title: Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2.
abstract: The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Annelien J M
authors.initials: AJ
authors.lastname: Zweemer
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Julia
authors.initials: J
authors.lastname: Bunnik
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Margo
authors.initials: M
authors.lastname: Veenhuizen
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Fabiana
authors.initials: F
authors.lastname: Miraglia
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Eelke B
authors.initials: EB
authors.lastname: Lenselink
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Maris
authors.initials: M
authors.lastname: Vilums
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Henk
authors.initials: H
authors.lastname: de Vries
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Arthur
authors.initials: A
authors.lastname: Gibert
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Stefanie
authors.initials: S
authors.lastname: Thiele
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.).
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands (A.J.M.Z., J.B., M.V., F.M., E.B.L., M.V., H.V., A.G., A.P.IJ., L.H.H.); Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.T., M.M.R.) l.h.heitman@lacdr.leidenuniv.nl.
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D000495:Allosteric Site; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D011759:Pyrrolidines / Q000494:pharmacology; D054390:Receptors, CCR2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C515334:CCR2 protein, human; C505875:INCB3344; D007093:Imidazoles; C532123:JNJ-27141491; D008024:Ligands; D011759:Pyrrolidines; D054390:Receptors, CCR2; D013449:Sulfonamides