Potent Biological Activity by a Synthetic Cu(I) Cationophore Redistributing Intracellular Copper Pools

Copper is an essential trace element for life and is present in the active site of various enzymes, whereas free copper is toxic to cells. Copper homeostasis is thus finely regulated, involving Cu(I) transporting membrane proteins Ctr1 and ATP7A/B. Disruption of copper homeostasis has been reported as a potential anticancer strategy. With this objective, we have developed a family of lipophilic compounds featuring two preorganized coordinating (benz)imidazole groups designed biomimetically to bind Cu(I) in a linear geometry and function as ionophores. The transport of Cu(I) cations across membranes by these compounds was first demonstrated in liposomes using an encapsulated Cu(I) sensitive fluorescent probe. Furthermore, six of the ionophores restored the growth of yeast cells lacking Ctr1, indicating their ability to also transport copper into cells. In hepatocarcinoma cells, four of these six ionophores exhibited potent anticancer activity, with IC₅₀ values ranging from 3 to 5 μM. These active ionophores share a relatively narrow lipophilicity range (cLogP = 7-9.2), underscoring the critical role of lipophilicity in their cellular activity. Further investigations of one of the most active compounds, named Cuphoralix, revealed no increase in intracellular Cu levels in hepatocytes but clear indications of metal-induced stress. State-of-the-art synchrotron X-ray fluorescence studies demonstrated that Cuphoralix alters the subcellular copper distribution, redistributing it from the vesicles to the cytosol. This redistribution likely accounts for the potent cytotoxicity of this novel class of Cu(I) ionophores, supporting further studies on their anticancer potential.