A range of biochemical and genetic studies indicate that aggregation of the Alzheimer's peptide, A?, plays a crucial role in the molecular etiology of Alzheimer's disease (AD). In particular, early steps of misfolding and oligomerization are thought to produce toxic species, which initiate a cascade of events ultimately leading to neurodegeneration. Inhibition of A? misfolding and oligomerization may provide a therapeutic strategy for the treatment of AD; however, finding compounds that specifically accomplish this goal without interfering with the folding of other proteins has remained a major challenge. To overcome this challenge, we propose to develop a rapid, reliable, and high throughput screen for specific inhibitors of A? misfolding and aggregation. This goal will be achieved thorough the following specific aims: ? ? (1) To construct and optimize a novel screen for inhibitors of A? aggregation. The proposed screen will use a fusion of A?42 to Green Fluorescent Protein (GFP). When fused to GFP, AB42 causes the entire fusion protein to misfold and aggregate, thereby preventing the fluorescence of the GFP portion of the protein. Compounds that inhibit A? aggregation will enable GFP to fold into its native structure, and will be identified by the resulting fluorescent signal. ? ? (2) To implement the screen and test its ability to distinguish inhibitors of aggregation from inactive compounds. This will be demonstrated using fluorescence to screen a pilot library of compounds. ? ? (3) To validate the screen using biochemical and biophysical methods. These studies will verify that compounds isolated by the A?42-GFP screen enhance solubility and inhibit aggregation of pure A?42. ? ? (4) To probe structure-activity relationships (SAR). Compounds isolated by the screen will be compared to related compounds in the libraries. These studies will elucidate the chemical features responsible for inhibition and will facilitate future lead optimization. ? ? (5) To adapt the screen for use in either cell-based or cell-free expression systems. Completion of these aims will produce a rapid method of screening for novel and specific inhibitors of aggregation, thereby identifying lead compounds for the development of new pharmaceuticals that prevent or delay the onset of Alzheimer's disease. ? ? ?
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