High-content fluorescent-based assay for screening activators of DNA damage checkpoint pathways.
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.
Bin Zhang
Boston Biomedical Incorporated, Norwood, Massachusetts 02062, USA.
Xiubin Gu
Uppalapati
Uma
U
Ashwell
Mark A
MA
Leggett
David S
DS
Li
Chiang J
CJ
D001681:Biological Assay; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D004396:Coloring Agents / Q000378:metabolism; D004249:DNA Damage; D004790:Enzyme Induction; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D010766:Phosphorylation; D011419:Propidium / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004396:Coloring Agents; D005456:Fluorescent Dyes; D007211:Indoles; D034741:RNA, Small Interfering; D015534:Trans-Activators; D011419:Propidium; C007293:DAPI; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases