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Unfolding the Hsp90 Foldasome: Structure-Activity Relationship Studies on EGCG and Development of Isoform-Selective Inhibitors

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Unfolding the Hsp90 Foldasome: Structure-Activity Relationship Studies on EGCG and Development of Isoform-Selective Inhibitors Unfolding the Hsp90 Foldasome: Structure-Activity Relationship Studies on EGCG and Development of Isoform-Selective Inhibitors By Anuj Khandelwal 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 ________________________________ Ryan A. Altman, Ph.D. ________________________________ Apurba Dutta, Ph.D. ________________________________ Paul R. Hanson, Ph.D. ________________________________ Rick T. Dobrowsky, Ph.D. Date Defended: June 9th, 2016 The Dissertation Committee for Anuj Khandelwal certifies that this is the approved version of the following dissertation: Unfolding the Hsp90 Foldasome: Structure-Activity Relationship Studies on EGCG and Development of Isoform-Selective Inhibitors ________________________________ Brian S. J. Blagg, Ph.D. Chairperson Date approved: June 13th, 2016 ii Abstract The 90 kDa heat shock proteins (Hsp90) are critical for the maintenance of cellular homeostasis and mitigate the effects of cellular stress and therefore, play an important role in cell survival. Hsp90, as a molecular chaperone, folds nascent polypeptides and denatured proteins to their biologically relevant conformations. Many of the proteins dependent upon Hsp90 are essential to the growth and proliferation of cancer cells. In fact, proteins associated with all ten hallmarks of cancer are dependent upon the Hsp90 protein folding machinery. Consequently, inhibition of Hsp90 represents a combinatorial approach for the treatment of cancer. 17 small molecule inhibitors of Hsp90 have entered clinical trials, all of which bind Hsp90 N-terminus and exhibit pan-inhibitory activity against the four Hsp90 isoforms: Hsp90, Hsp90, Grp94, and Trap1. However, lack of isoform selectivity with current clinical candidates appears detrimental as more than 20 clinical trials have failed, citing hepatotoxicity, cardiotoxicity, and peripheral neuropathy amongst other side effects. Additionally, pan-inhibition of Hsp90 induces the pro- survival heat shock response, requiring the escalation of patient doses to overcome increased Hsp90 expression. Therefore, alternative approaches for Hsp90 modulation are highly sought after. Isoform-selective inhibition of Hsp90 provides an opportunity to address the aforementioned detriments associated with pan-Hsp90 N-terminal inhibitors. Hydrolysis of ATP by the N-terminal nucleoside binding pocket is required for the maturation of client protein substrates, and all four Hsp90’s share >85% identity within this region. Consequently, the discovery of isoform-selective inhibitors has been challenging. Described herein is the rationale for development of isoform selective inhibitors and the identification of the first isoform selective inhibitors of Hsp90 and Hsp90-isoforms. iii Unlike the N-terminus, inhibition of the Hsp90 C-terminus does not induce the heat shock response and hence, C-terminal inhibitors manifest the desired cytotoxic affect against cancer cells. However, absence of a co-crystal structure and lack of lead compounds, have resulted in limited success towards the development of Hsp90 C-terminal inhibitors. Recently, EGCG, a green tea polyphenol, was shown to bind at the C-terminus of Hsp90. Structure activity relationships studies were conducted on EGCG for improved Hsp90 inhibition and are also presented. iv Acknowledgments I am extremely grateful to those who helped me throughout my graduate career. First, I would like to thank my advisor Dr. Brian S. J. Blagg to giving me the opportunity to work under his supervision. As a mentor, Brian allowed me to work independently but was always available to provide guidance, knowledge, motivation and inspiration when needed. I would like to acknowledge my family for their undying motivation and encouragement. I am beyond grateful to my parents as they continuously believed in me and supported my decision to pursue a career in research. I would not be where I am today without their support. I am also grateful to my wife Anshul Khandelwal for supporting my passion for research and showing great understanding as I finished my graduate career. I would also like to thank my fellow lab members Leah Forsberg, Vincent Crowley, Sanket Mishra, and Caitlin Kent, who were a great resource of scientific wisdom but also are great friends outside of laboratory, making my experience at KU enjoyable. Outside of the Blagg laboratory, I am thankful to other department faculty members and graduate students who greatly influenced my career. Specifically, Dr. Ryan Altman for mentoring and Brett Ambler for insightful scientific discussions. I must thank the department of Medicinal Chemistry for the exemplary training I received during my graduate career. I would also like to thank Dr. Apurba Dutta, Dr. Paul Hanson, Dr. Ryan Altman, and Dr. Rick Dobrowsky for serving on my committee and providing thoughtful suggestions. I dedicate this work to my uncle Subhash Khandelwal (1965-2010), who always inspired and motivated me to follow my dreams. v Table of Contents Chapter I Hsp90 and Its Natural Product-Based Inhibitors I. 1. Introduction……...………….………………….…………………………...………………..1 I.2. Hsp90 Structure and Function………………………….……………………………………..2 I.2.1 Hsp90 Chaperone Cycle……………………………..…….………………………....3 I.2.2 Inhibition of Hsp90 Chaperone Cycle………………………………………………..3 I.3. Hsp90 Inhibitors…………………..…………………………………………………………...6 I.3.1. N-Terminal inhibitors………………..………………………...……………………6 I.3.1.1 Geldanamycin-Based Inhibitors…………………………………………....6 I.3.1.2. Radicicol-Based Inhibitors……………………………………………….10 I.3.1.3. Chimeric Inhibitors………………………………………………………13 I.3.1.4. Purine-based Inhibitors…………………………………………………..15 I.3.1.5. Induction of Heat Shock Response……………………………………….17 I.3.2. C-Terminal Inhibitors…………………………………………..………………….18 I.3.2.1. Discovery of Hsp90 C-terminal Binding Site…………………………….18 I.3.2.2. Novobiocin……………………………………………………………….19 I.3.2.3. Silybin……………………………………………………………………21 I.3.3. Disruptors of Hsp90 Interaction with Co-Chaperones and Client Proteins…………22 I.3.3.1. Celastrol………………...………………………………………………..22 I.3.3.2. Gedunin…………………………………………………………………..24 I.3.3.3. Cruentaten A……………………………………………………………..24 I. 4. Conclusions & Future Direction……………………………………………………………..25 I. 5. References……..…………………………………………………………………………….26 vi Chapter II Synthesis and Structure−Activity Relationships of EGCG Analogues II.1. Introduction to EGCG………………………………………………………………………39 II.1.1. Binding of EGCG to Hsp90……………………………………………………….41 II.2. Proposed EGCG Analogues…………………………………………………………………42 II.3. Synthesis and Evaluation of Series I and Series II EGCG Analogues……………………….44 II.3.1 Synthesis of Series I Analogues……………………………………………………44 II.3.2. Synthesis of Series II Analogues…………………………........………………….46 II.3.3. Biological Evaluation of Series-I and Series-II Analogues……………….……….46 II.4. Synthesis and Evaluation of Series III Analogues…………………………………………..48 II.4.1. Syntheses of Series IIIa-c Analogues……………………………………..……….49 II.4.2. Biological Evaluation of Series IIIa-c Analogues…………………………….…..50 II.4.3. Synthesis of Series III-d Analogues……………………………………………....52 II.4.4. Biological Evaluation of Series III-d Analogues………………………………..…52 II.5. Optimization of Linker……………………………………………………………………...54 II.5.1. Synthesis of Amide Linker Analogues…………………………………………....54 II.5.2. Biological Evaluation of Amide Linker Analogues………………………………54 II.6. Western Blots Analyses…………………………………………………………………….55 II.7. Conclusion and Future Directions…………………………………………………………..57 II.8. Methods and Experiments…………………………………………………………………...58 II.9. References…………………………………………………………………………………153 Chapter III The Development of Grp94-Selective Inhibitors III.1. Introduction……………………………………………………………………………….160 vii III.2. Grp94……………………………………………………………………………………...161 III.3. Ligand Occupation of Grp94 N-Terminal ATP-Binding Pocket…………………………164 III.3.1 Binding of Geldanamycin to Grp94……………………………………………...164 III.3.2 Binding of NECA to Grp94……………………………………………………...165 III.3.3. Binding of Radamide Grp94…………………………………………………….168 III.4. Development of Grp94-Selective Inhibitors………………………………………………168 III.4.1 Development of Radamide Analogues as Grp94 Inhibitor……………………...169 III.4.1.1. Synthesis of Radamide Analogues…………………………………….170 III.4.1.2. Evaluation of Radamide Analogues…………………………………..171 III.4.2. Development of BnIm Analogues as Grp94 Inhibitors………………………….172 III.4.2.1. Synthesis of BnIm Analogues…………………………………………173 III.4.2.2. Evaluation of BnIm Analogues………………………………………..173 III.4.3. Development of Resorcinol-Isoindoline Class of Grp94 Inhibitors………..……176 III.4.3.1 Syntheses of Resorcinol-Isoindoline Analogues………………………177 III.4.3.2. Evaluation of Resorcinol-Isoindoline Class of Grp94 Inhibitors……...178 III.5. Conclusions and Future Directions………………………………………………………..181 III.6. Methods and Experiments………………………………………………………………...182 III.7. References………………………………………………………………………...............204 Chapter IV Development of the First Hsp90-Selective N-Terminal Inhibitor IV.1. Introduction……………………………………………………………………………….211 IV.1.1. Hsp90…………………………...……………………………………………. 213 IV.2. Differences in N-Terminal ATP-Binding Pocket of Hsp90 Isoforms……………………..214 IV.3. Development of Resorcinol Analogues Modified at the 4-Position……………………….215 viii IV.3.1. Design of Resorcinol Analogues Modified at the 4-Position for Selective Hsp90- Inhibition………………………………………………………………………………..215 IV.3.2. Synthesis
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