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; 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（由适用提供）：该提案的具体目的是使用我们开发的高吞吐量筛选测定法来查找TLR4信号传导的抑制剂。有许多疾病的病因涉及年代学或急性，但无菌的炎症。例子是动脉粥样硬化和肾脏移植。在其中一些疾病中，尤其是两种疾病，显然涉及由Toll样受体（TLR）信号引发的炎症。但是，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，并且可能是有用的，或者是用于发展热化合物的起点。我们已经开发了一种评估，当TLR4及其细胞内信号适配器MYD88助理时，该评估表现出正信号。当两个用TLR4和MyD88表示为嵌合蛋白的两个片段 - 乳糖酶的两个片段时，信号发展是重新构造 - 乳酰胺酶活性时，当两个嵌合蛋白相关时。该测定法测量� -lactamase活性表明TLR4 -MYD88关联。 TLR4 -MyD88关联抑制剂的抑制 - 乳酰胺酶活性信号检测。使用此测定法进行高吞吐量筛选后，我们将通过测定 - 乳糖酶抑制剂来消除假阳性命中。我们还将通过共免疫沉淀测定法抑制TLR4-MYD88关联的能力而主张积极的命中。最后，我们将评估MYD88与其他TLR的关联的积极命中的影响。有许多人类疾病，例如动脉粥样硬化和肾脏疾病，涉及由称为Toll样受体（TLR）的细胞表面蛋白的活性产生的注射。没有很好的候选药物可以阻止TLR的活性，这些活动可能在治疗这些疾病中有用。该项目将利用我们最近开发的评估来搜索此类化合物。