Targeting the EGFR G-quadruplex DNA in liver cancer: Discovery of alkynyl-modified phenanthroimidazole derivatives as potent stabilizers.

Targeting G-quadruplex (G4) DNA in oncogene promoters represents an emerging anticancer strategy. The epidermal growth factor receptor (EGFR) gene promoter contains a G4-forming sequence that epigenetically regulates EGFR transcription, making EGFR G4 DNA a promising therapeutic target. Here, we investigate the interaction of alkynyl-modified phenanthroimidazole derivatives (1-5) with this macromolecular structure. Through high-throughput molecular docking against multiple G4 targets (Bcl-2, c-Kit, EGFR, K-Ras), compound 5 (para-phenylethynyl-substituted) was identified as the most potent EGFR G4 DNA binder, with favorable binding energy and stable binding mode validated by molecular dynamics simulations. Biophysical analyses (UV-Vis/fluorescence titration, circular dichroism, isothermal titration calorimetry, PCR-stop assays) revealed that compound 5 binds EGFR G4 DNA with high affinity (Kd = 1.15 × 10-6 M), selectively stabilizes its parallel conformation, and inhibits DNA polymerase elongation. This stabilization leads to transcriptional repression of EGFR, as evidenced by dose-dependent downregulation of EGFR in HepG2 cells. Mechanistically, compound 5 induces G4-mediated DNA replication stress and damage, elevates ROS, activates p53 signaling, and modulates the PI3K/AKT/mTOR pathway, ultimately triggering ferroptosis. Notably, compound 5 exhibits superior anti-proliferative activity against HepG2 cells compared to doxorubicin (DOX) and Gefitinib, with markedly reduced cytotoxicity toward normal cells. In vivo efficacy was confirmed in a zebrafish xenograft model, where compound 5 significantly suppressed tumor proliferation. These findings establish compound 5 as a potent EGFR G4 DNA stabilizer that exerts multi-level anti-tumor effects through targeted modulation of this macromolecular structure, offering a promising lead for EGFR G4-directed liver cancer therapy.