Synthesis and anti-proliferative effect of novel 4-Aryl-1, 3-Thiazole-TPP conjugates via mitochondrial uncoupling process.

With the advent of mitochondrial targeting moiety such as triphenlyphosphonium cation (TPP+), targeting mitochondria in cancer cells has become a promising strategy for combating tumors. Herein, a series of novel 4-aryl-1,3-thiazole derivatives linked to TPP+ moiety were designed and synthesized. The cytotoxicity against a panel of four cancer cell lines was evaluated by CCK-8 assay. Most of these compounds exhibited moderate to good inhibitory activity over HeLa, PC-3 and HCT-15 cells while MCF-7 cells were less sensitive to most compounds. Among them, compound 12a exhibited a significant anti-proliferative activity against HeLa cells, and prompted for further investigation. Specifically, 12a decreased mitochondrial membrane potential and enhanced levels of reactive oxygen species (ROS). The flow cytometry analysis revealed that compound 12a could induce apoptosis and cell cycle arrest at G0/G1 phase in HeLa cells. In addition, mitochondrial bioenergetics assay revealed that 12a displayed mild mitochondrial uncoupling effect. Taken together, these findings suggest the therapeutic potential of compound 12a as an antitumor agent targeting mitochondria.

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

Oxidative stress mediates the impact of heatwaves on survival, growth and immune status in a lizard.

Climate change often includes increases in the occurrence of extreme environmental events. Among these, heatwaves affect the pace of life and performance of wildlife, particularly ectothermic animals, owing to their low thermoregulatory abilities. However, the underlying mechanisms by which this occurs remain unclear. Evidence shows that heatwaves alter the redox balance of ectotherms, and oxidative stress is a major mediator of life-history trade-offs. Therefore, oxidative stress may mediate the effect of extreme thermal conditions on the life histories of ectotherms. To test this hypothesis, a 2 × 2 experiment was conducted to manipulate the redox balance (through a mitochondrial uncoupler that alleviates oxidative stress) of the desert toad-headed agama (Phrynocephalus przewalskii) exposed to heatwave conditions. We recorded lizard growth and survival rates and quantified their redox and immune statuses. In control lizards (unmanipulated redox balance), heatwave conditions decreased growth and survival and induced oxidative damage and immune responses. By contrast, lizards with alleviated oxidative stress showed close-to-normal growth, survival, and immune status when challenged with heatwaves. These results provide mechanistic insight into the role of oxidative stress in mediating the effects of extreme temperatures on ectothermic vertebrates, which may have major eco-evolutionary implications.

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.

White to brown adipocyte transition mediated by Apigenin via VEGF-PRDM16 signaling.

The dysregulated energy metabolism in white adipose tissues results in derangement of biological signaling resulting in obesity. Lack of vascularization in these white adipose tissues is one of the major reasons for dysregulated energy metabolism. Not much work has been done in this direction to understand the role of angiogenesis in white adipose tissue metabolism. In the present study, we evaluated the effect of angiogenic modulator in the metabolism of white adipocyte (WAC). Bioactive Apigenin was selected and its angiogenic ability was studied. Apigenin was shown to be highly proangiogenic hence the effect of Apigenin on de novo and trans-differentiation of WAT was studied. Apigenin showed enhanced de novo differentiation and trans-differentiation of mouse WAC into brown-like phenotype. To understand the effect of Apigenin on adipose tissue vasculature, coculture studies were conducted. Cross talk between endothelial cell and adipocytes were observed in coculture studies. Gene expression studies of cocultured cells revealed that browning of WAC occurred by triggering the expression of Vascular endothelial growth factor A. The study provides a new insight for inducing metabolic shift in WACs by modulation of angiogenesis in WAC microenvironment by the upregulation of PRDM16 cascade to trigger browning for the treatment of obesity.

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.

Niclosamide Is a Much More Potent Toxicant of Mitochondrial Respiration than TFM in the Invasive Sea Lamprey (Petromyzon marinus).

Invasive sea lampreys in the Laurentian Great Lakes are controlled by applying TFM (3-trifluoromethyl-4-nitrophenol) and niclosamide to streams infested with their larvae. Both agents uncouple oxidative phosphorylation in the mitochondria, but TFM specifically targets lampreys, which have a lower capacity to detoxify the lampricide. Niclosamide lacks specificity and is more potent than TFM. However, its greater potency is poorly understood. We tested the hypothesis that niclosamide is a stronger uncoupler of mitochondrial oxidative phosphorylation than TFM by measuring oxygen consumption rates in isolated liver mitochondria exposed to physiologically relevant concentrations of TFM, niclosamide, or their mixture (100 TFM:1 niclosamide) at environmentally relevant temperatures (7, 13, and 25 °C). Niclosamide increased State 4 respiration and decreased the respiratory control ratio (RCR) at much lower concentrations than TFM. Calculations of the relative EC50 values, the amount of TFM or niclosamide required to decrease the RCR by 50%, indicated that niclosamide was 40-60 times more potent than TFM. Warmer temperature did not appear to decrease the sensitivity of mitochondria to niclosamide or TFM, as observed in the intact sea lamprey exposed to TFM in warmer waters. We conclude that the extreme sensitivity of mitochondria to niclosamide contributes to its greater in vivo toxicity in the whole animal.

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.

Sludge reduction and microbial community evolution of activated sludge induced by metabolic uncoupler o-chlorophenol in long-term anaerobic-oxic process.

Excess sludge management is a restrictive factor for the development of municipal wastewater treatment plants. The addition of metabolic uncouplers has been proven to be effective in sludge reduction. However, the long-term effect of metabolic uncoupler o-chlorophenol (oCP) on the biological wastewater treatment system operated in anaerobic-oxic mode is still unclear. To this end, two parallel reactors operated in anaerobic-oxic mode with and without 10 mg/L of oCP addition were investigated for 91 days. The results showed that 56.1 ± 2.3% of sludge reduction was achieved in the oCP-added system, and the nitrogen and phosphorus removal ability were negatively affected. Dosing oCP stimulated the formation of microbial products and increased the DNA concentration, but resulted in a decrease in the electronic transport activity of activated sludge. Microbial community analysis further demonstrated that a significant reduction of bacterial richness and diversity occurred after oCP dosing. However, after stopping oCP addition, the pollutant removal ability of activated sludge was gradually increased, but the sludge yield, as well as species richness and diversity, did not recover to the previous level. This study will provide insightful guidance on the long-term application of metabolic uncouplers in the activated sludge system.

2,4-Dinitrophenol as an Uncoupler Augments the Anthracyclines Toxicity against Prostate Cancer Cells.

One of the strategies for the treatment of advanced cancer diseases is targeting the energy metabolism of the cancer cells. The compound 2,4-DNP (2,4-dinitrophenol) disrupts the cell energy metabolism through the ability to decouple oxidative phosphorylation. The aim of the study was to determine the ability of 2,4-DNP to sensitize prostate cancer cells with different metabolic phenotypes to the action of known anthracyclines (doxorubicin and epirubicin). The synergistic effect of the anthracyclines and 2,4-DNP was determined using an MTT assay, apoptosis detection and a cell cycle analysis. The present of oxidative stress in cancer cells was assessed by CellROX, the level of cellular thiols and DNA oxidative damage. The study revealed that the incubation of LNCaP prostate cancer cells (oxidative phenotype) with epirubicin and doxorubicin simultaneously with 2,4-DNP showed the presence of a synergistic effect for both the cytostatics. Moreover, it contributes to the increased induction of oxidative stress, which results in a reduced level of cellular thiols and an increased number of AP sites in the DNA. The synergistic activity may consist of an inhibition of ATP synthesis and the simultaneous production of toxic amounts of ROS, destroying the mitochondria. Additionally, the sensitivity of the LNCaP cell line to the anthracyclines is relatively higher compared to the other two (PC-3, DU-145).

Stop the beat to see the rhythm: excitation-contraction uncoupling in cardiac research.

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. When compared with other uncoupling agents, blebbistatin is the most frequently used, as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue.

Ionophoric effects of the antitubercular drug bedaquiline.

Bedaquiline (BDQ), an inhibitor of the mycobacterial F1Fo-ATP synthase, has revolutionized the antitubercular drug discovery program by defining energy metabolism as a potent new target space. Several studies have recently suggested that BDQ ultimately causes mycobacterial cell death through a phenomenon known as uncoupling. The biochemical basis underlying this, in BDQ, is unresolved and may represent a new pathway to the development of effective therapeutics. In this communication, we demonstrate that BDQ can inhibit ATP synthesis in Escherichia coli by functioning as a H+/K+ ionophore, causing transmembrane pH and potassium gradients to be equilibrated. Despite the apparent lack of a BDQ-binding site, incorporating the E. coli Fo subunit into liposomes enhanced the ionophoric activity of BDQ. We discuss the possibility that localization of BDQ at F1Fo-ATP synthases enables BDQ to create an uncoupled microenvironment, by antiporting H+/K+ Ionophoric properties may be desirable in high-affinity antimicrobials targeting integral membrane proteins.

Effect of uncouplers of oxidative phosphorylation on metabolism of propionate in liver explants from dairy cows.

Our objective was to determine the effects of uncouplers of oxidative phosphorylation on the metabolism of propionate in liver tissue of dairy cows in the postpartum period. A total of 8 primiparous dairy cows were biopsied for liver tissue explants in 2 block-design experiments. Cows were 5.9 ± 2.8 (mean ± SD) days in milk, and the 2 experiments were run concurrently. Treatments for experiment 1 were 10 µM 2,4-dinitrophenol methyl ether (DNPME) or propylene carbonate (vehicle control). Treatments for experiment 2 were 5 mM sodium salicylate (SAL) or no treatment (control). Explants were incubated in 2.5 mM [13C3]propionate with treatments and terminated after 0.5, 15, and 60 min of exposure to tracer. Treatment with DNPME had no effects on measured metabolites compared with control. Treatment with SAL increased total 13C% enrichment of succinate (3.03 vs. 2.45%), but tended to decrease total 13C% enrichment of fumarate (2.86 vs. 3.10%) and decreased total 13C% enrichment of malate (3.96 vs. 4.58%) compared with the control. Treatment with DNPME appeared to have no effects on hepatic propionate metabolism, and treatment with SAL may impair the succinate dehydrogenase reaction.

Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice.

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp-/-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp-/- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp-/- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp-/- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp-/- mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp-/- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp-/- mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp-/- mice.

TBAJ-876 Displays Bedaquiline-Like Mycobactericidal Potency without Retaining the Parental Drug's Uncoupler Activity.

The diarylquinoline F1FO-ATP synthase inhibitor bedaquiline (BDQ) displays protonophore activity. Thus, uncoupling electron transport from ATP synthesis appears to be a second mechanism of action of this antimycobacterial drug. Here, we show that the new BDQ analogue TBAJ-876 did not retain the parental drug's protonophore activity. Comparative time-kill analyses revealed that both compounds exert the same bactericidal activity. These results suggest that the uncoupler activity is not required for the bactericidal activity of diarylquinolines.

Molecular hydrogen suppresses superoxide generation in the mitochondrial complex I and reduced mitochondrial membrane potential.

Molecular hydrogen (H2) is recognized as a medical gas applicable to numerous diseases including neurodegenerative diseases, metabolic disorders, and rheumatoid arthritis. Although the efficacy of H2 is reportedly attributed to its scavenging capability against the hydroxyl radical, the mechanisms underlying its therapeutic efficacy are not fully understood. Herein, we estimated the role of H2 in the energy converting system of the mitochondria, the source of reactive oxygen species. To investigate the effects of H2 on mitochondrial function, direction of electron flow, superoxide generation, and mitochondrial membrane potential were investigated. Forward electron transport (FET) or reverse electron transport (RET) was assessed by monitoring the decrease or increase of ß-nicotinamide adenine dinucleotide hydrate (NADH, - or +, µM, respectively) in the presence of ß-nicotinamide adenine dinucleotide (NAD+) and/or succinate in the isolated mitochondria. H2O2 converted from superoxide by superoxide dismutase (SOD) was measured to estimate electron leakage in the mitochondria. The effects of H2 on mitochondrial membrane potential were observed by staining cells with the fluorescence probe, teramethylrhodamine ethyl ester (TMRE). Despite the absence of succinate, a distinct RET was observed (from +0.0313 ± 0.0106 µM to +1.20 ± 0.302 µM) by adding 25 µM H2. In the presence of 5 µM NADH, RET by succinate inverted to FET from +1.62 ± 0.358 µM to -1.83 ± 0.191 µM, accompanied by a suppression of superoxide generated predominantly from complex I by 51.1%. H2 solely reduced mitochondrial membrane potential of the cultured cells by 11.3% as assessed by TMRE. The direction of electron flow was altered by H2 depending on the NAD+/NADH ratio, accompanied by suppression of superoxide generation H2 could suppress superoxide generation in complex I in vitro and reduce membrane potential in vivo. H2 may also neutralize semiquinone radicals to reduce superoxide produced in complex III. H2 may function as a rectifier of the electron flow affecting the mitochondrial membrane potential to suppress oxidative damage in mitochondria.

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.

Emodin, as a mitochondrial uncoupler, induces strong decreases in adenosine triphosphate (ATP) levels and proliferation of B16F10 cells, owing to their poor glycolytic reserve.

Many types of cancer cells show a characteristic increase in glycolysis, which is called the "Warburg effect." By screening plant extracts, we identified one that decreases cellular adenosine triphosphate (ATP) levels and suppresses proliferation of malignant melanoma B16F10 cells, but not of noncancerous MEF cells. We showed that its active ingredient is emodin, which showed strong antiproliferative effects on B16F10 cells both in vitro and in vivo. Moreover, we also found that emodin can function as a mitochondrial uncoupler. Consistently, three known mitochondrial uncouplers also displayed potent antiproliferative effects and preferential cellular ATP reduction in B16F10 cells, but not in MEF cells. These uncouplers provoked comparable mitochondrial uncoupling in both cell types, but they manifested dramatically different cellular effects. Namely in MEF cells, these uncouplers induced three to fivefold increases in glycolysis from the basal state, and this compensatory activation appeared to be responsible for the maintenance of cellular ATP levels. In contrast, B16F10 cells treated with the uncouplers showed less than a twofold enhancement of glycolysis, which was not sufficient to compensate for the decrease of ATP production. Together, these results raise the possibility that uncouplers could be effective therapeutic agents specifically for cancer cells with prominent "Warburg effect."

Anilinopyrazines as potential mitochondrial uncouplers.

Mitochondrial protonophores transport protons through the mitochondrial inner membrane into the matrix to uncouple nutrient oxidation from ATP production thereby decreasing the proton motive force. Mitochondrial uncouplers have beneficial effects of decrease reactive oxygen species generation and have the potential for treating diseases such as obesity, neurodegenerative diseases, non-alcoholic fatty liver disease (NAFLD), diabetes, and many others. In this study, we report the structure-activity relationship profile of the pyrazine scaffold bearing substituted aniline rings. Our work indicates that a trifluoromethyl group is best at the para position while the trifluoromethoxy group is preferred in the meta position of the aniline rings of 2,3-substituted pyrazines. As proton transport and cycling requires the formation of a negative charge that has to traverse the mitochondrial membrane, a stabilizing internal hydrogen bond is a key feature for efficient mitochondrial uncoupling activity.