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Carriemn 1.Pdf Discovery and Development of Small Molecule Probes for the SUMO protease SENP1, a Novel Target for Advanced Prostate Cancer Therapy by Carrie M. Johnson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2017 Doctoral Committee: Associate Professor Jorge A. Iñiguez-Lluhí, Chair Associate Professor Chris E. Canman Professor Scott D. Larsen Professor Diane M. Robins Professor John J. G. Tesmer Carrie Margaret Johnson [email protected] ORCID ID: 0000-0002-6922-0875 Acknowledgements First, I would like to thank my advisor, Dr. Jorge Iñiguez-Lluhí. His mentorship in the classroom and the lab have helped me improve as a scientist and as a person. I came in as a chemist with analytical and synthetic chemistry research experience and very little in research in biology. With his reputation for his quality teaching, I was able to directly benefit from his talents to learn pharmacology, protein biochemistry, molecular cloning, and numerous other skills and topics. I have benefitted tremendously from your teaching and training and your incredible ability to venture into completely new content areas to come up with interesting questions and potential applications to our work. Lastly, thank you for your kindness and patience as I had to find the tenacity to overcome a two-year series of knee injuries that affected more than just my physical abilities to do research. I am not sure if there are adequate words that fully encapsulate my gratitude for your efforts put into me, but I offer my sincerest thank you. To my thesis committee, for your dedication to helping see me succeed in my project by offering suggestions, and succeed in my career endeavors by intentionally discussing my interests at each committee meeting and individually. I especially want to thank Dr. Scott Larsen and his graduate student Jeffrey Zwicker for their dedicated efforts while collaborating on the project. To Dr. Diane Robins for your mentorship, support, conversation, and availability throughout the highs and lows of my graduate training. To Dr. Marcelo Murai, the research associate in the lab for the last three years. Thank you for your incredible support in both the lab and in life. I appreciate your ii willingness to take time out of your busy schedule to discuss experiments and techniques with me. I will miss your spontaneous little gifts of treats and snacks. You are a critical component of me getting to this point and I am so grateful for your friendship and mentorship. To my undergraduate mentors, Dr. Dwight Stoll, Dr. Amanda Nienow, Dr. Scott Bur, and Dr. Brenda Kelly, and Dr. Uttam Tambar, I never thought I was smart enough to major in chemistry let alone do research. With your support, you encouraged me to get in the lab and try it out and then later to pursue graduate school. Thank you for believing in me when I never would have. Additionally, thank you for your availability in continuing to serve as mentors beyond my bachelor’s degree. As the child of two teachers in a small community, I have long been aware of the importance of teachers throughout my academic path. From Kindergarten to 12th grade, rural Montevideo provided me with a great education to lay the foundation that allowed me to earn my PhD. I know teachers are not recognized and thanked enough for their dedication and skills in teaching students. Know that I am grateful for your sacrifices to my education. To my friends of graduate school, ice hockey, FEMMES, and beyond, thank you for your facilitation of incredible memories, your support as a surrogate family throughout my PhD training, and your listening ears and advice through both the cathartic and celebratory conversations. There are too many of you to name, but I gained the best friendships of my life in my time here thanks to you. I truly love this University and what it has fostered. Forever Go Blue! iii Finally, I would like to my thank family, including my parents Mark and Dana, my brothers Matthew and Eric, and their wives Julie and Becca, my grandparents, and distant family members Hannah Bolados and Elaine Miller. I am not sure where to really begin, since some of you date back to my beginning, but I know I am so blessed to have such a truly amazing family. Thank you for supporting a culture of activity that provides me with a lifetime of athletic outlets. Thank you for helping me navigate my entire academic career thus far, even if some of you did not always know what this latest step of pursuing a PhD entailed. Thank you for your dedication to my educational pursuits as you sacrificed personal time to talk to me or come visit. This dissertation and degree is your accomplishment as it is mine. Though there are many others left and this might be one of the longest acknowledgments for a dissertation, I am the product of countless individuals to whom I am grateful. They are too important and I receive great joy in attempting to note them all. iv TABLE OF CONTENTS Acknowledgements ........................................................................................................... ii List of Figures ........ ......................................................................................................... vii List of Tables ........ ........................................................................................................... ix Abstract .............. ............................................................................................................... x Chapter 1 Introduction to post-translational modification by SUMO and its roles in the modulation of function and disease ............................................................. 1 1. Overview ................................................................................................................. 1 2. Ubiquitin ................................................................................................................. 2 3. SUMO ..................................................................................................................... 6 4. SUMO Functions .................................................................................................. 13 5. SUMO and Transcription ...................................................................................... 20 6. SUMO and Nuclear Receptors .............................................................................. 22 7. SUMO and Prostate Cancer .................................................................................. 27 8. References ............................................................................................................. 33 Chapter 2 Generation of a robust FRET-based assay for high throughput screening and characterization of SENP enzymes ....................................................... 48 1. Introduction ........................................................................................................... 48 2. Materials and Methods .......................................................................................... 52 3. Results ................................................................................................................... 58 4. Discussion ............................................................................................................. 70 5. References ............................................................................................................. 73 Chapter 3 High Throughput Screening for SENP1 inhibitors and characterization of identified small molecule hits ....................................................... 76 1. Introduction ........................................................................................................... 76 2. Materials and Methods .......................................................................................... 81 3. Results ................................................................................................................... 86 4. Discussion ........................................................................................................... 100 5. References ........................................................................................................... 103 Chapter 4 Nucleotide-mediated inhibition of SENP1 ...................................... 106 1. Introduction ......................................................................................................... 106 2. Materials and Methods ........................................................................................ 108 3. Results ................................................................................................................. 110 v 4. Discussion ........................................................................................................... 121 5. References ........................................................................................................... 126 Chapter 5 Conclusions and future directions ................................................... 129 1. Overview ............................................................................................................. 129 2. Summary and impact of results ........................................................................... 130 3. Regulation of the SUMO and AR systems ......................................................... 132 4. Exploration of future prostate cancer therapeutics ............................................
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