P1-234: Production of stable oligomers allows HTS identification of small drug candidates targeting the toxic oligomers of Aβ1-42

Alzheimer's disease is associated with the formation and accumulation of toxic oligomers of Aβ in the brain. We aim to develop an oral drug that directly targets these oligomers. Thus far, such a direct approach has been hampered by the fact that assemblies of Aβ exist in a complex equilibrium. We set out to generate stable oligomers of Aβ1-42 and to develop oligomer specific drug screening assays. Oligomers were prepared by dissolving the Aβ peptide in an aqueous solution, stabilized and separated from monomers by SEC. Purified oligomers were structurally analyzed by SDS-PAGE, DLS, and electron microscopy. ELISA was performed to determine binding of Aβ to a recombinantly produced Aβ receptor. Functional analysis was performed in viability assays using PC12, SH-SY5H, primary rat cortical neuronal cells, and in an inflammatory assay with monocytic cells (THP-1). For HTS an Alphascreen (Perkin Elmer) based biochemical ligand proximity-assay between Aβ and the receptor was used to screen a compound library. When brought in proximity, upon binding of oligomer to the receptor light is emitted. Hits from primary screens were further validated in subsequent confirmation assays and IC50 values were determined. Oligomers were stable and found to be 8-10 nM in size. Oligomers bound an Aβ receptor with an affinity of 1.5 nM. The receptor did not interact with monomeric Aβ. The oligomers could be stored at 4 ËšC for at least one month without measurable structural differences. These oligomers induced toxicity in PC12, SH-SY5Y and primary rat neuronal cells, up to 85%. Moreover, these oligomers induced an inflammatory response in THP-1 cells as measured by NF-kB activity and cytokine production (TNF ±). An Alphascreen-based proximity assay was developed with a Z'> 0.8. HTS, with 100.000 compounds, resulted in > 100 confirmed hits, with an inhibition of at least 30%. Stable oligomers of Aβ1-42 can be prepared and used for drug screening and development. HTS using stable oligomers resulted in confirmed hits, which are attractive candidates for further development into an oral drug for the treatment of AD. The stabilizing technology provides opportunities for application with other oligomers involved in AD and/or other diseases.