Open AccessHigh-Content Fluorescent-Based Assay for Screening Activators of DNA Damage Checkpoint Pathways
Author(s) -
Bin Zhang,
Xiubin Gu,
Uma Uppalapati,
Mark A. Ashwell,
David Leggett,
Chiang J. Li
Publication year - 2008
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/1087057108318509
Subject(s) - dna damage , dna , genotoxicity , high content screening , checkpoint kinase 2 , g2 m dna damage checkpoint , dna repair , computational biology , chemistry , cell cycle checkpoint , microbiology and biotechnology , cell , biology , biochemistry , cell cycle , toxicity , organic chemistry
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.