Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors

Patrick Ross, Wilhelm Weihofen, Fai Siu, Amy Xie, Hetal Katakia, S. Kirk Wright, Ian Hunt, Richard K. Brown, Ernesto Freire

Research output: Contribution to journalArticlepeer-review

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 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 study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in 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 G-protein coupled receptors in vitro.

Original languageEnglish (US)
Pages (from-to)41-45
Number of pages5
JournalAnalytical biochemistry
Volume473
DOIs
StatePublished - Mar 15 2015

Keywords

  • Binding affinity determination
  • Binding thermodynamics
  • Chemical denaturation
  • GPCR

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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