HIGH THROUGHPUT SCREENING FOR TOLL-LIKE RECEPTORS INHIBITORS

高通量筛选 Toll 样受体抑制剂

• 批准号: 7568231
• 负责人: PETER S TOBIAS
• 金额: $ 47.38万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7568231
• 关键词: Adverse effects; Agonist; Autoimmunity; Biological Assay; Cardiovascular Diseases; Cells; Characteristics; Chimera organism; Complement; Data; Detection; Enzymes; Event; Evolution; Experimental Autoimmune Encephalomyelitis; Goals; Healed; Hela Cells; Immune response; In Vitro; Infection; Inflammation; Inflammatory Response; Injury; Lactamase; Lead; Libraries; Life; Ligand Binding; Mediating; Mediator of activation protein; Methods; Modeling; Molecular Bank; Mus; Natural Immunity; Pathogen detection; Pharmaceutical Preparations; Property; Protein Fragment; Receptor Activation; Reperfusion Injury; Research; Screening procedure; Septic Shock; Severities; Signal Transduction; Specificity; Sterility; System; TLR2 gene; TLR3 gene; TLR4 gene; Techniques; Testing; Therapeutic; Time; Tissues; Toll-like receptors; Uncertainty; United States National Institutes of Health; Work; base; cell type; detector; drug development; healing; high throughput screening; in vivo; inhibitor/antagonist; macrophage; monocyte; neutrophil; pathogen; public health relevance; receptor; renal ischemia; response; response to injury; stable cell line

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) are principal actors in innate immunity. Their detection of exogenous danger signals from pathogens as well as endogenous injury signals from host tissues initiates homeostatic inflammatory responses involving multiple cell types and the concomitant expression of many soluble mediators. Although these TLR mediated responses have obviously been positively selected by evolution, at times they have undesirable and even lethal side effects. Septic shock has long been recognized as a potentially lethal hyper response to infection. More recently, sterile injury such as renal ischemia reperfusion injury, systemic autoimmunity, experimental autoimmune encephalomyelitis, and cardiovascular disease have all been shown to have significant TLR involvement in that their severity is lessened by deficiency of one or more TLR. Intracellular signaling by most TLRs requires their association with MyD88. We have developed an assay which detects the association of TLRs with MyD88. The assay is based on the ability of ¿-lactamase (Bla) to be split into two inactive fragments. When these two fragments are brought into juxtaposition they will complement with each other to reform the active enzyme. This reassociation is readily detected through the use of a fluorescent Bla substrate. Thus in the assay we have already developed, TLR4 and MyD88 are expressed in HeLa cells as chimeras with fragments of Bla. When the TLR4 and MyD88 interact productively Bla activity is readily observed. A stable cell line expressing these chimeras of TLR4 and MyD88 was useful in high throughput screening of a 16,000 compound library in that it identified 5 compounds that were found to inhibit TLR4 - MyD88 association. Through this proposal we hope to expand our approach to identify inhibitors of signaling initiated by additional TLRs. Ultimately we hope that this work will lead to the development of drugs useful for therapeutic application as well as research. There should be no doubt that such drugs are needed. We propose three specific aims for this work: 1) To develop stable cell lines in which a MyD88 chimera associates with a chimera of TLR2 or TLR9 and leads to active Bla. 2) To demonstrate that the stable cell lines are useful for high throughput screening by screening a 16,000 compound subset of the NIH MLSCN library. 3) To characterize the compounds so identified as to their method of action, to define their efficacy in vitro, and to begin to characterize their efficacy in vivo. PUBLIC HEALTH RELEVANCE Inflammation, whether it is in response to infection or in response to injury, can be deleterious and in some cases even life threatening even though it is an essential part of the healing response. The goal of this application is to develop techniques for finding new inhibitors of inflammation.

描述（由适用提供）：类似收费的接收器（TLR）是先天免疫的主要参与者。他们检测病原体的外源危险信号以及宿主组织的内源性损伤信号启动了涉及多种细胞类型的稳态炎症反应以及许多固体介质的表达。尽管这些TLR介导的反应显然是通过进化肯定选择的，但有时它们具有不良甚至致命的副作用。长期以来，败血性休克被认为是对感染的潜在致命反应。最近，肾脏缺血再灌注损伤，全身性自身免疫性，实验性自身免疫性脑脊髓炎和心血管疾病等无菌损伤都被证明具有明显的TLR参与，因为它们的严重程度会因一个或更多TLR的缺乏而降低。大多数TLR的细胞内信号传导都需要它们与MyD88的关联。我们已经开发了一个集会，该组件检测到TLR与MyD88的关联。该测定基于� -lactamase（bla）分为两个非活动片段的能力。当这两个碎片并列时，他们将相互补充以改革活性酶。通过使用荧光BLA底物很容易检测到这种重新关联。在我们已经开发的测定中，TLR4和MYD88在HeLa细胞中表达为带有BLA片段的嵌合体。当TLR4和MyD88交互式BLA活性很容易观察到时。表达TLR4和MyD88的这些嵌合体的稳定细胞系可用于对16,000个化合物库的高吞吐量筛选，因为它鉴定了5种抑制TLR4 -MYD88关联的化合物。通过该提案，我们希望扩大我们的方法来识别其他TLR发起的信号传导抑制剂。最终，我们希望这项工作将导致对治疗有用的药物的开发。应用和研究。毫无疑问，需要这样的药物。我们提出了这项工作的三个特定目的：1）开发稳定的细胞系，其中myd88嵌合体与TLR2或TLR9的嵌合体相关联并导致活跃的BLA。 2）证明稳定的细胞系通过筛选NIH MLSCN库的16,000个化合物子集有用。 3）要表征如此鉴定出其作用方法的化合物，以在体外定义其效率，并开始表征其在体内的效率。公共卫生相关性炎症，无论是针对感染还是对伤害的响应，都可以删除，在某些情况下，即使这是康复反应的重要组成部分，甚至在某些情况下也要威胁生命。该应用的目的是开发用于寻找新的炎症抑制剂的技术。