HIGH THROUGHPUT SCREENING FOR TOLL-LIKE RECEPTORS INHIBITORS

描述（由申请人提供）：Toll样受体（TLR）是先天性免疫的主要参与者。它们对来自病原体的外源性危险信号以及来自宿主组织的内源性损伤信号的检测启动涉及多种细胞类型和许多可溶性介质的伴随表达的稳态炎症反应。虽然这些TLR介导的反应显然是通过进化积极选择的，但有时它们具有不期望的甚至致命的副作用。脓毒性休克长期以来被认为是一种潜在的致命的超反应感染。最近，无菌性损伤如肾缺血再灌注损伤、全身性自身免疫、实验性自身免疫性脑脊髓炎和心血管疾病都已显示出具有显著的TLR参与，因为它们的严重性通过一种或多种TLR的缺乏而减轻。大多数TLR的细胞内信号传导需要它们与MyD 88结合。我们已经开发了一种检测TLR与MyD 88的关联的测定法。该测定法基于戊-内酰胺酶（Bla）分裂成两个无活性片段的能力。当这两个片段并置时，它们将相互补充以改革活性酶。通过使用荧光Bla底物可以容易地检测到这种重新结合。因此，在我们已经开发的测定中，TLR 4和MyD 88在HeLa细胞中作为与Bla片段的嵌合体表达。当TLR 4和MyD 88有效地相互作用时，很容易观察到Bla活性。表达TLR 4和MyD 88的这些嵌合体的稳定细胞系可用于16，000种化合物文库的高通量筛选，因为其鉴定了5种发现抑制TLR 4-MyD 88缔合的化合物。通过这项提议，我们希望扩大我们的方法，以确定抑制剂的信号启动额外的TLR。最终，我们希望这项工作将导致药物的开发，用于治疗应用以及研究。毫无疑问，这些药物是必要的。我们提出了三个具体的目标，这项工作：1）开发稳定的细胞系，其中MyD 88嵌合体与TLR 2或TLR 9嵌合体缔合，并导致活性Bla。2)通过筛选NIH MLSCN文库的16，000个化合物子集，证明稳定细胞系可用于高通量筛选。3)表征如此鉴定的化合物的作用方法，确定其体外疗效，并开始表征其体内疗效。公共卫生相关性炎症，无论是对感染的反应还是对损伤的反应，都可能是有害的，在某些情况下甚至危及生命，尽管它是愈合反应的重要组成部分。该应用的目标是开发用于发现新的炎症抑制剂的技术。

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.