Unveiling the biological effects of DNA G-quadruplex ligands through multi-omics data integration.

G-quadruplexes (G4s) are non-canonical DNA structures that have proved to play a pivotal role in various biological processes, including telomere maintenance and gene expression regulation. Owing to their prevalence in tumor cells, G4s have emerged as promising targets for cancer therapy, with a substantial body of research demonstrating the potential of G4 ligands as anti-cancer tools. Nonetheless, a comprehensive multi-omics study to fully elucidate the mode of action of G-quadruplex ligands is still lacking. Such an investigation would be crucial for advancing the development of potent G4-based therapies against cancer. Herein, we employed a multi-omics approach, integrating transcriptomics, proteomics, and metabolomics, to identify key signaling pathways that mediate the anti-cancer effects of well-characterized G4-binding agents (berberine, pyridostatin and RHPS4) on human cervical adenocarcinoma (HeLa) cells. Particularly, we analyzed gene expression changes using RNA sequencing, quantified proteins by liquid-chromatography tandem mass spectrometry and examined metabolite levels via nuclear magnetic resonance. Our results revealed that, under the investigated experimental conditions, berberine treatment had only negligible cellular effects. In contrast, pyridostatin induced significant changes at the transcriptomic, proteomic, and metabolomic levels, decreasing the abundance of enzymes involved in cellular energy production, reducing the availability of precursors for lipid and nucleotide biosynthesis, and depleting essential cofactors and enzymes required for redox balance. Notably, RHPS4 could selectively disrupt mitochondrial activity, possibly through the specific stabilization of mitochondrial G-quadruplex structures. Overall, our findings provide a valuable multi-omics perspective on the cellular changes driven by G-quadruplex binders, that may accelerate the development of effective anti-cancer G4-targeted therapies.