Identification of Tetraazacyclic Compounds as Novel Potent Inhibitors Antagonizing RORγt Activity and Suppressing Th17 Cell Differentiation

CD4(+) T-helper cells that produce interleukin-17 (Th17 cells) are characterized as pathological T-helper cells in autoimmune diseases. Differentiation of human and mouse Th17 cells requires a key transcription regulator, retinoic acid receptor-related orphan receptor gamma t (ROR gamma t), which is a potential therapeutic target for autoimmune diseases. To develop a therapeutic agent for Th17-mediated autoimmune diseases, we have established a highthroughput screening (HTS) assay for candidate screening, in which the luciferase activity in ROR gamma t-LBD positive and negative Jurkat cells were analyzed to evaluate induction of ROR gamma t activity by compounds. This technique was applied to screen a commercially-available drug-like chemical compound library (Enamine) which contains 20155 compounds. The screening identified 17 compounds that can inhibit ROR gamma t function in the HTS screen system. Of these, three tetraazacyclic compounds can potently inhibit ROR gamma t activity, and suppress Th17 differentiation and IL-17 production. These three candidate compounds could significantly attenuate the expression of the Il17a by 65%-90%, and inhibit IL-17A secretion by 47%, 63%, and 74%, respectively. These compounds also exhibited a potent anti-ROR gamma t activity, with EC50 values of 0.25 mu M, 0.67 mu M and 2.6 mu M, respectively. Our data demonstrated the feasibility of targeting the ROR gamma t to inhibit Th17 cell differentiation and function with these tetraazacyclic compounds, and the potential to improve the structure of these compounds for autoimmune diseases therapeutics.