Work

Development of an HTS-Compatible Assay for the Discovery of ASK1 Signalosome Inhibitors Using AlphaScreen Technology

Year: 2014

Type: article

Abstract: 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.

Source: Assay and Drug Development Technologies

DOI: 10.1089/adt.2013.558

Authors: Emmanuel Sturchler, Weimin Chen, Timothy Spicer, Peter Hodder, Patricia McDonald, Derek Duckett

Language: English

Topic: Protein Kinase Regulation and GTPase Signaling

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: green

Funders: National Institute of General Medical Sciences, State of Florida

Awards: High Throughput Screening to Discover Chemical Probes of ASK1, G8879039376