Identification of a conserved chemokine receptor motif that enables ligand discrimination.

Extensive ligand-receptor promiscuity in the chemokine signaling system balances beneficial redundancy and specificity. However, this feature poses a major challenge to selectively modulate the system pharmacologically. Here, we identified a conserved cluster of three aromatic receptor residues that anchors the second extracellular loop (ECL2) to the top of receptor transmembrane helices (TM) 4 and 5 and enables recognition of both shared and specific characteristics of interacting chemokines. This cluster was essential for the activation of several chemokine receptors. Furthermore, characteristic motifs of the ß1 strand and 30s loop make the two main CC-chemokine subgroups-the macrophage inflammatory proteins (MIPs) and monocyte chemoattractant proteins (MCPs)-differentially dependent on this cluster in the promiscuous receptors CCR1, CCR2, and CCR5. The cluster additionally enabled CCR1 and CCR5 to discriminate between closely related MIPs based on the N terminus of the chemokine. G protein signaling and ß-arrestin2 recruitment assays confirmed the importance of the conserved cluster in receptor discrimination of chemokine ligands. This extracellular site may facilitate the development of chemokine-related therapeutics.

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

Extracellular disulfide bridges serve different purposes in two homologous chemokine receptors, CCR1 and CCR5.

In addition to the 7 transmembrane receptor (7TM)-conserved disulfide bridge between transmembrane (TM) helix 3 and extracellular loop (ECL)-2, chemokine receptors (CCR) contain a disulfide bridge between the N terminus and what previously was believed to be ECL-3. Recent crystal and NMR structures of the CXC chemokine receptors (CXCR) CXCR4 and CXCR1, combined with structural analysis of all endogenous chemokine receptors indicate that this chemokine receptor-conserved bridge in fact connects the N terminus to the top of TM-7. By employing chemokine ligands that mainly target extracellular receptor regions and small-molecule ligands that predominantly interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other chemokine receptors, high-affinity CCL3 chemokine binding was maintained in the absence of either bridge. In the highly related CCR5, a completely different dependency was observed as neither activation nor binding of the same chemokines was retained in the absence of either bridge. In contrast, both bridges were dispensable for activation by the same small molecules. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1 the preserved folding of ECL-2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup do not necessarily provide specific traits for the whole subgroup but rather provide unique traits to the single receptors.

Multistep continuous-flow synthesis in medicinal chemistry: discovery and preliminary structure-activity relationships of CCR8 ligands.

A three-step continuous-flow synthesis system and its application to the assembly of a new series of chemokine receptor ligands directly from commercial building blocks is reported. No scavenger columns or solvent switches are necessary to recover the desired test compounds, which were obtained in overall yields of 49-94%. The system is modular and flexible, and the individual steps of the sequence can be interchanged with similar outcome, extending the scope of the chemistry. Biological evaluation confirmed activity on the chemokine CCR8 receptor and provided initial structure-activity-relationship (SAR) information for this new ligand series, with the most potent member displaying full agonist activity with single-digit nanomolar potency. To the best of our knowledge, this represents the first published example of efficient use of multistep flow synthesis combined with biological testing and SAR studies in medicinal chemistry.

Modulation in selectivity and allosteric properties of small-molecule ligands for CC-chemokine receptors.

Among 18 human chemokine receptors, CCR1, CCR4, CCR5, and CCR8 were activated by metal ion Zn(II) or Cu(II) in complex with 2,2'-bipyridine or 1,10-phenanthroline with similar potencies (EC(50) from 3.9 to 172 µM). Besides being agonists, they acted as selective allosteric enhancers of CCL3. These actions were dependent on a conserved glutamic acid at TM-7 (VII:06/7.39). A screening of 20 chelator analogues in complex with Zn(II) identified compounds with increased potencies, with 7 reaching highest potency at CCR1 (EC(50) of 0.85 µM), 20 at CCR8 (0.39 µM), and 8 at CCR5 (1.0 µM). Altered selectivity for CCR1 and CCR8 over CCR5 (11, 12) and a receptor-dependent separation of allosteric from intrinsic properties were achieved (20). The pocket similarities of CCR1 and CCR8, contrary to CCR5 as proposed by the ligand screen, were elaborated by computational modeling. These studies facilitate exploration of chemokine receptors as possible targets for therapeutic intervention.