Journal of Biomolecular Screening

 

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First published on June 19, 2008, doi:10.1177/1087057108318509

Journal of Biomolecular Screening 2008;13:538.

A more recent version of this article appeared on July 1, 2008

Article

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

Bin Zhang, Xiubin Gu, Uma Uppalapati, Mark A. Ashwell, David S. Leggett, and Chang J. Li*

Boston Biomedical Incorporated

* To whom correspondence should be addressed. E-mail: cli{at}bostonbiomedical.com.


  Abstract
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. (Journal of Biomolecular Screening XXXX:xx-xx)


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