Modulation of mitochondrial energy balance in health and disease

Mitochondria are highly dynamic organelles and their function is crucial for the maintenance of cellular homeostasis. Alterations in mitochondrial homeostasis has emerged as a hallmark of several diseases, like cancer and genetic disorders. Mitochondrial fitness and its energetic state can impact cell viability and proliferation, and its modulation via pharmacological tools can be used to treat several mitochondria-related diseases. The potassium channel Kv1.3 is a voltage-gated potassium channel emerged as a novel oncological target. It is overexpressed in several tissues compared to normal ones, enhance cell proliferation, cell migration and metastasis. Direct inhibition of Kv1.3 using its known membrane-permeant inhibitor PAP-1 alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death. We generated two classes of PAP-1 derivatives in order to improve its effect. The first class comprises the mitochondria-targeted compounds PAPTP and PCARBTP. These molecules have a positively charged triphenyl-phosphonium group linked to the PAP-1 structure, so they can reach mitochondria in a more efficient way and can be used at lower concentrations compared to their precursor. We have demonstrated that these drugs selectively kill cancer cells in vitro, ex vivo and in vivo. The mechanism of selectivity involves both the overexpression of Kv1.3 and the high basal ROS level, present mainly in cancer cells. On the other hand, low, sub-lethal concentrations of PAPTP and PCARBTP cause a mild increase in mitochondrial ROS production, favoring cell survival and proliferation through the modulation of mitochondrial homeostasis. The second class of PAP-1 derivatives comprises the more soluble compounds PEGME and PTGME. Recapitulating the molecular mechanism of the mitochondria-targeted compounds, these more soluble derivatives act in vitro, ex vivo and in vivo. Moreover, PEGME and PTGME inhibited respiratory chain complex I (CI). To clarify this aspect, we revealed for the first time that mitoKv1.3 likely localizes in proximity of CI in the IMM, at the level of the supercomplexes. To detect what else the new mitochondria-targeted derivatives of PAP-1 make inside cancer cells at sub-lethal concentrations, we investigated the effects of mitoKv1.3 inhibition on Wnt signaling. We demonstrated that the reduction of mitochondrial ATP due to the use of mitochondria-affecting drugs or to genetic dysfunction due to complex III (CIII) deficiency, decreases calcium uptake into the endoplasmic reticulum (ER), leading to ER stress and to impaired Wnt signaling. Importantly, both the recovery of ATP level or the inhibition of ER stress restored Wnt activity downregulated by mitochondria-affected compounds. This research revealed for the first time an unexpected mechanism related to the control of Wnt signaling by mitochondrial ATP, opening a new possibility to use compounds affecting mitochondrial homeostasis to fight tumors, or to use mitochondrial ATP-increasing drugs to ameliorate patients’ symptoms. In the context of genetic dysfunction, we focused also on the use of the bacterial redox cycler to ameliorate CIII related disease. This redox cycler can mimic CIII activity, by accepting electrons from ubiquinol and reducing cytochrome c in vitro. We observed that sub-lethal doses of the drug recovers mitochondrial function both by increasing mitochondrial ATP production and by promoting mitohormesis, in vitro in CIII deficient cells from patients with CIII disease. Importantly, this redox cycler can increase also in vivo ATP production, and rescue respiration rate and locomotor ability in flies and zebrafish models of CIII deficiency. These results strongly suggest that the application of this drug in the sublethal concentration range might be a promising therapeutic tool against complex III diseases.