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: 7976276
• 负责人: PETER S TOBIAS
• 金额: $ 28.49万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976276
• 关键词: 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（由申请方提供）：先天性免疫应答对于启动对病原体的稳态炎症应答以及无菌性损伤非常重要。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的侵害。然而，炎症本身可以是病理性的。近年来，许多炎性综合征已被追溯到先天免疫系统的Nod样受体（NLR）分支的不适当激活。在某些情况下，这些来自病原体启动的激活，在其他情况下，它是不育性损伤，而在其他情况下，它是由于受体本身的遗传错误。到目前为止，还没有抑制NLR激活或信号传导的小分子。在这个提案中，我们描述了计划开发的检测，将允许高通量筛选抑制剂的NLR启动信号。NLR启动的信号传导依赖于与下游衔接子的相互作用。这些相互作用中的许多通过介导受体-衔接子相互作用的所谓CARD结构域发生。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，它们是稳定的，紧凑的，保守的结构。结构研究表明，Apaf 1-CARD与Procaspase 9-CARD的复合物具有两条蛋白质链的N和C末端，这些末端可以接近溶剂并且相当接近，这表明这些末端可以被修饰而不丧失相互作用的能力。在以前的工作中，我们已经利用了基于酶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，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两个其他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，如Apaf 1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，这确实发生在Apaf 1-CARD和Procaspase 9-CARD中。然后，我们将开发表达嵌合体的稳定细胞系，并使用它们来鉴定小分子文库中相互作用的抑制剂，这些小分子文库可以是市售的，也可以通过NIH分子文库筛选中心网络获得。抑制剂将首先表征其抑制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，但当它不适当地发生时，它可能导致病理损伤。该提案描述了一个系统的开发，以确定新的炎症抑制剂，最终可能是有用的药物开发以及基础研究。