High Throughput Screening Assays to Identify Inhibitors of TLR4 Signaling

描述（由申请人提供）：本文提出的工作旨在鉴定可用于验证有关动脉粥样硬化和阿尔茨海默病炎症起源的新假设的小分子。假设TLR 4与TLR 6和CD 36一起响应配体OxLDL和淀粉样蛋白-？（A？）促进炎症的发生，而炎症正是这些疾病的根源。由于所有这三种分子的多效性作用，基因缺失的标准技术不能用于验证这一假设。我们将开发酶片段互补分析使用的片段？-融合至TLR 4和TLR 6的内酰胺酶，以实现高通量筛选测定，从而鉴定具有抑制TLR 4-TLR 6相互作用潜力的小分子库。描述了反筛选测定以鉴定库中哪些化合物是TLR 4和TLR 6之间结合的特异性抑制剂，其也不抑制在除动脉粥样硬化和阿尔茨海默氏症之外的情况下重要的TLR 4和TLR 6的其他配对。这些经过验证的化合物将使进一步的研究能够探索这一假设。如果这一假设得到证实，它将为动脉粥样硬化和阿尔茨海默病的炎症确定一个新的范例，并可能导致新形式的药物治疗。 公共卫生相关性：动脉粥样硬化和阿尔茨海默病中的炎症是这些疾病病理后果的主要原因。这项工作将探索有关炎症起源的新想法。它将确定新的化合物来抑制模型系统中的炎症，并可能导致用于人类治疗的新药。

DESCRIPTION (provided by applicant): The work proposed here seeks to identify small molecules that could be used to validate a novel hypothesis about the origin of inflammation in atherosclerosis and Alzheimer's disease. The hypothesis is that TLR4 together with TLR6 and CD36 respond to the ligands OxLDL and amyloid-? (A?) to promote the inflammation that is at the root of these diseases. Because of the pleiotropic roles for all three of these molecules, standard techniques of gene deletion cannot be used to validate this hypothesis. We will develop enzyme fragment complementation assays using fragments of ?-lactamase fused to TLR4 and TLR6 to enable high throughput screening assays to identify a pool of small molecules with the potential to inhibit TLR4-TLR6 interactions. Counterscreening assays are described to identify which compounds in the pool are specific inhibitors of the binding between TLR4 and TLR6 that do not also inhibit other pairings of TLR4 and TLR6 that are important in contexts other than atherosclerosis and Alzheimer's. These validated compounds will enable further research to explore the hypothesis. If the hypothesis is validated, it will identify a new paradigm for inflammation in atherosclerosis and Alzheimer's disease and could lead to new forms of drug therapy. PUBLIC HEALTH RELEVANCE: Inflammation in atherosclerosis and Alzheimer's disease is a major cause of the pathological consequences of these diseases. This work will explore new ideas about the origin of the inflammation. It will identify new compounds to inhibit the inflammation in model systems and could lead to new drugs for human therapy.