A Screen for Small Molecule Inhibitors of Soluble Abeta Oligomer Assemebly

Soluble oligomeric forms of the Alzheimer's J3-peptide (AJ3(1-42)) are receiving increasing attention as potential mediators of the neurotoxic activity of AJ3and as a possible causative agent for Alzheimer's disease The search fm potentially therapeutic anti-oligomer compounds has been hampered by the lack of a high-throughput screening assay and artifactual interactions of compounds with antibodies. This exploratory proposal applies a novel, inexpensive assay format to screen for compounds that inhibit the formation of soluble AJ3oligomers or destabilize their structure, facilitating biological clearance. A small library of pharmaceutical agents will be screened to establish performance parameters followed by evaluation of a more extensive pharmacophore library to identify structures of potential interest. Specific Aim 1. To develop a high-throughput assay system to identify compounds to inhibit Aj3 oligomer formation. Preventing AJ3(1-42) oligomer assembly at an early stage will prevent stable toxic species from being formed. Oligomeric species of synthetic N-terminally biotinylated AJ3(1-42) will be quantified with a novel biotin-avidin single site binding assay. This method is preferable for primary screening over even oligomer-specific antibodies because it avoids a significant number of false positives, inactive compounds that interfere with antibody epitopes and antibodies. The procedure will be implemented on a Tecan Genesis LiHa/TeMo robotics system at UK. Specific Aim 2. To develop a high-throughput assay system to identify compounds to destabilize Aj3 oligomer structure. AJ3 oligomers are highly protease-resistant and thus poorly cleared from the brain. Compounds that destabilize oligomers allowing degradation by AJ3-metabolizing proteases would be useful in reducing oligomer levels and be of potential therapeutic value. Synthetic AJ3 oligomers formed in the presence of compound, or preformed and then treated with compound. will be screened with trypsin, and if positive, digested with insulin-degrading enzyme and neprilysin, proteases believed to degrade AJ3peptides in vivo. Selected positive compounds will be also tested for their effect on the protease sensitivity of soluble AJ3 oligomers extracted from AD brain employing a new highly sensitive single site immunoassay configured for the Luminex-1003 BeadlyteTM system. This method shows promise for the detection of oligomeric AJ3 in biological fluids. including animal models and clinical specimens to determine the effectiveness of anti-oligomer therapeutics. Specific Aim 3. To determine the performance of the assays developed in Specific Aims 1 and 2 on screening the LOPAC compound library and a targeted screen of the more extensive Hit-FinderTM pharmacophore collection. The LOPAC library will be used to troubleshoot the protocols optimized in Specific Aims 1 and 2 applied in a screening format. Results from this screen will be used to predict which compounds in the 16,000 compound Maybridge Hit-FinderTM collection may be active in blocking oligomer formation. Although the intent of this exploratory/developmental proposal is primarily to validate the assays, active compounds will provide tools that will be useful in probing the biological effects of AJ3 oligomers since the pharmaceutical agents that make up LOPAC are compatible with cellular systems. Anti-oligomer compounds could potentially block the cognitive impairment symptoms that oliqomers elicit in animal models and interfere with the proqression of Alzheimer's disease.