DESIGN, SYNTHESIS, AND EVALUATION OF CYSTEINE PROTEASE INHIBITORS A Dissertation Presented to The Academic Faculty By Sylvia Shadinger Bridges In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy in Chemistry Georgia Institute of Technology August, 2008 Copyright © Sylvia Shadinger Bridges 2008 DESIGN, SYNTHESIS, AND EVALUATION OF CYSTEINE PROTEASE INHIBITORS Approved by: Dr. James C. Powers, Advisor Dr. Donald Doyle School of Chemistry and School of Chemistry and Biochemistry Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Jonathan Glass Dr. Wendy L. Kelly Department of Neurology School of Chemistry and Emory University Biochemistry Georgia Institute of Technology Dr. Sheldon May School of Chemistry and Date Approved: May 23, 2008 Biochemistry Georgia Institute of Technology DEDICATION For my husband, Ben T. Bridges, and For my son, Wilson Bridges. iii ACKNOWLEDGEMENTS There are many people I have to thank for gifting me the tools necessary to complete this journey. First and foremost is Dr. James C. Powers, my advisor. Dr. Powers expertly walked the fine line between giving me the advice I needed to successfully complete my projects and prodding me to figure out my dilemmas myself. Both teaching methods have left me with skills that will serve me for a lifetime. His demonstration of how to develop and carryout a research project has been vital to my education. I would also like to thank my thesis committee members, Dr. Donald Doyle, Dr. Jonathan Glass, Dr. Wendy Kelly, and Dr. Sheldon May. Their comments and thoughtful evaluation of my work were vital to my success. For their time and energy spent reading and reviewing my work, I am grateful. To my co-workers in the lab, my day-to-day helpers and companions, I thank you for the hours spent teaching me and allowing me to teach you in turn. Juliana Asgian, Karen Ellis James, Brian Rukamp, Karrie Rukamp, Özlem Doğan Ekici, Marion Götz, and Amy Campbell were all essential in shaping both my research and who I am as a scientist. I am also thankful for research scientist Zhao Zhao Li for her excellent advice and knowledge in organic synthesis. For the many hours of commiseration, advice, training, and friendship I am very grateful. iv My success is in large part due to the loving support of my family. For my parents, Dr. and Mrs. Richard and Marilyn Shadinger, who raised me to believe that I could achieve anything I set my mind to, and proudly supported my dream of becoming a chemist, I am eternally grateful. My husband, Ben T. Bridges has been my constant supporter during this academic journey. His belief in me and my abilities has been the motivation that has pushed me past the bumps in the road to be the best that I can be. For his loving support I will always be thankful. And to my son, Wilson Bridges, whose sunny smile and joy for life has given me extra incentive to make this world a better place, Mommy loves you. v TABLE OF CONTENTS ACKNOWLEDGMENTS ……………………………………………………………….iv LIST OF TABLES………………………………………………………………………..xi LIST OF FIGURES…………...………………………………………………………....xii LIST OF ABBREVIATIONS...………………………………………………………....xiv SUMMARY………………………………...……………………………………………xx CHAPTER ONE. Introduction…….………………………………………..…………....1 Proteases…………………………………………………………………………..1 Protease Clans…………………………….……………………………………….1 Protease Nomenclature……………………………………………………………3 Cysteine Proteases………………………………………………………………...4 Clan CA Proteases and Specificities………………………………………………5 Papain……………………………………………………………………...5 Calpains……………………………………………………………………6 Cathepsin B………………………………………………………………..6 Clan CD Proteases and Specificities………………………………………………7 Caspases…………………………………………………………………...7 Gingipains…………………………………………………………………7 Clostripain…………………………………………………………………7 vi Inhibitor Design…………………………………………………………………...7 Irreversible Inhibitor Kinetics……………………………………………………..8 CHAPTER TWO. Aza-Peptide Epoxide and Epoxide Inhibitors of Calpains and other Clan CA Proteases ……………………………….…….....12 Introduction………………………………………………………………………12 Calpain Structure………………………………………………………...13 Calpain Inhibitors………………….……………………………………..13 Inhibitor Design………………………………………………………………….17 The Aza-Peptide Epoxide Design………………………………………..17 Epoxide Stereochemistry………………………………………………...19 Synthesis…………………………………………………………………………20 Results and Discussion…………………………………………………………..22 New Epoxide Inhibitor Design…………………………………………………..25 Synthesis ………………………………………………………………………...26 Results and Discussion…………………………………………………………..28 Conclusion……………………………………………………………………….31 Experimental……………………………………………………………………..32 Enzyme Assays…………………………………………………………..32 Calpains I and II………………………………………………….32 Cathepsin B………………………………………………………32 Papain…………………………………………………………….33 PC12 Cellular Assay with Paclitaxel…………………………………….33 Materials and Methods………………………………………………..….34 vii CHAPTER THREE. Solid Phase Synthesis of Aza-Peptide Michael Acceptor Inhibitors of Caspases……………...……………………….…61 Introduction………………………………………………………………………61 Caspases………………………………………………………………….61 Caspase Specificity………………………………………………………64 Caspase Structure………………………………………………………...65 Caspases in Diseases……………………………………………………..68 Caspase Inhibitors………………………………………………………..68 Caspase Inhibitors as Therapeutic Agents……………………………….69 Inhibitor Design………………………………………………………………….70 Aza-Peptide Michael Acceptor Inhibitors……………………………….70 New Aza-Peptide Michael Inhibitors: Scanning the P3’ Subsites of Caspases………………………………………………………………….72 Synthesis…………………………………………………………………………74 Results and Discussion…………………………………………………………..77 Selectivity of Aza-Peptide Michael Acceptor Inhibitors among Caspases………………………………………………….80 Caspases -3 and –7……………………….………………………80 Caspases -6 and –8……………………………………………….80 Stability of the Aza-Peptide Michael Acceptor Inhibitors……………….80 Conclusion…………………………………………………………………….…81 Experimental…………………………………………………………………..…81 Enzyme Assays…………………………………………………………..81 Caspase-3………………………………………………………...82 viii Caspase-6………………………………………………………...82 Caspase-7………………………………………………………...82 Caspase-8………………………………………………………...83 Materials and Methods…………………………………………………...83 CHAPTER FOUR. Aza-Peptide Epoxides and Michael Acceptors as Inhibitors of Gingipains R and K and Clostripain.………………………….92 Introduction………………………………………………………………………92 Gingipains………………………………………………………………..92 Gingipain Specificity…………………………………………….93 Gingipain Structure………………………………………………93 Gingipain Inhibitors……………………………………………...96 Clostripain………………………………………………………………..96 Clostripain Inhibitors…………………………………………….96 Inhibitor Design………………………………………………………………….97 Results and Discussion…………………………………………………………..98 Gingipain K……………………………………………………………..101 Gingipain R……………………………………………………………..103 Clostripain………………………………………………………………104 Specificity………………………………………………………………104 Conclusion……………………………………………………………………...105 Experimental……………………………………………………………………105 Enzyme Assays…………………………………………………………105 Gingipain R……………………………………………………..106 ix Gingipain K……………………………………………………..107 Clostripain………………………………………………………107 REFERENCES…………………………………………………………………………108 VITA…….……………………………………………………………………………...116 x LIST OF TABLES Table 2.1 Representative Calpain Inhibitors From the Literature………………….14 Table 2.2 Inhibition of Clan CA Proteases by Aza-Peptide Epoxides……………...23 Table 2.3 Inhibition of Clan CA Proteases by Aza-EP460 Analogs……………….24 Table 2.4 Inhibition of Clan CA Proteases by EP460 Analog Inhibitors…………..29 Table 3.1 Sequence Specificity of Caspases………………………………………..65 Table 3.2 Nonselectivity of Caspase Inhibitors…………………………………….69 Table 3.3 Representative Aza-Peptide Michael Acceptor Inhibitors for Caspases……………………………………………………………...71 Table 3.4 Inhibition of Caspases by Aza-Peptide Michael Acceptor P3’ Inhibitors…………………………………………………………….78 Table 4.1 Inhibition of Gingipains R and K and Clostripain by Aza-Peptide Epoxide and Michael Acceptor Inhibitors………………..……………...99 xi LIST OF FIGURES Figure 1.1 Cysteine Protease Clans…………………………………………………...2 Figure 1.2 Protease Nomenclature……………………………………………………4 Figure 1.3 Cysteine Protease Hydrolysis Mechanism………………………………...5 Figure 2.1 Structures of AK295, E64, and EP460…………………………………..15 Figure 2.2 Mechanism of Inhibition of Cysteine Proteases by Epoxide Inhibitors………………………………………………………..16 Figure 2.3 Aza-Peptide Epoxide Design…………………………………………….17 Figure 2.4 Aza-Peptide Epoxide Design for Calpains………………………………18 Figure 2.5 Aza-Ep460 Design……………………………………………………….19 Figure 2.6 Synthesis of Aza-Peptide Epoxide Inhibitors for Calpains………………21 Figure 2.7 Synthesis of Aza-EP460 Inhibitors for Calpains………………………...22 Figure 2.8 EP460 Inhibitor with Proposed Modification Site……………………….26 Figure 2.9 Synthesis of Epoxide Inhibitors Which Are Analogs of EP460…………27 Figure 3.1 Known Apoptotic Pathways Involving Caspases………………………..63 Figure 3.2 General Caspase Substrate Binding……………………………………...67 Figure 3.3 Aza-Peptide Michael Acceptor Inhibitors for Caspases…………………71 Figure 3.4 Fmoc Aza-Peptide Michael Acceptor Unit for Solid Phase Inhibitor Synthesis…………………………………………………………………73 Figure 3.5 Aza-Peptide Michael Acceptor Inhibitor Design………………………...74 Figure 3.6 Synthesis of Fmoc Aza-Peptide Michael Acceptor Unit for Solid Phase Inhibitor Synthesis………………………………………75 xii Figure 3.7 General Solid Phase Synthesis Method with the Wang Resin…………...76 Figure 4.1 Substrate Binding Mode of Gingipain R………………………………...95 Figure 4.2 Aza-Peptide Epoxide and Michael Acceptor Inhibitor Design for Gingipains and Clostripain…………………………………..98 Figure 4.3 Faces of Possible Thiol Attack for Double Bonds and Epoxides……………………………………………………………102