A cell-based NRG1-ERBB4 assay designed for high- throughput compound screening to identify small molecule modulators with relevance for schizophrenia Dissertation for the award of the degree ‘Doctor rerum naturalium’ of the Georg-August-Universität Göttingen submitted by Wilko Hinrichs from Norderney, Germany Göttingen, 30.09.2012 Prof. Klaus-Armin Nave Ph.D. (Reviewer) Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen Prof. Dr. Martin Göpfert (Reviewer) Department of Cellular Neurobiology, Schwann-Schleiden Research Center, Göttingen Prof. Dr. André Fischer (Reviewer) Laboratory for Aging and Cognitive Diseases, European Neuroscience Institute, Göttingen PD. Dr. Moritz Rossner Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen Prof. Dr. Thomas A. Bayer Department of Psychiatry, Division of Molecular Psychiatry, UKG Göttingen Dr. Judith Stegmüller Department of Cellular and Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen Declaration Herewith I declare that I prepared the PhD thesis entitled:‘A cell-based NRG1- ERBB4 assay designed for high-throughput compound screening to identify small molecule modulators with relevance for schizophrenia' on my own and with no other sources and aids than quoted. 2 “We are as forlorn as children lost in the woods. When you stand in front of me and look at me, what do you know of the griefs that are in me and what do I know of yours. And if I were to cast myself down before you and weep and tell you, what more would you know about me than you know about Hell when someone tells you it is hot and dreadful? For that reason alone we human beings ought to stand before one another as reverently, as reflectively, as lovingly, as we would before the entrance to Hell.” Franz Kafka 3 Acknowledgments First, I want to thank my supervisor PD. Dr. Moritz Rossner, for the great PhD project, his support, and all his brilliant ideas and good advice accumulated in this thesis. Equally, I want to thank my co-supervisor Dr. Michael Wehr, for his friendship, for his patience, for the great discussions, the help with the projects, especially screening and biochemistry, and the proof reading of my thesis. Dr. Sven Wichert for the help with the Hamilton robot, all bioinformatical questions, and help with all computer-related problems. Dr. Magdalena Brzozka for discussions, help with the corrections and the deeper insight into mouse behavioural studies. Dr. Elena Ciirdaeva for great support with cloning and supplies of DNA. All members of the Rossner group and the Department of Neurogenetics, for friendship, help in all lab problems concerning techniques, instruments, software, and discussion of projects. The members of the Lead Discovery Centre Dortmund, particularly Dr. Sascha Menninger for the great co-operation, and a deeper insight into HTP screening, lead discovery, and lead optimisation The GGNB-CMPB program, specifically Prof Michael Hörner, for the fruit-full discussions and seminar series and the GGNB staff for the help with the administrative challenges. I am grateful for my PhD committee, including Prof. Martin Göpfert , and Prof. André Fischer, and especially Prof. Klaus-Armin Nave for discussions, encouragements, and having me as PhD student in his lab. Further thanks are to my parents, without there love, support and encouragement this thesis would not be possible. Carolin, for her love and support in this part of my life. All friends, fencers, and tabletop-wargamers for the life abroad the PhD. 4 Table of Content Acknowledgments ............................................................................................ 4 1 Summary .................................................................................................. 12 2 Introduction ............................................................................................. 13 2.1 General characterisation of schizophrenia ................................................... 13 2.3 Positive symptoms ...................................................................................... 15 2.4 Negative symptoms ..................................................................................... 15 2.5 Cognitive deficits ......................................................................................... 15 2.6 Comorbidities .............................................................................................. 16 2.7 Residual symptoms ..................................................................................... 17 2.8 History of schizophrenia .............................................................................. 18 2.9 Morphologic findings in schizophrenia ......................................................... 19 2.10 Genetic studies ............................................................................................ 20 2.10.1 DISC1 .................................................................................................. 21 2.10.2 NRG1-ERBB4 ...................................................................................... 22 2.11 Environmental factors .................................................................................. 23 2.12 Two hit hypothesis ....................................................................................... 23 2.13 Findings in animal models of schizophrenia ................................................ 24 2.13.1 Dopamine hypothesis of schizophrenia ................................................ 24 2.14 NRG1-ERBB4 animal models ...................................................................... 25 2.15 NRG1-ERBB4: from proteins to network analysis ........................................ 28 2.15.1 Why addressing the NRG1-ERBB4 signalling system? ........................ 28 2.15.2 Protein-protein-interactions and NRG1-ERBB4 signalling .................... 28 2.15.3 Neuregulin1 ......................................................................................... 29 2.15.4 ERBB receptor tyrosine kinase family .................................................. 31 2.15.5 NRG1-ERBB4 signalling in the nervous system ................................... 35 2.15.6 Adapters of ERBB receptors ................................................................ 35 2.15.7 PI3K..................................................................................................... 36 2.15.8 STAT5 ............................................................................................... 37 2.15.9 SHC1 ................................................................................................... 37 2.15.10 GRB2 ................................................................................................... 37 2.15.11 SRC ..................................................................................................... 37 2.16 Downstream pathways ................................................................................ 38 5 2.16.1 PI3K/ AKT pathway .............................................................................. 38 2.16.2 MAPK /ERK pathway ........................................................................... 38 2.17 Localisation of NRG1-ERBB4 signalling in the neuronal architecture .......... 40 2.17.1 Glutamate hypothesis of SZ ................................................................. 40 2.17.2 NMDA .................................................................................................. 40 mGLu2/3 40 2.17.3 Hyper- and hypo-frontality in SZ .......................................................... 41 2.17.4 From neuronal circuits to single synapses ........................................... 41 2.17.5 Alterations found in the neuronal circuit ............................................... 42 2.17.6 NRG1-ERBB4 in the development of PV+ interneurons ....................... 43 2.18 Targeting NRG1-ERBB4 with protein-protein interaction assays ................. 45 2.18.1.1 First generation drugs ...................................................................... 45 2.18.1.2 Second generation drugs ................................................................. 45 2.18.2 Classic hit to lead discovery ................................................................. 46 2.18.2.1 Compound screens .......................................................................... 47 2.19 Cell based assays ....................................................................................... 47 2.19.1 Biochemical methods e.g. Co-IP .......................................................... 47 2.19.2 Affinity columns ................................................................................... 47 2.20 Protein fragment complementation assays .................................................. 48 2.20.1 Yeast two hybrid .................................................................................. 48 2.21 Split TEV ..................................................................................................... 48 2.22 z’-Factor ...................................................................................................... 49 2.22.1 The interpretation of the z’-factor: ........................................................ 49 2.23 Aim of the thesis .......................................................................................... 50 3 Chemicals and Reagents ........................................................................ 51 3.1 Laboratory suplies ......................................................................................