Mitochondria-targeted 1,4-naphthoquinone (SkQN) is a powerful prooxidant and cytotoxic agent.

An increase in the production of reactive oxygen species (ROS) in mitochondria due to targeted delivery of redox active compounds may be useful in studies of modulation of cell functions by mitochondrial ROS. Recently, the mitochondria-targeted derivative of menadione (MitoK3) was synthesized. However, MitoK3 did not induce mitochondrial ROS production and lipid peroxidation while exerting significant cytotoxic action. Here we synthesized 1,4-naphthoquinone conjugated with alkyltriphenylphosphonium (SkQN) as a prototype of mitochondria-targeted prooxidant, and its redox properties, interactions with isolated mitochondria, yeast cells and various human cell lines were investigated. According to electrochemical measurements, SkQN was more active redox agent and, due to the absence of methyl group in the naphthoquinone ring, more reactive as electrophile than MitoK3. SkQN (but not MitoK3) stimulated hydrogen peroxide production in isolated mitochondria. At low concentrations, SkQN stimulated state 4 respiration in mitochondria, decreased membrane potential, and blocked ATP synthesis, being more efficient uncoupler of oxidative phosphorylation than MitoK3. In yeast cells, SkQN decreased cell viability and induced oxidative stress and mitochondrial fragmentation. SkQN killed various tumor cells much more efficiently than MitoK3. Since many tumors are characterized by increased oxidative stress, the use of new mitochondria-targeted prooxidants may be a promising strategy for anticancer therapy.

Novel penetrating cations for targeting mitochondria.

Novel penetrating cations were used for the design of mitochondria-targeted compounds and tested in model lipid membranes, in isolated mitochondria and in living human cells in culture. Rhodamine-19, berberine and palmatine were conjugated by aliphatic linkers with plastoquinone possessing antioxidant activity. These conjugates (SkQR1,SkQBerb, SkQPalm) and their analogs lacking plastoquinol moiety (C12R1,C10Berb and C10Palm) penetrated bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria of living cells due to membrane potential negative inside. Reduced forms of SkQR1, SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In human fibroblasts SkQR1, SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by hydrogen peroxide. SkQR1 was effective at subnanomolar concentrations while SkQberb, SkQPalm and SkQ1 (prototypic conjugate of plastoquinone with dodecyltriphenylphosphonium) were effective at 10-times higher concentrations. The aliphatic conjugates of berberine and palmatine (as well as the conjugates of triphenylphosphonium) induced proton transport mediated by free fatty acids (FA) both in the model and mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. In contrast to the other cationic conjugates, SkQR1 and C12R1 induced FA-independent proton conductivity due to protonation/deprotonation of the rhodamine residue. This property in combination with the antioxidant activity probably makes rhodamine conjugates highly effective in protection against oxidative stress. The novel cationic conjugates described here are promising candidates for drugs against various pathologies and aging as mitochondria-targeted antioxidants and selective mild uncouplers.

Novel mitochondria-targeted antioxidants: plastoquinone conjugated with cationic plant alkaloids berberine and palmatine.

PURPOSE: To develop effective mitochondria-targeted antioxidants composed entirely of natural constituents. METHODS: Novel mitochondria-targeted antioxidants were synthesized containing plant electron carrier and antioxidant plastoquinone conjugated by nonyloxycarbonylmethyl residue with berberine or palmatine, penetrating cations of plant origin. These compounds, SkQBerb and SkQPalm, were tested in model planar phospholipid membranes and micelles, liposomes, isolated mitochondria and living cells. RESULTS: SkQBerb and SkQPalm penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in mitochondria isolated or in living human cells in culture. Reduced forms of SkQBerb and SkQPalm as well as C10Berb and C10Palm (SkQBerb and SkQPalm analogs lacking plastoquinol moiety) revealed radical scavenging activity in lipid micelles and liposomes, while oxidized forms were inactive. In isolated mitochondria and in living cells, berberine and palmatine moieties were not reduced, so antioxidant activity of C10Berb and C10Palm was not detected. SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations; their prooxidant effect was observed at 1,000 times higher concentrations. In human cell cuture, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. CONCLUSION: This is the first successful attempt to construct mitochondria-targeted antioxidants composed entirely of natural components, namely plastoquinone, nonyl, acetyl and berberine or palmatine residues.