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Glutamic Acid-Chelated Cobalt Stabilizes G-Quadruplexes and Selectively Suppresses Hepatocellular Carcinoma Growth
Kuan-Hao Lin1, Yu-Ju Lin1, Yu-Bin Hong1
1Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan.
Objectives:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
Glutamic acid-chelated cobalt (GACC) demonstrates G-quadruplex (G4) activity against hepatocellular carcinoma (HCC) with good safety in zebrafish models. AI-guided optimization yielded effective, less toxic drug combinations for HCC treatment.
Area of Science:
- Oncology
- Medicinal Chemistry
- Genomics
Background:
- Hepatocellular carcinoma (HCC) presents limited therapeutic options with significant toxicity concerns.
- G-quadruplex (G4) structures in oncogene promoters are promising yet challenging drug targets for HCC.
- Developing novel G4-active compounds with improved safety profiles is crucial.
Purpose of the Study:
- To evaluate glutamic acid-chelated cobalt (GACC) as a G4-active scaffold for anti-HCC efficacy and in vivo safety.
- To investigate the potential of AI-guided phenotypic response surface (PRS) for optimizing less toxic drug combinations.
- To elucidate the mechanism of GACC's action on HCC and its G4 targets.
Main Methods:
- Anticancer activity and IC50 values were determined in HCC cell lines and non-transformed hepatocytes.
- In vivo safety and efficacy were assessed using zebrafish embryo toxicity assays, xenograft models, and transgenic zebrafish models.
- Target engagement was evaluated through molecular assays including G4 DNA polymerase stop assays, immunofluorescence, and qPCR.
- AI-guided PRS was employed to optimize combinations of GACC with metformin and regorafenib.
Main Results:
- GACC exhibited significant HCC cell viability reduction (IC50 ~86-115 µM) with low embryotoxicity (IC50 6.87 mM).
- In vivo studies showed GACC reduced tumor burden by ~90% in zebrafish xenografts without observable hepatotoxicity, unlike sorafenib.
- GACC demonstrated G4-specific activity by increasing KRAS promoter polymerase stalling, enhancing nuclear G4 signals, and reducing KRAS transcripts.
- PRS identified an optimized triple combination that significantly reduced HCC viability while maintaining normal cell viability and zebrafish development.
Conclusions:
- GACC serves as a G4-active cobalt-glutamate scaffold demonstrating potent anti-HCC activity and favorable safety in preclinical zebrafish models.
- The integrated zebrafish and PRS workflow facilitates rational design of safer, effective combination therapies for HCC.
- This approach holds promise for developing novel, less toxic systemic options for hepatocellular carcinoma treatment.
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