Structure-activity relationships in peptide modulators of β-amyloid protein aggregation: variation in α,α-disubstitution results in altered aggregate size and morphology

Abstract

Neuronal cytotoxicity observed in Alzheimer's disease (AD) is linked to the aggregation of β-amyloid peptide (Aβ) into toxic forms. Increasing evidence points to oligomeric materials as the neurotoxic species, not Aβ fibrils; disruption or inhibition of Aβ self-assembly into oligomeric or fibrillar forms remains a viable therapeutic strategy to reduce Aβ neurotoxicity. We describe the synthesis and characterization of amyloid aggregation mitigating peptides (AAMPs) whose structure is based on the Aβ "hydrophobic core" Aβ(17-20), with α,α-disubstituted amino acids (ααAAs) added into this core as potential disrupting agents of fibril self-assembly. The number, positional distribution, and side-chain functionality of ααAAs incorporated into the AAMP sequence were found to influence the resultant aggregate morphology as indicated by ex situ experiments using atomic force microscopy (AFM) and transmission electron microscopy (TEM). For instance, AAMP-5, incorporating a sterically hindered ααAA with a diisobutyl side chain in the core sequence, disrupted Aβ(1-40) fibril formation. However, AAMP-6, with a less sterically hindered ααAA with a dipropyl side chain, altered fibril morphology, producing shorter and larger sized fibrils (compared with those of Aβ(1-40)). Remarkably, ααAA-AAMPs caused disassembly of existing Aβ fibrils to produce either spherical aggregates or protofibrillar structures, suggesting the existence of equilibrium between fibrils and prefibrillar structures.

Keywords: Alzheimer’s disease; amyloid aggregation mitigating peptides; fibrils; spherical aggregates; α,α-disubstituted amino acids; β-Amyloid.

Figure 1

Design of amyloid aggregation mitigating peptides (AAMPs) with α,α-disubstituted amino acids (ααAAs) as disrupters of Aβ assembly. AAMP-0 is the control peptide with no ααAAs. The ααAAs iBu (isobutyl-glycine), Bn (dibenzylglycine), and Pr (dipropylglycine) are analogs of l-natural amino acids leucine, phenylalanine, and alanine, respectively.

Figure 2

Assembly of Aβ₁₋₄₀ in the presence of ααAA-AAMPs. Time-dependent ThT fluorescence monitoring of Aβ₁₋₄₀ assembly in the presence or absence of the various AAMPs. Fluorescence (ThT) was set arbitrarily to 100% relative to Aβ_1−40. The ∗ denotes mitigators aged alone.

Figure 3

Aggregation of Aβ₁₋₄₀ with and without AAMPs: (A) fibrils formed by Aβ₁₋₄₀ alone after 1 week of incubation; (B) height distribution histogram for panel A; (C) fibril bundles formed by Aβ₁₋₄₀ alone after 3 months of incubation; (D) height distribution histogram for panel C; (E) protofibrils formed after 3 days of Aβ₁₋₄₀ aggregation mitigated by AAMP-0; (F) height distribution histogram for panel E; (G) fibril network formed after 1 week of Aβ₁₋₄₀ aggregation mitigation by AAMP-0; (H) height distribution histogram for panel G; (I) mixture of spherical and linear aggregates formed after 1 week of Aβ₁₋₄₀ aggregation with mitigation by AAMP-1; (J) height distribution histogram for panel I; (K) spherical assemblies and protofibrils observed after 3 months of Aβ₁₋₄₀ aggregation with mitigation by AAMP-1; (L) height distribution histogram for panel K.

Figure 4

Disruption of Aβ₁₋₄₀ fibril formation by AAMPs with two ααAAs: (A) globular aggregates formed after 7 days aging Aβ₁₋₄₀ in presence of AAMP-2; (B) corresponding height distribution analysis; (C) aggregates formed after 3 months aging Aβ₁₋₄₀ in presence of AAMP-2; (D) corresponding height distribution; (E) spherical aggregates formed after 1 week aging Aβ₁₋₄₀-AAMP-3 mixture; (F) height analysis for panel E; (G) spherical aggregates after 3 months aging Aβ₁₋₄₀-AAMP-3 mixture; (H) corresponding height analysis; (I) mixture of spherical aggregates and protofibrils formed after 7 days aging Aβ₁₋₄₀ in the presence of AAMP-4; (J) corresponding height distribution analysis for panel I; (K) after 3 months aging Aβ₁₋₄₀ in the presence of AAMP-4; (L) matching height distribution histogram.

Figure 5

Topographic AFM images showing disruption of Aβ₁₋₄₀ fibril formation by AAMPs with one ααAA: (A) view of nonfibrillic assemblies formed after 1 week Aβ aggregation mitigation by AAMP-5; (B) corresponding height distribution; (C) view after 3 months mitigation by AAMP-5; (D) height analysis for panel C; (E) progressive AFM view of rod-like fibrils detected after 1 week of aging Aβ₁₋₄₀-AAMP-6 mixture; (F) corresponding height distribution; (G) view after 90 days of aging Aβ₁₋₄₀-AAMP-6 mixture; (H) height distribution analysis for panel G; (I) view of spherical and protofibrils/fibrils (background) formed by Aβ₁₋₄₀-AAMP-7 mixture after 1 week aging; (J) corresponding height analysis; (K) view after 90 days aging of Aβ₁₋₄₀-AAMP-7 mixture; (L) height analysis for panel K; (M) view of spherical particles formed by Aβ₁₋₄₀ aggregation mitigation by AAMP-8; (N) corresponding height distribution; (O) view after 3 months aggregation mitigation by AAMP-8; (P) height analysis for panel O.

Figure 6

Disassembly of Aβ₁₋₄₀ preformed fibrils. ThT fluorescence of Aβ₁₋₄₀ fibril disassembly by the various AAMPs after 24 h incubation at 37 °C while shaking.

Figure 7

Disassembly of Aβ₁₋₄₀ preformed fibrils: (A) Aβ₁₋₄₀ fibrils; (B) corresponding histogram; (C) fibrils/protofibrils formed as a result of fibril disassembly after 24 h exposure to AAMP-0; (D) height analysis for panel C; (E) spherical aggregates induced by 24 h exposure to AAMP-1 fibril to induce disassembly; (F) height histogram analysis for panel E; (G) spherical particles formed after 24 h disassembly by AAMP-2; (H) height analysis for panel G; (I) spherical aggregates from disassembly by AAMP-3 after 24 h; (J) corresponding height analysis; (K) spherical assemblies formed from fibril disassembly by AAMP-4 after 24 h; (L) height histogram for panel K.

Figure 8

Disassembly of Aβ₁₋₄₀ preformed fibrils after 24 h exposure to certain AAMPs: (A) spherical aggregates induced by AAMP-5 fibril disassembly; (B) height histogram analysis for panel A; (C) fibrils/protofibrils formed as a result of fibril disassembly by AAMP-6; (D) height analysis for panel C; (E) spherical aggregates from disassembly by AAMP-7; (F) corresponding height analysis; (G) spherical assemblies formed from fibril disassembly by AAMP-8; (H) height histogram for panel G.