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.

1st Faculty of Medicine Charles University Katerinska 32 112 08 Prague Czech Republic

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Department Food Safety German Federal Institute for Risk Assessment Max Dohrn Str 8 10 Berlin 10589 Germany

Department of Pharmacology and Toxicology Faculty of Pharmacy in Hradec Kralove Charles University Akademika Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

DZIF Tübingen Partner Site University Hospital Tübingen 72076 Tuebingen Germany

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nám 2 166 10 Prague 6 Czech Republic

NMI Natural and Medical Sciences Institute at the University of Tuebingen Markwiesenstr 55 72770 Reutlingen Germany

See more in PubMed

Kamaraj R., Pavek P.. Novel antagonists reveal the mechanism of PXR inhibition. Trends Pharmacol. Sci. 2024;45(11):961–963. doi: 10.1016/j.tips.2024.10.002. PubMed DOI

Karpale M., Hukkanen J., Hakkola J.. Nuclear receptor PXR in drug-induced hypercholesterolemia. Cells. 2022;11(3):313. doi: 10.3390/cells11030313. PubMed DOI PMC

Karpale M., Kummu O., Kärkkäinen O., Lehtonen M., Näpänkangas J., Herfurth U. M., Braeuning A., Rysä J., Hakkola J.. Pregnane X receptor activation remodels glucose metabolism to promote NAFLD development in obese mice. Mol. Metab. 2023;76:101779. doi: 10.1016/j.molmet.2023.101779. PubMed DOI PMC

Chai S. C., Wright W. C., Chen T.. Strategies for developing pregnane X receptor antagonists: Implications from metabolism to cancer. Med. Res. Rev. 2020;40(3):1061–1083. doi: 10.1002/med.21648. PubMed DOI PMC

Delfosse V., Huet T., Harrus D., Granell M., Bourguet M., Gardia-Parege C., Chiavarina B., Grimaldi M., Le Mevel S., Blanc P.. et al. Mechanistic insights into the synergistic activation of the RXR-PXR heterodimer by endocrine disruptor mixtures. Proc. Natl. Acad. Sci. U.S.A. 2021;118(1):e2020551118. doi: 10.1073/pnas.2020551118. PubMed DOI PMC

Hall A., Chanteux H., Ménochet K., Ledecq M., Schulze M.-S. E. D.. Designing Out PXR Activity on Drug Discovery Projects: A Review of Structure-Based Methods, Empirical and Computational Approaches. J. Med. Chem. 2021;64(10):6413–6522. doi: 10.1021/acs.jmedchem.0c02245. PubMed DOI

Lin W., Huber A. D., Poudel S., Li Y., Seetharaman J., Miller D. J., Chen T.. Structure-guided approach to modulate small molecule binding to a promiscuous ligand-activated protein. Proc. Natl. Acad. Sci. U.S.A. 2023;120(10):e2217804120. doi: 10.1073/pnas.2217804120. PubMed DOI PMC

Kamaraj R., Drastik M., Maixnerova J., Pavek P.. Allosteric Antagonism of the Pregnane X Receptor (PXR): Current-State-of-the-Art and Prediction of Novel Allosteric Sites. Cells. 2022;11(19):2974. doi: 10.3390/cells11192974. PubMed DOI PMC

Lin W., Goktug A. N., Wu J., Currier D. G., Chen T.. High-Throughput Screening Identifies 1,4,5-Substituted 1,2,3-Triazole Analogs as Potent and Specific Antagonists of Pregnane X Receptor. Assay Drug Dev. Technol. 2017;15(8):383–394. doi: 10.1089/adt.2017.809. PubMed DOI PMC

Mustonen E.-K., Pantsar T., Rashidian A., Reiner J., Schwab M., Laufer S., Burk O.. Target Hopping from Protein Kinases to PXR: Identification of Small-Molecule Protein Kinase Inhibitors as Selective Modulators of Pregnane X Receptor from TüKIC Library. Cells. 2022;11(8):1299. doi: 10.3390/cells11081299. PubMed DOI PMC

Lin W., Wang Y.-M., Chai S. C., Lv L., Zheng J., Wu J., Zhang Q., Wang Y.-D., Griffin P. R., Chen T.. SPA70 is a potent antagonist of human pregnane X receptor. Nat. Commun. 2017;8(1):741. doi: 10.1038/s41467-017-00780-5. PubMed DOI PMC

Garcia-Maldonado E., Huber A. D., Chai S. C., Nithianantham S., Li Y., Wu J., Poudel S., Miller D. J., Seetharaman J., Chen T.. Chemical manipulation of an activation/inhibition switch in the nuclear receptor PXR. Nat. Commun. 2024;15(1):4054. doi: 10.1038/s41467-024-48472-1. PubMed DOI PMC

Li Y., Lin W., Chai S. C., Wu J., Annu K., Chen T.. Design and Optimization of 1H-1,2,3-Triazole-4-carboxamides as Novel, Potent, and Selective Inverse Agonists and Antagonists of PXR. J. Med. Chem. 2022;65(24):16829–16859. doi: 10.1021/acs.jmedchem.2c01640. PubMed DOI PMC

Huber A. D., Wright W. C., Lin W., Majumder K., Low J. A., Wu J., Buchman C. D., Pintel D. J., Chen T.. Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning. Cell. Mol. Life Sci. 2021;78(1):317–335. doi: 10.1007/s00018-020-03505-y. PubMed DOI PMC

Li Y., Lin W., Wright W. C., Chai S. C., Wu J., Chen T.. Building a Chemical Toolbox for Human Pregnane X Receptor Research: Discovery of Agonists, Inverse Agonists, and Antagonists Among Analogs Based on the Unique Chemical Scaffold of SPA70. J. Med. Chem. 2021;64(3):1733–1761. doi: 10.1021/acs.jmedchem.0c02201. PubMed DOI PMC

Huang H., Wang H., Sinz M., Zoeckler M., Staudinger J., Redinbo M. R., Teotico D. G., Locker J., Kalpana G. V., Mani S.. Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene. 2007;26(2):258–268. doi: 10.1038/sj.onc.1209788. PubMed DOI

Burk O., Kuzikov M., Kronenberger T., Jeske J., Keminer O., Thasler W. E., Schwab M., Wrenger C., Windshügel B.. Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles. Arch. Toxicol. 2018;92(4):1435–1451. doi: 10.1007/s00204-018-2165-4. PubMed DOI

Biswas A., Mani S., Redinbo M. R., Krasowski M. D., Li H., Ekins S.. Elucidating the ‘Jekyll and Hyde’ Nature of PXR: The Case for Discovering Antagonists or Allosteric Antagonists. Pharm. Res. 2009;26(8):1807–1815. doi: 10.1007/s11095-009-9901-7. PubMed DOI PMC

Huber A. D., Jung Y.-H., Li Y., Lin W., Wu J., Poudel S., Carrigan A. G., Mishra A., High A. A., Chen T.. First-in-Class Small Molecule Degrader of Pregnane X Receptor Enhances Chemotherapy Efficacy. J. Med. Chem. 2024;67(20):18549–18575. doi: 10.1021/acs.jmedchem.4c01926. PubMed DOI PMC

Huber A. D., Li Y., Lin W., Galbraith A. N., Mishra A., Porter S. N., Wu J., Florke Gee R. R., Zhuang W., Pruett-Miller S. M.. et al. SJPYT-195: A Designed Nuclear Receptor Degrader That Functions as a Molecular Glue Degrader of GSPT1. ACS Med. Chem. Lett. 2022;13(8):1311–1320. doi: 10.1021/acsmedchemlett.2c00223. PubMed DOI PMC

Bansard, L. ; Laconde, G. ; Delfosse, V. ; Huet, T. ; Ayeul, M. ; Rigal, E. ; Donati, Q. ; Gerbal-Chaloin, S. ; Daujat-Chavanieu, M. ; Legrand, B. . et al. A potent agonist-based PROTAC targeting Pregnane X Receptor that delays colon cancer relapse. bioRxiv, 2024. 10.1101/2024.06.18.599474. DOI

Mani S., Dou W., Redinbo M. R.. PXR antagonists and implication in drug metabolism. Drug Metab. Rev. 2013;45(1):60–72. doi: 10.3109/03602532.2012.746363. PubMed DOI PMC

Pascussi, J. M. ; Pannequin, J. ; Amblard-Caussil, M. ; Laconde, G. ; Gerbal-Chaloin, S. ; Chavanieu, A. ; Bourguet, W. ; Delfosse, V. . Bifunctional PROTAC-type compounds targeting PXR, method for preparing same, and therapeutic use thereof. WO2022243365, 2021.

Florke Gee R. R., Huber A. D., Wu J., Bajpai R., Loughran A. J., Pruett-Miller S. M., Chen T.. The F-box-only protein 44 regulates pregnane X receptor protein level by ubiquitination and degradation. Acta Pharm. Sin. B. 2023;13(11):4523–4534. doi: 10.1016/j.apsb.2023.07.014. PubMed DOI PMC

Mejdrová I., Dušek J., Škach K., Stefela A., Skoda J., Chalupský K., Dohnalová K., Pavkova I., Kronenberger T., Rashidian A.. et al. Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo­[1,2-a]­pyridine Structure. J. Med. Chem. 2023;66(4):2422–2456. doi: 10.1021/acs.jmedchem.2c01140. PubMed DOI PMC

Dusek J., Mejdrova I., Dohnalova K., Smutny T., Chalupsky K., Krutakova M., Skoda J., Rashidian A., Pavkova I., Skach K.. et al. The hypolipidemic effect of MI-883, the combined CAR agonist/ PXR antagonist, in diet-induced hypercholesterolemia model. Nat. Commun. 2025;16(1):1418. doi: 10.1038/s41467-025-56642-y. PubMed DOI PMC

Rashidian A., Mustonen E.-K., Kronenberger T., Schwab M., Burk O., Laufer S. A., Pantsar T.. Discrepancy in interactions and conformational dynamics of pregnane X receptor (PXR) bound to an agonist and a novel competitive antagonist. Comput. Struct. Biotechnol. J. 2022;20:3004–3018. doi: 10.1016/j.csbj.2022.06.020. PubMed DOI PMC

Lagorce D., Bouslama L., Becot J., Miteva M. A., Villoutreix B. O.. FAF-Drugs4: free ADME-tox filtering computations for chemical biology and early stages drug discovery. Bioinformatics. 2017;33(22):3658–3660. doi: 10.1093/bioinformatics/btx491. PubMed DOI

Lomize A. L., Hage J. M., Schnitzer K., Golobokov K., LaFaive M. B., Forsyth A. C., Pogozheva I. D.. PerMM: A Web Tool and Database for Analysis of Passive Membrane Permeability and Translocation Pathways of Bioactive Molecules. J. Chem. Inf. Model. 2019;59(7):3094–3099. doi: 10.1021/acs.jcim.9b00225. PubMed DOI PMC

Tang Z., Kang B., Li C., Chen T., Zhang Z.. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res. 2019;47(W1):W556–W560. doi: 10.1093/nar/gkz430. PubMed DOI PMC

Cao C., He M., Wang L., He Y., Rao Y.. Chemistries of bifunctional PROTAC degraders. Chem. Soc. Rev. 2022;51(16):7066–7114. doi: 10.1039/D2CS00220E. 10.1039/D2CS00220E. PubMed DOI

Szulc N. A., Stefaniak F., Piechota M., Soszyńska A., Piórkowska G., Cappannini A., Bujnicki J. M., Maniaci C., Pokrzywa W.. DEGRONOPEDIA: a web server for proteome-wide inspection of degrons. Nucleic Acids Res. 2024;52(W1):W221–W232. doi: 10.1093/nar/gkae238. PubMed DOI PMC

Honorato R. V., Trellet M. E., Jiménez-García B., Schaarschmidt J. J., Giulini M., Reys V., Koukos P. I., Rodrigues J. P. G. L. M., Karaca E., van Zundert G. C. P.. et al. The HADDOCK2.4 web server for integrative modeling of biomolecular complexes. Nat. Protoc. 2024;19(11):3219–3241. doi: 10.1038/s41596-024-01011-0. PubMed DOI

Zaidman D., Prilusky J., London N.. PRosettaC: Rosetta Based Modeling of PROTAC Mediated Ternary Complexes. J. Chem. Inf. Model. 2020;60(10):4894–4903. doi: 10.1021/acs.jcim.0c00589. PubMed DOI PMC

Laskowski R. A., Swindells M. B.. LigPlot+: Multiple Ligand–Protein Interaction Diagrams for Drug Discovery. J. Chem. Inf. Model. 2011;51(10):2778–2786. doi: 10.1021/ci200227u. PubMed DOI

Kamaraj R., Ghosh S., Das S., Sen S., Kumar P., Majumdar M., Dasgupta R., Mukherjee S., Das S., Ghose I.. et al. Targeted Protein Degradation (TPD) for Immunotherapy: Understanding Proteolysis Targeting Chimera-Driven Ubiquitin-Proteasome Interactions. Bioconjugate Chem. 2024;35(8):1089–1115. doi: 10.1021/acs.bioconjchem.4c00253. PubMed DOI PMC

Nguyen T. M., Sreekanth V., Deb A., Kokkonda P., Tiwari P. K., Donovan K. A., Shoba V., Chaudhary S. K., Mercer J. A. M., Lai S.. et al. Proteolysis-targeting chimeras with reduced off-targets. Nat. Chem. 2024;16(2):218–228. doi: 10.1038/s41557-023-01379-8. PubMed DOI PMC

Nowak R. P., DeAngelo S. L., Buckley D., He Z., Donovan K. A., An J., Safaee N., Jedrychowski M. P., Ponthier C. M., Ishoey M.. et al. Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat. Chem. Biol. 2018;14(7):706–714. doi: 10.1038/s41589-018-0055-y. PubMed DOI PMC

Treindl F., Ruprecht B., Beiter Y., Schultz S., Döttinger A., Staebler A., Joos T. O., Kling S., Poetz O., Fehm T.. et al. A bead-based western for high-throughput cellular signal transduction analyses. Nat. Commun. 2016;7(1):12852. doi: 10.1038/ncomms12852. PubMed DOI PMC

Bwayi M. N., Garcia-Maldonado E., Chai S. C., Xie B., Chodankar S., Huber A. D., Wu J., Annu K., Wright W. C., Lee H.-M.. et al. Molecular basis of crosstalk in nuclear receptors: heterodimerization between PXR and CAR and the implication in gene regulation. Nucleic Acids Res. 2022;50(6):3254–3275. doi: 10.1093/nar/gkac133. PubMed DOI PMC

Squires E. J., Sueyoshi T., Negishi M.. Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver. J. Biol. Chem. 2004;279(47):49307–49314. doi: 10.1074/jbc.M407281200. PubMed DOI

Poudel S., Huber A. D., Chen T.. Regulation of Nuclear Receptors PXR and CAR by Small Molecules and Signal Crosstalk: Roles in Drug Metabolism and Beyond. Drug Metab. Dispos. 2023;51(2):228–236. doi: 10.1124/dmd.122.000858. PubMed DOI PMC

Motta S., Callea L., Giani Tagliabue S., Bonati L.. Exploring the PXR ligand binding mechanism with advanced Molecular Dynamics methods. Sci. Rep. 2018;8(1):16207. doi: 10.1038/s41598-018-34373-z. PubMed DOI PMC

Smutny T., Smutna L., Lochman L., Kamaraj R., Kucera R., Pavek P.. Rifampicin and its derivatives: stability, disposition, and affinity towards pregnane X receptor employing 2D and 3D primary human hepatocytes. Biochem. Pharmacol. 2024;229:116500. doi: 10.1016/j.bcp.2024.116500. PubMed DOI

Krausova L., Stejskalova L., Wang H., Vrzal R., Dvorak Z., Mani S., Pavek P.. Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene. Biochem. Pharmacol. 2011;82(11):1771–1780. doi: 10.1016/j.bcp.2011.08.023. PubMed DOI PMC

Donovan K. A., An J., Nowak R. P., Yuan J. C., Fink E. C., Berry B. C., Ebert B. L., Fischer E. S.. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. eLife. 2018;7:e38430. doi: 10.7554/eLife.38430. PubMed DOI PMC

Meier F., Brunner A.-D., Frank M., Ha A., Bludau I., Voytik E., Kaspar-Schoenefeld S., Lubeck M., Raether O., Bache N.. et al. diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition. Nat. Methods. 2020;17(12):1229–1236. doi: 10.1038/s41592-020-00998-0. PubMed DOI

Demichev V., Messner C. B., Vernardis S. I., Lilley K. S., Ralser M.. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods. 2020;17(1):41–44. doi: 10.1038/s41592-019-0638-x. PubMed DOI PMC

Ritchie M. E., Phipson B., Wu D., Hu Y., Law C. W., Shi W., Smyth G. K.. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. doi: 10.1093/nar/gkv007. (acccessed 12/16/2024) PubMed DOI PMC

Khavrutskii L., Yeh J., Timofeeva O., Tarasov S. G., Pritt S., Stefanisko K., Tarasova N.. Protein Purification-libera Metodo di Binding Affinity Determinazione Thermophoresis microscala. J. Visualized Exp. 2013;15(78):e50541. doi: 10.3791/50541. PubMed DOI PMC

Friesner R. A., Murphy R. B., Repasky M. P., Frye L. L., Greenwood J. R., Halgren T. A., Sanschagrin P. C., Mainz D. T.. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein–Ligand Complexes. J. Med. Chem. 2006;49(21):6177–6196. doi: 10.1021/jm051256o. PubMed DOI

Kamaraj, R. ; Nencka, R. ; Pavek, P. . Chapter One - Computational strategies for the design of proteolysis targeting chimera degraders: Artificial intelligence enabled PROTAC design. In Annu. Rep. Med. Chem.; Nencka, R. , Ed.; Academic Press, 2024; Vol. 63, pp 1–37.

Yan Y., Tao H., He J., Huang S.-Y.. The HDOCK server for integrated protein–protein docking. Nat. Protoc. 2020;15(5):1829–1852. doi: 10.1038/s41596-020-0312-x. PubMed DOI

Smutny T., Dusek J., Hyrsova L., Nekvindova J., Horvatova A., Micuda S., Gerbal-Chaloin S., Pavek P.. The 3′-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids. Acta Pharm. Sin. B. 2020;10(1):136–152. doi: 10.1016/j.apsb.2019.09.010. PubMed DOI PMC

Saeed A. I., Sharov V., White J., Li J., Liang W., Bhagabati N., Braisted J., Klapa M., Currier T., Thiagarajan M.. et al. TM4: A Free, Open-Source System for Microarray Data Management and Analysis. BioTechniques. 2003;34(2):374–378. doi: 10.2144/03342mt01. PubMed DOI

Tang D., Chen M., Huang X., Zhang G., Zeng L., Zhang G., Wu S., Wang Y.. SRplot: A free online platform for data visualization and graphing. PLoS One. 2023;18(11):e0294236. doi: 10.1371/journal.pone.0294236. PubMed DOI PMC