Copper transport across cell membranes by calix[4]arene-based cationophores leading to potent biological activity

Lipid bilayers are impermeable to ions, including copper cations. Copper is an essential trace element for life, present in the active site of various enzymes, whereas free copper is detrimental inside cells. Copper homeostasis is thus finely controlled, involving Cu(I) transporting membrane proteins Ctr1 and ATP7A/B. Disruption of copper homeostasis has been reported as a potential anti-cancer strategy. With this objective, we have developed a series of lipophilic compounds with two copper coordinating (benz)imidazole groups that are able to function as ionophores, transporting copper cations across membranes. This was firstly demonstrated in liposomes with a Cu(I) sensitive fluorescent probe encapsulated. Secondly, five of these compounds were shown to restore the growth of yeast cells that had Ctr1 deleted, suggesting that these ionophores were able to transport copper into yeast cells. Thirdly, cytotoxicity studies in hepatocarcinoma cells highlighted the crucial role of the lipophilicity of Cu ionophores for their activity in cells. The impact of one of the most active compounds, named Cuphoralix, was further studied, showing no increase of intracellular Cu levels of the hepatocytes, but clear indications of metallic stress. Synchrotron X-ray fluorescence studies were then employed to study the effect of Cuphoralix on the subcellular copper distribution, revealing a redistribution of copper from vesicles to the cytosol. This explains the potent cytotoxicity of this novel class of copper ionophores, warranting further studies of their anti-cancer effects.