Ligand binding kinetics to evaluate the function and stability of A2AR in nanodiscs.

Abstract

G-protein-coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor, with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug-target residence time (1/k_off) has been suggested to improve predictions of in vivo success. Here, a ligand binding assay using fluorescence anisotropy was implemented to successfully determine on rates (k_on) and off rates (k_off) of labeled and unlabeled ligands binding to the adenosine A_2A receptor (A_2AR) purified into nanodiscs (A_2AR-NDs). The kinetic assay was used to determine the optimal storage conditions of A_2AR-NDs, where they were found to be stable for more than 6 months at -80°C. The binding assay was implemented to further understand receptor function by determining the effects of charged lipids on agonist binding kinetics, how sodium levels allosterically modulate A_2AR function, and how A_2AR protonation affects agonist binding.