High Throughput Screening for Toll-Like Receptors

描述(由申请人提供)：这项建议的具体目标是使用我们开发的高通量筛选试验来寻找TLR4信号的抑制剂。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，显然与Toll样受体(TLR)信号引发的炎症有关。然而，没有TLR信号的抑制剂可以作为TLRs活性的小分子抑制剂的模型，并且可能是有用的，或者作为治疗化合物开发的起点。我们已经开发了一种检测方法，当TLR4和它的细胞内信号转接器MyD88关联时，显示出阳性信号。当与TLR4和MyD88表达为嵌合蛋白的两个内酰胺酶片段在两个嵌合蛋白结合时重建内酰胺酶活性时，信号就会产生。因此，测定表明TLR4-MyD88关联的β-内酰胺酶活性。抑制β-内酰胺酶活性标志着检测到TLR4-MyD88结合的抑制物。在用这种方法进行高通量筛选后，我们将通过检测内酰胺酶抑制剂来消除假阳性。我们还将通过免疫共沉淀试验检测它们抑制TLR4-MyD88结合的能力的阳性HITS。最后，我们将评估积极命中对MyD88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，涉及到由称为Toll样受体(TLRs)的细胞表面蛋白的活动产生的炎症。目前还没有合适的候选药物来阻断TLRs的活性，这些药物可能对治疗这些疾病有用。这个项目将利用我们最近开发的一种分析方法来寻找这种化合物。

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.