(607d) Rosetta-Inspired Design of Conformationally Constrained Cylic Anti-Amyloid Peptides

Beta-amyloid oligomers are thought to be the most toxic species formed en route to fibril deposition in the Alzheimer’s brain, so sequestration of these low molecular weight aggregates may be therapeutic. Transthyretin (TTR), a tetrameric carrier protein of thyroxine and retinol that circulates in blood and cerebrospinal fluid, has been shown to be neuroprotective against beta-amyloid toxicity in a transgenic Alzheimer mouse model. TTR exerts its neuroprotective activity by sequestering beta-amyloid oligomers. The beta-amyloid binding domain on TTR has been identified as specific residues in strands G and H of the inner beta-sheet. A double-point mutant of transthyretin, mTTR, is stable as a monomer and is more potent than the wildtype (wt) TTR, because strands G and H are more solvent-exposed in mTTR than in wt. However, mTTR is susceptible to proteolysis, and binding of beta-amyloid to mTTR is significantly reduced in the presence of nonspecific proteins and lipids. We proposed that these concerns could be overcome by careful design of a small peptide mimic of the beta-amyloid binding domain of TTR. Linear peptides that mimicked the sequence of the beta-amyloid binding domain on mTTR were only moderately effective at sequestering beta-amyloid. Cyclization improved efficacy, greatly increased stability, and demonstrated less nonspecific interference compared to mTTR. However, the cyclic peptide was not as potent as mTTR. We hypothesized that the lower efficacy was a result of a higher degree of conformational flexibility in the cyclic peptide compared to mTTR. Using the ROSETTA software suite as an in silico predictor of cyclic peptide conformation stability and homogeneity, a screening procedure was developed to investigate putative structural enhancements, including insertion of beta-turn motifs and stabilization by disulfide linkages. The best candidates from this design approach were synthesized and analyzed for conformation, and their efficacy against beta-amyloid was compared to mTTR. This approach supports the use of ROSETTA simulations as a method to investigate sequence and structure effects on the conformation of cyclic peptides in the search for beta-amyloid-sequestering peptides. Such peptides can overcome the protease stability and nonspecific interference issues of the protein while retaining therapeutic efficacy.