Transfer of Copper between Bis(thiosemicarbazone) Ligands and Intracellular Copper-Binding Proteins. Insights into Mechanisms of Copper Uptake and Hypoxia Selectivity

Bis(thiosemicarbazonato) complexes CuII(Btsc) have attracted interest as promising metallodrugs and, in particular, as copper radiopharmaceuticals. Prototypes Cu(Atsm) and Cu(Gtsm) are membrane-permeable, but their metabolisms in cells are distinctly different: copper that is delivered by Cu(Gtsm) is trapped nonselectively in all cells, whereas copper that is delivered by Cu(Atsm) is retained selectively in hypoxic cells but is “washed out” readily in normal cells. We have studied copper-transfer reactions of these two complexes under various conditions, aiming to model their cellular chemistry. In Me2SO, both complexes exhibited reversible one-electron-reduction processes with Cu(Atsm) being more difficult to reduce than Cu(Gtsm) (E1/2′ = –0.60 and –0.44 V, respectively, vs AgCl/Ag). Upon introduction of an aqueous buffer into Me2SO, the electrochemical reduction remained chemically reversible for Cu(Atsm) but became irreversible for Cu(Gtsm). However, the estimated difference in their reduction potentials did not change. Chromophoric ligand anions bicinchonate (Bca) and bathocuproine disulfonate (Bcs) were used as CuI indicators to trace the destinations of copper in the reactions and to mimic cellular CuI-binding components (“sinks”). While both BtscH2 ligands have high affinities for CuI (KD in the picomolar range), they cannot compete with CuI sinks such as the copper-binding proteins Atx1 and Ctr1c (or a mimic such as Bcs). In the presence of these proteins, reduction of CuII(Btsc) leads to irreversible transfer of copper to the protein ligands. Endogenous reductants ascorbate and glutathione can reduce CuII(Gtsm) in the presence of such protein ligands but cannot reduce CuII(Atsm). These properties establish a strong correlation between the contrasting cellular retention properties of these complexes and their different reduction potentials. The endogenous reductants in normal cells appear to be able to reduce CuII(Gtsm) but not CuII(Atsm), allowing the latter to be washed out. The more reducing environment of hypoxic cells leads to reduction of CuII(Atsm) and retention of its copper.