Thermodynamics and Docking of Agonists to the β 2- Adrenoceptor Determined Using [ 3H](R,R′)-4-Methoxyfenoterol as the Marker Ligand

Abstract

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β₂-adrenergic receptor, we developed a new agonist radioligand for binding assays. [³H](R,R′)-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β₂-adrenoceptor (AR) population as determined with the antagonist [³H]CGP-12177. The β₂-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K_i values determined for fenoterol analogs model much better the cAMP activity of the β₂-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [³H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β₂-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β₂-adrenoceptor.