DESIGN AND CHARACTERIZATION OF CHEMICAL PROBES TARGETING THE CHROMODOMAINS OF POLYCOMB REPRESSIVE COMPLEX I Jacob I. Stuckey A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Pharmaceutical Sciences (Chemical Biology & Medicinal Chemistry) program in the Eshelman School of Pharmacy. Chapel Hill 2016 Approved by: Stephen V. Frye David S. Lawrence Albert A. Bowers Marcey L. Waters Brian D. Strahl © 2016 Jacob I. Stuckey ALL RIGHTS RESERVED ii ABSTRACT Jacob I. Stuckey: Design and characterization of chemical probes targeting the chromodomains of Polycomb Repressive Complex I (Under the direction of Stephen V. Frye) Eukaryotic DNA is wound around histone proteins to form repeating units of nucleosomes that are packaged into higher order structures to form chromosomes. Nucleosomal packaging is dynamically regulated to control the expression of the genome through regulating access of transcription factors to specific genes in response to stimuli. Critical for these processes are modifications to the N-terminal tails of histones. In particular, methylation of specific histone lysine residues has a profound impact on transcription. This “mark” recruits protein complexes that contain methyl-lysine “reader” modules that bind to the mark and facilitate transcriptional silencing or activation of the underlying gene. Mutation and misregulation of proteins that “write,” “read” and “erase” these marks play a foundational role in the development and progression of nearly all cancers. Chemical tools that antagonize these writers, readers and erasers have been developed in recent years. These tools have been valuable in explicating the biology of these proteins, and in some cases, as therapeutic agents. Development of antagonists of methyl-lysine readers has lagged compared to both methyl-lysine writers and erasers. Herein we describe the design, development and characterization of antagonists of the chromodomain reader modules contained in Polycomb Repressive Complex I (PRC1). PRC1 contains one of five chromodomains; CBX2, -4, -6, -7 or -8. PRC1 complexes containing any one of these proteins play critical roles in normal, growth and development. Accordingly, misregulation of PRC1 chromodomains contributes to the initiation and development of numerous cancers. We developed a series of potent, peptidic antagonists of PRC1 chromodomains and an expanded set of chemical tools around one of these molecules, UNC3866, which has the highest affinity for the chromodomains of CBX4 and -7. We demonstrated the ability of UNC3866 to inhibit proliferation in a metastatic prostate cancer cells through the induction of a senescent-like state. Further optimization of UNC3866 led to a second- generation antagonist of PRC1 chromodomains with a dramatically improved residence time for CBX7 iii and increased cellular potency. This work has produced a valuable tool set for studying CBX biology and laid the ground work for continued efforts to evaluate and improve CBX antagonists as potential therapeutic agents. iv DEDICATION To the glory of God in Christ Jesus v ACKNOWLEDGMENTS Nine years ago, I delivered a speech to my high school graduating class in which I quoted the wisdom of a respected teacher and coach who insisted that “when we crossed that stage, life was going to punch us in the face.” I continued by imploring my classmates that the difference between success and failure would be determined by those who chose to get back up when life knocks us down. What I didn’t realize when I delivered that speech so long ago, was just how relevant it was for the journey on which I was about to embark. Nothing about my life even remotely resembles what I pictured that day. Looking back at the course of my life, I am grateful to even be alive, let alone where I am today. One of my most beloved childhood heroes, Sylvester Stallone’s Rocky Balboa, put it this way, “You, me, or nobody is gonna hit as hard as life. But it ain’t about how hard you hit. It’s about how hard you can get hit and keep moving forward; how much you can take and keep moving forward.” I cannot adequately express my gratitude to my family for the many, many times that they have stood beside me and kept me moving forward through everything that life has thrown at me. I want to thank my beautiful wife, Jennifer, who has loved, supported and pushed forward me forward thru even the worst of times. To my parents, Charles and Lari Stuckey, who have believed in me and supported me throughout my life, even thru times when I didn’t believe in myself. To my father, who constantly challenged me to think for myself and to “be my own man.” To my mother, who is perhaps the most disciplined, hard-working and strongest person I’ve met. I have learned so much from you both. I am the person I am today because of you. To my brother and his wife, Joshua and Linsey Stuckey, for their love and support thru the years. Their two beautiful daughters, Claire and Cora Stuckey, who have brought me so many much-needed smiles. Despite anyone else may say, are the two most adorable little girls in the world. To Paul, Susan and Jonathan Fleetwood, who have welcomed me into their family with open arms. To my grandparents, Charles and Wilma Stuckey, and Leon and Lola Calvin. Nothing saddens me more than to not be able to share the joy of what I have accomplished with you. You are loved and missed and your memory is source of inspiration for me on a daily basis. Last but not least, to vi my dogs Lizzie, Carbon and Harley, all of whom are a constant reminder that even on my worst days, as long as I have my family, life is still pretty good. Numerous others, in addition to my family have been instrumental in seeing me thru to where I am now. To my undergraduate adviser, Dr. Joshua Ring, whose passion and enthusiasm for organic chemistry and biochemistry was instrumental in my decision to change my major to Chemistry at the start of my final year as an undergraduate and to pursue the graduate studies in Pharmaceutical Sciences. To the faculty in the Division of Chemical Biology and Medicinal Chemistry at UNC for the immense amount of time, money and resources you invested in me in order to develop and train me as an independent scientist. To my adviser and mentor, Dr. Stephen V. Frye, who has been an absolute joy to work with. I have learned so much from you both in what you have explicitly taught me and from the example you have set as a person, leader and scientist (in that order). To Dr. Lindsey James, who has been tremendous friend and teacher throughout my graduate studies. You are a truly rare person who is both a great scientist and a great person. I owe a great deal of my success as a graduate student to you and know that you will continue to be an outstanding mentor to many graduate students to come. Maybe most of all, thank you for seeming to be genuinely interested when I felt the need to brag about my nieces and show off pictures of just how cute they are. To my lab mates in the Center, for their guidance, patience and hard work throughout the years. Kim Barnash, who, I’m certain, made it her sole purpose on more than a few days, to keep me humble. It has been a genuine pleasure working with and learning from you. To Kyle Konze, for your friendship and support as we started our graduate careers together and somehow made it through. I want to thank Jarod Waybright for being a great person to bounce ideas off of and an even better person to grab lunch with and forget about science altogether for a little while. To Dr. Samantha Pattenden, Jacqueline Norris Drouin, Dr. Brandi Baughman and Stephanie Cholensky for all of your hard work, support and teaching that expanded my scientific experise far beyond what I ever expected. To Dr. Brad Dickson, whose own work was critical for my success as a graduate student and who was instrumental in my transition to an independent scientist and thinker. Finally, I want to thank my committee for their time, attention and input through the years, Dr. David Lawrence, Dr. Albert Bowers, Dr. Brian Strahl and Dr. Marcey Waters. vii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................................... x LIST OF FIGURES ..................................................................................................................................... xii LIST OF ABBREVIATIONS........................................................................................................................ xv CHAPTER I: INTRODUCTION ..................................................................................................................... 1 Chromatin regulation ........................................................................................................................ 1 Therapeutic interest in chromatin regulatory proteins ...................................................................... 4 The tractability of chemical probe discovery for readers of histone lysine methylation ................... 6 Screening for antagonists of methyl-lysine readers ......................................................................... 9 Examples of previous challenges and successes of small-molecule chemical probe discovery efforts targeting methyl-lysine readers ................................................................ 11 The CBX family of chromodomains ..............................................................................................