Development of an HTS-compatible assay for the discovery of ASK1 signalosome inhibitors using alphascreen technology.
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.
Department of Molecular Therapeutics, The Scripps Translational Science Institute , Jupiter, Florida.
Emmanuel
E
Sturchler
Weimin
W
Chen
Timothy
T
Spicer
Peter
P
Hodder
Patricia
P
McDonald
Derek
D
Duckett
D019921:Biotinylation; D004791:Enzyme Inhibitors; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D048669:MAP Kinase Kinase 6 / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries; D019311:Staurosporine; D019809:Streptavidin / Q000378:metabolism
D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D019809:Streptavidin; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D048669:MAP Kinase Kinase 6; C482566:MAP2K6 protein, human; D019311:Staurosporine