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- Gunnarsson, Anders, 1981, et al.
(författare)
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Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted beta-Secretase Using Single-Molecule Imaging
- 2015
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:8, s. 4100-4103
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Tidskriftsartikel (refereegranskat)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 beta-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 (K-d
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