Structure-Based Peptide Inhibitor Design of Amyloid-β Aggregation

Abstract

Many human neurodegenerative diseases are associated with amyloid fibril formation. Inhibition of amyloid formation is of importance for therapeutics of the related diseases. However, the development of selective potent amyloid inhibitors remains challenging. Here based on the structures of amyloid β (Aβ) fibrils and their amyloid-forming segments, we designed a series of peptide inhibitors using RosettaDesign. We further utilized a chemical scaffold to constrain the designed peptides into β-strand conformation, which significantly improves the potency of the inhibitors against Aβ aggregation and toxicity. Furthermore, we show that by targeting different Aβ segments, the designed peptide inhibitors can selectively recognize different species of Aβ. Our study developed an approach that combines the structure-based rational design with chemical modification for the development of amyloid inhibitors, which could be applied to the development of therapeutics for different amyloid-related diseases.

Keywords: Alzheimer’s disease; Aβ fibril; neurodegenerative diseases; protein misfolding; structure-based inhibitor design.

Figure 1

Structure-based peptide inhibitor design of amyloid β (Aβ) amyloid aggregation. (A) The fibril structure of full-length Aβ₄₂ determined by cryo-EM (PDB ID: 5OQV) is shown as black ribbons. The atomic crystal structures of peptides KLVFFA (PDB ID: 2Y2A, cyan) and GGVVIA (PDB ID: 2ONV, magenta) are aligned on one of the two protofilaments of the full-length Aβ₄₂ fibril structure and shown as sticks. (B) The fibril structure of full-length Aβ₄₀ determined by solid-state NMR (PDB ID: 2LMN) is shown as black ribbons. The atomic crystal structure of peptides KLVFFA is aligned on one of the two protofilaments of the full-length Aβ₄₀ fibril structure and shown as sticks. (C) Design strategy for peptide inhibitors of amyloid fibrils. The designing template is a five-stranded sheet extracted from the fibrillar structure of the targeting segment. Peptide inhibitors (in cyan) are designed to have the optimal interactions with the target via backbone hydrogen bonds (yellow dashed lines) and complementary side-chain interactions (shown as spheres and dots). Oxygen atoms are in red. Nitrogen atoms are in blue.

Figure 2

Inhibitory effects of designed peptides on Aβ₄₂ amyloid aggregation measured by the thioflavin T (ThT) fluorescence assay. (A) The ThT fluorescence curves of Aβ₄₂ in the presence of designed peptide inhibitors. The molar ratio of Aβ:peptide-inhibitor is 1:5. Three replicates were measured for each curve. The lag time of Aβ₄₂ aggregation in the presence of peptide inhibitors is compared in (B). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.

Figure 3

Design of macrocyclic peptide inhibitors. (A) The schematic shows that as the macrocyclic β-sheet mimic scaffold constrains the designed peptide sequence into a β-strand, the entropy loss is diminished during the process of target binding. “f” represents free peptide; “mc” represents macrocyclic peptide. The zoom-in view shows the structure model of a macrocyclic inhibitor binding to the targeting segment. The targeting segment is in magenta. The designed sequence is in cyan. The macrocyclic scaffold is in gray. H-bonds between the designed sequence and the targeting sequence are labeled by yellow dotted lines. (B) The 42-membered macrocyclic scaffold used in this study. The open strand (positions R1 to R5) accommodates the designed peptides in β-conformation. Two δ-linked ornithine turn units are in blue. The Hao unit in the blocking strand is in red. Sequences of R1-R7 are listed in the table below.

Figure 4

Inhibitory effects of designed macrocyclic peptides on Aβ₄₂ amyloid aggregation and cytotoxicity. (A) The designed macrocyclic peptides, in particular mcK6A1, mcG6A1 and mcG6A2, significantly inhibit the amyloid fibril formation of Aβ₄₂ in a dose-dependent manner. (B) Transmission electron microscopy (TEM) images of Aβ₄₂ (20 μM) after incubation without inhibitors (top) and with 1.0 equivalent of mcK6A1 (bottom) to Aβ monome for 15 h. The scale bars are 200 nm. (C) Inhibition of Aβ₄₂ oligomers. Aβ₄₂ oligomers formed after 7.5 h of incubation at a concentration of 5 μM (by Aβ₄₂ monomer equivalence) were invisible on the native gel with the addition of five molar excess of designed macrocyclic peptides. (D) The designed peptide inhibitors ameliorated Aβ₄₂ cytotoxicity to PC-12 cells. The first column is the cells treated with 0.1 mM NaOH and phosphate buffer saline (PBS) as a positive control. Error bars correspond to standard deviations three replicates of each experiment. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; n.s. represents “not significant”.

Figure 5

Specificity of designed macrocyclic peptides for the inhibition of Aβ₄₂ and Aβ₄₀ aggregation. (A) The sequences of Aβ₄₂ and Aβ₄₀. The amyloid-forming segment ¹⁶KLVFFA²¹ (highlighted in orange) is present in both Aβ₄₂ and Aβ₄₀, while segment ³⁷GGVVIA⁴² (highlighted in magenta) is present only in Aβ₄₂. The consensus sequence of Aβ₄₂ and Aβ₄₀ is highlighted in gray. (B) The structure models of mcG6A1 (cyan) in complex with ³⁷GGVVIA⁴² (magenta) and ¹⁶KLVFFA²¹ (orange), respectively. McG6A1 was designed based on the structure of GGVVIA. Residues Tyr and Phe of mcG6A1, and Ile41 of GGVVIA (highlighted with a gray frame) engage in van der Waals interactions at the inhibitor-target interface. In contrast, mcG6A1 designed for GGVVIA has no specific side-chain interactions, but merely non-specific back-bone interactions with KLVFFA. (C) The effects of mcK6A, mcG6A1 and mcG6A2 on Aβ₄₀ aggregation (30 μM by Aβ₄₀ monomer equivalence), measured by ThT assay. Error bars correspond to standard deviations of three replicates of each experiment. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; n.s. represents “not significant.” (D) TEM images of Aβ₄₀ (30 μM) after incubation without inhibitors (left), and with 1.0 equivalent of mcK6A1 to Aβ monome (right). The scale bars are 200 nm.