Abstract
Mitochondria are cellular powerhouses and are crucial for cell function. However, they are vulnerable to internal and external perturbagens that may impair mitochondrial function and eventually lead to cell death. In particular, small molecules may impact mitochondrial function, and therefore, their influence on mitochondrial homeostasis is at best assessed early on in the characterization of biologically active small molecules and drug discovery. We demonstrate that unbiased morphological profiling by means of the cell painting assay (CPA) can detect mitochondrial stress coupled with the induction of an integrated stress response. This activity is common for compounds addressing different targets, is not shared by direct inhibitors of the electron transport chain, and enables prediction of mitochondrial stress induction for small molecules that are profiled using CPA.