Abstract:We here report an experimentally verified binding mode for the known tripeptidomimetic CXCR4 antagonist KRH-1636 (1). A limited SAR study was first conducted based on the three functionalities of 1, followed by site-directed mutagenesis studies. The receptor mapping showed that both the potency and affinity of 1 were dependent on the transmembrane residues His 113 , Asp 171 , Asp 262 , and His 281 , and also suggested the involvement of

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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.Chemokines (chemotactic cytokines) regulate the differentiation, activation, and recruitment of leukocytes. They also play important roles in several physiological mechanisms outside the immune system such as organogenesis and angiogenesis (1, 2). With ϳ50 members, these cytokines exert their effects through chemokine receptors (23 members), which belong to class A of the family of seven-transmembrane (7TM) 2 G protein-coupled receptors (3). The implications of the chemokine system in a vast number of human diseases (3) have increased the interest in developing potent, selective, and clinically useful chemokine receptor antagonists.The binding of a chemokine to its cognate receptor is initially driven by electrostatic interactions between the overall positively charged chemokine and the negatively charged extracellular surface of the receptor. Then interactions between the chemokine N terminus and residues in the main binding pocket of the receptor trigger receptor activation (4 -6). In contrast, small molecule ligands bind deeper in the main binding pocket and constrain the receptors in either active or inactive conformations (7,8). Whereas most mapping studies of small molecules have focused on the transmembrane areas, newer studies as well as crystal structures of class A receptors suggest that extracellular receptor regions, in particular ex...

Edited by Henrik DohlmanThe small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors.CCR5 is one of 19 human chemokine receptors and thereby belongs to the protein family of seven-transmembrane helix (7TM) 2 G protein-coupled receptors (GPCRs). The human chemokine system additionally comprises around 50 endogenous chemokine ligands, which together with their receptors organize leukocyte trafficking. A chemokine receptor can have several chemokine ligands, and a single chemokine can bind to several receptors, properties that confer redundancy to the system (1). At the same time, the system's components are spatially and temporally organized and characterized by receptor, ligand, and tissue bias (2, 3), implying that a chemokine interacting with a given receptor in a certain tissue in fact relays a very specific and non-redundant signal (4). The chemokine system is investigated as a target for treating acute and chronic inflammations, allergies, and autoimmune diseases but also for cancer growth and metastasis, angiogenesis, and HIV infection (5).Chemokines are 8 -12-kDa large peptides that are divided into four groups according to the position of conserved cysteines: CC-chemokines (25 members), CXC-chemokine (18 members), XC-chemokines (XCL1 and XCL2), and CX 3 CL1 (1). These cysteines form disulfide bridges with cysteines in the chemokine core domain, which itself consists of an N-loop, a three-stranded ␤-sheet, and a C-terminal ␣-helix. The N-terminal residues in front of the first cysteine thereby remain unstructured and flexible (6). Recently, two crystal structures of chemokine receptors in complex with a chemokine ligand were revealed: CXCR4 in complex with the viral chemokine vMIP-II (7) and the viral chemokine receptor US28 in complex with CX 3 CL1 (8). These structures confirmed the overall binding mode of chemokines to their receptors, whereby the chemokine core interacts with extracellular receptor domains, such as the receptor N terminus and extracellular loop (ECL) 2, whereas the flexible chemokine N terminus protrudes into the

Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3 (1−70) affects its activity at CCR1. Truncated CCL3 (5−70) has 10-fold higher potency and enhanced efficacy in β-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP 3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chlorosubstituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu287 7.39 interacts with the N-terminal amine of truncated CCL3 (5−70) and with Zn 2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.

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 ß 1 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 β-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.

We here report the preparation of a new 2,6,8-trisubstituted bicyclic tripeptidomimetic scaffold through TFAmediated cyclization of a linear precursor containing three side chains. The introduction of a triphenylmethyl-protected thiol into carboxylic acid containing building blocks through sulfa Michael additions onto a,b-unsaturated hexafluoroisopropyl esters is described. The stereoselectivity of the bicycle

Here we report a series of close analogues of our recently published scaffold-based tripeptidomimetic CXCR4 antagonists, containing positively charged guanidino groups in R and R, and an aromatic group in R. While contraction/elongation of the guanidine carrying side chains (R and R) resulted in loss of activity, introduction of bromine in position 1 on the naphth-2-ylmethyl moiety (R) resulted in an EC of 61μM (mixture of diastereoisomers) against wild-type CXCR4; thus, the antagonistic activity of these tripeptidomimetics seems to be amenable to optimization of the aromatic moiety. Moreover, for analogues carrying a naphth-2-ylmethyl substituent, we observed that a Pictet-Spengler like cyclization side reaction depended on the nature of the R substituent.