Methylation of Phenyl Rings in Ester-Stabilized Phosphorus Ylides Vastly Enhances Their Protonophoric Activity.

We have recently discovered that ester-stabilized phosphorus ylides, resulting from deprotonation of a phosphonium salt such as [Ph3PCH2COOR], can transfer protons across artificial and biological membranes. To create more effective cationic protonophores, we synthesized similar phosphonium salts with one ((heptyloxycarbonylmethyl)(p-tolyl)bromide) or two ((butyloxycarbonylmethyl)(3,5-xylyl)osphonium bromide) methyl substituents in the phenyl groups. The methylation enormously augmented both protonophoric activity of the ylides on planar bilayer lipid membrane (BLM) and uncoupling of mammalian mitochondria, which correlated with strongly accelerated flip-flop of their cationic precursors across the BLM.

The transient character of mitochondrial uncoupling by the popular fungicide fluazinam is specific for liver.

The popular fungicide fluazinam is known to exhibit an unusual cyclic pattern of the protonophoric uncoupling activity in isolated rat liver mitochondria (RLM), with membrane deenergization followed by spontaneous recoupling in the minute scale, which is associated with glutathione conjugation of fluazinam catalyzed by glutathione-S-transferase (GST). Here, we compare the fluazinam effect on RLM with that on rat kidney (RKM) and heart (RHM) mitochondria by monitoring three bioenergetic parameters: oxygen consumption rate, mitochondrial membrane potential and reduction of nucleotides. Only in RLM, the uncoupling activity of fluazinam was transient, i.e. disappeared in a few minutes, whereas in RKM and RHM it was stable in this time scale. We attribute this difference to the increased activity of mitochondrial GST in liver. We report data on the detection of glutathione-fluazinam conjugates by mass-spectrometry, thin layer chromatography and capillary electrophoresis after incubation of fluazinam with RLM but not with RKM, which supports the assumption of the tissue specificity of the conjugation.

Alkyl esters of 7-hydroxycoumarin-3-carboxylic acid as potent tissue-specific uncouplers of oxidative phosphorylation: Involvement of ATP/ADP translocase in mitochondrial uncoupling.

An impressive body of evidence has been accumulated now on sound beneficial effects of mitochondrial uncouplers in struggling with the most dangerous pathologies such as cancer, infective diseases, neurodegeneration and obesity. To increase their efficacy while gaining further insight in the mechanism of the uncoupling action has been remaining a challenge. Encouraged by our previous promising results on lipophilic derivatives of 7-hydroxycoumarin-4-acetic acid (UB-4 esters), here, we use a 7-hydroxycoumarin-3-carboxylic acid scaffold to synthesize a new series of 7-hydroxycoumarin (umbelliferone, UB)-derived uncouplers of oxidative phosphorylation - alkyl esters of umbelliferone-3-carboxylic acid (UB-3 esters) with varying carbon chain length. Compared to the UB-4 derivatives, UB-3 esters proved to be stronger uncouplers: the most effective of them caused a pronounced increase in the respiration rate of isolated rat heart mitochondria (RHM) at submicromolar concentrations. Both of these series of UB derivatives exhibited a striking difference between their uncoupling patterns in mitochondria isolated from liver and heart or kidney, namely: a pronounced but transient decrease in membrane potential, followed by its recovery, was observed after the addition of these compounds to isolated rat liver mitochondria (RLM), while the depolarization of RHM and rat kidney mitochondria (RKM) was rather stable under the same conditions. Interestingly, partial reversal of this depolarization in RHM and RKM was caused by carboxyatractyloside, an inhibitor of ATP/ADP translocase, thereby pointing to the involvement of this mitochondrial membrane protein in the uncoupling activity of both UB-3 and UB-4 esters. The fast membrane potential recovery in RLM uncoupled by the addition of the UB esters was apparently associated with hydrolysis of these compounds, catalyzed by mitochondrial aldehyde dehydrogenase (ALDH2), being in high abundance in liver compared to other tissues. Protonophoric properties of the UB derivatives in isolated mitochondria were confirmed by measurements of RHM swelling in the presence of potassium acetate. In model bilayer lipid membranes (liposomes), proton-carrying activity of UB-3 esters was demonstrated by measuring fluorescence response of the pH-dependent dye pyranine. Electrophysiological experiments on identified neurons from Lymnaea stagnalis demonstrated low neurotoxicity of UB-3 esters. Resazurin-based cell viability assay showed low toxicity of UB-3 esters to HEK293 cells and primary human fibroblasts. Thus, the present results enable us to consider UB-3 esters as effective tissue-specific protonophoric mitochondrial uncouplers.

Antibiotic Pyrrolomycin as an Efficient Mitochondrial Uncoupler.

Pyrrolomycins C (Pyr_C) and D (Pyr_D) are antibiotics produced by Actinosporangium and Streptomyces. The mechanism of their antimicrobial activity consists in depolarization of bacterial membrane, leading to the suppression of bacterial bioenergetics through the uncoupling of oxidative phosphorylation, which is based on the protonophore action of these antibiotics [Valderrama et al., Antimicrob. Agents Chemother. (2019) 63, e01450]. Here, we studied the effect of pyrrolomycins on the isolated rat liver mitochondria. Pyr_C was found to be more active than Pyr_D and uncoupled mitochondria in the submicromolar concentration range, which was observed as the mitochondrial membrane depolarization and stimulation of mitochondrial respiration. In the case of mitoplasts (isolated mitochondria with impaired outer membrane integrity), the difference in the action of Pyr_C and Pyr_D was significantly less pronounced. By contrast, in inverted submitochondrial particles (SMPs), Pyr_D was more active as an uncoupler, which caused collapse of the membrane potential even at the nanomolar concentrations. The same ratio of the protonophoric activity of Pyr_D and Pyr_C was obtained by us on liposomes loaded with the pH indicator pyranine. The protonophore activity of Pyr_D in the planar bilayer lipid membranes (BLMs) was maximal at ~pH 9, i.e., at pH values close to pKa of this compound. Pyr_D functions as a typical anionic protonophore; its activity in the BLM could be reduced by the addition of the dipole modifier phloretin. The difference between the protonophore activity of Pyr_C and Pyr_D in the mitochondria and BLMs can be attributed to a higher ability of Pyr_C to penetrate the outer mitochondrial membrane.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

Bicarbonate suppresses mitochondrial membrane depolarization induced by conventional uncouplers.

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues. However, bicarbonate could not restore the ATP synthesis suppressed by DNP or CCCP in mitochondria. Bicarbonate neither altered the depolarizing action of DNP and FCCP on proteoliposomes with reconstituted cytochrome c oxidase, nor affected the protonophoric activity of DNP and FCCP in artificial lipid membranes as measured with pyranine-loaded liposomes, thereby showing that the bicarbonate-induced reversal of the depolarizing action of DNP and FCCP on mitochondria did not result from direct interaction of bicarbonate with the uncouplers.

A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted uncoupler and fluorescent neuro- and nephroprotector.

BACKGROUND: Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C10) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014). METHODS: Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively. RESULTS: We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C4-mitoFluo). MitoFluo, but not C4-mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C4-mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C4-mitoFluo. MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats. CONCLUSIONS: A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases. GENERAL SIGNIFICANCE: MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies.

Alkyl-substituted phenylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole as uncouplers of oxidative phosphorylation and antibacterial agents: involvement of membrane proteins in the uncoupling action.

In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work (Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines (NBD-Cn) were shown to exhibit uncoupling activity. It was concluded that despite a pKa value being about 10, the expected hindering of the uncoupling activity could be overcome by insertion of an alkyl chain. There is evidence in the literature that the introduction of an aryl substituent in the 4-amino NBD group shifts the pKa to neutral values. Here we report the data on the properties of a number of 4-arylamino derivatives of NBD, namely, alkylphenyl-amino-NBD (Cn-phenyl-NBD) with varying alkyl chain Cn. By measuring the electrical current across planar bilayer lipid membrane, the protonophoric activity of Cn-phenyl-NBD at neutral pH grew monotonously from C1- to C6-phenyl-NBD. All of these compounds increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Importantly, the uncoupling action of C6- and C4-phenyl-NBD was partially reversed by glutamate, diethyl pyrocarbonate (DEPC), 6-ketocholestanol, and carboxyatractyloside, thus pointing to the involvement of membrane proteins in the uncoupling activity of Cn-phenyl-NBD in mitochondria. The pronounced recoupling effect of DEPC, an inhibitor of an aspartate-glutamate carrier (AGC), and that of its substrates for the first time highlighted AGC participation in the action of potent uncouplers on mitochondria. C6-phenyl-NBD produced strong antimicrobial effect on Bacillus subtilis, which manifested itself in cell membrane depolarization and suppression of bacterial growth at submicromolar concentrations.

Weak C-H acids as protonophores can carry hydrogen ions through lipid membranes and mitochondria: a case of o-carborane.

ortho-Carborane (1,2-C2B10H12) was found to be a carrier of protons in both mitochondrial and artificial lipid membranes, suggesting that this dicarborane can reversibly release hydrogen ions and diffuse through the membranes in neutral and anionic forms. Similar to conventional uncouplers (e.g. 2,4-dinitrophenol), o-carborane stimulated mitochondrial respiration and decreased the membrane potential at concentrations of tens of micromoles. Protonophoric activity of o-carborane was observed both by a fluorometric assay using pyranine-loaded liposomes and electrical current measurements across planar lipid bilayers. Substantial contribution of the proton flux to the o-carborane-mediated current was proved by a shift of the zero current voltage upon imposing a pH gradient across the membrane. Meta-carborane (1,7-C2B10H12) lacked the protonophoric activity in line with its reduced C-H acidity. The results suggest that weak C-H acids can exhibit protonophoric activity in the biological environment. The finding of a new class of protonophoric compounds is of substantial interest due to promising anti-obesity and anti-diabetic properties of uncouplers.

Neuroprotective effect of glutamate-substituted analog of gramicidin A is mediated by the uncoupling of mitochondria.

BACKGROUND: Reactive oxygen species are grossly produced in the brain after cerebral ischemia and reperfusion causing neuronal cell death. Mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150mV. Therefore, limited uncoupling of oxidative phosphorylation could be beneficial for cells exposed to deleterious oxidative stress-associated conditions by preventing excessive generation of reactive oxygen species. METHODS: Protonophoric and uncoupling activities of different peptides were measured using pyranine-loaded liposomes and isolated mitochondria. To evaluate the effect of glutamate-substituted analog of gramicidin A ([Glu1]gA) administration on the brain ischemic damage, we employed the in vitro model of neuronal hypoxia using primary neuronal cell cultures and the in vivo model of cerebral ischemia induced in rats by the middle cerebral artery occlusion. RESULTS: [Glu1]gA was the most effective in proton-transferring activity among several N-terminally substituted analogs of gramicidin A tested in liposomes and rat brain and liver mitochondria. The peptides were found to be protective against ischemia-induced neuronal cell death and they lowered mitochondrial membrane potential in cultured neurons and diminished reactive oxygen species production in isolated brain mitochondria. The intranasal administration of [Glu1]gA remarkably diminished the infarct size indicated in MR-images of a brain at day 1 after the middle cerebral artery occlusion. In [Glu1]gA-treated rats, the ischemia-induced brain swelling and behavioral dysfunction were significantly suppressed. CONCLUSIONS: The glutamate-substituted analogs of gramicidin A displaying protonophoric and uncoupling activities protect neural cells and the brain from the injury caused by ischemia/reperfusion. GENERAL SIGNIFICANCE: [Glu1]gA may be potentially used as a therapeutic agent to prevent neuron damage after stroke.

Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations.

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector.

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.

Synthesis of Triphenylphosphonium-Linked Derivative of 3,5-Ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) via Knoevenagel Reaction Yields an Effective Mitochondria-Targeted Protonophoric Uncoupler.

Mitochondrial uncouplers are actively sought as potential therapeutics. Here, we report the first successful synthesis of mitochondria-targeted derivatives of the highly potent uncoupler 3,5-ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847), bearing a cationic alkyl(triphenyl)phosphonium (TPP) group. As a key step of the synthesis, we used condensation of a ketophenol with malononitrile via the Knoevenagel reaction. SF-C5-TPP with a pentamethylene linker between SF6847 and TPP, stimulating respiration and collapsing membrane potential of rat liver mitochondria at submicromolar concentrations, proved to be the most effective uncoupler of the series. SF-C5-TPP showed pronounced protonophoric activity on a model planar bilayer lipid membrane. Importantly, SF-C5-TPP exhibited rather low toxicity in fibroblast cell culture, causing mitochondrial depolarization in cells at concentrations that only slightly affected cell viability. SF-C5-TPP was more effective in decreasing the mitochondrial membrane potential in the cell culture than SF6847, in contrast to the case of isolated mitochondria. Like other zwitterionic uncouplers, SF-C5-TPP inhibited the growth of Bacillus subtilis in the micromolar concentration range.

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms.

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C4TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C4TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C4TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C4TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C4TPP-X series, with the exception of C4TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.

Selectivity of cation transport across lipid membranes by the antibiotic salinomycin.

The ionophoric antibiotic salinomycin is in the phase of preclinical tests against several types of malignant tumors including breast cancer. Notwithstanding, the data on its ion selectivity, although being critical for its therapeutic activity, are rather scarce. In the present work, we studied the ability of salinomycin to exert cation/H+-exchange across artificial bilayer lipid membranes (BLM) by measuring electrical potential on planar BLM in the presence of a protonophore and fluorescence responses of the pH-sensitive dye pyranine entrapped in liposomes. The following order of ion selectivity was obtained by these two methods: K+ > Na+ > Rb+ > Cs+ > Li+. Measurements of the monovalent cation-induced quenching of fluorescence of thallium ions in methanol showed that salinomycin effectively binds potassium and calcium but poorly binds sodium and lithium ions. At high concentrations, salinomycin transports Ca2+ through membranes of liposomes and mitochondria, as measured by using the calcium-sensitive dye Fluo-5 N. The data obtained can be used in the mechanistic studies of the anti-tumor activity of salinomycin and its selective cytotoxicity towards cancer stem cells.

Ester-stabilized phosphorus ylides as protonophores on bilayer lipid membranes, mitochondria and chloroplasts.

Triphenylphosphonium ylides are commonly used as key intermediates in the Wittig reaction. Based on the known acidities of stabilized ylide precursors, we proposed that a methylene group adjacent to phosphorus in these compounds can ensure proton shuttling across lipid membranes. Here, we synthesized (decyloxycarbonylmethyl)triphenylphosphonium bromide (CMTPP-C10) by reaction of triphenylphosphine with decyl bromoacetate. This phosphonium salt precursor of the ester-stabilized phosphorus ylide along with its octyl (CMTPP-C8) and dodecyl (CMTPP-C12) analogues was found to be a carrier of protons in mitochondrial, chloroplast and artificial lipid membranes, suggesting that it can reversibly release hydrogen ions and diffuse through the membranes in both zwitterionic (ylide) and cationic forms. The CMTPP-C10-mediated electrical current across planar bilayer lipid membranes exhibited pronounced proton selectivity. Similar to conventional protonophores, known to uncouple electron transport and ATP synthesis, CMTPP-Cn (n = 8, 10, 12) stimulated mitochondrial respiration, while decreasing membrane potential, at micromolar concentrations, thereby showing the classical uncoupling activity in mitochondria. CMTPP-C12 also caused dissipation of transmembrane pH gradient on chloroplast membranes. Importantly, CMTPP-C10 exhibited substantially lower toxicity in cell culture, than C12TPP. Thus, we report the finding of a new class of ylide-type protonophores, which is of substantial interest due to promising therapeutic properties of uncouplers.

Mitochondrial ATP Synthase and Mild Uncoupling by Butyl Ester of Rhodamine 19, C4R1.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the ß-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and ß-subunits, close to the anionic amino acid residues of the ß-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

Rate of translocation across lipid bilayer of triphenylphosphonium-linked salinomycin derivatives contributes significantly to their K+/H+ exchange activity on membranes.

Salinomycin (SAL), a polyether antibiotic exerting K+/H+-exchange on cellular membranes, effectively kills cancer stem cells. A series of cationic triphenylphosphonium (TPP+)-linked SAL derivatives were synthesized aiming to render them mitochondria-targeted. Remarkably, attaching a TPP+ moiety via a triazole linker at the C-20 position of SAL (compound 5) preserved the ion carrier potency of the antibiotic, while analogs with TPP+ linked at the C-1 position of SAL (6, 8) were ineffective. On planar bilayer lipid membranes (BLM), the SAL analogs 6 and 8 exhibited slow electrical current relaxation upon a voltage jump, similar to previously studied alkyl-TPP compounds. However, 5 demonstrated much faster current relaxation, which suggested its high permeability through BLM resulting in its pronounced potency to transport potassium and hydrogen ions across both artificial (liposomal) and mitochondrial membranes. SAL and 5 did not induce a steady-state electrical current through the planar lipid bilayer, thereby confirming that the transport mechanism is the electrically silent K+/H+ exchange. The ion exchange mediated by 5 in energized mitochondria was more active than that caused by SAL, which was apparently due to accumulation of 5 in mitochondria. Thus, compound 5 can be regarded as a promising lead compound for testing anticancer and antimicrobial activity.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria.

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

Membrane Permeability of Modified Butyltriphenylphosphonium Cations.

The alkyltriphenylphosphonium (TPP) group is the most widely used vector targeted to mitochondria. Previously, the length of the alkyl linker was varied as well as structural modifications in the TPP phenyl rings to obtain the optimal therapeutic effect of a pharmacophore conjugated with a lipophilic cation. In the present work, we synthesized butyltriphenylphosphonium cations halogenated and methylated in phenyl rings (C4TPP-X) and measured electrical current through a planar lipid bilayer in the presence of C4TPP-X. The permeability of C4TPP-X varied in the range of 6 orders of magnitude and correlates well with the previously measured translocation rate constant for dodecyltriphenylphosphonium analogues. The partition coefficient of the butyltriphenylphosphonium analogues obtained by calculating the difference in the free energy of cation solvation in water and octane using quantum chemical methods correlates well with the permeability values. Using an ion-selective electrode, a lower degree of accumulation of analogues with halogenated phenyl groups was found on isolated mitochondria of rat liver, which is in agreement with their permeability decrease. Our results indicate the translocation of the butyltriphenylphosphonium cations across the hydrophobic membrane core as rate-limiting stage in the permeability process rather than their binding/release to/from the membrane.