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细胞的分化中发挥核心作用，Th17细胞是分泌炎性细胞因子IL-17、IL-17F和IL-22的T辅助细胞的一个子集。最近发现Th17细胞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重要的致病功能，它们也与人类自身免疫性疾病和哮喘有关。在转化生长因子-β与IL-6、IL-21或IL-23联合作用下，在抗原刺激的NAVE辅助性T细胞中诱导ROR？T，然后引导Th17细胞因子的表达。我们已经证明，表达ROR？T缺陷的小鼠缺乏Th17细胞，并且对自身免疫性疾病具有抵抗力。因此，更好地了解ROR？T如何在诱导Th17谱系中发挥作用，可能会为开发炎症性疾病的新治疗方法提供重要信息。为了研究ROR？T的作用机制，我们建立了一种基于昆虫细胞的分析方法，以确定参与其转录活性的宿主因素，并在高通量分析中筛选小分子。Gal4-ROR？T融合蛋白在Gal4启动子调控下引导荧光素酶表达的细胞已被用于全基因组RNAi和小分子化合物筛选，以确定抑制或增强活性。筛选试验已经针对384孔的格式进行了优化，并在RNAi筛选中确定了多个候选基因。还成功地进行了具有有限小分子化合物文库的原理验证筛选。我们建议与NIH分子文库筛选网络合作进行高通量筛选，以确定调节ROR？T活性的化合物。然后，将在主筛选中检查影响转录活性的化合物，并在T细胞分化试验和其他二次筛选中检查活性，以确定它们是否直接作用于ROR？T，或者它们是否影响ROR？T定向转录的其他成分。特异性影响Th17细胞分化的化合物将在解剖这些炎性T细胞的转录程序方面非常有用，并将指导我们开发治疗自身免疫性疾病和移植物抗宿主疾病的新方法。

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.