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Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

Vitiligo is a hypopigmentary skin pathology resulting from the death of melanocytes due to the activity of CD8 + cytotoxic lymphocytes and overexpression of chemokines. These include CXCL9, CXCL10, and CXCL11 and its receptor CXCR3, both in peripheral cells of the immune system and in the skin of patients diagnosed with vitiligo. The three-dimensional structure of CXCR3 and CXCL9 has not been reported experimentally; thus, homology modeling and molecular dynamics could be useful for the study of this chemotaxis-promoter axis. In this work, a homology model of CXCR3 and CXCL9 and the structure of the CXCR3/Gα i/0 βγ complex with post-translational modifications of CXCR3 are reported for the study of the interaction of chemokines with CXCR3 through all-atom (AA-MD) and coarse-grained molecular dynamics (CG-MD) simulations. AA-MD and CG-MD simulations showed the first activation step of the CXCR3 receptor with all chemokines and the second activation step in the CXCR3-CXCL10 complex through a decrease in the distance between the chemokine and the transmembrane region of CXCR3 and the separation of the βγ complex from the α subunit in the G-protein. Additionally, a general protein-ligand interaction model was calculated, based on known antagonists binding to CXCR3. These results contribute to understanding the activation mechanism of CXCR3 and the design of new molecules that inhibit chemokine binding or antagonize the receptor, provoking a decrease of chemotaxis caused by the CXCR3/chemokines axis.

Chemokines bind to membrane-spanning chemokine receptors, which signal through G proteins and promote cell migration. However, atypical chemokine receptor 3 (ACKR3) does not appear to couple to G proteins, and instead of directly promoting cell migration, it regulates the extracellular concentration of chemokines that it shares with the G protein-coupled receptors (GPCRs) CXCR3 and CXCR4, thereby influencing the responses of these receptors. Understanding how these receptors bind their ligands is important for understanding these different processes. Here, we applied association and dissociation kinetic measurements coupled to β-arrestin recruitment assays to investigate ACKR3:chemokine interactions. Our results showed that CXCL12 binding is unusually slow and driven by the interplay between multiple binding epitopes. We also found that the amino terminus of the receptor played a key role in chemokine binding and activation by preventing chemokine dissociation. It was thought that chemokines initially bind receptors through interactions between the globular domain of the chemokine and the receptor amino terminus, which then guides the chemokine amino terminus into the transmembrane pocket of the receptor to initiate signaling. On the basis of our kinetic data, we propose an alternative mechanism in which the amino terminus of the chemokine initially forms interactions with the extracellular loops and transmembrane pocket of the receptor, which is followed by the receptor amino terminus wrapping around the core of the chemokine to prolong its residence time. These data provide insight into how ACKR3 competes and cooperates with canonical GPCRs in its function as a scavenger receptor., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Here we describe an experimental technique, termed plasmon waveguide resonance (PWR) spectroscopy that enables the characterization of molecular interactions occurring at the level of anisotropic thin films as lipid membranes and therein inserted or interacting molecules. PWR allows one to characterize such molecular interactions at different levels: (1) acquire binding curves and calculate dissociation constants; (2) obtain kinetic information; (3) obtain information about associated anisotropy changes and changes in membrane thickness; (4) obtain insight about lateral homogeneity (formation of domains). Points 1, 2, and 4 can be directly obtained from the data. Point 3 requires spectral fitting procedures so that the different optical parameters characterizing thin films as proteolipid membranes, namely refractive index and extinction coefficient for both p- (TM component of light that is parallel to the incident light) and s- (TE component of light that is perpendicular to the incident light) polarizations and thickness, can be determined. When applied to membrane proteins as the G-protein coupled receptor (GPCR) family, both ligand-induced conformational changes of the receptor can be followed as well as interactions with effectors (e.g., G-proteins). Additionally, by either altering the lipid composition in cellular membranes or specifically controlling its composition in the case of lipid model membranes with reconstituted proteins, the role of the lipid environment in receptor activation and signaling can be determined. Additionally, the eventual partition of receptors in different lipid microdomains (e.g., lipid rafts) can be followed. Such information can be obtained  ex cellulo with mammalian cell membrane fragments expressing the protein of interest and/or in vitro with lipid model systems where the protein under investigation has been reconstituted. Moreover, PWR can also be applied to directly follow the reconstitution of membrane proteins in lipid model membranes. The measurements are performed directly (no labeling of molecular partners), in real time and with very high sensitivity. Here we will discuss different aspects of GPCR activation and signaling where PWR brought important information in parallel with other approaches. The utility of PWR is not limited to GPCRs but can be applied to any membrane protein. PWR is also an excellent tool to characterize the interaction of membrane active molecules (as cell penetrating, antimicrobial, viral and amyloid peptides) with lipids. A brief section is dedicated to such applications, with particular emphasis on amyloid peptides. To finalize, as PWR is a homemade technology, ongoing instrument developments aiming at breaking current experimental limitations are briefly discussed, namely, the coupling of PWR with electrochemical measurements and the expansion of measurements from the visible to the infrared region.

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 β-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 β-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 β-arrestin-biased, but not a G protein-biased, agonist potentiated inflammation. T cell recruitment was increased by the β-arrestin-biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and β-arrestin in T cells. In mouse and human T cells, the β-arrestin-biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that β-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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The G-coupled receptors seen on the cell surface are composites with a lipid bilayer. The chemokines are kind of G-coupled receptor which majorly involved in the activation and downstream signalling of the cell. In general, many G-coupled receptors lack their 3D structures which become a hurdle in the drug designing process. In this study, comparative modelling of the CXCR3 receptor was carried out, structure evaluation was done using various tools and softwares. Additionally, molecular dynamics and docking were performed to prove the structural quality and architecture. Interestingly, the studies like toggle switch mechanism, lipid dynamics, virtual screening were carried out to find the potent antagonist for the CXCR3 receptor. During virtual screening 14,303 similar molecules were retrieved among them only four compounds have an ability to interact with a crucial amino acid residue of an antagonist. Hence, these screened compounds can serve as a drug candidate for a CXCR3 receptor, but further in vitro and in vivo studies are ought to do to prove its same efficacy.

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 ( N -1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d ]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)- N -[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[( N -{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d ]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([ 3 H] 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-methylpiperidin-4-yl)methyl]acetamide), we identified that F131 3.32 and Y308 7.43 contribute specifically to the binding pocket of BD064, whereas D186 4.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and β -arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F131 3.32 , S304 7.39 , and Y308 7.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D186 4.60 , W268 6.48 , and S304 7.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but β -arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first β-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.

The large yellow croaker (Larimichthys crocea) has a well-developed innate immune system. We studied a component of this system, chemokine receptor CXCR family. In this study, we report the full-length open reading frames, as well as the identification and characterization of the chemokine receptor genes CXCR2 (LycCXCR2), CXCR3 (LycCXCR3), and CXCR4 (LycCXCR4) of large yellow croaker. We report that LycCXCR3 and LycCXCR4 are evolving neutrally according to PAML analyses. Quantitative real-time PCR analysis revealed that CXCR transcripts were expressed in all examined tissues. The expression of chemokine receptors LycCXCR2, LycCXCR3, and LycCXCR4 was elevated in the kidney, spleen, and particularly the liver of the large yellow croaker after challenge with Vibrio anguillarum and polyinosinic:polycytidylic acid (poly I:C). These results suggest that LycCXCR2, LycCXCR3, and LycCXCR4 may be important immune-related genes, playing crucial roles in immune defence against bacterial infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chemokines and their receptors are known to play important roles in disease. More than 40 chemokine ligands and 20 chemokine receptors have been identified, but, to date, only two small molecule chemokine receptor antagonists have been approved by the FDA. The chemokine receptor CXCR3 was identified in 1996, and nearly 20 years later, new areas of CXCR3 disease biology continue to emerge. Several classes of small molecule CXCR3 antagonists have been developed, and two have shown efficacy in preclinical models of inflammatory disease. However, only one CXCR3 antagonist has been evaluated in clinical trials, and there remain many opportunities to further investigate known classes of CXCR3 antagonists and to identify new chemotypes. This Perspective reviews the known CXCR3 antagonists and considers future opportunities for the development of small molecules for clinical evaluation.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 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.

Background and Purpose: The chemokine receptor CXCR3 is implicated in a variety of clinically important diseases, notably rheumatoid arthritis and atherosclerosis. Consequently, antagonists of CXCR3 are of therapeutic interest. In this study, we set out to characterize binding sites of the specific low MW CXCR3 antagonist VUF10085 and the broad spectrum antagonist TAK-779 which blocks CXCR3 along with CCR2 and CCR5., Experimental Approach: Molecular modelling of CXCR3, followed by virtual ligand docking, highlighted several CXCR3 residues likely to contact either antagonist, notably a conserved aspartate in helix 2 (Asp-112(2:63) ), which was postulated to interact with the quaternary nitrogen of TAK-779. Validation of modelling was carried out by site-directed mutagenesis of CXCR3, followed by assays of cell surface expression, ligand binding and receptor activation., Key Results: Mutation of Asn-132(3.33) , Phe-207 and Tyr-271(6.51) within CXCR3 severely impaired both ligand binding and chemotactic responses, suggesting that these residues are critical for maintenance of a functional CXCR3 conformation. Contrary to our hypothesis, mutation of Asp-112(2:63) had no observable effects on TAK-779 activity, but clearly decreased the antagonist potency of VUF 10085. Likewise, mutations of Phe-131(3.32) , Ile-279(6.59) and Tyr-308(7.43) were well tolerated and were critical for the antagonist activity of VUF 10085 but not for that of TAK-779., Conclusions and Implications: This more detailed definition of a binding pocket within CXCR3 for low MW antagonists should facilitate the rational design of newer CXCR3 antagonists, with obvious clinical potential., (© 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

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.

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure-activity relationship and structure-function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of ortho-substituents play an important role in determining agonist efficacies. Compounds 38 (VUF11222) and 39 (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling.

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., (© 2012 John Wiley & Sons A/S.)

Dual-specific antibodies are characterized by an antigen-combining site mediating specific interactions with two different antigens. We have generated five dual-specific single chain variable fragments (scFv) that neutralize the activity of the two chemokines, CXCL9 and CXCL10, to bind to their receptor CXCR3. To better understand how these dual-specific scFvs bind these two chemokines that only share a 37% sequence identity, we mapped their epitopes on human CXCL9 and CXCL10 and identified serine 13 (Ser(13)) as a critical residue. It is conserved between the two chemokines but not in the third ligand for CXCR3, CXCL11. Furthermore, Ser(13) is exposed in the tetrameric structure of CXCL10, which is consistent with our finding that the scFvs are able to bind to CXCL9 and CXCL10 immobilized on glycosaminoglycans. Overall, the data indicate that these dual-specific scFvs bind to a conserved surface involved in CXCR3 receptor interaction for CXCL10 and CXCL9. Thus, structural mimicry between the two targets is likely to be responsible for the observed dual specificity of these antibody fragments.

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.

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.

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.

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.

A CXCR3 pocket capable of accommodating polycycloaliphatics was explored using a modular synthetic strategy. The systematic studies reveal that the tricyclic 2-adamantane and bicyclic (iso)bornyl group are efficiently recognized by CXCR3.

Chemokine receptors belong to the large family of 7-transmembrane (7TM) G-protein-coupled receptors. These receptors are targeted and activated by a variety of different ligands, indicating that activation is a result of similar molecular mechanisms but not necessarily similar modes of ligand binding. Attempts to unravel the activation mechanism of 7TM receptors have led to the conclusion that activation involves movements of the transmembrane segments VI and VII in particular, as recently gathered in the Global Toggle Switch Model. However, to understand the activation mechanism completely, more research has to be done in this field. Chemokine receptors are interesting tools in this matter. First, the chemokine system has a high degree of promiscuity that allows several chemokines to target one receptor in different ways, as well as a single chemokine ligand to target several receptors in different ways. Second, the endogenous ligands are large proteins that mainly activate their cognate receptors by interacting with various extracellular-located receptor regions. It is, however, also possible to introduce agonism of simple ligands like metal ions. Thus, the chemokine system offers the possibility to test and compare the activation profiles of several chemically diverse ligands. This also brings up the interesting discussion of allosterism, because small molecules in the chemokine field often interact with allosteric receptor sites.

The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T lymphocytes. We describe in this study the endocytosis of CXCR3 using T lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 down-regulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis, and transport through the endoplasmic reticulum and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C terminus revealed that internalization and degradation were independent of phosphorylation, ubiquitination, or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C terminus. Although freshly isolated T lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Because CXCR3(+) cells are themselves a source of IFN-gamma, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T lymphocyte recruitment.

Aim: To generate and identify the rabbit polyclonal antibodies against NH(2)-terminal peptides of CXCR3-B., Methods: Three peptides (aa. 1 to 19, aa. 17 to 35, and aa. 33 to 51) of human CXCR3-B NH(2)-terminus were synthesized by using standard Fmoc. The synthesized peptides were purified by reversed phase high-performance liquid chromatography (RP-HPLC), and cross-linked with keyhole limpet hemocyanin (KLH) by sodium metaperiodate. Rabbits were immunized with conjugated peptides for 3 times (400 microg/rabbit). The polyclonal antibodies were purified by Protein G from the collected antiserum., Results: NH(2)-terminal peptides of human CXCR3-B with the purity of 98%, 88.54%, and 80%, respectively, were prepared. The titers of purified polyclonal antibodies were 1:32,000 (0.1 mg/L), 1:4,000 (3 mg/L), and 1:1,000 (10 mg/L), respectively. Western blot results showed that the antibodies could recognize the protein with molecular weight of 50,000 in the total lysates of human fetal heart, whereas the antibodies against the peptides of No.1-19 amino acids could also recognize an additional protein (40,000). The antigens recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues., Conclusion: The polyclonal antibodies against NH(2)-terminal peptides generated and identified in the present work are specific to CXCR3-B protein and therefore can be useful tools for the functional study of human CXCR3-B.