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Shirish Shenolikar - One of the best experts on this subject based on the ideXlab platform.
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Protein Serine/Threonine Phosphatase Inhibitors and Human Disease
Handbook of Cell Signaling, 2010Co-Authors: Shirish Shenolikar, Matthew H. BrushAbstract:Publisher Summary This chapter focuses on new information on the mode of action of Phosphatase Inhibitors and discusses their potential contributions to the pathophysiology of human disease. Emerging studies suggest that some Phosphatase Inhibitors physically associate with cellular Phosphatase complexes composed of a catalytic subunit bound to one or more regulatory proteins that restrict the actions of the Phosphatases to control specific cellular events. The functions of these Phosphatase Inhibitors are in turn controlled by their expression, reversible phosphorylation, and dynamic association with target protein Phosphatases. In this manner, endogenous serine/threonine Phosphatase Inhibitors achieve both spatial and temporal control of phosphoproteins that regulate normal cell physiology, and growing evidence suggests that aberrant expression and activity of Phosphatase Inhibitors may be associated with many human diseases. The availability of protein and non-protein Inhibitors of protein serine/threonine Phosphatases has provided researchers with an extensive toolkit to study the role of Phosphatases and Phosphatase Inhibitors in modulating the mammalian physiology. Rapid progress is also being made in the understanding of cellular Phosphatase complexes and their functions. In addition, the mechanisms that regulate the expression and activity of Phosphatase Inhibitors and their mode of action in controlling specific pools of cellular protein Phosphatases are being actively investigated.
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UNIT 18.10 Use of Protein Phosphatase Inhibitors
2003Co-Authors: Douglas C Weiser, Shirish ShenolikarAbstract:Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein Phosphatase Inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting cellular phosphorylation activity with okadaic acid, microcystin-LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine Phosphatases.
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Use of protein Phosphatase Inhibitors.
Current protocols in molecular biology, 2003Co-Authors: Douglas C Weiser, Shirish ShenolikarAbstract:Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein Phosphatase Inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting cellular phosphorylation activity with okadaic acid, microcystin-LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine Phosphatases.
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Use of protein Phosphatase Inhibitors.
Current protocols in protein science, 2003Co-Authors: Douglas C Weiser, Shirish ShenolikarAbstract:Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein Phosphatase Inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting the cellular PP1/PP2A activity with okadaic acid, microcystin-LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine Phosphatases.
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Handbook of Cell Signaling - CHAPTER 105 – Serine/Threonine Phosphatase Inhibitor Proteins
Handbook of Cell Signaling, 2003Co-Authors: Shirish ShenolikarAbstract:Emerging evidence suggests that mammalian cells express a multitude of serine/threonine Phosphatase Inhibitors, many of which are not fully analyzed. Comparison of known serine/ threonine Phosphatase inhibitor proteins suggests that they utilize many different mechanisms to inhibit Phosphatases. Moreover, the Phosphatase Inhibitors may be regulated by hormone-induced changes in expression, alternate splicing, or reversible phosphorylation. Finally, inhibitor proteins most likely collaborate with other Phosphatase regulators to control unique Phosphatase populations and integrate multiple physiological signals that regulate protein phosphorylation. A number of protein kinases respond to changes in intracellular second messengers but only one serine/threonine Phosphatase, calcineurin (or PP2B), is activated by the second messenger calcium. This observation fostered the opinion that most Phosphatases are unregulated, and it was suggested that hormone-induced increases in protein kinase activity must be sufficiently high as to overcome the opposing actions of Phosphatases. It also meant that hormone signals are severely dampened and/or slowed by the constitutive activity of Phosphatases. However, it was found that Phosphatase Inhibitors allow cells to lower this barrier and accelerate or even amplify the kinase signals. The inherent appeal of this mechanism prompted an active search for hormone-regulated Phosphatase Inhibitors. Work over the last two decades has identified numerous gene products that regulate protein serine/threonine Phosphatases, firmly dismissing the idea of unregulated serine/threonine Phosphatases.
John S. Lazo - One of the best experts on this subject based on the ideXlab platform.
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in flow photooxygenation of aminothienopyridinones generates iminopyridinedione ptp4a3 Phosphatase Inhibitors
Organic and Biomolecular Chemistry, 2019Co-Authors: Nikhil R Tasker, John S. Lazo, Ettore J Rastelli, Isabella K Blanco, James C Burnett, Elizabeth R Sharlow, Peter WipfAbstract:A continuous flow photooxygenation of 7-aminothieno[3,2-c]pyridin-4(5H)-ones to produce 7-iminothieno[3,2-c]pyridine-4,6(5H,7H)-diones has been developed, utilizing ambient air as the sole reactant. N-H Imines are formed as the major products, and excellent functional group tolerance and conversion on gram-scale without the need for chromatographic purification allow for facile late-stage diversification of the aminothienopyridinone scaffold. Several analogs exhibit potent in vitro inhibition of the cancer-associated protein tyrosine Phosphatase PTP4A3, and the SAR supports an exploratory docking model.
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development and implementation of a 384 well homogeneous fluorescence intensity high throughput screening assay to identify mitogen activated protein kinase Phosphatase 1 dual specificity protein Phosphatase Inhibitors
Assay and Drug Development Technologies, 2007Co-Authors: Paul A Johnston, Peter Wipf, Caleb Foster, Tong Ying Shun, John J Skoko, Sunita N Shinde, John S. LazoAbstract:We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) Phosphatase-1 (MKP-1) dual-specificity Phosphatase Inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for Inhibitors of MKP-1 activity at 10 μM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by ≥50% at 10 μM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent Inhibitors of MKP-1 with 50% inhibitory concentration (IC50) values of <50 μM; four exhibited IC50 values <1.0 μM, six produced IC50 values in the 1–10 μM range, and 36 produced IC50 values in the 10–50 μM range. A clustering and cla...
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development and implementation of a 384 well homogeneous fluorescence intensity high throughput screening assay to identify mitogen activated protein kinase Phosphatase 1 dual specificity protein Phosphatase Inhibitors
Assay and Drug Development Technologies, 2007Co-Authors: Paul A Johnston, Peter Wipf, Caleb Foster, Tong Ying Shun, John J Skoko, Sunita Shinde, John S. LazoAbstract:We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) Phosphatase-1 (MKP-1) dual-specificity Phosphatase Inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for Inhibitors of MKP-1 activity at 10 microM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by > or =50% at 10 microM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent Inhibitors of MKP-1 with 50% inhibitory concentration (IC(50)) values of <50 microM; four exhibited IC(50) values <1.0 microM, six produced IC(50) values in the 1-10 microM range, and 36 produced IC(50) values in the 10-50 microM range. A clustering and classification analysis of the compound structures of the 46 confirmed MKP-1 Inhibitors produced 29 singleton structures and seven clusters of related structures. Some MKP-1 Inhibitors were members of structural classes or contained substructure pharmacophores that previously were reported to inhibit either MKP-1 or other protein tyrosine Phosphatases, validating the HTS assay. Importantly, we have identified several attractive and novel MKP-1 inhibitor structures that warrant further investigation as potential probes to study the biology of MKP-1 and its role in controlling the amplitude and/or duration of MAPK signaling, cell survival, and tumor progression.
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Discovery of Protein Kinase Phosphatase Inhibitors
Methods in molecular biology (Clifton N.J.), 2007Co-Authors: Andreas Vogt, John S. LazoAbstract:Dynamic protein phosphorylation, a major cellular regulatory system, is tightly controlled by coordinating the reversible action of protein kinases and Phosphatases. Recent evidence is consistent with sophisticated mechanisms that regulate both kinases and Phosphatases in the cell. Dual specificity Phosphatases, which act on phosphorylated serine, threonine, and tyrosine residues in proteins, are valid targets for drug discovery. Chemical complementation combines genetic manipulations with chemical biology and high-content multiparametric analyses and was developed as a screening approach to discover protein Phosphatase Inhibitors. Using a dual specificity mitogen-activated protein kinase Phosphatase as an example, a detailed protocol, discussion of issues relating to data analysis, and high-throughput implementation of the chemical complementation approach to drug discovery is presented.
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Cell-active dual specificity Phosphatase Inhibitors identified by high-content screening.
Chemistry & Biology, 2003Co-Authors: Andreas Vogt, Peter Wipf, Kathleen A. Cooley, Marni Brisson, Michael G Tarpley, John S. LazoAbstract:Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity Phosphatases (DSPases), but few Inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institute's 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase Phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B Phosphatases. The most potent inhibitor of MKP-3 had an IC50 of
Herbert Waldmann - One of the best experts on this subject based on the ideXlab platform.
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the therapeutic potential of Phosphatase Inhibitors
Current Opinion in Chemical Biology, 2009Co-Authors: Viktor V Vintonyak, Daniel Rauh, Andrey P Antonchick, Herbert WaldmannAbstract:Protein Phosphatases (PPs) constitute a large family of enzymes, which are crucial modulators of cellular phosphorylation events. Malfunction in PP activity has been associated with human diseases, including diabetes, obesity, cancer, and neurodegenerative and autoimmune disorders, and makes this class of enzymes attractive targets for chemical biology and medicinal chemistry research. A number of strategies are currently explored for the identification and development of various classes of PP modulators and have resulted in a plethora of chemically distinct Inhibitors. Limited selectivity and adverse pharmacological properties of PP Inhibitors are still major bottlenecks for further clinical development and resulted in only a few molecular entities currently in clinical trials.
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identification of Inhibitors for mycobacterial protein tyrosine Phosphatase b mptpb by biology oriented synthesis bios
Chemistry-an Asian Journal, 2007Co-Authors: Ivan R Correa, Andrea Norenmuller, Horstdieter Ambrosi, Sven Jakupovic, Krishna Saxena, Harald Schwalbe, Markus Kaiser, Herbert WaldmannAbstract:Protein Phosphatases have recently emerged as important targets for research in chemical biology and medicinal chemistry, and new classes of Phosphatase Inhibitors are in high demand. BIOS (biology-oriented synthesis) employs the criteria of relevance to nature and biological prevalidation for the design and synthesis of compound collections. In an application of the BIOS principle, an efficient solid-phase synthesis of highly substituted indolo[2,3-a]quinolizidines by using a vinylogous Mannich-Michael reaction in combination with phosgene- or acid-mediated ring closure was developed. Screening of this library for Phosphatase Inhibitors yielded a new inhibitor class for the Mycobacterium tuberculosis Phosphatase MptpB.
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3 substituted indolizine 1 carbonitrile derivatives as Phosphatase Inhibitors
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Timo Weide, Heino Prinz, Herbert Waldmann, Lars Arve, Horst KesslerAbstract:In the course of studies directed toward the discovery of novel scaffolds for medicinal application, we synthesized a series of 3-substituted indolizine-1-carbonitrile derivatives. Some of them displayed activity against MPtpA/MPtpB Phosphatases which are involved in infectious diseases. We report here the solid-phase synthesis and antiPhosphatase activity of a series of indolizines.
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The core structures of roseophilin and the prodigiosin alkaloids define a new class of protein tyrosine Phosphatase Inhibitors.
Chembiochem : a European journal of chemical biology, 2004Co-Authors: Alois Fürstner, Kerstin Reinecke, Heino Prinz, Herbert WaldmannAbstract:A common core. Protein Phosphatases, such as the dual-specificity Phosphatase VHR and the tyrosine Phosphatase PTP1B, are key regulators of innumerable cellular processes, and new classes of Phosphatase Inhibitors are in demand. The azafulvene-containing core structures of the roseophilin and prodigiosin alkaloids define such a new class
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solid phase synthesis of dysidiolide derived protein Phosphatase Inhibitors
Journal of the American Chemical Society, 2002Co-Authors: Dirk Brohm, Nicolas Philippe, Susanne Metzger, Ajay Bhargava, Oliver Muller, Folker Lieb, Herbert WaldmannAbstract:Biologically active natural products can be regarded as evolutionary selected and biologically validated starting points in structural space for the development of compound libraries. For libraries designed and synthesized around a given natural product, a higher hit rate and the identification of biologically relevant hits can be expected, justifying a probably higher investment in the development of the corresponding syntheses. This approach requires the development of complex multistep reaction sequences on the solid phase. Employing the protein Phosphatase Cdc25 inhibitor dysidiolide as an example, we demonstrate that this goal can be achieved successfully. The reaction sequences developed led to dysidiolide analogues in overall 8−12 linear steps with the longest sequence on the solid support amounting to up to 11 sequential transformations. The desired products were obtained in overall yields ranging from 6% to 27% and in multimilligram amounts starting from 100 mg of resin. The transformations appli...
S Suzuki - One of the best experts on this subject based on the ideXlab platform.
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calyculin a protein Phosphatase inhibitor enhances capacitation of human sperm
Fertility and Sterility, 1993Co-Authors: Satoru Furuya, Y Endo, Kazuoki Osumi, Shiro Nozawa, S SuzukiAbstract:Objective To examine the effects of protein Phosphatase Inhibitors on capacitation and protein phosphorylation in human sperm. Design The chlortetracycline (CTC) fluorescence assay was used to monitor capacitated sperm treated with or without protein Phosphatase Inhibitors. Capacitation was confirmed by the ability of sperm to undergo the acrosome reaction in response to Ca ++ ionophore A23187 or mouse zonae pellucidae. 32 P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effects of protein Phosphatase Inhibitors on protein phosphorylation. Results The treatment of sperm with calyculin A resulted in the following: [1] the rapid appearance of the clear perimeter pattern, specifically, distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece, in a dose-dependent manner in the 1 to 100 nM range; [2] an accelerated ability to undergo the acrosome reaction; and [3] an enhanced phosphorylation of sperm phosphoproteins in a dose-related fashion in the 1 to 100 nM range. A similar stimulatory effect was observed only with a 100-fold higher concentrations of okadaic acid, another protein Phosphatase inhibitor. Conclusion Our results strongly suggest that protein phosphorylation and dephosphorylation may be involved in the regulation of human sperm capacitation.
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Calyculin A, protein Phosphatase inhibitor, enhances capacitation of human sperm *
Fertility and sterility, 1993Co-Authors: Satoru Furuya, Y Endo, Kazuoki Osumi, Shiro Nozawa, Mikiko Oba, S SuzukiAbstract:Objective To examine the effects of protein Phosphatase Inhibitors on capacitation and protein phosphorylation in human sperm. Design The chlortetracycline (CTC) fluorescence assay was used to monitor capacitated sperm treated with or without protein Phosphatase Inhibitors. Capacitation was confirmed by the ability of sperm to undergo the acrosome reaction in response to Ca ++ ionophore A23187 or mouse zonae pellucidae. 32 P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effects of protein Phosphatase Inhibitors on protein phosphorylation. Results The treatment of sperm with calyculin A resulted in the following: [1] the rapid appearance of the clear perimeter pattern, specifically, distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece, in a dose-dependent manner in the 1 to 100 nM range; [2] an accelerated ability to undergo the acrosome reaction; and [3] an enhanced phosphorylation of sperm phosphoproteins in a dose-related fashion in the 1 to 100 nM range. A similar stimulatory effect was observed only with a 100-fold higher concentrations of okadaic acid, another protein Phosphatase inhibitor. Conclusion Our results strongly suggest that protein phosphorylation and dephosphorylation may be involved in the regulation of human sperm capacitation.
Hideaki Oikawa - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of specific protein Phosphatase Inhibitors, tautomycin and tautomycetin toward structure-activity relationship study.
Current medicinal chemistry, 2002Co-Authors: Hideaki OikawaAbstract:The recent progress in synthetic and SAR studies of the specific protein Phosphatase Inhibitors tautomycin and tautomycetin is reviewed. This article covers the total synthesis of tautomycin and synthetic studies of the spiroketal and the anhydride segments and tautomycetin, and SAR studies on PP inhibition and apoptosis-inducing activity of tautomycin.