Scutellarin as a specific GEN1 inhibitor synergizes with olaparib to induce synthetic lethality in human tumor cells.

The synthetic lethal interaction between poly(ADP-ribose) polymerase (PARP) inhibition and homologous recombination (HR)-deficiency, particularly in BRCA1/2-mutant tumors, has emerged as an effective strategy for cancer therapy. GEN1, a structure-selective endonuclease that resolves Holliday junctions (HJs) during HR, represents an attractive antitumor target. However, effective small-molecule GEN1 inhibitors have yet to be reported. Here, we established a fluorescence resonance energy transfer (FRET)-based high-throughput screening platform and identified the natural flavonoid scutellarin (SCU) as a specific inhibitor of human GEN1. SCU selectively inhibited GEN1 endonuclease activity against both 5'-flap and HJ DNA substrates, with no effect on other tested structure-selective endonucleases, such as SLX1-SLX4 and MUS81-EME1. Mechanistic studies showed that SCU does not disrupt GEN1-DNA binding but instead binds to the catalytic center of GEN1, thereby inhibiting its endonuclease activity. In HCT116 cells, SCU acted in synergy with the PARP inhibitor olaparib to markedly impair DNA double-strand break (DSB) repair, increase γH2AX accumulation, induce G2/M cell cycle arrest, and suppress cell migration. Notably, the SCU/olaparib combination produced robust synthetic lethality in both HCT116 and HeLa cells, resulting in substantial inhibition of cell viability and colony formation. Our study identifies SCU as a novel selective GEN1 inhibitor, and suggests a new therapeutic strategy that combines GEN1 inhibitors with PARP inhibitors for cancer treatment.