Rifamycin Structural Modifications Attenuate PXR Binding and CYP3A4 Induction

Rifamycins are a cornerstone of antimycobacterial therapy. However, their clinical use is limited by drug–drug interactions, arising from activation of the nuclear receptor pregnane X receptor (PXR). PXR activation induces expression of drug-metabolizing enzymes, including the cytochrome P450 3A4 isoform (CYP3A4), and accelerates clearance of coadministered medications. The structural understanding of rifamycin–PXR interactions remains limited. We designed a series of C25-modified rifabutin analogs and systematically evaluated their PXR binding, transcriptional activation, and pharmacological profile. Several analogs retained PXR binding affinity yet showed reduced CYP3A4 induction, exhibiting behavior consistent with antagonists or inverse agonists and revealing a disconnect between receptor binding and transcriptional activation. Molecular dynamics simulations indicated C25 modifications may disrupt positioning of the PXR α12 helix through steric interactions. These findings demonstrate conservative modifications of rifamycin can potentially convert PXR agonists into antagonists or inverse agonists and offer a structure-guided framework for developing rifamycins with attenuated CYP3A4 induction.