Distinct PXR Inverse Agonism Mechanisms Rely on Differential Ligand‐induced Co‐Regulator Recruitment

The pregnane X receptor (PXR) displays exceptional ligand promiscuity, yet the molecular basis of its antagonism remains poorly defined. PXR inhibition has traditionally been attributed to the displacement of coactivators from the activation function-2 (AF-2) surface. Here, we show that PXR antagonism is mechanistically heterogeneous, with distinct inhibitory mechanisms determined by ligand structure. Comparative analysis of imidazo[1,2-a]pyridine, MI891, and SPA70 antagonists reveals multiple non-canonical inhibitory mechanisms that decouple coregulator recruitment from transcriptional output. Notably, SPA70 preserves compatibility with coactivator recruitment while remaining transcriptionally inactive, consistent with concurrent corepressor engagement and a distinct coactivator-compatible antagonist state. In contrast, MI891 stabilizes a PXR conformation that favors NCoR1 and SMRT binding and impairs nuclear translocation. MD simulations and mutagenesis reveal ligand-specific perturbations of α12 and the α10/α11 region that underlie these effects. Together, these findings establish a ligand-encoded framework for PXR antagonism that extends beyond the classical on/off switch model.