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Isothermal Chemical Denaturation to Determine Binding Affinity of Small Molecules to G-Protein Coupled Receptors

Ross, Patrick and Weihofen, Wilhelm and Siu, Fai and Xie, Amy and Katakia, Hetal and Wright, Kirk and Hunt, Ian and Brown, Richard and Freire, Ernesto (2015) Isothermal Chemical Denaturation to Determine Binding Affinity of Small Molecules to G-Protein Coupled Receptors. Analytical Biochemistry, 473. pp. 41-45.

Abstract

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation (ICD) has been shown to be a valuable biophysical method to determine in a direct and label-free fashion the binding of ligands to soluble proteins. In this communication, the application of isothermal chemical denaturation is applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of nineteen small molecule agonists/antagonists of the A2aR were determined and found to be in strong agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically GPCRs in vitro.

Item Type: Article
Date Deposited: 13 Oct 2015 13:12
Last Modified: 13 Oct 2015 13:12
URI: https://oak.novartis.com/id/eprint/23780

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