A Small‐Molecule Protein–Protein Interaction Inhibitor of PARP1 That Targets Its BRCT Domain

Abstract

Poly(ADP-ribose)polymerase-1 (PARP1) is a BRCT-containing enzyme (BRCT = BRCA1 C-terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small-molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein-protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high-throughput microplate-based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)-gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein-protein interactions. As the first known cell-permeable small-molecule PPI inhibitor of PAPR1, we further established that (-)-gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage.

Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci

“…6, C and D). The requirement for the PARP-1 BRCT domain was further confirmed using the competitive BRCT domain inhibitor (±)-gossypol (Na et al, 2015), which inhibited PARP-1-dependent Sox2 binding to nucleosomes (Fig. 6, E and F).…”

“…The competitive BRCT domain inhibitor, (±)-gossypol (Na et al, 2015), was purchased from Sigma (G8761).…”

“…After 3 washes each with 1 ml of binding buffer, the beads were boiled in 2x SDS loading dye and the proteins were analyzed by Western blotting. In some cases, the Sox2 nucleosome binding assays were performed in the presence of 60 M of the BRCT inhibitor (±)-gossypol from Sigma-Aldrich (Na et al, 2015). …”

DNA repair targeted therapy: The past or future of cancer treatment?

“…NMS-P118 has an excellent absorption, distribution, metabolism, and excretion profile and high in vivo efficacy as a single agent in triple negative breast cancer with a BRCA1 mutation and also in combination with temozolomide in BRCA2 deficient pancreatic cancer xenograft models. Interestingly, the discovery of a naturally occurring (−)-gossypol as a protein-protein interaction inhibitor of the PARP1 BRCA1 C Terminus (BRCT) domain affords new opportunities for the development of highly potent and specific PARP1 inhibitors available (Na et al, 2015). …”

MSN‐on‐a‐Chip: Cell‐Based Screenings Made Possible on a Small‐Molecule Microarray of Native Natural Products

“…Finally, for HT screening of fixed cells, apoptotic HeLa cells resulting from release of STS from STS@MSN‐on‐a‐chip were successfully imaged in an immunofluorescence (IF) assay by using anti‐cleaved PARP‐1 antibody (Figure C, arrowed) . A similar live‐cell‐based apoptosis assay using commercially available cell‐permeable caspase‐3/7 activity probe was also established (Figure D) .…”