(6a) Design, Synthesis and Characterization of Transthyretin Mimetics for Inhibition of Beta-Amyloid Toxicity

Neuronal damage and destruction in Alzheimer’s disease has been linked to soluble oligomers of beta-amyloid. Transthyretin is a transport protein present in both serum and cerebrospinal fluid that protects against beta-amyloid toxicity, both in vitro and in vivo. Previously we showed that beta-amyloid oligomers bind to two regions on TTR: strand G (particularly L110) and the EF helix/loop (L82). We also showed that mutation of either leucine to alanine (L82A or L110A) significantly reduced beta-amyloid binding, and that these mutants were ineffective at protecting neurons against beta-amyloid toxicity. Here we present our efforts to design a peptidyl compound based on strand G that can mimic TTR’s ability to bind to beta-amyloid and prevent its toxicity. Beginning with the 16-mer corresponding to TTR residues 102-117, we used SPOT peptide arrays to determine the minimum length and to identify critical residues. 6- and 8-mers were too short to bind beta-amyloid; the minimum length required was 10 residues in our screen. Residues Ile107, Leu110, Leu111, Tyr114 and Tyr116 were identified as critical, because mutation led to loss of beta-amyloid binding. We synthesized several 12-mer and 16-mers, including a scrambled sequence peptide and a control in which Leu110 was mutated to alanine, and tested their ability to bind to beta-amyloid using Trp fluorescence, proteolytic fragmentation, and PICUP crosslinking assays. We also examined the role of charged residues at the N- and C-terminus on binding. We observed that active peptides self-assembled in the absence of beta-amyloid, and speculate that self-assembly may be necessary for binding activity. We tested for specificity by examining whether the strand G-based peptide also interacted with other amyloidogenic proteins, and evaluated their effect on beta-amyloid aggregation kinetics. Finally, we observed that our optimized peptide, but not the LtoA control mutant, was able to protect neurons against beta-amyloid toxicity. Thus, transthyretin’s binding and protective capability can be retained in a small designed compound.