The Development of Novel Cysteine Cross-Linkers and Their Application Towards Neurodegenerative Disorders
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The Development of Novel Cysteine Cross-linkers and Their Application Towards Neurodegenerative Disorders by Daniel Patrick Donnelly B.A. in English Literature, New York University B.S. in Chemistry, Salem State University A dissertation submitted to The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Doctor of Philosophy April 9th, 2019 Dissertation directed by Jeffrey N. Agar Associate Professor of Chemistry and Chemical Biology & Pharmaceutical Sciences 1 Dedication To my mother, Loretta Yannaco, my father, Robert Jude Donnelly (Jan 26, 1955-Dec 3, 2015), my sister and brother Claire Donnelly and Nicholas Rieber, and my soon-to-be wife, Rebecca Towers for your constant support over the years. I truly would not be where I am today without you. Thank you. 2 Acknowledgements I must first acknowledge my advisor, my mentor, my boss, and my friend, Jeffrey N. Agar, for his constant support and guidance throughout my Ph.D. The training, skillset, and knowledge I have gained under his mentorship is immeasurable. Jeff taught me that good research is thorough research, that publishing in high-impact journals is tedious but worth it, and that scientific problems can be approached from a number of angles. I am a better scientist as a result of his mentorship. I would like to thank the current and former Agar Lab members: Dr. Catherine M. Rawlins, for being an exceptional colleague, collaborator, and, most importantly, friend; Dr. Jeniffer V. Quijada, for being a mentor, teacher, and friend throughout my first years at Northeastern University; Nicholas D. Schmitt, for always being critical of our data and our writing and, consequently, making me a better scientist; Dr. Joseph P. Salisbury, for paving the way for my project and always allowing me to discuss ideas and problems throughout the years; Md Amin Hossain, for being an exceptional and supportive mentee; Richa Sarin, for being a friend in the lab and always offering your knowledge and constructive feedback; Krishna Aluri, for taking our project to new areas of chemistry; and Jeremy B. Conway for continuously coming back to the Agar Lab and supporting my research. You all have made my time at Northeastern University that much better. 3 Thank you to my collaborators, committee members, and mentors: Dr. Jared R. Auclair, for inspiring me to be a better experimentalist and giving me unwavering access to your resources and your knowledge; Dr. Steven A. Lopez, for teaching me a side of chemistry I thought I would never understand and being patient throughout the learning process; Dr. Roman Manestch, for diving headfirst into our project and always supporting my research; Dr. Alexander R. Ivanov, for being a welcoming face in the Barnett Institute and offering resources and support in the completion of my project. I would also like to thank my collaborators from the Consortium for Top-Down proteomics (of which there are too many to name). A special thanks goes to Jeremy Wolff of Bruker Daltronics for teaching me the ins and outs of the FT-ICR MS and supporting my research. 4 Abstract of Dissertation Neurodegenerative diseases often result from the aggregation of destabilized proteins in the central nervous system caused by genetic mutations or chemical modifications. Mutations in the gene responsible for the expression of Cu/Zn superoxide dismutase, for example, lead to proteins with aberrant conformations and are implicated in 2-7% of all amyotrophic lateral sclerosis (ALS) cases. These mutations destabilize the native homodimer of SOD1 and contribute to its dissociation into toxic monomers prone to self-assemble into higher-order aggregates. The development of pharmacological chaperones, small molecules that assist in the stabilization of nascent proteins, is an accepted therapeutic strategy. Past attempts to stabilize the native dimer of SOD1 through covalent cross-linking were successful in rescuing enzymatic activity of variant proteins but utilized toxic commercial cross-linkers. In this dissertation, a new class of compounds, cyclic disulfides and cyclic thiosulfinates, is introduced. Cyclic disulfides and cyclic thiosulfinates are the first reported cysteine-specific cross-linkers that target pairs of closely-spaced cysteine residues but avoid the formation of dead-end modification on lone cysteine residues (the reason all other cysteine cross-linkers are toxic in vivo). The chapters presented here explore the cross- linking mechanism of cyclic disulfides and cyclic thiosulfinates, identify the mechanistic driving force of cyclic thiosulfinate cross-linking rate-acceleration, apply this chemistry to ALS disease models (in vitro, in cellulo, and in vivo), and present newly established protocols to analyze intact proteins and protein modifications via mass spectrometry. 5 Table of Contents Dedication ....................................................................................................................................... 2 Acknowledgements ......................................................................................................................... 3 Abstract of Dissertation .................................................................................................................. 5 Table of Contents ............................................................................................................................ 6 List of Tables .................................................................................................................................. 9 List of Figures ............................................................................................................................... 10 Abbreviations ................................................................................................................................ 15 Chapter 1 ....................................................................................................................................... 21 Introduction ............................................................................................................................... 21 1.1 Proteinopathies and Neurodegeneration ................................................................... 21 1.2 Mechanism of Seeded Protein Aggregation and Neurotoxicity ............................... 23 1.3 Amyotrophic Lateral Sclerosis ................................................................................. 25 1.4 Aggregation of Superoxide Dismutase (SOD1) and ALS ........................................ 26 1.5 Protein Stabilization and Chemical/Pharmacological Chaperones ........................... 31 1.6 Tafamidis and the stabilization of Transthyretin ...................................................... 33 1.7 Identification of compounds that stabilize the SOD1 dimer ..................................... 36 1.8 Stabilization of SOD1 with homobifunctional maleimides ...................................... 39 1.9 Cyclic disulfides.............................................................................................................. 41 Chapter 2 ....................................................................................................................................... 44 Cyclic Thiosulfinates and Cyclic Disulfides Selectively Cross-Link Thiols While Avoiding Modification of Lone Thiols ..................................................................................................... 44 2.0 Statement of Contribution ............................................................................................... 45 2.1 Abstract ........................................................................................................................... 46 2.2 Introduction ..................................................................................................................... 47 2.3 Results ad Discussion ..................................................................................................... 51 2.4 Conclusion ...................................................................................................................... 55 2.5 Methods........................................................................................................................... 56 Chapter 3 ....................................................................................................................................... 69 Nucleophilic substitution reactions of cyclic thiosulfinates are accelerated by hyperconjugative interactions ................................................................................................................................ 69 3.0 Statement of Contribution ............................................................................................... 70 3.1 Abstract ........................................................................................................................... 71 3.2 Introduction ..................................................................................................................... 71 6 3.3 Results and Discussion ................................................................................................... 73 3.4 Methods........................................................................................................................... 86 3.5 Conclusion ...................................................................................................................... 87 Chapter 4 ......................................................................................................................................