Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Small Organic Molecules as Tunable Tools for Biology Unzue Lopez, Andrea Abstract: Drug discovery and development is a very challenging interdisciplinary endeavor that needs the contribution of medical doctors, biologists, chemists, X-ray crystallographers, and computer scientists, among many others, in order to be successful. The first part of this Ph. D. thesis focuses onthe development of EphB4 receptor tyrosine kinase inhibitors. EphB4 has been linked to angiogenesis, which involves the formation of new blood vessels supplying tumor cells with the necessary nutrients. Protein kinases play a key role in cell signaling by phosphorylating specific proteins and thus, the inhibition of their enzymatic activity by small organic molecules has been widely explored in drug design. In this work, the biological properties of an EphB4 inhibitor identified by computer simulations were improved bythe synthesis of several analogues. Their binding affinities were characterized by an array of biochemical and cell based assays, concluding with the validation of one of the most promising derivatives in an in vivo cancer xenograft model. The second part of the thesis deals with the development of novel bromodomain ligands starting from a micromolar potent in silico discovered hit. Bromodomain proteins are epigenetic readers that constitute an emerging topic in the field of drug discovery and are thus considered asvery attractive targets for the development of novel therapeutic drugs. A careful, structure-based design of analogues resulted in the discovery of nanomolar potent CREBBP ligands with an unprecedented selectivity profile among the bromodomain protein family. Moreover, the screening of the synthesized analogues against several cancer cell lines revealed leukemia as a possible therapeutical application for the developed compounds. The third aspect of this work deals with actin: a very attractive, but yet unexplored target in medicinal chemistry. Actin is a cytoskeletal protein that participates in many important cellular functions and has been linked to key pathogenic cellular processes such as angiogenesis, cell adhesion, cytokinesis and metastasis. A new computational approach to discover novel actin leads targeting the ATP binding site of actin resulted in the selection of promising compounds, which were synthesized and tested. The developed small organic molecules constitute valuable tools for the study of actin dynamics as they are able to modify the actin cytoskeleton in cells and moderately inhibit actin polymerization in vitro; thus becoming promising starting hits for the development of more potent actin binders. The last part of this Ph.D. thesis describes the synthesis of neuroprotective compounds by the development of fumaric acid and hydroxytyrosol conjugates, for which the corresponding receptor is unknown. The biological effects of the synthesized analogues are currently under investigation, but the synergistic effect of fumaric acid and hydroxytyrosol is expected to be beneficial in the contextof neuroprotection. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-106367 Dissertation Published Version Originally published at: Unzue Lopez, Andrea. Small Organic Molecules as Tunable Tools for Biology. 2015, University of Zurich, Faculty of Science. 2 Small Organic Molecules as Tunable Tools for Biology Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Andrea Unzue Lopez aus Spanien Promotionskomitee Prof. Dr. Cristina Nevado (Vorsitz und Leitung der Dissertation) Prof. Dr. Amedeo Caflisch Prof. Dr. Damian Brunner Zürich, 2015 CONTENTS Acknowledgments 1 Zusammenfassung 3 Summary 5 1 Introduction 7 1.1 Motivation ............................................................................................................ 7 1.1.1 Drug discovery process ................................................................................ 7 1.2 Challenges ............................................................................................................ 8 1.3 Outline of the thesis ............................................................................................. 9 1.4 List of publications and patents ......................................................................... 10 1.5 References .......................................................................................................... 11 2 Eph Tyrosine Kinase Inhibitors 13 2.1 Introduction ....................................................................................................... 13 2.1.1 Function, classification and structure of protein kinases ........................... 13 2.1.2 Protein kinases as drug targets and their binding mode ........................... 16 2.1.3 Eph receptor tyrosine kinases ................................................................... 23 2.2 Pyrrolo[3, 2-b]Quinoxaline Derivatives as Types I1/2 and II Eph Tyrosine Kinase Inhibitors ................................................................................................ 26 2.2.1 Crystal structures of type I inhibitors A and B with EphA3 .................... 27 2.2.2 Characterization of new type I1/2 and II inhibitors .................................... 29 2.2.3 Validation of type II binding by X-ray crystal structure determination ... 34 2.2.4 Selectivity and cellular activity ................................................................. 35 2.2.5 In vivo data ............................................................................................... 38 2.2.6 Conclusions ............................................................................................... 40 2.3 Experimental section .......................................................................................... 40 2.3.1 Chemistry. General methods. .................................................................... 40 2.3.2 Differential Scanning Fluorimetry ............................................................. 68 2.3.3 FRET based enzymatic assay ................................................................... 68 2.3.4 Surface Plasmon Resonance Measurements .............................................. 68 2.3.5 X-Ray Diffraction Analysis ....................................................................... 69 2.3.6 Selectivity profile ...................................................................................... 70 2.3.7 Cellular phosphorylation assays ................................................................ 71 2.3.8 In house cell culture and GI50 determination ............................................ 71 2.3.9 Antiproliferative activity against patient derived tumor cell lines ............ 72 2.3.10 Spheroid-based cellular angiogenesis assay .............................................. 72 2.3.11 In Vivo evaluation ................................................................................... 73 i 2.4 References .......................................................................................................... 74 3 Bromodomain Ligands 85 3.1 Introduction ....................................................................................................... 85 3.1.1 Epigenetics ................................................................................................ 85 3.1.2 Bromodomains: structure and function ..................................................... 88 3.1.3 Bromodomain ligands ................................................................................ 91 3.2 Fragment-based Design of Potent and Selective CREBBP Bromodomain Ligands: Polar Interactions with Arg1173 as a Tool Towards Selectivity ........ 108 3.3 Experimental section ........................................................................................ 116 3.3.1 Fragment-based high-throughput docking .............................................. 116 3.3.2 Finite-difference Poisson calculations ...................................................... 117 3.3.3 Fragment docking ................................................................................... 118 3.3.4 Synthetic methods ................................................................................... 118 3.3.5 Bromodomain expression and purification .............................................. 144 3.3.6 X-ray crystallography .............................................................................. 145 3.3.7 Thermal shift measurements ................................................................... 146 3.3.8 TR-FRET assays .................................................................................... 148 3.3.9 BROMOscan assays ................................................................................ 149 3.3.10 ITC Experiments ................................................................................... 150 3.3.11 Cell culture and cytotoxicity measurements .......................................... 152 3.3.12 Comparison table to known CREBBP ligands ...................................... 154 3.4 References ........................................................................................................ 154 4 Towards the Discovery of Small Molecule Actin Binders 167 4.1 Introduction ....................................................................................................