The Experts below are selected from a list of 189 Experts worldwide ranked by ideXlab platform
Rob Leurs - One of the best experts on this subject based on the ideXlab platform.
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pharmacological characterization of a small molecule agonist for the chemokine Receptor CXCR3
British Journal of Pharmacology, 2012Co-Authors: Danny J Scholten, Dennis Verzijl, Martine J Smit, Meritxell Canals, Maikel Wijtmans, S De Munnik, P Nguyen, Ijp De Esch, Henry F Vischer, Rob LeursAbstract:BACKGROUND AND PURPOSE The chemokine Receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACH Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 Receptor and in binding studies, with [125I]-CXCL10 and [125I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [35S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and β-arrestin recruitment. KEY RESULTS VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated Gi proteins with the same efficacy as CXCL11 in the [35S]-GTPγS binding and cAMP assay, while it recruited more β-arrestin1 and β-arrestin2 to CXCR3 Receptors than the chemokine. CONCLUSIONS AND IMPLICATIONS VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 Receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different Receptor and/or β-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists. LINKED ARTICLE This article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x
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synthesis and structure activity relationships of 3h quinazolin 4 ones and 3h pyrido 2 3 d pyrimidin 4 ones as CXCR3 Receptor antagonists
ChemInform, 2007Co-Authors: Stefania Storelli, Dennis Verzijl, Jawad Albadie, Niels Elders, Leontien Bosch, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Rob LeursAbstract:CXC chemokine Receptor-3 (CXCR3) is a G-protein coupled Receptor (GPCR) predominantly expressed on activated T lymphocytes that promote Th1 responses. Previously, we described the 3H-quinazolin-4-one containing VUF 5834 (decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide) as a small-molecule CXCR3 antagonist with submicromolar affinity and as a lead structure for the development of CXCR3 antagonists. More recently, the related 3H-pyrido[2,3-d]pyrimidin-4-one compounds AMG 487 and NBI-74330 have been reported as nanomolar CXCR3 antagonists and these ligands are currently under clinical investigation. The aim of this study is to link the structure-activity relationship (SAR) of the previously published class of 3H-quinazolin-4-one containing CXCR3 ligands with these novel clinical candidates. From the modification of the lead structure VUF 5834 emerged the importance of the (4-fluoro-3-(trifluoromethyl)phenyl)acetyl and the 3-methylen-pyridine as substituents to improve the affinity at the human CXCR3 Receptor, whereas other features are less important. The described molecules serve as tool to investigate the role of the CXCR3 Receptor in various inflammatory conditions.
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synthesis and structure activity relationship of 3 phenyl 3h quinazolin 4 one derivatives as CXCR3 chemokine Receptor antagonists
Bioorganic & Medicinal Chemistry Letters, 2005Co-Authors: Stefania Storelli, Dennis Verzijl, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Pauline Verdijk, Andrea Van De Stolpe, Cornelis P Tensen, Rob LeursAbstract:A series of 3-phenyl-3H-quinazolin-4-ones have been synthesized and tested for affinity and activity at the chemokine CXCR3 Receptor. The most potent compound (1d) has been evaluated using radioligand binding and calcium mobilization assays and is considered a useful tool for further characterization of the CXCR3 Receptor.
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human ip 9 a keratinocyte derived high affinity cxc chemokine ligand for the ip 10 mig Receptor CXCR3
Journal of Investigative Dermatology, 1999Co-Authors: Cornelis P Tensen, Rob Leurs, Jacoba Flier, Elizabeth M H Van Der Raaijhelmer, Shakun C Sampatsardjoepersad, Roel C Van Der Schors, Rik J Scheper, D M Boorsma, R WillemzeAbstract:Chemokines and their Receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common Receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-γ stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 Receptor, designated interferon-γ-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-γ-inducible protein-9, revealed that interferon-γ-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-γ-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as β-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-γ-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.
Alan Wells - One of the best experts on this subject based on the ideXlab platform.
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elr negative cxc chemokine cxcl11 ip 9 i tac facilitates dermal and epidermal maturation during wound repair
American Journal of Pathology, 2008Co-Authors: Cecelia C Yates, Diana Whaley, Amy Ychen, Priya Kulesekaran, Patricia A Hebda, Alan WellsAbstract:In skin wounds, the chemokine CXCR3 Receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis.
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interferon inducible protein 9 cxcl11 induced cell motility in keratinocytes requires calcium flux dependent activation of μ calpain
Molecular and Cellular Biology, 2005Co-Authors: Latha Satish, Harry C Blair, Angela Glading, Alan WellsAbstract:Regulated cell motility is critical to wound healing (25, 35). During repair, tissue deposition and remodeling by the immigrant fibroblasts and keratinocytes result in the regeneration of an intact skin barrier and functional organ. The cells from the remaining epidermal and dermal layers must proliferate and migrate to repopulate the nascent wound. The basal keratinocytes undergo a transition that enables such repopulation while the provisional matrix is invaded by fibroblasts as the first step in regenerating the future dermal layer. The numerous growth factors present throughout repair, including high levels of epidermal growth factor (EGF) Receptor (EGFR) ligands such as HB-EGF and transforming growth factor alpha, are thought to promote these mitogenic and motogenic responses (26, 35, 45, 50, 60). However, the process of cell repopulation is limited late in the process of healing to prevent fibroplasia and excess matrix deposition. Late in the repair process, members of the Alu-Leu-Arg (ELR)-negative family of CXC chemokines appear (13, 42). It has been proposed that the migration of fibroblasts and keratinocytes is controlled by the waves of these growth factors and chemokines produced throughout wound repair (9, 35, 42, 44, 60). This would include signals to promote as well as inhibit cell migration. Active cell locomotion requires the coordination of a number of cellular processes that should be common among cell types (18, 28). Thus, as numerous external signals can modulate cell motility, two key questions are which biochemical pathway is actuated to promote migration during regeneration and whether these differ between cell types. Any such signaling pathway needs to affect key biophysical processes. During cell migration, tail de-adhesion may be rate-limiting; in experimental models, failure to detach limits cell motility (23, 44). Activation of calpain (EC 3.4.22.17), an intracellular limited protease, is required for integrin-mediated tail de-adhesion on moderately and highly adhesive substrata (30, 37) and for growth factor-induced motility (17, 43). This intracellular protease is a key switch, as calpain inhibitors convert EGFR-mediated signals from cell motility to matrix contractility (1). Thus, calpain activators appear to shift a wide range of cells to motility-permissive adhesion regimens, while inhibitory signals for calpain block productive locomotion. As such, we proposed that keratinocyte motility was dependent on calpain activity. This requirement for calpain activity provides a target for regulating cell motility (18). Confounding any analysis, two calpain isoforms with seemingly identical target specificities are present in practically all cells (48). In vitro, calpain 1 (μ-calpain) is activated at nearly micromolar concentrations of calcium; calpain 2 (M-calpain) requires millimolar calcium levels. While calcium fluxes have been postulated to regulate μ-calpain, this has yet to be demonstrated conclusively in living cells (5), and the signaling cascade that triggers this isoform during cell locomotion remain undefined (30). Furthermore, the physiologically relevant activators of M-calpain are unknown since intracellular calcium levels fail to reach the nearly millimolar concentrations required in vitro (22). Still, we know that plasma membrane-localized M-calpain is activated subsequent to growth factor signaling by direct extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAP kinase) phosphorylation (12, 16, 17, 44). However, what signals and respective intracellular signaling pathways operate during reepithelialization by keratinocytes remains an open question (42). In an initial exploration (43, 44), we reported that ELR-negative CXC chemokines, present during the process of wound repair (13, 42), can block fibroblast motility by preventing activation of M-calpain and subsequent de-adhesion, demonstrating that this might be a physiologically operative pathway. Of this family of chemokines, interferon-inducible protein 10 (IP-10) appears to be produced by the neovasculature deep in the dermis (13). A related ELR-negative CXC chemokine, IP-9, also called beta-R1 (40), H174 (24), and I-TAC (10), is produced by basal keratinocytes in response to immune-mediated injuries (52). Previously, we demonstrated that IP-9 is a wound response factor (42). These ELR-negative CXC chemokines were originally found as modulators of cells of the hematopoietic lineage, but chemokine Receptors have been found on endothelial and epithelial cells (36, 38, 44, 47, 63). The ELR-negative members of the CXC family of chemokines, all of which bind to a common CXCR3 Receptor (2, 14, 27), inhibit endothelial cell proliferation, migration (21, 31, 51), and fibroblast migration (44). These appear to act dominantly over promitogenic and promotility chemokines and growth factors (43, 44). IP-9 is a wound response factor situated in the right place to limit fibroblast repopulation and promote the remodeling phase (42). However, with IP-9 being produced by keratinocytes in or near the wound bed, the situation for the reepithelializing keratinocytes is of great interest; would this chemokine, which serves to “mature” the dermis, also have the collateral effect of slowing reepithelialization? Here we report that keratinocyte motility is promoted by both EGF and IP-9 through their activation of calpains. Furthermore, IP-9 does not block EGF-induced motility in keratinocytes, opposite to its effects on fibroblasts. Keratinocyte motility induced by EGF requires the pathway previously described in fibroblasts, which culminates in activation of the calpain 2 isoform, M-calpain (17). In undifferentiated keratinocytes, IP-9 also activated calpain, though it was the μ-calpain isoform and not the M-calpain isoform that was both triggered and required. This chemokine activation occurred through a phospholipase Cβ (PLCβ)-mediated calcium flux in distinction to the ERK MAP kinase signaling cascade that growth factors utilize to activate M-calpain (17). These two pathways converge at cell-diminished cell adhesion to substratum as mirrored in vinculin aggregate disassembly and cleavage of the focal adhesion kinase (FAK). This is the first demonstration, to our knowledge, of different isoforms of calpain being activated by distinct signals in the same cell to accomplish the same phenotypic end point, cell migration. We also show for the first time that IP-9 increased the intracellular calcium flux and resulted in triggering of the μ-calpain isoform, in turn resulting in productive cell motility.
Dennis Verzijl - One of the best experts on this subject based on the ideXlab platform.
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pharmacological characterization of a small molecule agonist for the chemokine Receptor CXCR3
British Journal of Pharmacology, 2012Co-Authors: Danny J Scholten, Dennis Verzijl, Martine J Smit, Meritxell Canals, Maikel Wijtmans, S De Munnik, P Nguyen, Ijp De Esch, Henry F Vischer, Rob LeursAbstract:BACKGROUND AND PURPOSE The chemokine Receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACH Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 Receptor and in binding studies, with [125I]-CXCL10 and [125I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [35S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and β-arrestin recruitment. KEY RESULTS VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated Gi proteins with the same efficacy as CXCL11 in the [35S]-GTPγS binding and cAMP assay, while it recruited more β-arrestin1 and β-arrestin2 to CXCR3 Receptors than the chemokine. CONCLUSIONS AND IMPLICATIONS VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 Receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different Receptor and/or β-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists. LINKED ARTICLE This article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x
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synthesis and structure activity relationships of 3h quinazolin 4 ones and 3h pyrido 2 3 d pyrimidin 4 ones as CXCR3 Receptor antagonists
ChemInform, 2007Co-Authors: Stefania Storelli, Dennis Verzijl, Jawad Albadie, Niels Elders, Leontien Bosch, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Rob LeursAbstract:CXC chemokine Receptor-3 (CXCR3) is a G-protein coupled Receptor (GPCR) predominantly expressed on activated T lymphocytes that promote Th1 responses. Previously, we described the 3H-quinazolin-4-one containing VUF 5834 (decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide) as a small-molecule CXCR3 antagonist with submicromolar affinity and as a lead structure for the development of CXCR3 antagonists. More recently, the related 3H-pyrido[2,3-d]pyrimidin-4-one compounds AMG 487 and NBI-74330 have been reported as nanomolar CXCR3 antagonists and these ligands are currently under clinical investigation. The aim of this study is to link the structure-activity relationship (SAR) of the previously published class of 3H-quinazolin-4-one containing CXCR3 ligands with these novel clinical candidates. From the modification of the lead structure VUF 5834 emerged the importance of the (4-fluoro-3-(trifluoromethyl)phenyl)acetyl and the 3-methylen-pyridine as substituents to improve the affinity at the human CXCR3 Receptor, whereas other features are less important. The described molecules serve as tool to investigate the role of the CXCR3 Receptor in various inflammatory conditions.
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synthesis and structure activity relationship of 3 phenyl 3h quinazolin 4 one derivatives as CXCR3 chemokine Receptor antagonists
Bioorganic & Medicinal Chemistry Letters, 2005Co-Authors: Stefania Storelli, Dennis Verzijl, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Pauline Verdijk, Andrea Van De Stolpe, Cornelis P Tensen, Rob LeursAbstract:A series of 3-phenyl-3H-quinazolin-4-ones have been synthesized and tested for affinity and activity at the chemokine CXCR3 Receptor. The most potent compound (1d) has been evaluated using radioligand binding and calcium mobilization assays and is considered a useful tool for further characterization of the CXCR3 Receptor.
Martine J Smit - One of the best experts on this subject based on the ideXlab platform.
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pharmacological characterization of a small molecule agonist for the chemokine Receptor CXCR3
British Journal of Pharmacology, 2012Co-Authors: Danny J Scholten, Dennis Verzijl, Martine J Smit, Meritxell Canals, Maikel Wijtmans, S De Munnik, P Nguyen, Ijp De Esch, Henry F Vischer, Rob LeursAbstract:BACKGROUND AND PURPOSE The chemokine Receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACH Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 Receptor and in binding studies, with [125I]-CXCL10 and [125I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [35S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and β-arrestin recruitment. KEY RESULTS VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated Gi proteins with the same efficacy as CXCL11 in the [35S]-GTPγS binding and cAMP assay, while it recruited more β-arrestin1 and β-arrestin2 to CXCR3 Receptors than the chemokine. CONCLUSIONS AND IMPLICATIONS VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 Receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different Receptor and/or β-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists. LINKED ARTICLE This article is commented on by O'Boyle, pp. 895–897 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01759.x
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synthesis and structure activity relationships of 3h quinazolin 4 ones and 3h pyrido 2 3 d pyrimidin 4 ones as CXCR3 Receptor antagonists
ChemInform, 2007Co-Authors: Stefania Storelli, Dennis Verzijl, Jawad Albadie, Niels Elders, Leontien Bosch, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Rob LeursAbstract:CXC chemokine Receptor-3 (CXCR3) is a G-protein coupled Receptor (GPCR) predominantly expressed on activated T lymphocytes that promote Th1 responses. Previously, we described the 3H-quinazolin-4-one containing VUF 5834 (decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide) as a small-molecule CXCR3 antagonist with submicromolar affinity and as a lead structure for the development of CXCR3 antagonists. More recently, the related 3H-pyrido[2,3-d]pyrimidin-4-one compounds AMG 487 and NBI-74330 have been reported as nanomolar CXCR3 antagonists and these ligands are currently under clinical investigation. The aim of this study is to link the structure-activity relationship (SAR) of the previously published class of 3H-quinazolin-4-one containing CXCR3 ligands with these novel clinical candidates. From the modification of the lead structure VUF 5834 emerged the importance of the (4-fluoro-3-(trifluoromethyl)phenyl)acetyl and the 3-methylen-pyridine as substituents to improve the affinity at the human CXCR3 Receptor, whereas other features are less important. The described molecules serve as tool to investigate the role of the CXCR3 Receptor in various inflammatory conditions.
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synthesis and structure activity relationship of 3 phenyl 3h quinazolin 4 one derivatives as CXCR3 chemokine Receptor antagonists
Bioorganic & Medicinal Chemistry Letters, 2005Co-Authors: Stefania Storelli, Dennis Verzijl, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Pauline Verdijk, Andrea Van De Stolpe, Cornelis P Tensen, Rob LeursAbstract:A series of 3-phenyl-3H-quinazolin-4-ones have been synthesized and tested for affinity and activity at the chemokine CXCR3 Receptor. The most potent compound (1d) has been evaluated using radioligand binding and calcium mobilization assays and is considered a useful tool for further characterization of the CXCR3 Receptor.
Cornelis P Tensen - One of the best experts on this subject based on the ideXlab platform.
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synthesis and structure activity relationship of 3 phenyl 3h quinazolin 4 one derivatives as CXCR3 chemokine Receptor antagonists
Bioorganic & Medicinal Chemistry Letters, 2005Co-Authors: Stefania Storelli, Dennis Verzijl, Henk Timmerman, Martine J Smit, Iwan J P De Esch, Pauline Verdijk, Andrea Van De Stolpe, Cornelis P Tensen, Rob LeursAbstract:A series of 3-phenyl-3H-quinazolin-4-ones have been synthesized and tested for affinity and activity at the chemokine CXCR3 Receptor. The most potent compound (1d) has been evaluated using radioligand binding and calcium mobilization assays and is considered a useful tool for further characterization of the CXCR3 Receptor.
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human ip 9 a keratinocyte derived high affinity cxc chemokine ligand for the ip 10 mig Receptor CXCR3
Journal of Investigative Dermatology, 1999Co-Authors: Cornelis P Tensen, Rob Leurs, Jacoba Flier, Elizabeth M H Van Der Raaijhelmer, Shakun C Sampatsardjoepersad, Roel C Van Der Schors, Rik J Scheper, D M Boorsma, R WillemzeAbstract:Chemokines and their Receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common Receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-γ stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 Receptor, designated interferon-γ-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-γ-inducible protein-9, revealed that interferon-γ-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-γ-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as β-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-γ-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.
