Competition with glutathione explains the impact of lysosomal pH on the pro-oxidant and anticancer activity of the copper-phenanthroline complex

Several Cu-ligands, including 1,10-phenanthroline (Phen), have been investigated for anticancer purposes based on their capacity to bind excess Cu in cancer tissues and form redox active complexes able to catalyse the formation of reactive oxygen species (ROS), ultimately leading to oxidative stress and cell death. However, the stability and pro-oxidant activity of Cu-based drugs such as Cu-Phen2 is affected in most cell compartments (e.g. cytosol and nucleus) by the presence of compounds such as glutathione (GSH) and metallothioneins, which can reduce and dissociate Cu(II) from the ligand forming poorly redox-active Cu(I)-thiolate clusters. Here, cell culture studies suggested that lysosomal acidification may play a pivotal role in the anticancer activity of Cu-Phen2. In addition, ROS generation catalysed by Cu-Phen2 in the presence of GSH was shown to be remarkably accelerated at the acidic pH typical of lysosomes. The catalytic mechanism was thoroughly investigated by means of density functional theory (DFT) calculations, which disclose key reaction intermediate species, including a ternary Phen-Cu-GSH complex formed upon dissociation of one Phen ligand. Spectroscopic measurements (including low-temperature luminescence, UV-vis absorption and X-ray absorption spectroscopy) corroborated the formation of such a reactive intermediate ternary complex. Furthermore, they revealed that the faster ROS generation observed at lower pH is due to a pH-dependent competition between Phen and GSH for Cu, which results in a higher stability of Cu-Phen2 against dissociation and de-activation by GSH at lower pH. Overall, this study points to lysosomal targeting as an innovative and effective strategy to improve the stability and cytotoxic activity of Cu-based drugs.