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

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) 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) and off rates (k) of labeled and unlabeled ligands binding to the adenosine A receptor (AR) purified into nanodiscs (AR-NDs). The kinetic assay was used to determine the optimal storage conditions of AR-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 AR function, and how AR protonation affects agonist binding.