Self-association of β-amyloid (Aβ) into oligomers and fibrils is associated with Alzheimer's disease (AD), motivating the search for compounds that bind to and inhibit Aβ oligomerization and/or neurotoxicity. Peptides are an attractive class of such compounds, with potential advantages over small molecules in affinity and specificity. Self-complementation and peptide library screening are two strategies that have been employed in the search for peptides that bind to Aβ. Alternatively, one could design Aβ-binding peptides based on knowledge of complementary binding proteins. One candidate protein, transthyretin (TTR), binds Aβ, inhibits aggregation, and reduces its toxicity. Previously, strand G of TTR was identified as part of a specific Aβ binding domain, and G16, a 16-mer peptide with a sequence that spans strands G and H of TTR, was synthesized and tested. Although both TTR and G16 bound to Aβ, they differed significantly in their effect on Aβ aggregation, and G16 was less effective than TTR at protecting neurons from Aβ toxicity. G16 lacks the β-strand/loop/β-strand structure of TTR's Aβ binding domain. To enforce proper residue alignment, we transplanted the G16 sequence onto a β-hairpin template. Two peptides with 18 and 22 amino acids were synthesized using an orthogonally protected glutamic acid derivative, and an N-to-C cyclization reaction was carried out to further restrict conformational flexibility. The cyclized 22-mer (but not the noncyclized 22-mer nor the 18-mer) strongly suppressed Aβ aggregation into fibrils, and protected neurons against Aβ toxicity. The imposition of structural constraints generated a much-improved peptidomimetic of the Aβ binding epitope on TTR.

Self-association of β-amyloid (Aβ) into soluble oligomers and fibrillar aggregates is associated with Alzheimer's disease pathology, motivating the search for compounds that selectively bind to and inhibit Aβ oligomerization and/or neurotoxicity. Numerous small-molecule inhibitors of Aβ aggregation or toxicity have been reported in the literature. However, because of their greater size and complexity, peptides and peptidomimetics may afford improved specificity and affinity as Aβ aggregation modulators compared to small molecules. Two divergent strategies have been employed in the search for peptides that bind Aβ: (i) using recognition domains corresponding to sequences in Aβ itself (such as KLVFF) and (ii) screening random peptide-based libraries. In this study, we propose a third strategy, specifically, designing peptides that mimic binding domains of Aβ-binding proteins. Transthyretin, a plasma transport protein that is also relatively abundant in cerebrospinal fluid, has been shown to bind to Aβ, inhibit aggregation, and reduce its toxicity. Previously, we identified strand G of transthyretin as a specific Aβ binding domain. In this work we further explore and define the necessary features of this binding domain. We demonstrate that peptides derived from transthyretin bind Aβ and inhibit its toxicity. We also show that, although both transthyretin and transthyretin-derived peptides bind Aβ and inhibit toxicity, they differ significantly in their effect on Aβ aggregation.

Transthyretin (TTR) is a homotetrameric transport protein, assembled from monomers that each contain two four-stranded β-sheets and a short α-helix and loop. In the tetramer, the "inner" β-sheet forms a hydrophobic pocket while the helix and loop are solvent-exposed. β-Amyloid (Aβ) aggregates bind to TTR, and the level of binding is significantly reduced in mutants L82A (on the loop) and L110A (on the inner β-sheet). Protection against Aβ toxicity was demonstrated for wild-type TTR but not L82A or L110A, providing a direct link between TTR-Aβ binding and TTR-mediated cytoprotection. Protection is afforded at substoichiometric (1:100) TTR:Aβ molar ratios, and the level of binding of Aβ to TTR is highest for partially aggregated materials and decreased for freshly prepared or heavily aggregated Aβ, suggesting that TTR binds selectively to soluble toxic Aβ aggregates. A novel technique, nanoparticle tracking, is used to show that TTR arrests Aβ aggregation by both preventing formation of new aggregates and inhibiting growth of existing aggregates. TTR tetramers are normally quite stable; tetrameric structure is necessary for the protein's transport functions, and mutations that decrease tetramer stability have been linked to TTR amyloid diseases. However, TTR monomers bind more Aβ than do tetramers, presumably because the hydrophobic inner sheet is solvent-exposed upon tetramer disassembly. Wild-type and L110A tetramers, but not L82A, were destabilized upon being co-incubated with Aβ, suggesting that binding of Aβ to L82 triggers tetramer dissociation. Taken together, these results suggest a novel mechanism of action for TTR: the EF helix/loop "senses" the presence of soluble toxic Aβ oligomers, triggering destabilization of TTR tetramers and exposure of the hydrophobic inner sheet, which then "scavenges" these toxic oligomers and prevents them from causing cell death.

Transthyretin (TTR) binds to the Alzheimer-related peptide beta-amyloid (Aβ), and may protect against Aβ-induced neurotoxicity. In this work, the specific domains on TTR involved with binding to Aβ were probed. An array was constructed of peptides derived from overlapping sequences from TTR. Strong binding of Aβ to TIAALLSPYSYS (residues 106-117) was detected, corresponding to strand G on the inner β-sheet of TTR. Aβ bound weakly to four contiguous peptides spanning residues 59-83, which includes strand E through the E/F helix and loop. To further pinpoint specific residues on TTR involved with Aβ binding, nine alanine mutants were generated: I68A, I73A, K76A, L82A, I84A, S85A, L17A, T106A and L110A. Aβ binding was significantly inhibited only in L82A and L110A, indicating that Aβ binding to TTR is mediated through these bulky hydrophobic leucines. Aβ binding to L17A and S85A was significantly higher than to wild-type TTR. Enhancement of binding in L17A is postulated to arise from reduced steric restriction to the interior L110 site, since these two residues are adjacent in the native protein. The S85A mutation caused a reduction in TTR tetramer stability; increased Aβ binding is postulated to be a direct consequence of the reduced quaternary stability.