Glutamic Acid-Chelated Cobalt Stabilizes G-Quadruplexes and Selectively Suppresses Hepatocellular Carcinoma Growth.

Hepatocellular carcinoma (HCC) has limited systemic options with substantial toxicity. G-quadruplex (G4) structures in oncogene promoters are attractive but challenging drug targets. This study aimed to determine whether glutamic acid-chelated cobalt (GACC) is a G4-active scaffold with anti-HCC efficacy and favorable in vivo safety, and whether an AI-guided phenotypic response surface (PRS) can optimize less toxic combinations. Anticancer activity was tested in HCC cell lines (PLC/PRF/5, Hep3B, HepG2) and non-transformed THLE-2 hepatocytes (CCK-8, IC50). In vivo safety/efficacy were assessed in zebrafish embryo toxicity assays, a Hep3B xenograft model, and a tert-overexpressing transgenic zebrafish model, with hepatotoxicity monitored in a liver-fluorescent reporter line. Target engagement was examined by docking, native PAGE, a KRAS promoter G4 DNA polymerase stop assay, BG4 immunofluorescence, and KRAS qPCR. PRS was used to optimize GACC-metformin-regorafenib combinations. GACC reduced HCC viability (IC50 ~86-115 µM) and showed low embryotoxicity (IC50 6.87 mM). In zebrafish xenografts, GACC (50 µM) reduced Hep3B tumor fluorescence by ~90% without detectable hepatotoxicity, whereas sorafenib decreased liver size/fluorescence. In tert-overexpressing zebrafish, GACC suppressed proliferation and Wnt/β-catenin-associated transcripts and reduced mitotic figures and nuclear atypia. Mechanistically, GACC increased KRAS promoter polymerase stalling, enhanced nuclear G4 signal, and reduced KRAS transcripts. PRS identified an off-grid triple combination that reduced PLC/PRF/5 viability to 19% while maintaining THLE-2 viability at 52% and preserving zebrafish development. GACC is a G4-active cobalt-glutamate scaffold with anti-HCC activity and favorable zebrafish safety, and a zebrafish-plus-PRS workflow enables rational, less toxic combination design.