Soluble oligomeric forms of the Alzheimer's beta-peptide (Abeta(1-42)) are receiving increasing attention as potential mediators of the neurotoxic activity of Abeta and as a possible causative agent for Alzheimer's disease. The search for 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 Abeta oligomers 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 Abeta oligomer formation. Preventing Abeta(1-42) oligomer assembly at an early stage will prevent stable toxic species from being formed. Oligomeric species of synthetic N-terminally biotinylated Abeta(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 Abeta oligomer structure. Abeta oligomers are highly protease-resistant and thus poorly cleared from the brain. Compounds that destabilize oligomers allowing degradation by Abeta-metabolizing proteases would be useful in reducing oligomer levels and be of potential therapeutic value. Synthetic Abeta 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 Abeta peptides in vivo. Selected positive compounds will be also tested for their effect on the protease sensitivity of soluble Abeta oligomers extracted from AD brain employing a new highly sensitive single site immunoassay configured for the Luminex-100(r) Beadlyte(tm) system. This method shows promise for the detection of oligomeric Abeta 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-Finder(tm) 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-Finder(tm) 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 Abeta 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 oligomers elicit in animal models and interfere with the progression of Alzheimer's disease. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AG028816-01
Application #
7124441
Study Section
Special Emphasis Panel (ZRG1-BST-L (50))
Program Officer
Buckholtz, Neil
Project Start
2006-09-01
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
1
Fiscal Year
2006
Total Cost
$155,656
Indirect Cost
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Horton, William; Sood, Abha; Peerannawar, Swarada et al. (2017) Synthesis and application of ?-carbolines as novel multi-functional anti-Alzheimer's disease agents. Bioorg Med Chem Lett 27:232-236
Török, Béla; Sood, Abha; Bag, Seema et al. (2013) Diaryl hydrazones as multifunctional inhibitors of amyloid self-assembly. Biochemistry 52:1137-48
Bag, Seema; Ghosh, Sanjukta; Tulsan, Rekha et al. (2013) Design, synthesis and biological activity of multifunctional ?,?-unsaturated carbonyl scaffolds for Alzheimer's disease. Bioorg Med Chem Lett 23:2614-8
Török, Béla; Sood, Abha; Bag, Seema et al. (2012) Structure-activity relationships of organofluorine inhibitors of ?-amyloid self-assembly. ChemMedChem 7:910-9
LeVine 3rd, Harry; Lampe, Levi; Abdelmoti, Lina et al. (2012) Dihydroxybenzoic acid isomers differentially dissociate soluble biotinyl-A?(1-42) oligomers. Biochemistry 51:307-15
LeVine 3rd, Harry; Ding, Qunxing; Walker, John A et al. (2009) Clioquinol and other hydroxyquinoline derivatives inhibit Abeta(1-42) oligomer assembly. Neurosci Lett 465:99-103