Characterization of xenobiotic substrates and inhibitors of CYP26A1, CYP26B1 and CYP26C1 using computational modeling and in vitro analyses Robert Foti To cite this version: Robert Foti. Characterization of xenobiotic substrates and inhibitors of CYP26A1, CYP26B1 and CYP26C1 using computational modeling and in vitro analyses. Agricultural sciences. Université Nice Sophia Antipolis, 2016. English. NNT : 2016NICE4033. tel-01376678 HAL Id: tel-01376678 https://tel.archives-ouvertes.fr/tel-01376678 Submitted on 5 Oct 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Université de Nice-Sophia Antipolis Thèse pour obtenir le grade de DOCTEUR DE L’UNIVERSITE NICE SOPHIA ANTIPOLIS Spécialité : Interactions Moléculaires et Cellulaires Ecole Doctorale : Sciences de la Vie et de la Santé (SVS) Caractérisation des substrats xénobiotiques et des inhibiteurs des cytochromes CYP26A1, CYP26B1 et CYP26C1 par modélisation moléculaire et études in vitro présentée et soutenue publiquement par Robert S. Foti Le 4 Juillet 2016 Membres du jury Dr. Danièle Werck-Reichhart Rapporteur Dr. Philippe Roche Rapporteur Pr. Serge Antonczak Examinateur Dr. Philippe Breton Examinateur Pr. Philippe Diaz Examinateur Dr. Dominique Douguet Directrice de thèse 1 1. Table of Contents 1. Table of Contents ............................................................................................................................... 2 2. Acknowledgements ............................................................................................................................ 5 3. Funding Sources ................................................................................................................................ 6 4. List of Tables ....................................................................................................................................... 7 5. List of Figures ..................................................................................................................................... 8 6. List of Abbreviations ......................................................................................................................... 11 7. Chapter I: Introduction to Retinoic Acid Signaling and Cytochrome P450 26 ........................ 13 7.1. Introduction................................................................................................................................ 14 7.2. Retinoic Acid Signaling. ........................................................................................................... 16 7.3. Cytochrome P450. ................................................................................................................... 18 7.4. Role of CYP26. ......................................................................................................................... 20 7.5. CYP26 Pharmacology. ............................................................................................................ 22 7.6. CYP26 Homology Modeling. .................................................................................................. 23 8. Aims and Scope. .............................................................................................................................. 27 9. Chapter II: Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1 ......................................................................... 30 9.1. Introduction................................................................................................................................ 31 9.2. Materials and Methods. ........................................................................................................... 34 9.2.1. Materials. ........................................................................................................................... 34 9.2.2. Sequence Verification and Expression of CYP26B1. ................................................. 34 9.2.3. IC50 Determination for Retinoic Acid Receptor Agonists. ........................................... 35 9.2.4. Homology Modeling. ........................................................................................................ 36 9.2.5. Metabolic Profiling. ........................................................................................................... 39 9.2.6. Enzyme Kinetics. .............................................................................................................. 39 9.2.7. Tazarotenic Acid Phenotyping. ...................................................................................... 40 9.2.8. LC-MS/MS Analysis. ........................................................................................................ 40 9.2.9. Data Analysis. ................................................................................................................... 41 9.3. Results. ...................................................................................................................................... 43 9.3.1. Homology Modeling. ........................................................................................................ 43 9.3.2. Metabolic Profile. .............................................................................................................. 50 9.3.3. In Vitro Enzyme Kinetics. ................................................................................................ 53 2 9.3.4. Tazarotenic Acid Phenotyping. ...................................................................................... 59 9.4. Discussion ................................................................................................................................. 61 10. Chapter III: Comparison of the Ligand Binding Site of CYP2C8 with CYP26A1 and CYP26B1: A Structural Basis for the Identification of New Inhibitors of the Retinoic Acid Hydroxylases............................................................................................................................................. 67 10.1. Introduction ............................................................................................................................ 68 10.2. Materials and Methods. ....................................................................................................... 71 10.2.1. Materials. ....................................................................................................................... 71 10.2.2. Homology Modeling and Computational Docking Simulations. ............................ 71 10.2.3. In Vitro Inhibition Assays. ............................................................................................ 72 10.2.4. Spectral Binding Determination. ................................................................................ 74 10.2.5. Assessment of In Vitro Free Fraction. ....................................................................... 75 10.2.6. In Vitro Stability of Candesartan Cilexetil. ................................................................ 75 10.2.7. Calculation of Cmax,u / IC50. ...................................................................................... 76 10.2.8. Liquid Chromatography – Mass Spectrometry Analysis. ....................................... 77 10.3. Results ................................................................................................................................... 78 10.3.1. Evaluation of tazarotenic acid sulfoxide formation as a probe substrate of CYP26. 78 10.3.2. In Vitro Inhibition Screening and IC50 Determination. ............................................. 80 10.3.3. Computational Docking Simulations.......................................................................... 87 10.3.4. Spectral Binding Studies. ............................................................................................ 89 10.3.5. Calculation of Cmax,u / IC50. ........................................................................................... 92 10.4. Discussion. ............................................................................................................................ 92 11. Chapter IV: Contribution of CYP26 to the Metabolism and Clearance of Retinoic Acid Receptor Agonists and Antagonists .................................................................................................... 101 11.1. Introduction. ......................................................................................................................... 102 11.2. Materials and Methods. ..................................................................................................... 104 11.2.1. Materials. ..................................................................................................................... 104 11.2.2. In Vitro Clearance of Retinoic Acid Receptor Agonists and Antagonists by Recombinant CYP26s, CYP2C8 and CYP3A4. ........................................................................ 104 11.2.3.