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Journal of Biomolecular Screening
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High-Content Fluorescent-Based Assay for Screening Activators of DNA Damage Checkpoint Pathways

Bin Zhang

Boston Biomedical Incorporated, Norwood, Massachusetts

Xiubin Gu

Department of R & D Biology, ArQule Incorporated

Uma Uppalapati

Department of R & D Biology, ArQule Incorporated

Mark A. Ashwell

Department of R & D Chemistry, ArQule Incorporated, Woburn, Massachusetts

David S. Leggett

Boston Biomedical Incorporated, Norwood, Massachusetts

Chiang J. Li

Boston Biomedical Incorporated, Norwood, Massachusetts, cli{at}bostonbiomedical.com

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 EC 50 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. (Journal of Biomolecular Screening 2008:538-543)

Key Words: high-content analysis • Chk2 • fold induction • KS score • percentage of positively stained cells

This version was published on July 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 6, 538-543 (2008)
DOI: 10.1177/1087057108318509
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[Abstract] [PDF]