A mitochondria‐targeting and G‐quadruplex structure‐binding ligand inducing calcium overload and ferroptosis in human cancer cells

Background and Purpose Regulation of mitochondrial calcium overload and ferroptosis with mitochondria‐targeting ligands is an attractive anticancer strategy but it remains a challenge. The aim of the present study was to demonstrate that a mitochondria‐targeting and mtDNA G‐quadruplex‐binding ligand, BYB, induced mitochondrial calcium overload and ferroptosis in HeLa cells and showed potent in vitro and in vivo anticancer activity. Experimental Approach Cellular functions and molecular mechanism were studied using cell viability assay, live‐cell imaging, western blotting, immunofluorescence, cell uptake, cell cycle arrest and apoptosis analysis, mitochondrial metabolism analysis, Comet assay, and wound‐healing analysis. Pharmacokinetic studies were conducted in rat. In vivo antitumor activity was studied in a cervical cancer HeLa cell xenograft mouse model. Key Results Cellular results showed that BYB induced mitochondrial calcium overload, attributed to ligand‐induced mitochondrial dysfunction via the mechanism of inhibiting mitochondrial DNA replication and transcription. The expression of respiratory chain complexes was markedly downregulated in BYB‐treated HeLa cells. The respiratory chain function was also dysregulated. Mitophagy and mitochondrial calcium overload were induced in BYB‐treated HeLa cells. Mitochondrial calcium overload markedly induced mtROS production. The induced mtDNA stress activated cGAS‐STING pathway, leading to autophagy‐dependent ferroptosis. The antitumour efficacy of BYB, evaluated in a HeLa tumour xenograft mouse model, achieved over 60% tumour weight reduction. Conclusion and Implications BYB, via targeting mitochondria and mtDNA G‐quadruplexes, induced mitochondrial calcium overload and ferroptosis, exhibited high in vivo antitumour efficacy and low toxicity. It shows high potential to be a mitochondria‐targeting lead compound for chemical biology and drug discovery.