GPCR Close-Up: Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins.Liuet al.(p.232) used a protein-engineering strategy to produce a stabilized version of the human A_2Aadenosine receptor (A_2AAR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na⁺on ligand binding and how cholesterol may contribute to GPCR stabilization.

The cannabinoid CB₂receptor (CB₂R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB₂R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB₂R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB₂R agonists to study the role of CB₂R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

The crystal structure of CCR2 chemokine receptor in a complex with two different antagonists—one orthosteric the other allosteric—which functionally cooperate to inhibit CCR2. Chemokine receptors are a family of G-protein-coupled receptors that regulate the migration of immune cells; their function has been implicated in a range of diseases. Two groups reporting in this issue of Nature describe crystal structures of two different chemokine receptors bound to small-molecule inhibitors. Tracy Handel and colleagues describe the structure of CCR2—a promising drug target for autoimmune, inflammatory and metabolic diseases as well as cancer—bound to orthosteric (BMS-681) and allosteric (CCR2-RA-[R]) antagonists. Fiona Marshall and colleagues describe the structure of CCR9—involved in immune cell recruitment to the gut and a promising drug target in inflammatory bowel disease—in complex with the selective CCR9 antagonist vercirnon. Both CCR2 and CCR9 structures reveal an allosteric pocket on the cytoplasmic face of the receptor. This allosteric pocket appears to be highly druggable, and homologous pockets may be present on other chemokine receptors. CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov ) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

BACKGROUND AND PURPOSEThe adenosine A_2Areceptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A_2Areceptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand‐receptor interaction. The aim of this study was to assess the binding kinetics of A_2Areceptor agonists and explore a possible relationship with their functional efficacy.EXPERIMENTAL APPROACHWe set up, validated and optimized a kinetic radioligand binding assay (a so‐called competition association assay) at the A_2Areceptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A_2Areceptor agonists were determined in two different assays: a novel label‐free impedance‐based assay and a more traditional cAMP determination.KEY RESULTSA simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A_2Areceptor ligands at their receptor. A correlation was observed between the receptor residence time of A_2Areceptor agonists and their intrinsic efficacies in both functional assays. The affinity of A_2Areceptor agonists was not correlated to their functional efficacy.CONCLUSIONS AND IMPLICATIONSThis study indicates that the molecular basis of different agonist efficacies at the A_2Areceptor lies within their different residence times at this receptor.