Koyama Ryokichi, Fukuda Yasunori, Kamada Yusuke, Nakagawa Hideyuki, Witmer Darbi, Ambrus-Aikelin Geza, Sang Bi-Ching, Nakayama Masaharu, Iwata Hidehisa
1 Biomolecular Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited , Fujisawa, Japan .
2 Department of Structural Biology, Takeda California , San Diego, California.
Assay Drug Dev Technol. 2018 May/Jun;16(4):194-204. doi: 10.1089/adt.2018.852. Epub 2018 Jun 6.
The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.
维甲酸相关孤儿受体γT（RORγt）在Th17细胞增殖和功能中发挥重要作用。因此，RORγt反向激动剂被认为是治疗Th17介导的自身免疫性疾病（如类风湿性关节炎、哮喘、炎症性肠病和银屑病）的有效治疗剂。尽管RORγt具有组成性活性，但人们认识到该受体在生理上受各种胆固醇衍生物的调节。在本研究中，我们试图通过高通量筛选来鉴定RORγt反向激动剂。为此，我们比较了来自大肠杆菌的无载脂蛋白RORγt蛋白和来自昆虫细胞的胆固醇结合型RORγt蛋白。已知的RORγt反向激动剂TO901317对无载脂蛋白的IC显著低于对胆固醇结合型RORγt的IC。通过使用基于荧光的胆固醇结合测定法对无载脂蛋白进行高通量筛选，我们鉴定出化合物1为一种新型的胆固醇竞争性RORγt反向激动剂。化合物1抑制RORγt与TopFluor胆固醇的相互作用、共激活因子募集以及RORγt的转录活性。基于细胞的报告基因测定表明，通过脂质消耗处理，化合物1显示出更高的效力。总体而言，我们的研究结果表明，从RORγt蛋白中去除胆固醇适用于灵敏的高通量筛选以鉴定RORγt反向激动剂。
