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Jean Sevigny - One of the best experts on this subject based on the ideXlab platform.
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development of a selective and highly sensitive fluorescence assay for Nucleoside Triphosphate diphosphohydrolase1 ntpdase1 cd39
Analyst, 2018Co-Authors: Sangyong Lee, Jean Sevigny, Xihuan Luo, Vigneshwaran Namasivayam, Jennifer Geiss, Salahuddin Mirza, Julie Pelletier, Holger Stephan, Christa E. MüllerAbstract:Ecto-Nucleoside Triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5′-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z′-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 μM, and a kcat value of 119 × 10−3 s−1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 μM and 82.5 × 10−3 s−1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.
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exploration of carboxy pyrazole derivatives synthesis alkaline phosphatase nucleotide pyrophosphatase phosphodiesterase and Nucleoside Triphosphate diphosphohydrolase inhibition studies with potential anticancer profile
European Journal of Medicinal Chemistry, 2018Co-Authors: Pervaiz Ali Channar, Joanna Lecka, Jean Sevigny, Saira Afzal, Syeda Abida Ejaz, Aamer Saeed, Fayaz Ali Larik, Parvez Ali Mahesar, Mauricio F ErbenAbstract:Abstract In the present work we report the synthesis of new aryl pyrazole derivatives using 1,3-dicarbonyl motifs. The reaction was proceeded by the cyclization of pentane-2,4-dione (1a), 3-chloropentane-2,4-dione (1b) or ethyl 3-oxobutanoate (1c) with different aryl hydrazines. The products, which can be regarded as 1H-pyrazol-1-yl-one analogues (3a-f, 3g-o, 4a-c, 5a-b) and represent drug like molecules along with well-developed structure–activity relationships, were obtained in good to excellent yield. The structures of synthesized compounds were charcterized on the basis of FT-IR, 1H NMR, 13C NMR and mass spectroscopic data. Considering alkaline phosphatases (APs), nucleotide pyrophosphatases/phosphodiesterases (NPPs) and Nucleoside Triphosphate diphosphohydrolase as the molecular targets, the effects of these synthesized compounds were investigated on different isozymes of APs, NPPs and NTPDases. The data revealed that the synthesized compounds inhibited both enzymes but most of them inhibited tissue non-specific alkaline phosphatase (TNAP) more selectively. The antitumor activity results indicated that the synthesized derivatives have strong inhibitory effects on the growth of selected cell lines from different tissues such as breast, bone marrow and cervix (MCF-7, K-562 and Hela) but with varying intensities. Moreover the binding mode of interactions were explained on the basis of molecular docking and in-silico studies.
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Distinct roles of ecto-Nucleoside Triphosphate diphosphohydrolase-2 (NTPDase2) in liver regeneration and fibrosis
Purinergic Signalling, 2018Co-Authors: Linda Feldbrügge, Jean Sevigny, Z. Gordon Jiang, Eva Csizmadia, Shuji Mitsuhashi, Stephanie Tran, Sonja Rothweiler, Kahini A. Vaid, Moritz Schmelzle, Yury V. PopovAbstract:Ecto-Nucleoside Triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and modulating purinergic signaling. In the liver, NTPDase2 is reportedly expressed on portal fibroblasts, but its functional role in regulating tissue regeneration and fibrosis is incompletely understood. Here, we studied the role of NTPDase2 in several models of liver injury using global knockout mice. Liver regeneration and severity of fibrosis were analyzed at different time points after exposure to carbon tetrachloride (CCl_4) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or partial hepatectomy in C57BL/6 wild-type and globally NTPDase2-deficient ( Entpd2 null) mice. After chronic CCl_4 intoxication, Entpd2 null mice exhibit significantly more severe liver fibrosis, as assessed by collagen content and histology. In contrast, deletion of NTPDase2 does not have a substantial effect on biliary-type fibrosis in the setting of DDC feeding. In injured livers, NTPDase2 expression extends from the portal areas to fibrotic septae in pan-lobular (CCl_4-induced) liver fibrosis; the same pattern was observed, albeit to a lesser extent in biliary-type (DDC-induced) fibrosis. Liver regeneration after partial hepatectomy is not substantively impaired in global Entpd2 null mice. NTPDase2 protects from liver fibrosis resulting from hepatocellular injury induced by CCl_4. In contrast, Entpd2 deletion does not significantly impact fibrosis secondary to DDC injury or liver regeneration after partial hepatectomy. Our observations highlight mechanisms relating to purinergic signaling in the liver and indicate possible therapeutic avenues and new cellular targets to test in the management of hepatic fibrosis.
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extracellular atp selectively upregulates ecto Nucleoside Triphosphate diphosphohydrolase 2 and ecto 5 nucleotidase by rat cortical astrocytes in vitro
Journal of Molecular Neuroscience, 2015Co-Authors: Dusica Brisevac, Jean Sevigny, Marija Adzic, Danijela Laketa, Ana Parabucki, Milena Milosevic, Irena Lavrnja, Ivana Bjelobaba, Markus Kipp, Nadezda NedeljkovicAbstract:Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-Nucleoside Triphosphate diphosphohydrolases 1–3 (NTPDase1–3), ecto-nucleotide pyrophosphatase/phospodiesterases 1–3 (NPP1–3), and ecto-5′-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1–3, NPP1–3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis.
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Fluorescence polarization immunoassays for monitoring Nucleoside Triphosphate diphosphohydrolase (NTPDase) activity
Analyst, 2015Co-Authors: Amelie Fiene, Joanna Lecka, Jean Sevigny, Younis Baqi, Christa E. MüllerAbstract:The following members of the ecto-Nucleoside Triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z′-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z′-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.
Simon C Robson - One of the best experts on this subject based on the ideXlab platform.
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possible effects of microbial ecto Nucleoside Triphosphate diphosphohydrolases on host pathogen interactions
Microbiology and Molecular Biology Reviews, 2008Co-Authors: Fiona M Sansom, Simon C Robson, Elizabeth L HartlandAbstract:Summary: In humans, purinergic signaling plays an important role in the modulation of immune responses through specific receptors that recognize Nucleoside tri- and diphosphates as signaling molecules. Ecto-Nucleoside Triphosphate diphosphohydrolases (ecto-NTPDases) have important roles in the regulation of purinergic signaling by controlling levels of extracellular nucleotides. This process is key to pathophysiological protective responses such as hemostasis and inflammation. Ecto-NTPDases are found in all higher eukaryotes, and recently it has become apparent that a number of important parasitic pathogens of humans express surface-located NTPDases that have been linked to virulence. For those parasites that are purine auxotrophs, these enzymes may play an important role in purine scavenging, although they may also influence the host response to infection. Although ecto-NTPDases are rare in bacteria, expression of a secreted NTPDase in Legionella pneumophila was recently described. This ecto-enzyme enhances intracellular growth of the bacterium and potentially affects virulence. This discovery represents an important advance in the understanding of the contribution of other microbial NTPDases to host-pathogen interactions. Here we review other progress made to date in the characterization of ecto-NTPDases from microbial pathogens, how they differ from mammalian enzymes, and their association with organism viability and virulence. In addition, we postulate how ecto-NTPDases may contribute to the host-pathogen interaction by reviewing the effect of selected microbial pathogens on purinergic signaling. Finally, we raise the possibility of targeting ecto-NTPDases in the development of novel anti-infective agents based on potential structural and clear enzymatic differences from the mammalian ecto-NTPDases.
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ecto Nucleoside Triphosphate diphosphohydrolase 1 e ntpdase1 cd39 regulates neutrophil chemotaxis by hydrolyzing released atp to adenosine
Journal of Biological Chemistry, 2008Co-Authors: Ross Corriden, Simon C Robson, Yu Chen, Yoshiaki Inoue, Guido Beldi, Paul A Insel, Wolfgang G JungerAbstract:Polymorphonuclear neutrophils release ATP in response to stimulation by chemoattractants, such as the peptide N-formyl-methionyl-leucyl-phenylalanine. Released ATP and the hydrolytic product adenosine regulate chemotaxis of neutrophils by sequentially activating purinergic nucleotide and adenosine receptors, respectively. Here we show that that ecto-Nucleoside Triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) is a critical enzyme for hydrolysis of released ATP by neutrophils and for cell migration in response to multiple agonists (N-formyl-methionyl-leucyl-phenylalanine, interleukin-8, and C5a). Upon stimulation of human neutrophils or differentiated HL-60 cells in a chemotactic gradient, E-NTPDase1 tightly associates with the leading edge of polarized cells during chemotaxis. Inhibition of E-NTPDase1 reduces the migration speed of neutrophils but not their ability to detect the orientation of the gradient field. Studies of neutrophils from E-NTPDase1 knock-out mice reveal similar impairments of chemotaxis in vitro and in vivo. Thus, E-NTPDase1 plays an important role in regulating neutrophil chemotaxis by facilitating the hydrolysis of extracellular ATP.
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reconstitution of cd39 in liposomes amplifies Nucleoside Triphosphate diphosphohydrolase activity and restores thromboregulatory properties
Journal of Vascular Surgery, 2006Co-Authors: Carolyn A Haller, Simon C Robson, Wanxing Cui, Jing Wen, Elliot L ChaikofAbstract:Background CD39 (Nucleoside Triphosphate diphosphohydrolase [NTPDase-1]) expressed on the luminal surface of endothelial cells rapidly metabolizes extracellular adenosine Triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), and abrogates platelet reactivity. Optimization of CD39 enzymatic activity appears dependent upon the expression of both transmembrane domains within plasma membranes. Thus, motivation exists to examine therapeutic antiplatelet formulations that consist of liposomal CD39. Methods Full-length human CD39 was produced by using a yeast expression system, purified, and reconstituted within lipid vesicles. The catalytic efficiency (kcat/Km) of CD39-mediated phosphohydrolysis of ADP and ATP was determined both for detergent-solubilized and protein-reconstituted CD39 within lipid membranes. The capacity of CD39-containing lipid vesicles to inhibit platelet activation induced by ADP, collagen, or thrombin was determined in vitro by platelet aggregometry. A murine model of thromboplastin-induced thromboembolism was used to determine the effectiveness of intravenous liposomal CD39 in limiting platelet consumption and mortality. Results Reconstitution of human CD39 in lipid vesicles was associated with a decrease in Km of nearly an order of magnitude over the detergent-solubilized form. There was a concomitant increase in both ADPase and ATPase catalytic efficiencies (kcat/Km ADPase: sol CD39: 2.7 × 10 6 vs liposomal CD39: 1.4 × 10 7 min/ M; kcat/Km ATPase: sol CD39: 7.2 × 10 6 vs liposomal CD39: 2.0 ×10 7 min/M). Furthermore, CD39 lipid vesicles effectively inhibited platelet aggregation when activated by ADP, collagen, or thrombin, and also promoted platelet disaggregation (60.4% ± 6.1%). Treatment with CD39 lipid vesicles preserved platelet counts after thromboplastin injection (pretreatment, 906.8 ± 42.9 platelets/μm 3 ; empty vesicles, 278.6 ± 34.8 platelets/μm 3 ; CD39 vesicles, 563.6 ± 42.2 platelets/μm 3 ; n=10 mice/test group; P P ≤ .05; n=12 mice/experimental test group, n=15 mice/control test group). Conclusions Incorporation of solubilized CD39 into a lipid bilayer restores enzyme activity and optimizes thromboregulatory potential. Treatment with CD39 in liposomal formulations decreased mortality in a murine model of thromboplastin-induced thromboembolism by limiting intravascular platelet aggregation and thrombosis.
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Nucleoside Triphosphate diphosphohydrolase 2 ntpdase2 cd39l1 is the dominant ectonucleotidase expressed by rat astrocytes
Neuroscience, 2006Co-Authors: Marcia R Wink, Jean Sevigny, Elizandra Braganhol, Alessandra Sayuri Kikuchi Tamajusuku, Guido Lenz, Luiz F Zerbini, Towia A Libermann, Ana Maria Oliveira Battastini, Simon C RobsonAbstract:Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-Nucleoside Triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-Nucleoside Triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-Nucleoside Triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.
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cloning and characterization of mouse Nucleoside Triphosphate diphosphohydrolase 8
Biochemistry, 2004Co-Authors: Francois Bigonnesse, Joanna Lecka, Sebastien A Levesque, Filip Kukulski, Simon C Robson, Maria J G Fernandes, Jean SevignyAbstract:A novel mammalian plasma membrane bound Nucleoside Triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 14...
Chris Meier - One of the best experts on this subject based on the ideXlab platform.
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γ ketobenzyl modified Nucleoside Triphosphate prodrugs as potential antivirals
Journal of Medicinal Chemistry, 2020Co-Authors: Tobias Nack, Thiago Dinis De Oliveira, Stefan Weber, Dominique Schols, Jan Balzarini, Chris MeierAbstract:The antiviral activity of Nucleoside reverse transcriptase inhibitors is often hampered by insufficient phosphorylation. Nucleoside Triphosphate analogues are presented, in which the γ-phosphate wa...
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anti hiv active Nucleoside Triphosphate prodrugs
Journal of Medicinal Chemistry, 2020Co-Authors: Xiao Jia, Dominique Schols, Chris MeierAbstract:We disclose a study on Nucleoside Triphosphate (NTP) analogues in which the γ-phosphate is covalently modified by two different biodegradable masking units and d4T as Nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer (pH 7.3) and by enzyme-triggered reactions in human CD4+ T-lymphocyte CEM cell extracts. This allows the bypass of all steps normally needed in the intracellular phosphorylation. These TriPPPro-nucleotides comprising an acyloxybenzyl (AB; ester) or an alkoxycarbonyloxybenzyl (ACB; carbonate) in combination with an ACB moiety are described as NTP delivery systems. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes. γ-(AB)-d4TTPs are faster cleaved than γ-(ACB)-d4TTPs. In antiviral assays, the compounds are highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-).
Christa E. Müller - One of the best experts on this subject based on the ideXlab platform.
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development of a selective and highly sensitive fluorescence assay for Nucleoside Triphosphate diphosphohydrolase1 ntpdase1 cd39
Analyst, 2018Co-Authors: Sangyong Lee, Jean Sevigny, Xihuan Luo, Vigneshwaran Namasivayam, Jennifer Geiss, Salahuddin Mirza, Julie Pelletier, Holger Stephan, Christa E. MüllerAbstract:Ecto-Nucleoside Triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5′-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z′-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 μM, and a kcat value of 119 × 10−3 s−1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 μM and 82.5 × 10−3 s−1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.
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Fluorescence polarization immunoassays for monitoring Nucleoside Triphosphate diphosphohydrolase (NTPDase) activity
Analyst, 2015Co-Authors: Amelie Fiene, Joanna Lecka, Jean Sevigny, Younis Baqi, Christa E. MüllerAbstract:The following members of the ecto-Nucleoside Triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z′-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z′-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.
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Selective Nucleoside Triphosphate Diphosphohydrolase-2 (NTPDase2) Inhibitors : Nucleotide Mimetics Derived from Uridine-5'-carboxamide
Journal of Medicinal Chemistry, 2008Co-Authors: Andreas Brunschweiger, Jean Sevigny, Jamshed Iqbal, Frank Umbach, Anja B. Scheiff, Mercedes N. Munkonda, Aileen F. Knowles, Christa E. MüllerAbstract:Ecto-Nucleoside Triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases) dephosphorylate Nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present study we synthesized adenine and uracil nucleotide mimetics, in which the phosphate residues were replaced by phosphonic acid esters attached to the Nucleoside at the 5′-position by amide linkers. Among the synthesized uridine derivatives, we identified the first potent and selective inhibitors of human NTPDase2. The most potent compound was 19a (PSB-6426), which was a competitive inhibitor of NTPDase2 exhibiting a Ki value of 8.2 μM and selectivity versus other NTPDases. It was inactive toward uracil nucleotide-activated P2Y2, P2Y4, and P2Y6 receptor subtypes. Compound 19a was chemically and metabolically highly stable. In contrast to the few known (unselective) NTPDase inhibitors, 19a is an uncharged molecule and may be perorally bioavailable. NTPDase2 inhibitors have potential as novel cardioprotective...
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enzymatic properties of an ecto Nucleoside Triphosphate diphosphohydrolase from legionella pneumophila substrate specificity and requirement for virulence
Journal of Biological Chemistry, 2008Co-Authors: Fiona M Sansom, Christa E. Müller, Holger Stephan, Patrice Riedmaier, Hayley J Newton, Michelle A Dunstone, Emma Byres, Travis Clarke Beddoe, Jamie RossjohnAbstract:Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-Nucleoside Triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze Nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.
Joanna Lecka - One of the best experts on this subject based on the ideXlab platform.
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exploration of carboxy pyrazole derivatives synthesis alkaline phosphatase nucleotide pyrophosphatase phosphodiesterase and Nucleoside Triphosphate diphosphohydrolase inhibition studies with potential anticancer profile
European Journal of Medicinal Chemistry, 2018Co-Authors: Pervaiz Ali Channar, Joanna Lecka, Jean Sevigny, Saira Afzal, Syeda Abida Ejaz, Aamer Saeed, Fayaz Ali Larik, Parvez Ali Mahesar, Mauricio F ErbenAbstract:Abstract In the present work we report the synthesis of new aryl pyrazole derivatives using 1,3-dicarbonyl motifs. The reaction was proceeded by the cyclization of pentane-2,4-dione (1a), 3-chloropentane-2,4-dione (1b) or ethyl 3-oxobutanoate (1c) with different aryl hydrazines. The products, which can be regarded as 1H-pyrazol-1-yl-one analogues (3a-f, 3g-o, 4a-c, 5a-b) and represent drug like molecules along with well-developed structure–activity relationships, were obtained in good to excellent yield. The structures of synthesized compounds were charcterized on the basis of FT-IR, 1H NMR, 13C NMR and mass spectroscopic data. Considering alkaline phosphatases (APs), nucleotide pyrophosphatases/phosphodiesterases (NPPs) and Nucleoside Triphosphate diphosphohydrolase as the molecular targets, the effects of these synthesized compounds were investigated on different isozymes of APs, NPPs and NTPDases. The data revealed that the synthesized compounds inhibited both enzymes but most of them inhibited tissue non-specific alkaline phosphatase (TNAP) more selectively. The antitumor activity results indicated that the synthesized derivatives have strong inhibitory effects on the growth of selected cell lines from different tissues such as breast, bone marrow and cervix (MCF-7, K-562 and Hela) but with varying intensities. Moreover the binding mode of interactions were explained on the basis of molecular docking and in-silico studies.
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Fluorescence polarization immunoassays for monitoring Nucleoside Triphosphate diphosphohydrolase (NTPDase) activity
Analyst, 2015Co-Authors: Amelie Fiene, Joanna Lecka, Jean Sevigny, Younis Baqi, Christa E. MüllerAbstract:The following members of the ecto-Nucleoside Triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z′-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z′-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.
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cloning and characterization of mouse Nucleoside Triphosphate diphosphohydrolase 3
Biochemical Pharmacology, 2004Co-Authors: Elise G Lavoie, Joanna Lecka, Sebastien A Levesque, Filip Kukulski, Jean SevignyAbstract:Abstract We have cloned and characterized the Nucleoside Triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca2+>Mg2+) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis–Menten kinetics with apparent Kms of 11, 10, 19 and 27 μM, respectively. In agreement with the Km values, the pattern of triphosphoNucleoside hydrolysis showed a transient accumulation of the corresponding diphosphoNucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.
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cloning and characterization of mouse Nucleoside Triphosphate diphosphohydrolase 8
Biochemistry, 2004Co-Authors: Francois Bigonnesse, Joanna Lecka, Sebastien A Levesque, Filip Kukulski, Simon C Robson, Maria J G Fernandes, Jean SevignyAbstract:A novel mammalian plasma membrane bound Nucleoside Triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 14...
