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Chemical Genetics of Vertebrate Development by Charles H Williams

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Chemical Genetics of Vertebrate Development by Charles H Williams Chemical genetics of Vertebrate development By Charles H Williams III Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cell and Developmental Biology May, 2017 Nashville, Tennessee Approved: Charles C Hong, M.D., Ph.D. (Mentor) Ken Lau, Ph.D. David Bader, Ph.D. Craig Lindsley, Ph.D. Guoqiang Gu, Ph.D. (Chair) ACKNOWLEDGEMENTS I thank my committee members for their support during this thesis. Stephen Dalton for hiPSC-NCC work. Isabel Dominguez for dn-CK2α construct. Current and former Labmates Audrey frist, Johnathan Hempel, Adrian Cadar, Young Chun, Jamie Rickmyre and Erin Booton; Programs of Developmental biology, VICC, VICB, Department of Cardiology, and Cell and Developmental Biology. NIH for funding. ii ABSTRACT Small molecules have value in their ability to modulate protein activity in ways that are not accessible using conventional genetic methods, and their potential to be developed into therapeutics. As genetic causes of human disease are identified, many are found to play an integral role in embryonic development as well. The purpose of this dissertation is to utilize vertebrate embryonic development as a platform for discovery and characterization of chemical probes using phenotype guided development. I discovered three molecules, Eggmanone, Incaskin, and Ogremorphin based on ability to perturb development of a composite of 29 anatomical features. Using these anatomical features as a query against the wealth of reference genotype-phenotype data in ZFIN, I identified the targets of the small molecules as PDE4, CK2α, and GPR68. Using eggmanone, I characterize a novel role for PDE4 in regulation of HH signaling, and show that it could be a useful therapeutic target for smo inhibitor resistant cancers. Using incaskin, I show an unbiased phenotypic clustering methodology for target deconvolution; furthermore, I show that incaskin is a highly selective, potent CK2α inhibitor which provides a therapeutic approach for targeting cancers down stream of APC in Wnt signaling. Using ogremorphin, a first in class inhibitor of GPR68, I show that proton sensing GPR68 is critical neural crest migration. Furthermore, I show that proton-sensing GPR68 represents a novel chemically tractably therapeutic avenue for development of an anti-metastatic agent. This body of work contributes to novel discovery of small molecules, signaling, and developmental biology iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................ii ABSTRACT .................................................................................................................... iii LIST OF FIGURES......................................................................................................... vii LIST OF TABLES ........................................................................................................ viix 1. INTRODUCTION ......................................................................................................... 1 Phenotypic Screening ............................................................................................... 1 Phenotypic screening modalities .............................................................................. 3 Zebrafish screens ..................................................................................................... 7 Beyond discovery ..................................................................................................... 16 Next Steps: Genomics and drug discovery .................................................................. 17 Phenoclustering for target identification ............................................................... 20 In silico Clustering .................................................................................................. 21 In vitro Clustering ................................................................................................... 23 In vivo clustering .................................................................................................... 24 Advances for in vivo phenoclustering ..................................................................... 27 Signaling pathways ................................................................................................. 28 Hedgehog Pathway and inhibitors.......................................................................... 28 Wnt Pathway and inhibitors .................................................................................... 34 H+ extrusion and sensing ....................................................................................... 39 2. MATERIALS AND METHODS .................................................................................. 44 3. EGGMANONE .......................................................................................................... 59 Introduction .............................................................................................................. 59 iv Results ...................................................................................................................... 60 Discovery of eggmanone, a novel small molecule hedgehog inhibitor, from an in vivo chemical genetic screen ................................................................ 60 Eggmanone exerts its Hh-inhibitory effects downstream of Smo ........................... 62 Eggmanone differentially affects Gli transcription factors ....................................... 66 Eggmanone inhibits hedgehog signaling through antagonism of ............................... phosphodiesterase 4 .............................................................................................. 70 PDE4 modulates Hh signaling in vitro .................................................................... 71 Discussion ............................................................................................................... 72 4. ZePAC: ZEBRAFISH PHENOTYPIC ANATOMICAL CLUSTERING ..................... 74 Introduction .............................................................................................................. 74 Results ...................................................................................................................... 77 Pathway segregation based on genotype-phenotype association database .......... 77 Pheno-clustering of novel small molecule for target pathway identification ............ 80 Incaskin inhibits activation of β-catenin and nuclear subsequent nuclear translocation ........................................................................................................... 81 Incaskin is a CK2α inhibitor .................................................................................... 82 Genetic manipulation with CK2α modulates zebrafish dorsoventral patterning ...... 85 Discussion ................................................................................................................. 89 5. OGREMORPHEN ..................................................................................................... 92 Introduction .............................................................................................................. 92 Results ...................................................................................................................... 93 Ogremorphin inhibits GPR68 ................................................................................. 93 Genetic inhibition of GPR68 phenocopies Ogremorphin treatment ........................ 95 Proton efflux inhibition phenocopies Ogremorphin treatment ................................. 96 Ogremorphen inhibits neural crest migration ......................................................... 98 v Ogremorphen inhibits melanoma migration ......................................................... 101 GPR68 regulates cell adhesion ............................................................................ 102 Acidification acutely increases cellular motility through GPR68 ........................... 106 GPR68 variants associated with secondary metastasis ....................................... 108 Discussion ............................................................................................................. 110 6. SUMMARY AND FUTURE DIRECTIONS .............................................................. 113 REFERENCES ............................................................................................................ 115 vi LIST OF FIGURES Figure 1 Comparison of model organisms used in phenotypic screens. ......................... 7 Figure 2 Proposed zebrafish phenotypic screens incorporating human genome– phenome information to accelerate therapeutic discovery. ........................................... 16 Figure 3 Annotation of small molecule libraries through hierarchical clustering can be done in a number of models; in silico, in vitro and in vivo .............................................. 21 Figure 4 Hedgehog signaling ........................................................................................ 32 Figure 5 WNT signalling ................................................................................................ 37 Figure 6 Proton sensing mechanisms ........................................................................... 40 Figure 7 Eggmanone affects embryonic zebrafish patterning through inhibition of
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