title: Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted β-secretase using single-molecule imaging.
abstract: Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against β-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (Kd < nM) compounds.
authors: Anders
authors: A
authors: Gunnarsson
authors: Arjan
authors: A
authors: Snijder
authors: Jennifer
authors: J
authors: Hicks
authors: Jenny
authors: J
authors: Gunnarsson
authors: ‡Department of Applied Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden.
authors: Fredrik
authors: F
authors: Höök
authors: Stefan
authors: S
authors: Geschwindner
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D057054:Molecular Imaging; D012996:Solutions; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
chemical_list: D004791:Enzyme Inhibitors; D012996:Solutions; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human