title: Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors.
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.
authors.affiliation: AVIA Biosystems, Norton, MA 02766, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Ross
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Weihofen
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors.forename: Fai
authors.initials: F
authors.lastname: Siu
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Xie
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors.forename: Hetal
authors.initials: H
authors.lastname: Katakia
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors.forename: S Kirk
authors.initials: SK
authors.lastname: Wright
authors.affiliation: Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Hunt
authors.affiliation: AVIA Biosystems, Norton, MA 02766, USA.
authors.forename: Richard K
authors.initials: RK
authors.lastname: Brown
authors.affiliation: AVIA Biosystems, Norton, MA 02766, USA; Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA. Electronic address: ef@jhu.edu.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Freire
mesh_terms: D058908:Adenosine A2 Receptor Agonists / Q000378:metabolism; D058917:Adenosine A2 Receptor Antagonists / Q000378:metabolism; D001703:Biophysics / Q000379:methods*; D019791:Guanidine / Q000494:pharmacology; D008024:Ligands; D011485:Protein Binding; D011489:Protein Denaturation / Q000187:drug effects*; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature
chemical_list: D058908:Adenosine A2 Receptor Agonists; D058917:Adenosine A2 Receptor Antagonists; D008024:Ligands; D043705:Receptor, Adenosine A2A; D019791:Guanidine
keywords: Binding affinity determination; Binding thermodynamics; Chemical denaturation; GPCR