Development of an HTS-compatible assay for the discovery of ASK1 signalosome inhibitors using alphascreen technology. 2014

Emmanuel Sturchler, and Weimin Chen, and Timothy Spicer, and Peter Hodder, and Patricia McDonald, and Derek Duckett
Department of Molecular Therapeutics, The Scripps Translational Science Institute , Jupiter, Florida.

Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z' factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.

UI MeSH Term Description Entries
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D015398 Signal Transduction The intracellular transfer of information (biological activation/inhibition) through a signal pathway. In each signal transduction system, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) is mediated via the coupling of a receptor/enzyme to a second messenger system or to an ion channel. Signal transduction plays an important role in activating cellular functions, cell differentiation, and cell proliferation. Examples of signal transduction systems are the GAMMA-AMINOBUTYRIC ACID-postsynaptic receptor-calcium ion channel system, the receptor-mediated T-cell activation pathway, and the receptor-mediated activation of phospholipases. Those coupled to membrane depolarization or intracellular release of calcium include the receptor-mediated activation of cytotoxic functions in granulocytes and the synaptic potentiation of protein kinase activation. Some signal transduction pathways may be part of larger signal transduction pathways; for example, protein kinase activation is part of the platelet activation signal pathway. Cell Signaling,Receptor-Mediated Signal Transduction,Signal Pathways,Receptor Mediated Signal Transduction,Signal Transduction Pathways,Signal Transduction Systems,Pathway, Signal,Pathway, Signal Transduction,Pathways, Signal,Pathways, Signal Transduction,Receptor-Mediated Signal Transductions,Signal Pathway,Signal Transduction Pathway,Signal Transduction System,Signal Transduction, Receptor-Mediated,Signal Transductions,Signal Transductions, Receptor-Mediated,System, Signal Transduction,Systems, Signal Transduction,Transduction, Signal,Transductions, Signal
D048669 MAP Kinase Kinase 6 A mitogen-activated protein kinase kinase with specificity for P38 MITOGEN-ACTIVATED PROTEIN KINASES. MAP Kinase Kinase 6b,MAP Kinase Kinase 6c,MAP Kinase Kinase MEK6,MEK6 MAP Kinase Kinase,MEK6 Protein Kinase,MEK6b Protein Kinase,MEK6c Protein Kinase,MKK6b Protein Kinase,MKK6c Protein Kinase,Mitogen-Activated Protein Kinase Kinase 6,SAPKK3 Protein Kinase,Stress-Activated Protein Kinase Kinase-3,Mitogen Activated Protein Kinase Kinase 6,Protein Kinase, MEK6,Protein Kinase, MEK6b,Protein Kinase, MEK6c,Protein Kinase, MKK6b,Protein Kinase, MKK6c,Protein Kinase, SAPKK3,Stress Activated Protein Kinase Kinase 3
D048848 MAP Kinase Kinase Kinase 5 A 150-kDa MAP kinase kinase kinase that may play a role in the induction of APOPTOSIS. It has specificity for MAP KINASE KINASE 3; MAP KINASE KINASE 4; and MAP KINASE KINASE 6. ASK1 Protein Kinase,Apoptosis Signal-Regulating Kinase 1,MAP3K5 Protein Kinase,MAPK Kinase Kinase 5,MAPKKK5,Mitogen-Activated Kinase Kinase Kinase Kinase 5,Apoptosis Signal Regulating Kinase 1,Mitogen Activated Kinase Kinase Kinase Kinase 5,Protein Kinase, ASK1,Protein Kinase, MAP3K5
D054852 Small Molecule Libraries Large collections of small molecules (molecular weight about 600 or less), of similar or diverse nature which are used for high-throughput screening analysis of the gene function, protein interaction, cellular processing, biochemical pathways, or other chemical interactions. It includes virtual libraries. Chemical Libraries,Molecular Libraries, Small,Libraries, Chemical,Libraries, Small Molecular,Libraries, Small Molecule,Molecule Libraries, Small,Small Molecular Libraries
D057166 High-Throughput Screening Assays Rapid methods of measuring the effects of an agent in a biological or chemical assay. The assay usually involves some form of automation or a way to conduct multiple assays at the same time using sample arrays. High-Throughput Screening,High-Throughput Biological Assays,High-Throughput Chemical Assays,High-Throughput Screening Methods,Assay, High-Throughput Biological,Assay, High-Throughput Chemical,Assay, High-Throughput Screening,Biological Assay, High-Throughput,Chemical Assay, High-Throughput,High Throughput Biological Assays,High Throughput Chemical Assays,High Throughput Screening,High Throughput Screening Assays,High Throughput Screening Methods,High-Throughput Biological Assay,High-Throughput Chemical Assay,High-Throughput Screening Assay,High-Throughput Screening Method,High-Throughput Screenings,Screening Assay, High-Throughput,Screening Method, High-Throughput,Screening, High-Throughput
D057809 HEK293 Cells A cell line generated from human embryonic kidney cells that were transformed with human adenovirus type 5. 293T Cells,HEK 293 Cell Line,HEK 293 Cells,Human Embryonic Kidney Cell Line 293,Human Kidney Cell Line 293,293 Cell, HEK,293 Cells, HEK,293T Cell,Cell, 293T,Cell, HEK 293,Cell, HEK293,Cells, 293T,Cells, HEK 293,Cells, HEK293,HEK 293 Cell,HEK293 Cell
D019311 Staurosporine An indolocarbazole that is a potent PROTEIN KINASE C inhibitor which enhances cAMP-mediated responses in human neuroblastoma cells. (Biochem Biophys Res Commun 1995;214(3):1114-20) 8,12-Epoxy-1H,8H-2,7b,12a-triazadibenzo(a,g)cyclonona(cde)trinden-1-one, 2,3,9,10,11,12-hexahydro-9-methoxy-8-methyl-10-(methylamino)-, (8alpha,9beta,10beta,12alpha)-(+)-
D019809 Streptavidin A 60-kDa extracellular protein of Streptomyces avidinii with four high-affinity biotin binding sites. Unlike AVIDIN, streptavidin has a near neutral isoelectric point and is free of carbohydrate side chains. Strepavidin

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