Identification of a RAD52 Inhibitor Inducing Synthetic Lethality in BRCA2-Deficient Cancer Cells

Abstract

The breast cancer susceptibility gene 1/2 (BRCA1/2) is frequently mutated in many malignant tumors, such as breast cancer and ovarian cancer. Studies have demonstrated that inhibition of RAD52 gene function in BRCA2-deficient cancer causes synthetic lethality, suggesting a potential application of RAD52 in cancer-targeted therapy. In this study, we have performed a virtual screening by targeting the self-association domain (residues 85-159) of RAD52 with a library of 66,608 compounds and found one compound, C791-0064, that specifically inhibited the proliferation of BRCA2-deficient cancer cells. Our biochemical and cell-based experimental data suggested that C791-0064 specifically bound to RAD52 and disrupted the single-strand annealing activity of RAD52. Taken together, C791-0064 is a promising leading compound worthy of further exploitation in the context of BRCA-deficient targeted cancer therapy.

Keywords: BRCA2 deficiency; Rad52; molecular dynamics; self-association; synthetic lethality.

FIGURE 1

Human RAD52 self-association binding sites. (A) Functional domains of RAD52 protein. (B) Three-dimensional structure of RAD52 protein; the red box is the groove structure defined as the potential RAD52 self-association site. (C) Top 28 small molecules with the highest docking score bound to the self-association site of RAD52. The three colored structures represent three RAD52 monomers.

FIGURE 2

Backbone RMSD and RMSF values of RAD52–ligand complexes. (A) RMSD of the five RAD52–ligand complexes during a 27.5 ns simulation. (B) RMSFs of the amino acids within the RAD52 self-association domain during three equal portions of a 27.5 ns molecular dynamics simulation of RAD52–ligand complexes. (C–E) presented the enlarged fluctuation of three segments (Cys25-Gln40, Phe79-Gln91, and Cys108-Tyr126) containing the key amino acids in the RAD52 self-association binding sites involved in RAD52–ligand interaction.

FIGURE 3

Effect of the putative RAD52 inhibitors on the survival of BRCA2 wild-type and deficient cells. Capan-1(BRCA2-deficient) and BxPC3 (BRCA2 wild type) cells were treated with different concentrations of the five compounds for 72 h, (A) C791-0064, (B) E859-1790, (C) G672-0331, (D) F345-0611, and (E) G889-2311 (F) (n = 3). C791-0064 treatment of BRCA2-knockdown BxPC3 cells (sh-BRCA2-CDS and sh-BRCA2-UTR) and wild-type BxPC3 cells for 72 h (n = 3). CCK8 assay was performed to evaluate the cell survival. (G,H) Clonogenic assay performed with BRCA2 wild-type or BRCA2-knockdown cells and its quantification (n = 3). Error bars represent SDs. (I) siRNA knockdown of RAD52 in sh-BRCA2-CDS cells and treatment with C791-0064.

FIGURE 4

C791-0064 inhibited RAD52 self-association. (A) Purification of human RAD52 protein analyzed by SDS-PAGE. Lane 2, total cell lysate; lane 3, supernatant; lane 4, Ni sepharose flow-through; lane 5, Ni sepharose eluate; lane 6, heparin flow-through; lane 7, heparin wash; and lane 8, heparin eluate. (B) Western blot analysis of RAD52 expressing plasmid-transformed E. coli cell lysate and purified RAD52 protein with anti-Histag antibody. (C) Schematic illustration of the electrophoretic mobility shift assay (EMSA). (D) Western blot of the EMSA result detected with anti-Histag antibody. (E) Quantification of the relative amount of polymer protein; the values were normalized by setting the value of no C791-0064, glutaraldehyde, and ssDNA reaction to be 100% (n = 3, error bars stands for SD). (F,G) Single-strand annealing efficiency of RAD52 in the presence of C791-0064. ds, double strand; ss, single strand. (H,I) Microscale thermophoresis (MST) analysis of C791-0064 (H) and G672-0031 (I) binding to Rad52 protein.

FIGURE 5

C791-0064 caused apoptosis of BRCA2-deficient cells. (A) Flow cytometry detection of apoptosis after 40 μM of C791-0064 treatment of shBRCA2-CDS BxPC3 cells for 48 h. (B) Quantification of apoptotic cell percentage (n = 3). (C) Western blot analysis of shBRCA2-CDS BxPC3 cells treated with different concentrations of C791-0064 for 48 h. (D,E) Immunofluorescence staining of γ-H2AX in shBRCA2-CDS BxPC3 and BxPC3 cells treated with 40 μM of C791-0064 for 24 h and its quantification for cells positive forγ-H2AX foci (n = 3). Error bars represent for SD.