Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer

Abstract

PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

Figure 1

PARP-1 domain-domain contacts are necessary for DNA-dependent activity, but not high-affinity DNA binding. (A) Structural representation of activated PARP-1 highlighting the interdomain interfaces. (B) Schematic of full-length PARP-1. (C) DNA-dependent PARP-1 catalytic activity. (D) DNA binding affinities of full-length PARP-1 mutants.

Figure 2

Cellular localization and activity of PARP-1 mutants. (A) Immunofluorescent detection of PARP localization and PAR formation in response to H₂O₂ treatment. (B) Localization of GFP-PARP-1 to sites of laser-induced damage in HeLa cells. (C) NAD⁺ dependent release of PARP-1 mutants from DNA in the presence of NAD⁺ (5mM).

Figure 3

Functional consequences of inactivating PARP-1 interdomain communication. (A) Pancreatic cancer cells stably transfected with inactive PARP-1 mutants are sensitive to platinum-based agents compared to WT. (B) Western blot analysis of cells in panel (A) using anti-PARP (Trevigen) and anti-V5 (Invitrogen) antibodies. (C) Ligand-induced and control AR reporter activity were measured in transfected PARP-1^−/− MEFs by relative luciferase activity as result of 3 independent replicates ±SE. Results are normalized to the control set to “1”. PARP inhibitor ABT-888 used at 2.5 µM.

Figure 4

A high-throughput (HT) fluorescent polarization (FP) assay to detect allosteric PARP-1 inhibitors. (A) Schematic of PARP-1 proteins used in this assay (Zn1-Zn3 and WGR-CAT), mapped with mutation sites. (B) Dose response of WGR-CAT binding to fluorescein-labeled DNA alone or to fluorescein-labeled DNA saturated with Zn1-Zn3. (C) WGR-CAT binding in the FP assay in the presence of Zn1-Zn3 (top) or WGR-CAT (bottom) mutations. (D) Catalytic activity of Zn1-Zn3 and WGR-CAT combinations using the colorimetric assay with mutations made in Zn1-Zn3 (top) or WGR-CAT (bottom).