Amyloid fibrils results from a type of ordered polypeptide aggregation that is associated with ailments such as Alzheimer's disease (AD). Annually, millions of people in the United States alone develop and die from AD. Therefore, it is necessary to understand not only the process of amyloid formation, but also the causes of this specific type of aggregation. This study used ABeta(25-35) since it is a fragment of the Alzheimer?s peptide that behaves like the full length peptide found in patients with AD. To study the process of amyloid formation, several methods were used so that a more complete picture of the stepped aggregation process could be realized. Several oligomeric species were detected and described many of which could not have been observed without using the complete battery of methods utilized here. The oligomeric species detected included a novel 'rolled sheet' that appeared to be the immediate precursor of amyloid fibrils, and two supermolecular species that appear after amyloid fibrils were formed. In determining the causes of amyloid formation, two significant discoveries were made. First, by partial sequence randomization, truncation, and Ala scanning mutagenesis, the critical amyloidogenic region of ABeta(25-35) was found to be residues 30-35. This critical core region is important because it is thought to be the region that initiates amyloid formation, therefore knowing the residues involved in the region is a useful tool for developing methods of fibril formation prevention. Second, by inserting all naturally occurring amino acids into position 34 of ABeta(25-35), three distinct classes of variants were observed and the effect of several physiochemical properties on amyloidosis were examined. Hydrophobicity, solubility, and ?-strand propensity were found to affect aggregation to the greatest extent. Also within these two studies, our results suggest that early oligomers are the cytotoxic species as opposed to amyloid fibrils or other larger macromolecular assemblies.

<p>Therapeutic peptides have become a clinically and commercially important drug class providing novel treatment options in variety of disease areas. Today, more than 80 peptide drugs are marketed worldwide and hundreds more are in development. However, the development of peptide drugs can be hindered by their tendency to self-associate to form fibrils, an impurity that can affect potency and increase the potential for adverse immune responses in patients. Fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. From a pharmaceutical development perspective, early detection of instabilities can inform the development of mitigation strategies to minimize the risk of product failure and avoid costly delays in clinical development. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and formulations.</p> <p>The objective of this dissertation was to develop structurally modified fibrillation-resistant peptides based on a mechanistic understanding of the fibrillation process. The therapeutic peptides studied were human calcitonin (hCT), a glucagon/GLP-1 analog, and human insulin B-chain (INSB). Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) and other biophysical methods were used to provide mechanistic understanding of the intermolecular interactions and structural transitions during peptide fibrillation. Coupled with proteolytic digestion, pulsed HDX-MS of fibrillating peptides enabled identification of the residues involved in the early interactions leading to fibrillation based on their differential deuterium exchange rates. The high-resolution residue level information was used to make site-specific modifications to hCT, with phosphorylation in the central region resulting in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog under the stress conditions employed. Reversible ‘prodrug’ modifications such as phosphorylation can aid the rational design of fibrillation-resistant therapeutic peptides. Furthermore, the effects of structural modifications on peptide fibrillation were evaluated by reducing the Cys1-Cys7 disulfide bond in hCT, and by C-terminal amidation or substitution with a helix-stabilizing residue (α-aminoisobutyric acid, Aib) in the glucagon/GLP-1 analog peptide. Finally, studies of insulin B-chain probed fibrillation mechanisms of this therapeutically important peptide, contributing to our understanding of the mechanisms of insulin fibrillation with the broad goal of developing fibrillation-resistant, rapid-acting, monomeric insulin analogs. Overall, the results demonstrate that small structural changes can have significant effects on peptide fibrillation, that pulsed HDX-MS can be used to probe these effects, and that an understanding of these effects can inform the rational development of fibrillation-resistant peptide drugs. </p>