Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted beta-Secretase Using Single-Molecule Imaging
Artikel i vetenskaplig tidskrift, 2015

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.

Författare

Anders Gunnarsson

AstraZeneca AB

Arjan Snijder

AstraZeneca AB

J. Hicks

AstraZeneca AB

J. Gunnarsson

AstraZeneca AB

Fredrik Höök

Chalmers, Teknisk fysik, Biologisk fysik

S. Geschwindner

AstraZeneca AB

Analytical Chemistry

0003-2700 (ISSN) 1520-6882 (eISSN)

Vol. 87 4100-4103

Ämneskategorier

Analytisk kemi

DOI

10.1021/acs.analchem.5b00740