Copper-Catalyzed Glutathione Oxidation is Accelerated by the Anticancer Thiosemicarbazone Dp44mT and Further Boosted at Lower pH

Abstract

Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. Cu^II ions are very efficient oxidants of thiols, but with an excess of GSH, Cu^I_n(GS)_m clusters are formed, in which Cu^I is very slowly reoxidized by O₂ at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by Cu^II was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that Cu^II-Dp44mT catalyzes GSH oxidation faster than Cu^II alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by Cu^II-Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of Cu^II-Dp44mT on the hydrazinic nitrogen, which favors the rate-limiting reduction step without subsequent dissociation of the Cu^I intermediate. 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 Cu^II-Dp44mT.