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-spi-ro[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 (EC 50 from 11 to 87 nM) and with efficacies similar to that of the endogenous ago-nist CCL1, and it competed for 125 I-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-hy-droxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-meth-oxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1-yl)butanamide (LMD-268), and N-(1-(3-(2-methoxyphen-oxy)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. Chemokine receptors belong to the superfamily of rhodop-sin-like G protein-coupled 7TM receptors (Murphy et al., 2000). The chemokine ligands (chemotactic cytokines) are a family of large peptides (70-80 amino acids in length) composed of around 50 members. The CC-chemokines are characterized by the absence of an amino acid between the first two of four conserved cysteines and constitute the largest group (CCL1-28), whereas the CXC-chemokines constitute the other major group (CXCL1-16). Two additional chemo-kines, the XCL1 and the CX3CL1, have been described previously (Murphy et al., 2000). The chemokine system regulates the development, activation, and recruitment of leuko-cytes and plays important roles outside the immune system (for instance, on organogenesis, angiogenesis, and carcinogenesis) (Gerard and Rollins, 2001). CCR8 is selectively expressed on a subset of T-helper-2 (Th2) and regulatory T cells and is ...

Structural Analysis of Chemokine Receptor–Ligand Interactions

“…The Y42 1.39 A, Y113 3.32 A, or E286 7.39 A mutation data suggest that all four ligands share interactions with the minor pocket of CCR8 (Figure 20b). 59 The negative effect of the E286 7.39 A mutation supports a ligand binding mode in which the charged amine of the piperidine-ring of 71 – 75 forms an H-bond and ionic interaction with the carboxylate side chain of E286 7.39 . The differential effects of F254 6.51 A and L257 6.55 A mutants on the potency of 71 , 72 , 73 , 74 , and 75 suggest that the variable left-hand side of these ligands (Figure 20b) is targeting the major pocket.…”

“…A large amount of site-directed mutagenesis studies, covering almost all chemokine receptors (CCR1, 50,51 CCR2, 15,52,53,93,94 CCR3, 95 CCR5, 54−58,90,96−100 CCR8, 59 CCR9, 16 CXCR1, 101−103 CXCR2, 60,61 CXCR3, 63−67,104 CXCR4, 13,21,68−72,74,105−114 CXCR7, 115 and US28 75 ) have resulted in 2709 mutation data points, covering 343 different mutants (Figures 10–13, 18–20). About half of the mutation data (1389 data points, covering 238 different mutants) have resulted from studies with 63 unique small-molecule ligands (molecular weight ≤650), of which 46 are shown in Figures 11–13, 18–20.…”

“…63,67 Mutation of the residue at position 7.39 negatively affects binding affinity and/or potency of CXCL11 for CXCR3 (S304 7.39 ), 65 CXCL11 for ACKR3 (Q301 7.39 ), 115 CCL2 for CCR2, 53,94 CCL3 for CCR5, 58 and CCL5 for CCR5, 55 whereas mutation of E 7.39 does not affect binding affinity/potency of CXCL8 for CXCR1, 125,126 CXCL8 for CXCR2, 60,61 CXCL12 for ACKR3, 115 or CCL1 for CCR8. 59 The effects of single point mutations in the major binding pocket on chemokine ligand binding are receptor dependent (Figure 8), including W 6.48 , F/Y 6.51 , and residues at positions 6.55 and 7.43. For example, W248 6.48 A and Y251 6.51 A/I mutations affect CCL3 binding to CCR5 by 10-fold and 7-fold respectively 54 and Y271 6.51 A decreases the binding of CXCL11 to CXCR3 more than 10-fold.…”

“…Mutation of Y 1.39 , W 2.60 , and F 7.43 affects ligand binding to CXCR4 (Figure 11), 69 CCR5 (Figure 12), 54−57 CCR1 (section 5.6), 50,51 and CCR8 (section 5.8). 59 The 31 bound CXCR4, 16 bound CCR5, and 58 bound CCR2 crystal structures suggest that the highly conserved W 2.60 (Figure 2) plays an important role in ligand binding by forming hydrophobic/aromatic interactions (Figures 3, 5a), and this role is supported by mutation studies on CXCR4 binding to 1 , 69 2 , 69 and 13 (69) (Figure 11) and CCR5 binding to 16 , 54,55,100 17 (Vicriviroc), 55,100 19 , 55 and 21 (55,100) (Figure 12), whereas no mutation studies of this residue position have been reported for CCR2. The conserved Y 1.39 residue (Figure 2) does not interact with any of the cocrystallized CXCR4 ligands, but according to mutation data it is relevant for the binding of some CXCR4 small ligands, such as 1 , 69 2 , 69 and 13 (69) (Figure 11).…”

“…59 These analogues share a common scaffold including a positively charged amino within a piperidine-ring linked to a biphenyl ether on one side and to a variable group to the other site. The Y42 1.39 A, Y113 3.32 A, or E286 7.39 A mutation data suggest that all four ligands share interactions with the minor pocket of CCR8 (Figure 20b).…”