1 research outputs found
Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay
Small-molecule inhibitors of DNA
repair pathways are being intensively
investigated as primary and adjuvant chemotherapies. We report the
discovery that cardiac glycosides, natural products in clinical use
for the treatment of heart failure and atrial arrhythmia, are potent
inhibitors of DNA double-strand break (DSB) repair. Our data suggest
that cardiac glycosides interact with phosphorylated mediator of DNA
damage checkpoint protein 1 (phospho-MDC1) or E3 ubiquitin–protein
ligase ring finger protein 8 (RNF8), two factors involved in DSB repair,
and inhibit the retention of p53 binding protein 1 (53BP1) at the
site of DSBs. These observations provide an explanation for the anticancer
activity of this class of compounds, which has remained poorly understood
for decades, and provide guidance for their clinical applications.
This discovery was enabled by the development of the first high-throughput
unbiased cellular assay to identify new small-molecule inhibitors
of DSB repair. Our assay is based on the fully automated, time-resolved
quantification of phospho-SER139-H2AX (γH2AX) and 53BP1 foci,
two factors involved in the DNA damage response network, in cells
treated with small molecules and ionizing radiation (IR). This primary
assay is supplemented by robust secondary assays that establish lead
compound potencies and provide further insights into their mechanisms
of action. Although the cardiac glycosides were identified in an evaluation
of 2366 small molecules, the assay is envisioned to be adaptable to
larger compound libraries. The assay is shown to be compatible with
small-molecule DNA cleaving agents, such as bleomycin, neocarzinostatin
chromophore, and lomaiviticin A, in place of IR