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

基本信息

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
项目类别：
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

项目摘要

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对病原体和无菌损伤的稳态炎症反应很重要。他们还启动了自适应免疫系统的激活，以长期保护病原体。但是，炎症本身可以是病理性的。近年来，许多炎症综合症已被追溯到先天免疫系统的点状受体（NLR）分支的不适当激活。在某些情况下，这些是从病原体引发的激活的，在其他情况下是无菌损伤，而在其他情况下，这是由于受体本身的遗传误差所致。到目前为止，还没有抑制NLR激活或信号传导的小分子。在此提案中，我们描述了计划制定测定的计划，以允许对NLR启动信号的抑制剂进行高吞吐量筛选。 NLR启动信号传导取决于与下游适配器的相互作用。这些相互作用中的许多通过介导受体 - 适配器相互作用的所谓卡域进行。卡域已在结构和功能上广泛研究。这些研究表明它们是稳定，紧凑，保守的结构。方便的是，与procaspase 9卡中的结构策略库thatthatthatthatthatthatthatthatthapaf1卡具有n和c末端的n和c末端，这两个蛋白质的链条可用于溶剂，并且在相当接近的接近度中，这表明可以修改这些末端而不会失去能力相互作用。在先前的工作中，我们利用了基于酶B-内酰胺酶（BLA）的蛋白质片段互补系统。 BLA可以分为两个片段，不会自发地重组以形成活跃的BLA，但是当将这些片段融合到另外两个相互作用的蛋白质后将这些片段近接近时，这些片段将重新组合以形成活性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卡域（例如APAF1和Procaspase 9卡域），表达同一单元格中的两个嵌合蛋白，并测试BLA功能。我们的初步结果表明，APAF1卡和Procaspase 9卡确实发生了这种情况。然后，我们将开发出表达嵌合体的稳定细胞系，并使用它们来识别小分子库中相互作用的抑制剂，这些库是可商购的，或者通过NIH分子库筛选中心网络可用的。最初，抑制剂将以抑制卡卡相互作用，然后抑制各自NLR启动的信号传导的能力来表征。公共卫生相关性：炎症是对感染和组织损伤的稳态反应，但是当它发生不当时，会导致病理损害。该提案描述了一个系统的开发，该系统可以确定新的炎症抑制剂，最终可能对药物开发和基础研究有用。