Discovery Of RORγ Novel Small Molecule Inhibitors

Retinoic acid receptor-related orphan receptors(RORs) are ligand-regulated transcription factors that play multiple roles in a variety of physiological processes,including development, circadian rhythm, metabolic disorders, inflammation, and immunity. The ROR superfamily consists of three members, RORα, RORβ, and RORγ, which belongs to the nuclear receptors. RORα is widely expressed in liver,skeletal muscle, skin, lung, adipose tissue, kidney, thymus, brain and blood, related to glyconeogenesis, metabolism and atherosclerosis. RORβ is expressed exclusively in areas of the central nervous system(CNS), that are involved in the processing of sensory information, including spinal cord, thalamus and cerebellar cortices. RORγ is highly expressed in thymus, kidney, liver, heart, muscle, adipose tissue, testis, prostate and pancreas, and considered to be related to rheumatoid arthritis(RA), psoriasis, and multiple sclerosis(MS).Autoimmune disease is a kind of multifactorial disease that related to endocrine,environmental, genetic and other factors. Currently, the incidence rate of autoimmune disease is approximately 6% in the world. Autoimmune disease is a threat to human health and it cannot be easily cured. Th17 cells specifically produce interleukin-17(IL-17), which is known to enhance inflammatory processes. RORγ is considered to be the master regulator of the development of T helper 17 cells(Th17 cells), which play essential role in the development of autoimmune diseases and inflammation symptom. In Th17 cells, interleukin-17(IL-17) transcription is mediated by Th17-specific transcriptional regulator RORγ. Given its crucial role in suppression of IL-17 activity, RORγ is a promising therapeutic target for treating Th17-mediated autoimmune diseases.ROR receptors are still considered as ‘orphan’ receptors because the identification of their endogenous ligands has been controversial. Recent studies have demonstrated that these receptors are regulated by synthetic ligands. Since the identification of benzenesulfonamide liver X receptor(LXR) agonist T1317 as a RORγ inhibitors, several small molecule RORγ ligands have been disclosed in the literature. The Scripps Research and Glaxo Smith Kline(GSK) et al. developed many synthetic RORγ inhibitors, including SR1001, SR2211, SR1555, TMP778, etc. Inaddition, natural products digoxin and its less toxic analogues along with ursolic acid(UA) are RORγ selective inhibitors.This thesis aims at identification of RORγ new scaffold inhibitors. For this purpose, the following works have been done: N-terminal His6-fusion tag fusion human RORγ LBD wide type(WT) and human RORγ LBD mutant type proteins were expressed in E. coli BL21(DE3) competent cells with expression vector p ET24 a.The proteins were purified by a 5-m L Ni SO4-loaded His Trap HP column and a gel filtration column purification. The purified protein h RORγ LBD(WT) was used for binding assay, and the purified protein h RORγ LBD(C455E) was used for crystallization. We screened a small chemical library with approximately 1,150,000 compounds by structure-based virtual screening(SBVS) and ligand-based virtual screening(LBVS). 28 candidate compounds were assessed for their biological activity.The biological activity assay includes Alpha Screen assay, thermal shift assay(TSA),and luciferase assay. Among these assays, Alpha Screen assay measures the ability of a compound in disrupting the interaction between RORγ-LBD and its co-regulator. TSA can determine the thermodynamic stability of RORγ-LBD in the presence of a compound. Luciferase reporter assay can test the activity of a compound interfering RORγ-LBD transcription. Utilizing the bioassays, tertiary amines compound C11 was validated as a potential candidate compound. The biological activity assay results of C11 were 62.58 μM(Alpha Screen assay), 4.54 μM(Luciferase assay) and ΔTm=1.8oC(TSA). To improve the inhibitory activity, C11 was further optimized by medicinal chemistry. Among the 30 synthesized analogues of C11, 29, 36, 39 displayed significantly enhanced RORγ inhibition. The inhibitory activity of 29, 36, 39 were80-fold, 60-fold, 280-fold higher than that of the hit compound C11 in Alpha Screen assay. In this assay, the IC50 values of synthesized compound 29, 36, 39 were 0.77 μM,1.07 μM, and 0.22 μM, respectively. The selectivity in celluar luciferase reporter gene assay indicated that compounds 36 and 39 show higher selectivity than 26. Compared with 36 and 39, the inhibitory activity of 39 were 5-fold higher. Finally, compound 39 was used in co-crystal study with h RORγ LBD(C455E). The crystal structure of the h RORγ LBD(C455E) in complex with compound 39 was determined at 3.0 ?. The crystal structure is still in the data analysis. These key interactions would contribute to the high inhibition rate of compound 39.This study represents N-phenyl-2-(N-phenylphenylsulfonamido)acetamides as a new scaffold for developing potent small molecule RORγ inhibitors. Compound 39 displayed promising activities in all of the implied biochemical assays. Compound 39 is shown highly selectivity in transcriptional reporter assay. The co-crystal structure of compound 39 in complex with h RORγ LBD(C455E) provided useful insights into the binding interactions of new scaffold. Compound 39 represents a promising starting point for developing potent small molecule RORγ inhibitors. Current research progress has laid a solid foundation for the compound as a lead compound. The result provides a new idea for study nuclear receptor small molecule ligands, and promotes the process of anti-inflammatory drug discovery.