Inhibition of Amyloid β-Induced Lipid Membrane Permeation and Amyloid β Aggregation by K162

Abstract

Alzheimer's disease (AD) is characterized by progressive neurodegeneration associated with amyloid β (Aβ) peptide aggregation. The aggregation of Aβ monomers (AβMs) leads to the formation of Aβ oligomers (AβOs), the neurotoxic Aβ form, capable of permeating the cell membrane. Here, we investigated the effect of a fluorene-based active drug candidate, named K162, on both Aβ aggregation and AβO toxicity toward the bilayer lipid membrane (BLM). Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and molecular dynamics (MD) were employed to show that K162 inhibits AβOs-induced BLM permeation, thus preserving BLM integrity. In the presence of K162, only shallow defects on the BLM surface were formed. Apparently, K162 modifies Aβ aggregation by bypassing the formation of toxic AβOs, and only nontoxic AβMs, dimers (AβDs), and fibrils (AβFs) are produced. Unlike other Aβ toxicity inhibitors, K162 preserves neurologically beneficial AβMs. This unique K162 inhibition mechanism provides an alternative AD therapeutic strategy that could be explored in the future.

Keywords: Alzheimer’s disease; amyloid β; amyloid β aggregation; atomic force microscopy; membrane permeation; toxicity inhibition.

Scheme 1. Structural Formula of K162 (C₁₅H₁₄BrN)

Figure 1

(a) Impedance and (b) phase angle as a function of frequency for (black curve) fBLM-AβOs and (red curve) fBLM-AβOs-K162 in the PBS (0.01 M phosphate buffer, 0.0027 M KCl, and 0.137 M NaCl, pH = 7.4) solution at 0 V vs SCE. Symbols and curves of the same colors represent experimental data and results of fitting of parameters of the equivalent electrical circuits, shown as insets in Panel (a), to the EIS data, respectively, for the same measurement at a single potential. R_s and R_m – solution and membrane resistance, respectively; CPE_m and CPE_sp – constant-phase element for the membrane and submembrane (spacer) region, respectively.

Figure 2

AFM topography images of (a) BLM, (b) BLM in the presence of AβOs, and (c) BLM in the presence of AβOs and K162 in the PBS (0.01 M phosphate buffer, 0.0027 M KCl, and 0.137 M NaCl, pH = 7.4) solution at room temperature. Insets in Panels (a) and (b) show higher-resolution AFM images of the BLM in the absence of AβO, and the pores and AβO clusters formed in the BLM-AβOs, respectively. (d) Cross-sectional profile across the line in Panel (a) showing the BLM thickness. (e) Cross-sectional profile across the line in the inset in Panel (b) showing the pore depth and the membrane-protruding AβOs cluster’s height. (f) Cross-sectional profile across line 1 in Panel (c) displaying the depth of scratches and globular structures’ height. The inset in Panel (f) shows the cross-sectional profile along line 2 in Panel (c), displaying the height of the AβOs cluster located on top of the BLM-AβOs-K162. The height in all AFM images is scaled using the lowest point of the image as a reference.

Figure 3

AFM topography images of Aβ forms produced after (a, d) 0, (b, e) 24, and (c, f) 48 h of Aβ aggregation in the (a–c) absence and (d–f) presence of K162 in the PBS (0.01 M phosphate buffer, 0.0027 M KCl, and 0.137 M NaCl, pH = 7.4) solution at 4 °C. The corresponding height distributions of Aβ forms produced after (g, j) 0, (h, k) 24, and (i, l) 48 h of Aβ aggregation in the (g, h, i) absence and (j, k, l) presence of K162. Histograms corresponding to monomers, tetramers, and octamers of Aβ are colored in blue, red, and green, respectively.

Figure 4

Molecular dynamics modeled examples of configurations of the K162 complexes with (a) AβMs, (b) AβDs, (c) AβFs, and (d) K162. K162 molecules are represented as spheres and colored by the type of element, i.e., carbon is gray, bromine is pink, and nitrogen is blue. All Aβ forms are shown as ribbon structures, and their residues are colored by lipophilicity, i.e., hydrophilic residues are blue, neutral residues are white, and hydrophobic residues are red.

Scheme 2. Aβ Aggregation Pathways in the Absence (Grey Arrows) and Presence (Blue Arrows) of K162