Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted beta-Secretase Using Single-Molecule Imaging

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

Snijder, Arjan,1971AstraZeneca AB (författare)
Hicks, J.AstraZeneca AB (författare)
Gunnarsson, J.AstraZeneca AB (författare)
Höök, Fredrik,1966Chalmers tekniska högskola,Chalmers University of Technology(Swepub:cth)fredrikh (författare)
Geschwindner, S.AstraZeneca AB (författare)
AstraZeneca ABChalmers tekniska högskola (creator_code:org_t)

Ingår i:Analytical Chemistry: American Chemical Society (ACS)87:8, s. 4100-41030003-27001520-6882

Av författaren/redakt...: Gunnarsson, Ande ...; Snijder, Arjan, ...; Hicks, J.; Gunnarsson, J.; Höök, Fredrik, 1 ...; Geschwindner, S.

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