Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle.
Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.
Mirzadegan
T
T
Roche Bioscience, Palo Alto, CA 94304, USA. tara.mirzadegan@roche.com
Diehl
F
F
Ebi
B
B
Bhakta
S
S
Polsky
I
I
McCarley
D
D
Mulkins
M
M
Weatherhead
G S
GS
Lapierre
J M
JM
Dankwardt
J
J
Morgans
D
D
Wilhelm
R
R
Jarnagin
K
K
D000067956:Adenylyl Cyclase Inhibitors; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018946:Chemokine CCL5 / Q000037:antagonists & inhibitors; D054410:Chemokine CCL7; D002633:Chemotaxis; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D016207:Cytokines; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000037:antagonists & inhibitors; D018121:Receptors, Cytokine / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured
D000067956:Adenylyl Cyclase Inhibitors; D018946:Chemokine CCL5; D054410:Chemokine CCL7; D016207:Cytokines; D018076:DNA, Complementary; D008024:Ligands; D018945:Monocyte Chemoattractant Proteins; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D018698:Glutamic Acid; D000242:Cyclic AMP; D008156:Luciferases; D009584:Nitrogen; D002118:Calcium