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Nuska Tschammer - One of the best experts on this subject based on the ideXlab platform.
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the Chemokine Receptor CXCR3 isoform b drives breast cancer stem cells
Breast Cancer: Basic and Clinical Research, 2019Co-Authors: Nuska Tschammer, Namita Kundu, Regine Brox, Xiaoxuan Fan, Tyler Kochel, Jocelyn Reader, Amy M FultonAbstract:We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The Chemokine Receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC Chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this Receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24- phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.
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molecular mechanisms of biased and probe dependent signaling at cxc motif Chemokine Receptor CXCR3 induced by negative allosteric modulators
Molecular Pharmacology, 2018Co-Authors: Regine Brox, Markus R Heinrich, Paul Baumeister, Armin Buschauer, Lampros Milanos, Noureldin Saleh, Timothy Clark, Dagmar Hofmann, Nuska TschammerAbstract: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 ([3H]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 F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 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, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the Chemokine response. Notably, D1864.60, W2686.48, and S3047.39 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.
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identification of two distinct sites for antagonist and biased agonist binding to the human Chemokine Receptor CXCR3
Angewandte Chemie, 2016Co-Authors: Lampros Milanos, Nuska Tschammer, Noureldin Saleh, Ralf C Kling, Jonas Kaindl, Timothy ClarkAbstract:The Chemokine Receptor CXCR3 is a G protein-coupled Receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small-molecule allosteric CXCR3-agonists do not bind to the same allosteric binding pocket as 8-azaquinazolinone-based negative allosteric modulators. We have now performed molecular-dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3-binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a "shallow" and a second "deeper" pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively.
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development of photoactivatable allosteric modulators for the Chemokine Receptor CXCR3
ChemMedChem, 2016Co-Authors: Tizita Haimanot Admas, Viachaslau Bernat, Markus R Heinrich, Nuska TschammerAbstract: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.
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ligand biased and probe dependent modulation of Chemokine Receptor CXCR3 signaling by negative allosteric modulators
ChemMedChem, 2015Co-Authors: Viachaslau Bernat, Markus R Heinrich, Regine Brox, Yves Auberson, Nuska TschammerAbstract:Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled Receptors (GPCRs). The CXC Chemokine Receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate Receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC Chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.
Craig Gerard - One of the best experts on this subject based on the ideXlab platform.
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Memory T cells migrate to and reject vascularized cardiac allografts independent of the Chemokine Receptor CXCR3.
Transplantation, 2011Co-Authors: Martin H. Oberbarnscheidt, Craig Gerard, Jeffrey M. Walch, Amanda L. Williams, John T. Walters, Rosemary A. Hoffman, Anthony J. Demetris, Geoffrey Camirand, Fadi G. LakkisAbstract:Background. Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here, we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T-cell migration to vascularized allografts. Methods. CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3 ―/― and wildtype B6 mice and cotransferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hr later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues. Results. We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype versus CXCR3 ―/― CD4 and CD8 memory T cells to allografts could be detected in wildtype or aly/aly hosts. In the latter, wildtype and CXCR3 ―/― memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a Chemokine Receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3 ―/― memory T cells. Conclusions. CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell-homing to transplanted organs may be distinct from those required for their migration to sites of infection.
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Chemokine Receptor CXCR3 promotes growth of glioma
Carcinogenesis, 2011Co-Authors: Che Liu, Craig Gerard, Defang Luo, Brent A Reynolds, Geeta Meher, Alan R Katritzky, Cyrus P Bhadha, Jeffrey K HarrisonAbstract:Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3+, which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this Receptor may be a potential therapeutic target for treating human GBM.
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prolongation of cardiac and islet allograft survival by a blocking hamster anti mouse CXCR3 monoclonal antibody
Transplantation, 2008Co-Authors: Ravindra Uppaluri, Craig Gerard, Wayne W Hancock, Kathleen C F Sheehan, Liqing Wang, Jack D Bui, Joshua J Brotman, Robert D SchreiberAbstract:Background Acute allograft rejection requires a multifaceted immune response involving trafficking of immune cells into the transplant and expression of effector cell functions leading to graft destruction. The Chemokine Receptor CXCR3 and its ligands, CXCL9, CXCL10 and CXCL11, constitute an important pathway for effector cell recruitment post-transplant. However, analysis of CXCR3 expression and function has been hampered by a general lack of availability of a neutralizing anti-CXCR3 monoclonal antibody (mAb) for use in experimental models.
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gliadin induces an increase in intestinal permeability and zonulin release by binding to the Chemokine Receptor CXCR3
Gastroenterology, 2008Co-Authors: Karen M Lammers, Craig Gerard, Julie Brownley, Karen E Thomas, Prasad Rallabhandi, Terez Sheadonohue, Amir Tamiz, Sefik S Alkan, Sarah Netzelarnett, Toni M AntalisAbstract:Background & Aims: Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment. Methods: α-Gliadin affinity column was loaded with intestinal mucosal membrane lysates to identify the putative gliadin-binding moiety. In vitro experiments with Chemokine Receptor CXCR3 transfectants were performed to confirm binding of gliadin and/or 26 overlapping 20mer α-gliadin synthetic peptides to the Receptor. CXCR3 protein and gene expression were studied in intestinal epithelial cell lines and human biopsy specimens. Gliadin-CXCR3 interaction was further analyzed by immunofluorescence microscopy, laser capture microscopy, real-time reverse-transcription polymerase chain reaction, and immunoprecipitation/Western blot analysis. Ex vivo experiments were performed using C57BL/6 wild-type and CXCR3−/− mouse small intestines to measure intestinal permeability and zonulin release. Results: Affinity column and colocalization experiments showed that gliadin binds to CXCR3 and that at least 2 α-gliadin 20mer synthetic peptides are involved in this binding. CXCR3 is expressed in mouse and human intestinal epithelia and lamina propria. Mucosal CXCR3 expression was elevated in active celiac disease but returned to baseline levels following implementation of a gluten-free diet. Gliadin induced physical association between CXCR3 and MyD88 in enterocytes. Gliadin increased zonulin release and intestinal permeability in wild-type but not CXCR3−/− mouse small intestine. Conclusions: Gliadin binds to CXCR3 and leads to MyD88-dependent zonulin release and increased intestinal permeability.
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the Chemokine Receptor CXCR3 attenuates the control of chronic mycobacterium tuberculosis infection in balb c mice
Journal of Immunology, 2007Co-Authors: Soumya D Chakravarty, Craig Gerard, Joanne L Flynn, John ChanAbstract:The Chemokine Receptor CXCR3 plays a significant role in regulating the migration of Th1 cells. Given the importance of Th1 immunity in the control of tuberculous infection, the results of the present study demonstrating that CXCR3-deficient BALB/c mice are more resistant to Mycobacterium tuberculosis , compared with wild-type mice, is surprising. This enhanced resistance manifests in the chronic but not the acute phase of infection. Remarkable differences in the cellular composition of the pulmonic granuloma of the CXCR3−/− and wild-type mice were found, the most striking being the increase in the number of CD4+ T cells in the knockout strain. In the chronic phase of infection, the number of CD69-expressing CD4+ T lymphocytes in the lungs of CXCR3−/− mice was higher than in wild-type mice. Additionally, at 1 mo postinfection, the number of IFN-γ-producing CD4+ T cells in the lungs and mediastinal lymph nodes of the CXCR3-deficient strain was elevated compared with wild-type mice. Pulmonic expression of IFN-γ, IL-12, TNF-α, or NO synthase 2, the principal antimycobacterial factors, were equivalent in the two mouse strains. These results indicate that: 1) CXCR3 plays a role in modulating the cellular composition of tuberculous granuloma; 2) CXCR3 impairs antimycobacterial activity in chronic tuberculosis; and 3) in the absence of CXCR3, mice exhibit a heightened state of CD4+ T lymphocyte activation in the chronic phase of infection that is associated with enhanced CD4+ T cell priming. Therefore, CXCR3 can attenuate the host immune response to M. tuberculosis by adversely affecting T cell priming.
Regine Brox - One of the best experts on this subject based on the ideXlab platform.
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the Chemokine Receptor CXCR3 isoform b drives breast cancer stem cells
Breast Cancer: Basic and Clinical Research, 2019Co-Authors: Nuska Tschammer, Namita Kundu, Regine Brox, Xiaoxuan Fan, Tyler Kochel, Jocelyn Reader, Amy M FultonAbstract:We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The Chemokine Receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC Chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this Receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24- phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.
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molecular mechanisms of biased and probe dependent signaling at cxc motif Chemokine Receptor CXCR3 induced by negative allosteric modulators
Molecular Pharmacology, 2018Co-Authors: Regine Brox, Markus R Heinrich, Paul Baumeister, Armin Buschauer, Lampros Milanos, Noureldin Saleh, Timothy Clark, Dagmar Hofmann, Nuska TschammerAbstract: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 ([3H]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 F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 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, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the Chemokine response. Notably, D1864.60, W2686.48, and S3047.39 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.
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discovery and characterization of biased allosteric agonists of the Chemokine Receptor CXCR3
Journal of Medicinal Chemistry, 2016Co-Authors: Lampros Milanos, Regine Brox, Theresa Frank, Gasper Poklukar, Ralf Palmisano, Reiner Waibel, Jurgen Einsiedel, Maximilian Durr, Ivana Ivanovicburmazovic, Olav LarsenAbstract:In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to Receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the Chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce Receptor internalization or β-arrestin 2 recruitment. Concomitant structure–activity relationship studies indicated very steep structure–activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational...
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ligand biased and probe dependent modulation of Chemokine Receptor CXCR3 signaling by negative allosteric modulators
ChemMedChem, 2015Co-Authors: Viachaslau Bernat, Markus R Heinrich, Regine Brox, Yves Auberson, Nuska TschammerAbstract:Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled Receptors (GPCRs). The CXC Chemokine Receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate Receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC Chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.
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boronic acids as probes for investigation of allosteric modulation of the Chemokine Receptor CXCR3
ACS Chemical Biology, 2014Co-Authors: Viachaslau Bernat, Regine Brox, Tizita Haimanot Admas, Frank W Heinemann, Nuska TschammerAbstract: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. ...
Wayne W Hancock - One of the best experts on this subject based on the ideXlab platform.
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prolongation of cardiac and islet allograft survival by a blocking hamster anti mouse CXCR3 monoclonal antibody
Transplantation, 2008Co-Authors: Ravindra Uppaluri, Craig Gerard, Wayne W Hancock, Kathleen C F Sheehan, Liqing Wang, Jack D Bui, Joshua J Brotman, Robert D SchreiberAbstract:Background Acute allograft rejection requires a multifaceted immune response involving trafficking of immune cells into the transplant and expression of effector cell functions leading to graft destruction. The Chemokine Receptor CXCR3 and its ligands, CXCL9, CXCL10 and CXCL11, constitute an important pathway for effector cell recruitment post-transplant. However, analysis of CXCR3 expression and function has been hampered by a general lack of availability of a neutralizing anti-CXCR3 monoclonal antibody (mAb) for use in experimental models.
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glomerular infiltration by CXCR3 icos activated t cells in chronic allograft nephropathy with transplant glomerulopathy
American Journal of Transplantation, 2003Co-Authors: Enver Akalin, Steven Dikman, B Murphy, Jonathan S Bromberg, Wayne W HancockAbstract:The pathogeneses of chronic allograft nephropathy (CAN), a leading cause of allograft failure, and one of its complications, transplant glomerulopathy (TGP), are unknown. Immunohistologic analysis of human renal transplant biopsies showed expression of inducible costimulator (ICOS), the Chemokine Receptor CXCR3, and its ligands, Mig and IP-10, by intraglomerular and periglomerular leukocytes in biopsies with CAN and TGP but not CAN alone. ICOS and CXCR3 are both characteristics of activated, effector T cells, suggesting different pathogenetic mechanisms underlying TGP vs. CAN. We conclude that targeting of specific Chemokine and Chemokine Receptor pathways and/or ICOS may have clinical application in the prevention and treatment of TGP.
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expression of the Chemokine Receptor CXCR3 and its ligand ip 10 during human cardiac allograft rejection
Circulation, 2001Co-Authors: Michael Melter, Wayne W Hancock, Andrea Exeni, Marlies E J Reinders, James C Fang, Gearoid M Mcmahon, Peter Ganz, David M BriscoeAbstract:Background Chemokines play an essential role in regulating the infiltration of leukocytes into allografts in experimental models. Little is known of their expression or function after human cardiac transplantation. Methods and Results We analyzed 169 sequential human endomyocardial biopsies by immunocytochemistry for infiltration by CD3+ T cells and the expression of the Chemokine Receptors CCR1, CCR3, CCR5, and CXCR3. In both cross-sectional and longitudinal analyses, the expression of each of the Chemokine Receptors correlated with the degree of CD3+ T-cell infiltration. In particular, the expression of CXCR3 was temporally and spatially associated with CD3+ T-cell infiltrates and correlated with the histopathological diagnosis of acute rejection (OR, 11.73 and 4.05, respectively; P<0.001). Of 7 patients followed up longitudinally for 1 year, 4 with consecutive biopsies developed intimal thickening by intravascular ultrasound. In these patients, there was a trend for persistent expression of CD3- and CX...
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requirement of the Chemokine Receptor CXCR3 for acute allograft rejection
Journal of Experimental Medicine, 2000Co-Authors: Wayne W Hancock, Bao Lu, Vilmos Csizmadia, Kerrie L Faia, Jennifer A King, Stephen T Smiley, Mai Ling, Norma P Gerard, Craig GerardAbstract:Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein–coupled Receptors. However, in vitro data demonstrating the presence of multiple ligands for a given Chemokine Receptor, and often multiple Receptors for a given Chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the Chemokines interferon (IFN)-γ–inducible protein of 10 kD (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T cell α chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding Chemokine Receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3−/−) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3−/− allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR+/+ mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.
Markus R Heinrich - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanisms of biased and probe dependent signaling at cxc motif Chemokine Receptor CXCR3 induced by negative allosteric modulators
Molecular Pharmacology, 2018Co-Authors: Regine Brox, Markus R Heinrich, Paul Baumeister, Armin Buschauer, Lampros Milanos, Noureldin Saleh, Timothy Clark, Dagmar Hofmann, Nuska TschammerAbstract: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 ([3H]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 F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 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, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the Chemokine response. Notably, D1864.60, W2686.48, and S3047.39 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.
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development of photoactivatable allosteric modulators for the Chemokine Receptor CXCR3
ChemMedChem, 2016Co-Authors: Tizita Haimanot Admas, Viachaslau Bernat, Markus R Heinrich, Nuska TschammerAbstract: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.
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ligand biased and probe dependent modulation of Chemokine Receptor CXCR3 signaling by negative allosteric modulators
ChemMedChem, 2015Co-Authors: Viachaslau Bernat, Markus R Heinrich, Regine Brox, Yves Auberson, Nuska TschammerAbstract:Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled Receptors (GPCRs). The CXC Chemokine Receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate Receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC Chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.
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synthesis and application of the first radioligand targeting the allosteric binding pocket of Chemokine Receptor CXCR3
ChemMedChem, 2012Co-Authors: Viachaslau Bernat, Markus R Heinrich, Paul Baumeister, Armin Buschauer, Nuska TschammerAbstract:Strategies for the identification of allosteric modulators of Chemokine Receptors largely rely on various cell-based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium-labeled allosteric modulator of thehuman Chemokine Receptor CXCR3 (RAMX3, [3H]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). RAMX3 is chemically derived from 8-azaquinazolinone-type allosteric modulators and binds to the CXCR3 Receptor with a Kd value of 1.08 nM (specific activity: 80.4 Ci mmol-1). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and Chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8-azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 Receptor.