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.
LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein‐Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue‐6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue‐5/issuetoc
Summary
G‐protein‐coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. ...Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter‐metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.
Summary
This review addresses novel concepts of histamine H1‐receptor function and attempts to relate them to the anti‐inflammatory effects of H1‐antihistamines. Furthermore, the molecular mechanisms ...underlying the cardiotoxic effects of H1‐antihistamines are discussed.
H1‐receptors are G‐protein‐coupled‐receptors (GPCRs), the inactive and active conformations of which coexist in equilibrium. The degree receptor activation in the absence of histamine is its ‘constitutive activity’. In this two‐state model, histamine acts as an agonist by combining with and stabilizing the activated conformation of the H1‐receptor to shift the equilibrium towards the activated state. Drugs classified previously as antagonists act as either inverse agonists or neutral antagonists. Inverse agonists combine with and stabilize the inactive conformation of the receptor to shift the equilibrium towards the inactive state. Thus, they may down‐regulate constitutive receptor activity, even in the absence of histamine. Neutral antagonists combine equally with both conformations of the receptor, do not affect basal receptor activity but do interfere with agonist binding. All H1‐antihistamines examined to date are inverse agonists. As the term ‘H1‐receptor antagonists’ is obviously erroneous, we suggest that it be replaced by ‘H1‐antihistamines’.
The observations that H1‐receptors modulate NF‐κB activation and that there are complex interactions between GPCRs, has allowed us to postulate receptor dependent‐mechanisms for some anti‐inflammatory effects of H1‐antihistamines, e.g. inhibition of ICAM‐1 expression and the effects of bradykinin.
Finally, the finding that blockade of HERG1 K+ channels is the mechanism by which some H1‐antihistamines may cause cardiac arrhythmias has allowed the development of preclinical tests to predict such activity.
Background and Purpose
Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active ...molecules. G‐protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high‐resolution structural analyses of GPCR‐ligand complexes.
Experimental Approach
In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high‐resolution structural analyses of (hist)aminergic GPCR‐ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular.
Key Results
Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein‐ligand binding modes, receptor mutation studies, and ligand structure‐selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors.
Conclusions and Implications
We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure‐selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.
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 histamine H
4
receptor is the most recently identified of the G protein‐coupled histamine receptor family and binds several neuroactive drugs, including amitriptyline and ...clozapine. So far, H
4
receptors have been found only on haematopoietic cells, highlighting its importance in inflammatory conditions. Here we investigated the possibility that H
4
receptors may be expressed in both the human and mouse CNS.
Methods:
Immunological and pharmacological studies were performed using a novel anti‐H
4
receptor antibody in both human and mouse brains, and electrophysiological techniques in the mouse brain respectively. Pharmacological tools, selective for the H
4
receptor and patch clamp electrophysiology, were utilized to confirm functional properties of the H
4
receptor in layer IV of the mouse somatosensory cortex.
Results:
Histamine H
4
receptors were prominently expressed in distinct deep laminae, particularly layer VI, in the human cortex, and mouse thalamus, hippocampal CA4 stratum lucidum and layer IV of the cerebral cortex. In layer IV of the mouse somatosensory cortex, the H
4
receptor agonist 4‐methyl histamine (20 µmol·L
−1
) directly hyperpolarized neurons, an effect that was blocked by the selective H
4
receptor antagonist JNJ 10191584, and promoted outwardly rectifying currents in these cells. Monosynaptic thalamocortical CNQX‐sensitive excitatory postsynaptic potentials were not altered by 4‐methyl histamine (20 µmol·L
−1
) suggesting that H
4
receptors did not act as hetero‐receptors on thalamocortical glutamatergic terminals.
Conclusions and implications:
This is the first demonstration that histamine H
4
receptors are functionally expressed on neurons, which has major implications for the therapeutic potential of these receptors in neurology and psychiatry.
Summary
This review addresses novel concepts of histamine H
1
‐receptor function and attempts to relate them to the anti‐inflammatory effects of H
1
‐antihistamines. Furthermore, the molecular ...mechanisms underlying the cardiotoxic effects of H
1
‐antihistamines are discussed.
H
1
‐receptors are G‐protein‐coupled‐receptors (GPCRs), the inactive and active conformations of which coexist in equilibrium. The degree receptor activation in the absence of histamine is its ‘constitutive activity’. In this two‐state model, histamine acts as an agonist by combining with and stabilizing the activated conformation of the H
1
‐receptor to shift the equilibrium towards the activated state. Drugs classified previously as antagonists act as either inverse agonists or neutral antagonists. Inverse agonists combine with and stabilize the inactive conformation of the receptor to shift the equilibrium towards the inactive state. Thus, they may down‐regulate constitutive receptor activity, even in the absence of histamine. Neutral antagonists combine equally with both conformations of the receptor, do not affect basal receptor activity but do interfere with agonist binding. All H
1
‐antihistamines examined to date are inverse agonists. As the term ‘H
1
‐receptor antagonists’ is obviously erroneous, we suggest that it be replaced by ‘H
1
‐antihistamines’.
The observations that H
1
‐receptors modulate NF‐κB activation and that there are complex interactions between GPCRs, has allowed us to postulate receptor dependent‐mechanisms for some anti‐inflammatory effects of H
1
‐antihistamines, e.g. inhibition of ICAM‐1 expression and the effects of bradykinin.
Finally, the finding that blockade of HERG1 K
+
channels is the mechanism by which some H
1
‐antihistamines may cause cardiac arrhythmias has allowed the development of preclinical tests to predict such activity.
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 β‐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
The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a ...small‐molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11.
EXPERIMENTAL APPROACH
Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with 125I‐CXCL10 and 125I‐CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. 35S‐GTPγS binding was used to determine its potency to induce CXCR3‐mediated G protein activation and BRET‐based assays to investigate its effects on intracellular cAMP levels and β‐arrestin recruitment.
KEY RESULTS
VUF10661 acted as a partial agonist in CXCR3‐mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated Gi proteins with the same efficacy as CXCL11 in the 35S‐GTPγS binding and cAMP assay, while it recruited more β‐arrestin1 and β‐arrestin2 to CXCR3 receptors than the chemokine.
CONCLUSIONS AND IMPLICATIONS
VUF10661, like CXCL11, activates both G protein‐dependent and ‐independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or β‐arrestin conformations leading to differences in functional output. Such ligand‐biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.
LINKED ARTICLE
This article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476‐5381.2011.01759.x
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
Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and ...structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.
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•A structure-based virtual screening was used to find fragment-like CXCR4 ligands.•Several inhibitors show binding affinity comparable to hallmark antagonist AMD3100.•Key ligands have distinct competition modes with endogenous chemokine CXCL12.•SAR, 3D-QSAR and predicted binding modes of the hit compounds were analyzed.
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