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A NOVEL HSP90 INHIBITOR to DISRUPT HSP90/P50cdc37 COMPLEX for PANCREATIC CANCER THERAPY

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A NOVEL HSP90 INHIBITOR to DISRUPT HSP90/P50cdc37 COMPLEX for PANCREATIC CANCER THERAPY A NOVEL HSP90 INHIBITOR TO DISRUPT HSP90/p50CDC37 COMPLEX FOR PANCREATIC CANCER THERAPY by Tao Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmaceutical Sciences) in The University of Michigan 2011 Doctoral Committee: Associate Professor Duxin Sun, Chair Professor David E. Smith Professor Shaomeng Wang Assistant Professor Wei Cheng © Tao Zhang 2011 To my parents and my wife for their love, support and encouragement To my son for the sweet smile and so much fun every day ii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor Dr. Duxin Sun for his guidance, inspiration, and support throughout the years. His positive attitude to science and open mind to new findings have greatly impressed me and will benefit my whole life. He is my best supervisor and friend. I sincerely appreciated his support both scientifically and personally. I would like to thank my great dissertation committee, Dr. Wei Cheng, Dr. David E. Smith and Dr. Shaomeng Wang for the insightful comments on my research and career development. I also thank all the professors, colleagues and students for their help in my research. Thanks to Dr. Shaomeng Wang for providing the AT-406 project and Donna McEachern for the animal surgery. Thanks to the professors in other universities, Dr. Chang-Guo Zhan, Dr. David Toft, Dr. Thomas Ratajczak, Dr. Wei Li, Dr. Dan Bolon, David Z. D'Argenio, most of whom I have not seen yet but they have helped me generously in my project by providing either technical support or materials. Thanks to the senior students in our lab, Dr. Xianhua Cao, Dr. Lanyan Fang and Dr. Seth Gibbs, who have given me a lot of help when I first joined in the group. I also thank other students and colleagues in our lab, and in the pharmaceutics department of both The Ohio State University and The University of Michigan, Dr. Hao Cheng, Dr. Ping Chen, Zhiliang Xie, Josephine Aimiuwu, Chien-Ming Li, Peng Zou, Yanke Yu, Bryan Newman, Wenjun Ni, iii Dr. Huifei Cui, Shuwen Yu, Yanyan Li, Dr. Yiqun Jiang, Zhenkun Zhu, Hsui-Fang Lee, Dr. Wenpeng Zhang, Dr. Young Ho Seo, Dr. Xiaoqin Li, Yiling Liu, Xiaofei Zhang, Brunett Joseph, David John Maycock, Nan Zheng etc. The time I spent with those friends have made my graduate study so colorful and fulfilling. I would like to give my special thanks to my wife, Yanyan Li, for her immeasurable love and support. She has given up her opportunity to stay in her lab in the Food Science and Technology Department and followed me from The Ohio State University to The University of Michigan without the least hesitation during our lab’s transfer. For this I would also thank both my supervisor Dr. Duxin Sun and Yanyan’s supervisor Dr. Steven J. Schwartz who decided to co-advise Yanyan, which makes us live together in Ann Arbor. iv TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ............................................................................................................. xi LIST OF APPENDICES ................................................................................................... xii ABSTRACT ..................................................................................................................... xiii CHAPTER 1 Background and Introduction ....................................................................... 1 1.1 Current Status of Pancreatic Cancer ......................................................................... 1 1.2 Conventional Treatment Approaches for Pancreatic Cancer .................................... 1 1.2.1 Surgery ............................................................................................................... 2 1.2.2 Adjuvant chemotherapy ..................................................................................... 2 1.2.3 Radiation therapy ............................................................................................... 3 1.2.4 Combination of gemcitabine with cytotoxic agents ........................................... 3 1.3 Targeted Therapy ...................................................................................................... 5 1.3.1 The Ras pathway ................................................................................................ 6 1.3.2 The epidermal growth factor receptor (EGFR) pathway ................................... 7 1.3.3 Vascular endothelial growth factor (VEGF) ...................................................... 9 1.3.4 Matrix metalloproteinases (MMPs) ................................................................... 9 1.3.5 P13K-Akt mTOR pathway .............................................................................. 10 1.3.6 The cyclo-oxygenase (COX) pathway ............................................................. 10 1.3.7 Other potential therapeutic targets ................................................................... 11 1.4 Heat Shock Protein 90 (Hsp90) .............................................................................. 11 1.4.1 Introduction of Hsp90 ...................................................................................... 11 1.4.2 Hsp90 structure and protein folding mechanism ............................................. 12 1.4.3 Hsp90 N-terminal ATP binding site inhibitors ................................................ 15 1.4.4 Hsp90 C-terminal binding inhibitors ............................................................... 18 1.4.5 Inhibitors disrupting cochaperone/Hsp90 interactions .................................... 19 1.4.6 Inhibitors targeting client/Hsp90 associations ................................................. 20 1.4.7 Post-translational modifications of Hsp90 ....................................................... 21 v 1.5 Limitations of Classical Hsp90 Inhibitors .............................................................. 22 1.6 Evidence to Support Identifying Novel Hsp90 Inhibitors That Disrupt the Hsp90 and p50Cdc37 Interaction ............................................................................................. 24 1.7 Specific Aims .......................................................................................................... 26 1.8 References ............................................................................................................... 27 CHAPTER 2 A Novel Hsp90 Inhibitor to Disrupt Hsp90/p50Cdc37 Complex against Pancreatic Cancer Cells .................................................................................................... 42 2.1 Abstract ................................................................................................................... 42 2.2 Introduction ............................................................................................................. 43 2.3 Materials and Methods ............................................................................................ 45 2.3.1 Drugs and reagents ........................................................................................... 45 2.3.2 Molecular modeling ......................................................................................... 46 2.3.3 MTS assay ........................................................................................................ 48 2.3.4 Annexin V-EGFP assay ................................................................................... 48 2.3.5 ATP-Sepharose binding assay ......................................................................... 48 2.3.6 Western blotting and immunoprecipitation ..................................................... 49 2.3.7 Nude mice xenograft model ............................................................................. 49 2.3.8 RIP1-Tag2 transgenic mice model ................................................................... 50 2.4 Results ..................................................................................................................... 50 2.4.1 Molecular docking of celastrol for the interactions with Hsp90 ...................... 50 2.4.2 Molecular modeling of Hsp90/celastrol complex with p50Cdc37 .................. 51 2.4.3 Celastrol has no effect on ATP binding to Hsp90 ........................................... 52 2.4.4 Celastrol disrupts Hsp90/p50Cdc37 complex in pancreatic cancer cells ........ 53 2.4.5 p23 does not co-exist with p50Cdc37 or Hop in one complex ........................ 54 2.4.6 Celastrol decreases Hsp90 client protein levels ............................................... 55 2.4.7 Celastrol inhibits Panc-1 cell growth and induces apoptosis ........................... 56 2.4.8 Celastrol shows strong anticancer activity in vivo ........................................... 56 2.4.9 Celastrol is different from other proteasome inhibitors ................................... 57 2.5 Discussion ............................................................................................................... 58 2.6 Acknowledgments................................................................................................... 62 2.7 References ..............................................................................................................
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