title: 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 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 (125)I-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 [(3)H]INCB3344 and [(3)H]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.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands (A.J.M.Z., I.N., H.V., S.H., M.L.J.D., H.d.V., A.P.IJ., L.H.H.); Vertex Pharmaceuticals, Inc., San Diego, California (J.A., R.G., D.S., J.S.); and Division of Medicinal Chemistry, VU University Amsterdam, Amsterdam, The Netherlands (M.J.S.).
authors.forename: Annelien J M
authors.initials: AJ
authors.lastname: Zweemer
authors.forename: Indira
authors.initials: I
authors.lastname: Nederpelt
authors.forename: Hilde
authors.initials: H
authors.lastname: Vrieling
authors.forename: Sarah
authors.initials: S
authors.lastname: Hafith
authors.forename: Maarten L J
authors.initials: ML
authors.lastname: Doornbos
authors.forename: Henk
authors.initials: H
authors.lastname: de Vries
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Abt
authors.forename: Raymond
authors.initials: R
authors.lastname: Gross
authors.forename: Dean
authors.initials: D
authors.lastname: Stamos
authors.forename: John
authors.initials: J
authors.lastname: Saunders
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Smit
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: Ijzerman
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism / Q000494:pharmacology; D018925:Chemokines / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D011759:Pyrrolidines / Q000378:metabolism* / Q000494:pharmacology; D054390:Receptors, CCR2 / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D018932:Chemokine CCL2; D018925:Chemokines; C505875:INCB3344; D011759:Pyrrolidines; D054390:Receptors, CCR2