BACKGROUND The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. RESULTS We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β. CONCLUSION The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism.

中文翻译：

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用于诱导由合成苯并恶嗪酮配体触发的RORgamma特异性反向激动作用的结构决定因素。

背景技术核激素受体RORγ调节参与促炎性白介素IL-17产生的转录基因，促白细胞介素IL-17已与自身免疫性疾病如类风湿性关节炎，多发性硬化症和炎症性肠病相关。RORγ的这种转录活性是通过涉及RORγ配体结合域上的激活功能2（AF2）螺旋和共激活蛋白上保守的LXXLL螺旋基序的蛋白质-蛋白质相互作用来调节的。我们的目标是开发一种RORγ特异性反向激动剂，通过破坏与辅助激活剂蛋白作为治疗剂的蛋白相互作用来帮助下调促炎基因的转录。结果我们确定了一系列新的合成苯并恶嗪酮配体，它们在基于FRET的测定中具有激动剂（BIO592）和反向激动剂（BIO399）的作用方式。我们显示当反向激动剂BIO399结合时，RORγ的AF2螺旋对蛋白水解敏感。使用X射线晶体学，我们显示了苯并恶嗪酮激动剂BIO592的微小修饰如何触发RORγ的反向激动作用。使用体内报告基因测定，我们显示了反向激动剂BIO399在ROR亚家族成员α和β上显示出对RORγ的特异性。结论在我们的FRET分析中鉴定出的合成苯并嗪酮配体通过稳定或去稳定RORγ的激动剂构象而具有激动剂（BIO592）或反向激动剂（BIO399）的作用。RORγ的AF2螺旋的蛋白水解敏感性表明，它在扰动辅助激活蛋白结合位点的BIO399反向激动剂结合后不稳定。我们对BIO592激动剂和BIO399反向激动剂结构的结构研究确定RORγ上的残基Met358是RORγ特异性反向激动剂的触发因素。