Pregnane X Receptor (PXR) in Drug Metabolism: Mechanism, Role, and HepG2-Based Screening

The pregnane X receptor (PXR) is a ligand‑activated nuclear receptor that senses xenobiotics and controls expression of drug‑metabolizing enzymes, especially CYP3A4, it’s activation may lead to potential drug-drug interactions (DDIs). This blog discussed the mechanism, significance, and the study method of pregnane X receptor—a HepG2‑based PXR–CYP3A4 luciferase reporter line, and its applications.

What is Pregnane X Receptor (PXR)?

The pregnane X receptor (PXR) is a ligand-activated receptor, which is encoded by the NR1I2 gene ^[1]. The protein structure of PXR includes both DNA-binding and ligand-binding domains ^[2]. Among them, the flexibility of the ligand-binding domain enables PXR to respond to a wide variety of ligands ^[3].

Although PXR is found in many tissues and organs, it’s highly expressed in the human liver and intestine, which are critical sites for drug and xenobiotic metabolism ^[4]. PXR activation regulates the expression of downstream CYP450 enzymes, such as CYP3A4 ^[5, 6].

Overall, PXR serves as a “xenobiotic sensor”, functioning as a key regulator of drug metabolism and constituting a primary defense mechanism against drug exposure ^{[3, 5]}. The significance of studying PXR lies in its role in improving drug safety and reducing drug-drug interactions (DDIs) ^{[7, 8]}. Furthermore, PXR modulation offers novel therapeutic targets for metabolic diseases, cancers, and inflammatory diseases ^[9].

How Does Pregnane X Receptor Work?

Numerous studies reveal that pregnane X receptor can regulate CYP3A4 expression and thereby affect drug metabolism. How does PXR regulate CYP450? As a ligand-dependent transcription factor, PXR can be activated by many xenobiotics such as rifampicin. Upon binding to rifampicin, PXR will change its conformation and translocate to the cell nucleus ^[10]. Subsequently, PXR forms a heterodimer with the retinoid X receptor (RXR). This heterodimer directly upregulates the mRNA expression of CYP3A4 by binding to its promoter region (Figure 1) ^{[11, 12]}.

However, studies also suggest that PXR can bind to certain compounds in the cell nucleus directly without translocation ^[4]. The mechanism of intracellular PXR activity is not yet fully understood. Moreover, previous publications reported significant species-specific differences in PXR activation ^[12,13]. Therefore, these differences must be taken into consideration when scientists extrapolate PXR findings across species.

Figure 1. Mechanism of pregnane X receptor activation.

Significance of Studying Pregnane X Receptor in Drug R&D

It has been reported that CYP3A4 metabolizes nearly 50% of marketed drugs, and its expression is significantly regulated by PXR. When drugs or environmental compounds activate PXR, CYP3A4-mediated metabolism accelerates. For the co-administered drugs of CYP3A4 substrate (e.g., the antiviral drug Paxlovid), their systemic exposure may be decreased due to PXR activation, leading to diminished efficacy or treatment failures. Lei et al ^[14] demonstrated that PXR activation increased the metabolism of Nirmatrelvir (the active component in Paxlovid) and directly reduced the efficacy of this drug in transgenic mice expressing hPXR and CYP3A4. Additionally, herbal components and chemotherapeutic agents can cause adverse effects such as hyperglycemia or dyslipidemia due to PXR-mediated CYP3A4 upregulation ^[15].

Research on PXR is of great importance in drug development and clinical applications. Clearance of drug candidates may increase due to activating PXR and inducing metabolic enzymes, which may hinder translation of preclinical data to human settings. Additionally, scientists optimize drug design through structural modification to reduce PXR activity ^[5]. If ignored, it may obscure drug toxicity mechanisms ^[16].

HepG2 Cell Model in Accessing Pregnane X Receptor Response

Cell-based reporter gene systems are essential for high-throughput screening of PXR agonists. The HepG2 cell line is commonly used to generate the PXR luciferase reporter system. Briefly, HepG2 cells are transfected with the hPXR gene, the CYP3A4 promoter region, and a luciferase reporter gene to establish HepG2-PXR-p3A4 cell line ^[17]. This model is widely accepted and has been used for many years. In a previously published study, a PXR-overexpressing HepG2 cell line was used to screen 2816 clinically used drugs for their potential to activate PXR, and the results showed good correlation with those obtained from human primary hepatocytes. ^[18]

WuXi AppTec PXR-HepG2 Cell Line and Its Application

The characterization of the PXR-HepG2 cell line was performed by assessing the activation potential of 24 reported PXR agonists (commercially available). In the present study, all investigational compounds demonstrated more than a 2-fold increase in PXR response compared to the vehicle control group. Additionally, these compounds exhibited a concentration-dependent activation of PXR (Figure 2A). Moreover, the estimated EC₅₀ values in the present study are highly consistent with previously published data (Figure 2B).

Figure 2. WuXi AppTec PXR-HepG2 cell line characterization. A. Rifampicin activated PXR in a concentration-dependent manner after 24h or 48h of treatment in HepG2-PXR cells. B. Correlation Analysis of EC₅₀ Values: WuXi AppTec HepG2-PXR Cells Versus Literature Data (http://tripod.nih.gov/tox/). Cells were treated with investigational compounds for 24h.

Concluding Remarks

To accelerate the R&D process, rapid screening of PXR agonists followed by induction studies in primary hepatocytes is a promising strategy to reduce CYP450-mediated drug-drug interactions. If you are interested in PXR activation assays, please contact us to discuss how we can support your research program.

Authors: Yuxi Wang, Yudie Zhang, Yajuan Bi, Genfu Chen

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