Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

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.

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.

Nanoparticles of Push-Pull Triphenylamine-Based Molecules for Light-Controlled Stimulation of Neuronal Activity.

Organic semiconductor materials with a unique set of properties are very attractive for interfacing biological objects and can be used for noninvasive therapy or detection of biological signals. Here, we describe the synthesis and investigation of a novel series of organic push-pull conjugated molecules with the star-shaped architecture, consisting of triphenylamine as a branching electron donor core linked through the thiophene π-spacer to electron-withdrawing alkyl-dicyanovinyl groups. The molecules could form stable aqueous dispersions of nanoparticles (NPs) without the addition of any surfactants or amphiphilic polymer matrixes with the average size distribution varying from 40 to 120 nm and absorption spectra very similar to those of human eye retina pigments such as rods and green cones. Variation of the terminal alkyl chain length of the molecules forming NPs from 1 to 12 carbon atoms was found to be an efficient tool to modulate their lipophilic and biological properties. Possibilities of using the NPs as light nanoactuators in biological systems or as artificial pigments for therapy of degenerative retinal diseases were studied both on the model planar bilayer lipid membranes and on the rat cortical neurons. In the planar bilayer system, the photodynamic activity of these NPs led to photoinactivation of ion channels formed by pentadecapeptide gramicidin A. Treatment of rat cortical neurons with the NPs caused depolarization of cell membranes upon light irradiation, which could also be due to the photodynamic activity of the NPs. The results of the work gave more insight into the mechanisms of light-controlled stimulation of neuronal activity and for the first time showed that fine-tuning of the lipophilic affinity of NPs based on organic conjugated molecules is of high importance for creating a bioelectronic interface for biomedical applications.

Retinal-Based Anion Pump from the Cyanobacterium Tolypothrix campylonemoides.

In this work, TcaR rhodopsin from the cyanobacterium Tolypothrix campylonemoides was characterized. Analysis of the amino acid sequence of TcaR revealed that this protein possesses a TSD motif that differs by only one amino acid from the TSA motif of the known halorhodopsin chloride pump. The TcaR protein was expressed in E. coli, purified, and incorporated into proteoliposomes and nanodiscs. Functional activity was measured by electric current generation through the planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one side of the membrane surface, as well as by fluorescence using the voltage-dependent dye oxonol VI. We have shown that TcaR rhodopsin functions as a powerful anion pump. Our results show that the novel microbial anion transporter, TcaR, deserves deeper investigation and may be of interest both for fundamental studies of membrane proteins and as a tool for optogenetics.

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.

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.

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.

Mirror proteorhodopsins.

Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name "mirror proteorhodopsins", from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest.

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.

Photodynamic activity rather than drilling causes membrane damage by a light-powered molecular nanomotor.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.

Synchronization of opening and closing of two gramicidin A channels pulled together by a linker: possible relevance to channel clustering.

The linear 15-mer peptide gramicidin A (gA) produced by Bacillus brevis is known to form the simplest natural ion channel in lipid membranes representing a head-to-head transmembrane dimer. Its incorporation into a planar lipid bilayer manifests itself in regular electrical current transitions. If two gA subunits are tightly connected by a water-soluble, flexible linker of a certain length, the current transitions become heterogeneous: in a part of them, the amplitude is almost twofold higher than that of a single channel, thereby demonstrating the synchronous opening of two single channels. The lifetime, i.e. the open-state duration, of this dual channel is by several orders of magnitude longer than that of the single channel. Here, we used the ideas of the theory of excitons to hypothesize about the mechanism of synchronous opening and closing of two adjacent channels. Two independent (uncoupled) single channels can be described by two independent conformational coordinates q1 and q2, while two closely located channels can exhibit collective behavior, if the coupling between them produces mixing of the individual states (q1,0) and (0,q2). We suppose that a similar phenomenon can occur not only with synthetic derivatives of gA, but also with such natural channel-forming peptide antibiotics and toxins as alamethicin and syringomycin. In particular, channel clustering observed with these peptides may be also associated with formation of collective conductance states, resulting from mixing of their monomeric states, which allows us to explain the fact that clusters of these channels transmit ions and nonelectrolytes of the same size as the original single channels.

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.

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.

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.

Electrophysiological Characterization of Microbial Rhodopsin Transport Properties: Electrometric and ΔpH Measurements Using Planar Lipid Bilayer, Collodion Film, and Fluorescent Probe Approaches.

Electrophysiological approaches to the study of the activity of retinal-containing protein bacteriorhodopsin (bR) or other proteins of this family are based usually on measurements of electrical current through a planar bilayer lipid membrane (BLM) with proteoliposomes attached to the BLM surface at one side of the membrane. Here, we describe the measurements of the pumping activity of bR and channelrhodopsin 2 (ChR2) with special attention to the study of voltage dependence of the light-induced currents. Strong voltage dependence of ChR2 suggests light-triggered ion channel activity of ChR2. We also describe electrophysiological measurements with the help of collodion film instead of BLM for the measurements of fast stages of a rhodopsin photocycle as well as the estimation of the activity of proteoliposomes without a macro membrane using fluorescent probes such as oxonol VI or 9-aminoacridine.

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.

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.

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.

Alkyl esters of umbelliferone-4-acetic acid as protonophores in bilayer lipid membranes and ALDH2-dependent soft uncouplers in rat liver mitochondria.

A great variety of coumarin-related compounds, both natural and synthetic, being often brightly fluorescent, have shown themselves beneficial in medicine for both therapeutic and imaging purposes. Here, in search for effective uncouplers of oxidative phosphorylation, we synthesized a series of 7-hydroxycoumarin (umbelliferone, UB) derivatives combining rather high membrane affinity with the presence of a hydroxyl group deprotonable at physiological pH - alkyl esters of umbelliferone-4-acetic acid (UB-4 esters) differing in alkyl chain length. Addition of UB-4 esters to isolated rat liver mitochondria (RLM) resulted in their rapid depolarization, unexpectedly followed by membrane potential recovery on a minute time scale. According to TLC and HPLC data, incubation of RLM with UB-4 esters caused their hydrolysis, which led to disappearance of the uncoupling activity (recoupling). Both mitochondrial recoupling and hydrolysis of UB-4 esters were suppressed by inhibitors of mitochondrial aldehyde dehydrogenase (ALDH2), disulfiram and daidzin, thus pointing to the involvement of this enzyme in the recoupling of RLM incubated with UB-4 esters. The protonophoric mechanism of mitochondrial uncoupling by UB-4 esters was proved in experiments with artificial bilayer lipid membranes (BLM): these compounds induced proton-selective electrical current across planar BLM and caused dissipation of pH gradient on liposomes. UB-4 esters showed antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Mycobacterium smegmatis.