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Strategies for Modulating the Diverse Activities of Heat Shock Protein 70

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Strategies for Modulating the Diverse Activities of Heat Shock Protein 70 by Victoria A. Assimon A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2015 Doctoral Committee: Associate Professor Jason E. Gestwicki, Co-Chair Professor Anna K. Mapp, Co-Chair Professor Andrew P. Liebermann Professor David H. Sherman Professor Ronald W. Woodard © Victoria A. Assimon 2015 Dedication To my grandparents, Peter G. Assimon and Magdalini Morena. ii Acknowledgements First and foremost, I would like to thank my advisor, Professor Jason E. Gestwicki, for his patience, optimism, support, and for providing a unique interdisciplinary research environment. He has taught me a great deal about science, leadership, and collaboration. Jason has supported my intellectual curiosities, while always providing critical evaluation and direction. I feel privileged to have had the opportunity to learn from him and to have been a part of his laboratory. I would also like to thank all the members of the Gestwicki laboratory, both past and present. The work described in this thesis would not have been possible without their insights and contributions. The direct contributions of individuals have been noted in each chapter. Notable among these are Zapporah Young, Isabelle Taylor, and Xiaokai Li. Many others have continually inspired me with their passion for science, exceptional work ethic, and vibrant personalities: Tomoko Komiyama, Andrea Thompson, Matthew Smith, Leah Makley, Anne Gillies, Jennifer Rauch, Sharan Srinivasan, Bryan Dunyak, Laura Cesa, Sue-Ann Mok, Hao Shao, Jennifer Abrams, Rebecca Freilich, Kwadwo Opoku-Nsiah, and Matthew Ravalin. Additionally, I am indebted to my thesis committee, Professors Anna Mapp, Andrew Liebermann, David Sherman, and Ronald Woodard, for their insightful comments and encouragement. They have been generous with their time and have helped me to develop as a scientist. Finally, I would like to recognize Alex Martinko and my family for their constant love and support. iii Table of Contents Dedication ................................................................................................................................. ii Acknowledgements .................................................................................................................. iii List of Figures ......................................................................................................................... viii List of Tables ............................................................................................................................. xi List of Abbreviations ................................................................................................................ xii Abstract ................................................................................................................................... xv Chapter 1. Heat shock protein 70 complexes as drug targets 1.1 Abstract .................................................................................................................. 1 1.2 Diversity of Hsp70 functions ................................................................................. 1 1.3 Hsp70 as a therapeutic target ............................................................................... 2 1.4 Structure and function of Hsp70 and its complexes ............................................ 5 1.5 Approaches to targeting Hsp70 ............................................................................ 8 1.6 Opportunities for drugging J proteins and their interactions with Hsp70 ........ 10 1.7 Opportunities for drugging NEFs and their interactions with Hsp70 ................ 19 1.8 Opportunities for drugging TPR proteins and their interactions with Hsp70 ... 24 1.9 Analysis and prospectus ...................................................................................... 28 1.10 References.......................................................................................................... 31 iv 2. Specific binding of tetratricopeptide repeat (TPR) proteins to Hsp70 and Hsp90 is regulated by affinity and phosphorylation 2.1 Abstract ................................................................................................................ 42 2.2 Introduction ......................................................................................................... 43 2.3 Results and discussion ......................................................................................... 46 2.4 Conclusions .......................................................................................................... 55 2.5 Experimental procedures .................................................................................... 60 2.6 Appendix ............................................................................................................... 68 2.7 References ............................................................................................................ 75 3. Mutagenesis of Hsp70 reveals a complex and tunable allosteric network: Connecting drug binding sites to function and protein-protein interactions 3.1 Abstract ................................................................................................................ 80 3.2 Introduction ......................................................................................................... 80 3.3 Results and discussion ......................................................................................... 86 3.4 Conclusions .......................................................................................................... 96 3.5 Experimental procedures .................................................................................. 100 3.6 Appendix ............................................................................................................. 106 3.7 References .......................................................................................................... 114 4. Discovery of antibiotics targeting Hsp70 4.1 Abstract .............................................................................................................. 117 4.2 Introduction ....................................................................................................... 118 4.3 Results and discussion ....................................................................................... 121 v 4.4 Conclusions ........................................................................................................ 129 4.5 Experimental procedures .................................................................................. 130 4.6 Appendix ............................................................................................................. 139 4.7 References .......................................................................................................... 153 5. Conclusions and future directions: Progress towards understanding how to modulate the Hsp70 chaperone system 5.1 Abstract .............................................................................................................. 155 5.2 Conclusions ........................................................................................................ 156 5.3 Future directions ................................................................................................ 159 5.4 Broader implications .......................................................................................... 163 5.5 Final thoughts..................................................................................................... 164 5.6 References .......................................................................................................... 166 Appendix Concise synthesis of spergualin-inspired molecules with antibiotic activity A.1 Abstract .............................................................................................................. 168 A.2 The natural product spergualin has antibacterial activity and is difficult to synthesize ................................................................................................................. 169 A.3 The Ugi multicomponent reaction is an efficient way to assemble spergualin analogs ...................................................................................................................... 170 A.4 Results ................................................................................................................ 171 A.5 Discussion ........................................................................................................... 177 A.6 Materials and methods ..................................................................................... 179 vi A.7 Appendix ............................................................................................................ 186 A.8 References ......................................................................................................... 240 vii List of Figures 1.1 Hsp70 forms the core of a multi-protein complex and associates with numerous co- chaperones ............................................................................................................................... 3 1.2 The structure of Hsp70 ...................................................................................................... 5 1.3 The ATPase cycle of Hsp70 ...............................................................................................
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