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Lefleuganan, a clinical stage drug candidate for the treatment of cutaneous leishmaniasis, is a synthetic nonapeptide inspired by the natural antimicrobial peptide leucinostatin A, exhibiting excellent antiprotozoal activity in the low nM range. Lefleuganan shows strongly reduced acute toxicity, making it amenable for clinical use. Here, using a broad set of in vivo and in vitro measurements using purified enzymes, we find that leucinostatin A, but not lefleuganan, specifically targets the mitochondrial ATP synthase, inhibiting ATP synthesis by the human, bovine, and yeast enzyme in the nanomolar range. In a structure-activity relationship study covering the chemical space between the two compounds, hydroxyleucine at position 7 in leucinostatin A is identified as the key responsible moiety for specific ATP synthase inhibition and systemic toxicity. Our data suggest that efficient antiprotozoal activity of these class of compounds is mediated by efficient energetic uncoupling of negatively charged membranes.

Hypothesis: Trypanosomiasis is one of the main threats to human and animal health in African countries. Trypanosoma brucei can evade the host immune recognition by rapidly altering its variant surface glycoprotein (VSG). The ATP synthase F 1 subunit of the parasite exhibits extremely low similarity to that of its mammalian hosts, hypothetically making it an ideal target for the development of novel therapeutics., Experiments: Graphene quantum dots (GQDs) were synthesized, and their adhesion to T. brucei surface and internalization was observed microscopically. The activity of ATP synthase and mitochondrial membrane potential of T. brucei were measured after exposure to GQDs. Proteomics, biolayer interferometry, and molecular dynamic simulations were utilized to evaluate the interaction between GQDs with the target proteins., Findings: GQDs specifically adhered to the VSG of T. brucei and were conveyed inside the parasite via the VSG internalization pathway. The GQDs promoted intracellular ROS production, interacted with, and inhibited the activity of the p18 subunit of ATP synthase, disrupted parasite mitochondrial membrane potential. Additionally, the GQDs caused a decrease in aminoacyl - tRNA biosynthesis, and upregulated RNA and protein degradation pathways. The findings of this study offer a novel avenue for the target-oriented discovery of anti-trypanosome drugs., 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.)

Practical and conceptual barriers have kept human F-ATP synthase out of reach as a target for the treatment of human diseases. Although this situation has persisted for decades, it may change in the near future. In this review the principal functionalities of human F-ATP synthase--proton motive force / ATP interconversion, membrane bending and mitochondrial permeability transition--are surveyed in the context of their respective potential for pharmaceutical intervention. Further, the technical requirements necessary to allow drug designs that are effective at the multiple levels of functionality and modality of human F-ATP synthase are discussed. The structure-based development of gastric proton pump inhibitors is used to exemplify what might be feasible for human F-ATP synthase. And finally, four structural regions of the human F-ATP synthase are examined as potential sites for the development of structure based drug development., Competing Interests: Declaration of Competing Interest We hereby declare to have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. Despite their importance, there is a significant knowledge gap of druggable targets for inhibiting toxin production. To address this, we screened nonantibiotic phytochemicals to identify potential chemical genetic probes to discover antivirulence drug targets. This led to the identification of 18β-glycyrrhetinic acid (enoxolone), a licorice metabolite, as an inhibitor of TcdA and TcdB biosynthesis. Using affinity-based proteomics, potential targets were identified as ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade, which catalyzes conversion of adenine to hypoxanthine in the purine salvage pathway). To validate these targets, a multifaceted approach was adopted. Gene silencing of ade and atpA inhibited toxin biosynthesis, while surface plasmon resonance and isothermal titration calorimetry molecular interaction analyses revealed direct binding of enoxolone to Ade. Metabolomics demonstrated enoxolone induced the accumulation of adenosine, while depleting hypoxanthine and ATP in C. difficile. Transcriptomics further revealed enoxolone dysregulated phosphate uptake genes, which correlated with reduced cellular phosphate levels. These findings suggest that enoxolone's cellular action is multitargeted. Accordingly, supplementation with both hypoxanthine and triethyl phosphate, a phosphate source, was required to fully restore toxin production in the presence of enoxolone. In conclusion, through the characterization of enoxolone, we identified promising antivirulence targets that interfere with nucleotide salvage and ATP synthesis, which may also block toxin biosynthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase 1,2 . However, BDQ also inhibits human ATP synthase 3 . At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Mitochondrial dysfunction during ischemic stroke ultimately manifests as ATP depletion. Mitochondrial ATP synthase upon loss of mitochondrial membrane potential during ischemia rapidly hydrolyses ATP and thus contributes to ATP depletion. Increasing evidence suggests that inhibition of ATP synthase limits ATP depletion and is protective against ischemic tissue damage. Bedaquiline (BDQ) is an anti-microbial agent, approved for clinical use, that inhibits ATP synthase of Mycobacteria; however recently it has been shown to act on mitochondrial ATP synthase, inhibiting both ATP synthesis and hydrolysis in low micromolar concentrations. In this study, we investigated whether preconditioning with BDQ can alleviate ischemia/reperfusion-induced brain injury in Wistar rats after middle cerebral artery occlusion-reperfusion and whether it affects mitochondrial functions. We found that BDQ was effective in limiting necrosis and neurological dysfunction during ischemia-reperfusion. BDQ also caused inhibition of ATPase activity, mild uncoupling of respiration, and stimulated mitochondrial respiration both in healthy and ischemic mitochondria. Mitochondrial calcium retention capacity was unaffected by BDQ preconditioning. We concluded that BDQ has neuroprotective properties associated with its action on mitochondrial respiration and ATPase activity.

Disruption of retinal pigment epithelial (RPE) barrier integrity is involved in the pathology of several blinding retinal diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR), but the underlying causes and pathophysiology are not completely well-defined. Mitochondria dysfunction has often been considered as a potential candidate implicated in such a process. In this study, we aimed to dissect the role of different mitochondrial components; specifically, those of oxidative phosphorylation (OxPhos), in maintaining the barrier functionality of RPE. Electric cell-substrate impedance sensing (ECIS) technology was used to collect multi-frequency electrical impedance data to assess in real-time the barrier formation of the RPE cells. For this purpose, the human retinal pigment epithelial cell line-ARPE-19-was used and treated with varying concentrations of specific mitochondrial inhibitors that target different steps in OxPhos: Rotenone for complex I (the largest protein complex in the electron transport chain (ETC)); oligomycin for ATP synthase; and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) for uncoupling ATP synthesis from the accompanying ETC. Furthermore, data were modeled using the ECIS-Zθ software to investigate in depth the effects of these inhibitors on three separate barrier parameters: cell-cell interactions (R b ), cell-matrix interactions (α), and the cell membrane capacitance (C m ). The viability of ARPE-19 cells was determined by lactate dehydrogenase (LDH) Cytotoxicity Assay. The ECIS program's modeling demonstrated that FCCP and thus OxPhos uncoupling disrupt the barrier function in the ARPE-19 cells across all three components of the total resistance (Rb, α, and C m ) in a dose-dependent manner. On the other hand, oligomycin and thus ATP synthase inhibition mostly affects the ARPE-19 cells' attachment to their substrate evident by a significant decrease in α resistance in a dose-dependent manner, both at the end and throughout the duration of the experiment. On the contrary, rotenone and complex I inhibition mostly affect the ARPE-19 paracellular resistance R b in a dose-dependent manner compared to basolateral resistance α or C m . Our results clearly demonstrate differential roles for different mitochondrial components in maintaining RPE cell functionality in which uncoupling of OxPhos is a major contributing factor to the disruption barrier function. Such differences can be used in investigating gene expression as well as for screening of selective agents that improve the OxPhos coupling efficiency to be used in the therapeutic approach for treating RPE-related retinal diseases.

The spectacular success of the mycobacterial F 1 F 0 -ATP synthase inhibitor bedaquiline for the treatment of drug-resistant tuberculosis has generated wide interest in the development of other inhibitors of this enzyme. Work in this realm has included close analogues of bedaquiline with better safety profiles and 'bedaquiline-like' compounds, some of which show potent antibacterial activity in vitro although none have yet progressed to clinical trials. The search has lately extended to a range of new scaffolds as potential inhibitors, including squaramides, diaminoquinazolines, chloroquinolines, dihydropyrazolo[1,5-a]pyrazin-4-ones, thiazolidinediones, diaminopyrimidines and tetrahydroquinolines. Because of the ubiquitous expression of ATP synthase enzymes, there has also been interest in inhibitors of other bacterial ATP synthases, as well as inhibitors of human mitochondrial ATP synthase for cancer therapy. The latter encompass both complex natural products and simpler small molecules. The review seeks to demonstrate the breadth of the structural types of molecules able to effectively inhibit the function of variants of this intriguing enzyme.

Bone resorption by osteoclasts is an energy consuming activity, which depends on mitochondrial ATP. ATP5B, a mitochondrial ATP synthase beta subunit, is a catalytic core involved in producing ATP. Here, we investigated the contribution of ATP5B in osteoclast differentiation and joint destruction. ATP5B (LV-ATP5B) targeting or non-targeting (LV-NC) siRNA containing lentivirus particles were transduced into bone marrow macrophage derived osteoclasts or locally administered to arthritic mouse joints. Inhibition of ATP5B reduced the expression of osteoclast related genes and proteins, suppressed bone resorption by significantly impairing F-actin formation and decreased the levels of adhesion-associated proteins. In addition, ATP5B deficiency caused osteoclast mitochondrial dysfunction and, impaired the secretion of vacuole protons and MMP9. Importantly, inhibition of ATP5B expression, protected arthritis mice from joint destructions although serum levels of inflammatory mediators (TNF-α, IL-1β) and IgG2α antibodies were unaffected. These results demonstrate an essential function of ATP5B in osteoclast differentiation and bone resorption, and suggest it as a potential therapeutic target for protecting bones in RA., (Copyright © 2021. Published by Elsevier Ltd.)

Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated. Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models. Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro . Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo , MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway. Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

F 1 F O ATP synthase is responsible for the production of >95% of all ATP synthesis within the cell. Dysregulation of its expression, activity or localization is linked to various human diseases including cancer, diabetes, and Alzheimer's and Parkinson's disease. In addition, ATP synthase is a novel and viable drug target for the development of antimicrobials as evidenced by bedaquiline, which was approved in 2012 for the treatment of tuberculosis. Historically, natural products have been a rich source of ATP synthase inhibitors that help unravel the role of F 1 F O ATP synthase in cellular bioenergetics. During the last decade, new modulators of ATP synthase have been discovered through the isolation of novel natural products as well as through a ligand-based drug design process. In addition, new data has been obtained with regards to the structure and function of ATP synthase under physiological and pathological conditions. Crystal structure studies have provided a significant insight into the rotary function of the enzyme and may provide additional opportunities to design a new generation of inhibitors. This review provides an update on recently discovered ATP synthase modulators as well as an update on existing scaffolds., 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 © 2020 Elsevier Masson SAS. All rights reserved.)

Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis. However, Sirturo treatment leads to an increased risk of cardiac arrhythmias and death, raising the concern that this adverse effect results from inhibition at a secondary site. Here we show that BDQ is a potent inhibitor of the yeast and human mitochondrial ATP synthases. Single-particle cryo-EM reveals that the site of BDQ inhibition partially overlaps with that of the inhibitor oligomycin. Molecular dynamics simulations indicate that the binding mode of BDQ to this site is similar to that previously seen for a mycobacterial enzyme, explaining the observed lack of selectivity. We propose that derivatives of BDQ ought to be made to increase its specificity toward the mycobacterial enzyme and thereby reduce the side effects for patients that are treated with Sirturo.

Few discoveries have influenced drug discovery programs more than the finding that mitochondrial membranes undergo swings in permeability in response to cellular perturbations. The conductor of these permeability changes is the aptly named mitochondrial permeability transition pore which, although not yet precisely defined, is comprised of several integral proteins that differentially act to regulate the flux of ions, proteins and metabolic byproducts during the course of cellular physiological functions but also pathophysiological insults. Pursuit of the pore's exact identity remains a topic of keen interest, but decades of research have unearthed provocative functions for the integral proteins leading to their evaluation to develop novel therapeutics for a wide range of clinical indications. Chief amongst these targeted, integral proteins have been the Voltage Dependent Anion Channel (VDAC) and the F 1 F O ATP synthase. Research associated with the roles and ligands of VDAC has been extensive and we will expand upon 3 examples of ligand:VDAC interactions for consideration of drug discovery projects: Tubulin:VDAC1, Hexokinase I/II:VDAC1 and olesoxime:VDAC1. The discoveries that cyclosporine blocks mitochondrial permeability transition via binding to cyclophilin D, and that cyclophilin D is an important component of F 1 F O ATP synthase, has heightened interest in the F 1 F O ATP synthase as a focal point for drug discovery, and we will discuss 2 plausible campaigns associated with disease indications. To date no drug has emerged from prospective targeting these integral proteins; however, continued exploration such as the approaches suggested in this Commentary will increase the likelihood of providing important therapeutics for severely unmet medical needs., 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 © 2020 Elsevier Inc. All rights reserved.)

Modulation of prostate stromal cells (PrSCs) within tumor tissues is gaining attention for the treatment of solid tumors. Using our original in vitro coculture system, we previously reported that leucinostatin (LCS)-A, a peptide mycotoxin, inhibited prostate cancer DU-145 cell growth through reduction of insulin-like growth factor 1 (IGF-I) expression in PrSCs. To further obtain additional bioactive compounds from LCS-A, we designed and synthesized a series of LCS-A derivatives as compounds that target PrSCs. Among the synthesized LCS-A derivatives, LCS-7 reduced IGF-I expression in PrSCs with lower toxicity to PrSCs and mice than LCS-A. As LCS-A has been suggested to interact with mitochondrial adenosine triphosphate (ATP) synthase, a docking study was performed to elucidate the mechanism of reduced IGF-I expression in the PrSCs. As expected, LCS-A and LCS-7 directly interacted with mitochondrial ATP synthase, and like LCS-A and LCS-7, other mitochondrial ATP synthase inhibitors also reduced the expression of IGF-I by PrSCs. Furthermore, LCS-A and LCS-7 significantly decreased the growth of mouse xenograft tumors. Based on these data, we propose that the mitochondrial ATP synthases-IGF-I axis of PrSCs plays a critical role on cancer cell growth and inhibition could be a potential anticancer target for prostate cancer., (© 2020 UICC.)

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

Acquired resistance to HER2-targeted therapies occurs frequently in HER2 + breast tumors and new strategies for overcoming resistance are needed. Here, we report that resistance to trastuzumab is reversible, as resistant cells regained sensitivity to the drug after being cultured in drug-free media. RNA-sequencing analysis showed that cells resistant to trastuzumab or trastuzumab + pertuzumab in combination increased expression of oxidative phosphorylation pathway genes. Despite minimal changes in mitochondrial respiration, these cells exhibited increased expression of ATP synthase genes and selective dependency on ATP synthase function. Resistant cells were sensitive to inhibition of ATP synthase by oligomycin A, and knockdown of ATP5J or ATP5B, components of ATP synthase complex, rendered resistant cells responsive to a low dose of trastuzumab. Furthermore, combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant tumors in vivo . In conclusion, we identify a novel vulnerability of cells with acquired resistance to HER2-targeted antibody therapies and reveal a new therapeutic strategy to overcome resistance. SIGNIFICANCE: These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies., (©2019 American Association for Cancer Research.)

Background: Neonatal Encephalopathy (NE) is a mitochondrial ATP synthase (mATPase) disease, which results in the death of infants. The case presented here is reportedly caused by complex V deficiency as a result of mutation of Arginine to Cysteine at residue 329 in the mATPase. A recent breakthrough was the discovery of J147, which targets mATPase in the treatment of Alzheimer's disease. Based on the concepts of computational target-based drug design, this study investigated the possibility of employing J147 as a viable candidate in the treatment of NE., Objective/methods: The structural dynamic implications of this drug on the mutated enzyme are yet to be elucidated. Hence, integrative molecular dynamics simulations and thermodynamic calculations were employed to investigate the activity of J147 on the mutated enzyme in comparison to its already established inhibitory activity on the wild-type enzyme., Results: A correlated structural trend occurred between the wild-type and mutant systems whereby all the systems exhibited an overall conformational transition. Equal observations in favorable free binding energies further substantiated uniformity in the mobility, and residual fluctuation of the wild-type and mutant systems. The similarity in the binding landscape suggests that J147 could as well modulate mutant mATPase activity in addition to causing structural modifications in the wild-type enzyme., Conclusion: Findings suggest that J147 can stabilize the mutant protein and restore it to a similar structural state as the wild-type which depicts functionality. These details could be employed in drug design for potential drug resistance cases due to mATPase mutations that may present in the future., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 μM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.

Research has begun to elucidate the signal transduction pathway(s) that control cellular responses to changes in mitochondrial status. Important tools in such studies are chemical inhibitors used to initiate mitochondrial dysfunction. This study compares the effect of different inhibitors and treatment conditions on the transcript amount of nuclear genes specifically responsive to mitochondrial dysfunction in leaf of Nicotiana tabacum L. cv. Petit Havana. The Complex III inhibitors antimycin A (AA) and myxothiazol (MYXO), and the Complex V inhibitor oligomycin (OLIGO), each increased the transcript amount of the mitochondrial dysfunction genes. Transcript responses to OLIGO were greater during treatment in the dark than in the light, and the dark treatment resulted in cell death. In the dark, transcript responses to AA and MYXO were similar to one another, despite MYXO leading to cell death. In the light, transcript responses to AA and MYXO diverged, despite cell viability remaining high with either inhibitor. This divergent response may be due to differential signaling from the chloroplast because only AA also inhibited cyclic electron transport, resulting in a strong acceptor-side limitation in photosystem I. In the light, chemical inhibition of chloroplast electron transport reduced transcript responses to AA, while having no effect on the response to MYXO, and increasing the response to OLIGO. Hence, when studying mitochondrial dysfunction signaling, different inhibitor and treatment combinations differentially affect linked processes (e.g. chloroplast function and cell fate) that then contribute to measured responses. Therefore, inhibitor and treatment conditions should be chosen to align with specific study goals., (© 2018 Scandinavian Plant Physiology Society.)

The shortcomings of conventional tuberculosis treatments resulting from the development of drug resistance in Mycobacterium tuberculosis drive a need for the formulation of novel therapeutic agents. The diarylquinoline class of drugs such as bedaquiline was recently approved for the treatment of multidrug-resistant strains of tuberculosis, primarily targeting c and ε subunits of the ATP synthases. Yet resistance to bedaquiline has already been reported. Therefore, Rv1311 was used as the target for the identification of possible inhibitors against the M. tuberculosis. The structure of Rv1311 was predicted and common feature pharmacophore models were generated which facilitated the identification of potential inhibitors in the ZINC database. The activities of the selected molecules were compared with known inhibitors of the ATP synthase using quantitative structure-activity relationship. The ZINC classified inhibitors showed comparable predicted activities with that of known inhibitors. Furthermore, the inhibitory behavior of the studied drug molecules was experimentally determined using in vitro techniques and showed the minimum inhibitory concentration as low as 25 μM. The resulted outcomes provide a deeper insight into the structural basis of Rv1311 inhibitions and can facilitate the process of drug design against tuberculosis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Primary breast and prostate cancers can be cured, but metastatic disease cannot. Identifying cell factors that predict metastatic potential could guide both prognosis and treatment., Methods: We used Cell-SELEX to screen an RNA aptamer library for differential binding to prostate cancer cell lines with high vs. low metastatic potential. Mass spectroscopy, immunoblot, and immunohistochemistry were used to identify and validate aptamer targets. Aptamer properties were tested in vitro, in xenograft models, and in clinical biopsies. Gene expression datasets were queried for target associations in cancer., Results: We identified a novel aptamer (Apt63) that binds to the beta subunit of F 1 F o ATP synthase (ATP5B), present on the plasma membrane of certain normal and cancer cells. Apt63 bound to plasma membranes of multiple aggressive breast and prostate cell lines, but not to normal breast and prostate epithelial cells, and weakly or not at all to non-metastasizing cancer cells; binding led to rapid cell death. A single intravenous injection of Apt63 induced rapid, tumor cell-selective binding and cytotoxicity in MDA-MB-231 xenograft tumors, associated with endonuclease G nuclear translocation and DNA fragmentation. Apt63 was not toxic to non-transformed epithelial cells in vitro or adjacent normal tissue in vivo. In breast cancer tissue arrays, plasma membrane staining with Apt63 correlated with tumor stage (p < 0.0001, n = 416) and was independent of other cancer markers. Across multiple datasets, ATP5B expression was significantly increased relative to normal tissue, and negatively correlated with metastasis-free (p = 0.0063, 0.00039, respectively) and overall (p = 0.050, 0.0198) survival., Conclusion: Ecto-ATP5B binding by Apt63 may disrupt an essential survival mechanism in a subset of tumors with high metastatic potential, and defines a novel category of cancers with potential vulnerability to ATP5B-targeted therapy. Apt63 is a unique tool for elucidating the function of surface ATP synthase, and potentially for predicting and treating metastatic breast and prostate cancer.

The mitochondrial adenosine triphosphate (ATP) synthase produces most of the ATP required by mammalian cells. We isolated porcine tetrameric ATP synthase and solved its structure at 6.2-angstrom resolution using a single-particle cryo-electron microscopy method. Two classical V-shaped ATP synthase dimers lie antiparallel to each other to form an H-shaped ATP synthase tetramer, as viewed from the matrix. ATP synthase inhibitory factor subunit 1 (IF1) is a well-known in vivo inhibitor of mammalian ATP synthase at low pH. Two IF1 dimers link two ATP synthase dimers, which is consistent with the ATP synthase tetramer adopting an inhibited state. Within the tetramer, we refined structures of intact ATP synthase in two different rotational conformations at 3.34- and 3.45-Å resolution., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Some environmental pollutants impair mitochondria, which are of vital importance as energy factories in eukaryotic cells. Mitochondrial toxicity was quantified by measuring the change of the oxygen consumption rate (OCR) of HepG2 cells with the Agilent Seahorse XF e 96 Analyzer. Various mechanisms of mitochondrial toxicity, including inhibition of the electron transport chain or adenosine triphosphate (ATP) synthase as well as uncoupling of oxidative phosphorylation, were differentiated by dosing the sample in parallel with reference compounds following the OCR over time. These time-OCR traces were used to derive effect concentrations for 10% inhibition of the electron transport chain or 10% of uncoupling. The low effect level of 10% was necessary because environmental mixtures contain thousands of chemicals; only few of them interfere with mitochondria, but the others cause cytotoxicity. The OCR bioassay was validated with environmental pollutants of known mechanism of mitochondrial toxicity. Binary mixtures of uncouplers or inhibitors acted according to the mixture model of concentration addition. Uncoupling and/or inhibitory effects were detected in extracts of river water samples without apparent cytotoxicity. Uncoupling effects could only be quantified in water samples if inhibitory effects occurred at lower concentrations because no uncoupling can be detected without an appreciable membrane potential built up. The OCR bioassay can thus complement chemical analysis and in vitro bioassays for monitoring micropollutants in water. Environ Toxicol Chem 2019;00:1-12. © 2019 SETAC., (© 2019 SETAC.)

Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective antibacterial activity toward Streptococcus pneumoniae. Pneumococcal disease remains the leading source of bacterial pneumonia and meningitis worldwide. The structure-activity relationships of optochin were examined through modification to both the quinoline and quinuclidine subunits, which led to the identification of analogue 48 with substantially improved activity. Resistance and molecular modeling studies indicate that 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52 ion-binding site, while mechanistic studies demonstrated that 48 causes cytoplasmic acidification. Initial pharmacokinetic and drug metabolism analyses of optochin and 48 revealed limitations of these quinine analogues, which were rapidly cleared, resulting in poor in vivo exposure through hydroxylation pendants to the quinuclidine and O-dealkylation of the quinoline. Collectively, the results provide a foundation to advance 48 and highlight ATP synthase as a promising target for antibiotic development.

Although orphan G protein-coupled receptors (GPCRs) have been used as targets to discover unidentified natural ligands, increasing numbers of non-GPCRs have been found to mediate important biological functions. Bioinformatics of genome and cDNA resources predict putative bioactive peptides, demanding an alternative approach to efficiently unravel cell surface targets. In silico analysis of a full-length cDNA library previously allowed us to identify salusin-β, a parasympathomimetic/pro-atherosclerotic peptide with unique physicochemical properties. Here, we show that the β-chain of ATP synthase is a cell surface receptor for salusin-β by utilizing artificial liposomes embedded with endogenous membrane proteins directly transferred from animal tissues while retaining the ligand-binding capability. Conventional techniques using detergents identified a β-actin-profilin complex as membrane-associated salusin-β-binding proteins, but failed to identify the cell surface receptor. Since the α-chain of ATP synthase is a principal cell surface target for angiostatin, a potent endogenous angiogenesis inhibitor, we investigated whether salusin-β modulates angiogenesis. Salusin-β inhibited cell surface ATP synthase activity and prevented sarcoma cell-induced angiogenesis in an in vivo mouse air sac model. Therefore, salusin-β binds to membrane-bound ATP synthase and acts as an angiogenesis inhibitor. The current methodology allows the identification of novel cell surface targets, irrespective of the receptor structure.

Purpose: To identify retinal protein changes that mediate beneficial effects of intravitreal bevacizumab in experimental branch retinal vein occlusion (BRVO)., Methods: In six Danish Landrace pigs, BRVO was induced with argon laser in both eyes. After BRVO was induced, the right eye of each animal was given an intravitreal injection of bevacizumab while the left eye was treated with saline water. The retinas were collected 15 days after BRVO, and differentially expressed proteins were analyzed with tandem mass tags-based mass spectrometry. Validation of statistically significantly changed proteins was performed with immunohistochemistry and western blotting., Results: Fluorescein angiography showed no recanalization of the occluded vessels. A total of 4,013 proteins were successfully identified and quantified. Nine proteins were statistically significantly changed following bevacizumab intervention. In experimental BRVO, bevacizumab treatment resulted in upregulation of transthyretin (TTR) and pantothenate kinase 3. Bevacizumab downregulated protocadherin 7, protein FAM192A, and ATP synthase protein 8. Immunohistochemistry revealed that TTR was highly abundant in the choroid following bevacizumab intervention., Conclusions: Bevacizumab intervention in experimental BRVO resulted in an increased level of TTR. This is the second study in which we showed an increased retinal level of TTR following anti-vascular endothelial growth factor (VEGF) intervention in experimental BRVO. We hypothesize that there is an interaction between TTR and VEGF and that bevacizumab may exert a beneficial effect on the retina by upregulating TTR.

Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFκB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol, therefore we analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin (10 µM) inhibits the activity of ATP synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro and in vivo tumor models. The effects of curcumin on ATP synthase are independent of the inhibition of NFκB since the IκB Kinase inhibitor, SC-514, does not affect ATP synthase. The activities of the glycolytic enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type-specific manner. The energy impairment translates into decreased tumor cell viability. Moreover, curcumin induces apoptosis by promoting the generation of reactive oxygen species (ROS) and malondialdehyde (MDA), a marker of lipid oxidation, and autophagy, at least in part due to the activation of the AMP-activated protein kinase (AMPK). According to the in vitro anti-tumor effect, curcumin (30 mg/kg body weight) significantly delayed in vivo cancer growth likely due to an energy impairment but also through the reduction of tumor angiogenesis. These results establish the ATP synthase, a central enzyme of the cellular energy metabolism, as a target of the antitumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.

Enzymes in the respiratory chain are increasingly seen as potential targets against multi-drug resistance of human pathogens and cancerous cells. However, a detailed understanding of the mechanism and specificity determinants of known inhibitors is still lacking. Oligomycin, for example, has been known to be an inhibitor of the membrane motor of the mitochondrial ATP synthase for over five decades, and yet little is known about its mode of action at the molecular level. In a recent breakthrough, a crystal structure of the S. cerevisiae c-subunit ring with bound oligomycin revealed the inhibitor docked on the outer face of the proton-binding sites, deep into the transmembrane region. However, the structure of the complex was obtained in an organic solvent rather than detergent or a lipid bilayer, and therefore it has been unclear whether this mode of recognition is physiologically relevant. Here, we use molecular dynamics simulations to address this question and gain insights into the mechanism of oligomycin inhibition. Our findings lead us to propose that oligomycin naturally partitions into the lipid/water interface, and that in this environment the inhibitor can indeed bind to any of the c-ring proton-carrying sites that are exposed to the membrane, thereby becoming an integral component of the proton-coordinating network. As the c-ring rotates within the membrane, driven either by downhill proton permeation or ATP hydrolysis, one of the protonated, oligomycin-bound sites eventually reaches the subunit-a interface and halts the rotary mechanism of the enzyme., (Copyright © 2018. Published by Elsevier B.V.)

Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000 U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000 U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF 1 subunits, prevent the wasteful CCW F 1 F O -ATPase activity by blocking γ rotation at the α DP /β DP /γ interface of the F 1 portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F 1 -ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF 1 , it also binds to the α DP /β DP /γ interface of the F 1 of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the α DP /β DP /γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW F 1 F O -ATPase rotation without affecting the CW-F 1 F O -ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdF 1 F O -ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF 1 but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF 1 ) of this unique ATP synthase nanomotor, essential for life., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recent cardiology research studies have reported the role, function, and structure of the mitochondrial permeability transition pore (mPTP) and have shown that its opening plays a key role in the progression of myocardial cell death secondary to reperfusion. In this manuscript, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and describe the discovery, optimization, and structure-activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F 1 /F O -ATP synthase complex. We identified three potential compounds with good mPTP inhibitory activity and beneficial effects in a model of MI, including a decreased apoptotic rate in the whole heart and overall improvement of cardiac function upon administration during reperfusion. The selected compounds did not show off-target effects at the cellular and mitochondrial levels. Moreover, the compounds preserved the mitochondrial ATP content despite interacting with the ATP synthase complex.

Background: Many neuronal and glial diseases have been associated with changes in metabolism. Therefore, metabolic reprogramming has become an important area of research to better understand disease at the cellular level, as well as to identify targets for treatment. Model systems are ideal for interrogating metabolic questions in a tissue dependent context. However, while new tools have been developed to study metabolism in cultured cells there has been less progress towards studies in vivo and ex vivo., New Method: We have developed a method using newly designed tissue restraints to adapt the Agilent XFe96 metabolic analyzer for whole brain analysis. These restraints create a chamber for Drosophila brains and other small model system tissues to reside undisrupted, while still remaining in the zone for measurements by sensor probes., Results: This method generates reproducible oxygen consumption and extracellular acidification rate data for Drosophila larval and adult brains. Single brains are effectively treated with inhibitors and expected metabolic readings are observed. Measuring metabolic changes, such as glycolytic rate, in transgenic larval brains demonstrates the potential for studying how genotype affects metabolism., Comparison With Existing Methods and Conclusions: Current methodology either utilizes whole animal chambers to measure respiration, not allowing for targeted tissue analysis, or uses technically challenging MRI technology for in vivo analysis that is not suitable for smaller model systems. This new method allows for novel metabolic investigation of intact brains and other tissues ex vivo in a quick, and simplistic way with the potential for large-scale studies., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

Studies have demonstrated that zebrafish are powerful tools for monitoring environmental toxicity, including radiation hazard. Here we investigated the developmental toxicity of ionizing radiation (IR) in an in vivo embryonic zebrafish model. The effects of heavy ion ( 12 C 6+ ), proton, and X-ray radiation on early zebrafish embryos were determined. A similar dose-dependent decrease in the hatch and survival rate of zebrafish embryos was observed after exposure to these irradiations. Exposure of zebrafish embryos to 1-4 Gy IR caused significant loss of pigmentation. Quantitative real-time reverse transcription polymerase chain reaction, western blot analysis, and in situ hybridization (ISH) experiment revealed that atp5α1 was markedly upregulated in irradiated zebrafish embryos. In addition, IR resulted in a rapid decrease in total adenosine triphosphate (ATP) generation. With dual functions of synthesizing or hydrolyzing ATP, ATP synthase regulated H + transport crossing the mitochondrial inner. Administration of the mitochondrial ATP synthase inhibitor, oligomycin, partially restored pigmentation in irradiated zebrafish embryos, but the ATPase inhibitor, BTB06584, had no effect. Taken together, these results showed that IR exposure downregulated zebrafish pigmentation through regulation of H + ion transport in mitochondria., (© 2018 Wiley Periodicals, Inc.)

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague-Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Bcl-x L is an anti-apoptotic protein that localizes to the outer mitochondrial membrane and influences mitochondrial bioenergetics by controlling Ca 2+ influx into mitochondria. Here, we analyzed the effect of mitochondrial Bcl-x L on mitochondrial shape and function in knockout (KO), wild type and rescued mouse embryonic fibroblast cell lines. Mitochondria of KO cells were more fragmented, exhibited a reduced ATP concentration, and reduced oxidative phosphorylation (OXPHOS) suggesting an increased importance of ATP generation by other means. Under steady-state conditions, acidification of the growth medium as a readout for glycolysis was similar, but upon inhibition of ATP synthase with oligomycin, KO cells displayed an instant increase in glycolysis. In addition, forced energy production through OXPHOS by replacing glucose with galactose in the growth medium rendered KO cells more susceptible to mitochondrial toxins. KO cells had increased cellular reactive oxygen species and were more susceptible to oxidative stress, but had higher glutathione levels, which were however more rapidly consumed under conditions of oxidative stress. This coincided with an increased activity and protein abundance of the pentose phosphate pathway protein glucose-6-phosphate dehydrogenase, which generates NADPH necessary to regenerate reduced glutathione. KO cells were also less susceptible to pharmacological inhibition of the pentose phosphate pathway. We conclude that mitochondrial Bcl-x L is involved in maintaining mitochondrial respiratory capacity. Its deficiency causes oxidative stress, which is associated with an increased glycolytic capacity and balanced by an increased activity of the pentose phosphate pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Asthma is a complicated systemic disease of the airways, which is characterized by variable symptoms, including bronchial hyper‑responsive-ness, inflammation and airflow obstruction. The prevalence of asthma has increased 2‑3‑fold over recent decades in developed countries; however, the molecular mechanism of asthma remains unclear. In the current study, the expression of recombinant protein Dermatophagoides farinaeI (Derf I) was induced by isopropyl β‑D‑1‑thiogalactoside (IPTG) and purified using Ni‑NTA. Derf I, an important antigen of asthma, was used to establish the animal model of asthma. Airway hyper‑responsiveness was mea-sured using unrestrained whole‑body plethysmography with a four‑chamber system. Immunoglobulin (Ig)E, IgG and IgG2a were analyzed using indirect enzyme‑linked immunosorbent assay (ELISA). Proteomic technology was applied to detect the difference between the normal lung tissue and asthma lung tissue samples of the asthma model. Cytokines in bronchoalveolar lavage fluid and the splenocyte culture medium were measured by ELISA and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to detect the mRNA expression of ATP synthase, H+ transporting, mitochondrial F1 complex, β polypeptide (ATP5b). In addition, cell growth of arterial smooth muscle cells (ASMCs) was evaluated by MTT assay. In the current study, Derf I was successfully used to construct the animal model of asthma. Out of 23 proteins that exhibit 3‑fold upregulation or downregulation, ATP5b was chosen for further investigation. The data indicated that ATP5b was overexpressed in the asthma lung tissue when compared with the normal lung tissue. However, when ATP5b was knocked down, cell growth decreased. Therefore, overexpressed ATP5b leads to airway smooth muscle cell (ASMC) proliferation and finally to ASM thickening. Thus, to the best of our knowledge, this is the first study to report that the expression level of ATP5b was markedly increased in lung tissue samples of an asthma model compared with the tissue samples from normal lungs, which promoted ASMC proliferation and contributed to airway remodeling.

Mandelalides A-D (1-4) are macrocyclic polyketides known to have an unusual bioactivity profile influenced by compound glycosylation and growth phase of cultured cells. The isolation and characterization of additional natural congeners, mandelalides E-L (5-12), and the supply of synthetic compounds 1 and 12, as well as seco-mandelalide A methyl ester (13), have now facilitated mechanism of action and structure-activity relationship studies. Glycosylated mandelalides are effective inhibitors of aerobic respiration in living cells. Macrolides 1 and 2 inhibit mitochondrial function similar to oligomycin A and apoptolidin A, selective inhibitors of the mammalian ATP synthase (complex V). 1 inhibits ATP synthase activity from isolated mitochondria and triggers caspase-dependent apoptosis in HeLa cells, which are more sensitive to inhibition by 1 in the presence of the glycolysis inhibitor 2-deoxyglucose. Thus, mandelalide cytotoxicity depends on basal metabolic phenotype; cells with an oxidative phenotype are most likely to be inhibited by the mandelalides.

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.

Background: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints., Methods: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated., Results: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue., Conclusions: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

Introduction: The potential of a radioiodine-labeled, anti-adenosine triphosphate synthase monoclonal antibody (ATPS mAb) as a theragnostic agent for simultaneous cancer imaging and treatment was evaluated., Methods: Adenosine triphosphate synthase monoclonal antibody was labeled with radioiodine, then radiotracer uptake was measured in 6 different cancer cell lines. In vivo biodistribution was evaluated 24 and 48 hours after intravenous injection of 125 I-ATPS mAb into MKN-45 tumor-bearing mice (n = 3). For radioimmunotherapy, 18.5 MBq 131 I-ATPS mAb (n = 7), isotype immunoglobulin G (IgG) (n = 6), and vehicle (n = 6) were injected into MKN-45 tumor-bearing mice for 4 weeks, and tumor volume and percentage of tumor growth inhibition (TGI) were compared each week., Results: MKN-45 cells showed the highest in vitro cellular binding after 4 hours (0.00324 ± 0.00013%/μg), which was significantly inhibited by unlabeled ATPS mAb at concentrations of greater than 0.4 μM. The in vitro retention rate of 125 I-ATPS mAb in MKN-45 cells was 64.1% ± 1.0% at 60 minutes. The highest tumor uptake of 125 I-ATPS mAb in MKN-45 tumor-bearing mice was achieved 24 hours after injection (6.26% ± 0.47% injected dose [ID]/g), whereas tumor to muscle and tumor to blood ratios peaked at 48 hours. The 24-hour tumor uptake decreased to 3.43% ± 0.85% ID/g by blocking with unlabeled ATPS mAb. After 4 weeks of treatment, mice receiving 131 I-ATPS mAb had significantly smaller tumors (679.4 ± 232.3 mm 3 ) compared with control (1687.6 ± 420.4 mm 3 , P = .0431) and IgG-treated mice (2870.2 ± 484.1 mm 3 , P = .0010). The percentage of TGI of 131 I-ATPS mAb was greater than 50% during the entire study period (range: 53.7%-75.9%)., Conclusion: The specific binding and antitumor effects of radioiodinated ATPS mAb were confirmed in in vitro and in vivo models of stomach cancer.

A large conductance potassium (BKCa) channel opener, NS1619 (1,3-dihydro-1- [2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one), is well known for its protective effects against ischemia-reperfusion injury; however, the exact mode of its action remains unclear. The aim of this study was to characterize the effect of NS1619 on endothelial cells. The endothelial cell line EA.hy926, guinea pig hearts and submitochondrial particles isolated from the heart were used. In the isolated guinea pig hearts, which were perfused using the Langendorff technique, NS1619 caused a dose-dependent increase in coronary flow that was inhibited by L-NAME. In EA.hy926 cells, NS1619 also caused a dose-dependent increase in the intracellular calcium ion concentration [Ca(2+)]i, as measured using the FURA-2 fluorescent probe. Moreover, NS1619 decreased the oxygen consumption rate in EA.hy926 cells, as assessed using a Clark-type oxygen electrode. However, when NS1619 was applied in the presence of oligomycin, the oxygen consumption increased. NS1619 also decreased the mitochondrial membrane potential, as measured using a JC-1 fluorescent probe in the presence and absence of oligomycin. Additionally, the application of NS1619 to submitochondrial particles inhibited ATP synthase. In summary, NS1619 has pleiotropic actions on EA.hy926 cells and acts not only as an opener of the BKCa channel in EA.hy926 cells but also as an inhibitor of the respiratory chain component, sarcoplasmic reticulum ATPase, which leads to the release of Ca(2+) from the endoplasmic reticulum. Furthermore, NS1619 has the oligomycin-like property of inhibiting mitochondrial ATP synthase., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease associated with high morbidity and mortality rates. However, the exact regulatory mechanism of PAH is unknown. Although coupling factor 6 (CF6) is known to function as a repressor, its role in PAH has not been explored. Here, we investigated the involvement of endogenous CF6 in the development of PAH., Methods: PAH was induced with monocrotaline (MCT), as demonstrated by significant increases in pulmonary artery pressure and vessel wall thickness. The adeno-associated virus (AAV) carrying CF6 short hairpin RNA (shRNA) or control vector (2×10(10) gp) was intratracheally transfected into the lungs of rats 2 weeks before or after MCT injection., Results: A 2-6-fold increase in CF6 was observed in the lungs and circulation of the MCT-injected rats as confirmed by qRT-PCR and ELISA. Immunohistochemistry analysis revealed a small quantity of CF6 localized to endothelial cells (ECs) under physiological conditions spread to surrounding tissues in a paracrine manner in PAH lungs. Notably, CF6 shRNA effectively inhibited CF6 expression, abolished lung macrophage infiltration, reversed endothelial dysfunction and vascular remodeling, and ameliorated the severity of pulmonary hypertension and right ventricular dysfunction at 4 weeks both as a pretreatment and rescue intervention. In addition, the circulating and lung levels of 6-keto-PGF1a, a stable metabolite of prostacyclin, were reversed by CF6 inhibition, suggesting that the effect of CF6 inhibition may partly be mediated through prostacyclin., Conclusions: CF6 contributes to the pathogenesis of PAH, probably in association with downregulation of prostacyclin. The blockage of CF6 might be applied as a novel therapeutic approach for PAH and PA remodeling.

The mitochondrial permeability transition is an inner mitochondrial membrane event involving the opening of the permeability transition pore concomitant with a sudden efflux of matrix solutes and breakdown of membrane potential. The mitochondrial F(o)F(1) ATP synthase has been proposed as the molecular identity of the permeability transition pore. The likeliness of potential pore-forming sites in the mitochondrial F(o)F(1) ATP synthase is discussed and a new model, the death finger model, is described. In this model, movement of a p-side density that connects the lipid-plug of the c-ring with the distal membrane bending Fo domain allows reversible opening of the c-ring and structural cross-talk with OSCP and the catalytic (αβ)(3) hexamer. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016 The Author. Published by Elsevier B.V. All rights reserved.)