RT Journal Article SR Electronic T1 Multiple Binding Sites for Small-Molecule Antagonists at the CC Chemokine Receptor 2 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 551 OP 561 DO 10.1124/mol.113.086850 VO 84 IS 4 A1 Annelien J. M. Zweemer A1 Indira Nederpelt A1 Hilde Vrieling A1 Sarah Hafith A1 Maarten L. J. Doornbos A1 Henk de Vries A1 Jeffrey Abt A1 Raymond Gross A1 Dean Stamos A1 John Saunders A1 Martine J. Smit A1 Adriaan P. IJzerman A1 Laura H. Heitman YR 2013 UL http://molpharm.aspetjournals.org/content/84/4/551.abstract AB 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.