High Throughput Screening for Toll-Like Receptors

Toll 样受体的高通量筛选

• 批准号: 7304744
• 负责人: PETER S TOBIAS
• 金额: $ 2.5万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7304744
• 关键词: Acute; Atherosclerosis; Biological Assay; Cell Surface Proteins; Cells; Chimeric Proteins; Chronic; Co-Immunoprecipitations; Detection; Development; Disease; Etiology; Exhibits; Funding; Inflammation; Kidney Diseases; Kidney Transplantation; Lactamase; Measures; Modeling; Numbers; Pharmaceutical Preparations; Receptor Signaling; Screening procedure; Secure; Signal Transduction; Sterility; Therapeutic; Toll-like receptors; Validation; high throughput screening; human disease; inhibitor/antagonist; reconstitution; small molecule; toll-like receptor 4

DESCRIPTION (provided by applicant): The specific aim of this proposal is to use a high throughput screening assay that we have developed to find inhibitors of TLR4 signaling. There are a number of diseases whose etiology involves chronic or acute, but sterile, inflammation. Examples are atherosclerosis and kidney transplantation. In some of these diseases, notably the two just mentioned, inflammation initiated by Toll-like receptor (TLR) signaling is clearly involved. However, there are no inhibitors of TLR signaling that might serve as models for small molecule inhibitors of the activity of TLRs and which might be useful, or serve as starting points for development of therapeutic compounds. We have developed an assay that exhibits a positive signal when TLR4 and its intracellular signaling adapter MyD88 associate. The signal develops when two fragments of ¿-lactamase, expressed as chimeric proteins with TLR4 and MyD88, reconstitute ¿-lactamase activity when the two chimeric proteins associate. Thus the assay measures ¿-lactamase activity indicating TLR4 - MyD88 association. Inhibition of the ¿-lactamase activity signals detection of an inhibitor of TLR4 - MyD88 association. Following high throughput screening with this assay we will eliminate false positive hits by assaying for ¿-lactamase inhibitors. We will also assay positive hits for their ability to inhibit TLR4-MyD88 association by co-immunoprecipitation assays. Finally we will assess for effects of the positive hit on association of MyD88 with other TLRs. There are a number of human diseases, such as atherosclerosis and kidney disease, which involve inflammation generated by the activity of cell surface proteins called Toll-like receptors (TLRs). There are no good candidates for drugs that would block the activity of TLRs that could be useful in treating these diseases. This project will utilize an assay that we have recently developed to search for such compounds.

描述（由申请人提供）：本提案的具体目的是使用我们开发的高通量筛选试验来发现TLR 4信号传导抑制剂。有许多疾病的病因涉及慢性或急性但无菌的炎症。例如动脉粥样硬化和肾移植。在其中一些疾病中，特别是刚才提到的两种疾病，明显涉及由Toll样受体（TLR）信号传导引发的炎症。然而，没有TLR信号传导的抑制剂可以用作TLR活性的小分子抑制剂的模型，并且其可能是有用的，或者用作开发治疗性化合物的起点。我们已经开发了一种检测方法，当TLR 4和其细胞内信号转导衔接子MyD 88缔合时，该方法显示出阳性信号。当表达为TLR 4和MyD 88的嵌合蛋白的两个μ-内酰胺酶片段在两种嵌合蛋白结合时重建μ-内酰胺酶活性时，信号就会产生。因此，该测定测量指示TLR 4-MyD 88缔合的β-内酰胺酶活性。对β-内酰胺酶活性的抑制表明检测到TLR 4-MyD 88结合的抑制剂。在使用该测定进行高通量筛选后，我们将通过测定β-内酰胺酶抑制剂来消除假阳性命中。我们还将通过免疫共沉淀测定来测定阳性命中物抑制TLR 4-MyD 88缔合的能力。最后，我们将评估阳性命中对MyD 88与其他TLR关联的影响。有许多人类疾病，如动脉粥样硬化和肾脏疾病，其涉及由称为Toll样受体（TLR）的细胞表面蛋白的活性产生的炎症。目前还没有很好的候选药物可以阻断TLR的活性，从而对治疗这些疾病有用。该项目将利用我们最近开发的一种检测方法来寻找此类化合物。