Sub-stoichiometric inhibition of IAPP aggregation: a peptidomimetic approach to anti-amyloid agents

Abstract

Membrane-catalysed misfolding of islet amyloid polypeptide is associated with the death of β-cells in type II diabetes (T2D). Most active compounds so far reported require high doses for inhibition of membrane bound IAPP fibrillation. Here, we describe a naphthalimide-appended oligopyridylamide-based α-helical mimetic, DM 1, for targeting membrane bound IAPP. DM 1 completely inhibits the aggregation of IAPP at doses of 0.2 equivalents. DM 1 is also effective at similarly low doses for inhibition of seed-catalyzed secondary nucleation. An NMR based study demonstrates that DM 1 modulates IAPP self-assembly by stabilizing and/or perturbing the N-terminus helix conformation. DM 1 at substoichiometric doses rescues rat insulinoma cells from IAPP-mediated cytotoxicity. Most importantly, 0.2 equivalents of DM 1 disaggregate preformed oligomers and fibrils and can reverse cytotoxicity by modulating toxic preformed oligomers and fibrils of IAPP into non-toxic conformations.

Fig. 1. (a) The primary sequence of IAPP with amidation at the C-terminal and a conserved disulfide bond between cysteines at positions 2 and 7. (b) A helical wheel representation of the helical subdomain of IAPP. The blue, green, and pink colours represent the positively charged, polar, and hydrophobic residues, respectively. (c) α-Helical mimetic oligopyridylamide structure depicting a single conformation stabilized by intramolecular hydrogen bonding network (black dotted lines). It is representing the potential electrostatic and hydrophobic interactions between oligopyridylamide and IAPP. The IAPP peptide domain was extracted from PDB: 5MGQ.

Fig. 2. Chemical structure of compounds used in this study.

Fig. 3. Anti-amyloidogenic activity of DM 1 against IAPP self-assembly. (a) Comparison of t₅₀'s of IAPP aggregation in presence of the indicated ligands at a stoichiometric ratio of 1 : 0.2 (IAPP : ligand). Lipid free condition: IAPP = 25 μM in phosphate buffer (50 mM NaPi, 150 mM KCl, pH 7.4) [ThT] = 12.5 μM; lipid catalysed condition: IAPP = 15 μM in phosphate buffer (50 mM NaPi, 150 mM KCl, pH 7.4) including LUVs (DOPG : DOPC, 3 : 7, 750 μM, d = 100 nm). [ThT] = 7.5 μM. (b) ThT-based kinetic profile of lipid catalysed IAPP aggregation in the absence and presence of DM 1 at the indicated sub-stoichiometric ratios. Solid curves represent the average of three independent trials while the shaded regions represent the standard deviations of those measurements. (c) Statistical analysis of t₅₀'s of IAPP aggregation in the presence of different concentrations of DM 1. (d) TEM images of 15 μM IAPP after 2 h (top) and in presence of DM 1 (3 μM) after 8 h (bottom) under lipid catalysed condition. (e) Circular dichroism spectra of 15 μM IAPP in the absence (gray and purple curves) and presence (green and pink curves) of DM 1 in lipid catalysed conditions at different time intervals depicted in the figure.

Fig. 4. Top: HSQC NMR based binding characterization between DM 1 and IAPP. Overlay of ¹⁵N-IAPP (25 μM) in the absence (gray) and presence of 0.1 eq. and 1 eq. of DM 1 represented by green, dark red colour, respectively. Residues with highest changes, in terms of peak volume, are highlighted in dark red in membrane bound IAPP structure (inset). Bottom: Peak volume of different residues of recombinant ¹⁵N-IAPP (25 μM) at different concentration of DM 1. nd = peak not determined; d = peak disappeared upon addition of DM 1 to ¹⁵N-IAPP.

Fig. 5. Effect of DM 1 on the seed-catalysed processes, oligomerization, fibrillation of IAPP. (a) Inhibition of the secondary nucleation of IAPP aggregation by DM 1 under lipid catalysed conditions. ThT fluorescence based kinetic profiles of (i) 15 μM IAPP; (ii) 15 μM IAPP in presence of IAPP seed (5%, v/v) and (iii) 15 μM IAPP in presence of IAPP seed (5%, v/v) and 0.2 equivalents DM 1. (b) The effect of DM 1 on the preformed IAPP amyloid fibrils. ThT fluorescence based amyloid profile of 25 μM IAPP in the absence (gray) and presence of 0.2 equivalents of DM 1 added at different time points indicated by stars. Solid curves represent the average of three independent trials while the shaded regions represent the standard deviations of those measurements. (c) The TEM images were taken of all the samples after the completion of the reaction (at 8 h).

Fig. 6. Effect of DM 1 on the cytotoxicity mediated by IAPP. (a) Rescue of IAPP-mediated cytotoxicity by DM 1. Cytotoxicity of 10 μM IAPP applied to RIN-m cells in the absence and presence of different concentration of DM 1, measured using an MTS assay. Each experiment is the average of four on-plate repeats from each of three independently performed replicates. (b) Dose-dependent effect of DM 1 on 10 μM IAPP-induced toxicity in RIN-m cells. (c) The cell toxicity of IAPP (10 μM) fibrillation, mitigated by addition of DM 1 (2.5 μM) at different stages respectively.