Bile acid scaffold engineering reveals an androstane-triol derivative as a potent immunomodulator with therapeutic efficacy in EAE

BACKGROUND: Neuroinflammation driven by dysregulated adaptive and innate immune responses plays a central role in the pathogenesis of multiple sclerosis and related autoimmune disorders of the central nervous system. While bile acids are increasingly recognized as endogenous immunomodulators, their therapeutic exploitation has been limited by modest potency and incomplete mechanistic understanding. Here, we report the rational engineering of a bile acid-derived scaffold that yields a potent small molecule immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis, a preclinical model of multiple sclerosis. METHODS: A focused series of bile acid-based compounds was established, leading to the identification of an androstane-triol derivative, BA59. The immunomodulatory activity of BA59 was evaluated using in vitro T cell differentiation assays and ex vivo immune profiling in EAE. Therapeutic efficacy was assessed in mice with established EAE. Flow cytometry was used to characterize peripheral immune populations, including effector T cells, regulatory T cells, and antigen-presenting cells. Disease progression was monitored using clinical scoring and cumulative disease burden analyses. RESULTS: BA59 treatment significantly attenuated disease severity and cumulative disease burden when administered therapeutically after the onset of clinical signs. Immunophenotyping revealed that BA59 treatment shifted the peripheral immune profile toward a regulatory state. Specifically, we observed a reduction in pro-inflammatory populations relative to IL-10-producing T cells, alongside an expansion of CD39+ regulatory T cells and CD4 + T cells expressing the immune checkpoints CTLA-4, PD-1, and TIM-3. Furthermore, BA59 reprogrammed antigen-presenting cells toward a tolerogenic phenotype, characterized by enhanced PD-L1 expression. CONCLUSIONS: This study identifies BA59 as a first-in-class androstane-triol immunomodulator that ameliorates experimental autoimmune encephalomyelitis through coordinated regulation of T cell balance, immune checkpoints, and antigen-presenting cell function. Our findings highlight bile acid scaffold engineering as a viable strategy for developing small molecule therapeutics that reprogram neuroinflammatory immune circuits, offering a promising translational approach for multiple sclerosis and related neuroinflammatory diseases.