Imbalances in cellular redox state are frequently observed in cancer cells, and contribute significantly to cancer progression and apoptotic resistance. Hydrogen peroxide (H2
) is one reactive oxygen species (ROS) that is produced in excess within cancer cells. In this study, we investigated the mitochondrial glycerol-3-phosphate-dependent (GPD2) ROS production in PC-3 cells and demonstrated the importance of excessive H2
production on their survival. By exploiting the abnormal H2
production of PC-3 cells, we initiated a high-throughput screening of the Canadian Compound Collection
, composed of 29,586 small molecules, targeting the glycerophosphate-dependent H2
formation in PC-3 cells. Eighteen compounds were identified to have significant inhibitory activity. These compounds have not been previously characterized as inhibitors of the enzyme. Six of these compounds were further analyzed in PC-3 cells and dose response studies displayed an inhibitory and anti-oxidative potency that ranged from 1 µM to 30 µM. The results presented here demonstrate that inhibitors of mitochondrial GPD2 activity elicit anti-proliferative effects on cancer cells.