Decoding structural and dynamic determinants of Tropifexor-FXR binding: A comprehensive computational analysis.

Farnesoid X receptor (FXR) is a nuclear receptor considered a prominent therapeutic target associated with diseases such as non-alcoholic fatty liver disease, diabetes, and atherosclerosis. Tropifexor is a selective FXR agonist that shows promising activity against cholestatic liver diseases and non-alcoholic steatohepatitis. In this work, we performed comprehensive structural analyses and unbinding studies to investigate Tropifexor behaviour within the ligand binding domain using molecular dynamics (MD) simulations. Multiple repeats of 2 μs unbiased MD and WT-Metadynamics of the FXR-Tropifexor complex revealed salient aspects about ligand-induced agonism. Our analyses revealed that Tropifexor achieves its high FXR binding affinity and selectivity by simultaneously optimizing multiple interaction modes, including π-sulphur, π-π, and carbon-π contacts, which act as additional selectivity filters beyond conventional hydrogen bonding. The binding pocket volume expansion coupled with inter-helical distances fluctuations suggested a conformational wedge mechanism that stabilizes and activates the receptor through cooperative helical framework expansion. Furthermore, unbinding analyses revealed two distinct pathways. The most probable unbinding pathway (80% probability) embodies deep energy minima and significant dissociation barriers, which promote prolonged FXR receptor activation. The structural features elucidated in the current work serve as a rational blueprint for the discovery of next-generation FXR modulators with enhanced therapeutic indices.