens. However, inflammation can itself be pathologic. In recent years a number of inflammatory syndromes have been traced to inappropriate activation of the Nod-like Receptor (NLR) branch of the innate immune system. In some instances these derive from pathogen initiated activation, in others it is sterile injury, and in yet others it is due to genetic errors in the receptors themselves. As yet there are no small molecules that inhibit NLR activation or signaling. In this proposal we describe plans to develop assays that will permit high throughput screening for inhibitors of NLR initiated signaling. NLR initiated signaling depends on interaction with downstream adaptors. Many of these interactions occur through so-called CARD domains which mediate receptor - adaptor interaction. CARD domains have been widely studied both structurally and functionally. These studies suggest that they are stable, compact, conserved structures. Conveniently, structuralstudiesshowthattheApaf1-CARD in complex with the Procaspase 9-CARD has N and C termini of both protein's chains that are accessible to solvent and in fairly close proximity, suggesting that these termini could be modified without loss of capacity to interact. In previous work we have exploited a protein fragment complementation system based on the enzyme b-lactamase (Bla). Bla can be split into two fragments which will not spontaneously recombine to form active Bla, but when these fragments are brought into proximity after being fused to two other interacting proteins, the fragments will recombine to form active Bla. In this work we propose to fuse the fragments of Bla to different, interacting CARD domains, such as the Apaf1 and Procaspase 9 CARD domains, express the two chimeric proteins in the same cell, and test for Bla function. Our preliminary results show that this does occur with Apaf1-CARD and Procaspase 9-CARD. We will then develop stable cell lines expressing the chimeras and use them to identify inhibitors of the interaction in small molecule libraries that are either commercially available or available through the NIH Molecular Library Screening Center Network. The inhibitors will be initially characterized for their ability to inhibit the CARD- CARD interactions and then for their ability to inhibit signaling initiated by the respective NLR. PUBLIC HEALTH RELEVANCE: Inflammation is a homeostatic response to infection as well as tissue injury but when it occurs inappropriately it can cause pathologic damage. This proposal describes development of a system to identify new inhibitors of inflammation that may ultimately be useful for drug development as well as basic research.