HIGH THROUGHPUT SCREENING FOR TOLL-LIKE RECEPTORS INHIBITORS

描述(申请人提供)：Toll样受体(TLRs)是先天免疫的主要参与者。它们检测病原体的外源性危险信号和宿主组织的内源性损伤信号，启动涉及多种细胞类型的动态平衡炎症反应，并伴随着许多可溶性介质的表达。尽管这些TLR介导的反应显然是进化的积极选择，但它们有时会产生不良的、甚至是致命的副作用。感染性休克长期以来一直被认为是对感染的一种潜在的致命性高反应。最近，无菌损伤，如肾缺血再灌注损伤、全身自身免疫损伤、实验性自身免疫性脑脊髓炎和心血管疾病，都被证明与TLR有关，因为缺乏一个或多个TLR可以减轻其严重程度。大多数TLR的细胞内信号需要它们与MyD88相关联。我们已经开发了一种检测TLRs与MyD88关联的方法。这种分析是基于内酰胺酶(Bla)被分成两个非活性片段的能力。当这两个片段并置时，它们将相互补充，以改造活性酶。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，在我们已经建立的方法中，TLR4和MyD88在HeLa细胞中以嵌合体的形式与Bla片段一起表达。当TLR4和MyD88有效地相互作用时，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在16,000个化合物文库的高通量筛选中是有用的，因为它鉴定了5个被发现抑制TLR4-MyD88结合的化合物。通过这项提议，我们希望扩大我们的方法，以确定由额外的TLR启动的信号抑制物。最终，我们希望这项工作将导致对治疗应用和研究有用的药物的开发。毫无疑问，这类药物是必要的。我们提出了这项工作的三个具体目标：1)建立稳定的细胞系，其中MyD88嵌合体与TLR2或TLR9嵌合体结合，并导致活性Bla。2)通过筛选NIH MLSCN文库的16,000个化合物子集，证明稳定的细胞系对于高通量筛选是有用的。3)鉴定化合物的作用方法，确定它们的体外药效，并开始鉴定它们的体内药效。与公共卫生相关的炎症，无论是对感染的反应还是对伤害的反应，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。这项应用的目标是开发寻找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.