Development of bimolecular biosensors to detect chemicals with agonist activity toward rat PXR

The pregnane X receptor (PXR) exhibits broad ligand specificity and responds to diverse chemicals, making it an attractive target for evaluating the biological effects of exogenous chemicals. This study developed biosensors based on the integration of the PXR activation mechanism and NanoLuc Binary Technology to establish an in vitro assay for evaluating interactions between chemicals and rat PXR (rPXR). The biosensors comprised two molecules: rPXR ligand-binding domain (LBD)-SmBiT, in which the LBD is fused to the N-terminus of SmBiT, and LgBiT-LXXLL, in which a coactivator-derived LXXLL motif is fused to the C-terminus of LgBiT. The biosensors detected agonist-dependent interactions between PXR and its coactivator through NanoLuc luciferase reconstitution. The two selected bimolecular biosensors were generated using different optimization strategies to improve agonist responsiveness. They exhibited responses to the agonist pregnenolone 16α-carbonitrile at lower concentrations and largely similar response profiles for six additional agonists compared with a conventional p3A4-Luc reporter assay. Unlike conventional transcription-based assays, these biosensors enabled real-time detection of chemical-PXR interactions through enzyme reconstitution. Accordingly, this assay may provide a rapid and sensitive platform for evaluating chemical-induced PXR activation.