Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted β-secretase using
Anders Gunnarsson, Arjan Snijder, Jennifer Hicks
1‡Department of Applied Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden.
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.
