Combination of kinetically selected inhibitors in trans leads to highly effective inhibition of amyloid formation

Abstract

Amyloid formation plays a role in over 25 human disorders. A range of strategies have been applied to the problem of developing inhibitors of amyloid formation, but unfortunately, many inhibitors are effective only in molar excess and typically either lengthen the time to the onset of amyloid formation, (the lag time), while having modest effects on the total amount of amyloid fibrils produced, or decrease the amount of amyloid without significantly reducing the lag time. We demonstrate a general strategy whereby two moderate inhibitors of amyloid formation can be rationally selected via kinetic assays and combined in trans to yield a highly effective inhibitor which dramatically delays the time to the appearance of amyloid and drastically reduces the total amount of amyloid formed. A key feature is that the selection of the components of the mixture is based on their effect on the time course of amyloid formation rather than on just the amount of amyloid produced. The approach is validated using inhibitors of amyloid formation by islet amyloid polypeptide, the causative agent of amyloid formation in type 2 diabetes and the Alzheimer's disease Aβ peptide.

Figure 1

(A) The primary sequence of human IAPP. The polypeptide contains a disulfide bridge between Cy-2 and Cys-7 and the C-terminus is amidated. (B) The primary sequence of the 1-40 isoform of the Aβ polypeptide.

Figure 2

Synergistic inhibition of amyloid formation by IAPP. (A) The results of fluorescent detected thioflavin-T binding assays are displayed. Black, wild type IAPP; Red, a 1:1 mixture of wild type IAPP with G24P-IAPP; Blue, a 1:1 mixture of wild type IAPP with I26P-IAPP; Green, a 1:0.5:0.5 mixture of wild type IAPP with G24P-IAPP and I26P-IAPP. (B) TEM image of wild type IAPP alone. (C) TEM image of a 1:1 mixture of wild type IAPP and G24P-IAPP. (D) TEM image of a 1:1 mixture of wild type IAPP and IAPP-I26P. (E) TEM image of a 1:0.5:0.5 mixture of wild type IAPP, G24P-IAPP and I26P-IAPP. Scale bars represent 100 nm. Aliquots were removed from the kinetic experiments 600 minutes after amyloid formation was initiated and TEM images collected. The kinetic assays depicted in panel (A) were carried out in 20 mM Tris-HCl (pH 7.4), 2% HFIP (v/v) with continuous stirring at 25°C. The total concentration of inhibitor was the same in the 1:1 mixtures and in the 1:0.5:0.5 mixtures and was equal to 16 μM. Wild type IAPP was at 16 μM.

Figure 3

Synergistic inhibition of amyloid formation by the Aβ_1-40 polypeptide. (A) The results of fluorescent detected thioflavin-T binding assays are displayed. Black, Aβ_1-40; Red, a1:1 mixture of Aβ_1-40 with G24P-IAPP; Blue, a 1:1 mixture of Aβ_1-40 with I26P-IAPP; Green, a 1:0.5:0.5 mixture of Aβ_1-40 with G24P-IAPP and I26P-IAPP. (B) TEM image of Aβ_1-40 alone. (C) TEM image of a1:1 mixture of Aβ_1-40 and G24P-IAPP. (D) TEM image of a 1:1 mixture of Aβ_1-40 and I26P-IAPP. (E) TEM image of a 1:0.5:0.5 mixture of Aβ_1-40, G24P-IAPP and I26P-IAPP. Scale bars represent 100 nm. Aliquots were removed from the kinetic experiments 20 hours after amyloid formation was initiated and TEM images collected. The kinetic assays depicted in panel (A) were carried out in 100 mM Tris-HCl (pH 7.4) with continuous stirring at 25°C. The total concentration of inhibitor was the same in the 1:1 mixtures and in the 1:0.5:0.5 mixtures and was equal to 24 μM. Aβ_1-40 was at 24 μM.