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Chemical mitochondrial uncouplers are protonophoric, lipophilic small molecules that transport protons from the mitochondrial intermembrane space into the matrix independent of ATP synthase, thus uncoupling nutrient oxidation from ATP production. Our previous work identified BAM15 (IC 50  0.27 μM) as a potent and efficacious mitochondrial uncoupler with potential for obesity treatment. In this paper, we investigate in vitro and in vivo properties of hydroxylamine and hydrazine BAM15 derivatives and reveal the high uncoupling nature of these compounds. Our structure-activity relationship studies revealed that the hydroxylamine BAM15 analogs are more potent than hydrazine ones. For example, the most potent of the hydrazine series was 5a with an EC 50 value of 4.6 μM and 103 % activity of BAM15 while compound 4e was the best among the hydroxylamine series with EC 50 value of 340 nM and 118 % BAM15 mitochondrial uncoupling activity in rat L6 myoblasts. Pharmacokinetic profiling of 5a and 4e revealed low exposure (2-220 nM) and short half-life (15-27 min) in mice., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following competing financial interest(s): W.L.S. and K.L.H. are inventors of small molecule mitochondrial uncouplers that are licensed to Uncoupler Biosciences, Inc., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Optic neuritis is an inflammatory demyelinating disease of the optic nerve that often occurs in multiple sclerosis (MS) patients. Sixty percent of patients develop some level of permanent visual loss due to retinal ganglion cell (RGC) damage following optic neuritis, with no known treatment to prevent this loss. Prior studies showed that MP201, a prodrug of 2,4-dinitrophenol (DNP) administered in the experimental autoimmune encephalitis (EAE) mouse model of MS attenuated optic neuritis with preserved vision, increased retinal ganglion cell (RGC) survival, decreased axon loss, and reduced demyelination. Oral administration of MP201, which converts to active form DNP after entry in the portal vein, decreases mitochondrial-derived reactive oxygen species (ROS) and restores calcium homeostasis, which are both implicated in many neurodegenerative diseases. Due to the established therapeutic benefits of prodrug MP201 in EAE mice, we hypothesized that administration of DNP itself may also have significant potential for therapeutic effects. Here, effects of varying doses of DNP treatment in EAE mice were assessed by the extent of spinal cord paralysis, optokinetic response (OKR), RGC survival, and optic nerve demyelination and inflammation. Results show that daily oral doses of 5-10 mg/kg DNP initiated after onset of EAE can significantly reduce spinal cord paralysis, a marker of the EAE MS-like disease, by day 42 after disease induction. DNP treatment significantly reduces RGC loss induced by optic neuritis in EAE mice; however, effects of DNP do not significantly improve visual function, or optic nerve demyelination and inflammation. Current studies show DNP treatment promotes increased RGC survival, but continued inflammation and demyelination likely reduce visual function, suggesting future studies examining combination therapy of DNP with anti-inflammatory agents may be warranted.

Mitochondrial uncouplers dissipate proton gradients and deplete ATP production from oxidative phosphorylation (OXPHOS). While the growth of prostate cancer depends on OXPHOS-generated ATP, the oncogenic pathway mediated by the transcription factor E2F1 is crucial for the progression of this deadly disease. Here, we report that mitochondrial uncouplers, including tizoxanide (TIZ), the active metabolite of the Food and Drug Administration (FDA)-approved anthelmintic nitazoxanide (NTZ), inhibit E2F1-mediated expression of genes involved in cell cycle progression, DNA synthesis, and lipid synthesis. Consequently, NTZ/TIZ induces S-phase kinase-associated protein 2 (SKP2)-mediated G1 arrest while impeding DNA synthesis, lipogenesis, and the growth of prostate cancer cells. The anti-cancer activity of TIZ correlates with its OXPHOS-uncoupling activity. NTZ/TIZ appears to inhibit ATP production, thereby activating the AMP-activated kinase (AMPK)-p38 pathway, leading to cyclin D1 degradation, Rb dephosphorylation, and subsequent E2F1 inhibition. Our results thus connect OXPHOS uncoupling to the inhibition of an essential oncogenic pathway, supporting repositioning NTZ and other mitochondrial uncouplers for prostate cancer therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent patch-clamp studies of mitoplasts have challenged the traditional view that classical chemical uncoupling (by e.g. FCCP or DNP) is due to the protonophoric property of these substances themselves. These studies instead suggest that in brown-fat mitochondria, FCCP- and DNP-induced uncoupling is mediated through activation of UCP1 (and in other tissues by activation of the adenine nucleotide transporter). These studies thus advocate an entirely new paradigm for the interpretation of standard bioenergetic experiments. To examine whether these patch-clamp results obtained in brown-fat mitoplasts are directly transferable to classical isolated brown-fat mitochondria studies, we investigated the effects of FCCP and DNP in brown-fat mitochondria from wildtype and UCP1 KO mice, comparing the FCCP and DNP effects with those of a fatty acid (oleate), a bona fide activator of UCP1. Whereas the sensitivity of brown-fat mitochondria to oleate was much higher in UCP1-containing than in UCP1 KO mitochondria, there was no difference in sensitivity to FCCP and DNP between these mitochondria, neither in oxygen consumption rate nor in membrane potential studies. Correspondingly, the UCP1-dependent ability of GDP to competitively inhibit activation by oleate was not seen with FCCP and DNP. It would thus be premature to abandon the established bioenergetic interpretation of chemical uncoupler effects in classical isolated brown-fat mitochondria-and probably also generally in this type of mitochondrial study. Understanding the molecular and structural reasons for the different outcomes of mitoplast and mitochondrial studies is a challenging task., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Small molecule mitochondrial uncouplers have gained traction for their potential therapeutic use against metabolic dysfunction-associated steatohepatitis (MASH). Herein, we report a novel imidazo[4,5-b]pyridine scaffold derived from iterative modifications of the potent uncoupler BAM15. Our structure-activity relationship (SAR) study demonstrated that this promising scaffold has a range of tolerated substitutions that allows for the modulation of uncoupling activity and in vivo pharmacokinetic properties. Specifically, compound SHS206 displayed an EC 50 of 830 nM in L6 myoblasts and, importantly, showed no cytotoxicity in vitro or adverse effects in mice up to 1000 mg/kg. SHS206 was administered orally at 100 and 300 mg/kg in a GAN mouse model of MASH and was observed to lower liver triglyceride levels while food intake, body weight, temperature, organ weights, and cholesterol levels remained unaltered. Together, these findings illuminate imidazo[4,5-b]pyridine as a promising scaffold for the future development of mitochondrial uncouplers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Webster L. Santos reports a relationship with Uncoupler Biosciences, Inc. that includes: board membership and equity or stocks. Kyle L. Hoehn reports a relationship with Uncoupler Biosciences, Inc. that includes: board membership and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Mitochondrial uncouplers are small molecule protonophores that act to dissipate the proton motive force independent of adenosine triphosphate (ATP) synthase. Mitochondrial uncouplers such as BAM15 increase respiration and energy expenditure and have potential in treating a variety of metabolic diseases. In this study, we disclose the structure-activity relationship profile of 6-substituted [1,2,5]oxadiazolo[3,4- b ]pyridin-7-ol derivatives of BAM15. Utilizing an oxygen consumption rate assay as a measure of increased cellular respiration, SHO1122147 ( 7m ) displayed an EC 50 of 3.6 μM in L6 myoblasts. Pharmacokinetic studies indicated a half-life of 2 h, C max of 35 μM, and no observed adverse effects at 1,000 mg kg -1 dose in mice. In a Gubra-Amylin (GAN) mouse model of MASH, SHO1122147 was efficacious in decreasing body weight and liver triglyceride levels at 200 mg kg -1 day -1 without changes in body temperature. These findings indicate the potential of utilizing novel [1,2,5]oxadiazolo[3,4- b ]pyridin-7-ol mitochondrial uncouplers for treatment of fatty liver disease and obesity.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria, allowing for adjustments in cellular energy metabolism to maintain metabolic homeostasis. Small molecule uncouplers have been extensively studied for their potential to increase metabolic rate, and recent research has focused on developing safe and effective mitochondrial uncoupling agents for the treatment of obesity and cardiometabolic syndrome (CMS). Here, we provide a brief overview of CMS and cover the recent mechanisms by which chemical uncouplers regulate CMS-associated risk-factors and comorbidities, including dyslipidemia, insulin resistance, steatotic liver disease, type 2 diabetes, and atherosclerosis. Additionally, we review the current landscape of uncoupling agents, focusing on repurposed FDA-approved drugs and compounds in advanced preclinical or early-stage clinical development. Lastly, we discuss recent molecular insights by which chemical uncouplers enhance cellular energy expenditure, highlighting their potential as a new addition to the current CMS drug landscape, and outline several limitations that need to be addressed before these agents can successfully be introduced into clinical practice., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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). The mechanism by which BDCT induced two-phase changes in mitochondrial respiration (stimulation followed by deceleration) was unknown. To clarify this issue, we synthesized BDCT and its two analogues. Among these, 5-bromo-3,4-dinitro-N-(4-nitrophenyl)-2-thiophenamine (BDNT) appeared to be the most effective as a mitochondrial uncoupler, decreasing membrane potential and stimulating respiration at submicromolar concentrations. Importantly, BDNT exerted two-phase changes in both mitochondrial membrane potential and respiration rate of RLM, which were enhanced by the addition of glutathione (GSH) but inhibited by the compounds capable of GSH depleting, such as 1-chloro-2,4-dinitrobenzene (CDNB). By contrast, the phase of recoupling was not observed in rat heart mitochondria (RHM). Remarkably, BDNT elicited mitochondrial depolarization in primary human fibroblasts but not in cultured human liver (HepG2) cells. By detecting proton-selective electrical current through planar bilayer lipid membranes, we demonstrated the ability of BDCT and BDNT to transfer protons across membranes. BDNT proved to be an anionic protonophore with a pK a of 7.38. By using LC-MS and capillary electrophoresis, we directly showed the formation of BDNT conjugates with GSH upon incubation with RLM but not RHM. Therefore, we hypothesize that GST is involved in the disappearance of the anilinothiophene uncoupling activity in RLM, ensuring the tissue-specific behavior of the uncoupler., (© 2024 Federation of European Biochemical Societies.)

Mild mitochondrial uncoupling offers therapeutic benefits for various diseases like obesity by regulating cellular energy metabolism. However, effective chemical intervention tools for inducing mild mitochondria-targeted uncoupling are limited. Herein, we have developed a mitochondria-targeted H 2 S prodrug M1 with a unique property of on-demand photoactivated generation of H 2 S accompanied by self-reporting fluorescence for real-time tracking. Upon photoirradiation, M1 decomposes in mitochondria to generate H 2 S and a turn-on fluorescent coumarin derivative for the visualization and quantification of H 2 S. M1 is confirmed to induce reactive oxygen species (ROS)-dependent mild mitochondrial uncoupling, activating mitochondria-associated adenosine monophosphate-activated protein kinase (AMPK) to suppress palmitic acid (PA)-induced lipid deposition in hepatocytes. The uncoupling functions induced by M1 are strictly controlled in mitochondria, representing a fresh strategy to prevent lipid deposition and improve metabolic syndrome by increasing cellular energy expenditure.

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. Here, we report measurements of the CATR effect on the activity of a large number of conventional and novel uncouplers in isolated mammalian mitochondria. Despite the broad variety of chemical structures, CATR attenuated the uncoupling efficacy of all the anionic protonophores in rat heart mitochondria with high abundance of ANT, whereas the effect was much less pronounced or even absent, e.g. for SF6847, in rat liver mitochondria with low ANT content. The fact that the uncoupling action is tissue specific for a broad spectrum of anionic protonophores is highlighted here for the first time. Only with the cationic uncoupler ellipticine and the channel-forming peptide gramicidin A, no sensitivity to CATR was found even in rat heart mitochondria. By contrast, with the recently described ester-stabilized ylidic protonophores [Kirsanov et al. Bioelectrochemistry 2023], the stimulating effect of CATR was discovered both in liver and heart mitochondria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The isothiourea derivative NT-1505 is known as a neuroprotector and cognition enhancer in animal models of neurodegenerative diseases. Bearing in mind possible relation of the NT-1505-mediated neuroprotection to mitochondrial uncoupling activity, here, we examine NT-1505 effects on mitochondria functioning. At concentrations starting from 10 μM, NT-1505 prevented Ca 2+ -induced mitochondrial swelling, similar to common uncouplers. Alongside the inhibition of the mitochondrial permeability transition, NT-1505 caused a decrease in mitochondrial membrane potential and an increase in respiration rate in both isolated mammalian mitochondria and cell cultures, which resulted in the reduction of energy-dependent Ca 2+ uptake by mitochondria. Based on the oppositely directed effects of bovine serum albumin and palmitate, we suggest the involvement of fatty acids in the NT-1505-mediated mitochondrial uncoupling. In addition, we measured the induction of electrical current across planar bilayer lipid membrane upon the addition of NT-1505 to the bathing solution. Importantly, introduction of the palmitic acid into the lipid bilayer composition led to weak proton selectivity of the NT-1505-mediated BLM current. Thus, the present study revealed an ability of NT-1505 to cause moderate protonophoric uncoupling of mitochondria, which could contribute to the neuroprotective effect of this compound., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Elena Shevtsova reports financial support was provided by Russian Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

By uncoupling oxidative phosphorylation, 2,4-dinitrophenol (DNP) attenuates reactive oxygen species (ROS) biosynthesis, which are known to aggravate infectious myocarditis in Chagas disease. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. C56BL/6 mice uninfected and infected untreated and treated daily with 100 mg/kg benznidazole (Bz, reference drug), 5 and 10 mg/kg DNP by gavage for 11 days after confirmation of T. cruzi infection were investigated. Twenty-four hours ​after the last treatment, the animals were euthanized and the heart was collected for microstructural, immunological and biochemical analyses. T. cruzi inoculation induced systemic inflammation (e.g., cytokines and anti-T. cruzi IgG upregulation), cardiac infection (T. cruzi DNA), oxidative stress, inflammatory infiltrate and microstructural myocardial damage in untreated mice. DNP treatment aggravated heart infection and microstructural damage, which were markedly attenuated by Bz. DNP (10 mg/kg) was also effective in attenuating ROS (total ROS, H 2 O 2 , and O 2 - ), nitric oxide (NO), lipid (malondialdehyde - MDA) and protein (protein carbonyl - PCn) oxidation, TNF, IFN-γ, IL-10, and MCP-1/CCL2, anti-T. cruzi IgG, cardiac troponin I levels, as well as inflammatory infiltrate and cardiac damage in T. cruzi-infected mice. Our findings indicate that DNP aggravated heart infection and microstructural cardiomyocytes damage in infected mice. These responses were related to the antioxidant and anti-inflammatory properties of DNP, which favors infection by weakening the pro-oxidant and pro-inflammatory protective mechanisms of the infected host. Conversely, Bz-induced cardioprotective effects combined effective anti-inflammatory and antiparasitic responses, which protect against heart infection, oxidative stress, and microstructural damage in Chagas disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 ( C 200 ); (2) effective distribution coefficient ( E MW ) - 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 ( U M / U T ); (4) specific activity of reagents localized in the effective volume of mitochondria ( A M ). 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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondrial uncoupling by small molecule protonophores is a promising strategy for developing novel anticancer agents. Recently, aryl urea substituted fatty acids (aryl ureas) were identified as a new class of protonophoric anticancer agents. To mediate proton transport these molecules self-assemble into membrane-permeable anionic dimers in which intermolecular hydrogen bonds between the carboxylate and aryl-urea anion receptor delocalise the negative charge across the aromatic π-system. In this work, we extend the aromatic π-system by introducing a second phenyl substituent to the aryl urea scaffold and compare the proton transport mechanisms and mitochondrial uncoupling actions of these compounds to their monoaryl analogues. It was found that incorporation of meta-linked phenyl substituents into the aryl urea scaffold enhanced proton transport in vesicles and demonstrated superior capacity to depolarise mitochondria, inhibit ATP production and reduce the viability of MDA-MB-231 breast cancer cells. In contrast, diphenyl ureas linked through a 1,4-distribution across the phenyl ring displayed diminished proton transport activity, despite both diphenyl urea isomers possessing similar binding affinities for carboxylates. Mechanistic studies suggest that inclusion of a second aryl ring changes the proton transport mechanism, presumably due to steric factors that impose higher energy penalties for dimer formation., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

There has been renewed interest in using mitochondrial uncoupler compounds such as niclosamide and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) for the treatment of obesity, hepatosteatosis and diseases where oxidative stress plays a role. However, both FCCP and niclosamide have undesirable effects that are not due to mitochondrial uncoupling, such as inhibition of mitochondrial oxygen consumption by FCCP and induction of DNA damage by niclosamide. Through structure-activity analysis, we identified FCCP analogues that do not inhibit mitochondrial oxygen consumption but still provided good, although less potent, uncoupling activity. We also characterized the functional role of the niclosamide 4'-nitro group, the phenolic hydroxy group and the anilide amino group in mediating uncoupling activity. Our structural investigations provide important information that will aid further drug development., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In this paper we use simulation methods to study a hypothetical uncoupling agent as a therapy for dementia. We simulate the proliferation of mitochondrial deletion mutants amongst a population of wild-type in human neurons. Mitochondria play a key role in ATP generation. Clonal expansion can lead to the wild-type being overwhelmed by deletions such that a diminished population can no longer fulfil a cell's energy requirement, eventually leading to its demise. The intention of uncoupling is to reduce the formation of deletion mutants by reducing mutation rate. However, a consequence of uncoupling is that the energy production efficacy is also reduced which in turn increases wild-type copy number in order to compensate for the energy deficit. The results of this paper showed that uncoupling reduced the severity of dementia, however, there was some increase in cognitive dysfunction pre-onset of dementia. The effectiveness of uncoupling was dependent upon the timing of intervention relative to the onset of dementia and would necessitate predicting its onset many years in advance., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity., (© 2024. The Author(s).)

Prolonged severe hypoxia follows brief seizures and represents a mechanism underlying several negative postictal manifestations without interventions. Approximately 50% of the postictal hypoxia phenomenon can be accounted for by arteriole vasoconstriction. What accounts for the rest of the drop in unbound oxygen is unclear. Here, we determined the effect of pharmacological modulation of mitochondrial function on tissue oxygenation in the hippocampus of rats after repeatedly evoked seizures. Rats were treated with mitochondrial uncoupler 2,4 dinitrophenol (DNP) or antioxidants. Oxygen profiles were recorded using a chronically implanted oxygen-sensing probe, before, during, and after seizure induction. Mitochondrial function and redox tone were measured using in vitro mitochondrial assays and immunohistochemistry. Postictal cognitive impairment was assessed using the novel object recognition task. Mild mitochondrial uncoupling by DNP raised hippocampal oxygen tension and ameliorated postictal hypoxia. Chronic DNP also lowered mitochondrial oxygen-derived reactive species and oxidative stress in the hippocampus during postictal hypoxia. Uncoupling the mitochondria exerts therapeutic benefits on postictal cognitive dysfunction. Finally, antioxidants do not affect postictal hypoxia, but protect the brain from associated cognitive deficits. We provided evidence for a metabolic component of the prolonged oxygen deprivation that follow seizures and its pathological sequelae. Furthermore, we identified a molecular underpinning of this metabolic component, which involves excessive oxygen conversion into reactive species. Mild mitochondrial uncoupling may be a potential therapeutic strategy to treat the postictal state where seizure control is absent or poor., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 pK a 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.

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The finding that the most common mitochondrial DNA mutation m.11778G>A/ MT-ND4 (p.R340H) associated with Leber's hereditary optic neuropathy (LHON) induces rotenone resistance has produced a long-standing debate, because it contrasts structural evidence showing that the ND4 subunit is far away from the quinone-reaction site in complex I, where rotenone acts. However, recent cryo-electron microscopy data revealed that rotenone also binds to the ND4 subunit. We investigated the possible structural modifications induced by the LHON mutation and found that its amino acid replacement would disrupt a possible hydrogen bond between native R340 and Q139 in ND4, thereby destabilizing rotenone binding. Our analysis thus explains rotenone resistance in LHON patients as a biochemical signature of its pathogenic effect on complex I.