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Targeting Anti-Apoptotic Bcl-2 Proteins with Scyllatoxin-Based BH3 Domain Mimetics

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Targeting Anti-apoptotic Bcl-2 Proteins with Scyllatoxin-based BH3 Domain Mimetics A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Danushka M. Berugoda Arachchige May 2020 © 2020 Danushka M. Berugoda Arachchige. All Rights Reserved. 2 This dissertation titled Targeting Anti-apoptotic Bcl-2 Proteins with Scyllatoxin-bsed BH3 domain Mimetics by DANUSHKA M. BERUGODA ARACHCHIGE has been approved for the Department of Chemistry and Biochemistry and the College of Arts and Sciences by Justin M. Holub Associate Professor of Chemistry and Biochemistry Florenz Plassmann Dean, College of Arts and Sciences 3 ABSTRACT BERUGODA ARACHCHIGE, DANUSHKA M., Ph.D., May 2020, Chemistry Targeting Anti-apoptotic Bcl-2 Proteins with Scyllatoxin-based BH3 Domain Mimetics Director of Dissertation: Justin M. Holub Molecules that inhibit discrete protein-protein (PPIs) interactions hold immense promise to be developed as therapeutics or chemical genetics agents. The B cell lymphoma 2 (BCL-2) proteins are a family of evolutionarily-related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Proteins in the BCL-2 family are primarily categorized into three sub types: anti-apoptotic (pro-survival) proteins, pro-apoptotic (pro-death) proteins and BH3-only proteins. Overexpression of anti-apoptotic BCL-2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes or pathogenic cell survival. As a consequence, anti- apoptotic BCL-2 proteins have attracted considerable interest as therapeutic targets. Anti- apoptotic BCL-2 proteins bind to helical BH3 domains of pro-apoptotic proteins through a shallow, hydrophobic cleft. These pro-apoptotic proteins remain inactive when bound to anti-apoptotic members. BH3-only proteins are expressed under cell stress conditions to prevent interaction between ant-apoptotic BCL-2 and pro apoptotic proteins, which eventually leads to apoptosis initiation. Unfortunately, non-specific interactions between BCL-2 family members has made it difficult to elucidate specific mechanisms of BCL-2 signaling. Molecules that clearly define such interactions would be beneficial in the development of therapeutics that target specific proteins in the BCL-2 family. Scyllatoxin (ScTx) is a 31-amino acid protein isolated from scorpion venom that folds into α/β 4 structural motif stabilized by three disulfide linkages, Cys3-Cys21, Cys8-Cys26 and Cys12-Cys28. Previous work by our lab has shown that ScTx can be engineered to target anti-apoptotic BCL-2 proteins by grafting residues required for Bcl-2 recognition to the α helix of ScTx. These constructs also showed that the number of disulfides within the ScTx BH3 mimetics influence favorable Bcl-2 recognition. Notably, ScTx-BH3 domain mimetics containing three disulfides did not bind Bcl-2 in vitro, while ScTx-BH3 variants containing no disulfides bound Bcl-2 with sub micromolar affinity. These results strongly suggested that an induced-fit binding mechanism is required for favorable BH3:Bcl-2 interactions. In this work we expand on these studies by developing a library of structural variants of ScTx-Bax BH3 domain mimetics that vary in the number and position of disulfide linkages within the ScTx-BH3 sequence. In this work, we developed a library of ScTx-BH3 mimetics that contain 0, 1, 2 or 3 disulfides, surveying all possible combinations of native disulfide linkages. We generated this library using different synthetic schemes. For example, glutathione was used to generate ScTx-BH3 variants with three disulfide linkages, bis-disulfide peptides were formed using reactions with 2+ iodine and DMSO, and single disulfides were generated using Pt[(en)2Cl2] . Indeed, it is now possible to develop ScTx-BH3 proteins that contain all possible combinations of native disulfide linkages. Our results have revealed that the position and number of native disulfides have also profound influence on the proper folding of ScTx BH3 mimetics. ScTx BH3 mimetics with two or three disulfide linkages displayed similar folded structure reminiscent to wild type ScTx (wtScTx). ScTx BH3 mimetic with singly 5 disulfide between Cys8 and Cys26 of ScTx also showed similar folded structure reminiscent of wtScTx. Our results have also shown that the number and positioning of the disulfides significantly influences the ability for ScTx-BH3 domain mimetics for target Bcl-2 protein in vitro. Bis-disulfide ScTx BH3 mimetics did not target Bcl-2 efficiently while two single disulfide containing ScTx BH3 mimetics targeted Bcl-2 with sub micromolar affinities. Interestingly, these studies indicated that positioning the disulfide near the N- terminus (Cys3-Cys21) of the ScTx-BH3 helix completely abolished binding, while placing the disulfide in the middle (Cys8-Cys26) or near the C-terminus (Cys12-Cys28) of the helix resulted sub micromolar affinity for Bcl-2. Additionally, three ScTx-BH3 variants were shown efficient competing ability against natural BH3 domains for Bcl-2 in vitro. Notably, ScTx-BH3 peptides with a single disulfide at C8-C26 clearly showed favorable binding energetics compared to intrinsically disordered ScTx-BH3 peptide. Taken together, these structural variants of ScTx-Bax have provided insight into structural rearrangements and thermodynamic parameters for targeting the BH3:Bcl-2 interaction, and should facilitate the design of highly selective modulators of Bcl-2 activity. Furthermore, our results will have implications in the design and formulation of new small molecule and peptide-based therapeutics designed to target discrete BH3:Bcl-2 interactions. 6 DEDICATION I would like to dedicate this work to my parents, my family and all the past teachers. 7 ACKNOWLEDGMENTS First, I would like to acknowledge my advisor, Dr. Justin M. Holub for his support, patience, understanding, and his clear guidance. I also would like to thank my dissertation committee members, Dr. Michael Held, Dr. Jessica White and Dr. Rida Benhaddou for their time, efforts and their valuable comments to edit and improve my work. Special thank for Department of Chemistry and Biochemistry of Ohio University for my scholarships and TA assignments. I also would like to sincerely thank my lab mates: Ranju Pokhrel, Chang Xu, Nahar Khairun, Ryan Cannell, Emily Herrberg and all the past lab mates for their kindness, care, assistance, support and for creating a friendly and convenient research environment. Thanks to Dr. Michael Held group and Dr. Jennifer Hines group who always help me whenever I ask them for help. Special thanks to Ramin Rabbani from Dr. Masson’s group and Dr. Lingying Tong from Dr. Wu’s lab for assisting in my research. Finally, I would like to thank my family, my wife, and my friends for their love, support and for wishing me the success. 8 TABLE OF CONTENTS Page Abstract ...........................................................................................................................3 Dedication .......................................................................................................................6 Acknowledgments ...........................................................................................................7 List of Tables................................................................................................................. 11 List of Figures ............................................................................................................... 12 List of Abbreviations ..................................................................................................... 14 Chapter 1: Introduction .................................................................................................. 17 Protein-Protein Interactions...................................................................................... 17 PPI Categories ......................................................................................................... 19 Disease Conditions caused by Dysfunctional PPIs ................................................... 23 Targeting PPIs with Small Molecules ...................................................................... 26 Targeting PPIs using Proteins and Peptides .............................................................. 29 Synthetic and Natural Peptide Motifs as Therapeutic Agents for Targeting PPIs ...... 32 Miniature Proteins and PPIs ..................................................................................... 43 Protein Grafting on to Miniature Protein Scaffolds ................................................... 45 Potential Therapeutics from Miniature Protein Toxins ............................................. 46 Regulators of Intrinsic Apoptosis Pathway ............................................................... 48 Targeting Anti-apoptotic Bcl-2 Proteins using Scyllatoxin-based BH3 Domains ...... 51 Chapter 2: Role of single disulfide linkages in the folding and activity of Scyllatoxin based bh3 domain mimetics ........................................................................................... 55 Introduction ............................................................................................................. 55 Materials and Methods ............................................................................................. 61 Reagents and Chemicals ....................................................................................
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