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

Laboratory for Molecular Pharmacology, Department of Pharmacology, Copenhagen University, Copenhagen, Denmark (P.C.J., R.N., S.T., T.W.S., M.M.R.); and Millennium Pharmaceuticals, Massachusetts Institute of Technology, Cambridge, Boston, Massachusetts (A.E., G.Z., R.K., S.G.)

Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist, 8-[3-(2-methoxyphenoxy)benzyl]-1-phenethyl-1,3,8-triaza-spiro[4.5]decan-4-one (LMD-009), that also contains a centrally located, positively charged amine. LMD-009 selectively stimulated CCR8 among the 20 identified human chemokine receptors. It mediated chemotaxis, inositol phosphate accumulation, and calcium release with high potencies (EC₅₀ from 11 to 87 nM) and with efficacies similar to that of the endogenous agonist CCL1, and it competed for ¹²⁵I-CCL1 binding with an affinity of 66 nM. A series of 29 mutations targeting 25 amino acids broadly distributed in the minor and major ligand-binding pockets of CCR8 uncovered that the binding of LMD-009 and of four analogs [2-(1-(3-(2-methoxyphenoxy)benzyl)-4-hydroxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1yl)butanamide (LMD-268), and N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-1,2,3,4-tetrahydro-2-oxoquinoline-4-carboxamide (LMD-174)] included several key-residues for nonpeptide antagonists targeting CCR1, -2, and -5. It is noteworthy that a decrease in potency of nearly 1000-fold was observed for all five compounds for the Ala substitution of the anchor-point GluVII:06 (Glu²⁸⁶) and a gain-of-function of 19-fold was observed for LMD-009 (but not the four other analogs) for the Ala substitution of PheVI:16 (Phe²⁵⁴). These structural hallmarks were particularly important in the generation of a model of the molecular mechanism of action for LMD-009. In conclusion, we present the first molecular mapping of the interaction of a nonpeptide agonist with a chemokine receptor and show that the binding pocket of LMD-009 and of analogs overlaps considerably with the binding pockets of CC-chemokine receptor nonpeptide antagonists in general.

Address correspondence to: Mette M. Rosenkilde, Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, Copenhagen University, Blegdamsvej 2, 2200 Copenhagen, Denmark. E-mail: rosenkilde{at}molpharm.dk