Therapeutic strategies for the treatment of Amyotrophic Lateral Sclerosis (ALS) with mutations in Angiogenin and Superoxide Dismutase 1 by Krishna Chaitanya Aluri Bachelor of Pharmacy, Rajiv Gandhi University of Health Sciences Master of Science in Biopharmaceutical Science, Northeastern 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, 16, 2020 Dissertation directed by Jeffrey N. Agar Professor of Chemistry and Chemical Biology 1 Dedication “When I walk, I walk with you. Where I go, you're with me always.” ― Alice Hoffman, The Story Sisters, 2009. I dedicate this work to my family and friends. A special thanks to my parents Aluri Gopal Rao and Mallela Visalakshi; brother Venkata Vishnuvardan Aluri and wife Prathyusha Gundlapally for their inspiration and words of encouragement. I also dedicate this work to my friends Husain Attarwala, Arnik Shah, Aatman Doshi, Kirtika Asrani, Smith Patel and Ranjitha Gaddipati for their support. 2 Acknowledgments I would like to express my deep and sincere gratitude to Prof. Jeffrey N. Agar for continuous support and guidance. I would like to thank my fellow labmates Dr. Joseph P. Salisbury, Dr. Daniel P. Donnelly, Md. Amin Hossain, Durgalakshmi Sivasankar, and Nicholas D. Schmitt for their contributions and thoughtful discussions. I thank my thesis committee Prof. Alexander Ivanov, Prof. Ke Zhang, Dr. Jared R. Auclair, Dr. Roman Manetsch, and Dr. Saeho Chong for their insightful comments, time, and encouragement. I would like to thank our collaborators Dr. Jochen H.M. Prehn, Dr. Roman Manetsch, Dr. Adam Ekenseair, Matthew G. Dowgiallo, Brandon C. Miller, and Ninad Kanetkar for their contributions. Finally, I would like to thank my colleagues from Alnylam Pharmaceuticals for their support and encouragement throughout the process of completing my dissertation and organization Alnylam Pharmaceuticals for providing tuition assistance. 3 Abstract of Dissertation Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disorder. The cause of ALS is not completely understood. About 0.5–1% of ALS cases are associated with mutations in the angiogenin (ANG). These mutations are thought to cause disease through a loss of ANG function, but this hypothesis has not been evaluated statistically. In addition, the potential for ANG to promote disease has not been considered. With the goal of better defining the etiology of ANG- ALS, we assembled all clinical onset and disease duration data and determined if these were correlated with biochemical properties of ANG variants. Loss of ANG stability and ribonuclease activity were found to correlate with early ALS onset, confirming an aspect of the prevailing model of ANG-ALS. Conversely, loss of ANG stability and ribonuclease activity correlated with longer survival following diagnosis, which is inconsistent with the prevailing model. These results indicate that functional ANG appears to decrease the risk of developing ALS but exacerbate ALS once in progress. These findings are rationalized in terms of studies demonstrating that distinct mechanisms contribute to ALS onset and progression and propose that ANG replacement or stabilization would benefit pre-symptomatic ANG-ALS patients. However, this study challenges the prevailing hypothesis that augmenting ANG will benefit symptomatic ANG-ALS patients. Instead, our results suggest that the silencing of ANG activity may be beneficial for symptomatic ALS patients. ANG has an in vivo half-life of less than 2 hours, to use ANG as prophylactic for ANG-ALS multiple doses of ANG are required daily. To mitigate this problem, we evaluated cyclic thiosulfinates as a novel class of compounds for hydrogel synthesis to encapsulate ANG. We used cyclic thiosulfinates to cross-link PEG-thiol monomers and evaluated the safety of these hydrogels 4 in vitro. Using alkylated bovine serum albumin as a surrogate to ANG (which has no free cysteines to cross-link) we demonstrated diffusion mediated sustained protein release. Finally, 2% of ALS cases were associated with mutations in superoxide dismutase 1 (SOD1). SOD1 is a homodimeric protein, in ALS-SOD1 variants the dimer destabilizes forming monomers which are prone to aggregation and are associated with toxicity. Previous studies demonstrated dimer stabilization as a viable therapeutic strategy but the cross-linkers used often form dead-end modifications with lone thiols making them harmful. Using alpha and beta lipoic acid (routinely used as dietary supplements) we demonstrated cyclic disulfides and cyclic thiosulfinates can efficiently cross-link SOD1 monomers while avoiding dead-end modifications of lone thiols. 5 Table of Contents Dedication ................................................................................................................................................... 2 Acknowledgments....................................................................................................................................... 3 Abstract of Dissertation .............................................................................................................................. 4 Table of Contents ........................................................................................................................................ 6 List of Tables .............................................................................................................................................. 9 Table of Figures ........................................................................................................................................ 10 1. Introduction ........................................................................................................................................... 12 1.1 Amyotrophic Lateral Sclerosis .................................................................................................. 12 1.2 Epidemiology in ALS ................................................................................................................ 18 1.3 Role of angiogenin in ALS ........................................................................................................ 19 1.4 Controlled protein delivery and cross-linkers for hydrogel synthesis ....................................... 20 2. Loss of angiogenin function is related to earlier ALS onset and paradoxical increase in ALS duration ................................................................................................................................................................... 22 2.1 Statement of Contribution ......................................................................................................... 23 2.2 Introduction ............................................................................................................................... 24 2.2.1 Structure and function of angiogenin ..................................................................................... 24 2.2.2 Identification of angiogenin as ALS risk factor and current hypothesis ................................ 28 2.2.3 Physicochemical properties of proteins and role in disease pathophysiology ....................... 29 2.2.4 Statistical methods.................................................................................................................. 30 6 2.3 Results ....................................................................................................................................... 38 2.3.1 Loss of ANG stability correlates with faster ALS onset ........................................................ 42 2.3.2 Loss of ANG stability and ribonuclease activity correlate with longer ALS duration .......... 45 2.3.3 Additional hypothesis testing ................................................................................................. 49 2.3.4 Preclinical validation of a possible deleterious effect of ANG post-ALS onset. ................... 59 2.4 Discussion .................................................................................................................................. 60 3. Cyclic Thiosulfinates as a Novel Class of Disulfide Cleavable Cross-linkers for Facile Hydrogel Synthesis. .................................................................................................................................................. 67 3.1 Statement of contributions ......................................................................................................... 68 3.2 Introduction ............................................................................................................................... 69 3.2.1 Hydrogels ............................................................................................................................... 69 3.2.2 Crosslinkers in Hydrogel synthesis ........................................................................................ 70 3.2.3 Cyclic Thiosulfinates.............................................................................................................. 71 3.3 Results and Discussion .............................................................................................................. 72 3.3.1 Synthesis of 1,2-dithiane-1-oxide .......................................................................................... 72 3.3.2 Cross-linking of 4-arm PEG thiol (PEG-4SH) ......................................................................