High-throughput evaluation method for drug association with pregnane X receptor (PXR) using differential scanning...

Area of Science:

• Biochemistry
• Pharmacology
• Molecular Biology

Background:

• The pregnane xenobiotic receptor (PXR) is a critical regulator of drug metabolism and detoxification, primarily controlling the expression of cytochrome P450 3A (CYP3A) enzymes.
• PXR activation by diverse chemicals underscores its role as a master regulator of xenobiotic detoxification pathways in mammals.

Purpose of the Study:

• To develop and validate a rapid method for evaluating PXR-drug interactions using differential scanning fluorometry (DSF).
• To establish a new parameter for DSF analysis that accurately reflects ligand concentration and PXR activation.

Main Methods:

• Differential scanning fluorometry (DSF) was employed to assess PXR-drug interactions.
• A novel parameter, (dF/dT)(50), was defined to quantify ligand concentration based on fluorescence intensity and temperature changes during protein unfolding.
• Results were compared with traditional differential scanning calorimetry (DSC) measurements and EC(50) values.

Main Results:

• Standard DSF analysis was complicated by PXR's interaction with the fluorescence dye in both native and unfolded states, hindering direct measurement of transition midpoint shifts (ΔT(m)).
• The new parameter (dF/dT)(50) showed a strong correlation with EC(50) (r(2) = 0.84), outperforming the ΔT(m) correlation (r(2) = 0.71).
• The correlation of ΔT(m) from DSC with EC(50) (r(2) = 0.86) was comparable to the (dF/dT)(50) parameter.

Conclusions:

• The (dF/dT)(50) parameter enables DSF to serve as a rapid and effective tool for evaluating PXR-drug interactions.
• This DSF-based method can be utilized for prescreening potential drug ligands, identifying those most likely to induce CYP3A4 transcriptional activation.
• The findings facilitate faster drug discovery and development by enabling efficient identification of PXR modulators.