Investigating the Molecular Basis for the Constitutive Activity of the Nuclear Hormone Receptor CAR

Transcription

1 Investigating the Molecular Basis for the Constitutive Activity of the Nuclear Hormone Receptor CAR Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Mathematisch-Naturwissenschaftlich-Technischen Fakultät (mathematisch-naturwissenschaftlicher Bereich) der Martin-Luther-Universität Halle-Wittenberg von Herrn Björn Anselm Windshügel geb. am 1. Januar 1976 in Bietigheim-Bissingen Gutachter: 1. Prof. Dr. Wolfgang Sippl 2. PD Dr. Wolfgang Brandt 3. Prof. Dr. Antti Poso Halle (Saale), den 19. Juli 2006 urn:nbn:de:gbv: [

2 Acknowledgements The present work was carried out at the Institute of Pharmaceutical Chemistry of the Heinrich-Heine University Düsseldorf from 2002 to 2003 and at the Institute of Pharmaceutical Chemistry of the Martin-Luther-University Halle-Wittenberg from 2003 to In summer 2003 and 2004 I had the great opportunity to stay at the Pharmaceutical and Medicinal Chemistry (PMC) group of Prof. Dr. Antti Poso at the University of Kuopio, Finland. First of all, I d like to thank my supervisor Prof. Dr. Wolfgang Sippl for giving me the opportunity to join his group and to get in touch with the fascinating world of nuclear receptors. I d like to thank him for the support and the possibility for a stay abroad during my work. The support by Dr. Paavo Honkakoski and his group is greatly acknowledged. The comprehensive biological data from him and his co-workers Johanna Jyrkkärinne and Jenni Vanamo have contributed a lot to this work and provided the experimental basis for the hypothesis derived from my theoretical models. We had and still have a very fruitful cooperation that is going to reveal further secrets of CAR and even other nuclear receptors. I love to thank Prof. Dr. Antti Poso and his group for the very good cooperation and the great support during my stays in Kuopio. I really enjoyed it to work in the PMC group and I was happy to have the possibility to come back. I d like to thank Birgit Schlegel for her corrections and comments on the manuscripts making them much more understandable. Special thanks go to the other members of the Medicinal Chemistry group in Halle for the good working atmosphere and the helpful discussions I had especially with Sonja. I owe thanks to Rene for all the system administration and help with any kinds of computer problems. Also greatly acknowledged is the contribution of my family to this work by both, personal and financial support. Without you many things wouldn t have been possible.

3 I want to express my gratitude to the members of the groups of Prof. Dr. Langner and private lecturer Dr. Hilgeroth for the help at the beginning of my work in Halle and for the friendship. As the first contact persons for me you largely contributed that I could settle in quite easy and immediatly felt like home. I will definitely miss our BANG! sessions after lunch. Finally, I d like to thank Christine for her love and encouragement during the last year.

4 List of Original Publications This doctoral dissertation is based on the following publications, referred to in the text as Roman numerals I-III. I II Björn Windshügel, Johanna Jyrkkärinne, Antti Poso, Paavo Honkakoski and Wolfgang Sippl Molecular dynamics simulations of the human CAR ligandbinding domain: deciphering the molecular basis for constitutive activity J. Mol. Model., 11:69-79 (2005) Johanna Jyrkkärinne, Björn Windshügel, Janne Mäkinen, Markku Ylisirniö, Mikael Peräkylä, Antti Poso, Wolfgang Sippl and Paavo Honkakoski Amino acids important for ligand specificity of the human constitutive androstane receptor J. Biol. Chem., 280: (2005) III Björn Windshügel, Johanna Jyrkkärinne,Jenni Vanamo, Antti Poso, Paavo Honkakoski and Wolfgang Sippl Comparison of homology models and X-ray structures of the nuclear receptor CAR: Assessing the structural basis of constitutive activity J. Mol. Graph. Model., in press

5 Contents 1 Introduction Nuclear Hormone Receptors General Introduction Signaltransduction Structural Organisation N-Terminal Domain DNA Binding Domain Hinge Region Ligand Binding Domain The Subfamily NR1I Vitamin D Receptor Pregnane X Receptor Constitutive Androstane Receptor Signal Transduction CAR Ligands Regulation of Drug Metabolism Role in Bilirubin Clearance CAR and Energy Metabolism Adverse Effects Therapeutic Potential Aim of the Work Computational Methods Homology Modelling Template Selection Sequence-Structure Alignment i

6 CONTENTS ii Assignment of Side Chains Force Field Methods Energy Minimisation Molecular Dynamics Simulations Molecular Interaction Fields Molecular Docking Scoring Virtual Screening Homology Model Evaluation Generation of CAR Homology Models Homology Modelling (I, II) CAR Model (I) CAR/SRC-1 Model (I, II) CAR/NCoR Model (II) Model Refinement (I, II, III) Molecular Docking (I, II, III) The Mechanism of Constitutive Activity (I) Introduction Results Homology Modelling Constitutive Activity Co-Activator Binding Docking Studies Mutagenesis Studies Discussion Conclusions The Ligand Specificity of Human CAR (II) Introduction Results Modulation of Human CAR Activity Homology Models of Human CAR Basal Activities of Human CAR Mutants Docking and MD Simulation of Ligand Binding

7 CONTENTS iii Ligand Specificities of Human CAR Mutants Discussion Factors Contributing to Basal Activity of CAR Ligand Specificity of Human CAR Homology Model Evaluation (III) Introduction Results Quality of the Homology Model Reproducing Ligand Binding Modes The Basis for Constitutive Activity The Role of Helix X Discussion Conclusions Virtual Screening D Database Search Molecular Docking Re-Docking into X-ray Structures Results Discussion Conclusions & Outlook Summary 117 A Abbreviations and Units 150 A.1 Abbreviations A.2 Units B Amino acids 153