Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 13, Issue 6, Pages 538-543Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057108318509
Keywords
high-content analysis; Chk2; fold induction; KS score; percentage of positively stained cells
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Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC50 values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.
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