AlphaScreen™ Kinase HTS Platforms

Warner, Gregory J.; Illy, Chantal; Pedro, Liliana; Roby, Philippe; Bossé, Roger

doi:10.2174/0929867043455693

Cited by 45 publications

(30 citation statements)

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“…The application of AlphaScreen technology for highthroughput enzymatic assays has been described previously (Von Leoprechting et al, 2004;Warner et al, 2004;Burns et al, 2006). We adapted this assay format for the identification of inhibitors of PPM1D phosphatase activity.…”

Section: High-throughput Screen To Identify Chemical Inhibitors Of Ppm1dmentioning

confidence: 99%

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

et al. 2007

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The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. However, the issue of whether inhibition of PPM1D in human tumour cells that overexpress this protein compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of PPM1D can indeed reduce the viability of human tumour cells and that this effect is selective; tumour cell lines that overexpress PPM1D are sensitive to PPM1D inhibition whereas cell lines with normal levels are not. Loss of viability associated with PPM1D RNAi in human tumour cells occurs via the activation of the kinase P38. To identify chemical inhibitors of PPM1D, a high-throughput screening of a library of small molecules was performed. This strategy successfully identified a compound that selectively reduces viability of human tumour cell lines that overexpress PPM1D. As expected of a specific inhibitor, the toxicity to PPM1D overexpressing cell lines after inhibitor treatment is P38 dependent. These results further validate PPM1D as a therapeutic target and identify a proof-of-principle small molecule inhibitor.

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Section: High-throughput Screen To Identify Chemical Inhibitors Of Ppm1dmentioning

confidence: 99%

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

et al. 2007

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“…This has led to the development of a variety of assay formats for protein kinases [13]. These assays utilize various mechanisms to monitor kinase activity including production of ADP [14], protease sensitivity of phosphorylated peptides [15], time-resolved FRET [16,17], and the amplified luminescent proximity homogeneous assay format [18]. Although powerful, the broad applicability of these approaches may be limited due to the necessity of specialized instrumentation for data collection.…”

Section: Introductionmentioning

confidence: 99%

Design and evaluation of a real-time activity probe for focal adhesion kinase

Beck

Zhou

Casey

et al. 2015

Analytica Chimica Acta

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“…Traditional immunoblotting (Western blot) assays used to monitor ubiquitination were unsuitable for this purpose because they are difficult to standardize, not quantitative, and not amenable to genome-scale analysis. We elected to develop a ubiquitination assay based on luminescence (AlphaScreen TM technology) in order to achieve higher sensitivity, greater throughput, and the potential for automation (22,23). The assay measures the proximity of biotinylated ubiquitin (b-Ub) and GST-tagged substrate proteins (22,23) (Fig.…”

mentioning

confidence: 99%

“…We elected to develop a ubiquitination assay based on luminescence (AlphaScreen TM technology) in order to achieve higher sensitivity, greater throughput, and the potential for automation (22,23). The assay measures the proximity of biotinylated ubiquitin (b-Ub) and GST-tagged substrate proteins (22,23) (Fig. 1B).…”

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confidence: 99%

A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5

Kus

Gajadhar

Stanger

et al. 2005

Journal of Biological Chemistry

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Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.The ubiquitin pathway is conserved throughout eukaryotic evolution and is implicated in numerous cellular processes (1). Proteins modified by the ubiquitin pathway are processed for degradation, endocytosis, protein sorting, and subnuclear trafficking (2, 3). Ubiquitination is catalyzed by three enzymes termed E1 1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin protein ligase). E3 regulates the specificity of the reaction by binding directly to substrates (1, 3). The E3-substrate interaction is implicated in an increasing number of diseases, including neurodegeneration, immunological disorders, hypertension, and cancers. For example, numerous E3 enzymes such as Fbw7, Skp2, Mdm2, and VHL and their respective substrates, cyclin E, p27, p53, and HIF, have been linked to tumor progression (4, 5). The therapeutic importance of understanding ubiquitination has been underscored recently by the success of anticancer strategies that affect the ubiquitin pathway (6).Most, if not all, proteins are regulated by the ubiquitin pathway. A recent proteomic approach identified over a thousand proteins that are ubiquitinated in yeast under normal conditions (7). This study, which likely did not detect many nonabundant proteins or proteins that are ubiquitinated under specific conditions (e.g. stress and nutrition), underlines the breadth of the ubiquitin system. Current estimates also predict that there are hundreds of E3 enzymes in eukaryotic genomes (8) whose role is to ubiquitinate these proteins. Despite the biomedical importance of E3 enzymes and great advances in understanding the mechanics of the ubiquitin system, a very small fraction of E3 enzymes has been linked to specific substrates, and currently, the scarcity of identified E3-substrate pairs in the literature is a major bottleneck in the ubiquitin field.Rsp5 is a yeast E3 enzyme, and many of its substrates have not yet been characterized. It belongs to the Nedd4 family of E3 ligase...

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AlphaScreen™ Kinase HTS Platforms

Cited by 45 publications

References 0 publications

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D

Design and evaluation of a real-time activity probe for focal adhesion kinase

A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5

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