Investigation of the Hsp90 C-terminal Binding Site, Novel Inhibitors and Isoform-Dependent Client Proteins By Laura B. Peterson Submitted to the graduate degree program in Medicinal Chemistry and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________ Brian S. J. Blagg, Ph.D. Chairperson ________________________________ Thomas E. Prisinzano, Ph.D. ________________________________ Michael F. Rafferty, Ph.D. ________________________________ Jon A. Tunge, Ph.D. ________________________________ Rick T. Dobrowsky, Ph.D. Date Defended: May 30th, 2012 The Dissertation Committee for Laura B. Peterson certifies that this is the approved version of the following dissertation: Investigation of the Hsp90 C-terminal Binding Site, Novel Inhibitors and Isoform-Dependent Client Proteins ________________________________ Brian S. J. Blagg, Ph.D. Chairperson Date approved: June 1st, 2012 ii Abstract The heat shock proteins represent an important class of pro-survival proteins that are intimately involved in cell survival, adaptation to cellular stress, and protein management. Heat shock protein 90 kDa (Hsp90) is a molecular chaperone responsible for the post-translational maturation of nascent polypeptides. Many of the Hsp90-dependent client proteins are involved in oncogenic processes, and accordingly, Hsp90 has emerged as a promising target for anti-cancer therapies. Unfortunately, the clinical evaluation of Hsp90 inhibitors has been met with dosing, scheduling, and toxicity issues. The Hsp90 inhibitors that have reached clinical trials bind to the Hsp90 N-terminal ATP-binding site and demonstrate pan-Hsp90 inhibition, as they bind to and inhibit all four human Hsp90 isoforms. This characteristic may rationalize the undesired toxicities related to Hsp90 inhibition. Interestingly, the identification and characterization of isoform specific client proteins has not been extensively explored. In addition, N-terminal Hsp90 inhibition results in the induction of the heat shock response, whereby the expression of Hsp90 and other heat shock proteins is induced. This attribute of N-terminal inhibitors results in the clinically observed dosing and scheduling detriments as levels of Hsp90 increase with administration of the drug. Described herein is the design, synthesis and biological evaluation of novel Hsp90 inhibitors that avoid the above mentioned therapeutic liabilities of currently known Hsp90 inhibitors. The identification and characterization of an Hsp90-isoform dependent client protein and an Hsp90-isoform selective inhibitor is also presented. iii Acknowledgements For me, graduate school provided a chance for personal growth and a platform to challenge myself. Without the many mentors, family members, and friends I have made and maintained while at the University of Kansas, this experience would not have been nearly as fulfilling. Those people deserve my recognition and gratitude. First, thank you to my advisor, Dr. Brian S. J. Blagg. Brian was a constant source of knowledge, motivation, and inspiration and he helped me to grow as a scientist. I would also like to thank the other members of the Blagg Laboratory of all who greatly influenced my graduate career. Specifically, Adam Duerfeldt was an amazing colleague and friend who engaged in insightful discussions and generally made my time here more enjoyable. Outside of the Blagg Laboratory, thank you to Dr. Thomas Prisinzano and his lab members, all of whom greatly impacted my graduate school career. I would also like to thank Chrisostomos Prodromou for mentoring me during my research rotation at the Institute of Cancer Research. Besides support from my KU network, I would never be where I am today without my family. To my Mom and Dad who had high expectations for me and never let me settle for mediocrity and to my brother who is a great role model, friend, and my ultimate competition – thank you! Lastly, to my future husband, Alexander Grenning - who is my best friend and always pushes me to be better, his support and friendship means a tremendous amount to me. In addition to the amazing people I have had the opportunity to meet and work with over the last five years, I must thank the Department of Medicinal Chemistry, the Smissman Fellowship, the NIH Dynamic Aspects of Chemical Biology Training Grant, and the American Chemical Society Division of Medicinal Chemistry Pre-doctoral Fellowship for financial support. iv Table of Contents: Chapter I Introduction to Hsp90 Structure, Function, and Inhibition I. Introduction ..................................................................................................................................1 II. Heat Shock Protein 90 .................................................................................................................1 A. Hsp90 Structure......................................................................................................................2 B. Hsp90 Function ......................................................................................................................4 III. Heat Shock Protein 90 Inhibitors ...............................................................................................9 A. Therapeutic Opportunities ....................................................................................................10 A.1. Cancer ..........................................................................................................................10 A.2. Neurodegenerative Diseases ........................................................................................12 B. N-Terminal Inhibitors ..........................................................................................................16 C. C-Terminal Inhibitors ...........................................................................................................18 D. Other Inhibitors ....................................................................................................................19 IV. Clinical Detriments and Liabilities of Hsp90 Inhibition .........................................................20 A. Heat Shock Induction ...........................................................................................................20 B. Toxicities ..............................................................................................................................22 C. Other Considerations ............................................................................................................23 V. Conclusions and Future Directions of Hsp90 Inhibition ..........................................................24 VI. References................................................................................................................................25 Chapter II Exploration of the Hsp90 C-terminal Binding Site I. Introduction ................................................................................................................................37 A. C-terminal Domain Structure ...............................................................................................38 B. C-terminal Domain Function and Modulation .....................................................................41 v II. Development of a CoMFA for Hsp90 C-Terminal Inhibitors...................................................45 III. Development of a C-terminal Binding Site Model ..................................................................50 A. Identification of the Novobiocin Binding Site in Hsp90’s C-terminal Domain ..................50 B. Modeling of Novobiocin’s Conformation ............................................................................52 C. Modeling of the Hsp90 Novobiocin Binding Site ................................................................53 D. Correlation to Observed Effects Resulting from C-Terminal Inhibition .............................57 IV. Synthesis and Evaluation of a Series of Triazole Containing Novobiocin Analogues............60 A. Rationale for the Development of Triazole Containing Novobiocin Analogues .................60 B. Synthesis of Triazole Containing Novobiocin Analogues ...................................................61 C. Biological Evaluation of Triazole Containing Novobiocin Analogues ...............................63 V. Conclusions and Future Work...................................................................................................66 A. Structure Based Drug Design of Novel C-terminal Inhibitors .............................................67 B. Progress towards a C-terminal Inhibitor Co-Crystal Structure ............................................68 VI. Materials and Methods ............................................................................................................69 A. Development of a CoMFA for Hsp90 C-Terminal Inhibitors .............................................69 B. Identification of the Novobiocin Binding Site in Hsp90’s C-terminal Domain ..................75 C. Synthesis and Evaluation of a Series of Triazole Containing Novobiocin Analogues ........76 VII. References ..............................................................................................................................99 Chapter III Biological Evaluation of Isoform Selective Hsp90 Inhibitors and Characterization of Isoform Dependent Client Proteins I. Introduction ..............................................................................................................................104