The pregnane X receptor (PXR) is an important regulator of hepatic metabolism, yet mechanistic insights into the effects of pharmacological inhibition using PXR inverse agonists or antagonists on critical genes involved in both xenobiotic and endobiotic metabolism remain limited. Here, we discovered a novel PXR inverse agonist/antagonist, MI891, which binds to the ligand-binding domain of PXR. Furthermore, we computationally designed and synthesized the proteolysis-targeting chimera molecule, MI1013, based on the PXR antagonist SPA70, which degrades PXR in HepaRG hepatic cells. Using these tools, we investigated the regulation of key PXR target genes in HepaRG cells and human hepatocytes. Our findings indicate that PXR antagonism or degradation suppresses basal and rifampicin-induced expression of selected ADME genes. Moreover, the PXR antagonists and PROTAC degrader downregulate the expression of several key genes involved in gluconeogenesis, cholesterol homeostasis, bile acid synthesis, and proliferation in hepatocyte cells, suggesting their potential therapeutic applications for metabolic diseases.

• MeSH
• Hepatocytes metabolism   drug effects   MeSH
• Liver * metabolism   drug effects   MeSH
• Humans MeSH
• Drug Discovery * MeSH
• Pregnane X Receptor * antagonists & inhibitors   metabolism   MeSH
• Cell Proliferation drug effects   MeSH
• Gene Expression Regulation * drug effects   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Pregnane X Receptor * MeSH

Xenoreceptors of the nuclear receptor superfamily, such as pregnane X receptor (PXR), are liver-enriched ligand-activated transcription factors regarded as crucial sensors in xenobiotic exposure and detoxification. PXR controls transcription of many drug-handling genes and influx/efflux transporters, thus playing a crucial role in drug metabolism and excretion. Liver functions have been studied using primary human hepatocytes (PHHs), which, when conventionally cultured, undergo rapid de-differentiation, leaving them unsuitable for long-term studies. Recently, 3D PHHs called spheroids have emerged as an in vitro model that is similar to in vivo hepatocytes regarding phenotype and function and represents the first in vitro model to study the long-term regulation of drug-handling genes by PXR. In this study, we used mathematical modelling to analyze the long-term activation of PXR in 3D PHHs through expression kinetics of three key PXR-regulated drug-metabolizing enzymes, CYP3A4, CYP2C9, and CYP2B6 and the P-glycoprotein efflux transporter encoding gene, MDR1. PXR action in 3D PHHs was induced by the antibiotic rifampicin at two clinically relevant concentrations. The results confirmed that high rifampicin concentrations activated PXR nearly to its full capacity. The analysis indicated the highest PXR-induced transcription rate constant for CYP2B6. The rate constant dictating mRNA degradation associated with activated PXR was highest for CYP3A4. Moreover, we measured the metabolic activity of CYP3A4, CYP2C9, and CYP2B6 and quantified their metabolic rate constants. Metabolic activity rate constant of CYP3A4 was found to be the highest whereas that of CYP2B6 was found to be the lowest among the studied enzymes. Our results provide important insight into the regulation of PXR-target genes in 3D PHHs and show that mRNA expression and metabolic activity data can be combined with quantitative analysis to reveal the long-term action of PXR and its effects on drug-handling genes.

• MeSH
• Models, Biological MeSH
• Spheroids, Cellular * metabolism   MeSH
• Cytochrome P-450 CYP3A metabolism   genetics   MeSH
• Hepatocytes * metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Pregnane X Receptor * metabolism   MeSH
• Gene Expression Regulation * MeSH
• Rifampin pharmacology   MeSH
• Receptors, Steroid * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytochrome P-450 CYP3A MeSH
• Pregnane X Receptor * MeSH
• Rifampin MeSH
• Receptors, Steroid * MeSH