title: Molecular interaction of a potent nonpeptide agonist with the chemokine receptor CCR8.
abstract: 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 (Glu(286)) 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 (Phe(254)). 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.
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, Copenhagen University, Blegdamsvej 2, 2200 Copenhagen, Denmark.
authors.forename: Pia C
authors.initials: PC
authors.lastname: Jensen
authors.forename: Rie
authors.initials: R
authors.lastname: Nygaard
authors.forename: Stefanie
authors.initials: S
authors.lastname: Thiele
authors.forename: Amy
authors.initials: A
authors.lastname: Elder
authors.forename: Guoming
authors.initials: G
authors.lastname: Zhu
authors.forename: Roland
authors.initials: R
authors.lastname: Kolbeck
authors.forename: Shomir
authors.initials: S
authors.lastname: Ghosh
authors.forename: Thue W
authors.initials: TW
authors.lastname: Schwartz
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D054402:Chemokine CCL1; D019742:Chemokines, CC / Q000494:pharmacology; D002522:Chlorocebus aethiops; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054401:Receptors, CCR8; D019707:Receptors, Chemokine / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: C065971:CCL1 protein, human; C103330:CCR8 protein, human; D054402:Chemokine CCL1; D019742:Chemokines, CC; D054401:Receptors, CCR8; D019707:Receptors, Chemokine