Abstract
The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug-drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand-PXR interactions. Here we report the characterization of BODIPY FL-vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a Kd value of 673 nM. We provide evidence that BODIPY FL-vinblastine is a unique chemical entity different from either vinblastine or the fluorophore BODIPY FL in its function as a high-affinity human PXR ligand. We describe a BODIPY FL-vinblastine-based human PXR time-resolved fluorescence resonance energy transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL-vinblastine-based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR.