Site-directed Mutagenesis of CC Chemokine Receptor 1 Reveals the Mechanism of Action of UCB 35625, a Small Molecule Chemokine Receptor Antagonist

Abstract

The chemokine receptor CCR1 and its principal ligand, CCL3/MIP-1␣, have been implicated in the pathology of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and asthma. As such, these molecules are the focus of much research with the ultimate aim of developing novel therapies. We have described previously a non-competitive small molecule antagonist of CCR1 (UCB 35625), which we hypothesized interacted with amino acids located within the receptor transmembrane (TM) helices (Sabroe, I., Peck, M. J., Jan Van Keulen, B., Jorritsma, A., Simmons, G., Clapham, P. R., Williams, T. J., and Pease, J. E. (2000) J. Biol. Chem. 275, 25985-25992). Here we describe an approach to identifying the mechanism by which the molecule antagonizes CCR1. Thirty-three point mutants of CCR1 were expressed transiently in L1.2 cells, and the cells were assessed for their capacity to migrate in response to CCL3 in the presence or absence of UCB 35625. Cells expressing the mutant constructs Y41A (TM helix 1, or TM1), Y113A (TM3), and E287A (TM7) were responsive to CCL3 but resistant to the antagonist, consistent with a role for the TM helices in CCR1 interactions with UCB 35625. Subsequent molecular modeling successfully docked the compound with CCR1 and suggests that the antagonist ligates TM1, 2, and 7 of CCR1 and severely impedes access to TM2 and TM3, a region thought to be perturbed by the chemokine amino terminus during the process of receptor activation. Insights into the mechanism of action of these compounds may facilitate the development of more potent antagonists that show promise as future therapeutic agents in the treatment of inflammatory disease.

Molecular Interaction of a Potent Nonpeptide Agonist with the Chemokine Receptor CCR8

“…Because of the similarities in the pharmacophore of LMD-009 to many CC-chemokine receptor antagonists (Mirzadegan et al, 2000;Castonguay et al, 2003;Tsamis et al, 2003;de Mendonça et al, 2005;Maeda et al, 2006;Seibert et al, 2006;Seibert et al, 2006) as described above, it was tempting to believe that LMD-009 would in fact act as an antagonist for other CC-chemokine (or maybe even CXC-chemokine) receptors. We therefore repeated the activity screen of LMD-009, this time as antagonist.…”

“…Nonpeptide antagonists against CC-chemokine receptors usually share a common pharmacophore with a rather centrally located, positively charged amine Seibert et al, 2006). This amine has been shown to interact with a highly conserved Glu in the extracellular end of TM-VII (in position VII:06), whereas the flanking groups have been shown to interact with conserved aromatic residues, as well as other residues specific for each chemokine receptor (Mirzadegan et al, 2000;Castonguay et al, 2003;Tsamis et al, 2003;de Mendonça et al, 2005;Maeda et al, 2006;Seibert et al, 2006). Thus, all CC-chemokine receptor nonpeptide antagonists with characterized binding modes interact with GluVII:06 [except for the recently characterized binding pocket of BX471 in CCR1 (Vaidehi et al, 2006)] in addition to aromatic residues in the major binding pocket (composed of TM-III, TM-IV, TM-V, TM-VI, and TM-VII) and in the minor binding pocket (composed of TM-I, TM-II, TM-III, and TM-VII) ( Fig.…”

“…Thus, all CC-chemokine receptor nonpeptide antagonists with characterized binding modes interact with GluVII:06 [except for the recently characterized binding pocket of BX471 in CCR1 (Vaidehi et al, 2006)] in addition to aromatic residues in the major binding pocket (composed of TM-III, TM-IV, TM-V, TM-VI, and TM-VII) and in the minor binding pocket (composed of TM-I, TM-II, TM-III, and TM-VII) ( Fig. 1) (Mirzadegan et al, 2000;Castonguay et al, 2003;Tsamis et al, 2003;de Mendonça et al, 2005;Maeda et al, 2006;Seibert et al, 2006). Like most chemokine receptor nonpeptide ligands, LMD-009 contains a positively charged amine (the piperidine-ring of the spiro system).…”

Re-evaluation of Chicken CXCR1 Determines the True Gene Structure

“…pcDNA3 either unmodified or containing HA-tagged chCXCR1, HA-tagged CCR1 (24), and FLAG-tagged huCXCR1 constructs was introduced into L1.2 cells by electroporation (28). To enhance cell surface receptor expression, transient transfectants were cultured for 24 h in medium supplemented with 10 mM sodium butyrate (Sigma) prior to use.…”

“…The chCXCR1 cDNA was then excised from the TOPO vector with EcoRI (New England Biolabs) and XbaI (New England Biolabs) and then ligated into the same sites of a modified pcDNA3 vector containing a hemagglutinin epitope 5Ј to the multiple cloning site (24), to generate a recombinant chCXCR1 with an HA epitope at the 5Ј end.…”