Investigating the molecular basis for the constitutive activity of the nuclear hormone receptor CAR [Elektronische Ressource] / von Björn Anselm Windshügel
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Investigating the molecular basis for the constitutive activity of the nuclear hormone receptor CAR [Elektronische Ressource] / von Björn Anselm Windshügel

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163 pages
English
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Investigating the Molecular Basis for theConstitutive Activity of the NuclearHormone Receptor CARDissertationzur Erlangung des akademischen Gradesdoctor rerum naturalium (Dr. rer. nat.)vorgelegt derMathematisch Naturwissenschaftlich Technischen Fakult at¨(mathematisch naturwissenschaftlicher Bereich)der Martin Luther Universit at¨ Halle Wittenbergvon Herrn Bjorn Anselm Windshugel¨ ¨geb. am 1. Januar 1976 in Bietigheim BissingenGutachter:1. Prof. Dr. Wolfgang Sippl2. PD Dr. Wolfgang Brandt3. Prof. Dr. Antti PosoHalle (Saale), den 19. Juli 2006urn:nbn:de:gbv:3-000010669[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000010669]AcknowledgementsThe present work was carried out at the Institute of Pharmaceutical Chemistry ofthe Heinrich Heine University Dusseldorf¨ from 2002 to 2003 and at the Instituteof Pharmaceutical Chemistry of the Martin Luther University Halle Wittenbergfrom 2003 to 2006. In summer 2003 and 2004 I had the great opportunity to stayat the and Medicinal Chemistry (PMC) group of Prof. Dr. AnttiPoso at the University of Kuopio, Finland.First of all, I’d like to thank my supervisor Prof. Dr. Wolfgang Sippl for givingme the opportunity to join his group and to get in touch with the fascinatingworld of nuclear receptors. I’d like to thank him for the support and the possi bility for a stay abroad during my work.The support by Dr. Paavo Honkakoski and his group is greatly acknowl edged.

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Publié le 01 janvier 2006
Nombre de lectures 22
Langue English
Poids de l'ouvrage 5 Mo

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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 Fakult at¨
(mathematisch naturwissenschaftlicher Bereich)
der Martin Luther Universit at¨ Halle Wittenberg
von Herrn Bjorn Anselm Windshugel¨ ¨
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:3-000010669
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000010669]Acknowledgements
The present work was carried out at the Institute of Pharmaceutical Chemistry of
the Heinrich Heine University Dusseldorf¨ from 2002 to 2003 and at the Institute
of Pharmaceutical Chemistry of the Martin Luther University Halle Wittenberg
from 2003 to 2006. In summer 2003 and 2004 I had the great opportunity to stay
at the 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 possi
bility for a stay abroad during my work.
The support by Dr. Paavo Honkakoski and his group is greatly acknowl
edged. The comprehensive biological data from him and his co workers Jo
hanna Jyrkkarinne¨ 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 es
pecially 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.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.List of Original Publications
This doctoral dissertation is based on the following publications, referred to in
the text as Roman numerals I III.
I Bjorn¨ Windshugel,¨ Johanna Jyrkkarinne,¨ Antti Poso, Paavo
Honkakoski and Wolfgang Sippl
Molecular dynamics simulations of the human CAR ligand
binding domain: deciphering the molecular basis for con
stitutive activity
J. Mol. Model., 11:69 79 (2005)
II Johanna Jyrkkarinne,¨ Bjorn¨ Windshugel,¨ Janne Makinen,¨
Markku Ylisirnio,¨ Mikael Perakyl¨ a,¨ Antti Poso, Wolfgang
Sippl and Paavo Honkakoski
Amino acids important for ligand specificity of the human
constitutive androstane receptor
J. Biol. Chem., 280:5960 5971 (2005)
III Bjorn¨ Windshugel,¨ Johanna Jyrkkarinne,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 pressContents
1 Introduction 1
1.1 Nuclear Hormone Receptors . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 General Introduction . . . . . . . . . . . . . . . . . . . . . . 1
1.1.2 Signaltransduction . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.3 Structural Organisation . . . . . . . . . . . . . . . . . . . . 4
1.1.3.1 N Terminal Domain . . . . . . . . . . . . . . . . . 4
1.1.3.2 DNA Binding . . . . . . . . . . . . . . . 5
1.1.3.3 Hinge Region . . . . . . . . . . . . . . . . . . . . . 6
1.1.3.4 Ligand Binding Domain . . . . . . . . . . . . . . 6
1.2 The Subfamily NR1I . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2.1 Vitamin D Receptor . . . . . . . . . . . . . . . . . . . . . . 8
1.2.2 Pregnane X . . . . . . . . . . . . . . . . . . . . . . 8
1.2.3 Constitutive Androstane Receptor . . . . . . . . . . . . . . 10
1.2.3.1 Signal Transduction . . . . . . . . . . . . . . . . . 10
1.2.3.2 CAR Ligands . . . . . . . . . . . . . . . . . . . . . 12
1.2.3.3 Regulation of Drug Metabolism . . . . . . . . . . 13
1.2.3.4 Role in Bilirubin Clearance . . . . . . . . . . . . . 14
1.2.3.5 CAR and Energy Metabolism . . . . . . . . . . . 16
1.2.3.6 Adverse Eects . . . . . . . . . . . . . . . . . . . . 17
1.2.3.7 Therapeutic Potential . . . . . . . . . . . . . . . . 18
1.3 Aim of the Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2 Computational Methods 21
2.1 Homology Modelling . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.1 Template Selection . . . . . . . . . . . . . . . . . . . . . . . 22
2.1.2 Sequence Structure Alignment . . . . . . . . . . . . . . . . 23
iCONTENTS ii
2.1.3 Assignment of Side Chains . . . . . . . . . . . . . . . . . . 23
2.2 Force Field Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2.1 Energy Minimisation . . . . . . . . . . . . . . . . . . . . . . 27
2.2.2 Molecular Dynamics Simulations . . . . . . . . . . . . . . . 28
2.3 Molecular Interaction Fields . . . . . . . . . . . . . . . . . . . . . . 29
2.4 Docking . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.4.1 Scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.5 Virtual Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.6 Homology Model Evaluation . . . . . . . . . . . . . . . . . . . . . 33
3 Generation of CAR Homology Models 35
3.1 Homology Modelling (I, II) . . . . . . . . . . . . . . . . . . . . . . 35
3.1.1 CAR Model (I) . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.1.2 CAR/SRC 1 Model (I, II) . . . . . . . . . . . . . . . . . . . . 37
3.1.3 CAR/NCoR Model (II) . . . . . . . . . . . . . . . . . . . . . 38
3.2 Model Refinement (I, II, III) . . . . . . . . . . . . . . . . . . . . . . 38
3.3 Molecular Docking (I, II, III) . . . . . . . . . . . . . . . . . . . . . . 39
4 The Mechanism of Constitutive Activity (I) 40
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.1 Homology Modelling . . . . . . . . . . . . . . . . . . . . . 43
4.2.2 Constitutive Activity . . . . . . . . . . . . . . . . . . . . . . 45
4.2.3 Co Activator Binding . . . . . . . . . . . . . . . . . . . . . 49
4.2.4 Docking Studies . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2.5 Mutagenesis Studies . . . . . . . . . . . . . . . . . . . . . . 53
4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5 The Ligand Specificity of Human CAR (II) 60
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.2.1 Modulation of Human CAR Activity . . . . . . . . . . . . 63
5.2.2 Homology Models of Human CAR . . . . . . . . . . . . . . 65
5.2.3 Basal Activities of Human CAR Mutants . . . . . . . . . . 68
5.2.4 Docking and MD Simulation of Ligand Binding . . . . . . 71CONTENTS iii
5.2.5 Ligand Specificities of Human CAR Mutants . . . . . . . . 75
5.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
5.3.1 Factors Contributing to Basal Activity of CAR . . . . . . . 79
5.3.2 Ligand Specificity of Human CAR . . . . . . . . . . . . . . 83
6 Homology Model Evaluation (III) 85
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
6.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.2.1 Quality of the Homology Model . . . . . . . . . . . . . . . 89
6.2.2 Reproducing Ligand Binding Modes . . . . . . . . . . . . . 95
6.2.3 The Basis for Constitutive Activity . . . . . . . . . . . . . . 97
6.2.4 The Role of Helix X . . . . . . . . . . . . . . . . . . . . . . . 100
6.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
6.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7 Virtual Screening 108
7.1 3D Database Search . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.2 Molecular Docking . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

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