Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.727170
Title: Developing peptide-based inhibitors of amylin aggregation as a novel treatment for type 2 diabetes
Author: Obasse, Idira Christopher
ISNI:       0000 0004 6423 5415
Awarding Body: Lancaster University
Current Institution: Lancaster University
Date of Award: 2017
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Abstract:
Human islet amyloid polypeptide (hIAPP), also known as amylin, is the main constituent of the amyloid deposits present in approximately 95% of people with type 2 diabetes. Amylin aggregates into oligo-/polymeric sheet structures which are considered to be cytotoxic to pancreatic -cells. Inhibiting the early stages of amylin aggregation could provide a potential therapeutic strategy for the treatment of type 2 diabetes. In this study, overlapping peptides were designed to target the binding region (RLANFLVHSS, residues 11-20) of human amylin and their effects on amylin fibril formation were studied. The first generation of peptides, IO1, IO2, IO3, IO4, IO5, IO6 and IO7, showed less than 50% inhibition of amylin aggregation as observed using a Thioflavin T (Th-T) fluorescence assay. The next generation of peptides, IO8 and RI-IO8, were assessed using Th-T, Congo red and transmission electron microscopy (TEM) techniques. IO8 (H2N‑RGANFLVHGR-NH2) showed strong inhibitory effects on amylin aggregation, up to 71% inhibition, with TEM studies revealing a total inhibition of amylin fibril formation at a 1:1 molar ratio of peptide to amylin. MTS and LDH cytotoxicity studies revealed that IO8 protected human pancreatic islet β PANC-1 insulin producing cells from the toxic effects of human amylin. IO8 proved to be a significantly more potent inhibitor than the NMeG24 NMeI26 peptide reported in literature. In fact, contrary to reports in the literature, NMeG24 NMeI26 significantly enhanced amylin fibril formation. In addition, a homoarginine version of IO8, designed by replacing the arginine residues in IO8 (H2N-RGANFLVHGR-NH2) with homoarginine (H2N-HarGANFLVHGHar-NH2) also significantly impeded amylin fibril formation as observed by Th-T assay. To completely stabilise IO8 from proteolytic degradation, we designed RI-IO8 (Ac-rGhvlfnaGr-NH2), a retro-inverso peptide with L- replaced by D-amino acids. RI-IO8, however, significantly enhanced amylin fibril formation. Th-T experiments, Congo red assay and TEM revealed that two N-methylated versions of IO8, N1-IO8 (H2N-RGAmNFmLVmHGR-NH2) and N2-IO8 (H2N-RGANmFLmVHmR-NH2) significantly inhibited amylin aggregation by up to 85% and 87%, respectively, as observed by Th-T assay. TEM images revealed complete inhibition of fibril formation by N1-IO8 at a 1:1 molar ratio of peptide to amylin, and by N2-IO8 even at a 1:5 molar ratio of peptide to amylin. N1-IO8 and N2-IO8 were found to be stable against proteolytic enzymes, and in plasma, unlike IO8, and also protected human pancreatic islet β PANC-1 cells from the toxic effects of human amylin, and were themselves non-toxic to cells. These studies show that these N-methylated peptides, N1-IO8 and N2-IO8, are potent inhibitors of amylin aggregation and could be developed as a novel treatment for type 2 diabetes.
Supervisor: Allsop, David ; Parkin, Edward Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.727170  DOI:
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