Abstract
The pregnane xenobiotic receptor (PXR) is a key transcriptional regulator of cytochrome P450 (CYP) 3A, a crucial enzyme in the metabolism and detoxification of xenobiotics and endobiotics. PXR is activated by a wide variety of chemicals and serves as a master regulator of detoxification in mammals. Here, we report a fast evaluation method for PXR-drug interactions using differential scanning fluorometry (DSF). DSF analysis revealed that PXR associates with a fluorescence dye in the native state as well as in the unfolded state, which prevented precise evaluation of any shift in the transition midpoint (ΔTm) due to association with a drug. Hence, we defined a new parameter, (dF/dT)50, where F is fluorescence intensity and T is temperature, to describe the ligand concentration. (dF/dT)50 exhibited better correlation with EC50 (r2 = 0.84) than with ΔTm (r2 = 0.71). The correlation of ΔTm measured using differential scanning calorimetry (DSC) with EC50 (r2 = 0.86) was similar to the above (dF/dT)50 correlation. Therefore, the use of (dF/dT)50 enables DSF to be used for the rapid evaluation of PXR-drug interactions and could provide prescreening to narrow down the collection of candidate ligands that most likely result in transcriptional activation of CYP3A4.
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