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

描述(由申请人提供)：先天免疫反应对于启动对病原体和无菌损伤的动态平衡炎症反应是重要的。它们还启动适应性免疫系统的激活，以长期保护免受病原体侵袭。然而，炎症本身也可能是病理性的。近年来，一些炎症综合征被追溯到先天免疫系统的NOD样受体(NLR)分支的不适当激活。在一些情况下，这些是病原体启动的激活，在另一些情况下，它是无菌伤害，在另一些情况下，它是由于受体本身的遗传错误。到目前为止，还没有小分子抑制NLR的激活或信号传递。在这项提案中，我们描述了开发检测方法的计划，这些检测方法将允许高通量筛选NLR启动的信号传导抑制物。NLR发起的信令依赖于与下游适配器的相互作用。这些相互作用中的许多都是通过所谓的卡片域发生的，这些卡片域介导了受体-适配器的相互作用。卡片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Procaspase9-Card形成的复合体具有两个蛋白质链的N和C末端，这些末端可以被溶剂接触到，并且相当接近，这表明这些末端可以被修饰，而不会失去相互作用的能力。在以前的工作中，我们开发了一种基于酶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，但当这些片段与另外两个相互作用的蛋白质融合后靠近时，这些片段将重新结合形成活性Bla。在这项工作中，我们建议将Bla的片段融合到不同的相互作用的卡片域，如Apaf1和Procaspase 9卡片域，在同一细胞中表达两种嵌合蛋白，并测试Bla的功能。我们的初步结果表明，Apaf1-Card和Procaspase 9-Card确实会出现这种情况。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商业上可用的，要么是通过NIH分子文库筛选中心网络获得的。这些抑制剂最初的特征是它们抑制卡片-卡片相互作用的能力，然后是它们抑制由各自的NLR启动的信号传递的能力。 与公共卫生相关：炎症是对感染和组织损伤的一种动态平衡反应，但当它发生不当时，可能会造成病理损害。这项建议描述了一种系统的开发，以确定新的炎症抑制剂，这些药物最终可能对药物开发和基础研究有用。

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.