Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation.

The chemokine receptor CXCR3 plays a central role in inflammation by mediating effector/memory T cell migration in various diseases; however, drugs targeting CXCR3 and other chemokine receptors are largely ineffective in treating inflammation. Chemokines, the endogenous peptide ligands of chemokine receptors, can exhibit so-called biased agonism by selectively activating either G protein- or ß-arrestin-mediated signaling after receptor binding. Biased agonists might be used as more targeted therapeutics to differentially regulate physiological responses, such as immune cell migration. To test whether CXCR3-mediated physiological responses could be segregated by G protein- and ß-arrestin-mediated signaling, we identified and characterized small-molecule biased agonists of the receptor. In a mouse model of T cell-mediated allergic contact hypersensitivity (CHS), topical application of a ß-arrestin-biased, but not a G protein-biased, agonist potentiated inflammation. T cell recruitment was increased by the ß-arrestin-biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and ß-arrestin in T cells. In mouse and human T cells, the ß-arrestin-biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that ß-arrestin-biased signaling activated the kinase Akt, which promoted T cell migration. This study demonstrates that biased agonists of CXCR3 produce distinct physiological effects, suggesting discrete roles for different endogenous CXCR3 ligands and providing evidence that biased signaling can affect the clinical utility of drugs targeting CXCR3 and other chemokine receptors.

Conformational analysis of the human chemokine receptor CXCR3.

In the last years, some studies showed the patho-genetic role of CXCR3 bound to its ligands in many human inflammatory diseases and cancers. Thus, the blockage of the CXCR3 interaction site to its ligands is seen as a possible therapeutic target for the treatment of cancer. The presence of flexible regions in the chemokine receptors determines their capability to develop specific mechanisms of action. We have recently focused on the features of the N-terminal region of human CXCR3 free in solution, where we demonstrate the presence of numerous conformational ensembles, dynamically stabilized by H-bonds. Since up to now no structure was experimentally determined for CXCR3, we decided to approach the study of its conformational behavior by molecular dynamics simulations, in a lipid bilayer, surrounded of water, at neutral pH and 300K. Furthermore, we modeled the CXCR3/CXCL11 complex, where CXCL11 is one of its natural ligands. The aim of this work is to have a vision as realistic as possible in dynamic terms of the biological mechanism that drives the search for the ligand, its interaction and the formation of a stable complex between CXCR3 and CXCL11. Overall, our approach has been able to describe the structural events which dynamically characterize the molecular mechanisms involved in the binding of CXCR3 to CXCL11 and the critical role exerted by its N-terminal region in "hunting" and capturing the ligand.

Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR).

The chemokine receptor CXCR3 plays important roles in angiogenesis, inflammation and cancer. Activation studies and biological functions of CXCR3 are complex due to the presence of spliced isoforms. CXCR3-A is known as a pro-tumor receptor whereas CXCR3-B exhibits anti-tumor properties. Here, we focused on the conformational change of CXCR3-A and CXCR3-B after agonist or antagonist binding using Plasmon Waveguide Resonance (PWR). Agonist stimulation induced an anisotropic response with very distinct conformational changes for the two isoforms. The CXCR3 agonist bound CXCR3-A with higher affinity than CXCR3-B. Using various concentrations of SCH546738, a CXCR3 specific inhibitor, we demonstrated that low SCH546738 concentrations (≤1 nM) efficiently inhibited CXCR3-A but not CXCR3-B's conformational change and activation. This was confirmed by both, biophysical and biological methods. Taken together, our study demonstrates differences in the behavior of CXCR3-A and CXCR3-B upon ligand activation and antagonist inhibition which may be of relevance for further studies aimed at specifically inhibiting the CXCR3A isoform.

Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse Hippocampus abdominalis: Molecular perspectives and immune defensive role upon pathogenic stress.

CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse CXCR3 and CXCR4 (designated as HaCXCR3 and HaCXCR4) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. HaCXCR3 and HaCXCR4 encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that HaCXCR3 and HaCXCR4 were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney HaCXCR3 and HaCXCR4 mRNA expressions were differently modulated when challenged with Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses.

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3.

The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.

Metabolic profiling of ligands for the chemokine receptor CXCR3 by liquid chromatography-mass spectrometry coupled to bioaffinity assessment.

Chemokine receptors belong to the class of G protein-coupled receptors and are important in the host defense against infections and inflammation. However, aberrant chemokine signaling is linked to different disorders such as cancer, central nervous system and immune disorders, and viral infections [Scholten DJ et al. (2012) Br J Pharmacol 165(6):1617-1643]. Modulating the chemokine receptor function provides new ways of targeting specific diseases. Therefore, discovery and development of drugs targeting chemokine receptors have received considerable attention from the pharmaceutical industry in the past decade. Along with that, the determination of bioactivities of individual metabolites derived from lead compounds towards chemokine receptors is crucial for drug selectivity, pharmacodynamics, and potential toxicity issues. Therefore, advanced analytical methodologies are in high demand. This study is aimed at the optimization of a new analytical method for metabolic profiling with parallel bioaffinity assessment of CXCR3 ligands of the azaquinazolinone and piperazinyl-piperidine class and their metabolites. The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures. The bioaffinity assessment is performed "at-line" via high-resolution nanofractionation onto 96-well plates allowing direct integration of radioligand binding assays. This new method enables identification of metabolites from lead compounds with associated estimation of their individual bioaffinity. Moreover, the identification of the metabolite structures via accurate mass measurements and MS(2) allows the identification of liable metabolic "hotspots" for further lead optimization. The efficient combination of chemokine receptor ligand binding assays with analytical techniques, involving nanofractionation as linking technology, allows implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

CXCR3 in carcinoma progression.

CXCR3 is a G-protein coupled receptor which binds to ELR-negative CXC chemokines that have been found to impact immune responses, vascular develop, and wound repair. More recently, CXCR3 has been examined in the context of cancer and increased expression in many human tumors has been correlated with poor prognosis in breast, melanoma, colon and renal cancer patients. Three variants of CXCR3 are identified so far (CXCR3-A, CXCR3-B and CXCR3-alt) with the two primary ones, CXCR3-A and CXCR3-B, considered to induce opposite physiological functions. Generally, CXCR3-A, the predominant form in hematopoietic cells, appears to mediate tumor "go" signaling via promoting cell proliferation, survival, chemotaxis, invasion and metastasis; while CXCR3-B, the main form on formed elements including epithelial cells, appears to mediate tumor "stop" signaling via promoting growth suppression, apoptosis and vascular involution. Thus, aberrant expression of the isoforms CXCR3-A and CXCR3-B could affect tumor progression. In this review, we have discussed the profiles of CXCR3 variants and related signaling, as well as the role of CXCR3 variants in cancer.

Identifying modulators of CXC receptors 3 and 4 with tailored selectivity using multi-target docking.

The G protein-coupled receptors of the C-X-C subfamily form a group among the chemokine receptors whose endogenous ligands are peptides with a common Cys-X-Cys motif. The CXC chemokine receptors 3 and 4 (CXCR3, CXCR4), which are investigated in this study, are linked to severe diseases such as cancer, multiple sclerosis, and HIV infections. Of particular interest, this receptor pair potentially forms a target for a polypharmacological drug treatment. Considering known ligands from public databases, such dual binders have not been identified yet. We therefore applied large-scale docking to the structure of CXCR4 and a homology model of CXCR3 with the goal to predict such dual binders, as well as compounds selective for either one of the receptors. Using signaling and biochemical assays, we showed that more than 50% of these predictions were correct in each category, yielding ligands with excellent binding efficiencies. These results highlight that docking is a suitable tool for the identification of ligands with tailored binding profiles to GPCRs, even when using homology models. More importantly, we present novel CXCR3-CXCR4 dual modulators that might pave the road to understanding the mechanisms of polypharmacological inhibition of these receptors.

Characterization and expression analysis of an interferon-γ2 induced chemokine receptor CXCR3 in common carp (Cyprinus carpio L.).

Chemokine and chemokine receptor signalling pairs play a crucial role in regulation of cell migration, morphogenesis, and cell activation. Expressed in mammals on activated T and NK cells, chemokine receptor CXCR3 binds interferon-γ inducible chemokines CXCL9-11 and CCL21. Here we sequenced the carp CXCR3 chemokine receptor and showed its relationship to CXCR3a receptors found in other teleosts. We found high expression of the CXCR3 gene in most of the organs and tissues of the immune system and in immune-related tissues such as gills and gut, corroborating a predominantly immune-related function. The very high expression in gill and gut moreover indicates a role for CXCR3 in cell recruitment during infection. High in vivo expression of CXCR3 at later stages of inflammation, as well as its in vitro sensitivity to IFN-γ2 stimulation indicate that in carp, CXCR3 is involved in macrophage-mediated responses. Moreover, as expression of the CXCR3 and CXCb genes coincides in the focus of inflammation and as both the CXCb chemokines and the CXCR3 receptor are significantly up-regulated upon IFN-γ stimulation it is hypothesized that CXCb chemokines may be putative ligands for CXCR3.

The N-terminal region of CXCL11 as structural template for CXCR3 molecular recognition: synthesis, conformational analysis, and binding studies.

The chemokines and their receptors play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. The membrane receptor CXCR3 binds three chemokines, CXCL9, CXCL10, and CXCL11, and its involvement is recognized in many inflammatory diseases and cancers. Therefore, the inhibition of CXCR3 pathway through interactions with three ligands was indicated as putative therapeutic target for the treatment of these diseases, and some inhibitory compounds have already been described in the literature. Recently, we studied the interaction between CXCR3 and its three natural ligands and showed that three CXCR3 ligands bound the receptor mainly by their N-terminal regions using aromatic and electrostatic interactions, and, in particular, CXCL11 had the highest affinity for CXCR3. In light of these results, we focused our attention on what structural region(s) of CXCL11 interacted with CXCR3 and what were the structural features. Therefore, we have synthesized three peptides, corresponding to the N-terminal region of CXCL11, but with different aromatic amino acids, analyzed their conformations by circular dichroism, NMR, and molecular dynamics simulations, simulated their complexes with CXCR3 by docking methods, and validated these data by in vitro studies. The results showed that two peptides were able to bind CXCR3 and to mimic the molecular recognition of CXCL11 and demonstrated that N-terminal region of CXCL11 can be used as template and starting point to obtain new molecules by de novo design approaches.

CXCR3/ligands are significantly involved in the tumorigenesis of basal cell carcinomas.

Basal cell carcinoma (BCC) is the most common skin malignancy encountered worldwide. We hypothesized that CXC chemokines, small cytokines involved in inducing directed leukocyte chemotaxis, could play a key role in the modulation of BCC growth. In this study, quantitative RT-PCR revealed that the chemokines CXCL9, 10, 11, and their receptor CXCR3 were significantly upregulated by an average 22.6-fold, 9.2-fold, 26.6-fold, and 4.9-fold, respectively in BCC tissue samples as compared with nonlesional skin epithelium. Immunohistochemistry analysis revealed that CXCR3, CXCL10, and CXCL11, but not CXCL9, colocalized with cytokeratin 17 (K17) in BCC keratinocytes. In addition, CXCR3 and its ligands were expressed in cells of the surrounding BCC stroma. The chemokines and K17 were also expressed in cultured human immortalized HaCaT keratinocytes. Exposure of HaCaT cells or primary BCC-derived cells to CXCL11 peptides in vitro significantly increased cell proliferation. In primary BCC-derived cell cultures, addition of CXCL11 progressively selected for K17+/CXCR3+ co-expressing cells over time. The expression of CXCR3 and its ligands in human BCC keratinocytes, the enhancement of keratinocyte cell proliferation by CXCL11, and the homogeneity of K17+ BCC cells in human BCC-isolated cell population supported by CXCR3/CXCL11 signaling all suggest that CXCR3 and its ligands may be important autocrine and/or paracrine signaling mediators in the tumorigenesis of BCC.

A combined LS-SVM & MLR QSAR workflow for predicting the inhibition of CXCR3 receptor by quinazolinone analogs.

A novel QSAR workflow is constructed that combines MLR with LS-SVM classification techniques for the identification of quinazolinone analogs as "active" or "non-active" CXCR3 antagonists. The accuracy of the LS-SVM classification technique for the training set and test was 100% and 90%, respectively. For the "active" analogs a validated MLR QSAR model estimates accurately their I-IP10 IC(50) inhibition values. The accuracy of the QSAR model (R (2) = 0.80) is illustrated using various evaluation techniques, such as leave-one-out procedure (R(LOO2)) = 0.67) and validation through an external test set (R(pred2) = 0.78). The key conclusion of this study is that the selected molecular descriptors, Highest Occupied Molecular Orbital energy (HOMO), Principal Moment of Inertia along X and Y axes PMIX and PMIZ, Polar Surface Area (PSA), Presence of triple bond (PTrplBnd), and Kier shape descriptor ((1) kappa), demonstrate discriminatory and pharmacophore abilities.

Modelling of the membrane receptor CXCR3 and its complexes with CXCL9, CXCL10 and CXCL11 chemokines: putative target for new drug design.

The chemokines play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. These comprise over 50 proteins grouped into four classes, in basis to the arrangement of conserved cysteine residues within the sequence. CXCL9, CXCL10 and CXCL11 are the members of the family of ELR-CXC chemokines and bind the same CXCR3 receptor. During the past few years, several studies have demonstrated a pathogenetic role of CXCR3 and its ligands in many human inflammatory diseases. The blockade of CXCR3 interactions with its ligands has been suggested as a possible therapeutic target for the treatment of these diseases. Therefore, we modelled the three-dimensional structure of CXCL9 and CXCR3, and, successively, of the CXCL9/CXCR3 complex in comparison to CXCL10/CXCR3 and CXCL11/CXCR3 complexes. We have then shown the structural determinants of these interactions and their physico-chemical features. Finally, the interaction residues involved in the formation of the complexes have been highlighted and analyzed in order to be used for drug design.