Biosensor-based affinities and binding kinetics of small molecule antagonists to the adenosine A2A receptor reconstituted in HDL like particles

The options for investigating solubilised G protein-coupled receptors (GPCRs) by biophysical techniques have long been hampered by their instability. A thermostabilised adenosine A(2A) receptor expressed in insect cells, purified in detergent and reconstituted into high-density lipoprotein (HDL) particles was immobilised onto a Surface Plasmon Resonance sensor chip. This allowed measurement of affinities and kinetics for A(2A) antagonists with affinities ranging from 50 pM to almost 2 mu M. Compared with other formats, reproduction of affinities, and dissociation and association rate constants are good, reasonable and poor respectively, indicating stabilised receptors in HDL particles are useful for investigating specific aspects of GPCR-ligand interactions. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.