A novel inhibitor of amyloid ß (Aß) peptide aggregation: from high throughput screening to efficacy in an animal model of Alzheimer disease.

Compelling evidence indicates that aggregation of the amyloid ß (Aß) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aß42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aß42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aß42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aß42 aggregation and reducing Aß42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aß42. The ability of D737 to prevent Aß42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.

Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aß peptide.

The aggregation of polypeptides into amyloid fibrils is associated with a number of human diseases. Because these fibrils--or intermediates on the aggregation pathway--play important roles in the etiology of disease, considerable effort has been expended to understand which features of the amino acid sequence promote aggregation. One feature suspected to direct aggregation is the π-stacking of aromatic residues. Such π-stacking interactions have also been proposed as the targets for various aromatic compounds that are known to inhibit aggregation. In the case of Alzheimer's disease, the aromatic side chains Phe19 and Phe20 in the wild-type amyloid beta (Aß) peptide have been implicated. To explicitly test whether the aromaticity of these side chains plays a role in aggregation, we replaced these two phenylalanine side chains with leucines or isoleucines. These residues have similar sizes and hydrophobicities as Phe but are not capable of π-stacking. Thioflavin-T fluorescence and electron microscopy demonstrate that replacement of residues 19 and 20 by Leu or Ile did not prevent aggregation, but rather enhanced amyloid formation. Further experiments showed that aromatic inhibitors of aggregation are as effective against Ile- and Leu-substituted versions of Aß42 as they are against wild-type Aß. These results suggest that aromatic π-stacking interactions are not critical for Aß aggregation or for the inhibition of Aß aggregation.