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.

Small molecule microarrays enable the discovery of compounds that bind the Alzheimer's Aß peptide and reduce its cytotoxicity.

The amyloid-ß (Aß) aggregation pathway is a key target in efforts to discover therapeutics that prevent or delay the onset of Alzheimer's disease. Efforts at rational drug design, however, are hampered by uncertainties about the precise nature of the toxic aggregate. In contrast, high-throughput screening of compound libraries does not require a detailed understanding of the structure of the toxic species, and can provide an unbiased method for the discovery of small molecules that may lead to effective therapeutics. Here, we show that small molecule microarrays (SMMs) represent a particularly promising tool for identifying compounds that bind the Aß peptide. Microarray slides with thousands of compounds immobilized on their surface were screened for binding to fluorescently labeled Aß. Seventy-nine compounds were identified by the SMM screen, and then assayed for their ability to inhibit the Aß-induced killing of PC12 cells. Further experiments focused on exploring the mechanism of rescue for one of these compounds: Electron microscopy and Congo red binding showed that the compound enhances fibril formation, and suggest that it may rescue cells by accelerating Aß aggregation past an early toxic oligomer. These findings demonstrate that the SMM screen for binding to Aß is effective at identifying compounds that reduce Aß toxicity, and can reveal potential therapeutic leads without the biases inherent in methods that focus on inhibitors of aggregation.

New perspectives on multiple-copy, mean-field molecular dynamics methods.

Mean-field molecular dynamics (MD) techniques are designed to improve phase-space sampling in MD simulations. Reviewed here are theoretical and practical contributions from our group, and the ideas our contributions are based upon, beginning with the original time-dependent Hartree technique, locally enhanced sampling (LES), and a new, purely classical derivation of multiple-copy, mean-field equations of motion. This view is used to provide new insights into approximations inherent in LES, as well as the basis for a recently proposed method, ensembles eXtracted from atomic coordinate transformations (the EXACT approximation).

Structure-activity relationships for a series of compounds that inhibit aggregation of the Alzheimer's peptide, Aß42.

Inhibiting aggregation of the amyloid-beta (Aß) peptide may be an effective strategy for combating Alzheimer's disease. As the high-resolution structure of the toxic Aß aggregate is unknown, rational design of small molecule inhibitors is not possible, and inhibitors are best isolated by high-throughput screening. We applied high-throughput screening to a collection of 65,000 compounds to identify compound D737 as an inhibitor of Aß aggregation. D737 diminished the formation of oligomers and fibrils, and reduced Aß42-induced cytotoxicity. Most importantly, D737 increased the life span and locomotive ability of transgenic flies in a Drosophila melanogaster model of Alzheimer's disease (J Biol Chem, 287, 2012, 38992). To explore the chemical features that make D737 an effective inhibitor of Aß42 aggregation and toxicity, we tested a small collection of eleven analogues of D737. Overall, the ability of a compound to inhibit Aß aggregation was a good predictor of its efficacy in prolonging the life span and locomotive ability of transgenic flies expressing human Aß42 in the central nervous system. Two compounds (D744 and D830) with fluorine substitutions on an aromatic ring were effective inhibitors of Aß42 aggregation and increased the longevity of transgenic flies beyond that observed for the parent compound, D737.