Abstract

Summary G-quadruplex (G4) DNA structures, present in both nuclear and mitochondrial genomes, represent emerging therapeutic targets. Here, we developed and exploited a mitochondrial high-content profiling platform (Mito-HiCP), integrating automated microscopy with quantitative image analysis, to compare the effects of G4 ligands with cisplatin in cancer and normal cells. G4 ligands induce mitochondrial depolarization without reactive oxygen species (ROS) bursts, in contrast to the ROS-associated toxicity of cisplatin. Notably, the G4 ligand 360 A suppresses both mitochondrial and cytosolic ROS levels while retaining tumor selectivity, whereas its non-G4-binding isomer (EDL21) shows no effect, supporting a G4-dependent mechanism. Mechanistically, 360 A reduces mitochondrial DNA copy number, transcription, and electron transport chain protein levels. Using Mito-HiCP, we identify that G4 targeting uncouples mitochondrial membrane potential from ROS production. This reveals a mode of redox regulation that underlies selective anticancer activity and provides a framework to dissect mitochondrial responses and redox regulation across diverse therapeutic perturbations.