Multiple Binding Sites for Small-Molecule Antagonists at the CC Chemokine Receptor 2

Abstract

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 smallmolecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, all of which displaced the endogenous agonist 125

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

“…Interestingly, there are also examples of multiple allosteric sites on the same GPCR (Lazareno et al, 2002;de Kruijf et al, 2011;Noetzel et al, 2013;Zweemer et al, 2013), further highlighting the rich potential for allosteric targeting of this receptor family.…”

Discovery and Mapping of an Intracellular Antagonist Binding Site at the Chemokine Receptor CCR2

“…Nonspecific binding was determined with 100 nM unlabeled CCL2 or CCL3, and reactions were terminated as described before. [ 125 I]CCL2 binding assays on U2OS-CCR2 cell membranes were performed as described before (Zweemer et al, 2013). Briefly, the assay was performed in a 100 ml reaction volume containing 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl 2 , 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 15 mg of U2OS-CCR2 cell membrane protein at 37°C.…”

“…COS-7 cells and U2OS-CCR2 cells were cultured as described before (Thiele et al, 2011;Zweemer et al, 2013). CHO cells were cultured in Ham's F12 culture medium supplemented with 10% (v/v) newborn calf serum, penicillin (50 IU/ml), and streptomycin (50 mg/ml) at 37°C and 5% CO 2 .…”

“…Membranes were prepared as described before (Zweemer et al, 2013). Briefly, cells were scraped from 15-cm ø plates upon which the membranes and cytosolic fractions were separated during several centrifugation steps.…”

“…An example of such a CCR2 antagonist is INCB3344 (Fig. 1), to which we refer as an orthosteric antagonist, because it was previously reported to inhibit CCR2 in a competitive manner with respect to the chemokine ligand CCL2 (Shin et al, 2009;Zweemer et al, 2013). Interestingly, other classes of antagonists were discovered to bind at a different binding site than INCB3344 (Zweemer et al, 2013).…”