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Functional and Molecular Characterization of Hedgehog Signalling Regulation Functional and Molecular Characterization of Hedgehog Signalling Regulation by Celine Lacroix A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto © Copyright by Celine Lacroix 2016 Functional and Molecular Characterization of Hedgehog Signalling Regulation Celine Lacroix Doctor of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto 2016 Abstract Hedgehog (Hh) signalling plays a key role in controlling cell fate and patterning of tissues during development and also throughout adulthood for tissue homeostasis. The physiological importance of this pathway is further highlighted when mutations in Hh pathway components lead to developmental disorders and diseases: defects of the limbs, cranial structure or nervous system, and cancer in adults. Although the pathway is highly conserved between flies and humans, important divergences exist between them, including the necessity for Sufu to negatively regulate the Gli transcription factors, thought to mediate all Hedgehog transcriptional outputs. The first study presented in this thesis describes Zfp629, a new transcription factor first identified as a novel interactor of Sufu through a proteomics study. ChIP-seq analysis and validation confirmed that Zfp629 controlled the expression of Foxp2. Also a member of this pathway is Smoothened (Smo), a class F G-protein coupled receptor (GPCR). Several inhibitors have already been introduced to treat Hh-driven cancers such as medulloblastoma and basal cell carcinoma. However, resistance to these inhibitors rapidly develops thereby limiting their efficacy. The determination of SMO crystal structures enables structure-based discovery of new ligands with new chemotypes that will be critical to combat resistance. In the second study of this thesis, we docked 3.2 million available, lead-like molecules against SMO, looking for those with high physical complementarity to its structure; this represents the first such campaign ii against a class F GPCR. Twenty-one high-ranking compounds were selected for experimental testing, and four, representing three different chemotypes, were identified to antagonize SMO with IC50 values better than 50 µM. A second screen for analogs revealed another six molecules, with IC50 values in the 2.3 µM-22.4 µM range. Importantly, one of the most active of the new Smo antagonists continued to be efficacious against the D473H mutant of SMO, which confers clinical resistance to the antagonist vismodegib in cancer treatment. iii Acknowledgments The completion of this thesis was made possible by the support, guidance and contributions of several people. I would like to thank Dr. Carolyn Cummins, Dr. Chi-Chung Hui and Dr. Jason Matthews, my committee members, your insightful questions and discussions and your assistance allowed for this thesis to come to completion. To my supervisor, Dr. Stephane Angers, thank you for the opportunity, your guidance and for constantly challenging me. Thank you to the Angers lab, past and current members, for your suggestions, discussions and ideas. To Dr. Brian Shoichet and Inbar Fish, thank you for your patience with my relentless questioning and for your kindness. I would like to express my gratitude to the Faculty of Pharmacy and Graduate Department of Pharmaceutical Sciences for their support. Lastly, to my colleagues and friends: I could not have done this without your advice, friendship and encouragements. I have found in you several smart scientists, many more kind hearts and an amazing second family. I would like to thank my family and my partner for always being there for me, milles merci for helping me put things in perspective. iv Table of Contents List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Appendices ........................................................................................................................ xii List of Abbreviations ................................................................................................................... xiii Chapter 1 Introduction .................................................................................................................... 1 1 Introduction ................................................................................................................................ 1 1.1 Hedgehog Signalling ........................................................................................................... 1 1.2 Sufu ..................................................................................................................................... 5 1.2.1 Gli proteins .............................................................................................................. 6 1.2.2 Glis proteins ............................................................................................................ 7 1.2.3 Zic proteins ............................................................................................................. 9 1.3 Smoothened (Smo) .............................................................................................................. 9 1.3.1 Smo as a 7-transmembrane protein ....................................................................... 10 1.3.2 Smo in disease and as a drug target ...................................................................... 14 1.3.3 GPCR and in silico docking .................................................................................. 19 1.4 Research objectives ........................................................................................................... 19 1.4.1 Characterize a novel interactor of Sufu ................................................................ 19 1.4.2 Perform a structure-assisted docking screen to identify novel Smo ligands. ....... 20 Chapter 2 Material and Methods ................................................................................................... 21 2 Material and Methods .............................................................................................................. 21 2.1 ZFP629 results chapter ..................................................................................................... 21 2.2 Smo results chapter ........................................................................................................... 28 3 Results ...................................................................................................................................... 33 3.1 Results chapter 1: ZFP629 is a novel zinc finger transcription factor interacting with Sufu ................................................................................................................................... 33 v 3.1.1 ZFP629 is a novel interactor of Sufu .................................................................... 33 3.1.2 ZFP629 interaction with SUFU ............................................................................ 35 3.1.3 Zfp629 expression in the mouse cerebellum ......................................................... 38 3.1.4 ZFP629 can repress gene expression .................................................................... 42 3.1.5 ZFP629 interacts with proteins involved in transcription repression ................... 43 3.1.6 ZFP629 DNA-binding consensus sequence .......................................................... 46 3.1.7 Effect of knockdown of Zfp629 on gene expression in NIH3T3 .......................... 49 3.1.8 Chromatin Immunoprecipitation of ZFP629-3xFLAG ......................................... 55 3.1.9 Foxp2 expression is regulated by ZFP629 ............................................................ 60 3.2 Results chapter 2: Identification of novel Smoothened ligands using structure-based docking .............................................................................................................................. 63 3.2.1 Targeting the ligand binding site within the heptahelical domain of Smoothened ........................................................................................................... 63 3.2.2 Control docking screens for enrichment of ligand vs decoys. .............................. 63 3.2.3 Prospective full library docking screen – selection of 21 compounds .................. 64 3.2.4 Secondary screen identifies analogs. .................................................................... 69 3.2.5 The new antagonists exhibit efficacy at the chemoresistant Smo-D473H mutant. .................................................................................................................. 80 3.2.6 Library Bias .......................................................................................................... 85 4 Discussion and Conclusions ..................................................................................................... 86 4.1 ZFP629 Study ................................................................................................................... 86 4.1.1 ZFP629 is part of the SUFU protein complex ...................................................... 86 4.1.2 ZFP629 consensus sequence ................................................................................. 87 4.1.3
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