HIGH THROUGHPUT SCREENING FOR TOLL-LIKE RECEPTORS INHIBITORS

描述（由申请人提供）：Toll 样受体 (TLR) 是先天免疫的主要参与者。他们检测到来自病原体的外源危险信号以及来自宿主组织的内源损伤信号，从而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。尽管这些 TLR 介导的反应显然是通过进化积极选择的，但有时它们会产生不良甚至致命的副作用。脓毒性休克长期以来被认为是对感染的潜在致命性过度反应。最近，诸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等无菌性损伤均已被证明与 TLR 显着相关，因为一种或多种 TLR 的缺乏可减轻其严重程度。大多数 TLR 的细胞内信号传导需要与 MyD88 相关。我们开发了一种检测 TLR 与 MyD88 关联的检测方法。该测定基于 β-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，它们将相互补充以重新形成活性酶。通过使用荧光 Bla 底物可以很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，TLR4和MyD88在HeLa细胞中作为与Bla片段的嵌合体表达。当 TLR4 和 MyD88 有效相互作用时，很容易观察到 Bla 活性。表达这些 TLR4 和 MyD88 嵌合体的稳定细胞系可用于高通量筛选 16,000 个化合物库，因为它鉴定了 5 种被发现抑制 TLR4 - MyD88 关联的化合物。通过这项提案，我们希望扩展我们的方法来识别由其他 TLR 启动的信号传导抑制剂。最终，我们希望这项工作能够开发出可用于治疗应用和研究的药物。毫无疑问，需要这样的药物。我们提出了这项工作的三个具体目标：1）开发稳定的细胞系，其中 MyD88 嵌合体与 TLR2 或 TLR9 嵌合体结合并产生活性 Bla。 2) 通过筛选 NIH MLSCN 文库的 16,000 种化合物子集，证明稳定细胞系可用于高通量筛选。 3)表征所鉴定的化合物的作用方法，定义其体外功效，并开始表征其体内功效。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，尽管它是治愈反应的重要组成部分。该应用的目标是开发寻找新炎症抑制剂的技术。

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.