Protonophoric Uncoupler Research Articles

Exploring the role of mitochondrial uncoupling protein 4 in brain metabolism: implications for Alzheimer's disease.

The brain's high demand for energy necessitates tightly regulated metabolic pathways to sustain physiological activity. Glucose, the primary energy substrate, undergoes complex metabolic transformations, with mitochondria playing a central role in ATP production via oxidative phosphorylation. Dysregulation of this metabolic interplay is implicated in Alzheimer's disease (AD), where compromised glucose metabolism, oxidative stress, and mitochondrial dysfunction contribute to disease progression. This review explores the intricate bioenergetic crosstalk between astrocytes and neurons, highlighting the function of mitochondrial uncoupling proteins (UCPs), particularly UCP4, as important regulators of brain metabolism and neuronal function. Predominantly expressed in the brain, UCP4 reduces the membrane potential in the inner mitochondrial membrane, thereby potentially decreasing the generation of reactive oxygen species. Furthermore, UCP4 mitigates mitochondrial calcium overload and sustains cellular ATP levels through a metabolic shift from mitochondrial respiration to glycolysis. Interestingly, the levels of the neuronal UCPs, UCP2, 4 and 5 are significantly reduced in AD brain tissue and a specific UCP4 variant has been associated to an increased risk of developing AD. Few studies modulating the expression of UCP4 in astrocytes or neurons have highlighted protective effects against neurodegeneration and aging, suggesting that pharmacological strategies aimed at activating UCPs, such as protonophoric uncouplers, hold promise for therapeutic interventions in AD and other neurodegenerative diseases. Despite significant advances, our understanding of UCPs in brain metabolism remains in its early stages, emphasizing the need for further research to unravel their biological functions in the brain and their therapeutic potential.

Theoretical and Experimental Study of the Interaction of Protonophore Uncouplers and Decoupling Agents with Functionally Active Mitochondria.

The purpose of this work was to quantitatively characterize the effectiveness of oxidative phosphorylation uncouplers and decoupling agents in functionally active mitochondria, taking into account their content in the hydrophobic region of the inner membrane of these organelles. When conducting theoretical studies, it is accepted that uncouplers and decouplers occupy part of the volume of mitochondria to exhibit their activity, which is defined as the effective volume. The following quantities characterizing the action of these reagents are considered: (1) concentrations of reagents that cause double stimulation of mitochondrial respiration in state 4 ( ); (2) effective distribution coefficient ( ) - the ratio of the amount of reagents in the effective volume of mitochondria and the water volume; (3) the relative amount of reagents associated with the effective volume of mitochondria ( ); (4) specific activity of reagents localized in the effective volume of mitochondria ( ). We have developed methods for determining these values, based on an analysis of the dependence of the rate of mitochondrial respiration on the concentration of uncouplers and decoupling agents at two different concentrations of mitochondrial protein in the incubation medium. During experimental studies, we compared the effects of the classical protonophore uncouplers 2,4-dinitrophenol (DNP) and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the natural uncouplers lauric and palmitic acids, and the natural decouplers α,ω-tetradecanedioic (TDA) and α,ω-hexadecanedioic (HDA) acids that differ both in the structure of the molecule and in the degree of solubility in lipids. Using the developed methods, we have clarified the dependence of the degree of activity of these uncouplers and decoupling agents on the distribution of their molecules between the effective volume of mitochondria and the water volume.

Characterization of an in vitro model to study CD4+ T cells metabolism in dairy cows

Dairy cows are susceptible to several health disorders throughout their lactation. Objectives were to characterize an in vitro model to study bioenergetic measures in CD4+ T lymphocytes in dairy cows. Twenty-four healthy mid-lactating multiparous Holstein dairy cows were enrolled at a mean (±standard deviation) of 234 ± 22 d in milk (DIM). Cows were blocked according to DIM and blood was collected to isolate peripheral blood mononuclear cells followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The isolated CD4+ T lymphocytes from each cow were split into 2 tubes and randomly assigned to incubate in an assay medium as control (CON) or with a combination of phorbol myristate acetate and ionomycin (PMA+IMY) to evaluate metabolic function under resting and activated state. Mitochondrial and glycolytic functional kinetics were recorded in CD4+ T lymphocytes based on real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) under basal conditions and in response to complex V inhibitor (oligomycin), a protonophore uncoupler (BAM 15), and complex I and complex III inhibitors (rotenone and antimycin A). The mean (±SD) viability and purity of CD4+ T lymphocytes was 92.5 ± 2.9% and 95.2 ± 2.9%, respectively. The basal OCR in CD4+ T lymphocytes treated with PMA+IMY was greater than CON; nevertheless, the maximal respiration rate (CON = 58.0 vs. PMA+IMY = 47.3 ± 5.7 pmol/min) and sparing respiratory capacity rate (CON = 42.0 vs. PMA+IMY = 28.7 ± 4.2 pmol/min) were decreased in activated CD4+ T lymphocytes. The ECAR in CD4+ T lymphocytes increased progressively over time in PMA+IMY compared with CON, which indicated an increase in aerobic glycolysis in PMA+IMY compared with CON (CON = 46.9 vs. PMA+IMY = 86.4 ± 7.0 pmol/min). Activated CD4+ T lymphocytes exhibit a metabolic switch from oxidative phosphorylation to aerobic glycolysis, which may support rapid cell proliferation. The results observed in this experiment demonstrate the sensitivity of the technique to detect changes in metabolic function under different cellular conditions, providing a robust framework to study immuno-metabolism in dairy cattle.

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.

Oxidative Phosphorylation for Aerobic Survival, but Not for Growth: The Peculiar ‘Make-Accumulate-Consume’ Strategy in Zymomonas mobilis

Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, also including the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing cultures on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. Here, we show, for the first time, that in respiring, non-growing Z. mobilis cells receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. No improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, which is unable to oxidize ethanol. The ethanol effect is also hampered by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v), ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease in respiratory chain activity in response to stress conditions mitigates the production of reactive oxygen species.

Modulators reducing the efficiency of oxidative ATP synthesis in mitochondria: protonophore uncouplers, cyclic redox agents, and decouplers.

This work considers the main indicators of the oxidative phosphorylation efficiency in mitochondria: the ADP/O and H+/O ratios. Three groups of modulators that reduce the efficiency of oxidative phosphorylation are compared: protonophore uncouplers, cyclic redox compounds, and decouplers. It is noted that some of them are considered effective therapeutic agents. The paper analyzes the authors' original data on the mechanism of action of natural decouplers, represented by long-chain α,ω-dioic acids, as antioxidants. In conclusion, we discuss the hypothesis of their participation in the rescue of hepatocytes in various disorders of carbohydrate and lipid metabolism.

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.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane. IH results from the action of long-chain fatty acids (FA) on uncoupling protein 1 (UCP1) in brown fat and ADP/ATP carrier (AAC) in other tissues, but the underlying mechanism is poorly understood. Because no pharmacological activators of IH via UCP1 and AAC are known, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) that are thought to increase IH independently of membrane proteins. Protonophores are highly effective in combating obesity, insulin resistance, type II diabetes, and fatty liver in animal models, but because they indiscriminately increase H+ conductance across all biological membranes and have adverse side effects, their use in humans is restricted. Here we report the first direct measurement of IH induced by common protonophores and discover that their mechanism of action in mitochondria was significantly misunderstood. IH induced by DNP and other protonophores depends on AAC and UCP11. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain FA and find that they overlap with the putative ADP/ATP binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH via AAC. Thus, common protonophoric uncouplers emerge as the first synthetic activators of IH via UCP1 and AAC identified to date, paving the way for the development of new and more specific activators of these two proteins.

Protonophoric and mitochondrial uncoupling activity of aryl-carbamate substituted fatty acids.

Aryl-urea substituted fatty acids are protonophores and mitochondrial uncouplers that utilise a urea-based synthetic anion transport moiety to carry out the protonophoric cycle. Herein we show that replacement of the urea group with carbamate, a functional group not previously reported to possess anion transport activity, produces analogues that retain the activity of their urea counterparts. Thus, the aryl-carbamate substituted fatty acids uncouple oxidative phosphorylation and inhibit ATP production by collapsing the mitochondrial proton gradient. Proton transport proceeds via self-assembly of the deprotonated aryl-carbamates into membrane permeable dimeric species, formed by intermolecular binding of the carboxylate group to the carbamate moiety. These results highlight the anion transport capacity of the carbamate functional group.

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.

BAM15 Relieves Neurodegeneration in Aged Caenorhabditis elegans and Extends Lifespan.

BAM15 was recently screened as a protonophore uncoupler specifically for the mitochondrial membrane but not the plasma membrane. It is equally as potent as FCCP, but less toxic. Previously, mitochondrial uncoupling via DNP alleviates neurodegeneration in the nematode Caenorhabditis elegans during aging. Therefore, we investigated whether BAM15 uncouplers could phenotypically and functionally reduce neuronal defects in aged nematodes. We observed green fluorescence protein-tagged mechanosensory neurons and performed touch and chemotaxis assays during aging. Wild-type animals treated with both 50 µM BAM15 and 10 µM DNP showed reduced mechanosensory neuronal defects during aging, which correlates with the maintenance of touch responses and short-term memory during aging. Uncoupler mutant ucp-4 also responded the same way as the wild-type, reducing neurodegeneration in 50 µM BAM15 and 10 µM DNP-treated animals compared to the DMSO control. These results suggest that 50 µM BAM15 alleviates neurodegeneration phenotypically and functionally in C. elegans during aging, potentially through mitochondrial uncoupling. In accordance with the preserved neuronal shape and function in aged C. elegans, 50 µM BAM15 extended the mean lifespan of both wild-type and ucp-4 mutants.

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.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.

Comparative study of free respiration in liver mitochondria during oxidation of various electron donors and under conditions of shutdown of complex III of the respiratory chain

The present work shows that the rate of free respiration of liver mitochondria (in the absence of ATP synthesis (state 4) during the oxidation of succinate is 1.7 times higher than during the oxidation of glutamate with malate. In turn, in the case of oxidation of ferrocyanide with ascorbate, this value is 3.1 times greater than in the case of succinate oxidation. A similar pattern is also observed upon stimulation of free respiration by low concentrations (5 and 10 μM) of the protonophore uncoupler 2,4-dinitrophenol (DNP). It is found that the passive leakage rate of protons in state 4 is the same if the H+/O ratios are 10, 6, and 2 upon the oxidation of glutamate with malate, succinate, and ferrocyanide with ascorbate, respectively. At these values of the H+/O ratio, low concentrations of DNP stimulate passive proton leakage equally during the oxidation of these respiration substrates. In the case of succinate oxidation, bypassing complex III by N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to the maximum degree, as well as switching this complex completely to idle mode by α,ω-hexadecanedioic acid (HDA) cause a 3-fold stimulation of respiration in state 4. We conclude that at mitochondrial free respiration the values of the H+/2e− ratio for complexes I, III, and IV of the respiratory chain are 4, 4, and 2, respectively. It is assumed that the free respiration of mitochondria is carried out by simple diffusion of protons through the inner membrane, and the rate of this diffusion depends on the total number of protons released by the complexes of the electron transport chain into the intermembrane space.

Screening of 6000 Compounds for Uncoupling Activity: A Comparison Between a Mechanistic Biophysical Model and the Structural Alert Profiler Mitotox.

Protonophoric uncoupling of phosphorylation is an important factor when assessing chemicals for their toxicity, and has recently moved into focus in pharmaceutical research with respect to the treatment of diseases such as cancer, diabetes, or obesity. Reliably identifying uncoupling activity is thus a valuable goal. To that end, we screened more than 6000 anionic compounds for in vitro uncoupling activity, using a biophysical model based on ab initio COSMO-RS input parameters with the molecular structure as the only external input. We combined these results with a model for baseline toxicity (narcosis). Our model identified more than 1250 possible uncouplers in the screening dataset, and identified possible new uncoupler classes such as thiophosphoric acids. When tested against 423 known uncouplers and 612 known inactive compounds in the dataset, the model reached a sensitivity of 83% and a specificity of 96%. In a direct comparison, it showed a similar specificity than the structural alert profiler Mitotox (97%), but much higher sensitivity than Mitotox (47%). The biophysical model thus allows for a more accurate screening for uncoupling activity than existing structural alert profilers. We propose to use our model as a complementary tool to screen large datasets for protonophoric uncoupling activity in drug development and toxicity assessment.

Effects of Superoxide Dismutase Inhibitors and Glucose on Cell Death and Generation of Reactive Oxygen Species in Pea Leaves.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Triclosan depletes the membrane potential in Pseudomonas aeruginosa biofilms inhibiting aminoglycoside induced adaptive resistance.

Biofilm-based infections are difficult to treat due to their inherent resistance to antibiotic treatment. Discovering new approaches to enhance antibiotic efficacy in biofilms would be highly significant in treating many chronic infections. Exposure to aminoglycosides induces adaptive resistance in Pseudomonas aeruginosa biofilms. Adaptive resistance is primarily the result of active antibiotic export by RND-type efflux pumps, which use the proton motive force as an energy source. We show that the protonophore uncoupler triclosan depletes the membrane potential of biofilm growing P. aeruginosa, leading to decreased activity of RND-type efflux pumps. This disruption results in increased intracellular accumulation of tobramycin and enhanced antimicrobial activity in vitro. In addition, we show that triclosan enhances tobramycin effectiveness in vivo using a mouse wound model. Combining triclosan with tobramycin is a new anti-biofilm strategy that targets bacterial energetics, increasing the susceptibility of P. aeruginosa biofilms to aminoglycosides.

Discovery of Prenyltransferase Inhibitors with In Vitro and In Vivo Antibacterial Activity.

Cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS) and decaprenyl diphosphate synthase (DPPS) are essential enzymes in bacteria and are involved in cell wall biosynthesis. UPPS and DPPS are absent in the human genome, so they are of interest as targets for antibiotic development. Here, we screened a library of 750 compounds from National Cancer Institute Diversity Set V for the inhibition of Mycobacterium tuberculosis DPPS and found 17 hits, and then IC50s were determined using dose-response curves. Compounds were tested for growth inhibition against a panel of bacteria, for in vivo activity in a Staphylococcus aureus/Caenorhabditis elegans model, and for mammalian cell toxicity. The most active DPPS inhibitor was the dicarboxylic acid redoxal (compound 10), which also inhibited undecaprenyl diphosphate synthase (UPPS) as well as farnesyl diphosphate synthase. 10 was active against S. aureus, Clostridiodes difficile, Bacillus anthracis Sterne, and Bacillus subtilis, and there was a 3.4-fold increase in IC50 on addition of a rescue agent, undecaprenyl monophosphate. We found that 10 was also a weak protonophore uncoupler, leading to the idea that it targets both isoprenoid biosynthesis and the proton motive force. In an S. aureus/C. elegans in vivo model, 10 reduced the S. aureus burden 3 times more effectively than did ampicillin.

Protonophoric action of BAM15 on planar bilayers, liposomes, mitochondria, bacteria and neurons

Small molecules capable of uncoupling respiration and ATP synthesis in mitochondria are protective towards various cell malfunctions. Recently (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (BAM15), a new compound of this type, has become popular as a potent mitochondria-selective depolarizing agent producing minimal adverse effects. To validate protonophoric mechanism of BAM15 action, we examined its behavior in bilayer lipid membranes (BLM). BAM15 proved to be a typical anionic protonophore with the activity on planar membranes being suppressed upon decreasing membrane dipole potential. In both planar BLM and liposomes, BAM15 induced proton conductance with the potency close to that of the classical protonophoric uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). In isolated rat liver mitochondria (RLM), BAM15 caused membrane potential collapse, increased respiration rate and induced Ca2+ efflux at concentrations slightly higher than those for CCCP. Surprisingly, the uncoupling action of BAM15 on isolated RLM, in contrast to that of CCCP, was partially reversed by carboxyatractyloside (CATR), an inhibitor of adenine nucleotide translocase, thereby indicating involvement of this protein in the BAM15-induced uncoupling. BAM15 inhibited growth of Bacillus subtilis at micromolar concentrations. In electrophysiological experiments on molluscan neurons, BAM15 caused plasma membrane depolarization and suppression of electrical activity, but the effect developed more slowly than that of CCCP.

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.