LAUSR

The real‐time tracking of localization and dynamics of small molecules in organelles helps to understand their function and identification of their potential targets at subcellular resolution. To identify the mitochondrion‐targeting effects of small molecules (NA‐17 and NA‐2a) in cancer cells, we used mass spectrometry to study their distribution and accumulation in mitochondria and in the surrounding cytoplasm thus enabling tracing of action processes of therapeutic compounds. Colocalization analysis with the aid of imaging agents suggests that both NA‐17 and NA‐2a display mitochondrion‐targeting effects. However, MS analysis reveals that only NA‐2a displays both a mitochondrion‐targeting effect and an accumulation effect, whereas NA‐17 only distributes in the surrounding cytoplasm. A combination of mitochondrion imaging, immunoblotting, and MS analysis in mitochondria indicated that NA‐17 neither has the ability to enter mitochondria directly nor displays any mitochondrion‐targeting effect. Further studies revealed that NA‐17 could not enter into mitochondria even when the mitochondrial permeability in cells changed after NA‐17 treatment, as was evident from reactive oxygen species (ROS) generation and cytochrome c release. In the process of cellular metabolism, NA‐17 itself is firmly restricted to the cytoplasm during the metabolic process, but its metabolites containing fluorophores could accumulate in mitochondria for cell imaging. Our studies have furnished new insights into the drug metabolism processes.