Abstract

Elucidating the subcellular kinetics and target interaction mechanisms of small-molecule drugs is a key obstacle in pharmaceutical research. The therapeutic effects of bioactive small molecules from traditional Chinese medicine (TCM) often involve coordinated regulation of multiple subcellular sites. However, due to the limited spatiotemporal resolution ability of traditional omics methods, it is difficult to precisely analyze their dynamic action process. We developed an Imaging-Guided Drug Spatiotemporal Profiling (IGDSP) strategy. This approach combines technologies like super-resolution live cell imaging, subcellular structure separation, in situ drug-protein complex enrichment, and mass spectrometry identification. It aims to precisely analyze the distribution, dynamic migration trajectories, and key targets of drugs within living cells. Based on this strategy, we used Berberine (BBR), which has autofluorescence properties, as a model and successfully elucidated the time-resolved, three-stage trans-organelle trafficking pathway from mitochondria to the cytoplasm and finally to the nucleolus in HepG2 cells. We also elucidated the molecular mechanism underlying its antitumor activity, which follows the ordered pathway “mitochondrial stress-nuclear migration-autophagy activation.” BBR exerts antitumor effects via the “mitochondrion-nucleus axis”: mitochondrial stress initiates nuclear translocation, followed by nucleolar accumulation, engagement of NPM1, and concomitant activation of autophagy. The IGDSP strategy enhances the target specificity and spatiotemporal resolution of traditional methods, providing an effective approach for studying the multi-target and dynamic pharmacodynamic mechanisms of traditional Chinese medicine.