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Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3

  • Molecular Toxicology
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Abstract

Prediction of drug interactions, based on the induction of drug disposition, calls for the identification of chemicals, which activate xenosensing nuclear receptors. Constitutive androstane receptor (CAR) is one of the major human xenosensors; however, the constitutive activity of its reference variant CAR1 in immortalized cell lines complicates the identification of agonists. The exclusively ligand-dependent isoform CAR3 represents an obvious alternative for screening of CAR agonists. As CAR3 is even more abundant in human liver than CAR1, identification of its agonists is also of pharmacological value in its own right. We here established a cellular high-throughput screening assay for CAR3 to identify ligands of this isoform and to analyse its suitability for identifying CAR ligands in general. Proof-of-concept screening of 2054 drug-like compounds at 10 µM resulted in the identification of novel CAR3 agonists. The CAR3 assay proved to detect the previously described CAR1 ligands in the screened libraries. However, we failed to detect CAR3-selective compounds, as the four novel agonists, which were selected for further investigations, all proved to activate CAR1 in different cellular and in vitro assays. In primary human hepatocytes, the compounds preferentially induced the expression of the prototypical CAR target gene CYP2B6. Failure to identify CAR3-selective compounds was investigated by molecular modelling, which showed that the isoform-specific insertion of five amino acids did not impact on the ligand binding pocket but only on heterodimerization with retinoid X receptor. In conclusion, we demonstrate here the usability of CAR3 for screening compound libraries for the presence of CAR agonists.

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Acknowledgements

We greatly appreciate the expert technical assistance of K. Abuazi-Rincones. Primary human hepatocytes were kindly prepared by M. Demmel and colleagues in the Biobank. This study was supported by the Human Tissue and Cell Research (HTCR) Foundation, a non-profit foundation regulated by German civil law, which facilitates research with human tissue through the provision of an ethical and legal framework for sample collection and by the Robert Bosch Foundation, Stuttgart, Germany (M.S., O.B.). P. Pavek kindly provided the pEGFP-CAR1 + Ala plasmid.

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Correspondence to Björn Windshügel or Oliver Burk.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Keminer, O., Windshügel, B., Essmann, F. et al. Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3. Arch Toxicol 93, 2247–2264 (2019). https://doi.org/10.1007/s00204-019-02495-6

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  • DOI: https://doi.org/10.1007/s00204-019-02495-6

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