Application Note: Automation Of A Multiplexed Cell-Based Assay To Measure Simultaneously Induction Of Gene Expression And Activity Of Cytochrome P450 Isoform 3A4 By Small Molecule Compounds

By Brad Larson and Peter Banks, BioTek Instruments, Inc.

The pregnane X receptor (PXR) is a nuclear receptor which plays a part in the removal of foreign substances from the body. Upon detecting these xenobiotics, PXR responds by up regulating proteins that can help in the elimination process. One of the most important enzymes involved in the response is the cytochrome P450 3A4 enzyme. CYP3A4 is involved in the metabolism of the widest range of substrates, and metabolizes more than 50% of drugs on the market today. PXR upregulates the expression of this enzyme by binding to the response element of the promotor.

Many drugs upregulate the expression of the CYP3A4 gene via PXR. In contrast, a large set of drugs have also shown inhibitory effects on the CYP3A4 enzyme. The upregulation of the CYP3A4 gene or inhibition of the enzyme activity can result in adverse drug-drug interactions. Less understood and more difficult to assess is how certain drugs can induce the expression of the CYP3A4 gene and also inhibit the activity of the enzyme. The DPX2 cell line is a useful tool to assess the effects of a xenobiotic on gene induction and enzyme activity inhibition. The cell line is based on HepG2 cells, a human liver carcinoma cell line which contains human PXR and a luciferase-linked CYP3A4 promoter reporter construct. Upon activation, PXR binds to the native promoter and the luciferase-linked promoter. Increased CYP3A4 enzyme expressed from the native gene, as well as luciferase expressed from the reporter construct is then expressed.