PT  - JOURNAL ARTICLE
AU  - Annelien JM Zweemer
AU  - Indira Nederpelt
AU  - Hilde Vrieling
AU  - Sarah Hafith
AU  - Maarten LJ 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
TI  - Multiple Binding Sites for Small Molecule Antagonists at the Chemokine Receptor CCR2
AID  - 10.1124/mol.113.086850
DP  - 2013 Jul 22
TA  - Molecular Pharmacology
PG  - mol.113.086850
4099  - http://molpharm.aspetjournals.org/content/early/2013/07/22/mol.113.086850.short
4100  - http://molpharm.aspetjournals.org/content/early/2013/07/22/mol.113.086850.full
AB  - The chemokine receptor CCR2 is a G protein-coupled receptor that is primarily activated by the endogenous chemokine 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 of 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, which all displaced the endogenous agonist 125I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 and CCR2-RA, were radiolabeled in order to study the binding site in more 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 also examined CCR2 inhibition in multiple functional assays, including a novel label-free whole cell assay. INCB3344 was found to competitively inhibit CCL2-induced G protein activation, whereas CCR2-RA showed a non-competitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified in two groups with a different binding site and thereby a different mode of inhibition. We have provided further insights in CCR2 antagonism, which are important for the development of novel CCR2 inhibitors.