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

识别卡-卡相互作用抑制剂的高通量检测

• 批准号: 8143279
• 负责人: PETER S TOBIAS
• 金额: $ 23.5万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8143279
• 关键词: Acids; Address; Affect; Atherosclerosis; Autoimmunity; Basic Science; Biological Assay; Cell physiology; Cells; Chimera organism; Chimeric Proteins; Complex; Development; Disease; Enzymes; Family; Genes; Genetic; Homeostasis; Immune response; Immune system; Immunologic Receptors; Infection; Inflammation; Inflammatory; Inflammatory Response; Injury; Knowledge; Lactamase; Libraries; Ligand Binding Domain; Malignant Neoplasms; Mediating; Mediator of activation protein; Molecular Bank; Natural Immunity; Nucleic Acids; Pathologic; Property; Protein Fragment; Proteins; Reagent; Receptor Activation; Receptor Signaling; Reperfusion Injury; Research; Screening procedure; Signal Transduction; Solvents; Sterility; Structure; Syndrome; System; Testing; Tissues; Toll-like receptors; Tretinoin; United States National Institutes of Health; Work; adaptive immunity; assay development; base; cell type; cytokine; design; drug development; emergency service responder; extracellular; high throughput screening; inhibitor/antagonist; innate immune function; mutant; pathogen; pro-caspase-9; public health relevance; receptor; response; small molecule; small molecule libraries; stable cell line

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.

描述（由申请人提供）：先天免疫反应对于启动针对病原体的稳态炎症反应以及无菌性损伤很重要。它们还启动适应性免疫系统的激活，以长期防御病原体。然而，炎症本身可能是病理性的。近年来，许多炎症综合征已被追溯到先天免疫系统的 Nod 样受体 (NLR) 分支的不当激活。在某些情况下，这些源自病原体启动的激活，在其他情况下，这是无菌损伤，而在另一些情况下，则是由于受体本身的遗传错误造成的。迄今为止，还没有小分子能够抑制 NLR 激活或信号传导。在本提案中，我们描述了开发检测方法的计划，该检测方法将允许高通量筛选 NLR 启动信号传导的抑制剂。 NLR 发起的信号传导取决于与下游适配器的相互作用。许多这些相互作用是通过介导受体-适配器相互作用的所谓 CARD 结构域发生的。 CARD 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。方便地，结构研究表明，Apaf1-CARD 与 Procaspase 9-CARD 复合物的两条蛋白质链的 N 和 C 末端均可接触到溶剂，并且距离相当近，这表明这些末端可以在不损失相互作用能力的情况下进行修饰。在之前的工作中，我们开发了基于 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，这两个片段不会自发地重组形成活性Bla，但是当这些片段在与另外两个相互作用的蛋白质融合后靠近时，片段将重组形成活性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的 CARD 结构域，例如 Apaf1 和 Procaspase 9 CARD 结构域，在同一细胞中表达两种嵌合蛋白，并测试 Bla 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，并使用它们来识别小分子文库中相互作用的抑制剂，这些小分子文库可以是商业上可获得的，也可以通过 NIH 分子文库筛选中心网络获得。抑制剂首先将表征其抑制CARD-CARD相互作用的能力，然后表征其抑制由相应NLR引发的信号传导的能力。 公共卫生相关性：炎症是对感染和组织损伤的稳态反应，但如果发生不当，可能会导致病理损伤。该提案描述了开发一种系统来识别新的炎症抑制剂，该系统最终可能对药物开发和基础研究有用。