CGK733 binds to adenine nucleotide translocator 2 and modulates mitochondrial function and protein translation

Chemical genetics is a powerful strategy for dissecting biological mechanisms, and a crucial step in this approach is to identify the molecular target responsible for a compounds activity. CGK733, a compound with anti-proliferative activity, was once reported as an ATM/ATR inhibitor, although this activity has been debated, and its target and mode of action have remained unclear. Here, we show that CGK733 inhibits cell-cycle progression and global protein translation. Affinity purification identified adenine nucleotide translocator 2 (ANT2) as the primary target. CGK733 blocked ATP export from mitochondria and induced proton leak, thereby shifting ATP production from mitochondrial respiration to glycolysis and perturbing the TCA cycle. These mitochondrial alterations were accompanied by inactivation of the mTOR pathway and mild activation of the integrated stress response, resulting in translational inhibition. Collectively, our findings demonstrate that CGK733 acts mainly through ANT2-dependent mitochondrial modulation, revealing a mechanistic link between mitochondrial bioenergetics and translational control.