Lithocholic acid derivatives as potent modulators of the nuclear receptor RORγt.

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Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor found in various tissues that plays a crucial role in the differentiation and proliferation of T helper 17 (Th17) cells, as well as in their generation of the pro-inflammatory cytokine IL-17A. RORγt represents a promising therapeutic target for autoimmune diseases, metabolic disorders, and multiple tumors. Despite extensive research efforts focused on the development of small molecule RORγt modulators, no drug candidates have advanced to phase 3 clinical trials owing to a lack of efficacy or safety margin. This outcome highlights the unmet need to optimize small molecule drug candidates targeting RORγt to develop effective therapies for autoimmune and inflammatory diseases. In this study, we synthesized and evaluated 3-oxo-lithocholic acid amidates as a new class of RORγt modulators. Our evaluation entailed biophysical screening, cellular screening in different platforms, molecular docking, and in vitro pharmacokinetic profiling. The top compound from our study (3-oxo-lithocholic acid amidate, A2) binds to RORγt at an equilibrium dissociation constant (KD) of 16.5 ± 1.34 nM based on microscale thermophoresis (MST). Assessment of the efficacy of A2 in the cellular RORγt reporter luciferase assay revealed a half-maximal inhibitory concentration (IC50) value of 225 ± 10.4 nM. Unlike 3-oxo-lithocholic acid, A2 demonstrated the ability to reduce the IL-17A mRNA expression levels in EL4 cells with RORγt expression using quantitative reverse transcriptase PCR (RT-PCR). Validation of the desirable physicochemical properties and stability of A2 sets the stage for the preclinical evaluation of this new class of RORγt modulators in animal models of autoimmune diseases.