Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand

Background: The nuclear hormone receptor ROR gamma 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 ROR gamma is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of ROR gamma and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a ROR gamma 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 ROR gamma 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 ROR gamma. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for ROR gamma over ROR sub-family members alpha and beta. 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 ROR gamma. The proteolytic sensitivity of the AF2 helix of ROR gamma 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 ROR gamma as the trigger for ROR gamma specific inverse agonism.