Dataset Information

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

ABSTRACT: The constitutive androstane receptor (CAR) plays an important role in xenobiotic metabolism, energy homeostasis, and cell proliferation. Antagonism of the CAR represents a key strategy for studying its function and may have potential clinical applications. However, specific human CAR (hCAR) antagonists are limited and conflicting data on the activity of these compounds have been reported. 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a typical peripheral benzodiazepine receptor ligand, has been established as a potent hCAR deactivator in immortalized cells; whether it inhibits hCAR activity under physiologically relevant conditions remains unclear. Here, we investigated the effects of PK11195 on hCAR in metabolically competent human primary hepatocytes (HPH) and HepaRG cells. We show that although PK11195 antagonizes hCAR in HepG2 cells, it induces the expression of CYP2B6 and CYP3A4, targets of hCAR and the pregnane X receptor (PXR), in HPH, HepaRG, and PXR-knockout HepaRG cells. Utilizing a HPH-HepG2 coculture model, we demonstrate that inclusion of HPH converts PK11195 from an antagonist to an agonist of hCAR, and such conversion was attenuated by potent CYP3A4 inhibitor ketoconazole. Metabolically, we show that the N-desmethyl metabolite is responsible for PK11195-mediated hCAR activation by facilitating hCAR interaction with coactivators and enhancing hCAR nuclear translocation in HPHs. Structure-activity analysis revealed that N-demethylation alters the interaction of PK11195 with the binding pocket of hCAR to favor activation. Together, these results indicate that removal of a methyl group switches PK11195 from a potent antagonist of hCAR to an agonist in HPH and highlights the importance of physiologically relevant metabolism when attempting to define the biologic action of small molecules.

SUBMITTER: Mackowiak B

PROVIDER: S-EPMC5452073 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

Mackowiak Bryan B Li Linhao L Welch Matthew A MA Li Daochuan D Jones Jace W JW Heyward Scott S Kane Maureen A MA Swaan Peter W PW Wang Hongbing H

Molecular pharmacology 20170425 1

The constitutive androstane receptor (CAR) plays an important role in xenobiotic metabolism, energy homeostasis, and cell proliferation. Antagonism of the CAR represents a key strategy for studying its function and may have potential clinical applications. However, specific human CAR (hCAR) antagonists are limited and conflicting data on the activity of these compounds have been reported. 1-(2-chlorophenyl)-<i>N</i>-methyl-<i>N</i>-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a typical ...[more]

PMID: 28442602

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Project description:IntroductionHepatic drug metabolism by cytochrome P450 enzymes is altered by the nutritional status of patients. The expression of P450 enzymes is partly regulated by the constitutive androstane receptor (CAR). Fasting regulates the expression of both P450 enzymes and CAR and affects hepatic drug clearance. We hypothesized that the fasting-induced alterations in P450 mediated drug clearance are mediated by CAR.MethodsTo investigate this we used a drug cocktail validated in humans consisting of five widely prescribed drugs as probes for specific P450 enzymes: caffeine (CYP1A2), metoprolol (CYP2D6), omeprazole (CYP2C19), midazolam (CYP3A4) and s-warfarin (CYP2C9). This cocktail was administered to wild type (WT, C57Bl/6) mice or mice deficient for CAR (CAR-/-) that were either fed ad libitum or fasted for 24 hours. Blood was sampled at predefined intervals and drug concentrations were measured as well as hepatic mRNA expression of homologous/orthologous P450 enzymes (Cyp1a2, Cyp2d22, Cyp3a11, Cyp2c37, Cyp2c38 and Cyp2c65).ResultsFasting decreased Cyp1a2 and Cyp2d22 expression and increased Cyp3a11 and Cyp2c38 expression in both WT and CAR-/- mice. The decrease in Cyp1a2 was diminished in CAR-/- in comparison with WT mice. Basal Cyp2c37 expression was lower in CAR-/- compared to WT mice. Fasting decreased the clearance of all drugs tested in both WT and CAR-/- mice. The absence of CAR was associated with an decrease in the clearance of omeprazole, metoprolol and midazolam in fed mice. The fasting-induced reduction in clearance of s-warfarin was greater in WT than in CAR-/-. The changes in drug clearance correlated with the expression pattern of the specific P450 enzymes in case of Cyp1a2-caffeine and Cyp2c37-omeprazole.ConclusionWe conclude that CAR is important for hepatic clearance of several widely prescribed drugs metabolized by P450 enzymes. However the fasting-induced alterations in P450 mediated drug clearance are largely independent of CAR.

Project description:Background & aimsThe constitutive androstane receptor (CAR) is a nuclear receptor that binds diverse xenobiotics and whose activation leads to the modulation of the expression of target genes involved in xenobiotic detoxification and energy metabolism. Although CAR hepatic activity is considered to be higher in women than in men, its sex-dependent response to an acute pharmacological activation has seldom been investigated.MethodsThe hepatic transcriptome, plasma markers, and hepatic metabolome, were analysed in Car+/+ and Car-/- male and female mice treated either with the CAR-specific agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or with vehicle.ResultsAlthough 90% of TCPOBOP-sensitive genes were modulated in a sex-independent manner, the remaining 10% showed almost exclusive female liver specificity. These female-specific CAR-sensitive genes were mainly involved in xenobiotic metabolism, inflammation, and extracellular matrix organisation. CAR activation also induced higher hepatic oxidative stress and hepatocyte cytolysis in females than in males. Hepatic expression of flavin monooxygenase 3 (Fmo3) was almost abolished and was associated with a decrease in hepatic trimethylamine-N-oxide (TMAO) concentration in TCPOBOP-treated females. In line with a potential role in the control of TMAO homeostasis, CAR activation decreased platelet hyper-responsiveness in female mice supplemented with dietary choline.ConclusionsMore than 10% of CAR-sensitive genes are sex-specific and influence hepatic and systemic responses such as platelet aggregation. CAR activation may be an important mechanism of sexually-dimorphic drug-induced liver injury.Impact and implicationsCAR is activated by many drugs and pollutants. Its pharmacological activation had a stronger impact on hepatic gene expression and metabolism in females than in males, and had a specific impact on liver toxicity and trimethylamine metabolism. Sexual dimorphism should be considered when testing and/or prescribing xenobiotics known to activate CAR.

Dataset Information

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

Publications

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

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