In Vitro and Mechanistic Studies of an Antiamyloidogenic Self-Assembled Cyclic d,l-α-Peptide Architecture

Abstract

Misfolding of the Aβ protein and its subsequent aggregation into toxic oligomers are related to Alzheimer's disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic D,L-α-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of Aβ, thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic D,L-α-peptides and optimizing the activity of a lead peptide, we found one cyclic D,L-α-peptide (CP-2) that interacts strongly with Aβ and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, CP-2 inhibits the formation of Aβ aggregates, entirely disassembles preformed aggregated and fibrillar Aβ, and protects rat pheochromocytoma PC12 cells from Aβ toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying CP-2's antiamyloidogenic activity. NMR spectroscopy indicates that CP-2 interacts with Aβ through its self-assembled conformation and induces weak secondary structure in Aβ. Upon coincubation, CP-2 changes the aggregation pathway of Aβ and alters its oligomer distribution by stabilizing small oligomers (1-3 mers). Our results support studies suggesting that toxic early oligomeric states of Aβ may be composed of antiparallel β-peptide structures and that the interaction of Aβ with CP-2 promotes formation of more benign parallel β-structures. Further studies will show whether these kinds of abiotic cyclic D,L-α-peptides are also beneficial as an intervention in related in vivo models.

Anti-amyloidogenic Heterocyclic Peptides

“…In addition, the dot-blot immunoassay confirmed that the inhibitory effect of CP-2 was stronger toward the more toxic Aβ42. These findings collectively suggested that CP-2 interacted and altered the aggregation of Aβ to an "off-pathway" mechanism [38,58].…”

“…Reports have previously emphasized the importance of intermolecular hydrogen bonds and the cyclic nature of the macrocyclic subunit for self-assembly as well as antiviral and antibacterial activity of cyclic peptides [56,63]. The combined results of ThT, critical micelle concentration, and NMR analyses of CP-2 confirmed that self-assembly was essential for anti-amyloidogenic activity and that the bioactive form of CP-2 was probably selfassembled [58].…”

“…Based on the close structural and functional similarities between amyloids and cyclic D,L-α-peptides, we designed and synthesized a library of cyclic peptide hexamers and screened it for peptides that interact with Aβ and its aggregates [58]. The "one-bead-one-compound" combinatorial approach was employed to identify analogs exhibiting anti-amyloidogenic activity and probe their topological requirements in general.…”

“…Moreover, we showed that CP-2 decreased dose-dependently the Square brackets indicate a cyclic structure. J, CH, and Z denote norleucine, cyclohexylalanine, and 2-aminooctanoic acid, respectively [58,72] Anti-amyloidogenic Heterocyclic Peptides aggregation kinetics of Aβ40 and arrested completely the aggregation of Aβ42 at as high as a 1:1 Aβ:CP-2 concentration ratio, suggesting that CP-2 has a somewhat stronger inhibitory effect on the more neurotoxic Aβ42 peptide [58]. Most importantly, we demonstrated that CP-2 was recognized by the A11 conformational antibody, suggesting that it could be used as a structural mimic to cross-interact and interfere with the aggregation and toxicity of different amyloids [58,72,74].…”

“…Moreover, the results of photo-induced cross-linking of unmodified proteins (PICUP) suggested that CP-2 generated relatively stable co-aggregates with monomeric or small 1-3-mers of Aβ and shifted the aggregation equilibrium toward these early-assembled species [58].…”