title: High throughput screening of beta-amyloid secretion inhibitors using homogenous time-resolved fluorescence.
abstract: A cell-based assay using homogeneous time-resolved fluorescence has been developed for high throughput screening of putative beta-amyloid (Abeta)production inhibitors. In this assay, total Abeta is detected by simply adding two commercially available antibody complexes. The first was a biotinylated monoclonal antibody (4G8), specifically recognizing an epitope comprising the residues 17-24 of the Abetapeptide, complexed with europium cryptate-streptavidin conjugate. The second was a polyclonal antibody (BioS-N), raised against the N-terminus of the Abeta peptide, complexed with an allophycocyanin-anti rabbit antibody conjugate. Binding of the two complexes to the Abeta peptide brought europium cryptate (fluorescence donor) and allophycocyanin (fluorescence acceptor) into close proximity, consequently a fluorescent resonance energy transfer signal was produced upon excitation at 337 nm. The resulting fluorescence signal (665 nm) was then detected using a Discovery or a ViewLux reader. Detection of Abeta by the proposed method is possible at concentrations of approximately 1 nM. The method was employed for the detection of Abeta secreted from a stable transfected human neuroglioma cell line (H4) overexpressing a mutated form of the human amyloid precursor protein (APP695NL) and developed for robotic automation. At optimized conditions, signal-to-background ratios exceeding 5 and Z' factors around 0.7 were achieved in a 384-well format. High throughput screening of 56,913 potential Abeta production inhibitors led to identification of new non-cytotoxic and cell permeable compounds with potencies in the submicromolar range.
authors.affiliation: Discovery Partners International, Integrated Drug Discovery Division, Allschwil, Switzerland.
authors.forename: Hugo
authors.initials: H
authors.lastname: Albrecht
authors.forename: Peter
authors.initials: P
authors.lastname: Zbinden
authors.forename: Andrea
authors.initials: A
authors.lastname: Rizzi
authors.forename: Gino
authors.initials: G
authors.lastname: Villetti
authors.forename: Benedetta
authors.initials: B
authors.lastname: Riccardi
authors.forename: Paola
authors.initials: P
authors.lastname: Puccini
authors.forename: Silvia
authors.initials: S
authors.lastname: Catinella
authors.forename: Bruno P
authors.initials: BP
authors.lastname: Imbimbo
mesh_terms: D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry
chemical_list: D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes