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

A. Gunnarsson ; A. Snijder ; J. Hicks ; J. Gunnarsson ; Fredrik Höök (Institutionen för teknisk fysik, Biologisk fysik) ; S. Geschwindner
Analytical Chemistry (0003-2700). Vol. 87 (2015), 8, p. 4100-4103.
[Artikel, refereegranskad vetenskaplig]

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 < nM) compounds.



Denna post skapades 2015-05-21.
CPL Pubid: 217392

 

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Institutioner (Chalmers)

Institutionen för teknisk fysik, Biologisk fysik (2007-2015)

Ämnesområden

Analytisk kemi

Chalmers infrastruktur