Spontaneous reversal of small molecule-induced mitochondrial uncoupling: the case of anilinothiophenes.

Tissue specificity can render mitochondrial uncouplers more promising as leading compounds for creating drugs against serious diseases. In search of tissue-specific uncouplers, we address anilinothiophenes as possible glutathione-S-transferase substrates (GST). Earlier, 'cyclic' uncoupling activity was reported for 5-bromo-N-(4-chlorophenyl)-3,4-dinitro-2-thiophenamine (BDCT) in isolated rat liver mitochondria (RLM).

Mitochondrial uncoupling caused by a wide variety of protonophores is differently sensitive to carboxyatractyloside in rat heart and liver mitochondria.

Mitochondrial uncoupling by small-molecule protonophores is generally accepted to proceed via transmembrane proton shuttling. The idea of facilitating this process by the adenine nucleotide translocase ANT originated primarily from the partial reversal of the DNP-induced mitochondrial uncoupling by the ANT inhibitor carboxyatractyloside (CATR). Recently, the sensitivity to CATR was also observed for the action of such potent OxPhos uncouplers as BAM15, SF6847, FCCP and niclosamide.

Synthesis of Triphenylphosphonium-Linked Derivative of 3,5-Di-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-di-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.

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.

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-C) by reaction of triphenylphosphine with decyl bromoacetate.

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.

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.

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler.

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1-2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol.

Lipophilic ion aromaticity is not important for permeability across lipid membranes.

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (CTCHP) and butyltriphenylphosphonium (CTPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria.

Lipophilic Cations Rescue the Growth of Yeast under the Conditions of Glycolysis Overflow.

Chemicals inducing a mild decrease in the ATP/ADP ratio are considered as caloric restriction mimetics as well as treatments against obesity. Screening for such chemicals in animal model systems requires a lot of time and labor. Here, we present a system for the rapid screening of non-toxic substances causing such a de-energization of cells.

Fluorescein Derivatives as Antibacterial Agents Acting via Membrane Depolarization.

Appending a lipophylic alkyl chain by ester bond to fluorescein has been previously shown to convert this popular dye into an effective protonophoric uncoupler of oxidative phosphorylation in mitochondria, exhibiting neuro- and nephroprotective effects in murine models. In line with this finding, we here report data on the pronounced depolarizing effect of a series of fluorescein decyl esters on bacterial cells. The binding of the fluorescein derivatives to cells was monitored by fluorescence microscopy and fluorescence correlation spectroscopy (FCS).