Copper-catalysed glutathione oxidation is accelerated by the anticancer thiosemicarbazone Dp44mT and further boosted at lower pH

Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining the redox state of many biomolecules, in detoxification and in antioxidant defence. The thiols of two GSH molecules can be oxidized to the disulphide GSSG. The cytosolic GSH/GSSG ratio is very high (> 100), and its decrease can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, the formed CuIn(GS)m clusters are only slowly re-oxidized by O2 at pH 7.4, and even more slowly at lower pH. Here, the oxidation of GSH by CuII was investigated in the presence of the anticancer thiosemicarbazone Dp44mT. The results showed that CuII-Dp44mT oxidizes GSH faster than CuII alone at pH 7.4, and hence accelerates the production of the very reactive hydroxyl radicals. Interestingly, the GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes, where CuII-Dp44mT was previously shown to accumulate and induce lysosomal membrane permeabilization. To decipher this unusually faster thiol oxidation a lower pH, Density Functional Theory (DFT) calculations and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favours the rate-limiting reduction step without subsequent dissociation of the CuI-complex. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of Dp44mT.