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 inflam matory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-t ransmembrane receptor superfamily, These studies identified a family of ant agonists, spiropiperidines. One of the more potent compounds blocks MCP-1 b inding to CCR2 with a K-d of 60 nM, but it is unable to block binding to CX CR1, CCR1, or CCR3, These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis, Th e 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 re sidue contributes little to MCP-1 binding to CCR2, The basic nitrogen prese nt in the spiropiperidine compounds may be the interaction partner for Glu( 291), be cause the basicity of this nitrogen was essential for affinity; fu rthermore, a different class of antagonists, a class that does not have a b asic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(29 1). In addition to the CCR2 receptor, spiropiperidine compounds have affini ty for several biogenic amine receptors, Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position sim ilar to the acidic residue contributed from transmembrane-3 of biogenic ami ne 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 wit hin the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling thus the small molecul e ligands act as antagonists. An acidic residue in transmembrane region 7 i s found in most chemokine receptors and is rare in other serpentine recepto rs. The model of the binding site may suggest ways to make new small molecu le chemokine receptor antagonists, and it may rationalize the design of mor e potent and selective antagonists.