Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay

Abstract

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.

Synergistic potentiation of (−)-lomaiviticin A cytotoxicity by the ATR inhibitor VE-821

“…We monitored the production and colocalization of phosphor-SER139 H2AX (γH2AX) 31 and 53BP1 32 foci as an indicator of DSB formation, as previously described. 2a,2b,8d In the presence of 1 alone (up to 250 pM) γH2AX and 53BP1 foci were observed, as expected. However, in the presence of 7 (10 μM), pan-nuclear staining of nuclear γH2AX and the formation of apoptotic bodies (also displaying pan-staining) was observed, which is indicative of apoptosis.…”

“…The application of DNA repair inhibitors as chemo- and radiosensitizers is an active area of research, and several combinations of repair inhibitors and DNA damaging agents [small molecules, ionizing radiation (IR)] are undergoing clinical evaluation. 8 Synergistic potentiation of 1 by DNA repair inhibitors could decrease required doses, resulting in fewer off-target effects and higher therapeutic indices across a range of tumor types, including DSB repair-proficient tumors. Herein, we report an evaluation of combinations of 1 and six inhibitors of DNA DSB repair.…”

Local DNA Repair Inhibition for Sustained Radiosensitization of High-Grade Gliomas

“…We utilized a live-cell, dual HR and NHEJ repair developed by our group, which measures repair activity at two site-specific, intrachromosomally-integrated DSBs (schematic shown in Fig.2a) (21). This cell-based assay is ideal for the comparison of DSB repair inhibition by free drugs versus NPs, because the DSB kinetics can be precisely controlled during exposure to slow-release NPs, and the assay can be miniaturized to test a range of drugs, variable doses, and formulations in a single experiment (33, 34). The HR repair pathway is active in the S and G2 phases of the cell cycle, and thus is the primary form of DNA repair in mitotically active cells such as tumor cells, while the canonical NHEJ pathway is most active in G0 and G1 phases and is the dominant mode of DNA repair in quiescent cells such as neurons (35).…”

Non-homologous End Joining Inhibitor SCR-7 to Exacerbate Low-dose Doxorubicin Cytotoxicity in HeLa Cells

“…Additionally, SCR-7 and other small-molecule inhibitors to DSB repair have been reported as viable options in primary and adjuvant chemotherapies for several cancer types. There are reports on cardiac glycosides acting as potential inhibitors of DNA DSB repair by phosphorylating DNA damage checkpoint protein 1 (phospho-MDC1) or E3 ubiqui-tin-protein ligase ring finger protein 8 (RNF8) 20. In a recent paper, Wang et al27 reported that inhibition of CtIP (RBBP8), a DSB repair protein, sensitizes breast carcinoma to PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888) therapy.…”