Development of Chemical Tactics to Study Fundamental Aspects of Pathogenic Factors Found in Neurodegenerative Diseases
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Disclaimer Doctoral Dissertation Development of Chemical Tactics to Study Fundamental Aspects of Pathogenic Factors Found in Neurodegenerative Diseases Juhye Kang Department of Chemistry Graduate School of UNIST 2019 Development of Chemical Tactics to Study Fundamental Aspects of Pathogenic Factors Found in Neurodegenerative Diseases Juhye Kang Department of Chemistry Graduate School of UNIST II Abstract Amyloidogenic peptides are considered central pathological factors in neurodegenerative diseases; however, their roles in the pathologies of the diseases have not been fully understood. Amyloidogenic peptides have indicated their multiple faces on formation, aggregation, and accumulation that portray the complexity of the intrinsically disordered protein stemming from their convoluted structure and heterogeneous nature. Although the latest research suggests oligomeric forms of amyloidogenic peptides as toxic species towards neurodegeneration, their direct involvements in the pathologies and the corresponding mechanistic details remain rudimentary. Therefore, novel chemical approaches to modify amyloidogenic peptides at the molecular level would be useful in advancing our comprehension of those aspects and the subject. In Chapter 1, we briefly introduce outline and discuss possible therapeutic targets in Alzheimer’s disease. In Chapter 2, the design of an Ir(III) complex, Ir-1, is described as a chemical tool for oxidizing amyloidogenic peptides upon photoactivation and subsequently modulating their aggregation pathways. Biochemical and biophysical investigations illuminate that the oxidation of representative amyloidogenic peptides [i.e., amyloid- (A), -synuclein, and human islet amyloid polypeptide] is promoted by light-activated Ir-1, which can alter the conformations and aggregation pathways of the peptides. In Chapter 3, effective chemical strategies for modifications of A peptides implemented by a single Ir(III) complex are illustrated. Such peptide variations can be achieved by our rationally designed Ir(III) complexes leading to the significant modulation of the aggregation pathways of A40 and A42 as well as the production of toxic A species. Amyloidogenic peptides can coordinate to metal ions, including Zn(II), which can subsequently affect the peptides' aggregation and toxicity, leading to neurodegeneration. Unfortunately, the detection of metal–amyloidogenic peptide complexation has been very challenging. In Chapter 4, we report the development and utilization of a probe (A-1) capable of monitoring metal–A complexation based on Förster resonance energy transfer (FRET). Moreover, as the FRET signal of Zn(II)-added A-1 is drastically changed when the interaction between Zn(II) and A-1 is disrupted, the Zn(II)-treated probe can be used for screening a chemical library to determine effective inhibitors against metal–A interaction. Overall, we demonstrate chemical tactics for modifications of amyloidogenic peptides in an effective and manageable manner utilizing the coordination capacities and/or photophysical properties of chemical reagents. Our approaches will provide the foundation for developing effective and efficient methods for elucidating fundamental properties of pathological factors at the molecular level and assisting in identifying therapeutic candidates against neurodegenerative disorders. V Table of Contents Abstract ·········································································································· V Table of Contents ····························································································· VI List of Figures ·································································································· X List of Tables ································································································· XIII List of Schemes ······························································································ XIV List of Abbreviations ························································································ XV Chapter 1. Potential Pathological Factors in Alzheimer’s Disease 1.1. Introduction ······························································································· 2 1.1.1. Neurodegenerative Diseases ····································································· 2 1.2. Alzheimer’s Disease (AD) ·············································································· 4 1.2.1. Amyloid- (A) and Tau ········································································· 5 1.2.2. Metal Ions ·························································································· 8 1.2.3. Oxidative Stress ················································································· 19 1.2.4. Cholinesterases (ChEs) ········································································· 22 1.2.5. Additional Targets ··············································································· 24 1.3. Conclusions ····························································································· 26 1.4. Acknowledgments ····················································································· 27 1.5. References ······························································································ 27 Chapter 2. An Iridium(III) Complex as a Photoactivatable Tool for Oxidation of Amyloidogenic Peptides with Subsequent Modulation of Peptide Aggregation 2.1. Introduction ····························································································· 41 2.2. Results and Discussion ················································································ 42 2.2.1. Rational Design of Ir-1 for Oxidation of Amyloidogenic Peptides ······················· 42 2.2.2. Oxidative Modifications of Amyloidogenic Peptides by Ir-1 ····························· 43 2.2.3. Identification of Oxidation Sites in Amyloidogenic Peptides ······························ 47 2.2.4. Aggregation Behavior Influenced upon Oxidation of Amyloidogenic Peptides by Light- Activated Ir-1 ···················································································· 51 2.2.4.1. Change in the Size Distribution ························································· 51 2.2.4.2. Morphological Changes of Amyloidogenic Peptides ································ 54 2.3. Conclusions ····························································································· 54 2.4. Experimental Section ·················································································· 54 2.4.1. Materials and Methods ········································································· 55 VI 2.4.2. Synthesis of Ir-1 ················································································ 56 2.4.3. Photophysical Properties of Ir-1 ······························································ 56 2.4.4. Electrospray Ionization Ion Mobility Mass Spectrometry (ESI-IM-MS) ················ 58 2.4.5. Mass Spectrometric Analyses ·································································· 59 2.4.6. Peptide Aggregation Experiments ····························································· 59 2.4.7. 2D NMR Spectroscopy ········································································· 60 2.4.8. Gel Electrophoresis with Western Blotting (Gel/Western Blot) ··························· 60 2.4.9. Transmission Electron Microscopy (TEM) ·················································· 61 2.5. Acknowledgments ····················································································· 61 2.6. References ······························································································ 61 Chapter 3. Chemical Strategies to Modify Amyloidogenic Peptides by Iridium(III) Complexes: Coordination and Photo-induced Oxidation 3.1. Introduction ····························································································· 66 3.2. Results and Discussion ················································································ 66 3.2.1. Rational Strategies for Peptide Modifications by Ir(III) Complexes ······················ 66 3.2.2. Coordination-Dependent Photophysical Properties and Singlet Oxygen Production of Ir(III) Complexes ······················································································· 68 3.2.3. Photoirradiation-Dependent Peptide Modifications by Ir(III) Complexes ··············· 71 3.2.4. Effects of Peptide Modifications Triggered by Ir(III) Complexes on A Aggregation · 74 3.2.5. Cytotoxicity of A Species Generated upon Incubation with Ir(III) Complexes ········ 77 3.2.6. Ternary Complexation with A and Intramolecular and Intermolecular A Oxidation 79 3.3. Conclusions ····························································································· 80 3.4. Experimental Section ·················································································· 81 3.4.1. Materials and Methods ········································································· 81 3.4.2. Synthesis of Ir(III)