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Background and Purpose The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via β-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in β-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or β-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling. Experimental Approach We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi protein signalling or β-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure–activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling. Key Results One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and β-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were β-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as β-arrestin2-biased indolecarboxamides to induce β-arrestin2 recruitment could be correlated with different ligand features and hH4R regions. Conclusion and Implications Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and β-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-β-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. Experimental Approach We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and β-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. Key Results VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C983.36 prevented this covalent interaction. Conclusion and Implications VUF14480 is thought to bind covalently to the hH4 receptor-C983.36 residue and partially induce hH4 receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

BACKGROUND AND PURPOSE The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers. EXPERIMENTAL APPROACH CXCR3–CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity. KEY RESULTS We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific b-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation. CONCLUSIONS AND IMPLICATIONS This study indicates that heteromeric CXCR3–CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities

Background and Purpose Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR. Experimental Approach We used transfection strategies in HEK293 and human T cells. Key Results Co-expression of hCCX-CKR completely inhibits hCXCR3-induced chemotaxis. We found that hCCX-CKR forms complexes with hCXCR3, suggesting a relationship between CCX-CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX-CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX-CKR-induced inhibition of chemotaxis. Conclusions and Implications These findings suggest that hCCX-CKR prevents hCXCR3-induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

Most cells express a panel of different G protein-coupled receptors (GPCRs) allowing them to respond to at least a corresponding variety of extracellular ligands. In order to come to an integrative well-balanced functional response these ligand-receptor pairs can often cross-regulate each other. Although most GPCRs are fully capable to induce intracellular signalling upon agonist binding on their own, many GPCRs, if not all, appear to exist and function in homomeric and/or heteromeric assemblies for at least some time. Such heteromeric organization offers unique allosteric control of receptor pharmacology and function between the protomers and might even unmask 'new' features. However, it is important to realize that some functional consequences that are proposed to originate from heteromeric receptor interactions may also be observed due to intracellular crosstalk between signalling pathways of non-associated GPCRs. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.