Caffeic acid phenethyl ester, an active component of propolis extract, inhibits Yipoxygenase in the micromolar concentration range. The inhibition is of an uncompetitive type, i.e. the inhibitor binds to the enzyme-substrate complex but not to the free enzyme. Caffeic acid phenethyl ester also exhibits antioxidant properties. At a concentration of 10 PM, it completely blocks production of reactive oxygen species in human neutrophils and the xanthinelxanthine oxidase system.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

A unique phenomenon of mitochondria-targeted protonophores is described.ItconsistsinatransmembraneH þ -conductingfattyacidcycling mediated by penetrating cations such as 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C 12 TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δψ) of the Nernstian value (about 60 mV Δψ at ΔpH ¼ 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C 12 TPP, but not cetyltrimethylammonium, potentiatedfattyacid-induced(i)uncouplingofrespirationandphosphorylation, and (ii) inhibition of H 2 O 2 formation. In intact yeast cells, C 12 TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanidephenylhydrazone)stimulatedrespiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.mild uncoupling | membrane | Mitochondria-targeted uncoupler | penetrating ion | antioxidant S ome decrease in mitochondrial membrane potential (Δψ) in the resting state may be favorable in treating obesity and hypothyroidism as well as in preventing senescence and certain types of cancer [for reviews, see refs. 1, 2]. In the first two cases, Δψ lowering stimulates respiratory metabolism. As to senescence and cancer, such an effect seems to be related to a decrease in production of reactive oxygen species (ROS) in mitochondria. ROS, in turn, were assumed to mediate senescence and some steps of cancerogenesis (2, 3). As was shown in our group (4), there is a very steep dependence of mitochondrial ROS formation on Δψ. Small (10-15%) lowering of Δψ resulted in ten-fold decrease in the ROS production rate (4). In isolated mitochondria, this can be achieved by adding a low concentration of a protonophorous uncoupler (4-6). This approach, called "mild uncoupling" (4, 6), was recently used by Padalko (7) and by Kowaltowski and coworkers (8) to prolong the lifespan of Drosophila and mice, respectively. However, long-term treatment of animals with uncouplers results in toxic side effects (9).In this paper, we put forward an alternative approach based on the use of synthetic cations that easily penetrate through biological membranes. Penetrating ions were suggested by our group to reveal electric potential difference across mitochondrial membrane (9, 10). In tetraphenylphosphonium (TPP), a typical representative of such ions, the positive charge is strongly displaced over four phenyl residues. As a result, water dipoles cannot be held by t...

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273-1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663-668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH2 interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ- being reduced back to SkQH2 by heme bH of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C9 or C13 of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (capital ES, Cyrillic12TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since capital ES, Cyrillic12TPP is specifically accumulated in mitochondria and increases the H+ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by capital ES, Cyrillic12TPP.

Accumulating evidence suggests that, during translation, nascent chains can form specific interactions with ribosomal exit tunnel to regulate translation and promote initial folding events. The clinically important macrolide antibiotics bind within the exit tunnel and inhibit translation by preventing progression of the nascent chain and inducing peptidyl-tRNA drop-off. Here, we have synthesized amino acid- and peptide-containing macrolides, which are used to demonstrate that distinct amino acids and peptides can establish interaction with components of the ribosomal tunnel and enhance the ribosome-binding and inhibitory properties of the macrolide drugs, consistent with the concept that the exit tunnel is not simply a Teflon-like channel. Surprisingly, we find that macrolide antibiotics do not inhibit translation of all nascent chains similarly, but rather exhibit polypeptide-specific inhibitory effects, providing a change to our general mechanistic understanding of macrolide inhibition.