Developing peptide-based inhibitors of amylin aggregation as a novel
      treatment for type 2 diabetes
Creators: name: Obasse, Idira Christopher
Publisher: name: Lancaster University
Year: 2017

description: 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.