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 O 2 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 ratelimiting 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.

Dual Role of Glutathione as a Reducing Agent and Cu-Ligand Governs the ROS Production by Anticancer Cu-Thiosemicarbazone Complexes

“…The Cu I center coordinated to two Cys – adopts a linear geometry and the adduct is only slightly, by 0.4 kcal/mol, more stable than the preceding minimum. As seen in the case of Cu II -Dp44mT, from the geometrical arrangement of the deprotonated cysteines around the reduced copper, it is possible to draw the conclusion that one electron flows from the unbound cysteine to copper through a bridge formed with one of the cysteines coordinated to the metal, forming a disulfide radical anion (the calculated spin density of 4B-3AP product is depicted in Figure ).…”

“…In order to find a possible explanation of this difference in behavior, we focused our attention on the difference in hard/soft character of the two ligands and their copper complexes and invoked the HSAB principle. , The binding of water to the metal center in the Cu II -3AP complex and, on the contrary, its detachment and substitution with a Cys – unit in the Cu II -Dp44mT are an indication of the softer character of the copper center and, in turn, of the ligand, in the latter case. Also the difficulty of the Cu center in the Cu-Dp44mT complex to break the bond with the S donor atom of the ligand clearly indicates a softer character of the system . As a consequence, the reduction of the Cu II -3AP complex is accompanied by the Cu I transfer from the harder ligand to the softer cysteines.…”

“…In the presence of O 2 , Cu II -3AP undergoes reduction by GSH and partial dissociation, whereas Cu II -Dp44mT forms a kinetically stable yet redox-active GS – -Cu II -Dp44mT complex. − However, a direct comparison of the ROS production by these two Cu-TSC complexes has never been reported. Since we recently showed that the reduction of Cu II is the rate-limiting step of the ROS production by Cu-TSC complexes, Cu II -3AP, in spite of its lower cytotoxicity, is predicted to produce ROS faster than Cu II -Dp44mT. Here, we assessed the ROS generation and GSH oxidation by these two Cu II -TSC complexes showing, instead, that Cu II -Dp44mT produces ROS faster than Cu II -3AP does.…”

“…Notably, in anaerobic conditions, physiologically relevant concentrations of GSH reduced Cu II -3AP much faster than Cu II -Dp44mT. In the presence of O 2 , Cu II -3AP undergoes reduction by GSH and partial dissociation, whereas Cu II -Dp44mT forms a kinetically stable yet redox-active GS – -Cu II -Dp44mT complex. − However, a direct comparison of the ROS production by these two Cu-TSC complexes has never been reported. Since we recently showed that the reduction of Cu II is the rate-limiting step of the ROS production by Cu-TSC complexes, Cu II -3AP, in spite of its lower cytotoxicity, is predicted to produce ROS faster than Cu II -Dp44mT.…”

A vinyl-decorated covalent organic framework for ferroptotic cancer therapy via visible-light-triggered cysteine depletion

“…9,10 However, GSH depletion-promoted antitumor therapies are hindered by the high GSH concentrations (5-10 mM) in tumor cells, the sustained reduction of oxidized glutathione (GSSG), and the de novo synthesis of GSH, which may lead to therapeutic failure. 11,12 More seriously, in tumor cells, there are alternative antioxidant mechanisms that support cell survival under GSH depletion, such as thioredoxin reductases, 13 deubiquitinases, 14 and nuclear factor erythroid 2-related factor 2, 15,16 making GSH depletion de facto insufficient to induce cell death in most tumor cell lines and solid tumors. 14 Cysteine-a sulfhydryl-containing amino acid-is not only the rate-limiting precursor for the synthesis of GSH but also an important sulfur source for the synthesis of coenzyme A 17 and is essential for maintaining redox homeostasis and promoting cell proliferation.…”