Discovery Of Small Molecules As Ligands Of Orphan Nuclear Receptor RoRγ

Retinoic acid-related orphan receptor γ(RORγ) is a kind of liganddependent orphan receptor.RORγ is well known as a master regulator of Interleukin17(IL-17)-producing helper T(Th17)cell development. RORγ plays a key role in development and metabolic homeostasis.Th17celldevelopment and IL-17production are inhibited through RORγ repression. So RORγ is apromising therapeutic target for treating Th17-mediated autoimmune disease, suchas multiplesclerosis and rheumatoid arthritis. Although significant progress has been made in developingRORγ antagonists, the identification of novel, non-steroidal RORγ antagonists for therapeuticuse still remains an urgent need. Until now, there is no medicine working for RORγ in market.In the present project, our aim is to find out promising drug candidate for autoimmune diseases.The main work of this study is as follows:(1)24novelstructures were selected from300,000compounds library and purchasedthroughstructure-based virtual screening.(2) Protein expression and purification of the human RORγ-LBD.The human RORγ-LBD（residues，262-507）was expressed in Escherichia coli(E.coli)BL21.Cells were lysed by high-pressure and centrifuged. Cell were harvested and then purifiedby5-mL NiSO4-loaded His-Trap HP column(GE Healthcare,Piscataway,NJ). A gel filtrationcolumn (HiLoad S75,16/60, GE Healthcare) was used for a second purification for high purityprotein.(3) Biological assay for activityTo test compound activity, AlphaScreen assay and luciferase dual reporter assay were usedfor all the compounds. For the screened24compounds from SPECS, compound S4wasdemonstrated as hit compound with micromolar level activity (AlphaScreen IC50=13.83μM，Luciferase IC50=11.84μM). Then a series of compounds with similar structure were purchasedor synthetized based on the hit compound S4. Four compounds showed significantly improvedactivity. The IC50values of compound1(AlphaScreen IC50=3.83μM，Luciferase IC50=0.14μM)and compound4(AlphaScreen IC50=5.88μM，Luciferase IC50=0.09μM) are similar to positivecontrol compound T1317(AlphaScreen IC50=2.01μM，Luciferase IC50=0.53μM). (4) Protein crystallizationVapor-diffusion method with sitting drops was used for protein-ligand complex crystallization.We haven’t obtained the crystal for the current study compound and RORγ. However,theprotein thermal stability and crystallization condition are analyzed through thermal stabilityshift assay(TSA). This provided important information for future crystallization.In summary, we have established a platform for protein expression, purification,crystallization and activity testing.4compounds were obtained starting from virtual screeninghit. Protein-ligand complex crystallization is still in progress.