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Philip Cohen - One of the best experts on this subject based on the ideXlab platform.
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the selectivity of Protein Kinase Inhibitors a further update
Biochemical Journal, 2007Co-Authors: Jenny Bain, Hilary Mclauchlan, Matthew Elliott, Lorna Plater, Natalia Shpiro, James C Hastie, Iva V Klevernic, Simon J C Arthur, Dario R Alessi, Philip CohenAbstract:The specificities of 65 compounds reported to be relatively specific Inhibitors of Protein Kinases have been profiled against a panel of 70–80 Protein Kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule Inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated Protein Kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-Kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK Kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (Protein Kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated Protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase Kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 Kinase), D4476 to inhibit CK1 (casein Kinase 1), VX680 to inhibit Aurora Kinases, and roscovitine as a pan-CDK (cyclin-dependent Kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated Kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule Inhibitors of Protein Kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular Protein Kinases in cellular processes.
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The selectivity of Protein Kinase Inhibitors; a further update
Biochemical Journal, 2007Co-Authors: Jenny Bain, Simon Arthur, Hilary Mclauchlan, Lorna Plater, Natalia Shpiro, Iva V Klevernic, Matt Elliott, James Hastie, Dario Alessi, Philip CohenAbstract:The specificities of 65 compounds reported to be relatively specific Inhibitors of Protein Kinases have been profiled against a panel of 70-80 Protein Kinases. Based on this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small molecule Inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess of the physiological roles of p38 MAPK isoforms, PI-103 and wortmannin to be used in parallel to inhibit PI 3-Kinase, PP1 or PP2 to be used in parallel with Src inhibitor-1 to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB/AKT, rapamycin to inhibit TORC1, CT 99021 to inhibit GSK3, BI-D1870 and SL0101 or FMK to be used in parallel to inhibit RSK, D4476 to inhibit CK1, VX680 to inhibit Aurora Kinases and roscovitine as a pan CDK inhibitor. We have also identified harmine as a very potent and specific inhibitor of DYRK1A in vitro. The results have further emphasised the need for considerable caution in using small molecule Inhibitors of Protein Kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular Protein Kinases in cellular processes.
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the specificities of Protein Kinase Inhibitors an update
Biochemical Journal, 2003Co-Authors: Jenny Bain, Hilary Mclauchlan, Matthew Elliott, Philip CohenAbstract:We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective Inhibitors of particular serine/threonine-specific Protein Kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of Protein Kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more Protein Kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent Protein Kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase Kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 Protein Kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated Kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated Kinase (PRAK; IC(50)=1.0 microM).
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specificity and mechanism of action of some commonly used Protein Kinase Inhibitors
Biochemical Journal, 2000Co-Authors: Stephen P Davies, Helen Reddy, Matilde Caivano, Philip CohenAbstract:The specificities of 28 commercially available compounds reported to be relatively selective Inhibitors of particular serine/threonine-specific Protein Kinases have been examined against a large panel of Protein Kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many Protein Kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective Inhibitors, but still inhibited two or more Protein Kinases with similar potency. LY 294002 was found to inhibit casein Kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-Kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated Protein Kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK Kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective Inhibitors affected at least one additional Protein Kinase. Our results demonstrate that the specificities of Protein Kinase Inhibitors cannot be assessed simply by studying their effect on Kinases that are closely related in primary structure. We propose guidelines for the use of Protein Kinase Inhibitors in cell-based assays.
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the development and therapeutic potential of Protein Kinase Inhibitors
Current Opinion in Chemical Biology, 1999Co-Authors: Philip CohenAbstract:A number of small cell-permeant Protein Kinase Inhibitors have been developed that have the potential for treating a variety of diseases. The basis for the specificity of several of these drugs has been elucidated and a number are undergoing human clinical trials.
Jenny Bain - One of the best experts on this subject based on the ideXlab platform.
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the selectivity of Protein Kinase Inhibitors a further update
Biochemical Journal, 2007Co-Authors: Jenny Bain, Hilary Mclauchlan, Matthew Elliott, Lorna Plater, Natalia Shpiro, James C Hastie, Iva V Klevernic, Simon J C Arthur, Dario R Alessi, Philip CohenAbstract:The specificities of 65 compounds reported to be relatively specific Inhibitors of Protein Kinases have been profiled against a panel of 70–80 Protein Kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule Inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated Protein Kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-Kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK Kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (Protein Kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated Protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase Kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 Kinase), D4476 to inhibit CK1 (casein Kinase 1), VX680 to inhibit Aurora Kinases, and roscovitine as a pan-CDK (cyclin-dependent Kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated Kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule Inhibitors of Protein Kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular Protein Kinases in cellular processes.
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The selectivity of Protein Kinase Inhibitors; a further update
Biochemical Journal, 2007Co-Authors: Jenny Bain, Simon Arthur, Hilary Mclauchlan, Lorna Plater, Natalia Shpiro, Iva V Klevernic, Matt Elliott, James Hastie, Dario Alessi, Philip CohenAbstract:The specificities of 65 compounds reported to be relatively specific Inhibitors of Protein Kinases have been profiled against a panel of 70-80 Protein Kinases. Based on this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small molecule Inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess of the physiological roles of p38 MAPK isoforms, PI-103 and wortmannin to be used in parallel to inhibit PI 3-Kinase, PP1 or PP2 to be used in parallel with Src inhibitor-1 to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB/AKT, rapamycin to inhibit TORC1, CT 99021 to inhibit GSK3, BI-D1870 and SL0101 or FMK to be used in parallel to inhibit RSK, D4476 to inhibit CK1, VX680 to inhibit Aurora Kinases and roscovitine as a pan CDK inhibitor. We have also identified harmine as a very potent and specific inhibitor of DYRK1A in vitro. The results have further emphasised the need for considerable caution in using small molecule Inhibitors of Protein Kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular Protein Kinases in cellular processes.
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the specificities of Protein Kinase Inhibitors an update
Biochemical Journal, 2003Co-Authors: Jenny Bain, Hilary Mclauchlan, Matthew Elliott, Philip CohenAbstract:We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective Inhibitors of particular serine/threonine-specific Protein Kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of Protein Kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more Protein Kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent Protein Kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase Kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 Protein Kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated Kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated Kinase (PRAK; IC(50)=1.0 microM).
Carrow I Wells - One of the best experts on this subject based on the ideXlab platform.
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new tools for evaluating Protein tyrosine sulfation tyrosylProtein sulfotransferases tpsts are novel targets for raf Protein Kinase Inhibitors
Biochemical Journal, 2018Co-Authors: Dominic P Byrne, Krithika Ramakrishnan, Claire E Eyers, Carrow I Wells, Pawin Ngamlert, David H Drewry, William J Zuercher, Neil G Berry, David G Fernig, Patrick A EyersAbstract:Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular Protein-Protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylProtein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine in a Protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule Inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the Kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based Inhibitors of the Ser/Thr Kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar Inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF Inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST Inhibitors in vitro We propose that target-validated Protein Kinase Inhibitors could be repurposed, or redesigned, as more-specific TPST Inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF Protein Kinase Inhibitors.
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new tools for carbohydrate sulfation analysis heparan sulfate 2 o sulfotransferase hs2st is a target for small molecule Protein Kinase Inhibitors
Biochemical Journal, 2018Co-Authors: Dominic P Byrne, Yong Li, Krithika Ramakrishnan, Igor L Barsukov, Edwin A Yates, Claire E Eyers, Dulce Papygarcia, Sandrine Chantepie, Vijayakanth Pagadala, Carrow I WellsAbstract:Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST Inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the Protein Kinase inhibitor rottlerin and oxindole-based RAF Kinase Inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl Protein sulfotranferases are also inhibited by a variety of Protein Kinase Inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and Protein) sulfation in cells, and that Protein Kinase Inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.
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new tools for carbohydrate sulphation analysis heparan sulphate 2 o sulphotranserase hs2st is a target for small molecule Protein Kinase Inhibitors
bioRxiv, 2018Co-Authors: Dominic P Byrne, Krithika Ramakrishnan, Igor L Barsukov, Edwin A Yates, Claire E Eyers, Dulce Papygarcia, Sandrine Chantepie, Vijayakanth Pagadala, Jian Liu, Carrow I WellsAbstract:Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2 O sulphotransferase (HS2ST), which transfers sulphate from the cofactor PAPS (3 phosphoadenosine- 5 phosphosulphate) to the 2 O position of iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST Inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the Protein Kinase inhibitor rottlerin and oxindole-based RAF Kinase Inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protien Sulpha Transferases (TPSTs) are also inhibited by a variety of Protein Kinase Inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and Protein) sulphation in cells, and that Protein Kinase Inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.
Hilary Mclauchlan - One of the best experts on this subject based on the ideXlab platform.
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the selectivity of Protein Kinase Inhibitors a further update
Biochemical Journal, 2007Co-Authors: Jenny Bain, Hilary Mclauchlan, Matthew Elliott, Lorna Plater, Natalia Shpiro, James C Hastie, Iva V Klevernic, Simon J C Arthur, Dario R Alessi, Philip CohenAbstract:The specificities of 65 compounds reported to be relatively specific Inhibitors of Protein Kinases have been profiled against a panel of 70–80 Protein Kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule Inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated Protein Kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-Kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK Kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (Protein Kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated Protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase Kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 Kinase), D4476 to inhibit CK1 (casein Kinase 1), VX680 to inhibit Aurora Kinases, and roscovitine as a pan-CDK (cyclin-dependent Kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated Kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule Inhibitors of Protein Kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular Protein Kinases in cellular processes.
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The selectivity of Protein Kinase Inhibitors; a further update
Biochemical Journal, 2007Co-Authors: Jenny Bain, Simon Arthur, Hilary Mclauchlan, Lorna Plater, Natalia Shpiro, Iva V Klevernic, Matt Elliott, James Hastie, Dario Alessi, Philip CohenAbstract:The specificities of 65 compounds reported to be relatively specific Inhibitors of Protein Kinases have been profiled against a panel of 70-80 Protein Kinases. Based on this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small molecule Inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess of the physiological roles of p38 MAPK isoforms, PI-103 and wortmannin to be used in parallel to inhibit PI 3-Kinase, PP1 or PP2 to be used in parallel with Src inhibitor-1 to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB/AKT, rapamycin to inhibit TORC1, CT 99021 to inhibit GSK3, BI-D1870 and SL0101 or FMK to be used in parallel to inhibit RSK, D4476 to inhibit CK1, VX680 to inhibit Aurora Kinases and roscovitine as a pan CDK inhibitor. We have also identified harmine as a very potent and specific inhibitor of DYRK1A in vitro. The results have further emphasised the need for considerable caution in using small molecule Inhibitors of Protein Kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular Protein Kinases in cellular processes.
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the specificities of Protein Kinase Inhibitors an update
Biochemical Journal, 2003Co-Authors: Jenny Bain, Hilary Mclauchlan, Matthew Elliott, Philip CohenAbstract:We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective Inhibitors of particular serine/threonine-specific Protein Kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of Protein Kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more Protein Kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent Protein Kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase Kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 Protein Kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated Kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated Kinase (PRAK; IC(50)=1.0 microM).
Dominic P Byrne - One of the best experts on this subject based on the ideXlab platform.
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new tools for evaluating Protein tyrosine sulfation tyrosylProtein sulfotransferases tpsts are novel targets for raf Protein Kinase Inhibitors
Biochemical Journal, 2018Co-Authors: Dominic P Byrne, Krithika Ramakrishnan, Claire E Eyers, Carrow I Wells, Pawin Ngamlert, David H Drewry, William J Zuercher, Neil G Berry, David G Fernig, Patrick A EyersAbstract:Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular Protein-Protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylProtein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine in a Protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule Inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the Kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based Inhibitors of the Ser/Thr Kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar Inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF Inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST Inhibitors in vitro We propose that target-validated Protein Kinase Inhibitors could be repurposed, or redesigned, as more-specific TPST Inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF Protein Kinase Inhibitors.
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new tools for carbohydrate sulfation analysis heparan sulfate 2 o sulfotransferase hs2st is a target for small molecule Protein Kinase Inhibitors
Biochemical Journal, 2018Co-Authors: Dominic P Byrne, Yong Li, Krithika Ramakrishnan, Igor L Barsukov, Edwin A Yates, Claire E Eyers, Dulce Papygarcia, Sandrine Chantepie, Vijayakanth Pagadala, Carrow I WellsAbstract:Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST Inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the Protein Kinase inhibitor rottlerin and oxindole-based RAF Kinase Inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl Protein sulfotranferases are also inhibited by a variety of Protein Kinase Inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and Protein) sulfation in cells, and that Protein Kinase Inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.
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new tools for carbohydrate sulphation analysis heparan sulphate 2 o sulphotranserase hs2st is a target for small molecule Protein Kinase Inhibitors
bioRxiv, 2018Co-Authors: Dominic P Byrne, Krithika Ramakrishnan, Igor L Barsukov, Edwin A Yates, Claire E Eyers, Dulce Papygarcia, Sandrine Chantepie, Vijayakanth Pagadala, Jian Liu, Carrow I WellsAbstract:Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2 O sulphotransferase (HS2ST), which transfers sulphate from the cofactor PAPS (3 phosphoadenosine- 5 phosphosulphate) to the 2 O position of iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST Inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the Protein Kinase inhibitor rottlerin and oxindole-based RAF Kinase Inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protien Sulpha Transferases (TPSTs) are also inhibited by a variety of Protein Kinase Inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and Protein) sulphation in cells, and that Protein Kinase Inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.
