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 (<b>CP-2</b>) that interacts strongly with Aβ and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, <b>CP-2</b> 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 <b>CP-2</b>’s antiamyloidogenic activity. NMR spectroscopy indicates that <b>CP-2</b> interacts with Aβ through its self-assembled conformation and induces weak secondary structure in Aβ. Upon coincubation, <b>CP-2</b> 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 <b>CP-2</b> 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