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Research ArticleArticle

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

Annelien J.M. Zweemer, Julia Bunnik, Margo Veenhuizen, Fabiana Miraglia, Eelke B. Lenselink, Maris Vilums, Henk de Vries, Arthur Gibert, Stefanie Thiele, Mette M. Rosenkilde, Adriaan P. IJzerman and Laura H. Heitman
Molecular Pharmacology October 2014, 86 (4) 358-368; DOI: https://doi.org/10.1124/mol.114.093328
Annelien J.M. Zweemer
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.)
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Julia Bunnik
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.)
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Margo Veenhuizen
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.)
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Fabiana Miraglia
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.)
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Eelke B. Lenselink
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.)
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Maris Vilums
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.)
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Henk de Vries
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.)
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Arthur Gibert
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.)
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Stefanie Thiele
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.)
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Mette M. Rosenkilde
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.)
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Adriaan P. IJzerman
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.)
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Laura H. Heitman
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.)
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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 Y7.53 and phenylalanine F8.50 of the NPxxYx(5,6)F motif, as well as V6.36 at the bottom of TM-VI and K8.49 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.

Footnotes

    • Received April 17, 2014.
    • Accepted July 14, 2014.
  • This study was performed and financially supported within the framework of Top Institute Pharma, project number D1-301.

  • dx.doi.org/10.1124/mol.114.093328.

  • ↵Embedded ImageThis article has supplemental material available at mol.aspetjournals.org

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 86 (4)
Molecular Pharmacology
Vol. 86, Issue 4
1 Oct 2014
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Research ArticleArticle

Intracellular Binding Site at the Chemokine Receptor CCR2

Annelien J.M. Zweemer, Julia Bunnik, Margo Veenhuizen, Fabiana Miraglia, Eelke B. Lenselink, Maris Vilums, Henk de Vries, Arthur Gibert, Stefanie Thiele, Mette M. Rosenkilde, Adriaan P. IJzerman and Laura H. Heitman
Molecular Pharmacology October 1, 2014, 86 (4) 358-368; DOI: https://doi.org/10.1124/mol.114.093328

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Research ArticleArticle

Intracellular Binding Site at the Chemokine Receptor CCR2

Annelien J.M. Zweemer, Julia Bunnik, Margo Veenhuizen, Fabiana Miraglia, Eelke B. Lenselink, Maris Vilums, Henk de Vries, Arthur Gibert, Stefanie Thiele, Mette M. Rosenkilde, Adriaan P. IJzerman and Laura H. Heitman
Molecular Pharmacology October 1, 2014, 86 (4) 358-368; DOI: https://doi.org/10.1124/mol.114.093328
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