IDENTIFICATION AND CHARACTERIZATION OF SPECIFIC INHIBITORS OF THE EYA2 PHOSPHATASE by AARON B. KRUEGER B.S., North Dakota State University, 2006 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Structural Biology and Biochemistry 2013 This thesis for the Doctor of Philosophy degree by Aaron B. Krueger has been approved for the Structural Biology and Biochemistry by Robert Hodges, Chair Heide Ford Changwei Liu Steve Nordeen Rui Zhao, Advisor Date ___10/21/13____________ ii Krueger, Aaron B. (Ph.D., Structural Biology and Biochemistry) Identification and characterization of specific inhibitors of the Eya2 phosphatase Thesis directed by Associate Professor Rui Zhao. ABSTRACT Eya proteins are transcriptional co-activators of the homeobox gene Six1 and contain a unique protein tyrosine phosphatase activity. Eya and Six1 are required for normal development and are down-regulated in most adult tissues. However, Six1 and Eya are re-expressed in a large number of breast tumors and play a causal role in the initiation and development of these tumors. Eya’s unique phosphatase activity has been shown to be important for the transformation, migration, invasion, and metastasis of breast cancer cells. Because of its mechanistically unique phosphatase activity and its important role in the establishment and spread of breast cancer, I targeted Eya for anti-breast cancer therapy. To this end, I developed an HTS strategy to identify inhibitors of Eya2’s phosphatase activity. In collaboration with the NIH Chemical Genomics Center, I screened over 330,000 compounds and have identified a series of compounds effective at inhibiting Eya2’s phosphatase activity. Cell-based migration assays using MCF10A breast cells have demonstrated an import role for Eya2’s phosphatase activity and that these identified phosphatase inhibitors can effectively inhibit migration of these cells. Using biochemical, biophysical, and structural approaches, I further characterized these inhibitors as reversible, iii mixed mode inhibitors that likely bind at an allosteric site, with a KD of 2.0 µM. I have demonstrated that these inhibitors are specific towards Eya2 over other mechanistically similar phosphatases including its highly-conserved family member, Eya3. These characterizations are leading us to develop a model of how the inhibitor could specifically interact and inhibit Eya2 which will help us to further improve potency to make a viable anti-breast cancer drug. The form and content of this abstract are approved. I recommend its publication. Approved: Rui Zhao iv DEDICATION I dedicate this work to ‘the’ wife Katie who happily provided strength and support throughout the entire process, to my parents who kept me curious and never said no, and to my friends who kept me sane when I otherwise would not be. v ACKNOWLEDGMENTS I would first like to thank the members of my committee for their many insightful questions and advice through the progression of the project. I would like to thank the prior and past members of the Zhao lab for the fun times, the assistance provided, and support given. I would like to especially thank Dr. Zhao for allowing me the opportunity to join her lab and for the years of teaching me to become a successful scientist. I would like to thank our collaborators whose contributions were instrumental in allowing the project to progress. Specifically, I would like to thank members of the NIH Chemical Genomics Center for their contributions to the high throughput screening, of the University of Colorado BioPhysics CORE for their assistance and guidance, Philip Reigan and lab for assistance in modeling, Elan Eisenmesser and lab for assistance with NMR experiments, and Heide Ford and lab for essential contributions. Without these people, I would be nowhere. Finally, for the years of guidance and support, I would like to thank the fellow students, faculty, and staff of the Department of Biochemistry and Molecular Genetics, and specifically those of the Structural Biology and Biochemistry Program. vi CONTENTS CHAPTER I. INTRODUCTION ............................................................................................... 1 Transcription complexes as therapeutic targets ................................... 1 The Six1/Eya transcriptional complex ................................................... 2 Six1 and Eya in cancer ......................................................................... 6 The transcription activator Eya is a HAD family phosphatase .............. 7 Functions of Eya’s protein tyrosine phosphatase activity ................... 15 Targeting protein tyrosine phosphatases for therapeutic drug design 17 Targeting the Eya protein tyrosine phosphatase activity .................... 21 II. THE IDENTIFICATION OF EYA2 PHOSPHATASE INHIBITORS .................. 23 Abstract .............................................................................................. 23 Introduction ......................................................................................... 24 Results ............................................................................................... 27 Design of assays suitable for HTS ................................................ 27 Known phosphatase inhibitors do not significantly inhibit Eya2’s phosphatase activity ...................................................................... 31 The OMFP-based phosphatase assay is suitable for HTS ............ 32 A large scale primary screen identified a class of structurally related initial hits ....................................................................................... 35 The best compounds from this series are active in a pH2AX-based secondary phosphatase assay ...................................................... 38 The N-arylidenebenzohydrazide compounds do not inhibit other cellular phosphatases ................................................................... 40 Discussion .......................................................................................... 41 Methods .............................................................................................. 45 vii Protein expression and purification ............................................... 45 pNPP-based Eya phosphatase assay ........................................... 45 OMFP-based Eya phosphatase assay .......................................... 46 Miniaturized Eya phosphatase assay for HTS .............................. 46 Compound library and instruments for liquid handling .................. 47 HTS data analysis ......................................................................... 48 pH2AX-based Eya phosphatase assay ......................................... 48 Phosphatase assays of PTP1B, PPM1A, and Scp1 ...................... 49 III. DETERMINING THE MECHANISM OF ACTION OF EYA2 INHIBITORS..... 51 Abstract .............................................................................................. 51 Introduction ......................................................................................... 52 Results ............................................................................................... 55 HTS identified a new chemical series that specifically inhibit Eya2’s phosphatase activity. ..................................................................... 55 Enzyme kinetic analyses suggest that the N- arylidenebenzohydrazide compounds are mixed mode inhibitors. 58 Attempt to determine the binding mechanism using X-ray crystallography. ............................................................................. 60 Attempt to determine the binding mechanism using NMR. ........... 61 Compounds likely do not bind in the active site and do not require Mg2+ for function. ........................................................................... 65 Compounds likely bind to an allosteric site as determined by mutational analysis. ....................................................................... 72 Evaluate the effect of inhibitors on cellular phenotypes. ............... 81 Discussion .......................................................................................... 83 Methods .............................................................................................. 90 Protein expression and purification ............................................... 90 viii Eya2 phosphatase assays ............................................................ 91 Eya2 kinetic experiments .............................................................. 91 Isothermal titration calorimetry ...................................................... 92 Circular dichroism ......................................................................... 93 NMR spectroscopy ........................................................................ 93 UV-Vis spectra analysis of selected hydrazides ............................ 94 Molecular docking ......................................................................... 94 Motility assay................................................................................. 95 Cell lines ....................................................................................... 95 IV. DEVELOPMENT OF ASSAYS TARGETING OTHER ASPECTS OF SIX1 ACTION .............................................................................................................. 96 Abstract .............................................................................................