A Two-Step Strategy for Structure–Activity Relationship Studies of N-Methylated Aβ42 C-Terminal Fragments as Aβ42 Toxicity Inhibitors

Neurotoxic Aβ42 oligomers are believed to be the main cause of Alzheimer’s disease. Previously, we found that the C-terminal fragments (CTFs), Aβ(30–42) and Aβ(31–42) were the most potent inhibitors of Aβ42 oligomerization and toxicity in a series of Aβ(x–42) peptides, (x=28–39). Therefore, we chose these peptides as leads for further development. These CTFs are 12/13-amino-acid long, hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the N-terminus resulted in toxic peptides. Therefore, next we incorporated N-methyl amino acids, which are known to increase the solubility of such peptides by disrupting β-sheet formation. Focusing on Aβ(31–42), we used a two-step N-methyl (N-Me) amino acid substitution strategy to study the structural factors controlling inhibition of Aβ42-induced toxicity. First, each residue was substituted by N-Me-alanine (N-Me-A). In the next step, in positions where substitution produced a significant effect, we restored the original side-chain. This strategy allowed exploring the role of both side-chain structure and N-Me substitution in inhibitory activity. We found that the introduction of N-Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aβ(31–42). In particular, N-Me amino acid substitution at positions 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aβ42 toxicity by short-peptides is highly structure-specific, providing basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.