Pin1 Inhibitors: Towards Understanding the Enzymatic Mechanism Guoyan Xu Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University In the partial fulfillment of the requirement for the degree of Doctor of Philosophy In Chemistry Felicia A Etzkorn, Chair David G. I. Kingston Neal Castagnoli Paul R. Carlier Brian E. Hanson May 6, 2010 Blacksburg, Virginia Keywords: Pin1, anti-cancer drug target, transition-state analogues, ketoamides, ketones, reduced amides, PPIase assay, inhibition Pin1 Inhibitors: Towards Understanding the Enzymatic Mechanism Guoyan Xu Abstract An important role of Pin1 is to catalyze the cis-trans isomerization of pSer/Thr- Pro bonds; as such, it plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including Cdc25, c- Jun, and p53. The expression of Pin1 correlates with cyclin D1 levels, which contributes to cancer cell transformation. Overexpression of Pin1 promotes tumor growth, while its inhibition causes tumor cell apoptosis. Because Pin1 is overexpressed in many human cancer tissues, including breast, prostate, and lung cancer tissues, it plays an important role in oncogenesis, making its study vital for the development of anti-cancer agents. Many inhibitors have been discovered for Pin1, including 1) several classes of designed inhibitors such as alkene isosteres, non-peptidic, small molecular Pin1 inhibitors, and indanyl ketones, and 2) several natural products such as juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen. These Pin1 inhibitors show promise in the development of novel diagnostic and therapeutic anticancer drugs due to their ability to block cell cycle progression. In order to develop potent Pin1 inhibitors, the concept of transition-state analogues was used for the design of three classes of compounds: ketoamide, ketone, and reduced amide analogues. Specifically, a convergent synthesis of !-ketoamide inhibitors of Pin1 was developed. An !-hydroxyorthothioester derivative of Ser was reacted directly with an aminyl synthon. The reaction was catalyzed by HgO and HgCl2 to form an !- hydroxyamide. Hydrolysis and coupling were combined in one step in 80% yield. Two diastereomers of a phospho-Ser-Pro !-ketoamide analogue were synthesized. The resulting IC50 values of 100 µM and 200 µM were surprisingly weak for the Pin1 peptidyl-prolyl isomerase. Diastereomeric ketones were synthesized by coupling cyclohexenyl lithium to the serine Weinreb amide, via the Michael addition of a carboxylate synthon. The IC50 values of the two ketone diastereomers were determined to be 260 µM and 61 µM, respectively. Five reduced amide inhibitors for Pin1 were synthesized through a selective reduction using borane. The most potent inhibitor was found to be Fmoc–pSer– ![CH2N]-Pro–tryptamine, which had an IC50 value of 6.3 µM. This represents a 4.5-fold better inhibition for Pin1 than a comparable cis-amide alkene isostere. The co-crystal structure of Ac–pSer–![CH2N]-Pro–tryptamine bound to Pin1 was determined to 1.76 Å resolution. Towards understanding the two proposed mechanisms of Pin1 catalysis, nucleophilic-additition mechanism and twisted-amide mechanism, three classes of Pin1 inhibitors (ketoamide, ketone, and reduced amide analogues) involving a total of nine compounds were synthesized and evaluated. The weak inhibitory activities of ketoamide and ketone analogues do not support the nucleophilic-addition mechanism, while the twisted-amide mechanism of Pin1 catalysis is promising based on the reduced amide inhibitors with good potencies. III ACKNOWLEDGEMENTS I would like to thank my committee members, Dr. Felicia Etzkorn, Dr. David Kingston, Dr. Neal Castagnoli, Dr. Paul Carlier and Dr. Brian Hanson for their help and support throughout my graduate studies at Virginia Tech. I would especially like to acknowledge my advisor, Dr. Felicia Etzkorn, for her unfailing guidance and advice— both with my research and with matters outside the laboratory. I have her to thank for making me a better researcher and for helping me to interact more effectively with colleagues and faculty. I also would like to extend my heartfelt thanks to my current and former colleagues: Dr. Xiaodong Wang, Dr. Tao Liu, Dr. Song Zhao, Dr. Nan Dai, Ms. Ana Mercedes-Camacho, Ms. Onyi Freeman, Ms. Jiajia Li, Ms. Fan Shuang, Mr. Jian Wu, Dr. Yanpeng Hou, Dr. Wujun Fu, Dr. Ting Cai, Dr. Jun Qi, and Dr. Yanxing Li. Not only did their suggestions and input assist in advancing my research in innumerable ways, but they also helped to make my time here at Virginia Tech so enjoyable and memorable. Lastly, I want to thank my family, my parents, and my sister and brother. Their constant encouragement and support were very important to me—even from so far away! Most importantly, I would like to thank my husband, Mr. Xingguo Chen, for his support during our lives and while we worked together to achieve our education goals. IV Table of Contents Chapter 1: Pin1 as an anticancer drug target..................................................................... 1! Abstract........................................................................................................... 1! Pin1 binds pSer/Thr-Pro motifs ...................................................................... 2! Pin1 enzymatic activity................................................................................... 2! Regulation of entry into mitosis...................................................................... 3! Other cell cycle checkpoints ........................................................................... 4! DNA damage response ................................................................................... 5! Pin1 in human cancers .................................................................................... 5! Breast cancer................................................................................................... 6! Hepatocellular carcinoma (HCC) ................................................................... 6! Non-small cell lung cancer (NSCLC)............................................................. 7! Esophageal squamous cell carcinoma (SCC) ................................................. 7! Cervical cancer................................................................................................ 7! Colorectal cancer ............................................................................................ 7! Prostate cancer (PCa)...................................................................................... 8! Thyroid tumors................................................................................................ 8! Oral squamous cell carcinoma (OSCC).......................................................... 8! Alzheimer’s disease ........................................................................................ 9! Inhibitors of Pin1 as promising therapies for cancer ...................................... 9! Designed inhibitors ......................................................................................... 9! Peptides and peptidomimetics......................................................................... 9! Cell permeable inhibitors.............................................................................. 11! V Reduced amide inhibitors ............................................................................. 12! Indanyl ketone inhibitors .............................................................................. 12! Spiroketones.................................................................................................. 13! Perhydropyrrolizines..................................................................................... 14! WW Domain Ligands ................................................................................... 14! Natural and library screened inhibitors......................................................... 15! Juglone .......................................................................................................... 15! Pepticinnamin E derivatives ......................................................................... 16! PiB and its derivatives .................................................................................. 17! Conclusions................................................................................................... 17! References..................................................................................................... 18! Chapter 2: "-Ketoamide inhibitors ................................................................................. 27! Part A: "-Ketoamide Inhibitory Activities and Synthetic Methods ................ 27! 2.1. Introduction............................................................................................ 27! 2.2. "-Ketoamide inhibitory activities.......................................................... 29! 2.2.1. HIV and FIV protease inhibitors........................................................ 29! 2.2.2. Serine protease inhibitors.................................................................... 31! 2.2.3. Cysteine protease inhibitors................................................................ 41! 2.2.4. Aminopeptidase inhibitors.................................................................. 46! 2.2.5. Histone deacetylase inhibitors ............................................................ 46! 2.2.6.