Mechanism and Development of Peptide-Based Inhibitors to Human Islet Amyloid Polypeptide (hIAPP) Self-Assembly

Amyloid fibrils formed by of hIAPP1-37 (also known as amylin) has been linked to type-II diabetes mortalities and its formation was found to be related to the three aromatic residues in hIAPP1-37. In this dissertation, the role of aromatic amino acids, particularly that of Phe-23, and its various interactions to the self-assembly of human islet amyloid polypeptide (hIAPP)22-29 were investigated. Using a variety of spectroscopic techniques with emphasis to vibrational spectroscopy (FT-IR and Raman spectroscopies) in conjunction with computational methods, different factors leading to aggregation as well as its inhibition were identified. Among the driving forces identified are aromatic-π stacking between Phe residues which upon disruption by utilizing specially designed peptide-based inhibitors can lead to the abolition of aggregation propensity of hIAPP. In this work, three classes of peptide-based inhibitors were developed. The first group of inhibitors take advantage of the aromaticity of the Phe-23 residue of the hIAPP­22-29 fragment that undergoes π-stacking upon aggregation. These peptides disrupt the aromaticity by introducing electron donating group (EDG) on Phe-23. Although these peptides showed no inherent amyloidogenic properties, their effectivity towards inhibition of full-length hIAPP aggregation was weak. The second batch of inhibitors, also based on hIAPP22-29 where benzenecarboxylic acid groups were conjugated to the N-terminus of the peptide fragment, showed a more promising inhibitory property towards full-length hIAPP aggregation. These peptides were designed to inhibit aggregation by interfering with the formation of the β-turn intermediate during the amyloid formation and at the same time providing electrostatic repulsion between amylin monomers. Although some of the conjugates showed inhibition, others showed promotion of amyloid formation. The third batch of peptide-based inhibitors were designed to exploit cationic-π interactions, a common interaction between aromatic and charged amino acid residues in polypeptides and proteins. In this batch of peptides, some inhibition was achieved primarily on the basis of increased solubility brought about by the peptide inhibitors binding to hIAPP via hydrophobic interactions

Peptide Conjugates of Benzene Carboxylic Acids as Agonists and Antagonists of Amylin Aggregation

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37 residue peptide hormone that is stored and co-secreted with insulin. hIAPP plays a pivotal role in type 2 diabetes and is the major component of amyloid deposits found in the pancreas of patients afflicted with the disease. The self-assembly of hIAPP and the formation of amyloid is linked to the death of insulin producing β-cells. Recent findings suggest that soluble hIAPP oligomers are the cytotoxic species responsible for β-cell loss whereas amyloid fibrils themselves may indeed be innocuous. Potential avenues of therapeutic intervention include the development of compounds that prevent hIAPP self-assembly as well as those that reduce or eliminate lag time and rapidly accelerate the formation of amyloid fibrils. Both of these approaches minimize temporal exposure to soluble cytotoxic hIAPP oligomers. Toward this end our laboratory has pursued an electrostatic repulsion approach to the development of potential inhibitors and modulators of hIAPP self-assembly. Peptide conjugates were constructed in which benzene carboxylic acids of varying charge were employed as electrostatic disrupting elements and appended to the N-terminal of the hIAPP_22–29 (NFGAILSS) self-recognition sequence. The self-assembly kinetics of conjugates were characterized by turbidity measurements and the structure of aggregates probed by Raman and CD spectroscopy while the morphology was assessed using transmission electron microscopy. Several benzene carboxylic acid peptide conjugates failed to self-assemble and some were found to inhibit the aggregation of full-length amylin while others served to enhance the rate of amyloid formation and/or increase the yield of amyloid produced. Studies reveal that the geometric display of free carboxylates on the benzene ring of the conjugates plays an important role in the activity of conjugates. In addition, a number of free benzene carboxylic acids were found to modulate amylin self-assembly on their own. The results of these investigations confirm the viability of the electrostatic repulsion approach to the modulation of amyloid formation and may aid the design and development of potential therapeutic agents