3 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
hBfl-1/hNOXA Interaction Studies Provide New Insights on the Role of Bfl‑1 in Cancer Cell Resistance and for the Design of Novel Anticancer Agents
Upregulation
of antiapoptotic Bcl-2 proteins in certain tumors confers cancer cell
resistance to chemotherapy or radiations. Members of the antiapoptotic
Bcl-2 proteins, including Bcl-2, Mcl-1, Bcl-xL, Bcl-w, and Bfl-1,
inhibit apoptosis by selectively binding to conserved α-helical
regions, named BH3 domains, of pro-apoptotic proteins such as Bim,
tBid, Bad, or NOXA. Five antiapoptotic proteins have been identified
that interact with various selectivity with BH3 containing pro-apoptotic
counterparts. Cancer cells present various and variable levels of
these proteins, making the design of effective apoptosis based therapeutics
challenging. Recently, BH3 profiling was introduced as a method to
classify cancer cells based on their ability to resist apoptosis following
exposure to selected BH3 peptides. However, these studies were based
on binding affinities measured with model BH3 peptides and Bcl-2-proteins
taken from mouse sequences. While the majority of these interactions
are conserved between mice and humans, we found surprisingly that
human NOXA binds to human Bfl-1 potently and covalently <i>via</i> conserved Cys residues, with over 2 orders of magnitude increased
affinity over hMcl-1. Our data suggest that some assumptions of the
original BH3 profiling need to be revisited and that perhaps further
targeting efforts should be redirected toward Bfl-1, for which no
suitable specific inhibitors or pharmacological tools have been reported.
In this regard, we also describe the initial design and characterizations
of novel covalent BH3-based agents that potently target Bfl-1. These
molecules could provide a novel platform on which to design effective
Bfl-1 targeting therapeutics