Reactive Cysteines in Proteins Are the Dominant Reductants for Platinum(IV) Prodrug Activation in Live Cells

The intracellular reduction of Pt(IV) prodrugs is crucial for their anticancer efficacy. However, the major components responsible for the reduction of Pt(IV) complexes within live cells remain elusive. Herein, we developed an aminoluciferin‐functionalized Pt(IV) complex, Pt‐Luc, that can be used as a bioluminescent reporter for real‐time monitoring of Pt(IV) reduction in live cancer cells by capturing immediate bioluminescent signals from the released aminoluciferin. Utilizing this powerful reporter, we found that the reduction of Pt(IV) prodrugs in live cancer cells significantly slows down when cysteine levels are reduced, while the levels of glutathione do not impact the reduction rate. Further investigation reveals that reactive cysteines in proteins, rather than small‐molecule thiols, play a primary role in reducing the Pt(IV) complex. In vivo studies reveal a substantial 63% decrease in bioluminescence from Pt‐Luc in thiol‐blocking tumors in mice, reinforcing the pivotal role of reactive cysteines in Pt(IV) reduction. This study provides valuable insights into the activation mechanisms of Pt(IV) prodrugs in live cells and in vivo, enhancing our understanding of prodrug activation beyond buffer systems or fixed cells.