Screen for Small Molecule Compounds that Modulate the Transcriptional Activity of

描述（由申请人提供）：孤儿核受体ROR？t在Th17细胞的分化中起核心作用，Th17细胞是t辅助细胞的一个亚群，分泌炎症细胞因子IL-17、IL-17F和IL-22。Th17细胞最近被证明在多种小鼠自身免疫性疾病模型和移植物抗宿主病中具有主要的致病功能，并且它们也与人类自身免疫性疾病和哮喘有关。ROR吗?在TGF-存在的情况下，t在抗原刺激的na - ve t辅助细胞中诱导。与IL-6、IL-21或IL-23结合，然后指导Th17谱系细胞因子的表达。我们已经证明小鼠的ROR？它缺乏Th17细胞，对自身免疫性疾病具有抵抗力。更好地理解ROR？因此，t在诱导Th17谱系中的功能可能为开发炎症性疾病的新治疗方法提供重要信息。研究ROR的作用机制。最后，我们开发了一种基于昆虫细胞的检测方法，以确定参与其转录活性的宿主因子，并在高通量检测中筛选小分子。细胞中Gal4-ROR？t融合蛋白在Gal4启动子的调控下指导荧光素酶的表达，已被用于全基因组RNAi和小分子化合物筛选，以确定抑制或增强活性。筛选试验已优化为384孔格式，并在RNAi筛选中确定了多个候选基因。具有有限小分子化合物库的原理验证筛选也已成功进行。我们建议与NIH分子文库筛选网络合作，进行高通量筛选，以鉴定调节ROR活性的化合物。在初级筛选中影响转录活性的化合物将在T细胞分化试验和其他次级筛选中检查活性，以确定它们是否直接作用于ROR？或者它们是否会影响ROR的其他组件？t-directed转录。特异性影响Th17细胞分化的化合物将在解剖这些炎症T细胞的转录程序方面发挥重要作用，并将指导我们开发自身免疫性疾病和移植物抗宿主病的新治疗方法。

DESCRIPTION (provided by applicant): The orphan nuclear receptor ROR?t has a central role in the differentiation of Th17 cells, a subset of T helper cells that secrete the inflammatory cytokines IL-17, IL-17F, and IL-22. Th17 cells have recently been shown to have major pathogenic functions in a variety of murine autoimmune disease models and in graft versus host disease, and they have also been implicated in human autoimmune disease and asthma. ROR?t is induced in antigen-stimulated na¿ve T helper cells in the presence of TGF-??combined with IL-6, IL-21, or IL-23, and thereafter directs the expression of the Th17 lineage cytokines. We have shown that mice defective for expression of ROR?t lack Th17 cells and are resistant to autoimmune disease. A better understanding of how ROR?t functions in inducing the Th17 lineage is therefore likely to provide important information for developing new therapeutic approaches for inflammatory diseases. To study the mechanism of action of ROR?t, we have developed an insect cell-based assay to identify host factors involved in its transcriptional activity and to screen for small molecules in high throughput assays. Cells in which a Gal4-ROR?t fusion protein directs expression of luciferase under regulation of the Gal4 promoter have been used in genome-wide RNAi and small molecule compound screens to identify inhibitory or enhancing activities. The screening assay has been optimized for a 384-well format, and multiple candidate genes have been identified in the RNAi screen. A proof-of-principle screen with a limited small molecule compound library has also been successfully performed. We propose to collaborate with the NIH Molecular Libraries Screening Network to perform a high throughput screen to identify compounds that modulate the activity of ROR?t. Compounds that affect transcriptional activity in the primary screen will then be examined for activity in T cell differentiation assays and other secondary screens to determine if they act directly on ROR?t or if they affect other components of ROR?t-directed transcription. Compounds that specifically affect Th17 cell differentiation will be of great utility in dissecting the transcriptional program of these inflammatory T cells and will guide us in developing new therapeutic approaches for autoimmune disease and graft versus host disease.