TY - JOUR T1 - Multiple Binding Sites for Small-Molecule Antagonists at the CC Chemokine Receptor 2 JF - Molecular Pharmacology JO - Mol Pharmacol SP - 551 LP - 561 DO - 10.1124/mol.113.086850 VL - 84 IS - 4 AU - Annelien J. M. Zweemer AU - Indira Nederpelt AU - Hilde Vrieling AU - Sarah Hafith AU - Maarten L. J. Doornbos AU - Henk de Vries AU - Jeffrey Abt AU - Raymond Gross AU - Dean Stamos AU - John Saunders AU - Martine J. Smit AU - Adriaan P. IJzerman AU - Laura H. Heitman Y1 - 2013/10/01 UR - http://molpharm.aspetjournals.org/content/84/4/551.abstract N2 - The chemokine receptor CCR2 is a G protein–coupled receptor that is activated primarily by the endogenous CC chemokine ligand 2 (CCL2). Many different small-molecule antagonists have been developed to inhibit this receptor, as it is involved in a variety of diseases characterized by chronic inflammation. Unfortunately, all these antagonists lack clinical efficacy, and therefore a better understanding of their mechanism of action is warranted. In this study, we examined the pharmacological properties of small-molecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, all of which displaced the endogenous agonist 125I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 [N-(2-(((3S,4S)-1-((1r,4S)-4-(benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexyl)-4-ethoxypyrrolidin-3-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide] and CCR2-RA, were radiolabeled to study the binding site in greater detail. We discovered that [3H]INCB3344 and [3H]CCR2-RA bind to distinct binding sites at CCR2, the latter being the first allosteric radioligand for CCR2. Besides the binding properties of the antagonists, we examined CCR2 inhibition in multiple functional assays, including a novel label-free whole-cell assay. INCB3344 competitively inhibited CCL2-induced G protein activation, whereas CCR2-RA showed a noncompetitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified into two groups with different binding sites and thereby different modes of inhibition. We have provided further insights in CCR2 antagonism, and these insights are important for the development of novel CCR2 inhibitors. ER -