After conversion to their active forms by the liver, ticlopidine and clopidogrel exert antiplatel... more After conversion to their active forms by the liver, ticlopidine and clopidogrel exert antiplatelet effects through irreversible inhibition of the P2Y12 receptor. Concentrations of nucleotides such as ADP, the physiological agonist at platelet P2Y1 and P2Y12 receptors, are regulated by vascular ectonucleotidases, mainly nucleoside triphosphate diphosphohydrolase (NTPDase)1 and ecto-5′-nucleotidase. Here we evaluate the effect of these pro-drugs on vascular ectonucleotidase activity and on the natural function of these enzymes in regulating platelet aggregation. Nucleotidase assays were performed by HPLC and by Pi determination, using human umbilical vein endothelial cells (HUVEC) and protein extracts from transfected COS-7 cells as sources of enzymes. Platelet aggregation was assayed using human platelet-rich plasma. Each pro-drug inhibited endothelial ectonucleotidase activities and decreased their ability to block platelet aggregation in vitro. At their therapeutic concentrations, ticlopidine (60 mM) and clopidogrel (20 mM) inhibited ADP hydrolysis by HUVEC by about 80%, and AMP hydrolysis by one-third. Accordingly, these compounds showed a mixed-type inhibition of recombinant human NTPDase1 with an apparent Ki (Ki,app) of 10 mM (clopidogrel) and 14 mM (ticlopidine). Recombinant rat ecto-5′-nucleotidase, but not its human orthologue, was inhibited by ticlopidine with a Ki,app of 4.5 mM. These pro-drugs facilitated platelet aggregation via the inhibition of vascular NTPDase1 in vitro. Further studies should be performed to assess whether this effect also occurs in vivo, especially at the beginning of treatment, before sufficient levels of active metabolites are produced by the liver.
The aim of this research work is the synthesis of sulfamoyl-benzamides as a selective inhibitor f... more The aim of this research work is the synthesis of sulfamoyl-benzamides as a selective inhibitor for h-NTPDases. Sulfonamides are synthesized in aqueous medium from chlorosulfonylbenzoic acid while carboxamides are synthesized using carbodiimide coupling decorated with different biologically relevant substituents such as n-butyl, cyclopropyl, benzylamine, morpholine, and substituted anilines. In addition, sulfonamide-carboxamide derivatives were synthesized having the same substituents on either side. These compounds were screened against h-NTPDase activity, a main family of ectonucleotidases. Among the eight discovered isoforms of the h-NTPDases, four isoforms, h-NTPDase1, -2, -3, and -8, are involved in various physiological and pathological functions, for instance thrombosis, diabetes, inflammation, and cancer. The compound N-(4-bromophenyl)-4-chloro-3-(morpholine-4-carbonyl) benzenesulfonamide (3i) was found to be the most potent inhibitor of h-NTPDase1 with an IC 50 value of 2.88 ± 0.13 mM. Similarly, the compounds N-(4-methoxyphenyl)-3-(morpholinosulfonyl)benzamide (3f), 5-(N-benzylsulfamoyl)-2-chloro-N-(4-methoxyphenyl)benzamide (3j) and 2-chloro-N-cyclopropyl-5-(N-cyclopropylsulfamoyl)benzamide (4d) reduced the activity of the h-NTPDases2 with IC 50 in submicromolar concentrations. Against the h-NTPDase3, 3i was the potent compound with an IC 50 concentration of 0.72 ± 0.11 mM. The h-NTPDase8 was selectively blocked by the most potent inhibitor 2-chloro-5-(N-cyclopropylsulfamoyl)benzoic acid (2d) with (IC 50 = 0.28 ± 0.07 mM). Moreover, the molecular docking studies of the potent inhibitors showed significant interactions with the amino acids of the respective h-NTPDase homology model proteins.
Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collec... more Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5′-nucleotidase (5′NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility.
To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a ... more To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5Ј-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (Ϫ14%; PϽ0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl 3 ) was significantly reduced by 20% in cd73 Ϫ/Ϫ mice (PϽ0.05). Bleeding time after tail tip resection tended to be shorter in cd73 Ϫ/Ϫ mice (Ϫ35%). In vivo platelet cAMP levels were 0.96Ϯ0.46 in WT versus 0.68Ϯ0.27 pmol/10 6 cells in cd73 Ϫ/Ϫ mice (PϽ0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73 Ϫ/Ϫ compared with WT littermates (WT 98% versus cd73 Ϫ/Ϫ 245%; PϽ0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73 Ϫ/Ϫ mice (PϽ0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo. (Circ Res. 2004;95:814-821.
Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plas... more Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ectonucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of co-expressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ERtargeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.
Journal of Pharmacology and Experimental Therapeutics, Jul 12, 2007
Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigm... more Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigment epithelial (RPE) cells that may influence their relationship with the adjacent photoreceptors. The ectoenzyme nucleoside-triphosphate diphosphohy-drolase-1 (NTPDase1) catalyzes the dual dephosphorylation of ATP and ADP to AMP. Although NTPDase1 can consequently control the balance between ATP and adenosine, it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts an increase in enzyme activity in response to enhanced exposure to substrate. This study asked whether exposure to ATP increases NTPDase1 activity in RPE cells. Although levels of NTPDase1 mRNA and protein in cultured human ARPE-19 cells were generally low under control conditions, exposure to slowly hydrolyzable ATPγS led to a time-dependent increase in NTPDase1 mRNA that was accompanied by a rise in levels of the functional 78-kDa protein. Neither NTPDase2 nor NTPDase3 mRNA message was elevated by ATPγS. The ATPase activity of cells increased in parallel, indicating the up-regulation of NTPDase1 was functionally relevant. The up-regulation of NTPDase1 protein was partially blocked by P2Y 1 receptor inhibitors MRS2179 (N 6 -methyl-2′-deoxyadenosine-3′, 5′-bisphosphate) and MRS2500 [2-iodo-N 6 -methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′bisphosphate] and increased by P2Y 1 receptor agonist MRS2365 [(N)-methanocarba-2MeSADP]. In conclusion, prolonged exposure to extracellular ATPγS increased NTPDase1 message and protein levels and increased ecto-ATPase activity. This up-regulation reflects a feedback circuit, mediated at least in part by the P2Y 1 receptor, to regulate levels of extracellular purines in subretinal space. NTPDase1 levels may thus serve as an index for increased extracellular ATP levels under certain pathologic conditions, although other mechanisms could also contribute. Extracellular ATP and adenosine act at distinct receptors to mediate discrete actions in many tissues . The two transmitters can act as a balanced pair, with diverse effects resulting from their activation of different receptors (Zhang et al., 2006b).
Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohy... more Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/ phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K m values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes' varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.
Bile duct epithelia are the target of a number of "cholangiopathies" characterized by disordered ... more Bile duct epithelia are the target of a number of "cholangiopathies" characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTP-Dase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative realtime reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NT-PDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTP-Dase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.
Metalloenzymes have an important role in the regulation of many biological functions. Overexpress... more Metalloenzymes have an important role in the regulation of many biological functions. Overexpressed and/or reduced secretion of such enzymes lead to different complications of clinical interest. The metal ions present in enzymes control the structure, folding, and functions of such proteins. The protein data bank (PDB) revealed that over 50% of proteins contain metal ions . The development of metalloenzyme inhibitors are of interest in the treatment of various diseases. The interaction of ligands, i.e., compounds as inhibitors with target proteins via active sites provide a means of curing diseases. Most aptly, the inhibitors reported by academic or pharmaceutical usage of small molecules as inhibitors provide a rapid and viable way to treat diseases. Urease is a ubiquitous metalloenzyme, produced by various cell types from plants, fungi, and bacteria, etc., that bears a nickel atom in its active pocket. It hydrolyzes the urea into ammonia and carbamate which further decompose to ammonia and CO 2 . The overexpression of urease was known to be linked with ulcers, hepatic coma, and formation of urinary stones . Carbonic anhydrase with zinc metal ion catalyze the hydration of CO 2 with water to produce hydrogen carbonate and H + ions (Alvarez-Leefmans and Delpire, 2009). The hydration reaction involves the nucleophilic attack of the metal-bounded hydroxy (OH) group with the carbon (C) atom of carbon dioxide species . The coordination of carbonic anhydrases (CAs) with metal ion occurs at active sites via binding with histidine, cysteine, and/or glutamine residues to form a tetrahedral shape . The inhibitors of CAs have been employed as diuretic and antiglaucoma agents as well as anti-obesity and anticancer agents . Furthermore, ubiquitous ecto-nucleotidases such as 1) nucleoside triphosphate diphosphohydrolases (NTPDases), 2) nucleotide pyrophosphatase/phosphodiesterases (NPPs), 3) alkaline phosphatases (APs or ALPs), and 4) ecto-5′-nucleotidase (e5′NT) are all responsible for the integrity of proper cell functioning . The NPPs possess zinc (Zn 2+ ) metal ion at active sites while the e5′NT has additionally magnesium ion (Mg 2+ ) at the active site. The overexpression of surface-located ecto-enzymes hydrolyzing nucleotides causes various complications which affect different functions such as cell proliferation, apoptosis as well as degenerative, neurological, and immunological responses. In the current issue, Baqi et al. reported the use of anthraquinone derivatives as NTPDase inhibitors which showed selectivity towards NTPDase2 and -3. The compound, 1-amino-4-(9-phenanthrylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, with an IC 50 value of 539 nM was found to be a potent inhibitor of NTPDase2, while the anthraquinone, 1-amino-4-[3-(4,6-dichlorotriazin-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence ... more Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5′-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.
Protein Engineering Design & Selection, May 27, 2010
We adapted the method of epitope mapping by sitedirected masking, which was described for purifie... more We adapted the method of epitope mapping by sitedirected masking, which was described for purified soluble antigens Proc. Natl Acad. Sci. USA, 103,[9172][9173][9174][9175][9176][9177], to map the binding site of an inhibitory monoclonal antibody on the cell surface protein ecto-nucleotidase NTPDase3. Using homology modeling, we built a 3D structure of NTPDase3 and designed 21 single cysteine mutations distributed over the surface of the enzyme. The mutant proteins were expressed in cells, biotinylated with a cysteine-specific reagent, and then extracted with detergent and immobilized on streptavidin-coated plates. Tethering NTPDase3 via cysteine residues located in a surface patch near the active site cleft masked the epitope and blocked antibody binding, as evaluated by enzyme inhibition assay and by ELISA. We then constructed 18 single alanine substitution mutations within the defined patch and found that W403A, D414A, E415A and R419A decreased the inhibitory effect of the antibody, whereas the double mutation W403A/R419A abolished both antibody binding and enzyme inhibition, suggesting the critical role of these residues for interaction with the antibody. Lack of competition between the antibody and a non-hydrolyzable substrate analog AMPPCP, as well as location of the epitope adjacent to the active site, suggest a noncompetitive mechanism of inhibition by steric hindrance. The described technique should be useful for systematic epitope mapping in cell membrane proteins for which either a 3D structure is available, or a sufficiently accurate 3D model can be obtained by homology modeling.
The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stel... more The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.
Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, ... more Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcification during bone formation and growth. The elevated level of TNAP has been linked to vascular calcification and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of APs. Herein, a series of tricyclic coumarin sulphonate 1a-za with known antiproliferative activity, was evaluated for AP inhibition against human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). The methylbenzenesulphonate derivative 1f (IC 50 ¼ 0.38 ± 0.01 lM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC 50 ¼ 0.45 ± 0.02 lM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investigated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study was also supported by the docking studies to rationalise the most possible binding site interactions of the identified inhibitors with the targeted enzymes.
Background & Aims-Contribution of hepatic stellate cells (HSCs), portal fibroblasts (PFs), and me... more Background & Aims-Contribution of hepatic stellate cells (HSCs), portal fibroblasts (PFs), and mesothelial cells (MCs) to myofibroblasts is not fully understood due to insufficient availability of markers and isolation methods. The present study aimed to isolate these cells, characterize their phenotypes, and examine their contribution to myofibroblasts in liver fibrosis. Methods-Liver fibrosis was induced in Collagen1a1-green fluorescent protein (Col1a1 GFP ) mice by bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, or CCl 4 injections. Combining vitamin A (VitA) lipid autofluorescence and expression of GFP and glycoprotein M6a (GPM6A), we separated HSCs, PFs, and MCs from normal and fibrotic livers by fluorescence-activated cell sorting (FACS).
IntroductionThe tumor microenvironment (TME) of glioblastoma (GB) is characterized by an increase... more IntroductionThe tumor microenvironment (TME) of glioblastoma (GB) is characterized by an increased infiltration of immunosuppressive cells that attenuate the antitumor immune response. The participation of neutrophils in tumor progression is still controversial and a dual role in the TME has been proposed. In this study, we show that neutrophils are reprogrammed by the tumor to ultimately promote GB progression.MethodsUsing in vitro and in vivo assays, we demonstrate the existence of bidirectional GB and neutrophil communication, directly promoting an immunosuppressive TME. Results and discussionNeutrophils have shown to play an important role in tumor malignancy especially in advanced 3D tumor model and Balb/c nude mice experiments, implying a time- and neutrophil concentration-dependent modulation. Studying the tumor energetic metabolism indicated a mitochondria mismatch shaping the TME secretome. The given data suggests a cytokine milieu in patients with GB that favors the recrui...
Ecto-5′-nucleotidase (e5′NT), a membrane-bound enzyme and an essential member of ecto-nucleotidas... more Ecto-5′-nucleotidase (e5′NT), a membrane-bound enzyme and an essential member of ecto-nucleotidases which regulates extracellular purinergic signalling. Their upregulation results in various disease conditions, for example, inflammation, hypoxia and cancer. Therefore, efforts have been made to synthesize potent and selective inhibitors of e5′NT. Here we have synthesized, characterized and evaluated six thiazole derivatives (3a–3f) as potent e5′NT inhibitors. Among all derivatives, the compound ( E )-1-(4-methyl-2-(2-(pyridin-3-ylmethylene)hydrazinyl) thiazol-5-yl)ethanone (3a) exhibited maximum inhibition towards both human and rat enzymes. However, their potency against h -e5′NT was 24-fold higher than r -e5′NT. Only two compounds exhibited inhibitory behaviour towards r -e5′NT. The molecular structures of these derivatives were confirmed with the help of solid-state characterization through NMR ( 1 H and 13 C), FTIR and elemental analysis. Additionally, molecular docking was also ...
A new fluorescence-based assay is useful for monitoring CD39 reactions and enables low picomolar ... more A new fluorescence-based assay is useful for monitoring CD39 reactions and enables low picomolar detection of nucleotides.
European journal of medicinal chemistry, Jan 20, 2018
Alkaline Phosphatases (APs) play a key role in maintaining a ratio of phosphate to inorganic pyro... more Alkaline Phosphatases (APs) play a key role in maintaining a ratio of phosphate to inorganic pyrophosphate (Pi/PPi) and thus regulate extracellular matrix calcification during bone formation and growth. Among different isozymes of AP, aberrant increase in the level of tissue non-specific alkaline phosphatase (TNAP) is strongly associated with vascular calcification and end-stage renal diseases. In this context, we synthesized a novel series of fluorinated pyrimidone derivatives, i.e., 2-bromo-7-trifluoromethyl-5-oxo-5H-1,3,4-thiadiazolepyrimidones. The bromine functionality was further used for derivatisation by nucleophilic aromatic substitution using amines as nucleophiles as well as by Palladium catalysed Suzuki-Miyaura reactions. The synthesized derivatives were found potent but non-selective inhibitors of both isozymes of AP. Arylated thiadiazolopyrimidones exhibited stronger inhibitory activities than 2-amino-thiadiazolopyrimidones. The binding modes and possible interactions ...
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Papers by Jean Sévigny