Mitochondria and mitochondrial DNA as relevant targets for environmental contaminants.

PubMed

Roubicek, Deborah A; Souza-Pinto, Nadja C de

2017-11-01

The mitochondrial DNA (mtDNA) is a closed circular molecule that encodes, in humans, 13 polypeptides components of the oxidative phosphorylation complexes. Integrity of the mitochondrial genome is essential for mitochondrial function and cellular homeostasis, and mutations and deletions in the mtDNA lead to oxidative stress, mitochondrial dysfunction and cell death. In vitro and in situ studies suggest that when exposed to certain genotoxins, mtDNA accumulates more damage than nuclear DNA, likely owing to its organization and localization in the mitochondrial matrix, which tends to accumulate lipophilic, positively charged molecules. In that regard, several relevant environmental and occupational contaminants have physical-chemical characteristics that indicate that they might accumulate in mitochondria and target mtDNA. Nonetheless, very little is known so far about mtDNA damage and mitochondrial dysfunction due to environmental exposure, either in model organisms or in humans. In this article, we discuss some of the characteristics of mtDNA which render it a potentially relevant target for damage by environmental contaminants, as well as possible functional consequences of damage/mutation accumulation. In addition, we review the data available in the literature focusing on mitochondrial effects of the most common classes of environmental pollutants. From that, we conclude that several lines of experimental evidence support the idea that mitochondria and mtDNA are susceptible and biologically relevant targets for pollutants, and more studies, including mechanistic ones, are needed to shed more light into the contribution of mitochondrial dysfunction to the environmental and human health effects of chemical exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

Mitochondrial Calcium Transport in Trypanosomes

PubMed Central

Docampo, Roberto; Vercesi, Anibal E.; Huang, Guozhong

2014-01-01

The biochemical peculiarities of trypanosomes were fundamental for the recent molecular identification of the long-sought channel involved in mitochondrial Ca2+ uptake, the mitochondrial Ca2+ uniporter or MCU. This discovery led to the finding of numerous regulators of the channel, which form a high molecular weight complex with MCU. Some of these regulators have been bioinformatically identified in trypanosomes, which are the first eukaryotic organisms described for which MCU is essential. In trypanosomes MCU is important for buffering cytosolic Ca2+ changes and for activation of the bioenergetics of the cells. Future work on this pathway in trypanosomes promises further insight into the biology of these fascinating eukaryotes, as well as the potential for novel target discovery. PMID:25218432

Chemoprevention of obesity by dietary natural compounds targeting mitochondrial regulation.

PubMed

Lai, Ching-Shu; Wu, Jia-Ching; Ho, Chi-Tang; Pan, Min-Hsiung

2017-06-01

Mitochondria are at the center stage in the control of energy homeostasis in many organs and tissues including adipose tissue. Recently, abundant evidence from experimental studies has clearly supported the strong correlation between mitochondrial dysfunction in adipocytes and obesity. Various physiological conditions such as excessive nutrition, genetic factors, hypoxia, and toxins disrupt mitochondrial function by impairing mitochondrial biogenesis, dynamics, and oxidative capacity. Mitochondrial dysfunction in adipocytes could have an impact on differentiation, adipogenesis, insulin sensitivity, and the significant alteration in their metabolic function, which ultimately results in obesity and type 2 diabetes. Numerous dietary natural compounds are the subject of research for the prevention and treatment of obesity through reprogramming multiple metabolic pathways. Some of them have the potential against obesity by modulating insulin signaling, decreasing oxidative damage, downregulating adipokines secretion, and increasing mitochondrial DNA that improves mitochondrial function and thus maintain metabolic homeostasis. Here, we focus on and summarize and briefly discuss the currently known targets and the mitochondria-targeting effects of dietary natural compounds in the intervention of obesity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mitochondrial Gene Therapy: Advances in Mitochondrial Gene Cloning, Plasmid Production, and Nanosystems Targeted to Mitochondria.

PubMed

Coutinho, Eduarda; Batista, Cátia; Sousa, Fani; Queiroz, João; Costa, Diana

2017-03-06

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes

NASA Astrophysics Data System (ADS)

Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi

2012-08-01

Mitochondrial gene therapy has the potential for curing a variety of diseases that are associated with mitochondrial DNA mutations and/or defects. To achieve this, it will be necessary to deliver therapeutic agents into the mitochondria in diseased cells. A number of mitochondrial drug delivery systems have been reported to date. However, reports of mitochondrial-targeted DNA delivery are limited. To achieve this, the therapeutic agent must be taken up by the cell (1), after which, the multi-processes associated with intracellular trafficking must be sophisticatedly regulated so as to release the agent from the endosome and deliver it to the cytosol (2) and to pass through the mitochondrial membrane (3). We report herein on the mitochondrial delivery of oligo DNA as a model therapeutic using a Dual Function (DF)-MITO-Porter, an innovative nano carrier designed for mitochondrial delivery. The critical structural elements of the DF-MITO-Porter include mitochondria-fusogenic inner envelopes and endosome-fusogenic outer envelopes, modified with octaarginine which greatly assists in cellular uptake. Inside the cell, the carrier passes through the endosomal and mitochondrial membranes via step-wise membrane fusion. When the oligo DNA was packaged in the DF-MITO-Porter, cellular uptake efficiency was strongly enhanced. Intracellular observation using confocal laser scanning microscopy showed that the DF-MITO-Porter was effectively released from endosomes. Moreover, the findings confirmed that the mitochondrial targeting activity of the DF-MITO-Porter was significantly higher than that of a carrier without outer endosome-fusogenic envelopes. These results support the conclusion that mitochondrial-targeted DNA delivery using a DF-MITO-Porter can be achieved when intracellular trafficking is optimally regulated.

Selective Mitochondrial Targeting Exerts Anxiolytic Effects In Vivo.

PubMed

Nussbaumer, Markus; Asara, John M; Teplytska, Larysa; Murphy, Michael P; Logan, Angela; Turck, Christoph W; Filiou, Michaela D

2016-06-01

Current treatment strategies for anxiety disorders are predominantly symptom-based. However, a third of anxiety patients remain unresponsive to anxiolytics highlighting the need for more effective, mechanism-based therapeutic approaches. We have previously compared high vs low anxiety mice and identified changes in mitochondrial pathways, including oxidative phosphorylation and oxidative stress. In this work, we show that selective pharmacological targeting of these mitochondrial pathways exerts anxiolytic effects in vivo. We treated high anxiety-related behavior (HAB) mice with MitoQ, an antioxidant that selectively targets mitochondria. MitoQ administration resulted in decreased anxiety-related behavior in HAB mice. This anxiolytic effect was specific for high anxiety as MitoQ treatment did not affect the anxiety phenotype of C57BL/6N and DBA/2J mouse strains. We furthermore investigated the molecular underpinnings of the MitoQ-driven anxiolytic effect and found that MitoQ treatment alters the brain metabolome and that the response to MitoQ treatment is characterized by distinct molecular signatures. These results indicate that a mechanism-driven approach based on selective mitochondrial targeting has the potential to attenuate the high anxiety phenotype in vivo, thus paving the way for translational implementation as long-term MitoQ administration is well-tolerated with no reported side effects in mice and humans.

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Characterization of mitochondrial ferritin in Drosophila.

PubMed

Missirlis, Fanis; Holmberg, Sara; Georgieva, Teodora; Dunkov, Boris C; Rouault, Tracey A; Law, John H

2006-04-11

Mitochondrial function depends on iron-containing enzymes and proteins, whose maturation requires available iron for biosynthesis of iron-sulfur clusters and heme. Little is known about how mitochondrial iron homeostasis is maintained, although the recent discovery of a mitochondrial ferritin in mammals and plants has uncovered a potential key player in the process. Here, we show that Drosophila melanogaster expresses mitochondrial ferritin from an intron-containing gene. It has high similarity to the mouse and human mitochondrial ferritin sequences and, as in mammals, is expressed mainly in testis. This ferritin contains a putative mitochondrial targeting sequence and an epitope-tagged version localizes to mitochondria in transfected cells. Overexpression of mitochondrial ferritin fails to alter both total-body iron levels and iron that is bound to secretory ferritins. However, the viability of iron-deficient flies is compromised by overexpression of mitochondrial ferritin, suggesting that it may sequester iron at the expense of other important cellular functions. The conservation of mitochondrial ferritin in an insect species underscores the importance of this iron-storage molecule.

Discovery of novel drugs for promising targets.

PubMed

Martell, Robert E; Brooks, David G; Wang, Yan; Wilcoxen, Keith

2013-09-01

Once a promising drug target is identified, the steps to actually discover and optimize a drug are diverse and challenging. The goal of this study was to provide a road map to navigate drug discovery. Review general steps for drug discovery and provide illustrating references. A number of approaches are available to enhance and accelerate target identification and validation. Consideration of a variety of potential mechanisms of action of potential drugs can guide discovery efforts. The hit to lead stage may involve techniques such as high-throughput screening, fragment-based screening, and structure-based design, with informatics playing an ever-increasing role. Biologically relevant screening models are discussed, including cell lines, 3-dimensional culture, and in vivo screening. The process of enabling human studies for an investigational drug is also discussed. Drug discovery is a complex process that has significantly evolved in recent years. © 2013 Elsevier HS Journals, Inc. All rights reserved.

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting.

PubMed

Lin, Pei-Hsuan; Lin, Hsien-Yi; Kuo, Cheng-Chin; Yang, Liang-Tung

2015-06-24

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3's action remain unanswered. We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants. Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on

Mitochondrial-targeted antioxidants represent a promising approach for prevention of cisplatin-induced nephropathy

PubMed Central

Mukhopadhyay, Partha; Horváth, Béla; Zsengellér, Zsuzsanna; Zielonka, Jacek; Tanchian, Galin; Holovac, Eileen; Kechrid, Malek; Patel, Vivek; Stillman, Isaac E.; Parikh, Samir M.; Joseph, Joy; Kalyanaraman, Balaraman; Pacher, Pál

2011-01-01

Cisplatin is a widely used anti-neoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show that mitochondrial dysfunction is not only a feature of cisplatin nephrotoxicity, but that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially-targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatin’s anti-neoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Since similar compounds appear to be safe in humans, mitochondrially-targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity. PMID:22120494

Selective Mitochondrial Targeting Exerts Anxiolytic Effects In Vivo

PubMed Central

Nussbaumer, Markus; Asara, John M; Teplytska, Larysa; Murphy, Michael P; Logan, Angela; Turck, Christoph W; Filiou, Michaela D

2016-01-01

Current treatment strategies for anxiety disorders are predominantly symptom-based. However, a third of anxiety patients remain unresponsive to anxiolytics highlighting the need for more effective, mechanism-based therapeutic approaches. We have previously compared high vs low anxiety mice and identified changes in mitochondrial pathways, including oxidative phosphorylation and oxidative stress. In this work, we show that selective pharmacological targeting of these mitochondrial pathways exerts anxiolytic effects in vivo. We treated high anxiety-related behavior (HAB) mice with MitoQ, an antioxidant that selectively targets mitochondria. MitoQ administration resulted in decreased anxiety-related behavior in HAB mice. This anxiolytic effect was specific for high anxiety as MitoQ treatment did not affect the anxiety phenotype of C57BL/6N and DBA/2J mouse strains. We furthermore investigated the molecular underpinnings of the MitoQ-driven anxiolytic effect and found that MitoQ treatment alters the brain metabolome and that the response to MitoQ treatment is characterized by distinct molecular signatures. These results indicate that a mechanism-driven approach based on selective mitochondrial targeting has the potential to attenuate the high anxiety phenotype in vivo, thus paving the way for translational implementation as long-term MitoQ administration is well-tolerated with no reported side effects in mice and humans. PMID:26567514

Cell-permeable, mitochondrial-targeted, peptide antioxidants.

PubMed

Szeto, Hazel H

2006-04-21

Cellular oxidative injury has been implicated in aging and a wide array of clinical disorders including ischemia-reperfusion injury; neurodegenerative diseases; diabetes; inflammatory diseases such as atherosclerosis, arthritis, and hepatitis; and drug-induced toxicity. However, available antioxidants have not proven to be particularly effective against many of these disorders. A possibility is that some of the antioxidants do not reach the relevant sites of free radical generation, especially if mitochondria are the primary source of reactive oxygen species (ROS). The SS (Szeto-Schiller) peptide antioxidants represent a novel approach with targeted delivery of antioxidants to the inner mitochondrial membrane. The structural motif of these SS peptides centers on alternating aromatic residues and basic amino acids (aromatic-cationic peptides). These SS peptides can scavenge hydrogen peroxide and peroxynitrite and inhibit lipid peroxidation. Their antioxidant action can be attributed to the tyrosine or dimethyltyrosine residue. By reducing mitochondrial ROS, these peptides inhibit mitochondrial permeability transition and cytochrome c release, thus preventing oxidant-induced cell death. Because these peptides concentrate >1000-fold in the inner mitochondrial membrane, they prevent oxidative cell death with EC50 in the nM range. Preclinical studies support their potential use for ischemia-reperfusion injury and neurodegenerative disorders. Although peptides have often been considered to be poor drug candidates, these small peptides have excellent "druggable" properties, making them promising agents for many diseases with unmet needs.

Highly selective luminescent nanostructures for mitochondrial imaging and targeting

NASA Astrophysics Data System (ADS)

Fanizza, E.; Iacobazzi, R. M.; Laquintana, V.; Valente, G.; Caliandro, G.; Striccoli, M.; Agostiano, A.; Cutrignelli, A.; Lopedota, A.; Curri, M. L.; Franco, M.; Depalo, N.; Denora, N.

2016-02-01

Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents.Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino

Drug Discovery for Neglected Diseases: Molecular Target-Based and Phenotypic Approaches

PubMed Central

2013-01-01

Drug discovery for neglected tropical diseases is carried out using both target-based and phenotypic approaches. In this paper, target-based approaches are discussed, with a particular focus on human African trypanosomiasis. Target-based drug discovery can be successful, but careful selection of targets is required. There are still very few fully validated drug targets in neglected diseases, and there is a high attrition rate in target-based drug discovery for these diseases. Phenotypic screening is a powerful method in both neglected and non-neglected diseases and has been very successfully used. Identification of molecular targets from phenotypic approaches can be a way to identify potential new drug targets. PMID:24015767

Bioenergetic Effects of Mitochondrial-Targeted Coenzyme Q Analogs in Endothelial Cells

PubMed Central

Fink, Brian D.; Herlein, Judith A.; Yorek, Mark A.; Fenner, Amanda M.; Kerns, Robert J.

2012-01-01

Mitochondrial-targeted analogs of coenzyme Q (CoQ) are under development to reduce oxidative damage induced by a variety of disease states. However, there is a need to understand the bioenergetic effects of these agents and whether or not these effects are related to redox properties, including their known pro-oxidant effects. We examined the bioenergetic effects of two mitochondrial-targeted CoQ analogs in their quinol forms, mitoquinol (MitoQ) and plastoquinonyl-decyl-triphenylphosphonium (SkQ1), in bovine aortic endothelial cells. We used an extracellular oxygen and proton flux analyzer to assess mitochondrial action at the intact-cell level. Both agents, in dose-dependent fashion, reduced the oxygen consumption rate (OCR) directed at ATP turnover (OCRATP) (IC50 values of 189 ± 13 nM for MitoQ and 181 ± 7 for SKQ1; difference not significant) while not affecting or mildly increasing basal oxygen consumption. Both compounds increased extracellular acidification in the basal state consistent with enhanced glycolysis. Both compounds enhanced mitochondrial superoxide production assessed by using mitochondrial-targeted dihydroethidium, and both increased H2O2 production from mitochondria of cells treated before isolation of the organelles. The manganese superoxide dismutase mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin did not alter or actually enhanced the actions of the targeted CoQ analogs to reduce OCRATP. In contrast, N-acetylcysteine mitigated this effect of MitoQ and SkQ1. In summary, our data demonstrate the important bioenergetic effects of targeted CoQ analogs. Moreover, these effects are mediated, at least in part, through superoxide production but depend on conversion to H2O2. These bioenergetic and redox actions need to be considered as these compounds are developed for therapeutic purposes. PMID:22661629

Bioenergetic effects of mitochondrial-targeted coenzyme Q analogs in endothelial cells.

PubMed

Fink, Brian D; Herlein, Judith A; Yorek, Mark A; Fenner, Amanda M; Kerns, Robert J; Sivitz, William I

2012-09-01

Mitochondrial-targeted analogs of coenzyme Q (CoQ) are under development to reduce oxidative damage induced by a variety of disease states. However, there is a need to understand the bioenergetic effects of these agents and whether or not these effects are related to redox properties, including their known pro-oxidant effects. We examined the bioenergetic effects of two mitochondrial-targeted CoQ analogs in their quinol forms, mitoquinol (MitoQ) and plastoquinonyl-decyl-triphenylphosphonium (SkQ1), in bovine aortic endothelial cells. We used an extracellular oxygen and proton flux analyzer to assess mitochondrial action at the intact-cell level. Both agents, in dose-dependent fashion, reduced the oxygen consumption rate (OCR) directed at ATP turnover (OCR(ATP)) (IC₅₀ values of 189 ± 13 nM for MitoQ and 181 ± 7 for SKQ1; difference not significant) while not affecting or mildly increasing basal oxygen consumption. Both compounds increased extracellular acidification in the basal state consistent with enhanced glycolysis. Both compounds enhanced mitochondrial superoxide production assessed by using mitochondrial-targeted dihydroethidium, and both increased H₂O₂ production from mitochondria of cells treated before isolation of the organelles. The manganese superoxide dismutase mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin did not alter or actually enhanced the actions of the targeted CoQ analogs to reduce OCR(ATP). In contrast, N-acetylcysteine mitigated this effect of MitoQ and SkQ1. In summary, our data demonstrate the important bioenergetic effects of targeted CoQ analogs. Moreover, these effects are mediated, at least in part, through superoxide production but depend on conversion to H₂O₂. These bioenergetic and redox actions need to be considered as these compounds are developed for therapeutic purposes.

The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats.

PubMed

Fouret, Gilles; Tolika, Evanthia; Lecomte, Jérôme; Bonafos, Béatrice; Aoun, Manar; Murphy, Michael P; Ferreri, Carla; Chatgilialoglu, Chryssostomos; Dubreucq, Eric; Coudray, Charles; Feillet-Coudray, Christine

2015-10-01

Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats. To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production. These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats. Copyright © 2015 Elsevier B.V. All rights reserved.

Repositioning of antibiotic levofloxacin as a mitochondrial biogenesis inhibitor to target breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Min; Li, Ruishu, E-mail: liruishu2016@yahoo.com; Zhang, Juan

Targeting mitochondrial biogenesis has become a potential therapeutic strategy in cancer due to their unique metabolic dependencies. In this study, we show that levofloxacin, a FDA-approved antibiotic, is an attractive candidate for breast cancer treatment. This is achieved by the inhibition of proliferation and induction of apoptosis in a panel of breast cancer cell lines while sparing normal breast cells. It also acts synergistically with conventional chemo drug in two independent in vivo breast xenograft mouse models. Importantly, levofloxacin inhibits mitochondrial biogenesis as shown by the decreased level of mitochondrial respiration, membrane potential and ATP. In addition, the anti-proliferative and pro-apoptoticmore » effects of levofloxacin are reversed by acetyl-L-Carnitine (ALCAR, a mitochondrial fuel), confirming that levofloxacin's action in breast cancer cells is through inhibition of mitochondrial biogenesis. A consequence of mitochondrial biogenesis inhibition by levofloxacin in breast cancer cells is the deactivation of PI3K/Akt/mTOR and MAPK/ERK pathways. We further demonstrate that breast cancer cells have increased mitochondrial biogenesis than normal breast cells, and this explains their different sensitivity to levofloxacin. Our work suggest that levofloxacin is a useful addition to breast cancer treatment. Our work also establish the essential role of mitochondrial biogenesis on the activation of PI3K/Akt/mTOR and MAPK/ERK pathways in breast cancer cells. - Highlights: • Levofloxacin targets a panel of breast cancer cell lines in vitro and in vivo. • Levofloxacin acts synergistically with 5-Fluorouracil in breast cancer. • Levofloxacin targets breast cancer cells via inhibiting mitochondrial biogenesis. • Breast cancer cells have increased mitochondrial biogenesis than normal cells. • Mitochondrial biogenesis inhibition lead to deactivation of PI3K/Akt/mTOR pathway.« less

Mitochondrial metals as a potential therapeutic target in neurodegeneration

PubMed Central

Grubman, A; White, A R; Liddell, J R

2014-01-01

Transition metals are critical for enzyme function and protein folding, but in excess can mediate neurotoxic oxidative processes. As mitochondria are particularly vulnerable to oxidative damage due to radicals generated during ATP production, mitochondrial biometal homeostasis must therefore be tightly controlled to safely harness the redox potential of metal enzyme cofactors. Dysregulation of metal functions is evident in numerous neurological disorders including Alzheimer's disease, stroke, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Friedrich's ataxia. This review describes the mitochondrial metal defects in these disorders and highlights novel metal-based therapeutic approaches that target mitochondrial metal homeostasis in neurological disorders. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24206195

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

PubMed

Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

2016-12-01

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer.

PubMed

Tian, Shenglan; Chen, Heng; Tan, Wei

2018-05-23

Targeting mitochondrial respiration has been documented as an effective therapeutic strategy in cancer. However, the impact of mitochondrial respiration inhibition on cervical cancer cells are not well elucidated. Using a panel of cervical cancer cell lines, we show that an existing drug atovaquone is active against the cervical cancer cells with high profiling of mitochondrial biogenesis. Atovaquone inhibited proliferation and induced apoptosis with varying efficacy among cervical cancer cell lines regardless of HPV infection, cellular origin and their sensitivity to paclitaxel. We further demonstrated that atovaquone acts on cervical cancer cells via inhibiting mitochondrial respiration. In particular, atovaquone specifically inhibited mitochondrial complex III but not I, II or IV activity, leading to respiration inhibition and energy crisis. Importantly, we found that the different sensitivity of cervical cancer cell lines to atovaquone were due to their differential level of mitochondrial biogenesis and dependency to mitochondrial respiration. In addition, we demonstrated that the in vitro observations were translatable to in vivo cervical cancer xenograft mouse model. Our findings suggest that the mitochondrial biogenesis varies among patients with cervical cancer. Our work also suggests that atovaquone is a useful addition to cervical cancer treatment, particularly to those with high dependency on mitochondrial respiration. Copyright © 2018 Elsevier Inc. All rights reserved.

Mitochondrial targeting of electron scavenging antioxidants: Regulation of selective oxidation vs random chain reactions

PubMed Central

Kagan, Valerian E.; Wipf, Peter; Stoyanovsky, Detcho; Greenberger, Joel S.; Borisenko, Grigory; Belikova, Natalia A.; Yanamala, Naveena; Samhan Arias, Alejandro K.; Tungekar, Muhammad A.; Jiang, Jianfei; Tyurina, Yulia Y.; Ji, Jing; Klein-Seetharaman, Judith; Pitt, Bruce R.; Shvedova, Anna A; Bayır, Hülya

2009-01-01

Effective regulation of highly compartmentalized production of reactive oxygen species and peroxidation reactions in mitochondria requires targeting of small molecule antioxidants and antioxidant enzymes into the organelles. This review describes recently developed approaches to mitochondrial targeting of small biologically active molecules based on: (i) preferential accumulation in mitochondria because of their hydrophobicity and positive charge (hydrophobic cations), (ii) binding with high affinity to an intra-mitochondrial constituent, and (iii) metabolic conversions by specific mitochondrial enzymes to reveal an active entity. In addition, targeted delivery of antioxidant enzymes via expression of leader-sequences directing the proteins into mitochondria is considered. Examples of successful antioxidant and anti-apoptotic protection based on the ability of targeted cargoes to inhibit cytochrome c-catalyzed peroxidation of a mitochondria-specific phospholipid cardiolipin, in vitro and in vivo are presented. Particular emphasis is placed on the employment of triphenylphosphonium- and hemi-gramicidin S-moieties as two effective vehicles for mitochondrial delivery of antioxidants. PMID:19716396

Pharmacological modulation of mitochondrial calcium homeostasis.

PubMed

Arduino, Daniela M; Perocchi, Fabiana

2018-01-10

Mitochondria are pivotal organelles in calcium (Ca 2+ ) handling and signalling, constituting intracellular checkpoints for numerous processes that are vital for cell life. Alterations in mitochondrial Ca 2+ homeostasis have been linked to a variety of pathological conditions and are critical in the aetiology of several human diseases. Efforts have been taken to harness mitochondrial Ca 2+ transport mechanisms for therapeutic intervention, but pharmacological compounds that direct and selectively modulate mitochondrial Ca 2+ homeostasis are currently lacking. New avenues have, however, emerged with the breakthrough discoveries on the genetic identification of the main players involved in mitochondrial Ca 2+ influx and efflux pathways and with recent hints towards a deep understanding of the function of these molecular systems. Here, we review the current advances in the understanding of the mechanisms and regulation of mitochondrial Ca 2+ homeostasis and its contribution to physiology and human disease. We also introduce and comment on the recent progress towards a systems-level pharmacological targeting of mitochondrial Ca 2+ homeostasis. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Targeting Mitochondrial Calcium Handling and Reactive Oxygen Species in Heart Failure.

PubMed

Dietl, Alexander; Maack, Christoph

2017-08-01

In highly prevalent cardiac diseases, new therapeutic approaches are needed. Since the first description of oxidative stress in heart failure, reactive oxygen species (ROS) have been considered as attractive drug targets. Though clinical trials evaluating antioxidant vitamins as ROS-scavenging agents yielded neutral results in patients at cardiovascular risk, the knowledge of ROS as pathophysiological factors has considerably advanced in the past few years and led to novel treatment approaches. Here, we review recent new insights and current strategies in targeting mitochondrial calcium handling and ROS in heart failure. Mitochondria are an important ROS source, and more recently, drug development focused on targeting mitochondria (e.g. by SS-31 or MitoQ). Important advancement has also been made to decipher how the matching of energy supply and demand through calcium (Ca 2+ ) handling impacts on mitochondrial ROS production and elimination. This opens novel opportunities to ameliorate mitochondrial dysfunction in heart failure by targeting cytosolic and mitochondrial ion transporters to improve this matching process. According to this approach, highly specific substances as the preclinical CGP-37157, as well as the clinically used ranolazine and empagliflozin, provide promising results on different levels of evidence. Furthermore, the understanding of redox signalling relays, resembled by catalyst-mediated protein oxidation, is about to change former paradigms of ROS signalling. Novel methods, as redox proteomics, allow to precisely analyse key regulatory thiol switches, which may induce adaptive or maladaptive signalling. Additionally, the generation of genetically encoded probes increased the spatial and temporal resolution of ROS imaging and opened a new methodological window to subtle, formerly obscured processes. These novel insights may broaden our understanding of why previous attempts to target oxidative stress have failed, and at the same time provide us

Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

PubMed Central

Fink, Brian D.; O'Malley, Yunxia; Dake, Brian L.; Ross, Nicolette C.; Prisinzano, Thomas E.; Sivitz, William I.

2009-01-01

Background Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. Methods and Results To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. Conclusions In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells

Mitochondrial targeted coenzyme Q, superoxide, and fuel selectivity in endothelial cells.

PubMed

Fink, Brian D; O'Malley, Yunxia; Dake, Brian L; Ross, Nicolette C; Prisinzano, Thomas E; Sivitz, William I

2009-01-01

Previously, we reported that the "antioxidant" compound "mitoQ" (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over

Development of pharmacological strategies for mitochondrial disorders

PubMed Central

Kanabus, M; Heales, S J; Rahman, S

2014-01-01

Mitochondrial diseases are an unusually genetically and phenotypically heterogeneous group of disorders, which are extremely challenging to treat. Currently, apart from supportive therapy, there are no effective treatments for the vast majority of mitochondrial diseases. Huge scientific effort, however, is being put into understanding the mechanisms underlying mitochondrial disease pathology and developing potential treatments. To date, a variety of treatments have been evaluated by randomized clinical trials, but unfortunately, none of these has delivered breakthrough results. Increased understanding of mitochondrial pathways and the development of many animal models, some of which are accurate phenocopies of human diseases, are facilitating the discovery and evaluation of novel prospective treatments. Targeting reactive oxygen species has been a treatment of interest for many years; however, only in recent years has it been possible to direct antioxidant delivery specifically into the mitochondria. Increasing mitochondrial biogenesis, whether by pharmacological approaches, dietary manipulation or exercise therapy, is also currently an active area of research. Modulating mitochondrial dynamics and mitophagy and the mitochondrial membrane lipid milieu have also emerged as possible treatment strategies. Recent technological advances in gene therapy, including allotopic and transkingdom gene expression and mitochondrially targeted transcription activator-like nucleases, have led to promising results in cell and animal models of mitochondrial diseases, but most of these techniques are still far from clinical application. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24116962

Targeting cysteine proteases in trypanosomatid disease drug discovery.

PubMed

Ferreira, Leonardo G; Andricopulo, Adriano D

2017-12-01

Chagas disease and human African trypanosomiasis are endemic conditions in Latin America and Africa, respectively, for which no effective and safe therapy is available. Efforts in drug discovery have focused on several enzymes from these protozoans, among which cysteine proteases have been validated as molecular targets for pharmacological intervention. These enzymes are expressed during the entire life cycle of trypanosomatid parasites and are essential to many biological processes, including infectivity to the human host. As a result of advances in the knowledge of the structural aspects of cysteine proteases and their role in disease physiopathology, inhibition of these enzymes by small molecules has been demonstrated to be a worthwhile approach to trypanosomatid drug research. This review provides an update on drug discovery strategies targeting the cysteine peptidases cruzain from Trypanosoma cruzi and rhodesain and cathepsin B from Trypanosoma brucei. Given that current chemotherapy for Chagas disease and human African trypanosomiasis has several drawbacks, cysteine proteases will continue to be actively pursued as valuable molecular targets in trypanosomatid disease drug discovery efforts. Copyright © 2017. Published by Elsevier Inc.

Off-Target Effects of Drugs that Disrupt Human Mitochondrial DNA Maintenance

PubMed Central

Young, Matthew J.

2017-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus (HIV) the cause of acquired immunodeficiency syndrome. Development of severe mitochondrial toxicity has been well documented in patients infected with HIV and administered NRTIs. In vitro biochemical experiments have demonstrated that the replicative mitochondrial DNA (mtDNA) polymerase gamma, Polg, is a sensitive target for inhibition by metabolically active forms of NRTIs, nucleotide reverse transcriptase inhibitors (NtRTIs). Once incorporated into newly synthesized daughter strands NtRTIs block further DNA polymerization reactions. Human cell culture and animal studies have demonstrated that cell lines and mice exposed to NRTIs display mtDNA depletion. Further complicating NRTI off-target effects on mtDNA maintenance, two additional DNA polymerases, Pol beta and PrimPol, were recently reported to localize to mitochondria as well as the nucleus. Similar to Polg, in vitro work has demonstrated both Pol beta and PrimPol incorporate NtRTIs into nascent DNA. Cell culture and biochemical experiments have also demonstrated that antiviral ribonucleoside drugs developed to treat hepatitis C infection act as off-target substrates for POLRMT, the mitochondrial RNA polymerase and primase. Accompanying the above-mentioned topics, this review examines: (1) mtDNA maintenance in human health and disease, (2) reports of DNA polymerases theta and zeta (Rev3) localizing to mitochondria, and (3) additional drugs with off-target effects on mitochondrial function. Lastly, mtDNA damage may induce cell death; therefore, the possibility of utilizing compounds that disrupt mtDNA maintenance to kill cancer cells is discussed. PMID:29214156

Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy.

PubMed

Yang, Yuhui; Karakhanova, Svetlana; Hartwig, Werner; D'Haese, Jan G; Philippov, Pavel P; Werner, Jens; Bazhin, Alexandr V

2016-12-01

Mitochondria are indispensable for energy metabolism, apoptosis regulation, and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression, and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the "vicious cycle" between mitochondria, ROS, genomic instability, and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria-targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria-targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria-targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria-targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. J. Cell. Physiol. 231: 2570-2581, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mitochondrial-Based Therapeutics for the Treatment of Spinal Cord Injury: Mitochondrial Biogenesis as a Potential Pharmacological Target

PubMed Central

Scholpa, Natalie E.

2017-01-01

Spinal cord injury (SCI) is characterized by an initial trauma followed by a progressive cascade of damage referred to as secondary injury. A hallmark of secondary injury is vascular disruption leading to vasoconstriction and decreased oxygen delivery, which directly reduces the ability of mitochondria to maintain homeostasis and leads to loss of ATP-dependent cellular functions, calcium overload, excitotoxicity, and oxidative stress, further exacerbating injury. Restoration of mitochondria dysfunction during the acute phases of secondary injury after SCI represents a potentially effective therapeutic strategy. This review discusses the past and present pharmacological options for the treatment of SCI as well as current research on mitochondria-targeted approaches. Increased antioxidant activity, inhibition of the mitochondrial permeability transition, alternate energy sources, and manipulation of mitochondrial morphology are among the strategies under investigation. Unfortunately, many of these tactics address single aspects of mitochondrial dysfunction, ultimately proving largely ineffective. Therefore, this review also examines the unexplored therapeutic efficacy of pharmacological enhancement of mitochondrial biogenesis, which has the potential to more comprehensively improve mitochondrial function after SCI. PMID:28935700

Mitochondrial-Based Therapeutics for the Treatment of Spinal Cord Injury: Mitochondrial Biogenesis as a Potential Pharmacological Target.

PubMed

Scholpa, Natalie E; Schnellmann, Rick G

2017-12-01

Spinal cord injury (SCI) is characterized by an initial trauma followed by a progressive cascade of damage referred to as secondary injury. A hallmark of secondary injury is vascular disruption leading to vasoconstriction and decreased oxygen delivery, which directly reduces the ability of mitochondria to maintain homeostasis and leads to loss of ATP-dependent cellular functions, calcium overload, excitotoxicity, and oxidative stress, further exacerbating injury. Restoration of mitochondria dysfunction during the acute phases of secondary injury after SCI represents a potentially effective therapeutic strategy. This review discusses the past and present pharmacological options for the treatment of SCI as well as current research on mitochondria-targeted approaches. Increased antioxidant activity, inhibition of the mitochondrial permeability transition, alternate energy sources, and manipulation of mitochondrial morphology are among the strategies under investigation. Unfortunately, many of these tactics address single aspects of mitochondrial dysfunction, ultimately proving largely ineffective. Therefore, this review also examines the unexplored therapeutic efficacy of pharmacological enhancement of mitochondrial biogenesis, which has the potential to more comprehensively improve mitochondrial function after SCI. U.S. Government work not protected by U.S. copyright.

Mitochondrial Flash: Integrative Reactive Oxygen Species and pH Signals in Cell and Organelle Biology

PubMed Central

Gong, Guohua; Wang, Xianhua; Wei-LaPierre, Lan; Cheng, Heping; Dirksen, Robert

2016-01-01

Abstract Significance: Recent breakthroughs in mitochondrial research have advanced, reshaped, and revolutionized our view of the role of mitochondria in health and disease. These discoveries include the development of novel tools to probe mitochondrial biology, the molecular identification of mitochondrial functional proteins, and the emergence of new concepts and mechanisms in mitochondrial function regulation. The discovery of “mitochondrial flash” activity has provided unique insights not only into real-time visualization of individual mitochondrial redox and pH dynamics in live cells but has also advanced understanding of the excitability, autonomy, and integration of mitochondrial function in vivo. Recent Advances: The mitochondrial flash is a transient and stochastic event confined within an individual mitochondrion and is observed in a wide range of organisms from plants to Caenorhabditis elegans to mammals. As flash events involve multiple transient concurrent changes within the mitochondrion (e.g., superoxide, pH, and membrane potential), a number of different mitochondrial targeted fluorescent indicators can detect flash activity. Accumulating evidence indicates that flash events reflect integrated snapshots of an intermittent mitochondrial process arising from mitochondrial respiration chain activity associated with the transient opening of the mitochondrial permeability transition pore. Critical Issues: We review the history of flash discovery, summarize current understanding of flash biology, highlight controversies regarding the relative roles of superoxide and pH signals during a flash event, and bring forth the integration of both signals in flash genesis. Future Directions: Investigations using flash as a biomarker and establishing its role in cell signaling pathway will move the field forward. Antioxid. Redox Signal. 25, 534–549. PMID:27245241

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: therapeutic potential of mitochondrially-targeted antioxidants

PubMed Central

Mukhopadhyay, Partha; Horváth, Bėla; Zsengellėr, Zsuzsanna; Bátkai, Sándor; Cao, Zongxian; Kechrid, Malek; Holovac, Eileen; Erdėlyi, Katalin; Tanchian, Galin; Liaudet, Lucas; Stillman, Isaac E.; Joseph, Joy; Kalyanaraman, Balaraman; Pacher, Pál

2012-01-01

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury, however its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explored the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2 hours of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute pro-inflammatory response (TNF-α, MIP-1αCCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6 hours of reperfusion and peaking at 24 hours). Mitochondrially-targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage. PMID:22683818

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins.

PubMed

Tkatch, Tatiana; Greotti, Elisa; Baranauskas, Gytis; Pendin, Diana; Roy, Soumitra; Nita, Luliaoana I; Wettmarshausen, Jennifer; Prigge, Matthias; Yizhar, Ofer; Shirihai, Orian S; Fishman, Daniel; Hershfinkel, Michal; Fleidervish, Ilya A; Perocchi, Fabiana; Pozzan, Tullio; Sekler, Israel

2017-06-27

Key mitochondrial functions such as ATP production, Ca 2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH + ) across the inner membrane. Although several drugs can modulate ΔμH + , their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψ m ) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca 2+ dynamics, and respiratory metabolism. By directly modulating Δψ m , the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Mitochondrial chaperones may be targets for anti-cancer drugs

Cancer.gov

Scientists at NCI have found that a mitochondrial chaperone protein, TRAP1, may act indirectly as a tumor suppressor as well as a novel target for developing anti-cancer drugs. Chaperone proteins, such as TRAP1, help other proteins adapt to stress, but sc

Application of chemical biology in target identification and drug discovery.

PubMed

Zhu, Yue; Xiao, Ting; Lei, Saifei; Zhou, Fulai; Wang, Ming-Wei

2015-09-01

Drug discovery and development is vital to the well-being of mankind and sustainability of the pharmaceutical industry. Using chemical biology approaches to discover drug leads has become a widely accepted path partially because of the completion of the Human Genome Project. Chemical biology mainly solves biological problems through searching previously unknown targets for pharmacologically active small molecules or finding ligands for well-defined drug targets. It is a powerful tool to study how these small molecules interact with their respective targets, as well as their roles in signal transduction, molecular recognition and cell functions. There have been an increasing number of new therapeutic targets being identified and subsequently validated as a result of advances in functional genomics, which in turn led to the discovery of numerous active small molecules via a variety of high-throughput screening initiatives. In this review, we highlight some applications of chemical biology in the context of drug discovery.

Advancing cancer drug discovery towards more agile development of targeted combination therapies.

PubMed

Carragher, Neil O; Unciti-Broceta, Asier; Cameron, David A

2012-01-01

Current drug-discovery strategies are typically 'target-centric' and are based upon high-throughput screening of large chemical libraries against nominated targets and a selection of lead compounds with optimized 'on-target' potency and selectivity profiles. However, high attrition of targeted agents in clinical development suggest that combinations of targeted agents will be most effective in treating solid tumors if the biological networks that permit cancer cells to subvert monotherapies are identified and retargeted. Conventional drug-discovery and development strategies are suboptimal for the rational design and development of novel drug combinations. In this article, we highlight a series of emerging technologies supporting a less reductionist, more agile, drug-discovery and development approach for the rational design, validation, prioritization and clinical development of novel drug combinations.

Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

PubMed

Hu, Hongtao; Li, Mo

2016-09-09

Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting mitochondrially mediated plasticity to develop improved therapeutics for bipolar disorder.

PubMed

de Sousa, Rafael T; Machado-Vieira, Rodrigo; Zarate, Carlos A; Manji, Husseini K

2014-10-01

Bipolar disorder (BPD) is a severe illness with few treatments available. Understanding BPD pathophysiology and identifying potential relevant targets could prove useful for developing new treatments. Remarkably, subtle impairments of mitochondrial function may play an important role in BPD pathophysiology. This article focuses on human studies and reviews evidence of mitochondrial dysfunction in BPD as a promising target for the development of new, improved treatments. Mitochondria are crucial for energy production, generated mainly through the electron transport chain (ETC) and play an important role in regulating apoptosis and calcium (Ca²⁺) signaling as well as synaptic plasticity. Mitochondria move throughout the neurons to provide energy for intracellular signaling. Studies showed polymorphisms of mitochondria-related genes as risk factors for BPD. Postmortem studies in BPD also show decreased ETC activity/expression and increased nitrosative and oxidative stress (OxS) in patient brains. BPD has been also associated with increased OxS, Ca²⁺ dysregulation and increased proapoptotic signaling in peripheral blood. Neuroimaging studies consistently show decreased energy levels and pH in brains of BPD patients. Targeting mitochondrial function, and their role in energy metabolism, synaptic plasticity and cell survival, may be an important avenue for development of new mood-stabilizing agents.

Targeting mitochondrially mediated plasticity to develop improved therapeutics for bipolar disorder

PubMed Central

de Sousa, Rafael T; Machado-Vieira, Rodrigo; Zarate, Carlos A

2014-01-01

Introduction Bipolar disorder (BPD) is a severe illness with few treatments available. Understanding BPD pathophysiology and identifying potential relevant targets could prove useful for developing new treatments. Remarkably, subtle impairments of mitochondrial function may play an important role in BPD pathophysiology. Areas covered This article focuses on human studies and reviews evidence of mitochondrial dysfunction in BPD as a promising target for the development of new, improved treatments. Mitochondria are crucial for energy production, generated mainly through the electron transport chain (ETC) and play an important role in regulating apoptosis and calcium (Ca2+) signaling as well as synaptic plasticity. Mitochondria move throughout the neurons to provide energy for intracellular signaling. Studies showed polymorphisms of mitochondria-related genes as risk factors for BPD. Postmortem studies in BPD also show decreased ETC activity/expression and increased nitrosative and oxidative stress (OxS) in patient brains. BPD has been also associated with increased OxS, Ca2+ dysregulation and increased proapoptotic signaling in peripheral blood. Neuroimaging studies consistently show decreased energy levels and pH in brains of BPD patients. Expert opinion Targeting mitochondrial function, and their role in energy metabolism, synaptic plasticity and cell survival, may be an important avenue for development of new mood-stabilizing agents. PMID:25056514

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry*

PubMed Central

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.; Monroe, Matthew E.; Orton, Daniel J.; Ibrahim, Yehia M.; Gritsenko, Marina A.; Clauss, Therese R. W.; Shukla, Anil K.; Moore, Ronald J.; Purvine, Samuel O.; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S.; Smith, Richard D.

2016-01-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. PMID:27670688

The HK2 Dependent "Warburg Effect" and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate.

PubMed

Lis, Paweł; Dyląg, Mariusz; Niedźwiecka, Katarzyna; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2016-12-15

This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the "Warburg" and "Crabtree" effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the "Warburg effect" and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the "Warburg effect", and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the "Warburg effect", i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as "multiple myeloma". Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.

Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

PubMed

Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

2014-05-01

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Blueprint for antimicrobial hit discovery targeting metabolic networks.

PubMed

Shen, Y; Liu, J; Estiu, G; Isin, B; Ahn, Y-Y; Lee, D-S; Barabási, A-L; Kapatral, V; Wiest, O; Oltvai, Z N

2010-01-19

Advances in genome analysis, network biology, and computational chemistry have the potential to revolutionize drug discovery by combining system-level identification of drug targets with the atomistic modeling of small molecules capable of modulating their activity. To demonstrate the effectiveness of such a discovery pipeline, we deduced common antibiotic targets in Escherichia coli and Staphylococcus aureus by identifying shared tissue-specific or uniformly essential metabolic reactions in their metabolic networks. We then predicted through virtual screening dozens of potential inhibitors for several enzymes of these reactions and showed experimentally that a subset of these inhibited both enzyme activities in vitro and bacterial cell viability. This blueprint is applicable for any sequenced organism with high-quality metabolic reconstruction and suggests a general strategy for strain-specific antiinfective therapy.

Targeted mitochondrial uncoupling beyond UCP1 - The fine line between death and metabolic health.

PubMed

Ost, Mario; Keipert, Susanne; Klaus, Susanne

2017-03-01

In the early 1930s, the chemical uncoupling agent 2,4-dinitrophenol (DNP) was promoted for the very first time as a powerful and effective weight loss pill but quickly withdrawn from the market due to its lack of tissue-selectivity with resulting dangerous side effects, including hyperthermia and death. Today, novel mitochondria- or tissue-targeted chemical uncouplers with higher safety and therapeutic values are under investigation in order to tackle obesity, diabetes and fatty liver disease. Moreover, in the past 20 years, transgenic mouse models were generated to understand the molecular and metabolic consequences of targeted uncoupling, expressing functional uncoupling protein 1 (UCP1) ectopically in white adipose tissue or skeletal muscle. Similar to the action of chemical mitochondrial uncouplers, UCP1 protein dissipates the proton gradient across the inner mitochondrial membrane, thus allowing maximum activity of the respiratory chain and compensatory increase in oxygen consumption, uncoupled from ATP synthesis. Consequently, targeted mitochondrial uncoupling in adipose tissue and skeletal muscle of UCP1-transgenic mice increased substrate metabolism and ameliorates obesity, hypertriglyceridemia and insulin resistance. Further, muscle-specific decrease in mitochondrial efficiency promotes a cell-autonomous and cell-non-autonomous adaptive metabolic remodeling with increased oxidative stress tolerance. This review provides an overview of novel chemical uncouplers as well as the metabolic consequences and adaptive processes of targeted mitochondrial uncoupling on metabolic health and survival. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry.

PubMed

Burnum-Johnson, Kristin E; Nie, Song; Casey, Cameron P; Monroe, Matthew E; Orton, Daniel J; Ibrahim, Yehia M; Gritsenko, Marina A; Clauss, Therese R W; Shukla, Anil K; Moore, Ronald J; Purvine, Samuel O; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S; Smith, Richard D

2016-12-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A mitochondrial-targeted ubiquinone modulates muscle lipid profile and improves mitochondrial respiration in obesogenic diet-fed rats.

PubMed

Coudray, Charles; Fouret, Gilles; Lambert, Karen; Ferreri, Carla; Rieusset, Jennifer; Blachnio-Zabielska, Agnieszka; Lecomte, Jérôme; Ebabe Elle, Raymond; Badia, Eric; Murphy, Michael P; Feillet-Coudray, Christine

2016-04-14

The prevalence of the metabolic syndrome components including abdominal obesity, dyslipidaemia and insulin resistance is increasing in both developed and developing countries. It is generally accepted that the development of these features is preceded by, or accompanied with, impaired mitochondrial function. The present study was designed to analyse the effects of a mitochondrial-targeted lipophilic ubiquinone (MitoQ) on muscle lipid profile modulation and mitochondrial function in obesogenic diet-fed rats. For this purpose, twenty-four young male Sprague-Dawley rats were divided into three groups and fed one of the following diets: (1) control, (2) high fat (HF) and (3) HF+MitoQ. After 8 weeks, mitochondrial function markers and lipid metabolism/profile modifications in skeletal muscle were measured. The HF diet was effective at inducing the major features of the metabolic syndrome--namely, obesity, hepatic enlargement and glucose intolerance. MitoQ intake prevented the increase in rat body weight, attenuated the increase in adipose tissue and liver weights and partially reversed glucose intolerance. At the muscle level, the HF diet induced moderate TAG accumulation associated with important modifications in the muscle phospholipid classes and in the fatty acid composition of total muscle lipid. These lipid modifications were accompanied with decrease in mitochondrial respiration. MitoQ intake corrected the lipid alterations and restored mitochondrial respiration. These results indicate that MitoQ protected obesogenic diet-fed rats from some features of the metabolic syndrome through its effects on muscle lipid metabolism and mitochondrial activity. These findings suggest that MitoQ is a promising candidate for future human trials in the metabolic syndrome prevention.

Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

PubMed

DeBalsi, Karen L; Wong, Kari E; Koves, Timothy R; Slentz, Dorothy H; Seiler, Sarah E; Wittmann, April H; Ilkayeva, Olga R; Stevens, Robert D; Perry, Christopher G R; Lark, Daniel S; Hui, Simon T; Szweda, Luke; Neufer, P Darrell; Muoio, Deborah M

2014-03-21

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle*

PubMed Central

DeBalsi, Karen L.; Wong, Kari E.; Koves, Timothy R.; Slentz, Dorothy H.; Seiler, Sarah E.; Wittmann, April H.; Ilkayeva, Olga R.; Stevens, Robert D.; Perry, Christopher G. R.; Lark, Daniel S.; Hui, Simon T.; Szweda, Luke; Neufer, P. Darrell; Muoio, Deborah M.

2014-01-01

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIPSKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIPSKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability. PMID:24482226

Mitochondrial Targeting of Vitamin E Succinate Enhances Its Pro-apoptotic and Anti-cancer Activity via Mitochondrial Complex II*

PubMed Central

Dong, Lan-Feng; Jameson, Victoria J. A.; Tilly, David; Cerny, Jiri; Mahdavian, Elahe; Marín-Hernández, Alvaro; Hernández-Esquivel, Luz; Rodríguez-Enríquez, Sara; Stursa, Jan; Witting, Paul K.; Stantic, Bela; Rohlena, Jakub; Truksa, Jaroslav; Kluckova, Katarina; Dyason, Jeffrey C.; Ledvina, Miroslav; Salvatore, Brian A.; Moreno-Sánchez, Rafael; Coster, Mark J.; Ralph, Stephen J.; Smith, Robin A. J.; Neuzil, Jiri

2011-01-01

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC50 of 80 μm, whereas the electron transfer from CII to CIII was inhibited with IC50 of 1.5 μm. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser68 within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug. PMID:21059645

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer

PubMed Central

Oran, Amanda R.; Adams, Clare M.; Zhang, Xiao-yong; Gennaro, Victoria J.; Pfeiffer, Harla K.; Mellert, Hestia S.; Seidel, Hans E.; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R.; Shen, Chen; Rigoutsos, Isidore; King, Michael P.; Cotney, Justin L.; Arnold, Jamie J.; Sharma, Suresh D.; Martinez, Ubaldo E.; Vakoc, Christopher R.; Chodosh, Lewis A.; Thompson, James E.; Bradner, James E.; Cameron, Craig E.; Shadel, Gerald S.; Eischen, Christine M.; McMahon, Steven B.

2016-01-01

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors. PMID:27590350

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

PubMed

Oran, Amanda R; Adams, Clare M; Zhang, Xiao-Yong; Gennaro, Victoria J; Pfeiffer, Harla K; Mellert, Hestia S; Seidel, Hans E; Mascioli, Kirsten; Kaplan, Jordan; Gaballa, Mahmoud R; Shen, Chen; Rigoutsos, Isidore; King, Michael P; Cotney, Justin L; Arnold, Jamie J; Sharma, Suresh D; Martinez-Outschoorn, Ubaldo E; Vakoc, Christopher R; Chodosh, Lewis A; Thompson, James E; Bradner, James E; Cameron, Craig E; Shadel, Gerald S; Eischen, Christine M; McMahon, Steven B

2016-11-08

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Blueprint for antimicrobial hit discovery targeting metabolic networks

PubMed Central

Shen, Y.; Liu, J.; Estiu, G.; Isin, B.; Ahn, Y-Y.; Lee, D-S.; Barabási, A-L.; Kapatral, V.; Wiest, O.; Oltvai, Z. N.

2010-01-01

Advances in genome analysis, network biology, and computational chemistry have the potential to revolutionize drug discovery by combining system-level identification of drug targets with the atomistic modeling of small molecules capable of modulating their activity. To demonstrate the effectiveness of such a discovery pipeline, we deduced common antibiotic targets in Escherichia coli and Staphylococcus aureus by identifying shared tissue-specific or uniformly essential metabolic reactions in their metabolic networks. We then predicted through virtual screening dozens of potential inhibitors for several enzymes of these reactions and showed experimentally that a subset of these inhibited both enzyme activities in vitro and bacterial cell viability. This blueprint is applicable for any sequenced organism with high-quality metabolic reconstruction and suggests a general strategy for strain-specific antiinfective therapy. PMID:20080587

Mitochondrial abnormalities in Alzheimer’s disease: Possible targets for therapeutic intervention

PubMed Central

Silva, Diana F.; Selfridge, J. Eva; Lu, Jianghua; Lezi, E; Cardoso, Sandra M.; Swerdlow, Russell H.

2013-01-01

Mitochondria from persons with Alzheimer’s disease (AD) differ from those of age-matched, control subjects. Differences in mitochondrial morphology and function are well-documented, and are not brain-limited. Some of these differences are present during all stages of AD, and are even seen in individuals who are without AD symptoms and signs but who have an increased risk of developing AD. This chapter considers the status of mitochondria in AD subjects, the potential basis for AD subject mitochondrial perturbations, and the implications of these perturbations. Data from multiple lines of investigation, including epidemiologic, biochemical, molecular, and cytoplasmic hybrid studies are reviewed. The possibility that mitochondria could potentially constitute a reasonable AD therapeutic target is discussed, as are several potential mitochondrial medicine treatment strategies. PMID:22840745

Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis.

PubMed

Ayotte, Yann; Bilodeau, François; Descoteaux, Albert; LaPlante, Steven R

2018-05-02

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment-based phenotypic lead discovery (FPLD) combines aspects of traditional fragment-based lead discovery (FBLD), which involves the screening of small-molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug-like compounds in cell-based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low-molecular-weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone-marrow-derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space.

PubMed

Newman, Laura E; Schiavon, Cara; Kahn, Richard A

2016-01-01

We describe the construction and uses of a series of plasmids for directing expression to varied levels of exogenous proteins targeted to the mitochondrial matrix or intermembrane space. We found that the level of protein expression achieved, the kinetics of expression and mitochondrial import, and half-life after import can each vary with the protein examined. These factors should be considered when directing localization of an exogenous protein to mitochondria for rescue, proteomics, or other approaches. We describe the construction of a collection of plasmids for varied expression of proteins targeted to the mitochondrial matrix or intermembrane space, using previously defined targeting sequences and strength CMV promoters. The limited size of these compartments makes them particularly vulnerable to artifacts from over-expression. We found that different proteins display different kinetics of expression and import that should be considered when analyzing results from this approach. Finally, this collection of plasmids has been deposited in the Addgene plasmid repository to facilitate the ready access and use of these tools.

Genetics of Mitochondrial Disease.

PubMed

Saneto, Russell P

2017-01-01

Mitochondria are intracellular organelles responsible for adenosine triphosphate production. The strict control of intracellular energy needs require proper mitochondrial functioning. The mitochondria are under dual controls of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mitochondrial dysfunction can arise from changes in either mtDNA or nDNA genes regulating function. There are an estimated ∼1500 proteins in the mitoproteome, whereas the mtDNA genome has 37 proteins. There are, to date, ∼275 genes shown to give rise to disease. The unique physiology of mitochondrial functioning contributes to diverse gene expression. The onset and range of phenotypic expression of disease is diverse, with onset from neonatal to seventh decade of life. The range of dysfunction is heterogeneous, ranging from single organ to multisystem involvement. The complexity of disease expression has severely limited gene discovery. Combining phenotypes with improvements in gene sequencing strategies are improving the diagnosis process. This chapter focuses on the interplay of the unique physiology and gene discovery in the current knowledge of genetically derived mitochondrial disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Imbalanced target prediction with pattern discovery on clinical data repositories.

PubMed

Chan, Tak-Ming; Li, Yuxi; Chiau, Choo-Chiap; Zhu, Jane; Jiang, Jie; Huo, Yong

2017-04-20

Clinical data repositories (CDR) have great potential to improve outcome prediction and risk modeling. However, most clinical studies require careful study design, dedicated data collection efforts, and sophisticated modeling techniques before a hypothesis can be tested. We aim to bridge this gap, so that clinical domain users can perform first-hand prediction on existing repository data without complicated handling, and obtain insightful patterns of imbalanced targets for a formal study before it is conducted. We specifically target for interpretability for domain users where the model can be conveniently explained and applied in clinical practice. We propose an interpretable pattern model which is noise (missing) tolerant for practice data. To address the challenge of imbalanced targets of interest in clinical research, e.g., deaths less than a few percent, the geometric mean of sensitivity and specificity (G-mean) optimization criterion is employed, with which a simple but effective heuristic algorithm is developed. We compared pattern discovery to clinically interpretable methods on two retrospective clinical datasets. They contain 14.9% deaths in 1 year in the thoracic dataset and 9.1% deaths in the cardiac dataset, respectively. In spite of the imbalance challenge shown on other methods, pattern discovery consistently shows competitive cross-validated prediction performance. Compared to logistic regression, Naïve Bayes, and decision tree, pattern discovery achieves statistically significant (p-values < 0.01, Wilcoxon signed rank test) favorable averaged testing G-means and F1-scores (harmonic mean of precision and sensitivity). Without requiring sophisticated technical processing of data and tweaking, the prediction performance of pattern discovery is consistently comparable to the best achievable performance. Pattern discovery has demonstrated to be robust and valuable for target prediction on existing clinical data repositories with imbalance and

Mitochondrial Metabolism as a Treatment Target in Anaplastic Thyroid Cancer

PubMed Central

Johnson, Jennifer M; Lai, Stephen Y.; Cotzia, Paolo; Cognetti, David; Luginbuhl, Adam; Pribitkin, Edmund A.; Zhan, Tingting; Mollaee, Mehri; Domingo-Vidal, Marina; Chen, Yunyun; Campling, Barbara; Bar-Ad, Voichita; Birbe, Ruth; Tuluc, Madalina; Outschoorn, Ubaldo Martinez; Curry, Joseph

2015-01-01

Aims Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically Monocarboxylate Transporter 1 (MCT1) and Translocase of the Outer Mitochondrial Membrane Member 20 (TOMM20). Methods Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer (PTC), and 8 non-cancerous thyroid) and 9 ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. Results MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (p<0.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (p<0.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (p<0.001). These xenograft tumors have high 13C- pyruvate uptake. Conclusions Anaplastic thyroid cancer has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target. PMID:26615136

Mitochondrial Metabolism as a Treatment Target in Anaplastic Thyroid Cancer.

PubMed

Johnson, Jennifer M; Lai, Stephen Y; Cotzia, Paolo; Cognetti, David; Luginbuhl, Adam; Pribitkin, Edmund A; Zhan, Tingting; Mollaee, Mehri; Domingo-Vidal, Marina; Chen, Yunyun; Campling, Barbara; Bar-Ad, Voichita; Birbe, Ruth; Tuluc, Madalina; Martinez Outschoorn, Ubaldo; Curry, Joseph

2015-12-01

Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically monocarboxylate transporter 1 (MCT1) and translocase of the outer mitochondrial membrane member 20 (TOMM20). Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer [PTC], and eight non-cancerous thyroid) and nine ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (P<.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (P<.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (P<.001). These xenograft tumors have high (13)C- pyruvate uptake. ATC has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Drug discovery strategies to outer membrane targets in Gram-negative pathogens.

PubMed

Brown, Dean G

2016-12-15

This review will cover selected recent examples of drug discovery strategies which target the outer membrane (OM) of Gram-negative bacteria either by disruption of outer membrane function or by inhibition of essential gene products necessary for outer membrane assembly. Significant advances in pathway elucidation, structural biology and molecular inhibitor designs have created new opportunities for drug discovery within this target-class space. Copyright © 2016 Elsevier Ltd. All rights reserved.

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.

Current proteomics approaches are comprised of both broad discovery measurements as well as more quantitative targeted measurements. These two different measurement types are used to initially identify potentially important proteins (e.g., candidate biomarkers) and then enable improved quantification for a limited number of selected proteins. However, both approaches suffer from limitations, particularly the lower sensitivity, accuracy, and quantitation precision for discovery approaches compared to targeted approaches, and the limited proteome coverage provided by targeted approaches. Herein, we describe a new proteomics approach that allows both discovery and targeted monitoring (DTM) in a single analysis using liquid chromatography, ion mobility spectrometrymore » and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled peptides for target ions are spiked into tryptic digests and both the labeled and unlabeled peptides are broadly detected using LC-IMS-MS instrumentation, allowing the benefits of discovery and targeted approaches. To understand the possible improvement of the DTM approach, it was compared to LC-MS broad measurements using an accurate mass and time tag database and selected reaction monitoring (SRM) targeted measurements. The DTM results yielded greater peptide/protein coverage and a significant improvement in the detection of lower abundance species compared to LC-MS discovery measurements. DTM was also observed to have similar detection limits as SRM for the targeted measurements indicating its potential for combining the discovery and targeted approaches.« less

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

PubMed Central

Pokrzywinski, Kaytee L.; Biel, Thomas G.; Kryndushkin, Dmitry; Rao, V. Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis. PMID:28030582

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

PubMed

Pokrzywinski, Kaytee L; Biel, Thomas G; Kryndushkin, Dmitry; Rao, V Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

Mitochondrial Dynamics in Mitochondrial Diseases

PubMed Central

Suárez-Rivero, Juan M.; Villanueva-Paz, Marina; de la Cruz-Ojeda, Patricia; de la Mata, Mario; Cotán, David; Oropesa-Ávila, Manuel; de Lavera, Isabel; Álvarez-Córdoba, Mónica; Luzón-Hidalgo, Raquel; Sánchez-Alcázar, José A.

2016-01-01

Mitochondria are very versatile organelles in continuous fusion and fission processes in response to various cellular signals. Mitochondrial dynamics, including mitochondrial fission/fusion, movements and turnover, are essential for the mitochondrial network quality control. Alterations in mitochondrial dynamics can cause neuropathies such as Charcot-Marie-Tooth disease in which mitochondrial fusion and transport are impaired, or dominant optic atrophy which is caused by a reduced mitochondrial fusion. On the other hand, mitochondrial dysfunction in primary mitochondrial diseases promotes reactive oxygen species production that impairs its own function and dynamics, causing a continuous vicious cycle that aggravates the pathological phenotype. Mitochondrial dynamics provides a new way to understand the pathophysiology of mitochondrial disorders and other diseases related to mitochondria dysfunction such as diabetes, heart failure, or Hungtinton’s disease. The knowledge about mitochondrial dynamics also offers new therapeutics targets in mitochondrial diseases. PMID:28933354

Potential biological targets for bioassay development in drug discovery of Sturge-Weber syndrome.

PubMed

Mohammadipanah, Fatemeh; Salimi, Fatemeh

2018-02-01

Sturge-Weber Syndrome (SWS) is a neurocutaneous disease with clinical manifestations including ocular (glaucoma), cutaneous (port-wine birthmark), neurologic (seizures), and vascular problems. Molecular mechanisms of SWS pathogenesis are initiated by the somatic mutation in GNAQ. Therefore, no definite treatments exist for SWS and treatment options only mitigate the intensity of its clinical manifestations. Biological assay design for drug discovery against this syndrome demands comprehensive knowledge on mechanisms which are involved in its pathogenesis. By analysis of the interrelated molecular targets of SWS, some in vitro bioassay systems can be allotted for drug screening against its progression. Development of such platforms of bioassay can bring along the implementation of high-throughput screening of natural or synthetic compounds in drug discovery programs. Regarding the fact that study of molecular targets and their integration in biological assay design can facilitate the process of effective drug discovery; some potential biological targets and their respective biological assay for SWS drug discovery are propounded in this review. For this purpose, some biological targets for SWS drug discovery such as acetylcholinesterase, alkaline phosphatase, GABAergic receptors, Hypoxia-Inducible Factor (HIF)-1α and 2α are suggested. © 2017 John Wiley & Sons A/S.

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

PubMed Central

Jhun, Bong Sook; Xu, Shangcheng; Hurst, Stephen; Raffaello, Anna; Liu, Xiaoyun; Yi, Bing; Zhang, Huiliang; Gross, Polina; Mishra, Jyotsna; Ainbinder, Alina; Kettlewell, Sarah; Smith, Godfrey L.; Dirksen, Robert T.; Wang, Wang; Rizzuto, Rosario

2014-01-01

Abstract Aims: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Innovation: Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879. PMID:24800979

Mitochondrial permeability transition pore: a promising target for the treatment of Parkinson's disease.

PubMed

Rasheed, Md Zeeshan; Tabassum, Heena; Parvez, Suhel

2017-01-01

Among the neurodegenerative diseases (ND), Parkinson's disease affects 6.3 million people worldwide characterized by the progressive loss of dopaminergic neurons in substantia nigra. The mitochondrial permeability transition pore (mtPTP) is a non-selective voltage-dependent mitochondrial channel whose opening modifies the permeability properties of the mitochondrial inner membrane. It is recognized as a potent pharmacological target for diseases associated with mitochondrial dysfunction and excessive cell death including ND such as Parkinson's disease (PD). Imbalance in Ca 2+ concentration, change in mitochondrial membrane potential, overproduction of reactive oxygen species (ROS), or mutation in mitochondrial genome has been implicated in the pathophysiology of the opening of the mtPTP. Different proteins are released by permeability transition including cytochrome c which is responsible for apoptosis. This review aims to discuss the importance of PTP in the pathophysiology of PD and puts together different positive as well as negative aspects of drugs such as pramipexole, ropinirole, minocyclin, rasagilin, and safinamide which act as a blocker or modifier for mtPTP. Some of them may be detrimental in their neuroprotective nature.

Emerging Mitochondrial Therapeutic Targets in Optic Neuropathies.

PubMed

Lopez Sanchez, M I G; Crowston, J G; Mackey, D A; Trounce, I A

2016-09-01

Optic neuropathies are an important cause of blindness worldwide. The study of the most common inherited mitochondrial optic neuropathies, Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA) has highlighted a fundamental role for mitochondrial function in the survival of the affected neuron-the retinal ganglion cell. A picture is now emerging that links mitochondrial dysfunction to optic nerve disease and other neurodegenerative processes. Insights gained from the peculiar susceptibility of retinal ganglion cells to mitochondrial dysfunction are likely to inform therapeutic development for glaucoma and other common neurodegenerative diseases of aging. Despite it being a fast-evolving field of research, a lack of access to human ocular tissues and limited animal models of mitochondrial disease have prevented direct retinal ganglion cell experimentation and delayed the development of efficient therapeutic strategies to prevent vision loss. Currently, there are no approved treatments for mitochondrial disease, including optic neuropathies caused by primary or secondary mitochondrial dysfunction. Recent advances in eye research have provided important insights into the molecular mechanisms that mediate pathogenesis, and new therapeutic strategies including gene correction approaches are currently being investigated. Here, we review the general principles of mitochondrial biology relevant to retinal ganglion cell function and provide an overview of the major optic neuropathies with mitochondrial involvement, LHON and ADOA, whilst highlighting the emerging link between mitochondrial dysfunction and glaucoma. The pharmacological strategies currently being trialed to improve mitochondrial dysfunction in these optic neuropathies are discussed in addition to emerging therapeutic approaches to preserve retinal ganglion cell function. Copyright © 2016 Elsevier Inc. All rights reserved.

Drug-Target Kinetics in Drug Discovery.

PubMed

Tonge, Peter J

2018-01-17

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.

Drug–Target Kinetics in Drug Discovery

PubMed Central

2017-01-01

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure–kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug–target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug–target kinetics into predictions of drug activity. PMID:28640596

Systems biology-embedded target validation: improving efficacy in drug discovery.

PubMed

Vandamme, Drieke; Minke, Benedikt A; Fitzmaurice, William; Kholodenko, Boris N; Kolch, Walter

2014-01-01

The pharmaceutical industry is faced with a range of challenges with the ever-escalating costs of drug development and a drying out of drug pipelines. By harnessing advances in -omics technologies and moving away from the standard, reductionist model of drug discovery, there is significant potential to reduce costs and improve efficacy. Embedding systems biology approaches in drug discovery, which seek to investigate underlying molecular mechanisms of potential drug targets in a network context, will reduce attrition rates by earlier target validation and the introduction of novel targets into the currently stagnant market. Systems biology approaches also have the potential to assist in the design of multidrug treatments and repositioning of existing drugs, while stratifying patients to give a greater personalization of medical treatment. © 2013 Wiley Periodicals, Inc.

Targeting mitochondrial function and proteostasis to mitigate dynapenia.

PubMed

Musci, Robert V; Hamilton, Karyn L; Miller, Benjamin F

2018-01-01

Traditionally, interventions to treat skeletal muscle aging have largely targeted sarcopenia-the age-related loss of skeletal muscle mass. Dynapenia refers to the age-related loss in skeletal muscle function due to factors outside of muscle mass, which helps to inform treatment strategies for aging skeletal muscle. There is evidence that mechanisms to maintain protein homeostasis and proteostasis, deteriorate with age. One key mechanism to maintain proteostasis is protein turnover, which is an energetically costly process. When there is a mismatch between cellular energy demands and energy provision, inelastic processes related to metabolism are maintained, but there is competition for the remaining energy between the elastic processes of somatic maintenance and growth. With aging, mitochondrial dysfunction reduces ATP generation capacity, constraining the instantaneous supply of energy, thus compromising growth and somatic maintenance processes. Further, with age the need for somatic maintenance increases because of the accumulation of protein damage. In this review, we highlight the significant role mitochondria have in maintaining skeletal muscle proteostasis through increased energy provision, protein turnover, and substrate flux. In addition, we provide evidence that improving mitochondrial function could promote a cellular environment that is conducive to somatic maintenance, and consequently for mitigating dynapenia. Finally, we highlight interventions, such as aerobic exercise, that could be used to improve mitochondrial function and improve outcomes related to dynapenia.

Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches

DTIC Science & Technology

2010-07-01

1-0431 TITLE: Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches PRINCIPAL INVESTIGATOR...June 2010 4. TITLE AND SUBTITLE Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic 5a. CONTRACT NUMBER...1-0430; W81XWH-08-1-0431; Grant sponsor: NIH/NCRR COBRE Grant; Grant number: 1P20RR020171; Grant sponsor: NIH/NIDDK Grant; Grant number: R01DK053525

Novel Dual Mitochondrial and CD44 Receptor Targeting Nanoparticles for Redox Stimuli-Triggered Release

NASA Astrophysics Data System (ADS)

Wang, Kaili; Qi, Mengjiao; Guo, Chunjing; Yu, Yueming; Wang, Bingjie; Fang, Lei; Liu, Mengna; Wang, Zhen; Fan, Xinxin; Chen, Daquan

2018-02-01

In this work, novel mitochondrial and CD44 receptor dual-targeting redox-sensitive multifunctional nanoparticles (micelles) based on oligomeric hyaluronic acid (oHA) were proposed. The amphiphilic nanocarrier was prepared by (5-carboxypentyl)triphenylphosphonium bromide (TPP), oligomeric hyaluronic acid (oHA), disulfide bond, and curcumin (Cur), named as TPP-oHA-S-S-Cur. The TPP targeted the mitochondria, the antitumor drug Cur served as a hydrophobic core, the CD44 receptor targeting oHA worked as a hydrophilic shell, and the disulfide bond acted as a connecting arm. The chemical structure of TPP-oHA-S-S-Cur was characterized by 1HNMR technology. Cur was loaded into the TPP-oHA-S-S-Cur micelles by self-assembly. Some properties, including the preparation of micelles, morphology, redox sensitivity, and mitochondrial targeting, were studied. The results showed that TPP-oHA-S-S-Cur micelles had a mean diameter of 122.4 ± 23.4 nm, zeta potential - 26.55 ± 4.99 mV. In vitro release study and cellular uptake test showed that TPP-oHA-S-S-Cur micelles had redox sensibility, dual targeting to mitochondrial and CD44 receptor. This work provided a promising smart multifunctional nanocarrier platform to enhance the solubility, decrease the side effects, and improve the therapeutic efficacy of anticancer drugs.

Mitochondrial respiratory chain complexes as sources and targets of thiol-based redox-regulation.

PubMed

Dröse, Stefan; Brandt, Ulrich; Wittig, Ilka

2014-08-01

The respiratory chain of the inner mitochondrial membrane is a unique assembly of protein complexes that transfers the electrons of reducing equivalents extracted from foodstuff to molecular oxygen to generate a proton-motive force as the primary energy source for cellular ATP-synthesis. Recent evidence indicates that redox reactions are also involved in regulating mitochondrial function via redox-modification of specific cysteine-thiol groups in subunits of respiratory chain complexes. Vice versa the generation of reactive oxygen species (ROS) by respiratory chain complexes may have an impact on the mitochondrial redox balance through reversible and irreversible thiol-modification of specific target proteins involved in redox signaling, but also pathophysiological processes. Recent evidence indicates that thiol-based redox regulation of the respiratory chain activity and especially S-nitrosylation of complex I could be a strategy to prevent elevated ROS production, oxidative damage and tissue necrosis during ischemia-reperfusion injury. This review focuses on the thiol-based redox processes involving the respiratory chain as a source as well as a target, including a general overview on mitochondria as highly compartmentalized redox organelles and on methods to investigate the redox state of mitochondrial proteins. This article is part of a Special Issue entitled: Thiol-Based Redox Processes. Copyright © 2014 Elsevier B.V. All rights reserved.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.

PubMed

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-05-01

Mitochondrial calcium ([Ca 2+ ] m ) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca 2+ ] m uptake upon SK channel activation as detected by time lapse mitochondrial Ca 2+ measurements with the Ca 2+ -binding mitochondria-targeted aequorin and FRET-based [Ca 2+ ] m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca 2+ ] m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

PubMed Central

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-01-01

Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death. PMID:28282037

Potential biological targets for bioassay development in drug discovery of Sturge-Weber syndrome.

PubMed

Mohammadipanah, Fatemeh; Salimi, Fatemeh

2017-04-29

Sturge-Weber Syndrome (SWS) is among the neurocutaneous diseases, which has several clinical manifestations of ocular (glaucoma), cutaneous (port-wine stain), neurological (seizures) and vascular problems. Molecular mechanisms of SWS pathogenesis are initiated by the somatic mutation in GNAQ. Therefore, no definite treatments exist for the SWS and treatment options only mitigate the intensity of its clinical manifestations. Biological assay design for drug discovery against this syndrome demands comprehensive knowledge on mechanisms which are involved in its pathogenesis. By analysis of the interrelated molecular targets of SWS, some in vitro bioassay systems can be allotted for drug screening against this syndrome. Development of such platforms of bioassay can bring along the implementation of high throughput screening of natural or synthetic compounds in drug discovery programs. Regarding the fact that study of biological targets and their integration in biological assay design can facilitate the process of effective drug discovery; some potential biological targets and their respective biological assay for SWS drug discovery are propounded in this review. For this purpose, some biological targets for SWS drug discovery such as acetylcholine esterase, alkaline phosphatase, gamma-aminobutyricacidergic, Hypoxia-Inducible Factor (HIF) -1α and 2α are suggested. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Application of industrial scale genomics to discovery of therapeutic targets in heart failure.

PubMed

Mehraban, F; Tomlinson, J E

2001-12-01

In recent years intense activity in both academic and industrial sectors has provided a wealth of information on the human genome with an associated impressive increase in the number of novel gene sequences deposited in sequence data repositories and patent applications. This genomic industrial revolution has transformed the way in which drug target discovery is now approached. In this article we discuss how various differential gene expression (DGE) technologies are being utilized for cardiovascular disease (CVD) drug target discovery. Other approaches such as sequencing cDNA from cardiovascular derived tissues and cells coupled with bioinformatic sequence analysis are used with the aim of identifying novel gene sequences that may be exploited towards target discovery. Additional leverage from gene sequence information is obtained through identification of polymorphisms that may confer disease susceptibility and/or affect drug responsiveness. Pharmacogenomic studies are described wherein gene expression-based techniques are used to evaluate drug response and/or efficacy. Industrial-scale genomics supports and addresses not only novel target gene discovery but also the burgeoning issues in pharmaceutical and clinical cardiovascular medicine relative to polymorphic gene responses.

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

PubMed Central

Comte, Caroline; Tonin, Yann; Heckel-Mager, Anne-Marie; Boucheham, Abdeldjalil; Smirnov, Alexandre; Auré, Karine; Lombès, Anne; Martin, Robert P.; Entelis, Nina; Tarassov, Ivan

2013-01-01

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases. PMID:23087375

Mitochondrial biogenesis: pharmacological approaches.

PubMed

Valero, Teresa

2014-01-01

Organelle biogenesis is concomitant to organelle inheritance during cell division. It is necessary that organelles double their size and divide to give rise to two identical daughter cells. Mitochondrial biogenesis occurs by growth and division of pre-existing organelles and is temporally coordinated with cell cycle events [1]. However, mitochondrial biogenesis is not only produced in association with cell division. It can be produced in response to an oxidative stimulus, to an increase in the energy requirements of the cells, to exercise training, to electrical stimulation, to hormones, during development, in certain mitochondrial diseases, etc. [2]. Mitochondrial biogenesis is therefore defined as the process via which cells increase their individual mitochondrial mass [3]. Recent discoveries have raised attention to mitochondrial biogenesis as a potential target to treat diseases which up to date do not have an efficient cure. Mitochondria, as the major ROS producer and the major antioxidant producer exert a crucial role within the cell mediating processes such as apoptosis, detoxification, Ca2+ buffering, etc. This pivotal role makes mitochondria a potential target to treat a great variety of diseases. Mitochondrial biogenesis can be pharmacologically manipulated. This issue tries to cover a number of approaches to treat several diseases through triggering mitochondrial biogenesis. It contains recent discoveries in this novel field, focusing on advanced mitochondrial therapies to chronic and degenerative diseases, mitochondrial diseases, lifespan extension, mitohormesis, intracellular signaling, new pharmacological targets and natural therapies. It contributes to the field by covering and gathering the scarcely reported pharmacological approaches in the novel and promising field of mitochondrial biogenesis. There are several diseases that have a mitochondrial origin such as chronic progressive external ophthalmoplegia (CPEO) and the Kearns- Sayre syndrome (KSS

Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

PubMed

Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

2015-09-09

Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI.

Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.

PubMed

Kitagaki, Hiroshi

2009-05-29

Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains.

Mitochondrial proteome disruption in the diabetic heart through targeted epigenetic regulation at the mitochondrial heat shock protein 70 (mtHsp70) nuclear locus.

PubMed

Shepherd, Danielle L; Hathaway, Quincy A; Nichols, Cody E; Durr, Andrya J; Pinti, Mark V; Hughes, Kristen M; Kunovac, Amina; Stine, Seth M; Hollander, John M

2018-06-01

>99% of the mitochondrial proteome is nuclear-encoded. The mitochondrion relies on a coordinated multi-complex process for nuclear genome-encoded mitochondrial protein import. Mitochondrial heat shock protein 70 (mtHsp70) is a key component of this process and a central constituent of the protein import motor. Type 2 diabetes mellitus (T2DM) disrupts mitochondrial proteomic signature which is associated with decreased protein import efficiency. The goal of this study was to manipulate the mitochondrial protein import process through targeted restoration of mtHsp70, in an effort to restore proteomic signature and mitochondrial function in the T2DM heart. A novel line of cardiac-specific mtHsp70 transgenic mice on the db/db background were generated and cardiac mitochondrial subpopulations were isolated with proteomic evaluation and mitochondrial function assessed. MicroRNA and epigenetic regulation of the mtHsp70 gene during T2DM were also evaluated. MtHsp70 overexpression restored cardiac function and nuclear-encoded mitochondrial protein import, contributing to a beneficial impact on proteome signature and enhanced mitochondrial function during T2DM. Further, transcriptional repression at the mtHsp70 genomic locus through increased localization of H3K27me3 during T2DM insult was observed. Our results suggest that restoration of a key protein import constituent, mtHsp70, provides therapeutic benefit through attenuation of mitochondrial and contractile dysfunction in T2DM. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

PubMed

Chernyavsky, Alex; Chen, Yumay; Wang, Ping H; Grando, Sergei A

2015-11-01

The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity. Copyright © 2015 Elsevier B.V. All rights reserved.

Atypical case of Wolfram syndrome revealed through targeted exome sequencing in a patient with suspected mitochondrial disease

PubMed Central

2012-01-01

Background Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci. Case Presentation Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome. Conclusion This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients. PMID:22226368

Mitoketoscins: Novel mitochondrial inhibitors for targeting ketone metabolism in cancer stem cells (CSCs)

PubMed Central

Ozsvari, Bela; Sotgia, Federica; Simmons, Katie; Trowbridge, Rachel; Foster, Richard; Lisanti, Michael P.

2017-01-01

Previous studies have now well-established that epithelial cancer cells can utilize ketone bodies (3-hydroxybutyrate and aceto-acetate) as mitochondrial fuels, to actively promote tumor growth and metastatic dissemination. The two critical metabolic enzymes implicated in this process are OXCT1 and ACAT1, which are both mitochondrial proteins. Importantly, over-expression of OXCT1 or ACAT1 in human breast cancer cells is sufficient to genetically drive tumorigenesis and/or lung metastasis, validating that they indeed behave as metabolic “tumor promoters”. Here, we decided to target these two enzymes, which give cancer cells the ability to recycle ketone bodies into Acetyl-CoA and, therefore, to produce increased ATP. Briefly, we used computational chemistry (in silico drug design) to select a sub-set of potentially promising compounds that spatially fit within the active site of these enzymes, based on their known 3D crystal structures. These libraries of compounds were then phenotypically screened for their effects on total cellular ATP levels. Positive hits were further validated by metabolic flux analysis. Our results indicated that four of these compounds effectively inhibited mitochondrial oxygen consumption. Two of these compounds also induced a reactive glycolytic phenotype in cancer cells. Most importantly, using the mammosphere assay, we showed that these compounds can be used to functionally inhibit cancer stem cell (CSC) activity and propagation. Finally, our molecular modeling studies directly show how these novel compounds are predicted to bind to the active catalytic sites of OXCT1 and ACAT1, within their Coenzyme A binding site. As such, we speculate that these mitochondrial inhibitors are partially mimicking the structure of Coenzyme A. Thus, we conclude that OXCT1 and ACAT1 are important new therapeutic targets for further drug development and optimization. We propose that this new class of drugs should be termed “mitoketoscins”, to reflect

System-level multi-target drug discovery from natural products with applications to cardiovascular diseases.

PubMed

Zheng, Chunli; Wang, Jinan; Liu, Jianling; Pei, Mengjie; Huang, Chao; Wang, Yonghua

2014-08-01

The term systems pharmacology describes a field of study that uses computational and experimental approaches to broaden the view of drug actions rooted in molecular interactions and advance the process of drug discovery. The aim of this work is to stick out the role that the systems pharmacology plays across the multi-target drug discovery from natural products for cardiovascular diseases (CVDs). Firstly, based on network pharmacology methods, we reconstructed the drug-target and target-target networks to determine the putative protein target set of multi-target drugs for CVDs treatment. Secondly, we reintegrated a compound dataset of natural products and then obtained a multi-target compounds subset by virtual-screening process. Thirdly, a drug-likeness evaluation was applied to find the ADME-favorable compounds in this subset. Finally, we conducted in vitro experiments to evaluate the reliability of the selected chemicals and targets. We found that four of the five randomly selected natural molecules can effectively act on the target set for CVDs, indicating the reasonability of our systems-based method. This strategy may serve as a new model for multi-target drug discovery of complex diseases.

Targeting Metabolic Plasticity in Breast Cancer Cells via Mitochondrial Complex I Modulation

PubMed Central

Xu, Qijin; Biener-Ramanujan, Eva; Yang, Wei; Ramanujan, V Krishnan

2016-01-01

Purpose Heterogeneity commonly observed in clinical tumors stems both from the genetic diversity as well as from the differential metabolic adaptation of multiple cancer types during their struggle to maintain uncontrolled proliferation and invasion in vivo. This study aims to identify a potential metabolic window of such adaptation in aggressive human breast cancer cell lines. Methods With a multidisciplinary approach using high resolution imaging, cell metabolism assays, proteomic profiling and animal models of human tumor xenografts and via clinically-relevant, pharmacological approach for modulating mitochondrial complex I function in human breast cancer cell lines, we report a novel route to target metabolic plasticity in human breast cancer cells. Results By a systematic modulation of mitochondrial function and by mitigating metabolic switch phenotype in aggressive human breast cancer cells, we demonstrate that the resulting metabolic adaptation signatures can predictably decrease tumorigenic potential in vivo. Proteomic profiling of the metabolic adaptation in these cells further revealed novel protein-pathway interactograms highlighting the importance of antioxidant machinery in the observed metabolic adaptation. Conclusions Improved metabolic adaptation potential in aggressive human breast cancer cells contribute to improving mitochondrial function and reducing metabolic switch phenotype –which may be vital for targeting primary tumor growth in vivo. PMID:25677747

Pharmacological approaches to restore mitochondrial function

PubMed Central

Andreux, Pénélope A.; Houtkooper, Riekelt H.; Auwerx, Johan

2014-01-01

Mitochondrial dysfunction is not only a hallmark of rare inherited mitochondrial disorders, but is also implicated in age-related diseases, including those that affect the metabolic and nervous system, such as type 2 diabetes and Parkinson’s disease. Numerous pathways maintain and/or restore proper mitochondrial function, including mitochondrial biogenesis, mitochondrial dynamics, mitophagy, and the mitochondrial unfolded protein response. New and powerful phenotypic assays in cell-based models, as well as multicellular organisms, have been developed to explore these different aspects of mitochondrial function. Modulating mitochondrial function has therefore emerged as an attractive therapeutic strategy for a range of diseases, which has spurred active drug discovery efforts in this area. PMID:23666487

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

PubMed Central

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Multiplexed Thiol Reactivity Profiling for Target Discovery of Electrophilic Natural Products.

PubMed

Tian, Caiping; Sun, Rui; Liu, Keke; Fu, Ling; Liu, Xiaoyu; Zhou, Wanqi; Yang, Yong; Yang, Jing

2017-11-16

Electrophilic groups, such as Michael acceptors, expoxides, are common motifs in natural products (NPs). Electrophilic NPs can act through covalent modification of cysteinyl thiols on functional proteins, and exhibit potent cytotoxicity and anti-inflammatory/cancer activities. Here we describe a new chemoproteomic strategy, termed multiplexed thiol reactivity profiling (MTRP), and its use in target discovery of electrophilic NPs. We demonstrate the utility of MTRP by identifying cellular targets of gambogic acid, an electrophilic NP that is currently under evaluation in clinical trials as anticancer agent. Moreover, MTRP enables simultaneous comparison of seven structurally diversified α,β-unsaturated γ-lactones, which provides insights into the relative proteomic reactivity and target preference of diverse structural scaffolds coupled to a common electrophilic motif and reveals various potential druggable targets with liganded cysteines. We anticipate that this new method for thiol reactivity profiling in a multiplexed manner will find broad application in redox biology and drug discovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

The targeted anti-oxidant MitoQ causes mitochondrial swelling and depolarization in kidney tissue.

PubMed

Gottwald, Esther M; Duss, Michael; Bugarski, Milica; Haenni, Dominik; Schuh, Claus D; Landau, Ehud M; Hall, Andrew M

2018-04-01

Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

The mitochondrially targeted antioxidant MitoQ protects the intestinal barrier by ameliorating mitochondrial DNA damage via the Nrf2/ARE signaling pathway.

PubMed

Hu, Qiongyuan; Ren, Jianan; Li, Guanwei; Wu, Jie; Wu, Xiuwen; Wang, Gefei; Gu, Guosheng; Ren, Huajian; Hong, Zhiwu; Li, Jieshou

2018-03-14

Disruption of the mucosal barrier following intestinal ischemia reperfusion (I/R) is life threatening in clinical practice. Mitochondrial dysfunction and oxidative stress significantly contribute to the early phase of I/R injury and amplify the inflammatory response. MitoQ is a mitochondrially targeted antioxidant that exerts protective effects following I/R injury. In the present study, we aimed to determine whether and how MitoQ protects intestinal epithelial cells (IECs) from I/R injury. In both in vivo and in vitro studies, we found that MitoQ pretreatment downregulated I/R-induced oxidative stress and stabilized the intestinal barrier, as evidenced by MitoQ-treated I/R mice exhibiting attenuated intestinal hyperpermeability, inflammatory response, epithelial apoptosis, and tight junction damage compared to controls. Mechanistically, I/R elevated mitochondrial 8-hydroxyguanine content, reduced mitochondrial DNA (mtDNA) copy number and mRNA transcription levels, and induced mitochondrial disruption in IECs. However, MitoQ pretreatment dramatically inhibited these deleterious effects. mtDNA depletion alone was sufficient to induce apoptosis and mitochondrial dysfunction of IECs. Mitochondrial transcription factor A (TFAM), a key activator of mitochondrial transcription, was significantly reduced during I/R injury, a phenomenon that was prevented by MitoQ treatment. Furthermore, we observed that thee protective properties of MitoQ were affected by upregulation of cellular antioxidant genes, including HO-1, NQO-1, and γ-GCLC. Transfection with Nrf2 siRNA in IECs exposed to hypoxia/reperfusion conditions partially blocked the effects of MitoQ on mtDNA damage and mitochondrial oxidative stress. In conclusion, our data suggest that MitoQ exerts protective effect on I/R-induced intestinal barrier dysfunction.

Succinyl-CoA Synthetase is a Phosphate Target for the Activation of Mitochondrial Metabolism

PubMed Central

Phillips, Darci; Aponte, Angel M.; French, Stephanie A.; Chess, David J.; Balaban, Robert S.

2009-01-01

Succinyl-CoA synthetase (SCS) is the only mitochondrial enzyme capable of ATP production via substrate level phosphorylation in the absence of oxygen, but it also plays a key role in the citric acid cycle, ketone metabolism and heme synthesis. Inorganic phosphate (Pi) is a signaling molecule capable of activating oxidative phosphorylation at several sites, including NADH generation and as a substrate for ATP formation. In this study it was shown that Pi-binds porcine heart SCS α-subunit (SCSα) in a non-covalent manner and enhances its enzymatic activity, thereby providing a new target for Pi-activation in mitochondria. Coupling 32P-labeling of intact mitochondria with SDS gel electrophoresis revealed that 32P-labeling of SCSα was enhanced in substrate-depleted mitochondria. Using mitochondrial extracts and purified bacterial SCS (BSCS) it was shown that this enhanced 32P-labeling resulted from a simple binding of 32P, not covalent protein phosphorylation. The ability of SCSα to retain its 32P throughout the SDS denaturing gel process was unique over the entire mitochondrial proteome. In vitro studies also revealed a Pi-induced activation of SCS activity by more than 2-fold when mitochondrial extracts and purified BSCS were incubated with mM concentrations of Pi. Since 32P-binding to SCSα was increased in substrate-depleted mitochondria, where matrix Pi concentration is increased, we conclude that SCS activation by Pi-binding represents another mitochondrial target for the Pi-induced activation of oxidative phosphorylation and anaerobic ATP production in energy-limited mitochondria. PMID:19527071

Innovative Methodology in the Discovery of Novel Drug Targets in the Free-Living Amoebae

PubMed

Baig, Abdul Mannan

2018-04-25

Despite advances in drug discovery and modifications in the chemotherapeutic regimens, human infections caused by free-living amoebae (FLA) have high mortality rates (~95%). The FLA that cause fatal human cerebral infections include Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. Novel drug-target discovery remains the only viable option to tackle these central nervous system (CNS) infection in order to lower the mortality rates caused by the FLA. Of these FLA, N. fowleri causes primary amoebic meningoencephalitis (PAM), while the A. castellanii and B. Mandrillaris are known to cause granulomatous amoebic encephalitis (GAE). The infections caused by the FLA have been treated with drugs like Rifampin, Fluconazole, Amphotericin-B and Miltefosine. Miltefosine is an anti-leishmanial agent and an experimental anti-cancer drug. With only rare incidences of success, these drugs have remained unsuccessful to lower the mortality rates of the cerebral infection caused by FLA. Recently, with the help of bioinformatic computational tools and the discovered genomic data of the FLA, discovery of newer drug targets has become possible. These cellular targets are proteins that are either unique to the FLA or shared between the humans and these unicellular eukaryotes. The latter group of proteins has shown to be targets of some FDA approved drugs prescribed in non-infectious diseases. This review out-lines the bioinformatic methodologies that can be used in the discovery of such novel drug-targets, their chronicle by in-vitro assays done in the past and the translational value of such target discoveries in human diseases caused by FLA. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery

PubMed Central

Gordon, Laurie J.; Wayne, Gareth J.; Almqvist, Helena; Axelsson, Hanna; Seashore-Ludlow, Brinton; Treyer, Andrea; Lundbäck, Thomas; West, Andy; Hann, Michael M.; Artursson, Per

2017-01-01

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery. PMID:28701380

Discovery of novel drug targets and their functions using phenotypic screening of natural products.

PubMed

Chang, Junghwa; Kwon, Ho Jeong

2016-03-01

Natural products are valuable resources that provide a variety of bioactive compounds and natural pharmacophores in modern drug discovery. Discovery of biologically active natural products and unraveling their target proteins to understand their mode of action have always been critical hurdles for their development into clinical drugs. For effective discovery and development of bioactive natural products into novel therapeutic drugs, comprehensive screening and identification of target proteins are indispensable. In this review, a systematic approach to understanding the mode of action of natural products isolated using phenotypic screening involving chemical proteomics-based target identification is introduced. This review highlights three natural products recently discovered via phenotypic screening, namely glucopiericidin A, ecumicin, and terpestacin, as representative case studies to revisit the pivotal role of natural products as powerful tools in discovering the novel functions and druggability of targets in biological systems and pathological diseases of interest.

Is antioxidant potential of the mitochondrial targeted ubiquinone derivative MitoQ conserved in cells lacking mtDNA?

PubMed

Lu, Chao; Zhang, Dawei; Whiteman, Matthew; Armstrong, Jeffrey S

2008-03-01

MitoQ has been developed as a mitochondrial targeted antioxidant for diseases associated with oxidative stress. Here we show that MitoQ blocks the generation of reactive oxygen species (ROS) and mitochondrial protein thiol oxidation, and preserves mitochondrial function and ultrastructure after glutathione (GSH) depletion. Furthermore, the antioxidant effect of MitoQ is conserved in cells lacking mitochondrial DNA, indicating that its antioxidant properties do not depend on a functional electron transport chain (ETC). Our results elucidate the antioxidant mechanism of MitoQ and suggest that it may be a useful therapeutic for disorders associated with a dysfunctional ETC and increased ROS production.

Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism

PubMed Central

Cheng, Gang; Zielonka, Jacek; McAllister, Donna; Hardy, Micael; Ouari, Olivier; Joseph, Joy; Dwinell, Michael B.; Kalyanaraman, Balaraman

2015-01-01

One of the proposed mechanisms for tumor proliferation involves redox signaling mediated by reactive oxygen species such as superoxide and hydrogen peroxide generated at moderate levels. Thus, the antiproliferative and anti-tumor effects of certain antioxidants were attributed to their ability to mitigate intracellular reactive oxygen species (ROS). Recent reports support a role for mitochondrial ROS in stimulating tumor cell proliferation. In this study, we compared the antiproliferative effects and the effects on mitochondrial bioenergetic functions of a mitochondria-targeted cationic carboxyproxyl nitroxide (Mito-CP), exhibiting superoxide dismutase (SOD)-like activity and a synthetic cationic acetamide analog (Mito-CP-Ac) lacking the nitroxide moiety responsible for the SOD activity. Results indicate that both Mito-CP and Mito-CP-Ac potently inhibited tumor cell proliferation. Both compounds altered mitochondrial and glycolytic functions, and intracellular citrate levels. Both Mito-CP and Mito-CP-Ac synergized with 2-deoxy-glucose (2-DG) to deplete intracellular ATP, inhibit cell proliferation and induce apoptosis in pancreatic cancer cells. We conclude that mitochondria-targeted cationic agents inhibit tumor proliferation via modification of mitochondrial bioenergetics pathways rather than by dismutating and detoxifying mitochondrial superoxide. PMID:26004344

Targeted overexpression of mitochondrial catalase prevents radiation-induced cognitive dysfunction.

PubMed

Parihar, Vipan K; Allen, Barrett D; Tran, Katherine K; Chmielewski, Nicole N; Craver, Brianna M; Martirosian, Vahan; Morganti, Josh M; Rosi, Susanna; Vlkolinsky, Roman; Acharya, Munjal M; Nelson, Gregory A; Allen, Antiño R; Limoli, Charles L

2015-01-01

Radiation-induced disruption of mitochondrial function can elevate oxidative stress and contribute to the metabolic perturbations believed to compromise the functionality of the central nervous system. To clarify the role of mitochondrial oxidative stress in mediating the adverse effects of radiation in the brain, we analyzed transgenic (mitochondrial catalase [MCAT]) mice that overexpress human catalase localized to the mitochondria. Compared with wild-type (WT) controls, overexpression of the MCAT transgene significantly decreased cognitive dysfunction after proton irradiation. Significant improvements in behavioral performance found on novel object recognition and object recognition in place tasks were associated with a preservation of neuronal morphology. While the architecture of hippocampal CA1 neurons was significantly compromised in irradiated WT mice, the same neurons in MCAT mice did not exhibit extensive and significant radiation-induced reductions in dendritic complexity. Irradiated neurons from MCAT mice maintained dendritic branching and length compared with WT mice. Protected neuronal morphology in irradiated MCAT mice was also associated with a stabilization of radiation-induced variations in long-term potentiation. Stabilized synaptic activity in MCAT mice coincided with an altered composition of the synaptic AMPA receptor subunits GluR1/2. Our findings provide the first evidence that neurocognitive sequelae associated with radiation exposure can be reduced by overexpression of MCAT, operating through a mechanism involving the preservation of neuronal morphology. Our article documents the neuroprotective properties of reducing mitochondrial reactive oxygen species through the targeted overexpression of catalase and how this ameliorates the adverse effects of proton irradiation in the brain.

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease

PubMed Central

Yin, Xiangling; Manczak, Maria; Reddy, P. Hemachandra

2016-01-01

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. PMID:26908605

Disruption of mitochondrial function as mechanism for anti-cancer activity of a novel mitochondriotropic menadione derivative.

PubMed

Teixeira, José; Amorim, Ricardo; Santos, Katia; Soares, Pedro; Datta, Sandipan; Cortopassi, Gino A; Serafim, Teresa L; Sardão, Vilma A; Garrido, Jorge; Borges, Fernanda; Oliveira, Paulo J

2018-01-15

Menadione, also known as vitamin K 3 , is a 2-methyl-1,4 naphthoquinone with a potent cytotoxic activity mainly resulting from its quinone redox-cycling with production of reactive oxygen species (ROS). Although increased ROS generation is considered a relevant mechanism in cancer cell death, it may not be sufficiently effective to kill cancer cells due to phenotypic adaptations. Therefore, combining ROS-generating agents with other molecules targeting important cancer cell phenotypes can be an effective therapeutic strategy. As mitochondrial dysfunction has been implicated in many human diseases, including cancer, we describe here the discovery of a mitochondrial-directed agent (MitoK 3 ), which was developed by conjugating a TPP cation to the C3 position of the menadione's naphthoquinone ring, increasing its selective accumulation in mitochondria, as well as led to alterations of its redox properties and consequent biological outcome. MitoK 3 disturbed the mitochondrial bioenergetic apparatus, with subsequent loss of mitochondrial ATP production. The combinatory strategy of MitoK 3 with anticancer agent doxorubicin (DOX) resulted in a degree of cytotoxicity higher than those of the individual molecules, as the combination triggered tumour apoptotic cell death evident by caspase 3/9 activities, probably through mitochondrial destabilization or by interference with mitochondrial redox processes. The results of this investigation support the importance of drug discovery process in developing molecules that can be use as adjuvant therapy in patients with specific cancer subtypes. Copyright © 2017 Elsevier B.V. All rights reserved.

Mitochondrial multifaceted dysfunction in schizophrenia; complex I as a possible pathological target.

PubMed

Ben-Shachar, Dorit

2017-09-01

Mitochondria are key players in various essential cellular processes beyond being the main energy supplier of the cell. Accordingly, they are involved in neuronal synaptic transmission, neuronal growth and sprouting and consequently neuronal plasticity and connectivity. In addition, mitochondria participate in the modulation of gene transcription and inflammation as well in physiological responses in health and disease. Schizophrenia is currently regarded as a neurodevelopmental disorder associated with impaired immune system, aberrant neuronal differentiation and abnormalities in various neurotransmitter systems mainly the dopaminergic, glutaminergic and GABAergic. Ample evidence has been accumulated over the last decade indicating a multifaceted dysfunction of mitochondria in schizophrenia. Indeed, mitochondrial deficit can be of relevance for the majority of the pathologies observed in this disease. In the present article, we overview specific deficits of the mitochondria in schizophrenia, with a focus on the first complex (complex I) of the mitochondrial electron transport chain (ETC). We argue that complex I, being a major factor in the regulation of mitochondrial ETC, is a possible key modulator of various functions of the mitochondria. We review biochemical, molecular, cellular and functional evidence for mitochondrial impairments and their possible convergence to impact in-vitro neuronal differentiation efficiency in schizophrenia. Mitochondrial function in schizophrenia may advance our knowledge of the disease pathophysiology and open the road for new treatment targets for the benefit of the patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

PubMed

Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

2012-05-01

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

Mitochondrial transcription in mammalian cells

PubMed Central

Shokolenko, Inna N.; Alexeyev, Mikhail F.

2017-01-01

As a consequence of recent discoveries of intimate involvement of mitochondria with key cellular processes, there has been a resurgence of interest in all aspects of mitochondrial biology, including the intricate mechanisms of mitochondrial DNA maintenance and expression. Despite four decades of research, there remains a lot to be learned about the processes that enable transcription of genetic information from mitochondrial DNA to RNA, as well as their regulation. These processes are vitally important, as evidenced by the lethality of inactivating the central components of mitochondrial transcription machinery. Here, we review the current understanding of mitochondrial transcription and its regulation in mammalian cells. We also discuss key theories in the field and highlight controversial subjects and future directions as we see them. PMID:27814650

Hot-spot analysis for drug discovery targeting protein-protein interactions.

PubMed

Rosell, Mireia; Fernández-Recio, Juan

2018-04-01

Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

Mitochondrial impairment contributes to cocaine-induced cardiac dysfunction: Prevention by the targeted antioxidant MitoQ.

PubMed

Vergeade, Aurélia; Mulder, Paul; Vendeville-Dehaudt, Cathy; Estour, François; Fortin, Dominique; Ventura-Clapier, Renée; Thuillez, Christian; Monteil, Christelle

2010-09-01

The goal of this study was to assess mitochondrial function and ROS production in an experimental model of cocaine-induced cardiac dysfunction. We hypothesized that cocaine abuse may lead to altered mitochondrial function that in turn may cause left ventricular dysfunction. Seven days of cocaine administration to rats led to an increased oxygen consumption detected in cardiac fibers, specifically through complex I and complex III. ROS levels were increased, specifically in interfibrillar mitochondria. In parallel there was a decrease in ATP synthesis, whereas no difference was observed in subsarcolemmal mitochondria. This uncoupling effect on oxidative phosphorylation was not detectable after short-term exposure to cocaine, suggesting that these mitochondrial abnormalities were a late rather than a primary event in the pathological response to cocaine. MitoQ, a mitochondrial-targeted antioxidant, was shown to completely prevent these mitochondrial abnormalities as well as cardiac dysfunction characterized here by a diastolic dysfunction studied with a conductance catheter to obtain pressure-volume data. Taken together, these results extend previous studies and demonstrate that cocaine-induced cardiac dysfunction may be due to a mitochondrial defect. Copyright 2010 Elsevier Inc. All rights reserved.

The Tuberculosis Drug Discovery and Development Pipeline and Emerging Drug Targets

PubMed Central

Mdluli, Khisimuzi; Kaneko, Takushi; Upton, Anna

2015-01-01

The recent accelerated approval for use in extensively drug-resistant and multidrug-resistant-tuberculosis (MDR-TB) of two first-in-class TB drugs, bedaquiline and delamanid, has reinvigorated the TB drug discovery and development field. However, although several promising clinical development programs are ongoing to evaluate new TB drugs and regimens, the number of novel series represented is few. The global early-development pipeline is also woefully thin. To have a chance of achieving the goal of better, shorter, safer TB drug regimens with utility against drug-sensitive and drug-resistant disease, a robust and diverse global TB drug discovery pipeline is key, including innovative approaches that make use of recently acquired knowledge on the biology of TB. Fortunately, drug discovery for TB has resurged in recent years, generating compounds with varying potential for progression into developable leads. In parallel, advances have been made in understanding TB pathogenesis. It is now possible to apply the lessons learned from recent TB hit generation efforts and newly validated TB drug targets to generate the next wave of TB drug leads. Use of currently underexploited sources of chemical matter and lead-optimization strategies may also improve the efficiency of future TB drug discovery. Novel TB drug regimens with shorter treatment durations must target all subpopulations of Mycobacterium tuberculosis existing in an infection, including those responsible for the protracted TB treatment duration. This review summarizes the current TB drug development pipeline and proposes strategies for generating improved hits and leads in the discovery phase that could help achieve this goal. PMID:25635061

Targeted Overexpression of Mitochondrial Catalase Prevents Radiation-Induced Cognitive Dysfunction

PubMed Central

Parihar, Vipan K.; Allen, Barrett D.; Tran, Katherine K.; Chmielewski, Nicole N.; Craver, Brianna M.; Martirosian, Vahan; Morganti, Josh M.; Rosi, Susanna; Vlkolinsky, Roman; Acharya, Munjal M.; Nelson, Gregory A.; Allen, Antiño R.

2015-01-01

Abstract Aims: Radiation-induced disruption of mitochondrial function can elevate oxidative stress and contribute to the metabolic perturbations believed to compromise the functionality of the central nervous system. To clarify the role of mitochondrial oxidative stress in mediating the adverse effects of radiation in the brain, we analyzed transgenic (mitochondrial catalase [MCAT]) mice that overexpress human catalase localized to the mitochondria. Results: Compared with wild-type (WT) controls, overexpression of the MCAT transgene significantly decreased cognitive dysfunction after proton irradiation. Significant improvements in behavioral performance found on novel object recognition and object recognition in place tasks were associated with a preservation of neuronal morphology. While the architecture of hippocampal CA1 neurons was significantly compromised in irradiated WT mice, the same neurons in MCAT mice did not exhibit extensive and significant radiation-induced reductions in dendritic complexity. Irradiated neurons from MCAT mice maintained dendritic branching and length compared with WT mice. Protected neuronal morphology in irradiated MCAT mice was also associated with a stabilization of radiation-induced variations in long-term potentiation. Stabilized synaptic activity in MCAT mice coincided with an altered composition of the synaptic AMPA receptor subunits GluR1/2. Innovation: Our findings provide the first evidence that neurocognitive sequelae associated with radiation exposure can be reduced by overexpression of MCAT, operating through a mechanism involving the preservation of neuronal morphology. Conclusion: Our article documents the neuroprotective properties of reducing mitochondrial reactive oxygen species through the targeted overexpression of catalase and how this ameliorates the adverse effects of proton irradiation in the brain. Antioxid. Redox Signal. 22, 78–91. PMID:24949841

Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

PubMed Central

Calvo, Sarah E.; Compton, Alison G.; Hershman, Steven G.; Lim, Sze Chern; Lieber, Daniel S.; Tucker, Elena J.; Laskowski, Adrienne; Garone, Caterina; Liu, Shangtao; Jaffe, David B.; Christodoulou, John; Fletcher, Janice M.; Bruno, Damien L; Goldblatt, Jack; DiMauro, Salvatore; Thorburn, David R.; Mootha, Vamsi K.

2012-01-01

Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. While in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic utility, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease, who were refractory to traditional molecular diagnosis. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with over 100 causal genes identified to date. We performed “MitoExome” sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Since patients and controls harbored a comparable number of such heterozygous alleles, we could not prioritize dominant acting genes. However, patients showed a five-fold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23/42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. 13 patients (31%) had mutations in nuclear genes never linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by cDNA complementation and evidence from multiple patients, respectively. The results underscore the immediate potential and challenges of deploying NGS in clinical settings. PMID:22277967

Aging Biology and Novel Targets for Drug Discovery

PubMed Central

McLachlan, Andrew J.; Quinn, Ronald J.; Simpson, Stephen J.; de Cabo, Rafael

2012-01-01

Despite remarkable technological advances in genetics and drug screening, the discovery of new pharmacotherapies has slowed and new approaches to drug development are needed. Research into the biology of aging is generating many novel targets for drug development that may delay all age-related diseases and be used long term by the entire population. Drugs that successfully delay the aging process will clearly become “blockbusters.” To date, the most promising leads have come from studies of the cellular pathways mediating the longevity effects of caloric restriction (CR), particularly target of rapamycin and the sirtuins. Similar research into pathways governing other hormetic responses that influence aging is likely to yield even more targets. As aging becomes a more attractive target for drug development, there will be increasing demand to develop biomarkers of aging as surrogate outcomes for the testing of the effects of new agents on the aging process. PMID:21693687

Microfluidics for Drug Discovery and Development: From Target Selection to Product Lifecycle Management

PubMed Central

Kang, Lifeng; Chung, Bong Geun; Langer, Robert; Khademhosseini, Ali

2009-01-01

Microfluidic technologies’ ability to miniaturize assays and increase experimental throughput have generated significant interest in the drug discovery and development domain. These characteristics make microfluidic systems a potentially valuable tool for many drug discovery and development applications. Here, we review the recent advances of microfluidic devices for drug discovery and development and highlight their applications in different stages of the process, including target selection, lead identification, preclinical tests, clinical trials, chemical synthesis, formulations studies, and product management. PMID:18190858

Mitochondria-targeted molecules MitoQ and SS31 reduce mutant huntingtin-induced mitochondrial toxicity and synaptic damage in Huntington's disease.

PubMed

Yin, Xiangling; Manczak, Maria; Reddy, P Hemachandra

2016-05-01

The objective of this study was to determine the protective effects of the mitochondria-targeted molecules MitoQ and SS31 in striatal neurons that stably express mutant huntingtin (Htt) (STHDhQ111/Q111) in Huntington's disease (HD). We studied mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, mitochondrial function, and ultra-structural changes in MitoQ- and SS31-treated mutant Htt neurons relative to untreated mutant Htt neurons. We used gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods. In the MitoQ- and SS31-treated mutant Htt neurons, fission genes Drp1 and Fis1 were down-regulated, and fusion genes Mfn1, Mfn2 and Opa1 were up-regulated relative to untreated neurons, suggesting that mitochondria-targeted molecules reduce fission activity. Interestingly, the mitochondrial biogenesis genes PGC1α, PGC1β, Nrf1, Nrf2 and TFAM were up-regulated in MitoQ- and SS31-treated mutant Htt neurons. The synaptic genes synaptophysin and PSD95 were up-regulated, and mitochondrial function was normal in the MitoQ- and SS31-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with the mRNA findings. TEM studies revealed decreased numbers of structurally intact mitochondria in MitoQ- and SS31-treated mutant Htt neurons. These findings suggest that mitochondria-targeted molecules MitoQ and SS31 are protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons, and these mitochondria-targeted molecules are potential therapeutic molecules for the treatment of HD neurons. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Sensors, transmitters, and targets in mitochondrial oxygen shortage-a hypoxia-inducible factor relay story.

PubMed

Dehne, Nathalie; Brüne, Bernhard

2014-01-10

Cells sense and respond to a shortage of oxygen by activating the hypoxia-inducible transcription factors HIF-1 and HIF-2 and evoking adaptive responses. Mitochondria are at the center of a hypoxia sensing and responding relay system. Under normoxia, reactive oxygen species (ROS) and nitric oxide (NO) are HIF activators. As their individual flux rates determine their diffusion-controlled interaction, predictions how these radicals affect HIF appear context-dependent. Considering that the oxygen requirement for NO formation limits its role in activating HIF to conditions of ambient oxygen tension. Given the central role of mitochondrial complex IV as a NO target, especially under hypoxia, allows inhibition of mitochondrial respiration by NO to spare oxygen thus, raising the threshold for HIF activation. HIF targets seem to configure a feedback-signaling circuit aimed at gradually adjusting mitochondrial function. In hypoxic cancer cells, mitochondria redirect Krebs cycle intermediates to preserve their biosynthetic ability. Persistent HIF activation lowers the entry of electron-delivering compounds into mitochondria to reduce Krebs cycle fueling and β-oxidation, attenuates the expression of electron transport chain components, limits mitochondria biosynthesis, and provokes their removal by autophagy. Mitochondria can be placed central in a hypoxia sensing-hypoxia responding circuit. We need to determine to which extent and how mitochondria contribute to sense hypoxia, explore whether modulating their oxygen-consuming capacity redirects hypoxic responses in in vivo relevant disease conditions, and elucidate how the multiple HIF targets in mitochondria shape conditions of acute versus chronic hypoxia.

Physical consequences of the mitochondrial targeting of single-walled carbon nanotubes probed computationally

NASA Astrophysics Data System (ADS)

Chistyakov, V. A.; Zolotukhin, P. V.; Prazdnova, E. V.; Alperovich, I.; Soldatov, A. V.

2015-06-01

Experiments by F. Zhou and coworkers (2010) [16] showed that mitochondria are the main target of the cellular accumulation of single-walled carbon nanotubes (SWCNTs). Our in silico experiments, based on geometrical optimization of the system consisting of SWCNT+proton within Density Functional Theory, revealed that protons can bind to the outer side of SWCNT so generating a positive charge. Calculation results allow one to propose the following mechanism of SWCNTs mitochondrial targeting. SWCNTs enter the space between inner and outer membranes of mitochondria, where the excess of protons has been formed by diffusion. In this compartment SWCNTs are loaded with protons and acquire positive charges distributed over their surface. Protonation of hydrophobic SWCNTs can also be carried out within the mitochondrial membrane through interaction with the protonated ubiquinone. Such "charge loaded" particles can be transferred as "Sculachev ions" through the inner membrane of the mitochondria due to the potential difference generated by the inner membrane. Physiological consequences of the described mechanism are discussed.

A phosphorescent rhenium(I) histone deacetylase inhibitor: mitochondrial targeting and paraptosis induction.

PubMed

Ye, Rui-Rong; Tan, Cai-Ping; Lin, Yan-Nan; Ji, Liang-Nian; Mao, Zong-Wan

2015-05-14

In this report, we designed a histone deacetylase-targeted phosphorescent Re(I) complex ReLMito. Colocalization studies suggested that ReLMito could specially localize to mitochondria. We also demonstrated that ReLMito could induce paraptosis in cancer cells. These features endowed the complex with potential to induce and monitor mitochondrial morphological changes during the paraptosis simultaneously.

Sorafenib and FH535 in combination act synergistically on hepatocellular carcinoma by targeting cell bioenergetics and mitochondrial function.

PubMed

Turcios, Lilia; Vilchez, Valery; Acosta, Luis F; Poyil, Pratheeshkumar; Butterfield, David Allan; Mitov, Mihail; Marti, Francesc; Gedaly, Roberto

2017-06-01

Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Small Interfering RNA Targeting Mitochondrial Calcium Uniporter Improves Cardiomyocyte Cell Viability in Hypoxia/Reoxygenation Injury by Reducing Calcium Overload.

PubMed

Oropeza-Almazán, Yuriana; Vázquez-Garza, Eduardo; Chapoy-Villanueva, Héctor; Torre-Amione, Guillermo; García-Rivas, Gerardo

2017-01-01

Intracellular Ca 2+ mishandling is an underlying mechanism in hypoxia/reoxygenation (H/R) injury that results in mitochondrial dysfunction and cardiomyocytes death. These events are mediated by mitochondrial Ca 2+ ( m Ca 2+ ) overload that is facilitated by the mitochondrial calcium uniporter (MCU) channel. Along this line, we evaluated the effect of siRNA-targeting MCU in cardiomyocytes subjected to H/R injury. First, cardiomyocytes treated with siRNA demonstrated a reduction of MCU expression by 67%, which resulted in significant decrease in mitochondrial Ca 2+ transport. siRNA treated cardiomyocytes showed decreased mitochondrial permeability pore opening and oxidative stress trigger by Ca 2+ overload. Furthermore, after H/R injury MCU silencing decreased necrosis and apoptosis levels by 30% and 50%, respectively, and resulted in reduction in caspases 3/7, 9, and 8 activity. Our findings are consistent with previous conclusions that demonstrate that MCU activity is partly responsible for cellular injury induced by H/R and support the concept of utilizing siRNA-targeting MCU as a potential therapeutic strategy.

Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy.

PubMed

Fiesel, Fabienne C; James, Elle D; Hudec, Roman; Springer, Wolfdieter

2017-12-05

Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson's disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.

In silico pharmacology for drug discovery: applications to targets and beyond

PubMed Central

Ekins, S; Mestres, J; Testa, B

2007-01-01

Computational (in silico) methods have been developed and widely applied to pharmacology hypothesis development and testing. These in silico methods include databases, quantitative structure-activity relationships, similarity searching, pharmacophores, homology models and other molecular modeling, machine learning, data mining, network analysis tools and data analysis tools that use a computer. Such methods have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. The first part of this review discussed the methods that have been used for virtual ligand and target-based screening and profiling to predict biological activity. The aim of this second part of the review is to illustrate some of the varied applications of in silico methods for pharmacology in terms of the targets addressed. We will also discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. Our conclusion is that the in silico pharmacology paradigm is ongoing and presents a rich array of opportunities that will assist in expediating the discovery of new targets, and ultimately lead to compounds with predicted biological activity for these novel targets. PMID:17549046

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease.

PubMed

Solesio, María E; Prime, Tracy A; Logan, Angela; Murphy, Michael P; Del Mar Arroyo-Jimenez, María; Jordán, Joaquín; Galindo, María F

2013-01-01

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50nM, 30min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress. Copyright © 2012 Elsevier B.V. All rights reserved.

Mitochondrial dynamics and Parkinson's disease: focus on parkin.

PubMed

Lim, Kah-Leong; Ng, Xiao-Hui; Grace, Lim Gui-Yin; Yao, Tso-Pang

2012-05-01

Parkinson's disease (PD) is a prevalent neurodegenerative disease affecting millions of individuals worldwide. Despite intensive efforts devoted to drug discovery, the disease remains incurable. To provide more effective medical therapy for PD, better understanding of the underlying causes of the disease is clearly necessary. A broad range of studies conducted over the past few decades have collectively implicated aberrant mitochondrial homeostasis as a key contributor to the development of PD. Supporting this, mutations in several PD-linked genes are directly or indirectly linked to mitochondrial dysfunction. In particular, recent discoveries have identified parkin, whose mutations are causative of recessive parkinsonism, as a key regulator of mitochondrial homeostasis. Parkin appears to be involved in the entire spectrum of mitochondrial dynamics, including organelle biogenesis, fusion/fission, and clearance via mitophagy. How a single protein can regulate such diverse mitochondrial events is as intriguing as it is amazing; the mechanism underlying this is currently under intense research. Here, we provide an overview of mitochondrial dynamics and its relationship with neurodegenerative diseases and discuss current evidence and controversies surrounding the role of parkin in mitochondrial quality control and its relevance to PD pathogenesis. Although the emerging field of parkin-mediated mitochondrial quality control has proven to be exciting, it is important to recognize that PD pathogenesis is likely to involve an intricate network of interacting pathways. Elucidating the reciprocity of pathways, particularly how other PD-related pathways potentially influence mitochondrial homeostasis, may hold the key to therapeutic development.

Mitochondrial permeability transition in cardiac ischemia–reperfusion: whether cyclophilin D is a viable target for cardioprotection?

PubMed Central

Jang, Sehwan; Parodi-Rullan, Rebecca; Khuchua, Zaza; Kuznetsov, Andrey V.

2018-01-01

Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia–reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia–reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors. PMID:28378042

Surveillance theory applied to virus detection: a case for targeted discovery

USGS Publications Warehouse

Bogich, Tiffany L.; Anthony, Simon J.; Nichols, James D.

2013-01-01

Virus detection and mathematical modeling have gone through rapid developments in the past decade. Both offer new insights into the epidemiology of infectious disease and characterization of future risk; however, modeling has not yet been applied to designing the best surveillance strategies for viral and pathogen discovery. We review recent developments and propose methods to integrate viral and pathogen discovery and mathematical modeling through optimal surveillance theory, arguing for a more targeted approach to novel virus detection guided by the principles of adaptive management and structured decision-making.

Measurement of Mitochondrial Cholesterol Import Using a Mitochondria-Targeted CYP11A1 Fusion Construct.

PubMed

Kennedy, Barry E; Charman, Mark; Karten, Barbara

2017-01-01

All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimer's, and Niemann-Pick Type C Disease. The mechanisms of mitochondrial cholesterol import are not fully elucidated yet, and may vary in different cell types and environmental conditions. Measuring cholesterol trafficking to the mitochondrial membranes is technically challenging because of its low abundance; for example, traditional pulse-chase experiments with isotope-labeled cholesterol are not feasible. Here, we describe improvements to a method first developed by the Miller group at the University of California to measure cholesterol trafficking to the inner mitochondrial membrane (IMM) through the conversion of cholesterol to pregnenolone. This method uses a mitochondria-targeted, ectopically expressed fusion construct of CYP11A1, ferredoxin reductase and ferredoxin. Pregnenolone is formed exclusively from cholesterol at the IMM, and can be analyzed with high sensitivity and specificity through ELISA or radioimmunoassay of the medium/buffer to reflect mitochondrial cholesterol import. This assay can be used to investigate the effects of genetic or pharmacological interventions on mitochondrial cholesterol import in cultured cells or isolated mitochondria.

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.

PubMed

Kvirkvelia, Nino; McMenamin, Malgorzata; Warren, Marie; Jadeja, Ravirajsinh N; Kodeboyina, Sai Karthik; Sharma, Ashok; Zhi, Wenbo; O'Connor, Paul M; Raju, Raghavan; Lucas, Rudolf; Madaio, Michael P

2018-05-04

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Natural products used as a chemical library for protein-protein interaction targeted drug discovery.

PubMed

Jin, Xuemei; Lee, Kyungro; Kim, Nam Hee; Kim, Hyun Sil; Yook, Jong In; Choi, Jiwon; No, Kyoung Tai

2018-01-01

Protein-protein interactions (PPIs), which are essential for cellular processes, have been recognized as attractive therapeutic targets. Therefore, the construction of a PPI-focused chemical library is an inevitable necessity for future drug discovery. Natural products have been used as traditional medicines to treat human diseases for millennia; in addition, their molecular scaffolds have been used in diverse approved drugs and drug candidates. The recent discovery of the ability of natural products to inhibit PPIs led us to use natural products as a chemical library for PPI-targeted drug discovery. In this study, we collected natural products (NPDB) from non-commercial and in-house databases to analyze their similarities to small-molecule PPI inhibitors (iPPIs) and FDA-approved drugs by using eight molecular descriptors. Then, we evaluated the distribution of NPDB and iPPIs in the chemical space, represented by the molecular fingerprint and molecular scaffolds, to identify the promising scaffolds, which could interfere with PPIs. To investigate the ability of natural products to inhibit PPI targets, molecular docking was used. Then, we predicted a set of high-potency natural products by using the iPPI-likeness score based on a docking score-weighted model. These selected natural products showed high binding affinities to the PPI target, namely XIAP, which were validated in an in vitro experiment. In addition, the natural products with novel scaffolds might provide a promising starting point for further medicinal chemistry developments. Overall, our study shows the potency of natural products in targeting PPIs, which might help in the design of a PPI-focused chemical library for future drug discovery. Copyright © 2017 Elsevier Inc. All rights reserved.

The N-terminus of survivin is a mitochondrial-targeting sequence and Src regulator

PubMed Central

Dunajová, Lucia; Cash, Emily; Markus, Robert; Rochette, Sophie; Townley, Amelia R.

2016-01-01

ABSTRACT Survivin (also known as BIRC5) is a cancer-associated protein that exists in several locations in the cell. Its cytoplasmic residence in interphase cells is governed by CRM1 (also known as XPO1)-mediated nuclear exportation, and its localisation during mitosis to the centromeres and midzone microtubules is that of a canonical chromosomal passenger protein. In addition to these well-established locations, survivin is also a mitochondrial protein, but how it gets there and its function therein is presently unclear. Here, we show that the first ten amino acids at the N-terminus of survivin are sufficient to target GFP to the mitochondria in vivo, and ectopic expression of this decapeptide decreases cell adhesion and accelerates proliferation. The data support a signalling mechanism in which this decapeptide regulates the tyrosine kinase Src, leading to reduced focal adhesion plaques and disruption of F-actin organisation. This strongly suggests that the N-terminus of survivin is a mitochondrial-targeting sequence that regulates Src, and that survivin acts in concert with Src to promote tumorigenesis. PMID:27246243

Design of a Covert RFID Tag Network for Target Discovery and Target Information Routing

PubMed Central

Pan, Qihe; Narayanan, Ram M.

2011-01-01

Radio frequency identification (RFID) tags are small electronic devices working in the radio frequency range. They use wireless radio communications to automatically identify objects or people without the need for line-of-sight or contact, and are widely used in inventory tracking, object location, environmental monitoring. This paper presents a design of a covert RFID tag network for target discovery and target information routing. In the design, a static or very slowly moving target in the field of RFID tags transmits a distinct pseudo-noise signal, and the RFID tags in the network collect the target information and route it to the command center. A map of each RFID tag’s location is saved at command center, which can determine where a RFID tag is located based on each RFID tag’s ID. We propose the target information collection method with target association and clustering, and we also propose the information routing algorithm within the RFID tag network. The design and operation of the proposed algorithms are illustrated through examples. Simulation results demonstrate the effectiveness of the design. PMID:22163693

Design of a covert RFID tag network for target discovery and target information routing.

PubMed

Pan, Qihe; Narayanan, Ram M

2011-01-01

Radio frequency identification (RFID) tags are small electronic devices working in the radio frequency range. They use wireless radio communications to automatically identify objects or people without the need for line-of-sight or contact, and are widely used in inventory tracking, object location, environmental monitoring. This paper presents a design of a covert RFID tag network for target discovery and target information routing. In the design, a static or very slowly moving target in the field of RFID tags transmits a distinct pseudo-noise signal, and the RFID tags in the network collect the target information and route it to the command center. A map of each RFID tag's location is saved at command center, which can determine where a RFID tag is located based on each RFID tag's ID. We propose the target information collection method with target association and clustering, and we also propose the information routing algorithm within the RFID tag network. The design and operation of the proposed algorithms are illustrated through examples. Simulation results demonstrate the effectiveness of the design.

Euglena gracilis and Trypanosomatids possess common patterns in predicted mitochondrial targeting presequences.

PubMed

Krnáčová, Katarína; Vesteg, Matej; Hampl, Vladimír; Vlček, Čestmír; Horváth, Anton

2012-10-01

Euglena gracilis possessing chloroplasts of secondary green algal origin and parasitic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania major belong to the protist phylum Euglenozoa. Euglenozoa might be among the earliest eukaryotic branches bearing ancestral traits reminiscent of the last eukaryotic common ancestor (LECA) or missing features present in other eukaryotes. LECA most likely possessed mitochondria of endosymbiotic α-proteobacterial origin. In this study, we searched for the presence of homologs of mitochondria-targeted proteins from other organisms in the currently available EST dataset of E. gracilis. The common motifs in predicted N-terminal presequences and corresponding homologs from T. brucei, T. cruzi and L. major (if found) were analyzed. Other trypanosomatid mitochondrial protein precursor (e.g., those involved in RNA editing) were also included in the analysis. Mitochondrial presequences of E. gracilis and these trypanosomatids seem to be highly variable in sequence length (5-118 aa), but apparently share statistically significant similarities. In most cases, the common (M/L)RR motif is present at the N-terminus and it is probably responsible for recognition via import apparatus of mitochondrial outer membrane. Interestingly, this motif is present inside the predicted presequence region in some cases. In most presequences, this motif is followed by a hydrophobic region rich in alanine, leucine, and valine. In conclusion, either RR motif or arginine-rich region within hydrophobic aa-s present at the N-terminus of a preprotein can be sufficient signals for mitochondrial import irrespective of presequence length in Euglenozoa.

Mitochondrially Targeted α-Tocopheryl Succinate Is Antiangiogenic: Potential Benefit Against Tumor Angiogenesis but Caution Against Wound Healing

PubMed Central

Kluckova, Katarina; Zobalova, Renata; Goodwin, Jacob; Tilly, David; Stursa, Jan; Pecinova, Alena; Philimonenko, Anatoly; Hozak, Pavel; Banerjee, Jaideep; Ledvina, Miroslav; Sen, Chandan K.; Houstek, Josef; Coster, Mark J.

2011-01-01

Abstract Aims A plausible strategy to reduce tumor progress is the inhibition of angiogenesis. Therefore, agents that efficiently suppress angiogenesis can be used for tumor suppression. We tested the antiangiogenic potential of a mitochondrially targeted analog of α-tocopheryl succinate (MitoVES), a compound with high propensity to induce apoptosis. Results MitoVES was found to efficiently kill proliferating endothelial cells (ECs) but not contact-arrested ECs or ECs deficient in mitochondrial DNA, and suppressed angiogenesis in vitro by inducing accumulation of reactive oxygen species and induction of apoptosis in proliferating/angiogenic ECs. Resistance of arrested ECs was ascribed, at least in part, to the lower mitochondrial inner transmembrane potential compared with the proliferating ECs, thus resulting in the lower level of mitochondrial uptake of MitoVES. Shorter-chain homologs of MitoVES were less efficient in angiogenesis inhibition, thus suggesting a molecular mechanism of its activity. Finally, MitoVES was found to suppress HER2-positive breast carcinomas in a transgenic mouse as well as inhibit tumor angiogenesis. The antiangiogenic efficacy of MitoVES was corroborated by its inhibitory activity on wound healing in vivo. Innovation and Conclusion We conclude that MitoVES, a mitochondrially targeted analog of α-tocopheryl succinate, is an efficient antiangiogenic agent of potential clinical relevance, exerting considerably higher activity than its untargeted counterpart. MitoVES may be helpful against cancer but may compromise wound healing. Antioxid. Redox Signal. 15, 2923–2935. PMID:21902599

Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches

DTIC Science & Technology

2012-07-01

1-0431 TITLE: Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches PRINCIPAL INVESTIGATOR...July 2012 2. REPORT TYPE Final 3. DATES COVERED (From - To) 1 July 2008 – 30 June 2012 4. TITLE AND SUBTITLE Biomarker Discovery and Mechanistic...Department of Defense Synergistic Idea Development Award W81XWH-08-1-0430 (to H.Z) and W81XWH-08-1-0431 (to N.K.), an NIH/NCRR COBRE grant 1P20RR020171 (to

Targeting mitochondrial STAT3 with the novel phospho-valproic acid (MDC-1112) inhibits pancreatic cancer growth in mice.

PubMed

Mackenzie, Gerardo G; Huang, Liqun; Alston, Ninche; Ouyang, Nengtai; Vrankova, Kvetoslava; Mattheolabakis, George; Constantinides, Panayiotis P; Rigas, Basil

2013-01-01

New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%-97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-xL, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.

Role of mitochondrial dysfunction and altered autophagy in cardiovascular aging and disease: from mechanisms to therapeutics

PubMed Central

Marzetti, Emanuele; Csiszar, Anna; Dutta, Debapriya; Balagopal, Gauthami; Calvani, Riccardo

2013-01-01

Advanced age is associated with a disproportionate prevalence of cardiovascular disease (CVD). Intrinsic alterations in the heart and the vasculature occurring over the life course render the cardiovascular system more vulnerable to various stressors in late life, ultimately favoring the development of CVD. Several lines of evidence indicate mitochondrial dysfunction as a major contributor to cardiovascular senescence. Besides being less bioenergetically efficient, damaged mitochondria also produce increased amounts of reactive oxygen species, with detrimental structural and functional consequences for the cardiovascular system. The age-related accumulation of dysfunctional mitochondrial likely results from the combination of impaired clearance of damaged organelles by autophagy and inadequate replenishment of the cellular mitochondrial pool by mitochondriogenesis. In this review, we summarize the current knowledge about relevant mechanisms and consequences of age-related mitochondrial decay and alterations in mitochondrial quality control in the cardiovascular system. The involvement of mitochondrial dysfunction in the pathogenesis of cardiovascular conditions especially prevalent in late life and the emerging connections with neurodegeneration are also illustrated. Special emphasis is placed on recent discoveries on the role played by alterations in mitochondrial dynamics (fusion and fission), mitophagy, and their interconnections in the context of age-related CVD and endothelial dysfunction. Finally, we discuss pharmacological interventions targeting mitochondrial dysfunction to delay cardiovascular aging and manage CVD. PMID:23748424

Fruitful research: drug target discovery for neurodegenerative diseases in Drosophila.

PubMed

Konsolaki, Mary

2013-12-01

Although vertebrate model systems have obvious advantages in the study of human disease, invertebrate organisms have contributed enormously to this field as well. The conservation of genome structure and physiology among organisms poses unexpected peculiarities, and the redundancy in certain gene families or the presence of polymorphisms that can slightly alter gene expression can, in certain instances, bring invertebrate systems, such as Drosophila, closer to humans than mice and vice versa. This necessitates the analysis of disease pathways in multiple model organisms. The author highlights findings from Drosophila models of neurodegenerative diseases that have occurred in the past few years. She also highlights and discusses various molecular, genetic and genomic tools used in flies, as well as methods for generating disease models. Finally, the author describes Drosophila models of Alzheimer's, Parkinson's tri-nucleotide repeat diseases, and Fragile X syndrome and summarizes insights in disease mechanisms that have been discovered directly in fly models. Full genome genetic screens in Drosophila can lead to the rapid identification of drug target candidates that can be subsequently validated in a vertebrate system. In addition, the Drosophila models of neurodegeneration may often show disease phenotypes that are absent in equivalent mouse models. The author believes that the extensive contribution of Drosophila to both new disease drug target discovery, in addition to target validation, makes them indispensible to drug discovery and development.

Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei: TARGETING SIGNAL AND DEPENDENCE ON A UNIQUE BIOGENESIS FACTOR.

PubMed

Bruggisser, Julia; Käser, Sandro; Mani, Jan; Schneider, André

2017-02-24

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei , is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death.

PubMed

Bernardinelli, Emanuele; Costa, Roberta; Scantamburlo, Giada; To, Janet; Morabito, Rossana; Nofziger, Charity; Doerrier, Carolina; Krumschnabel, Gerhard; Paulmichl, Markus; Dossena, Silvia

2017-01-01

Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis.

Gallic acid targets acute myeloid leukemia via Akt/mTOR-dependent mitochondrial respiration inhibition.

PubMed

Gu, Ruixin; Zhang, Minqin; Meng, Hu; Xu, Dandan; Xie, Yonghua

2018-06-05

Gallic acid is one of the many phenolic acids that can be found in dietary substances and traditional medicine herbs. The anti-cancer activities of gallic acid have been shown in various cancers but its underlying molecular mechanisms are not well understood. In this study, we show Akt/mammalian target of rapamycin (mTOR)-dependent inhibition of mitochondrial respiration as a mechanism of gallic acid's action in acute myeloid leukemia (AML). Gallic acid significantly induces apoptosis of AML cell lines, primary mononuclear cells (MNC) and CD34 stem/progenitors isolated form AML patients via caspase-dependent pathway. It also significantly enhances two standard AML chemotherapeutic agents' efficacy in vitro cell culture system and in vivo xenograft model. Gallic acid inhibits dose- and time-dependent mitochondrial respiration, leading to decreased ATP production and oxidative stress. Overexpression of constitutively active Akt restores gallic acid-mediated inhibition of mTOR signaling, mitochondrial dysfunction, energy crisis and apoptosis. Our results demonstrate that mitochondrial respiration inhibition by gallic acid is a consequence of Akt/mTOR signaling suppression. Our findings suggest that combination therapy with gallic acid may enhance antileukemic efficacy of standard chemotherapeutic agents in AML. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Abnormal Mitochondrial Dynamics and Synaptic Degeneration as Early Events in Alzheimer’s Disease: Implications to Mitochondria-Targeted Antioxidant Therapeutics

PubMed Central

Reddy, P. Hemachandra; Tripathy, Raghav; Troung, Quang; Thirumala, Karuna; Reddy, Tejaswini P.; Anekonda, Vishwanath; Shirendeb, Ulziibat P.; Calkins, Marcus J.; Reddy, Arubala P.; Mao, Peizhong; Manczak, Maria

2011-01-01

Synaptic pathology and mitochondrial oxidative damage are early events in Alzheimer’s disease (AD) progression. Loss of synapses and synaptic damage are the best correlate of cognitive deficits found in AD patients. Recent research on amyloid bet (Aβ) and mitochondria in AD revealed that Aβ accumulates in synapses and synaptic mitochondria, leading to abnormal mitochondrial dynamics and synaptic degeneration in AD neurons. Further, recent studies using live-cell imaging and primary neurons from amyloid beta precursor protein (AβPP) transgenic mice revealed that reduced mitochondrial mass, defective axonal transport of mitochondria and synaptic degeneration, indicating that Aβ is responsible for mitochondrial and synaptic deficiencies. Tremendous progress has been made in studying antioxidant approaches in mouse models of AD and clinical trials of AD patients. This article highlights the recent developments made in Aβ-induced abnormal mitochondrial dynamics, defective mitochondrial biogenesis, impaired axonal transport and synaptic deficiencies in AD. This article also focuses on mitochondrial approaches in treating AD, and also discusses latest research on mitochondria-targeted antioxidants in AD. PMID:22037588

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

Decaprenyl-phosphoryl-ribose 2'-epimerase (DprE1): challenging target for antitubercular drug discovery.

PubMed

Gawad, Jineetkumar; Bonde, Chandrakant

2018-06-23

Tuberculosis has proved harmful to the entire history of mankind from past several decades. Decaprenyl-phosphoryl-ribose 2'-epimerase (DprE1) is a recent target which was identified in 2009 but unfortunately it is neither explored nor crossed phase II. In past several decades few targets were identified for effective antitubercular drug discovery. Resistance is the major problem for effective antitubercular drug discovery. Arabinose is constituent of mycobacterium cell wall. Biosynthesis of arabinose is FAD dependant two step epimerisation reaction which is catalysed by DprE1 and DprE2 flavoprotein enzymes. The current review is mainly emphases on DprE1 as a perspective challenge for further research.

Chemical proteomics for target discovery of head-to-tail cyclized mini-proteins

NASA Astrophysics Data System (ADS)

Hellinger, Roland; Thell, Kathrin; Vasileva, Mina; Muhammad, Taj; Gunasekera, Sunithi; Kümmel, Daniel; Göransson, Ulf; Becker, Christian W.; Gruber, Christian W.

2017-10-01

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomal synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target of the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

BCL-2 Antagonism to Target the Intrinsic Mitochondrial Pathway of Apoptosis

PubMed Central

Gibson, Christopher J.; Davids, Matthew S.

2015-01-01

Despite significant improvements in treatment, cure rates for many cancers remain suboptimal. The rise of cytotoxic chemotherapy has led to curative therapy for a subset of cancers, though intrinsic treatment resistance is difficult to predict for individual patients. The recent wave of molecularly targeted therapies has focused on druggable activating mutations, and is thus limited to specific subsets of patients. The lessons learned from these two disparate approaches suggest the need for therapies that borrow aspects of both, targeting biological properties of cancer that are at once distinct from normal cells and yet common enough to make the drugs widely applicable across a range of cancer subtypes. The intrinsic mitochondrial pathway of apoptosis represents one such promising target for new therapies, and successfully targeting this pathway has the potential to alter the therapeutic landscape of therapy for a variety of cancers. Here, we discuss the biology of the intrinsic pathway of apoptosis, an assay known as BH3 profiling that can interrogate this pathway, early attempts to target BCL-2 clinically, and the recent promising results with the BCL-2 antagonist venetoclax (ABT-199) in clinical trials in hematologic malignancies. PMID:26567361

Molecular targets for flavivirus drug discovery

PubMed Central

Sampath, Aruna; Padmanabhan, R.

2009-01-01

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research. PMID:18796313

Discovery of non-peptidic small molecule inhibitors of cyclophilin D as neuroprotective agents in Aβ-induced mitochondrial dysfunction

NASA Astrophysics Data System (ADS)

Park, Insun; Londhe, Ashwini M.; Lim, Ji Woong; Park, Beoung-Geon; Jung, Seo Yun; Lee, Jae Yeol; Lim, Sang Min; No, Kyoung Tai; Lee, Jiyoun; Pae, Ae Nim

2017-10-01

Cyclophilin D (CypD) is a mitochondria-specific cyclophilin that is known to play a pivotal role in the formation of the mitochondrial permeability transition pore (mPTP).The formation and opening of the mPTP disrupt mitochondrial homeostasis, cause mitochondrial dysfunction and eventually lead to cell death. Several recent studies have found that CypD promotes the formation of the mPTP upon binding to β amyloid (Aβ) peptides inside brain mitochondria, suggesting that neuronal CypD has a potential to be a promising therapeutic target for Alzheimer's disease (AD). In this study, we generated an energy-based pharmacophore model by using the crystal structure of CypD—cyclosporine A (CsA) complex and performed virtual screening of ChemDiv database, which yielded forty-five potential hit compounds with novel scaffolds. We further tested those compounds using mitochondrial functional assays in neuronal cells and identified fifteen compounds with excellent protective effects against Aβ-induced mitochondrial dysfunction. To validate whether these effects derived from binding to CypD, we performed surface plasmon resonance (SPR)—based direct binding assays with selected compounds and discovered compound 29 was found to have the equilibrium dissociation constants (KD) value of 88.2 nM. This binding affinity value and biological activity correspond well with our predicted binding mode. We believe that this study offers new insights into the rational design of small molecule CypD inhibitors, and provides a promising lead for future therapeutic development.

The mitochondria targeted antioxidant MitoQ protects against fluoroquinolone-induced oxidative stress and mitochondrial membrane damage in human Achilles tendon cells.

PubMed

Lowes, Damon A; Wallace, Carol; Murphy, Michael P; Webster, Nigel R; Galley, Helen F

2009-04-01

Tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress. This study investigated whether ciprofloxacin and moxifloxacin cause oxidative stress and mitochondrial damage in cultured normal human Achilles' tendon cells and whether an antioxidant targeted to mitochondria (MitoQ) would protect against such damage better than a non-mitochondria targeted antioxidant. Human tendon cells from normal Achilles' tendons were exposed to 0-0.3 mM antibiotic for 24 h and 7 days in the presence of 1 microM MitoQ or an untargeted form, idebenone. Both moxifloxacin and ciprofloxacin resulted in up to a 3-fold increase in the rate of oxidation of dichlorodihydrofluorescein, a marker of general oxidative stress in tenocytes (p<0.0001) and loss of mitochondrial membrane permeability (p<0.001). In cells treated with MitoQ the oxidative stress was less and mitochondrial membrane potential was maintained. Mitochondrial damage to tenocytes during fluoroquinolone treatment may be involved in tendinitis and tendon rupture.

Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

PubMed

Odeh, Ahmad M; Craik, James D; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T

2014-01-01

Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.

Drug target ontology to classify and integrate drug discovery data.

PubMed

Lin, Yu; Mehta, Saurabh; Küçük-McGinty, Hande; Turner, John Paul; Vidovic, Dusica; Forlin, Michele; Koleti, Amar; Nguyen, Dac-Trung; Jensen, Lars Juhl; Guha, Rajarshi; Mathias, Stephen L; Ursu, Oleg; Stathias, Vasileios; Duan, Jianbin; Nabizadeh, Nooshin; Chung, Caty; Mader, Christopher; Visser, Ubbo; Yang, Jeremy J; Bologa, Cristian G; Oprea, Tudor I; Schürer, Stephan C

2017-11-09

One of the most successful approaches to develop new small molecule therapeutics has been to start from a validated druggable protein target. However, only a small subset of potentially druggable targets has attracted significant research and development resources. The Illuminating the Druggable Genome (IDG) project develops resources to catalyze the development of likely targetable, yet currently understudied prospective drug targets. A central component of the IDG program is a comprehensive knowledge resource of the druggable genome. As part of that effort, we have developed a framework to integrate, navigate, and analyze drug discovery data based on formalized and standardized classifications and annotations of druggable protein targets, the Drug Target Ontology (DTO). DTO was constructed by extensive curation and consolidation of various resources. DTO classifies the four major drug target protein families, GPCRs, kinases, ion channels and nuclear receptors, based on phylogenecity, function, target development level, disease association, tissue expression, chemical ligand and substrate characteristics, and target-family specific characteristics. The formal ontology was built using a new software tool to auto-generate most axioms from a database while supporting manual knowledge acquisition. A modular, hierarchical implementation facilitate ontology development and maintenance and makes use of various external ontologies, thus integrating the DTO into the ecosystem of biomedical ontologies. As a formal OWL-DL ontology, DTO contains asserted and inferred axioms. Modeling data from the Library of Integrated Network-based Cellular Signatures (LINCS) program illustrates the potential of DTO for contextual data integration and nuanced definition of important drug target characteristics. DTO has been implemented in the IDG user interface Portal, Pharos and the TIN-X explorer of protein target disease relationships. DTO was built based on the need for a formal semantic

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal.

PubMed

Unchwaniwala, Nuruddin; Sherer, Nathan M; Loeb, Daniel D

2016-10-01

To understand subcellular sites of hepatitis B virus (HBV) replication, we visualized core (Cp), polymerase (Pol), and pregenomic RNA (pgRNA) in infected cells. Interestingly, we found that the majority of Pol localized to the mitochondria in cells undergoing viral replication. The mitochondrial localization of Pol was independent of both the cell type and other viral components, indicating that Pol contains an intrinsic mitochondrial targeting signal (MTS). Neither Cp nor pgRNA localized to the mitochondria during active replication, suggesting a role other than DNA synthesis for Pol at the mitochondria. The Pol of duck hepatitis B virus (DHBV) also localized to the mitochondria. This result indicates that localization of Pol to mitochondria is likely a feature of all hepadnaviruses. To map the MTS within HBV Pol, we generated a series of Pol-green fluorescent protein (Pol-GFP) fusions and found that a stretch spanning amino acids (aa) 141 to 160 of Pol was sufficient to target GFP to the mitochondria. Surprisingly, deleting aa 141 to 160 in full-length Pol did not fully ablate Pol's mitochondrial localization, suggesting that additional sequences are involved in mitochondrial targeting. Only by deleting the N-terminal 160 amino acids in full-length Pol was mitochondrial localization ablated. Crucial residues for pgRNA packaging are contained within aa 141 to 160, indicating a multifunctional role of this region of Pol in the viral life cycle. Our studies show an unexpected Pol trafficking behavior that is uncoupled from its role in viral DNA synthesis. Chronic infection by HBV is a serious health concern. Existing therapies for chronically infected individuals are not curative, underscoring the need for a better understanding of the viral life cycle to develop better antiviral therapies. To date, the most thoroughly studied function of Pol is to package the pgRNA and reverse transcribe it to double-stranded DNA within capsids. This study provides evidence for

Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites

PubMed Central

Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

2015-01-01

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. DOI: http://dx.doi.org/10.7554/eLife.11553.001 PMID:26609810

Mitochondrial targeting sequence variants of the CHCHD2 gene are a risk for Lewy body disorders

PubMed Central

Ogaki, Kotaro; Koga, Shunsuke; Heckman, Michael G.; Fiesel, Fabienne C.; Ando, Maya; Labbé, Catherine; Lorenzo-Betancor, Oswaldo; Moussaud-Lamodière, Elisabeth L.; Soto-Ortolaza, Alexandra I.; Walton, Ronald L.; Strongosky, Audrey J.; Uitti, Ryan J.; McCarthy, Allan; Lynch, Timothy; Siuda, Joanna; Opala, Grzegorz; Rudzinska, Monika; Krygowska-Wajs, Anna; Barcikowska, Maria; Czyzewski, Krzysztof; Puschmann, Andreas; Nishioka, Kenya; Funayama, Manabu; Hattori, Nobutaka; Parisi, Joseph E.; Petersen, Ronald C.; Graff-Radford, Neill R.; Boeve, Bradley F.; Springer, Wolfdieter; Wszolek, Zbigniew K.; Dickson, Dennis W.

2015-01-01

Objective: To assess the role of CHCHD2 variants in patients with Parkinson disease (PD) and Lewy body disease (LBD) in Caucasian populations. Methods: All exons of the CHCHD2 gene were sequenced in a US Caucasian patient-control series (878 PD, 610 LBD, and 717 controls). Subsequently, exons 1 and 2 were sequenced in an Irish series (355 PD and 365 controls) and a Polish series (394 PD and 350 controls). Immunohistochemistry and immunofluorescence studies were performed on pathologic LBD cases with rare CHCHD2 variants. Results: We identified 9 rare exonic variants of unknown significance. These variants were more frequent in the combined group of PD and LBD patients compared to controls (0.6% vs 0.1%, p = 0.013). In addition, the presence of any rare variant was more common in patients with LBD (2.5% vs 1.0%, p = 0.050) compared to controls. Eight of these 9 variants were located within the gene's mitochondrial targeting sequence. Conclusions: Although the role of variants of the CHCHD2 gene in PD and LBD remains to be further elucidated, the rare variants in the mitochondrial targeting sequence may be a risk factor for Lewy body disorders, which may link CHCHD2 to other genetic forms of parkinsonism with mitochondrial dysfunction. PMID:26561290

2013 Philip S. Portoghese Medicinal Chemistry Lectureship: Drug Discovery Targeting Allosteric Sites†

PubMed Central

2015-01-01

The identification of sites on receptors topographically distinct from the orthosteric sites, so-called allosteric sites, has heralded novel approaches and modes of pharmacology for target modulation. Over the past 20 years, our understanding of allosteric modulation has grown significantly, and numerous advantages, as well as caveats (e.g., flat structure–activity relationships, species differences, “molecular switches”), have been identified. For multiple receptors and proteins, numerous examples have been described where unprecedented levels of selectivity are achieved along with improved physiochemical properties. While not a panacea, these novel approaches represent exciting opportunities for tool compound development to probe the pharmacology and therapeutic potential of discrete molecular targets, as well as new medicines. In this Perspective, in commemoration of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship (LindsleyC. W.Adventures in allosteric drug discovery. Presented at the 246th National Meeting of the American Chemical Society, Indianapolis, IN, September 10, 2013; The 2013 Portoghese Lectureship), several vignettes of drug discovery campaigns targeting novel allosteric mechanisms will be recounted, along with lessons learned and guidelines that have emerged for successful lead optimization. PMID:25180768

Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity.

PubMed

Schirris, Tom J J; Ritschel, Tina; Herma Renkema, G; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

2015-09-29

Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

Mitochondrial Function in Allergic Disease.

PubMed

Iyer, Divyaanka; Mishra, Navya; Agrawal, Anurag

2017-05-01

The connections between allergy, asthma and metabolic syndrome are becoming increasingly clear. Recent research suggests a unifying mitochondrial link between the diverse phenotypes of these interlinked morbidities. The scope of this review is to highlight cellular mechanisms, epidemiology and environmental allergens influencing mitochondrial function and its importance in allergy and asthma. We briefly also consider the potential of mitochondria-targeted therapies in prevention and cure. Recent research has shown allergy, asthma and metabolic syndrome to be linked to mitochondrial dysfunction. Environmental pollutants and allergens are observed to cause mitochondrial dysfunction, primarily by inducing oxidative stress and ROS production. Malfunctioning mitochondria change the bioenergetics of the cell and its metabolic profile to favour systemic inflammation, which drives all three types of morbidities. Given the existing experimental evidence, approaches targeting mitochondria (e.g. antioxidant therapy and mitochondrial replacement) are being conducted in relevant disease models-with some progressing towards clinical trials, making mitochondrial function the focus of translational therapy research in asthma, allergy and linked metabolic syndrome.

Mitochondrial targeting of HIF-1α inhibits hypoxia-induced apoptosis independently of its transcriptional activity.

PubMed

Li, Hong-Sheng; Zhou, Yan-Ni; Li, Lu; Li, Sheng-Fu; Long, Dan; Chen, Xue-Lu; Zhang, Jia-Bi; Li, You-Ping; Feng, Li

2018-04-25

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates adaptive responses to hypoxia by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to hypoxia. However, the transcriptional and non-transcriptional mechanisms by which HIF-1α regulates mitochondria under hypoxia are poorly understood. Here, we examined the subcellular localization of HIF-1α in human cells and identified a small fraction of HIF-1α that translocated to the mitochondria after exposure to hypoxia or hypoxia-mimicking pharmacological agents. To probe the function of this HIF-1α population, we ectopically expressed a mitochondrial-targeted form of HIF-1α (mito-HIF-1α). Expression of mito-HIF-1α was sufficient to attenuate apoptosis induced by exposure to hypoxia or H 2 O 2 -induced oxidative stress. Moreover, mito-HIF-1α expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia. However, these functions of mito-HIF-1α were independent of its conventional transcriptional activity. Finally, the livers of mice with CCl 4 -induced fibrosis showed a progressive increase in HIF-1α association with the mitochondria, indicating the clinical relevance of this finding. These data suggested that mitochondrial HIF-1α protects against apoptosis independently of its well-known role as a transcription factor. Copyright © 2018. Published by Elsevier Inc.

Mitigating peroxynitrite mediated mitochondrial dysfunction in aged rat brain by mitochondria-targeted antioxidant MitoQ.

PubMed

Maiti, Arpan Kumar; Spoorthi, B C; Saha, Nimai Chandra; Panigrahi, Ashis Kumar

2018-05-17

Although reactive oxygen species mediated oxidative stress is a well-documented mechanism of aging, recent evidences indicate involvement of nitrosative stress in the same. As mitochondrial dysfunction is considered as one of the primary features of aging, the present study was designed to understand the involvement of nitrosative stress by studying the impact of a mitochondria-targeted antioxidant MitoQ, a peroxynitrite (ONOO - ) scavenger, on mitochondrial functions. Four groups of rats were included in this study: Group I: Young-6 months (-MitoQ), Group II: Aged-22 months (- MitoQ), Group III: Young-6 months (+ MitoQ), Group IV: Aged-22 months (+ MitoQ). The rats belonging to group III and IV were treated with oral administration of MitoQ (500 μM) daily through drinking water for 5 weeks. MitoQ efficiently suppressed synaptosomal lipid peroxidation and protein oxidation accompanied by diminution of nitrite production and protein bound 3-nitrotyrosine. MitoQ normalized enhanced caspase 3 and 9 activities in aged rat brains and efficiently reversed ONOO - mediated mitochondrial complex I and IV inhibition, restored mitochondrial ATP production and lowered mitochondrial membrane potential loss. To ascertain these findings, a mitochondrial in vitro model (iron/ascorbate) was used involving different free radical scavengers and anti-oxidants. MitoQ provided better protection compared to mercaptoethylguanidine, N-nitro-L-arginine-methyl ester and superoxide dismutase establishing the predominancy of ONOO - in the process compared to • NO and O 2 •- . These results clearly highlight the involvement of nitrosative stress in aging process with MitoQ having therapeutic potential to fight against ONOO - mediated aging deficits.

Protective effect of mitochondrial-targeted antioxidant MitoQ against iron ion 56Fe radiation induced brain injury in mice.

PubMed

Gan, Lu; Wang, Zhenhua; Si, Jing; Zhou, Rong; Sun, Chao; Liu, Yang; Ye, Yancheng; Zhang, Yanshan; Liu, Zhiyuan; Zhang, Hong

2018-02-15

Exposure to iron ion 56 Fe radiation (IR) during space missions poses a significant risk to the central nervous system and radiation exposure is intimately linked to the production of reactive oxygen species (ROS). MitoQ is a mitochondria-targeted antioxidant that has been shown to decrease oxidative damage and lower mitochondrial ROS in a number of animal models. Therefore, the present study aimed to investigate role of the mitochondrial targeted antioxidant MitoQ against 56 Fe particle irradiation-induced oxidative damage and mitochondria dysfunction in the mouse brains. Increased ROS levels were observed in mouse brains after IR compared with the control group. Enhanced ROS production leads to disruption of cellular antioxidant defense systems, mitochondrial respiration dysfunction, altered mitochondria dynamics and increased release of cytochrome c (cyto c) from mitochondria into cytosol resulting in apoptotic cell death. MitoQ reduced IR-induced oxidative stress (decreased ROS production and increased SOD, CAT activities) with decreased lipid peroxidation as well as reduced protein and DNA oxidation. MitoQ also protected mitochondrial respiration after IR. In addition, MitoQ increased the expression of mitofusin2 (Mfn2) and optic atrophy gene1 (OPA1), and decreased the expression of dynamic-like protein (Drp1). MitoQ also suppressed mitochondrial DNA damage, cyto c release, and caspase-3 activity in IR-treated mice compared to the control group. These results demonstrate that MitoQ may protect against IR-induced brain injury. Copyright © 2018 Elsevier Inc. All rights reserved.

Rheb and mammalian target of rapamycin in mitochondrial homoeostasis

PubMed Central

Groenewoud, Marlous J.; Zwartkruis, Fried J. T.

2013-01-01

Mitochondrial dysfunction has been associated with various diseases, such as cancer, myopathies, neurodegeneration and obesity. Mitochondrial homoeostasis is achieved by mechanisms that adapt the number of mitochondria to that required for energy production and for the supply of metabolic intermediates necessary to sustain cell growth. Simultaneously, mitochondrial quality control mechanisms are in place to remove malfunctioning mitochondria. In the cytoplasm, the protein complex mTORC1 couples growth-promoting signals with anabolic processes, in which mitochondria play an essential role. Here, we review the involvement of mTORC1 and Rheb in mitochondrial homoeostasis. The regulatory processes downstream of mTORC1 affect the glycolytic flux and the rate of mitophagy, and include regulation of the transcription factors HIF1α and YY1/PGC-1α. We also discuss how mitochondrial function feeds back on mTORC1 via reactive oxygen species signalling to adapt metabolic processes, and highlight how mTORC1 signalling is integrated with the unfolded protein response in mitochondria, which in Caenorhabditis elegans is mediated via transcription factors such as DVE-1/UBL-5 and ATFS-1. PMID:24352740

NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana.

PubMed

Ye, Weihua; Spånning, Erika; Unnerståle, Sofia; Gotthold, David; Glaser, Elzbieta; Mäler, Lena

2012-10-01

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. © 2012 The Authors Journal compilation © 2012 FEBS.

Efficient Mitochondrial Glutamine Targeting Prevails Over Glioblastoma Metabolic Plasticity.

PubMed

Oizel, Kristell; Chauvin, Cynthia; Oliver, Lisa; Gratas, Catherine; Geraldo, Fanny; Jarry, Ulrich; Scotet, Emmanuel; Rabe, Marion; Alves-Guerra, Marie-Clotilde; Teusan, Raluca; Gautier, Fabien; Loussouarn, Delphine; Compan, Vincent; Martinou, Jean-Claude; Vallette, François M; Pecqueur, Claire

2017-10-15

Purpose: Glioblastoma (GBM) is the most common and malignant form of primary human brain tumor in adults, with an average survival at diagnosis of 18 months. Metabolism is a new attractive therapeutic target in cancer; however, little is known about metabolic heterogeneity and plasticity within GBM tumors. We therefore aimed to investigate metabolic phenotyping of primary cultures in the context of molecular tumor heterogeneity to provide a proof of concept for personalized metabolic targeting of GBM. Experimental Design: We have analyzed extensively several primary GBM cultures using transcriptomics, metabolic phenotyping assays, and mitochondrial respirometry. Results: We found that metabolic phenotyping clearly identifies 2 clusters, GLN High and GLN Low , mainly based on metabolic plasticity and glutamine (GLN) utilization. Inhibition of glutamine metabolism slows the in vitro and in vivo growth of GLN High GBM cultures despite metabolic adaptation to nutrient availability, in particular by increasing pyruvate shuttling into mitochondria. Furthermore, phenotypic and molecular analyses show that highly proliferative GLN High cultures are CD133 neg and display a mesenchymal signature in contrast to CD133 pos GLN Low GBM cells. Conclusions: Our results show that metabolic phenotyping identified an essential metabolic pathway in a GBM cell subtype, and provide a proof of concept for theranostic metabolic targeting. Clin Cancer Res; 23(20); 6292-304. ©2017 AACR . ©2017 American Association for Cancer Research.

The mitochondrial permeability transition pore: Molecular nature and role as a target in cardioprotection

PubMed Central

Bernardi, Paolo; Di Lisa, Fabio

2015-01-01

The mitochondrial permeability transition (PT) – an abrupt increase permeability of the inner membrane to solutes – is a causative event in ischemia–reperfusion injury of the heart, and the focus of intense research in cardioprotection. The PT is due to opening of the PT pore (PTP), a high conductance channel that is critically regulated by a variety of pathophysiological effectors. Very recent work indicates that the PTP forms from the F-ATP synthase, which would switch from an energy-conserving to an energy-dissipating device. This review provides an update on the current debate on how this transition is achieved, and on the PTP as a target for therapeutic intervention. This article is part of a Special Issue entitled "Mitochondria: from basic mitochondrial biology to cardiovascular disease". PMID:25268651

Mitochondrial targeted catalase suppresses invasive breast cancer in mice.

PubMed

Goh, Jorming; Enns, Linda; Fatemie, Soroosh; Hopkins, Heather; Morton, John; Pettan-Brewer, Christina; Ladiges, Warren

2011-05-23

Treatment of invasive breast cancer has an alarmingly high rate of failure because effective targets have not been identified. One potential target is mitochondrial generated reactive oxygen species (ROS) because ROS production has been associated with changes in substrate metabolism and lower concentration of anti-oxidant enzymes in tumor and stromal cells and increased metastatic potential. Transgenic mice expressing a human catalase gene (mCAT) were crossed with MMTV-PyMT transgenic mice that develop metastatic breast cancer. All mice (33 mCAT positive and 23 mCAT negative) were terminated at 110 days of age, when tumors were well advanced. Tumors were histologically assessed for invasiveness, proliferation and metastatic foci in the lungs. ROS levels and activation status of p38 MAPK were determined. PyMT mice expressing mCAT had a 12.5 per cent incidence of high histological grade primary tumor invasiveness compared to a 62.5 per cent incidence in PyMT mice without mCAT. The histological grade correlated with incidence of metastasis with 56 per cent of PyMT mice positive for mCAT showing evidence of pulmonary metastasis compared to 85.4 per cent of PyMT mice negative for mCAT with pulmonary metastasis (p ≤ 0.05). PyMT tumor cells expressing mCAT had lower ROS levels and were more resistant to hydrogen peroxide-induced oxidative stress than wild type tumor cells, suggesting that mCAT has the potential of quenching intracellular ROS and subsequent invasive behavior. The metastatic tumor burden in PyMT mice expressing mCAT was 0.1 mm2/cm2 of lung tissue compared with 1.3 mm2/cm2 of lung tissue in PyMT mice expressing the wild type allele (p ≤ 0.01), indicating that mCAT could play a role in mitigating metastatic tumor progression at a distant organ site. Expression of mCAT in the lungs increased resistance to hydrogen peroxide-induced oxidative stress that was associated with decreased activation of p38MAPK suggesting ROS signaling is dependent on p38MAPK for

Calcium Homeostatasis and Mitochondrial Dysfunction in Dopaminergic Neurons of the Substantia Nigra

DTIC Science & Technology

2010-03-01

discovery that calcium entry through L-type channels during normal pacemaking elevates the sensitivity of SNc dopaminergic neurons to toxins; • the...discovery that L-type calcium channels participate in but are not necessary for pacemaking; • the discovery that serum concentration of the...FDA approved doses; • the discovery that calcium entry through L-type channels during pacemaking elevates mitochondrial oxidant stress and leads

Designing multi-targeted agents: An emerging anticancer drug discovery paradigm.

PubMed

Fu, Rong-Geng; Sun, Yuan; Sheng, Wen-Bing; Liao, Duan-Fang

2017-08-18

The dominant paradigm in drug discovery is to design ligands with maximum selectivity to act on individual drug targets. With the target-based approach, many new chemical entities have been discovered, developed, and further approved as drugs. However, there are a large number of complex diseases such as cancer that cannot be effectively treated or cured only with one medicine to modulate the biological function of a single target. As simultaneous intervention of two (or multiple) cancer progression relevant targets has shown improved therapeutic efficacy, the innovation of multi-targeted drugs has become a promising and prevailing research topic and numerous multi-targeted anticancer agents are currently at various developmental stages. However, most multi-pharmacophore scaffolds are usually discovered by serendipity or screening, while rational design by combining existing pharmacophore scaffolds remains an enormous challenge. In this review, four types of multi-pharmacophore modes are discussed, and the examples from literature will be used to introduce attractive lead compounds with the capability of simultaneously interfering with different enzyme or signaling pathway of cancer progression, which will reveal the trends and insights to help the design of the next generation multi-targeted anticancer agents. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Mitochondrial peptides modulate mitochondrial function during cellular senescence.

PubMed

Kim, Su-Jeong; Mehta, Hemal H; Wan, Junxiang; Kuehnemann, Chisaka; Chen, Jingcheng; Hu, Ji-Fan; Hoffman, Andrew R; Cohen, Pinchas

2018-06-10

Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

Improved health-span and lifespan in mtDNA mutator mice treated with the mitochondrially targeted antioxidant SkQ1

PubMed Central

Shabalina, Irina G.; Vyssokikh, Mikhail Yu.; Gibanova, Natalia; Csikasz, Robert I.; Edgar, Daniel; Hallden-Waldemarson, Anne; Rozhdestvenskaya, Zinaida; Bakeeva, Lora E.; Vays, Valeria B.; Pustovidko, Antonina V.; Skulachev, Maxim V.; Cannon, Barbara; Skulachev, Vladimir P.; Nedergaard, Jan

2017-01-01

MtDNA mutator mice exhibit marked features of premature aging. We find that these mice treated from age of ≈100 days with the mitochondria-targeted antioxidant SkQ1 showed a delayed appearance of traits of aging such as kyphosis, alopecia, lowering of body temperature, body weight loss, as well as ameliorated heart, kidney and liver pathologies. These effects of SkQ1 are suggested to be related to an alleviation of the effects of an enhanced reactive oxygen species (ROS) level in mtDNA mutator mice: the increased mitochondrial ROS released due to mitochondrial mutations probably interact with polyunsaturated fatty acids in cardiolipin, releasing malondialdehyde and 4-hydroxynonenal that form protein adducts and thus diminishes mitochondrial functions. SkQ1 counteracts this as it scavenges mitochondrial ROS. As the results, the normal mitochondrial ultrastructure is preserved in liver and heart; the phosphorylation capacity of skeletal muscle mitochondria as well as the thermogenic capacity of brown adipose tissue is also improved. The SkQ1-treated mice live significantly longer (335 versus 290 days). These data may be relevant in relation to treatment of mitochondrial diseases particularly and the process of aging in general. PMID:28209927

Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin.

PubMed

Chowdhury, Shreya Roy; Ray, Upasana; Chatterjee, Bishnu P; Roy, Sib S

2017-05-04

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for

Fragment-based drug discovery and its application to challenging drug targets.

PubMed

Price, Amanda J; Howard, Steven; Cons, Benjamin D

2017-11-08

Fragment-based drug discovery (FBDD) is a technique for identifying low molecular weight chemical starting points for drug discovery. Since its inception 20 years ago, FBDD has grown in popularity to the point where it is now an established technique in industry and academia. The approach involves the biophysical screening of proteins against collections of low molecular weight compounds (fragments). Although fragments bind to proteins with relatively low affinity, they form efficient, high quality binding interactions with the protein architecture as they have to overcome a significant entropy barrier to bind. Of the biophysical methods available for fragment screening, X-ray protein crystallography is one of the most sensitive and least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. Fragment-based screening using X-ray crystallography is therefore an efficient method for identifying binding hotspots on proteins, which can then be exploited by chemists and biologists for the discovery of new drugs. The use of FBDD is illustrated here with a recently published case study of a drug discovery programme targeting the challenging protein-protein interaction Kelch-like ECH-associated protein 1:nuclear factor erythroid 2-related factor 2. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Mitochondrial neuronal uncoupling proteins: a target for potential disease-modification in Parkinson's disease

PubMed Central

2012-01-01

This review gives a brief insight into the role of mitochondrial dysfunction and oxidative stress in the converging pathogenic processes involved in Parkinson's disease (PD). Mitochondria provide cellular energy in the form of ATP via oxidative phosphorylation, but as an integral part of this process, superoxides and other reactive oxygen species are also produced. Excessive free radical production contributes to oxidative stress. Cells have evolved to handle such stress via various endogenous anti-oxidant proteins. One such family of proteins is the mitochondrial uncoupling proteins (UCPs), which are anion carriers located in the mitochondrial inner membrane. There are five known homologues (UCP1 to 5), of which UCP4 and 5 are predominantly expressed in neural cells. In a series of previous publications, we have shown how these neuronal UCPs respond to 1-methyl-4-phenylpyridinium (MPP+; toxic metabolite of MPTP) and dopamine-induced toxicity to alleviate neuronal cell death by preserving ATP levels and mitochondrial membrane potential, and reducing oxidative stress. We also showed how their expression can be influenced by nuclear factor kappa-B (NF-κB) signaling pathway specifically in UCP4. Furthermore, we previously reported an interesting link between PD and metabolic processes through the protective effects of leptin (hormone produced by adipocytes) acting via UCP2 against MPP+-induced toxicity. There is increasing evidence that these endogenous neuronal UCPs can play a vital role to protect neurons against various pathogenic stresses including those associated with PD. Their expression, which can be induced, may well be a potential therapeutic target for various drugs to alleviate the harmful effects of pathogenic processes in PD and hence modify the progression of this disease. PMID:23210978

Trends in GPCR drug discovery: new agents, targets and indications.

PubMed

Hauser, Alexander S; Attwood, Misty M; Rask-Andersen, Mathias; Schiöth, Helgi B; Gloriam, David E

2017-12-01

G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly due to their substantial involvement in human pathophysiology and their pharmacological tractability. Here, we report an up-to-date analysis of all GPCR drugs and agents in clinical trials, which reveals current trends across molecule types, drug targets and therapeutic indications, including showing that 475 drugs (~34% of all drugs approved by the US Food and Drug Administration (FDA)) act at 108 unique GPCRs. Approximately 321 agents are currently in clinical trials, of which ~20% target 66 potentially novel GPCR targets without an approved drug, and the number of biological drugs, allosteric modulators and biased agonists has increased. The major disease indications for GPCR modulators show a shift towards diabetes, obesity and Alzheimer disease, although several central nervous system disorders are also highly represented. The 224 (56%) non-olfactory GPCRs that have not yet been explored in clinical trials have broad untapped therapeutic potential, particularly in genetic and immune system disorders. Finally, we provide an interactive online resource to analyse and infer trends in GPCR drug discovery.

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

PubMed

Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-03-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Mitochondrial medicine for neurodegenerative diseases.

PubMed

Du, Heng; Yan, Shirley ShiDu

2010-05-01

Mitochondrial dysfunction has been reported in a wide array of neurological disorders ranging from neuromuscular to neurodegenerative diseases. Recent studies on neurodegenerative diseases have revealed that mitochondrial pathology is generally found in inherited or sporadic neurodegenerative diseases and is believed to be involved in the pathophysiological process of these diseases. Commonly seen types of mitochondrial dysfunction in neurodegenerative diseases include excessive free radical generation, lowered ATP production, mitochondrial permeability transition, mitochondrial DNA lesions, perturbed mitochondrial dynamics and apoptosis. Mitochondrial medicine as an emerging therapeutic strategy targeted to mitochondrial dysfunction in neurodegenerative diseases has been proven to be of value, though this area of research is still at in its early stage. In this article, we report on recent progress in the development of several mitochondrial therapies including antioxidants, blockade of mitochondrial permeability transition, and mitochondrial gene therapy as evidence that mitochondrial medicine has promise in the treatment of neurodegenerative diseases. 2010 Elsevier Ltd. All rights reserved.

Cancer metabolism: strategic diversion from targeting cancer drivers to targeting cancer suppliers.

PubMed

Kim, Soo-Youl

2015-03-01

Drug development groups are close to discovering another pot of gold-a therapeutic target-similar to the success of imatinib (Gleevec) in the field of cancer biology. Modern molecular biology has improved cancer therapy through the identification of more pharmaceutically viable targets, and yet major problems and risks associated with late-phase cancer therapy remain. Presently, a growing number of reports have initiated a discussion about the benefits of metabolic regulation in cancers. The Warburg effect, a great discovery approximately 70 years ago, addresses the "universality" of cancer characteristics. For instance, most cancer cells prefer aerobic glycolysis instead of mitochondrial respiration. Recently, cancer metabolism has been explained not only by metabolites but also through modern molecular and chemical biological techniques. Scientists are seeking context-dependent universality among cancer types according to metabolic and enzymatic pathway signatures. This review presents current cancer metabolism studies and discusses future directions in cancer therapy targeting bio-energetics, bio-anabolism, and autophagy, emphasizing the important contribution of cancer metabolism in cancer therapy.

A Bioinorganic Approach to Fragment-Based Drug Discovery Targeting Metalloenzymes.

PubMed

Cohen, Seth M

2017-08-15

Metal-dependent enzymes (i.e., metalloenzymes) make up a large fraction of all enzymes and are critically important in a wide range of biological processes, including DNA modification, protein homeostasis, antibiotic resistance, and many others. Consequently, metalloenzymes represent a vast and largely untapped space for drug development. The discovery of effective therapeutics that target metalloenzymes lies squarely at the interface of bioinorganic and medicinal chemistry and requires expertise, methods, and strategies from both fields to mount an effective campaign. In this Account, our research program that brings together the principles and methods of bioinorganic and medicinal chemistry are described, in an effort to bridge the gap between these fields and address an important class of medicinal targets. Fragment-based drug discovery (FBDD) is an important drug discovery approach that is particularly well suited for metalloenzyme inhibitor development. FBDD uses relatively small but diverse chemical structures that allow for the assembly of privileged molecular collections that focus on a specific feature of the target enzyme. For metalloenzyme inhibition, the specific feature is rather obvious, namely, a metal-dependent active site. Surprisingly, prior to our work, the exploration of diverse molecular fragments for binding the metal active sites of metalloenzymes was largely unexplored. By assembling a modest library of metal-binding pharmacophores (MBPs), we have been able to find lead hits for many metalloenzymes and, from these hits, develop inhibitors that act via novel mechanisms of action. A specific case study on the use of this strategy to identify a first-in-class inhibitor of zinc-dependent Rpn11 (a component of the proteasome) is highlighted. The application of FBDD for the development of metalloenzyme inhibitors has raised several other compelling questions, such as how the metalloenzyme active site influences the coordination chemistry of bound

Using in Vitro Evolution and Whole Genome Analysis To Discover Next Generation Targets for Antimalarial Drug Discovery

PubMed Central

2018-01-01

Although many new anti-infectives have been discovered and developed solely using phenotypic cellular screening and assay optimization, most researchers recognize that structure-guided drug design is more practical and less costly. In addition, a greater chemical space can be interrogated with structure-guided drug design. The practicality of structure-guided drug design has launched a search for the targets of compounds discovered in phenotypic screens. One method that has been used extensively in malaria parasites for target discovery and chemical validation is in vitro evolution and whole genome analysis (IVIEWGA). Here, small molecules from phenotypic screens with demonstrated antiparasitic activity are used in genome-based target discovery methods. In this Review, we discuss the newest, most promising druggable targets discovered or further validated by evolution-based methods, as well as some exceptions. PMID:29451780

Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS

NASA Astrophysics Data System (ADS)

Garabedian, Alyssa; Benigni, Paolo; Ramirez, Cesar E.; Baker, Erin S.; Liu, Tao; Smith, Richard D.; Fernandez-Lima, Francisco

2018-05-01

In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150-250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMS-CID-TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10-200 nM range, while simultaneously achieving discovery measurements of not initially targeted peptides as markers from the same proteins and, eventually, other proteins. [Figure not available: see fulltext.

Discovery of peptide drug carrier candidates for targeted multi-drug delivery into prostate cancer cells.

PubMed

Bashari, O; Redko, B; Cohen, A; Luboshits, G; Gellerman, G; Firer, M A

2017-11-01

Metastatic castration-resistant prostate cancer (mCRPC) remains essentially incurable. Targeted Drug Delivery (TDD) systems may overcome the limitations of current mCRPC therapies. We describe the use of strict criteria to isolate novel prostate cancer cell targeting peptides that specifically deliver drugs into target cells. Phage from a libraries displaying 7mer peptides were exposed to PC-3 cells and only internalized phage were recovered. The ability of these phage to internalize into other prostate cancer cells (LNCaP, DU-145) was validated. The displayed peptides of selected phage clones were synthesized and their specificity for target cells was validated in vitro and in vivo. One peptide (P12) which specifically targeted PC-3 tumors in vivo was incorporated into mono-drug (Chlorambucil, Combretastatin or Camptothecin) and dual-drug (Chlorambucil/Combretastatin or Chlorambucil/Camptothecin) PDCs and the cytotoxic efficacy of these conjugates for target cells was tested. Conjugation of P12 into dual-drug PDCs allowed discovery of new drug combinations with synergistic effects. The use of strict selection criteria can lead to discovery of novel peptides for use as drug carriers for TDD. PDCs represent an effective alternative to current modes of free drug chemotherapy for prostate cancer. Copyright © 2017. Published by Elsevier B.V.

Resveratrol induces mitochondrial biogenesis in endothelial cells.

PubMed

Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-07-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Resveratrol induces mitochondrial biogenesis in endothelial cells

PubMed Central

Csiszar, Anna; Labinskyy, Nazar; Pinto, John T.; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-01-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1α, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. PMID:19429820

CaMKII determines mitochondrial stress responses in heart

PubMed Central

Joiner, Mei-ling A.; Koval, Olha M.; Jingdong, Li; He, B. Julie; Allamargot, Chantal; Gao, Zhan; Luczak, Elizabeth D.; Hall, Duane D.; Fink, Brian D.; Chen, Biyi; Yang, Jinying; Moore, Steven A.; Scholz, Thomas D.; Strack, Stefan; Mohler, Peter J.; Sivitz, William I.; Song, Long-Sheng; Anderson, Mark E.

2012-01-01

Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry, causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm)1,2. However, the signaling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU)3–5 are not known. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is activated in ischemia reperfusion (I/R), myocardial infarction (MI) and neurohumoral injury, common causes of myocardial death and heart failure, suggesting CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A (CsA), an mPTP antagonist with clinical efficacy in I/R injury6, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to I/R injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition are resistant to I/R injury, MI and neurohumoral injury, suggesting pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry and suggest mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure dysfunction in response to common experimental forms of pathophysiological stress. PMID:23051746

Mitochondrial targeted catalase suppresses invasive breast cancer in mice

PubMed Central

2011-01-01

Background Treatment of invasive breast cancer has an alarmingly high rate of failure because effective targets have not been identified. One potential target is mitochondrial generated reactive oxygen species (ROS) because ROS production has been associated with changes in substrate metabolism and lower concentration of anti-oxidant enzymes in tumor and stromal cells and increased metastatic potential. Methods Transgenic mice expressing a human catalase gene (mCAT) were crossed with MMTV-PyMT transgenic mice that develop metastatic breast cancer. All mice (33 mCAT positive and 23 mCAT negative) were terminated at 110 days of age, when tumors were well advanced. Tumors were histologically assessed for invasiveness, proliferation and metastatic foci in the lungs. ROS levels and activation status of p38 MAPK were determined. Results PyMT mice expressing mCAT had a 12.5 per cent incidence of high histological grade primary tumor invasiveness compared to a 62.5 per cent incidence in PyMT mice without mCAT. The histological grade correlated with incidence of metastasis with 56 per cent of PyMT mice positive for mCAT showing evidence of pulmonary metastasis compared to 85.4 per cent of PyMT mice negative for mCAT with pulmonary metastasis (p ≤ 0.05). PyMT tumor cells expressing mCAT had lower ROS levels and were more resistant to hydrogen peroxide-induced oxidative stress than wild type tumor cells, suggesting that mCAT has the potential of quenching intracellular ROS and subsequent invasive behavior. The metastatic tumor burden in PyMT mice expressing mCAT was 0.1 mm2/cm2 of lung tissue compared with 1.3 mm2/cm2 of lung tissue in PyMT mice expressing the wild type allele (p ≤ 0.01), indicating that mCAT could play a role in mitigating metastatic tumor progression at a distant organ site. Expression of mCAT in the lungs increased resistance to hydrogen peroxide-induced oxidative stress that was associated with decreased activation of p38MAPK suggesting ROS signaling

Mitochondrial Agents for Bipolar Disorder.

PubMed

Pereira, Círia; Chavarria, Victor; Vian, João; Ashton, Melanie Maree; Berk, Michael; Marx, Wolfgang; Dean, Olivia May

2018-03-27

Bipolar disorder is a chronic and often debilitating illness. Current treatment options (both pharmaco- and psychotherapy) have shown efficacy, but for many leave a shortfall in recovery. Advances in the understanding of the pathophysiology of bipolar disorder suggest that interventions that target mitochondrial dysfunction may provide a therapeutic benefit. This review explores the current and growing theoretical rationale as well as existing preclinical and clinical data for those therapies aiming to target the mitochondrion in bipolar disorder. A Clinicaltrials.gov and ANZCTR search was conducted for complete and ongoing trials on mitochondrial agents used in psychiatric disorders. A PubMed search was also conducted for literature published between January 1981 and July 2017. Systematic reviews, randomized controlled trials, observational studies, case series, and animal studies with an emphasis on agents affecting mitochondrial function and its role in bipolar disorder were included. The search was augmented by manually searching the references of key papers and related literature. The results were presented as a narrative review. Mitochondrial agents offer new horizons in mood disorder treatment. While some negative effects have been reported, most compounds are overall well tolerated and have generally benign side-effect profiles. The study of neuroinflammation, neurodegeneration, and mitochondrial function has contributed the understanding of bipolar disorder's pathophysiology. Agents targeting these pathways could be a potential therapeutic strategy. Future directions include identification of novel candidate mitochondrial modulators as well as rigorous and well-powered clinical trials.

The p38α mitogen-activated protein kinase as a central nervous system drug discovery target

PubMed Central

Borders, Aaron S; de Almeida, Lucia; Van Eldik, Linda J; Watterson, D Martin

2008-01-01

Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38α mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38α MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38α MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38α MAPK in neurodegenerative disorders. PMID:19090985

The p38alpha mitogen-activated protein kinase as a central nervous system drug discovery target.

PubMed

Borders, Aaron S; de Almeida, Lucia; Van Eldik, Linda J; Watterson, D Martin

2008-12-03

Protein kinases are critical modulators of a variety of cellular signal transduction pathways, and abnormal phosphorylation events can be a cause or contributor to disease progression in a variety of disorders. This has led to the emergence of protein kinases as an important new class of drug targets for small molecule therapeutics. A serine/threonine protein kinase, p38alpha mitogen-activated protein kinase (MAPK), is an established therapeutic target for peripheral inflammatory disorders because of its critical role in regulation of proinflammatory cytokine production. There is increasing evidence that p38alpha MAPK is also an important regulator of proinflammatory cytokine levels in the central nervous system, raising the possibility that the kinase may be a drug discovery target for central nervous system disorders where cytokine overproduction contributes to disease progression. Development of bioavailable, central nervous system-penetrant p38alpha MAPK inhibitors provides the required foundation for drug discovery campaigns targeting p38alpha MAPK in neurodegenerative disorders.

Mitochondrial Ion Channels in Cancer Transformation

PubMed Central

Madamba, Stephen M.; Damri, Kevin N.; Dejean, Laurent M.; Peixoto, Pablo M.

2015-01-01

Cancer transformation involves reprograming of mitochondrial function to avert cell death mechanisms, monopolize energy metabolism, accelerate mitotic proliferation, and promote metastasis. Mitochondrial ion channels have emerged as promising therapeutic targets because of their connection to metabolic and apoptotic functions. This mini review discusses how mitochondrial channels may be associated with cancer transformation and expands on the possible involvement of mitochondrial protein import complexes in pathophysiological process. PMID:26090338

Nanotized PPARα Overexpression Targeted to Hypertrophied Myocardium Improves Cardiac Function by Attenuating the p53-GSK3β-Mediated Mitochondrial Death Pathway.

PubMed

Rana, Santanu; Datta, Ritwik; Chaudhuri, Ratul Datta; Chatterjee, Emeli; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2018-05-09

Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac

Multi-omics Reveal Specific Targets of the RNA-Binding Protein Puf3p and Its Orchestration of Mitochondrial Biogenesis.

PubMed

Lapointe, Christopher P; Stefely, Jonathan A; Jochem, Adam; Hutchins, Paul D; Wilson, Gary M; Kwiecien, Nicholas W; Coon, Joshua J; Wickens, Marvin; Pagliarini, David J

2018-01-24

Coenzyme Q (CoQ) is a redox-active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a multi-omic strategy to identify mRNAs that not only bind Puf3p but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Puf3p target: Puf3p regulates the abundance of Coq5p and prevents its detrimental hyperaccumulation, thereby enabling efficient CoQ production. More broadly, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly (pathways essential to prime mitochondrial biogenesis). Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis, and they demonstrate the power of multi-omics for defining genuine targets of RBPs. Copyright © 2017 Elsevier Inc. All rights reserved.

Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis.

PubMed

Hudson, Matthew B; Smuder, Ashley J; Nelson, W Bradley; Wiggs, Michael P; Shimkus, Kevin L; Fluckey, James D; Szeto, Hazel H; Powers, Scott K

2015-01-01

Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

PubMed Central

Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

1986-01-01

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

PubMed

Raza Shaikh, Saame; Brown, David A

2013-01-01

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

PubMed Central

Sacoman, Juliana L.; Dagda, Raul Y.; Burnham-Marusich, Amanda R.; Dagda, Ruben K.; Berninsone, Patricia M.

2017-01-01

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics. PMID:28100784

Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

PubMed

Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

2014-06-01

Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

PubMed

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarroya, Joan, E-mail: joanvillarroya@gmail.com; Institut de Recerca l'Hospital de la Santa Creu i Sant Pau, Barcelona; Lara, Mari-Carmen

Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed themore » first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory

Therapeutic target discovery using Boolean network attractors: improvements of kali

PubMed Central

Guziolowski, Carito

2018-01-01

In a previous article, an algorithm for identifying therapeutic targets in Boolean networks modelling pathological mechanisms was introduced. In the present article, the improvements made on this algorithm, named kali, are described. These improvements are (i) the possibility to work on asynchronous Boolean networks, (ii) a finer assessment of therapeutic targets and (iii) the possibility to use multivalued logic. kali assumes that the attractors of a dynamical system, such as a Boolean network, are associated with the phenotypes of the modelled biological system. Given a logic-based model of pathological mechanisms, kali searches for therapeutic targets able to reduce the reachability of the attractors associated with pathological phenotypes, thus reducing their likeliness. kali is illustrated on an example network and used on a biological case study. The case study is a published logic-based model of bladder tumorigenesis from which kali returns consistent results. However, like any computational tool, kali can predict but cannot replace human expertise: it is a supporting tool for coping with the complexity of biological systems in the field of drug discovery. PMID:29515890

Strategies for Discovery of Small Molecule Radiation Protectors and Radiation Mitigators

PubMed Central

Greenberger, Joel S.; Clump, David; Kagan, Valerian; Bayir, Hülya; Lazo, John S.; Wipf, Peter; Li, Song; Gao, Xiang; Epperly, Michael W.

2011-01-01

Mitochondrial targeted radiation damage protectors (delivered prior to irradiation) and mitigators (delivered after irradiation, but before the appearance of symptoms associated with radiation syndrome) have been a recent focus in drug discovery for (1) normal tissue radiation protection during fractionated radiotherapy, and (2) radiation terrorism counter measures. Several categories of such molecules have been discovered: nitroxide-linked hybrid molecules, including GS-nitroxide, GS-nitric oxide synthase inhibitors, p53/mdm2/mdm4 inhibitors, and pharmaceutical agents including inhibitors of the phosphoinositide-3-kinase pathway and the anti-seizure medicine, carbamazepine. Evaluation of potential new radiation dose modifying molecules to protect normal tissue includes: clonogenic radiation survival curves, assays for apoptosis and DNA repair, and irradiation-induced depletion of antioxidant stores. Studies of organ specific radioprotection and in total body irradiation-induced hematopoietic syndrome in the mouse model for protection/mitigation facilitate rational means by which to move candidate small molecule drugs along the drug discovery pipeline into clinical development. PMID:22655254

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery.

PubMed

Vanwetswinkel, Sophie; Heetebrij, Robert J; van Duynhoven, John; Hollander, Johan G; Filippov, Dmitri V; Hajduk, Philip J; Siegal, Gregg

2005-02-01

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.

Cancer: Mitochondrial Origins.

PubMed

Stefano, George B; Kream, Richard M

2015-12-01

The primacy of glucose derived from photosynthesis as an existential source of chemical energy across plant and animal phyla is universally accepted as a core principle in the biological sciences. In mammalian cells, initial processing of glucose to triose phosphate intermediates takes place within the cytosolic glycolytic pathway and terminates with temporal transport of reducing equivalents derived from pyruvate metabolism by membrane-associated respiratory complexes in the mitochondrial matrix. The intra-mitochondrial availability of molecular oxygen as the ultimate electron acceptor drives the evolutionary fashioned chemiosmotic production of ATP as a high-efficiency biological process. The mechanistic bases of carcinogenesis have demonstrated profound alteration of normative mitochondrial function, notably dysregulated respiratory processes. Accordingly, the classic Warburg effect functionally links aerobic glycolysis, aberrant production and release of lactate, and metabolic down-regulation of mitochondrial oxidative processes with the carcinogenetic phenotype. We surmise, however, that aerobic fermentation by cancer cells may also represent a developmental re-emergence of an evolutionarily conserved early phenotype, which was "sidelined" with the emergence of mitochondrial oxidative phosphorylation as a primary mechanism for ATP production in normal cells. Regardless of state-dependent physiological status in mixed populations of cancer cells, it has been established that mitochondria are functionally linked to the initiation of cancer and its progression. Biochemical, molecular, and physiological differences in cancer cell mitochondria, notably mtDNA heteroplasmy and allele-specific expression of selected nuclear genes, may represent major focal points for novel targeting and elimination of cancer cells in metastatic disease afflicting human populations. To date, and despite considerable research efforts, the practical realization of advanced mitochondrial

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.

PubMed

Gong, Guohua; Liu, Xiaoyun; Zhang, Huiliang; Sheu, Shey-Shing; Wang, Wang

2015-10-01

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions. Copyright © 2015 the American Physiological Society.

Application of Combination High-Throughput Phenotypic Screening and Target Identification Methods for the Discovery of Natural Product-Based Combination Drugs.

PubMed

Isgut, Monica; Rao, Mukkavilli; Yang, Chunhua; Subrahmanyam, Vangala; Rida, Padmashree C G; Aneja, Ritu

2018-03-01

Modern drug discovery efforts have had mediocre success rates with increasing developmental costs, and this has encouraged pharmaceutical scientists to seek innovative approaches. Recently with the rise of the fields of systems biology and metabolomics, network pharmacology (NP) has begun to emerge as a new paradigm in drug discovery, with a focus on multiple targets and drug combinations for treating disease. Studies on the benefits of drug combinations lay the groundwork for a renewed focus on natural products in drug discovery. Natural products consist of a multitude of constituents that can act on a variety of targets in the body to induce pharmacodynamic responses that may together culminate in an additive or synergistic therapeutic effect. Although natural products cannot be patented, they can be used as starting points in the discovery of potent combination therapeutics. The optimal mix of bioactive ingredients in natural products can be determined via phenotypic screening. The targets and molecular mechanisms of action of these active ingredients can then be determined using chemical proteomics, and by implementing a reverse pharmacokinetics approach. This review article provides evidence supporting the potential benefits of natural product-based combination drugs, and summarizes drug discovery methods that can be applied to this class of drugs. © 2017 Wiley Periodicals, Inc.

Mitochondrial electron transport chain identified as a novel molecular target of SPIO nanoparticles mediated cancer-specific cytotoxicity.

PubMed

He, Chengyong; Jiang, Shengwei; Jin, Haijing; Chen, Shuzhen; Lin, Gan; Yao, Huan; Wang, Xiaoyong; Mi, Peng; Ji, Zhiliang; Lin, Yuchun; Lin, Zhongning; Liu, Gang

2016-03-01

Superparamagnetic iron oxide nanoparticles (SPIONs) are highly cytotoxic and target cancer cells with high specificity; however, the mechanism by which SPIONs induce cancer cell-specific cytotoxicity remains unclear. Herein, the molecular mechanism of SPION-induced cancer cell-specific cytotoxicity to cancer cells is clarified through DNA microarray and bioinformatics analyses. SPIONs can interference with the mitochondrial electron transport chain (METC) in cancer cells, which further affects the production of ATP, mitochondrial membrane potential, and microdistribution of calcium, and induces cell apoptosis. Additionally, SPIONs induce the formation of reactive oxygen species in mitochondria; these reactive oxygen species trigger cancer-specific cytotoxicity due to the lower antioxidative capacity of cancer cells. Moreover, the DNA microarray and gene ontology analyses revealed that SPIONs elevate the expression of metallothioneins in both normal and cancer cells but decrease the expression of METC genes in cancer cells. Overall, these results suggest that SPIONs induce cancer cell death by targeting the METC, which is helpful for designing anti-cancer nanotheranostics and evaluating the safety of future nanomedicines. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.

PubMed

Lombardo, Tomás; Folgar, Martín Gil; Salaverry, Luciana; Rey-Roldán, Estela; Alvarez, Elida M; Carreras, María C; Kornblihtt, Laura; Blanco, Guillermo A

2018-05-01

Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 μM low-dose range (LDR) of CCCP. Within the 20-50 μM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage. © 2017

Dietary fat and hepatic lipogenesis: mitochondrial citrate carrier as a sensor of metabolic changes.

PubMed

Ferramosca, Alessandra; Zara, Vincenzo

2014-05-01

Citrate carrier (CIC) is an integral protein of the inner mitochondrial membrane that has a fundamental role in hepatic intermediary metabolism. Its primary function is to catalyze the transport of citrate from mitochondria, where this molecule is formed, to cytosol, where this molecule is used for fatty acid (FA) and cholesterol synthesis. Therefore, mitochondrial CIC acts upstream of cytosolic lipogenic reactions, and its regulation is particularly important in view of the modulation of hepatic lipogenesis. Although a great deal of data are currently available on the dietary modulation of cytosolic lipogenic enzymes, little is known about the nutritional regulation of CIC transport activity. In this review, we describe the differential effects of distinct FAs present in the diet on the activity of mitochondrial CIC. In particular, polyunsaturated FAs were powerful modulators of the activity of mitochondrial CIC by influencing its expression through transcriptional and posttranscriptional mechanisms. On the contrary, saturated and monounsaturated FAs did not influence mitochondrial CIC activity. Moreover, variations in CIC activity were connected to similar alterations in the metabolic pathways to which the transported citrate is channeled. Therefore, CIC may be considered as a sensor for changes occurring inside the hepatocyte and may represent an important target for the regulation of hepatic lipogenesis. The crucial role of this protein is reinforced by the recent discovery of its involvement in other cellular processes, such as glucose-stimulated insulin secretion, inflammation, tumorigenesis, genome stability, and sperm metabolism. © 2014 American Society for Nutrition.

Targeting Endoplasmic Reticulum and/or Mitochondrial Ca2+ Fluxes as Therapeutic Strategy for HCV Infection.

PubMed

Scrima, Rosella; Piccoli, Claudia; Moradpour, Darius; Capitanio, Nazzareno

2018-01-01

Chronic hepatitis C is characterized by metabolic disorders and by a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that can in the long term lead to liver cirrhosis and hepatocellular carcinoma. Several lines of evidence suggest that mitochondrial dysfunctions play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most in vitro studies performed so far have shown that several of the hepatitis C virus (HCV) proteins also localize to mitochondria, but the consequences of these interactions on mitochondrial functions remain contradictory and need to be confirmed in the context of productively replicating virus and physiologically relevant in vitro and in vivo model systems. In the past decade we have been proposing a temporal sequence of events in the HCV-infected cell whereby the primary alteration is localized at the mitochondria-associated ER membranes and causes release of Ca 2+ from the ER, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen and nitrogen species and a progressive metabolic adaptive response consisting in decreased oxidative phosphorylation and enhanced aerobic glycolysis and lipogenesis. Here we resume the major results provided by our group in the context of HCV-mediated alterations of the cellular inter-compartmental calcium flux homeostasis and present new evidence suggesting targeting of ER and/or mitochondrial calcium transporters as a novel therapeutic strategy.

Targeting endoplasmic reticulum and/or mitochondrial Ca2+ fluxes as therapeutic strategy for HCV infection

NASA Astrophysics Data System (ADS)

Scrima, Rosella; Piccoli, Claudia; Moradpour, Darius; Capitanio, Nazzareno

2018-03-01

Chronic hepatitis C is characterized by metabolic disorders and by a microenvironment in the liver dominated by oxidative stress, inflammation and regeneration processes that can in the long term lead to liver cirrhosis and hepatocellular carcinoma. Several lines of evidence suggest that mitochondrial dysfunctions play a central role in these processes. However, how these dysfunctions are induced by the virus and whether they play a role in disease progression and neoplastic transformation remains to be determined. Most in vitro studies performed so far have shown that several of the hepatitis C virus (HCV) proteins also localize to mitochondria, but the consequences of these interactions on mitochondrial functions remain contradictory and need to be confirmed in the context of productively replicating virus and physiologically relevant in vitro and in vivo model systems. In the past decade we have been proposing a temporal sequence of events in the HCV-infected cell whereby the primary alteration is localized at the mitochondria-associated ER membranes and causes release of Ca2+ from the ER, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen and nitrogen species and a progressive metabolic adaptive response consisting in decreased oxidative phosphorylation and enhanced aerobic glycolysis and lipogenesis. Here we resume the major results provided by our group in the context of HCV-mediated alterations of the cellular inter-compartmental calcium flux homeostasis and present new evidence suggesting targeting of ER and/or mitochondrial calcium transporters as a novel therapeutic strategy.

Mitochondrial DNA Targets Increase Sensitivity of Malaria Detection Using Loop-Mediated Isothermal Amplification ▿

PubMed Central

Polley, Spencer D.; Mori, Yasuyoshi; Watson, Julie; Perkins, Mark D.; González, Iveth J.; Notomi, Tsugunori; Chiodini, Peter L.; Sutherland, Colin J.

2010-01-01

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per μl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per μl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays. PMID:20554824

Functional Properties of the Mitochondrial Carrier System.

PubMed

Taylor, Eric B

2017-09-01

The mitochondrial carrier system (MCS) transports small molecules between mitochondria and the cytoplasm. It is integral to the core mitochondrial function to regulate cellular chemistry by metabolism. The mammalian MCS comprises the transporters of the 53-member canonical SLC25A family and a lesser number of identified noncanonical transporters. The recent discovery and investigations of the mitochondrial pyruvate carrier (MPC) illustrate the diverse effects a single mitochondrial carrier may exert on cellular function. However, the transport selectivities of many carriers remain unknown, and most have not been functionally investigated in mammalian cells. The mechanisms coordinating their function as a unified system remain undefined. Increased accessibility to molecular genetic and metabolomic technologies now greatly enables investigation of the MCS. Continued investigation of the MCS may reveal how mitochondria encode complex regulatory information within chemical thermodynamic gradients. This understanding may enable precision modulation of cellular chemistry to counteract the dysmetabolism inherent in disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis.

PubMed

Karnewar, Santosh; Vasamsetti, Sathish Babu; Gopoju, Raja; Kanugula, Anantha Koteswararao; Ganji, Sai Krishna; Prabhakar, Sripadi; Rangaraj, Nandini; Tupperwar, Nitin; Kumar, Jerald Mahesh; Kotamraju, Srigiridhar

2016-04-11

Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE(-/-) mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE(-/-) mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects.

NMR approaches in structure-based lead discovery: Recent developments and new frontiers for targeting multi-protein complexes

PubMed Central

Dias, David M.; Ciulli, Alessio

2014-01-01

Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets. PMID:25175337

NMR approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes.

PubMed

Dias, David M; Ciulli, Alessio

2014-01-01

Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example.

PubMed

Xie, Yiyue; Dahlin, Jayme L; Oakley, Aaron J; Casarotto, Marco G; Board, Philip G; Baell, Jonathan B

2018-05-10

Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance

PubMed Central

Fazakerley, Daniel J; Chaudhuri, Rima; Yang, Pengyi; Maghzal, Ghassan J; Thomas, Kristen C; Krycer, James R; Humphrey, Sean J; Parker, Benjamin L; Fisher-Wellman, Kelsey H; Meoli, Christopher C; Hoffman, Nolan J; Diskin, Ciana; Burchfield, James G; Cowley, Mark J; Kaplan, Warren; Modrusan, Zora; Kolumam, Ganesh; Yang, Jean YH; Chen, Daniel L; Samocha-Bonet, Dorit; Greenfield, Jerry R; Hoehn, Kyle L

2018-01-01

Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered mitochondrial oxidants and insulin resistance while CoQ supplementation in either insulin-resistant cell models or mice restored normal insulin sensitivity. Specifically, lowering of mitochondrial CoQ caused insulin resistance in adipocytes as a result of increased superoxide/hydrogen peroxide production via complex II. These data suggest that mitochondrial CoQ is a proximal driver of mitochondrial oxidants and insulin resistance, and that mechanisms that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance. PMID:29402381

Mitochondrial inheritance in budding yeasts: towards an integrated understanding.

PubMed

Solieri, Lisa

2010-11-01

Recent advances in yeast mitogenomics have significantly contributed to our understanding of the diversity of organization, structure and topology in the mitochondrial genome of budding yeasts. In parallel, new insights on mitochondrial DNA (mtDNA) inheritance in the model organism Saccharomyces cerevisiae highlighted an integrated scenario where recombination, replication and segregation of mtDNA are intricately linked to mitochondrial nucleoid (mt-nucleoid) structure and organelle sorting. In addition to this, recent discoveries of bifunctional roles of some mitochondrial proteins have interesting implications on mito-nuclear genome interactions and the relationship between mtDNA inheritance, yeast fitness and speciation. This review summarizes the current knowledge on yeast mitogenomics, mtDNA inheritance with regard to mt-nucleoid structure and organelle dynamics, and mito-nuclear genome interactions. Copyright © 2010 Elsevier Ltd. All rights reserved.

Adaptations Required for Mitochondrial Import following Mitochondrial to Nucleus Gene Transfer of Ribosomal Protein S101[w

PubMed Central

Murcha, Monika W.; Rudhe, Charlotta; Elhafez, Dina; Adams, Keith L.; Daley, Daniel O.; Whelan, James

2005-01-01

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphiphatic α-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal. PMID:16040655

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells.

PubMed

Figarola, James L; Singhal, Jyotsana; Tompkins, Joshua D; Rogers, George W; Warden, Charles; Horne, David; Riggs, Arthur D; Awasthi, Sanjay; Singhal, Sharad S

2015-12-18

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. © 2015 by The American

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells*

PubMed Central

Figarola, James L.; Singhal, Jyotsana; Tompkins, Joshua D.; Rogers, George W.; Warden, Charles; Horne, David; Riggs, Arthur D.; Awasthi, Sanjay; Singhal, Sharad S.

2015-01-01

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. PMID:26534958

Target-decoy Based False Discovery Rate Estimation for Large-scale Metabolite Identification.

PubMed

Wang, Xusheng; Jones, Drew R; Shaw, Timothy I; Cho, Ji-Hoon; Wang, Yuanyuan; Tan, Haiyan; Xie, Boer; Zhou, Suiping; Li, Yuxin; Peng, Junmin

2018-05-23

Metabolite identification is a crucial step in mass spectrometry (MS)-based metabolomics. However, it is still challenging to assess the confidence of assigned metabolites. In this study, we report a novel method for estimating false discovery rate (FDR) of metabolite assignment with a target-decoy strategy, in which the decoys are generated through violating the octet rule of chemistry by adding small odd numbers of hydrogen atoms. The target-decoy strategy was integrated into JUMPm, an automated metabolite identification pipeline for large-scale MS analysis, and was also evaluated with two other metabolomics tools, mzMatch and mzMine 2. The reliability of FDR calculation was examined by false datasets, which were simulated by altering MS1 or MS2 spectra. Finally, we used the JUMPm pipeline coupled with the target-decoy strategy to process unlabeled and stable-isotope labeled metabolomic datasets. The results demonstrate that the target-decoy strategy is a simple and effective method for evaluating the confidence of high-throughput metabolite identification.

The Emory Chemical Biology Discovery Center: leveraging academic innovation to advance novel targets through HTS and beyond.

PubMed

Johns, Margaret A; Meyerkord-Belton, Cheryl L; Du, Yuhong; Fu, Haian

2014-03-01

The Emory Chemical Biology Discovery Center (ECBDC) aims to accelerate high throughput biology and translation of biomedical research discoveries into therapeutic targets and future medicines by providing high throughput research platforms to scientific collaborators worldwide. ECBDC research is focused at the interface of chemistry and biology, seeking to fundamentally advance understanding of disease-related biology with its HTS/HCS platforms and chemical tools, ultimately supporting drug discovery. Established HTS/HCS capabilities, university setting, and expertise in diverse assay formats, including protein-protein interaction interrogation, have enabled the ECBDC to contribute to national chemical biology efforts, empower translational research, and serve as a training ground for young scientists. With these resources, the ECBDC is poised to leverage academic innovation to advance biology and therapeutic discovery.

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

PubMed Central

Kessler, Daniel; Papatheodorou, Panagiotis; Stratmann, Tina; Dian, Elke Andrea; Hartmann-Fatu, Cristina; Rassow, Joachim; Bayer, Peter; Mueller, Jonathan Wolf

2007-01-01

Background The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study. Results Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions. Conclusion Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae. PMID:17875217

Pro-oxidant mitochondrial matrix-targeted ubiquinone MitoQ10 acts as anti-oxidant at retarded electron transport or proton pumping within Complex I.

PubMed

Plecitá-Hlavatá, Lydie; Jezek, Jan; Jezek, Petr

2009-01-01

Oxidative stress of mitochondrial origin, i.e. elevated mitochondrial superoxide production, belongs to major factors determining aging and oxidative-stress-related diseases. Antioxidants, such as the mitochondria-targeted coenzyme Q, MitoQ(10), may prevent or cure these pathological conditions. To elucidate pro- and anti-oxidant action of MitoQ(10), we studied its effects on HepG2 cell respiration, mitochondrial network morphology, and rates of superoxide release (above that neutralized by superoxide dismutase) to the mitochondrial matrix (J(m)). MitoSOX Red fluorescence confocal microscopy monitoring of J(m) rates showed pro-oxidant effects of 3.5-fold increased J(m) with MitoQ(10). MitoQ(10) induced fission of the mitochondrial network which was recovered after 24h. In rotenone-inhibited HepG2 cells (i.e., already under oxidative stress) MitoQ(10) sharply decreased rotenone-induced J(m), but not together with the Complex II inhibitor thenoyltrifluoroacetone. Respiration of HepG2 cells and isolated rat liver mitochondria with MitoQ(10) increased independently of rotenone. The increase was prevented by thenoyltrifluoroacetone. These results suggest that MitoQ(10) accepts electrons prior to the rotenone-bound Q-site, and the Complex II reverse mode oxidizes MitoQ(10)H(2) to regenerate MitoQ(10). Consequently, MitoQ(10) has a pro-oxidant role in intact cells, whereas it serves as an antioxidant when Complex I-derived superoxide generation is already elevated due to electron flow retardation. Moreover, unlike mitochondrial uncoupling, MitoQ(10) exerted its antioxidant role when Complex I proton pumping was retarded by a hydrophobic amiloride, 5-(N-ethyl-N-isopropyl) amiloride. Consequently, MitoQ(10) may be useful in the treatment of diseases originating from impairment of respiratory chain Complex I due to oxidatively damaged mitochondrial DNA, when its targeted delivery to pathogenic tissues is ensured.

Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins.

PubMed

Martí, María C; Olmos, Enrique; Calvete, Juan J; Díaz, Isabel; Barranco-Medina, Sergio; Whelan, James; Lázaro, Juan J; Sevilla, Francisca; Jiménez, Ana

2009-06-01

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

Mitochondrial Permeability Transition in Pathogenesis of Hemorrhagic Injury: Targeted Therapy with Minocycline

DTIC Science & Technology

2012-03-01

minocy- cline treatment (Figures 1-4). Minocycline also improved mitochondrial function as assessed by intravital multiphoton imaging of the...will make direct measurements by intravital multiphoton microscopy to determine whether onset of the mitochondrial permeability transition and...oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization were assessed by intravital microscopy. After H/R with vehicle or

Dietary Fat and Hepatic Lipogenesis: Mitochondrial Citrate Carrier as a Sensor of Metabolic Changes1

PubMed Central

Ferramosca, Alessandra; Zara, Vincenzo

2014-01-01

Citrate carrier (CIC) is an integral protein of the inner mitochondrial membrane that has a fundamental role in hepatic intermediary metabolism. Its primary function is to catalyze the transport of citrate from mitochondria, where this molecule is formed, to cytosol, where this molecule is used for fatty acid (FA) and cholesterol synthesis. Therefore, mitochondrial CIC acts upstream of cytosolic lipogenic reactions, and its regulation is particularly important in view of the modulation of hepatic lipogenesis. Although a great deal of data are currently available on the dietary modulation of cytosolic lipogenic enzymes, little is known about the nutritional regulation of CIC transport activity. In this review, we describe the differential effects of distinct FAs present in the diet on the activity of mitochondrial CIC. In particular, polyunsaturated FAs were powerful modulators of the activity of mitochondrial CIC by influencing its expression through transcriptional and posttranscriptional mechanisms. On the contrary, saturated and monounsaturated FAs did not influence mitochondrial CIC activity. Moreover, variations in CIC activity were connected to similar alterations in the metabolic pathways to which the transported citrate is channeled. Therefore, CIC may be considered as a sensor for changes occurring inside the hepatocyte and may represent an important target for the regulation of hepatic lipogenesis. The crucial role of this protein is reinforced by the recent discovery of its involvement in other cellular processes, such as glucose-stimulated insulin secretion, inflammation, tumorigenesis, genome stability, and sperm metabolism. PMID:24829468

Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

2013-01-01

Background Drug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties. Methods An updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available. Results The updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated. Conclusion The updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics. PMID:23537208

New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

PubMed Central

Marty, Naomi J.; Teresinski, Howard J.; Hwang, Yeen Ting; Clendening, Eric A.; Gidda, Satinder K.; Sliwinska, Elwira; Zhang, Daiyuan; Miernyk, Ján A.; Brito, Glauber C.; Andrews, David W.; Dyer, John M.; Mullen, Robert T.

2014-01-01

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins. PMID:25237314

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

PubMed

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

From Pathways to Targets: Understanding the Mechanisms behind Polyglutamine Disease

PubMed Central

Weber, Jonasz Jeremiasz; Sowa, Anna Sergeevna

2014-01-01

The history of polyglutamine diseases dates back approximately 20 years to the discovery of a polyglutamine repeat in the androgen receptor of SBMA followed by the identification of similar expansion mutations in Huntington's disease, SCA1, DRPLA, and the other spinocerebellar ataxias. This common molecular feature of polyglutamine diseases suggests shared mechanisms in disease pathology and neurodegeneration of disease specific brain regions. In this review, we discuss the main pathogenic pathways including proteolytic processing, nuclear shuttling and aggregation, mitochondrial dysfunction, and clearance of misfolded polyglutamine proteins and point out possible targets for treatment. PMID:25309920

Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease.

PubMed

Boczonadi, Veronika; King, Martin S; Smith, Anthony C; Olahova, Monika; Bansagi, Boglarka; Roos, Andreas; Eyassu, Filmon; Borchers, Christoph; Ramesh, Venkateswaran; Lochmüller, Hanns; Polvikoski, Tuomo; Whittaker, Roger G; Pyle, Angela; Griffin, Helen; Taylor, Robert W; Chinnery, Patrick F; Robinson, Alan J; Kunji, Edmund R S; Horvath, Rita

2018-03-08

PurposeTo understand the role of the mitochondrial oxodicarboxylate carrier (SLC25A21) in the development of spinal muscular atrophy-like disease.MethodsWe identified a novel pathogenic variant in a patient by whole-exome sequencing. The pathogenicity of the mutation was studied by transport assays, computer modeling, followed by targeted metabolic testing and in vitro studies in human fibroblasts and neurons.ResultsThe patient carries a homozygous pathogenic variant c.695A>G; p.(Lys232Arg) in the SLC25A21 gene, encoding the mitochondrial oxodicarboxylate carrier, and developed spinal muscular atrophy and mitochondrial myopathy. Transport assays show that the mutation renders SLC25A21 dysfunctional and 2-oxoadipate cannot be imported into the mitochondrial matrix. Computer models of central metabolism predicted that impaired transport of oxodicarboxylate disrupts the pathways of lysine and tryptophan degradation, and causes accumulation of 2-oxoadipate, pipecolic acid, and quinolinic acid, which was confirmed in the patient's urine by targeted metabolomics. Exposure to 2-oxoadipate and quinolinic acid decreased the level of mitochondrial complexes in neuronal cells (SH-SY5Y) and induced apoptosis.ConclusionMitochondrial oxodicarboxylate carrier deficiency leads to mitochondrial dysfunction and the accumulation of oxoadipate and quinolinic acid, which in turn cause toxicity in spinal motor neurons leading to spinal muscular atrophy-like disease.GENETICS in MEDICINE advance online publication, 8 March 2018; doi:10.1038/gim.2017.251.

Mitochondrial targeted curcumin exhibits anticancer effects through disruption of mitochondrial redox and modulation of TrxR2 activity.

PubMed

Jayakumar, Sundarraj; Patwardhan, Raghavendra S; Pal, Debojyoti; Singh, Babita; Sharma, Deepak; Kutala, Vijay Kumar; Sandur, Santosh Kumar

2017-12-01

Mitocurcumin is a derivative of curcumin, which has been shown to selectively enter mitochondria. Here we describe the anti-tumor efficacy of mitocurcumin in lung cancer cells and its mechanism of action. Mitocurcumin, showed 25-50 fold higher efficacy in killing lung cancer cells as compared to curcumin as demonstrated by clonogenic assay, flow cytometry and high throughput screening assay. Treatment of lung cancer cells with mitocurcumin significantly decreased the frequency of cancer stem cells. Mitocurcumin increased the mitochondrial reactive oxygen species (ROS), decreased the mitochondrial glutathione levels and induced strand breaks in the mitochondrial DNA. As a result, we observed increased BAX to BCL-2 ratio, cytochrome C release into the cytosol, loss of mitochondrial membrane potential and increased caspase-3 activity suggesting that mitocurcumin activates the intrinsic apoptotic pathway. Docking studies using mitocurcumin revealed that it binds to the active site of the mitochondrial thioredoxin reductase (TrxR2) with high affinity. In corroboration with the above finding, mitocurcumin decreased TrxR activity in cell free as well as the cellular system. The anti-cancer activity of mitocurcumin measured in terms of apoptotic cell death and the decrease in cancer stem cell frequency was accentuated by TrxR2 overexpression. This was due to modulation of TrxR2 activity to NADPH oxidase like activity by mitocurcumin, resulting in higher ROS accumulation and cell death. Thus, our findings reveal mitocurcumin as a potent anticancer agent with better efficacy than curcumin. This study also demonstrates the role of TrxR2 and mitochondrial DNA damage in mitocurcumin mediated killing of cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Oxidative stress in duckweed (Lemna minor L.) induced by glyphosate: Is the mitochondrial electron transport chain a target of this herbicide?

PubMed

Gomes, Marcelo Pedrosa; Juneau, Philippe

2016-11-01

We investigated the physiological responses of Lemna minor plants exposed to glyphosate. The deleterious effects of this herbicide on photosynthesis, respiration, and pigment concentrations were related to glyphosate-induced oxidative stress through hydrogen peroxide (H 2 O 2 ) accumulation. By using photosynthetic and respiratory electron transport chain (ETC) inhibitors we located the primary site of reactive oxygen species (ROS) production in plants exposed to 500 mg glyphosate l -1 . Inhibition of mitochondrial ETC Complex I by rotenone reduced H 2 O 2 concentrations in glyphosate-treated plants. Complex III activity was very sensitive to glyphosate which appears to act much like antimycin A (an inhibitor of mitochondrial ETC Complex III) by shunting electrons from semiquinone to oxygen, with resulting ROS formation. Confocal evaluations for ROS localization showed that ROS are initially produced outside of the chloroplasts upon initial glyphosate exposure. Our results indicate that in addition to interfering with the shikimate pathway, glyphosate can induce oxidative stress in plants through H 2 O 2 formation by targeting the mitochondrial ETC, which would explain its observed effects on non-target organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ.

PubMed

Stucki, David M; Ruegsegger, Céline; Steiner, Silvio; Radecke, Julika; Murphy, Michael P; Zuber, Benoît; Saxena, Smita

2016-08-01

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1(154Q/2Q) mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1(154Q/2Q) mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as prevented oxidative stress-induced DNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

PubMed Central

Cline, Susan D.

2012-01-01

How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

Redox Regulation of Mitochondrial Function

PubMed Central

Handy, Diane E.

2012-01-01

Abstract Redox-dependent processes influence most cellular functions, such as differentiation, proliferation, and apoptosis. Mitochondria are at the center of these processes, as mitochondria both generate reactive oxygen species (ROS) that drive redox-sensitive events and respond to ROS-mediated changes in the cellular redox state. In this review, we examine the regulation of cellular ROS, their modes of production and removal, and the redox-sensitive targets that are modified by their flux. In particular, we focus on the actions of redox-sensitive targets that alter mitochondrial function and the role of these redox modifications on metabolism, mitochondrial biogenesis, receptor-mediated signaling, and apoptotic pathways. We also consider the role of mitochondria in modulating these pathways, and discuss how redox-dependent events may contribute to pathobiology by altering mitochondrial function. Antioxid. Redox Signal. 16, 1323–1367. PMID:22146081

Plant uncoupling mitochondrial proteins.

PubMed

Vercesi, Aníbal Eugênio; Borecký, Jiri; Maia, Ivan de Godoy; Arruda, Paulo; Cuccovia, Iolanda Midea; Chaimovich, Hernan

2006-01-01

Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide-sensitive free fatty acid-activated H(+) flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (Deltamu(H)+) and production of reactive oxygen species.

Estrogen receptor-β in mitochondria: implications for mitochondrial bioenergetics and tumorigenesis.

PubMed

Liao, Tien-Ling; Tzeng, Chii-Ruey; Yu, Chao-Lan; Wang, Yi-Pei; Kao, Shu-Huei

2015-09-01

Estrogen enhances mitochondrial function by enhancing mitochondrial biogenesis and sustaining mitochondrial energy-transducing capacity. Shifts in mitochondrial bioenergetic pathways from oxidative phosphorylation to glycolysis have been hypothesized to be involved in estrogen-induced tumorigenesis. Studies have shown that mitochondria are an important target of estrogen. Estrogen receptor-β (ERβ) has been shown to localize to mitochondria in a ligand-dependent or -independent manner and can affect mitochondrial bioenergetics and anti-apoptotic signaling. However, the functional role of mitochondrial ERβ in tumorigenesis remains unclear. Clinical studies of ERβ-related tumorigenesis have shown that ERβ stimulates mitochondrial metabolism to meet the high energy demands of processes such as cell proliferation, cell survival, and transformation. Thus, in elucidating the precise role of mitochondrial ERβ in cell transformation and tumorigenesis, it will be particularly valuable to explore new approaches for the development of medical treatments targeting mitochondrial ERβ-mediated mitochondrial function and preventing apoptosis. © 2015 New York Academy of Sciences.

Common effects of lithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrial damage.

PubMed

Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K

2009-07-01

Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

The effect of mitochondrial calcium uniporter on mitochondrial fission in hippocampus cells ischemia/reperfusion injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Lantao; Li, Shuhong; Wang, Shilei, E-mail: wshlei@aliyun.com

The mitochondrial calcium uniporter (MCU) transports free Ca{sup 2+} into the mitochondrial matrix, maintaining Ca{sup 2+} homeostasis, thus regulates the mitochondrial morphology. Previous studies have indicated that there was closely crosstalk between MCU and mitochondrial fission during the process of ischemia/reperfusion injury. This study constructed a hypoxia reoxygenation model using primary hippocampus neurons to mimic the cerebral ischemia/reperfusion injury and aims to explore the exactly effect of MCU on the mitochondrial fission during the process of ischemia/reperfusion injury and so as the mechanisms. Our results found that the inhibitor of the MCU, Ru360, decreased mitochondrial Ca{sup 2+} concentration, suppressed themore » expression of mitochondrial fission protein Drp1, MIEF1 and Fis1, and thus improved mitochondrial morphology significantly. Whereas spermine, the agonist of the MCU, had no significant impact compared to the I/R group. This study demonstrated that the MCU regulates the process of mitochondrial fission by controlling the Ca{sup 2+} transport, directly upregulating mitochondrial fission proteins Drp1, Fis1 and indirectly reversing the MIEF1-induced mitochondrial fusion. It also provides new targets for brain protection during ischemia/reperfusion injury. - Highlights: • We study MCU with primary neuron culture. • MCU induces mitochondrial fission. • MCU reverses MIEF1 effect.« less

Mitochondrial dysfunction and organophosphorus compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karami-Mohajeri, Somayyeh; Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Kerman University of Medical Sciences, Kerman; Abdollahi, Mohammad, E-mail: Mohammad.Abdollahi@UToronto.Ca

2013-07-01

Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen frommore » dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. - Highlights: • As a non-cholinergic mechanism of toxicity, mitochondria is a target for OPs. • OPs affect action of complexes I, II, III, IV and V in the mitochondria. • OPs reduce mitochondrial ATP. • OPs promote oxidative and genotoxic damage via release of cytochrome C from mitochondria. • OP-induced mitochondrial dysfunction can be restored by increasing the cytosolic ATP.« less

A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

PubMed Central

Moraes, Carlos T

2014-01-01

When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO). PMID:24623377

Target discovery focused approaches to overcome bottlenecks in the exploitation of antimycobacterial natural products.

PubMed

Baptista, Rafael; Bhowmick, Sumana; Nash, Robert J; Baillie, Les; Mur, Luis Aj

2018-04-01

Tuberculosis is a major global health hazard. The search for new antimycobacterials has focused on such as screening combinational chemistry libraries or designing chemicals to target predefined pockets of essential bacterial proteins. The relative ineffectiveness of these has led to a reappraisal of natural products for new antimycobacterial drug leads. However, progress has been limited, we suggest through a failure in many cases to define the drug target and optimize the hits using this information. We highlight methods of target discovery needed to develop a drug into a candidate for clinical trials. We incorporate these into suggested analysis pipelines which could inform the research strategies to accelerate the development of new drug leads from natural products.

Disrupting mitochondrial Ca2+ homeostasis causes tumor-selective TRAIL sensitization through mitochondrial network abnormalities.

PubMed

Ohshima, Yohei; Takata, Natsuhiko; Suzuki-Karasaki, Miki; Yoshida, Yukihiro; Tokuhashi, Yasuaki; Suzuki-Karasaki, Yoshihiro

2017-10-01

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent with high tumor-selective cytotoxicity. The congenital and acquired resistance of some cancer types including malignant melanoma and osteosarcoma impede the current TRAIL therapy of these cancers. Since fine tuning of the intracellular Ca2+ level is essential for cell function and survival, Ca2+ dynamics could be a promising target for cancer treatment. Recently, we demonstrated that mitochondrial Ca2+ removal increased TRAIL efficacy toward malignant melanoma and osteosarcoma cells. Here we report that mitochondrial Ca2+ overload leads to tumor-selective sensitization to TRAIL cytotoxicity. Treatment with the mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157 and oxidative phosphorylation inhibitor antimycin A and FCCP resulted in a rapid and persistent mitochondrial Ca2+ rise. These agents also increased TRAIL sensitivity in a tumor-selective manner with a switching from apoptosis to a nonapoptotic cell death. Moreover, we found that mitochondrial Ca2+ overload led to increased mitochondrial fragmentation, while mitochondrial Ca2+ removal resulted in mitochondrial hyperfusion. Regardless of their reciprocal actions on the mitochondrial dynamics, both interventions commonly exacerbated TRAIL-induced mitochondrial network abnormalities. These results expand our previous study and suggest that an appropriate level of mitochondrial Ca2+ is essential for maintaining the mitochondrial dynamics and the survival of these cells. Thus, disturbing mitochondrial Ca2+ homeostasis may serve as a promising approach to overcome the TRAIL resistance of these cancers with minimally compromising the tumor-selectivity.

Mitochondrial Control by DRP1 in Brain Tumor Initiating Cells

PubMed Central

Xie, Qi; Wu, Qiulian; Horbinski, Craig M.; Flavahan, William A.; Yang, Kailin; Zhou, Wenchao; Dombrowski, Stephen M.; Huang, Zhi; Fang, Xiaoguang; Shi, Yu; Ferguson, Ashley N.; Kashatus, David F.; Bao, Shideng; Rich, Jeremy N.

2015-01-01

Brain tumor initiating cells (BTICs) coopt the neuronal high affinity GLUT3 glucose transporter to withstand metabolic stress. Here, we investigated another mechanism critical to brain metabolism, mitochondrial morphology. BTICs displayed mitochondrial fragmentation relative to non-BTICs, suggesting that BTICs have increased mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), was activated in BTICs and inhibited in non-BTICs. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and AMPK targeting rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca2+–calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTICs, suggesting tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlates with poor prognosis in glioblastoma, suggesting mitochondrial dynamics may represent a therapeutic target for BTICs. PMID:25730670

Drug discovery in tuberculosis. New drug targets and antimycobacterial agents.

PubMed

Campaniço, André; Moreira, Rui; Lopes, Francisca

2018-04-25

Tuberculosis (TB) remains a major health problem worldwide. The infectious agent, Mycobacterium tuberculosis, has a unique ability to survive within the host, alternating between active and latent disease states, and escaping the immune system defences. The extended duration of anti-TB regimens and the increasing prevalence of multidrug- (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains have created an urgent need for new antibiotics active against drug-resistant organisms and that can shorten standard therapy. However, despite success in identifying active compounds through phenotypic screens, the conversion of hits into novel chemical series and ultimately into clinical candidates is hampered by the poor efficacy in eliminating M. tuberculosis within different host compartments, including macrophages, as well as a lack of knowledge about the specific target(s) inhibited and/or upregulated. The current status of anti-TB lead generation has much improved over the last decade, as exemplified by the recent approval of bedaquiline and delamanid to treat MDR-TB and XDR-TB. This review provides a critical analysis on the strategies used to progress hit compounds into viable lead candidates, and how emerging targets may play a role in TB drug discovery in the near future. Four new relevant targets are addressed: the enoyl-acyl carrier protein reductase, InhA; the transmembrane transport protein large, MmpL3; the decaprenylphospho-beta-d-ribofuranose 2-oxidase, DprE1; and the ubiquinol-cytochrome C reductase, QcrB. Validated hit compounds for each target are presented and explored, and the medicinal chemistry strategies to expand SAR around novel chemotypes analyzed. In addition, very recent emerging targets are also discussed. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

PubMed

de Moura, Michelle Barbi; Uppala, Radha; Zhang, Yuxun; Van Houten, Bennett; Goetzman, Eric S

2014-01-01

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

Emodin targets mitochondrial cyclophilin D to induce apoptosis in HepG2 cells.

PubMed

Zhang, Ling; He, Dian; Li, Kun; Liu, Hongli; Wang, Baitao; Zheng, Lifang; Li, Jiazhong

2017-06-01

Emodin has demonstrated potent anticancer activity in human hepatocarcinoma cells and animal models, however, the cellular targets of emodin have not been fully defined. Here we report that emodin induces the dysfunction of mitochondria and the apoptosis in HepG2 cells through an enrichment in mitochondria. Specifically, A mitochondrial matrix protein (cyclophilin D, CyPD) is involved in emodin-induced apoptosis, and the inhibitor of CyPD (cyclosporin A) could almost completely suppressing the apoptosis; Moreover, as the expression of CyPD could be effectively inhibited by antioxidant N-acetyl-l-cysteine and epidermal growth factor (the activator of ERK), reactive oxygen species and ERK might be involved in the relevant role of CyPD. A further molecule-docking discloses the existence of three hydrogen-bonds in CyPD-emodin complex. Thus, target localization and CyPD in mitochondria provides an insight into the action of emodin in the treatment of liver cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies.

PubMed

Moussaud, Simon; Malany, Siobhan; Mehta, Alka; Vasile, Stefan; Smith, Layton H; McLean, Pamela J

2015-05-01

Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garabedian, Alyssa; Benigni, Paolo; Ramirez, Cesar E.

Abstract. In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150–250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMSCID- TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptidemore » biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10–200 nM range, while simultaneously achieving discovery measurements« less

The enemy within: Targeting host-parasite interaction for antileishmanial drug discovery.

PubMed

Lamotte, Suzanne; Späth, Gerald F; Rachidi, Najma; Prina, Eric

2017-06-01

The state of antileishmanial chemotherapy is strongly compromised by the emergence of drug-resistant Leishmania. The evolution of drug-resistant phenotypes has been linked to the parasites' intrinsic genome instability, with frequent gene and chromosome amplifications causing fitness gains that are directly selected by environmental factors, including the presence of antileishmanial drugs. Thus, even though the unique eukaryotic biology of Leishmania and its dependence on parasite-specific virulence factors provide valid opportunities for chemotherapeutical intervention, all strategies that target the parasite in a direct fashion are likely prone to select for resistance. Here, we review the current state of antileishmanial chemotherapy and discuss the limitations of ongoing drug discovery efforts. We finally propose new strategies that target Leishmania viability indirectly via mechanisms of host-parasite interaction, including parasite-released ectokinases and host epigenetic regulation, which modulate host cell signaling and transcriptional regulation, respectively, to establish permissive conditions for intracellular Leishmania survival.

Natural Products and Their Mimics as Targets of Opportunity for Discovery

PubMed Central

2017-01-01

Diverse structural types of natural products and their mimics have served as targets of opportunity in our laboratory to inspire the discovery and development of new methods and strategies to assemble polyfunctional and polycyclic molecular architectures. Furthermore, our efforts toward identifying novel compounds having useful biological properties led to the creation of new targets, many of which posed synthetic challenges that required the invention of new methodology. In this Perspective, selected examples of how we have exploited a diverse range of natural products and their mimics to create, explore, and solve a variety of problems in chemistry and biology will be discussed. The journey was not without its twists and turns, but the unexpected often led to new revelations and insights. Indeed, in our recent excursion into applications of synthetic organic chemistry to neuroscience, avoiding the more-traveled paths was richly rewarding. PMID:28738152

SWATHtoMRM: Development of High-Coverage Targeted Metabolomics Method Using SWATH Technology for Biomarker Discovery.

PubMed

Zha, Haihong; Cai, Yuping; Yin, Yandong; Wang, Zhuozhong; Li, Kang; Zhu, Zheng-Jiang

2018-03-20

The complexity of metabolome presents a great analytical challenge for quantitative metabolite profiling, and restricts the application of metabolomics in biomarker discovery. Targeted metabolomics using multiple-reaction monitoring (MRM) technique has excellent capability for quantitative analysis, but suffers from the limited metabolite coverage. To address this challenge, we developed a new strategy, namely, SWATHtoMRM, which utilizes the broad coverage of SWATH-MS technology to develop high-coverage targeted metabolomics method. Specifically, SWATH-MS technique was first utilized to untargeted profile one pooled biological sample and to acquire the MS 2 spectra for all metabolites. Then, SWATHtoMRM was used to extract the large-scale MRM transitions for targeted analysis with coverage as high as 1000-2000 metabolites. Then, we demonstrated the advantages of SWATHtoMRM method in quantitative analysis such as coverage, reproducibility, sensitivity, and dynamic range. Finally, we applied our SWATHtoMRM approach to discover potential metabolite biomarkers for colorectal cancer (CRC) diagnosis. A high-coverage targeted metabolomics method with 1303 metabolites in one injection was developed to profile colorectal cancer tissues from CRC patients. A total of 20 potential metabolite biomarkers were discovered and validated for CRC diagnosis. In plasma samples from CRC patients, 17 out of 20 potential biomarkers were further validated to be associated with tumor resection, which may have a great potential in assessing the prognosis of CRC patients after tumor resection. Together, the SWATHtoMRM strategy provides a new way to develop high-coverage targeted metabolomics method, and facilitates the application of targeted metabolomics in disease biomarker discovery. The SWATHtoMRM program is freely available on the Internet ( http://www.zhulab.cn/software.php ).

Cardiac-Targeted Transgenic Mutant Mitochondrial Enzymes

PubMed Central

Kohler, James J.; Hosseini, Seyed H.; Green, Elgin; Hoying-Brandt, Amy; Cucoranu, Ioan; Haase, Chad P.; Russ, Rodney; Srivastava, Jaya; Ivey, Kristopher; Ludaway, Tomika; Kapoor, Victor; Abuin, Allison; Shapoval, Alexsey; Santoianni, Robert; Saada, Ann; Elpeleg, Orly; Lewis, William

2009-01-01

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (pol γ) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol γ and mtDNA replication. Cardiac “dominant negative” murine transgenes (TGs; Pol γ Y955G, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in “2 × 2” studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs (∼50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass (≈50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy. PMID:18446447

NMR-based platform for fragment-based lead discovery used in screening BRD4-targeted compounds

PubMed Central

Yu, Jun-lan; Chen, Tian-tian; Zhou, Chen; Lian, Fu-lin; Tang, Xu-long; Wen, Yi; Shen, Jing-kang; Xu, Ye-chun; Xiong, Bing; Zhang, Nai-xia

2016-01-01

Aim: Fragment-based lead discovery (FBLD) is a complementary approach in drug research and development. In this study, we established an NMR-based FBLD platform that was used to screen novel scaffolds targeting human bromodomain of BRD4, and investigated the binding interactions between hit compounds and the target protein. Methods: 1D NMR techniques were primarily used to generate the fragment library and to screen compounds. The inhibitory activity of hits on the first bromodomain of BRD4 [BRD4(I)] was examined using fluorescence anisotropy binding assay. 2D NMR and X-ray crystallography were applied to characterize the binding interactions between hit compounds and the target protein. Results: An NMR-based fragment library containing 539 compounds was established, which were clustered into 56 groups (8–10 compounds in each group). Eight hits with new scaffolds were found to inhibit BRD4(I). Four out of the 8 hits (compounds 1, 2, 8 and 9) had IC50 values of 100–260 μmol/L, demonstrating their potential for further BRD4-targeted hit-to-lead optimization. Analysis of the binding interactions revealed that compounds 1 and 2 shared a common quinazolin core structure and bound to BRD4(I) in a non-acetylated lysine mimetic mode. Conclusion: An NMR-based platform for FBLD was established and used in discovery of BRD4-targeted compounds. Four potential hit-to-lead optimization candidates have been found, two of them bound to BRD4(I) in a non-acetylated lysine mimetic mode, being selective BRD4(I) inhibitors. PMID:27238211

Quantitative proteomic analysis reveals novel mitochondrial targets of estrogen deficiency in the aged female rat heart.

PubMed

Lancaster, T S; Jefferson, S J; Hunter, J Craig; Lopez, Veronica; Van Eyk, J E; Lakatta, E G; Korzick, D H

2012-10-17

The incidence of myocardial infarction rises sharply at menopause, implicating a potential role for estrogen (E(2)) loss in age-related increases in ischemic injury. We aimed to identify quantitative changes to the cardiac mitochondrial proteome of aging females, based on the hypothesis that E(2) deficiency exacerbates age-dependent disruptions in mitochondrial proteins. Mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary-intact or ovariectomized (OVX) rats were labeled with 8plex isobaric tags for relative and absolute quantification (iTRAQ; n = 5-6/group). Groups studied were adult, adult OVX, aged, and aged OVX. In vivo coronary artery ligation and in vitro mitochondrial respiration studies were also performed in a subset of rats. We identified 965 proteins across groups and significant directional changes in 67 proteins of aged and/or aged OVX; 32 proteins were unique to aged OVX. Notably, only six proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C). Proteins affected by aging were primarily related to cellular metabolism, oxidative stress, and cell death. The largest change occurred in monoamine oxidase-A (MAO-A), a source of oxidative stress. While acute MAO-A inhibition induced mild uncoupling in aged mitochondria, reductions in infarct size were not observed. Age-dependent alterations in mitochondrial signaling indicate a highly selective myocardial response to E(2) deficiency. The combined proteomic and functional approaches described here offer possibility of new protein targets for experimentation and therapeutic intervention in the aged female population.

Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice.

PubMed

Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M; Gorodnya, Olena M; Ruchko, Mykhaylo V; Potter, Barry J; Wilson, Glenn L; Gillespie, Mark N; Parker, James C

2013-02-15

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

Synergistic Protective Effects of Mitochondrial Division Inhibitor 1 and Mitochondria-Targeted Small Peptide SS31 in Alzheimer's Disease.

PubMed

Reddy, P Hemachandra; Manczak, Maria; Yin, XiangLing; Reddy, Arubala P

2018-01-01

The purpose of our study was to determine the synergistic protective effects of mitochondria-targeted antioxidant SS31 and mitochondria division inhibitor 1 (Mdivi1) in Alzheimer's disease (AD). Using biochemical methods, we assessed mitochondrial function by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity, mitochondrial ATP, and GTPase Drp1 enzymatic activity in mutant AβPP cells. Using biochemical methods, we also measured cell survival and apoptotic cell death. Amyloid-β (Aβ) levels were measured using sandwich ELISA, and using real-time quantitative RT-PCR, we assessed mtDNA (mtDNA) copy number in relation to nuclear DNA (nDNA) in all groups of cells. We found significantly reduced levels of Aβ40 and Aβ42 in mutant AβPP cells treated with SS31, Mdivi1, and SS31+Mdivi1, and the reduction of Aβ42 levels were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. The levels of mtDNA copy number and cell survival were significantly increased in SS31, Mdivi1, and SS31+Mdivi1 treated mutant AβPP cells; however, the increased levels of mtDNA copy number and cell survival were much higher in SS31+Mdivi1 treated cells than individual treatments of SS31 and Mdivi1. Mitochondrial dysfunction is significantly reduced in SS31, Mdivi1, and SS31+Mdivi1 treated mutant AβPP cells; however, the reduction is much higher in cells treated with both SS31+Mdvi1. Similarly, GTPase Drp1 activity is reduced in all treatments, but reduced much higher in SS31+Mdivi1 treated cells. These observations strongly suggest that combined treatment of SS31+Mdivi1 is effective than individual treatments of SS31 and Mdivi1. Therefore, we propose that combined treatment of SS31+Mdivi1 is a better therapeutic strategy for AD. Ours is the first study to investigate combined treatment of mitochondria-targeted antioxidant SS31 and mitochondrial division inhibitor 1 in AD neurons.

Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs.

PubMed

Ortiz, Diana; Forquer, Isaac; Boitz, Jan; Soysa, Radika; Elya, Carolyn; Fulwiler, Audrey; Nilsen, Aaron; Polley, Tamsen; Riscoe, Michael K; Ullman, Buddy; Landfear, Scott M

2016-08-01

Endochin-like quinolones (ELQs) are potent and specific inhibitors of cytochrome bc1 from Plasmodium falciparum and Toxoplasma gondii and show promise for novel antiparasitic drug development. To determine whether the mitochondrial electron transport chain of Leishmania parasites could be targeted similarly for drug development, we investigated the activity of 134 structurally diverse ELQs. A cohort of ELQs was selectively toxic to amastigotes of Leishmania mexicana and L. donovani, with 50% inhibitory concentrations (IC50s) in the low micromolar range, but the structurally similar hydroxynaphthoquinone buparvaquone was by far the most potent inhibitor of electron transport, ATP production, and intracellular amastigote growth. Cytochrome bc1 is thus a promising target for novel antileishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for development of enhanced therapeutics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Folate-mediated mitochondrial targeting with doxorubicin-polyrotaxane nanoparticles overcomes multidrug resistance

PubMed Central

Yan, Fengjiao; Sun, Mingna; Du, Lingran; Peng, Wei; Li, Qiuli; Feng, Yinghong; Zhou, Yi

2015-01-01

Resistance to treatment with anticancer drugs is a significant obstacle and a fundamental cause of therapeutic failure in cancer therapy. Functional doxorubicin (DOX) nanoparticles for targeted delivery of the classical cytotoxic anticancer drug DOX to tumor cells, using folate-terminated polyrotaxanes along with dequalinium, have been developed and proven to overcome this resistance due to specific molecular features, including a size of approximately 101 nm, a zeta potential of 3.25 mV and drug-loading content of 18%. Compared with free DOX, DOX hydrochloride, DOX nanoparticles, and targeted DOX nanoparticles, the functional DOX nanoparticles exhibited the strongest anticancer efficacy in vitro and in the drug-resistant MCF-7/ Adr (DOX) xenograft tumor model. More specifically, the nanoparticles significantly increased the intracellular uptake of DOX, selectively accumulating in mitochondria and the endoplasmic reticulum after treatment, with release of cytochrome C as a result. Furthermore, the caspase-9 and caspase-3 cascade was activated by the functional DOX nanoparticles through upregulation of the pro-apoptotic proteins Bax and Bid and suppression of the antiapoptotic protein Bcl-2, thereby enhancing apoptosis by acting on the mitochondrial signaling pathways. In conclusion, functional DOX nanoparticles may provide a strategy for increasing the solubility of DOX and overcoming multidrug-resistant cancers. PMID:25605018

Insights into Integrated Lead Generation and Target Identification in Malaria and Tuberculosis Drug Discovery

PubMed Central

2017-01-01

Conspectus New, safe and effective drugs are urgently needed to treat and control malaria and tuberculosis, which affect millions of people annually. However, financial return on investment in the poor settings where these diseases are mostly prevalent is very minimal to support market-driven drug discovery and development. Moreover, the imminent loss of therapeutic lifespan of existing therapies due to evolution and spread of drug resistance further compounds the urgency to identify novel effective drugs. However, the advent of new public–private partnerships focused on tropical diseases and the recent release of large data sets by pharmaceutical companies on antimalarial and antituberculosis compounds derived from phenotypic whole cell high throughput screening have spurred renewed interest and opened new frontiers in malaria and tuberculosis drug discovery. This Account recaps the existing challenges facing antimalarial and antituberculosis drug discovery, including limitations associated with experimental animal models as well as biological complexities intrinsic to the causative pathogens. We enlist various highlights from a body of work within our research group aimed at identifying and characterizing new chemical leads, and navigating these challenges to contribute toward the global drug discovery and development pipeline in malaria and tuberculosis. We describe a catalogue of in-house efforts toward deriving safe and efficacious preclinical drug development candidates via cell-based medicinal chemistry optimization of phenotypic whole-cell medium and high throughput screening hits sourced from various small molecule chemical libraries. We also provide an appraisal of target-based screening, as invoked in our laboratory for mechanistic evaluation of the hits generated, with particular focus on the enzymes within the de novo pyrimidine biosynthetic and hemoglobin degradation pathways, the latter constituting a heme detoxification process and an associated

Use of eQTL Analysis for the Discovery of Target Genes Identified by GWAS

DTIC Science & Technology

2013-04-01

the biologic pathways affected by these inherited factors, and ultimately to identify targets for disease prediction, risk stratification and...quality using an Agilent chip technology. Cases having a RIN number of 7.0 or greater were considered good quality. Once completed, the optimum set of...AD_________________ Award Number: W81XWH-11-1-0261 TITLE: Use of eQTL Analysis for the Discovery of

Emerging techniques for the discovery and validation of therapeutic targets for skeletal diseases.

PubMed

Cho, Christine H; Nuttall, Mark E

2002-12-01

Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure-activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis

Mitochondrial dynamics in Parkinson's disease

PubMed Central

Van Laar, Victor S.; Berman, Sarah B.

2009-01-01

The unique energy demands of neurons require well-orchestrated distribution and maintenance of mitochondria. Thus, dynamic properties of mitochondria, including fission, fusion, trafficking, biogenesis, and degradation, are critical to all cells, but may be particularly important in neurons. Dysfunction in mitochondrial dynamics has been linked to neuropathies and is increasingly being linked to several neurodegenerative diseases, but the evidence is particularly strong, and continuously accumulating, in Parkinson's disease (PD). The unique characteristics of neurons that degenerate in PD may predispose those neuronal populations to susceptibility to alterations in mitochondrial dynamics. In addition, evidence from PD-related toxins supports that mitochondrial fission, fusion, and transport may be involved in pathogenesis. Furthermore, rapidly increasing evidence suggests that two proteins linked to familial forms of the disease, parkin and PINK1, interact in a common pathway to regulate mitochondrial fission/fusion. Parkin may also play a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Taken together, the current data suggests that mitochondrial dynamics may play a role in PD pathogenesis, and a better understanding of mitochondrial dynamics within the neuron may lead to future therapeutic treatments for PD, potentially aimed at some of the earliest pathogenic events. PMID:19332061

The enemy within: Targeting host–parasite interaction for antileishmanial drug discovery

PubMed Central

Späth, Gerald F.; Rachidi, Najma; Prina, Eric

2017-01-01

The state of antileishmanial chemotherapy is strongly compromised by the emergence of drug-resistant Leishmania. The evolution of drug-resistant phenotypes has been linked to the parasites’ intrinsic genome instability, with frequent gene and chromosome amplifications causing fitness gains that are directly selected by environmental factors, including the presence of antileishmanial drugs. Thus, even though the unique eukaryotic biology of Leishmania and its dependence on parasite-specific virulence factors provide valid opportunities for chemotherapeutical intervention, all strategies that target the parasite in a direct fashion are likely prone to select for resistance. Here, we review the current state of antileishmanial chemotherapy and discuss the limitations of ongoing drug discovery efforts. We finally propose new strategies that target Leishmania viability indirectly via mechanisms of host–parasite interaction, including parasite-released ectokinases and host epigenetic regulation, which modulate host cell signaling and transcriptional regulation, respectively, to establish permissive conditions for intracellular Leishmania survival. PMID:28594938

Enhancing mitochondrial proteostasis reduces amyloid-β proteotoxicity.

PubMed

Sorrentino, Vincenzo; Romani, Mario; Mouchiroud, Laurent; Beck, John S; Zhang, Hongbo; D'Amico, Davide; Moullan, Norman; Potenza, Francesca; Schmid, Adrien W; Rietsch, Solène; Counts, Scott E; Auwerx, Johan

2017-12-14

Alzheimer's disease is a common and devastating disease characterized by aggregation of the amyloid-β peptide. However, we know relatively little about the underlying molecular mechanisms or how to treat patients with Alzheimer's disease. Here we provide bioinformatic and experimental evidence of a conserved mitochondrial stress response signature present in diseases involving amyloid-β proteotoxicity in human, mouse and Caenorhabditis elegans that involves the mitochondrial unfolded protein response and mitophagy pathways. Using a worm model of amyloid-β proteotoxicity, GMC101, we recapitulated mitochondrial features and confirmed that the induction of this mitochondrial stress response was essential for the maintenance of mitochondrial proteostasis and health. Notably, increasing mitochondrial proteostasis by pharmacologically and genetically targeting mitochondrial translation and mitophagy increases the fitness and lifespan of GMC101 worms and reduces amyloid aggregation in cells, worms and in transgenic mouse models of Alzheimer's disease. Our data support the relevance of enhancing mitochondrial proteostasis to delay amyloid-β proteotoxic diseases, such as Alzheimer's disease.

Mitochondrial Approaches to Protect Against Cardiac Ischemia and Reperfusion Injury

PubMed Central

Camara, Amadou K. S.; Bienengraeber, Martin; Stowe, David F.

2011-01-01

The mitochondrion is a vital component in cellular energy metabolism and intracellular signaling processes. Mitochondria are involved in a myriad of complex signaling cascades regulating cell death vs. survival. Importantly, mitochondrial dysfunction and the resulting oxidative and nitrosative stress are central in the pathogenesis of numerous human maladies including cardiovascular diseases, neurodegenerative diseases, diabetes, and retinal diseases, many of which are related. This review will examine the emerging understanding of the role of mitochondria in the etiology and progression of cardiovascular diseases and will explore potential therapeutic benefits of targeting the organelle in attenuating the disease process. Indeed, recent advances in mitochondrial biology have led to selective targeting of drugs designed to modulate or manipulate mitochondrial function, to the use of light therapy directed to the mitochondrial function, and to modification of the mitochondrial genome for potential therapeutic benefit. The approach to rationally treat mitochondrial dysfunction could lead to more effective interventions in cardiovascular diseases that to date have remained elusive. The central premise of this review is that if mitochondrial abnormalities contribute to the etiology of cardiovascular diseases (e.g., ischemic heart disease), alleviating the mitochondrial dysfunction will contribute to mitigating the severity or progression of the disease. To this end, this review will provide an overview of our current understanding of mitochondria function in cardiovascular diseases as well as the potential role for targeting mitochondria with potential drugs or other interventions that lead to protection against cell injury. PMID:21559063

Mitochondrial Contribution to Parkinson's Disease Pathogenesis

PubMed Central

Schapira, Anthony H. V.; Gegg, Matthew

2011-01-01

The identification of the etiologies and pathogenesis of Parkinson's disease (PD) should play an important role in enabling the development of novel treatment strategies to prevent or slow the progression of the disease. The last few years have seen enormous progress in this respect. Abnormalities of mitochondrial function and increased free radical mediated damage were described in post mortem PD brain before the first gene mutations causing familial PD were published. Several genetic causes are now known to induce loss of dopaminergic cells and parkinsonism, and study of the mechanisms by which these mutations produce this effect has provided important insights into the pathogenesis of PD and confirmed mitochondrial dysfunction and oxidative stress pathways as central to PD pathogenesis. Abnormalities of protein metabolism including protein mis-folding and aggregation are also crucial to the pathology of PD. Genetic causes of PD have specifically highlighted the importance of mitochondrial dysfunction to PD: PINK1, parkin, DJ-1 and most recently alpha-synuclein proteins have been shown to localise to mitochondria and influence function. The turnover of mitochondria by autophagy (mitophagy) has also become a focus of attention. This review summarises recent discoveries in the contribution of mitochondrial abnormalities to PD etiology and pathogenesis. PMID:21687805

MEDICI: Mining Essentiality Data to Identify Critical Interactions for Cancer Drug Target Discovery and Development | Office of Cancer Genomics

Cancer.gov

Protein-protein interactions (PPIs) mediate the transmission and regulation of oncogenic signals that are essential to cellular proliferation and survival, and thus represent potential targets for anti-cancer therapeutic discovery. Despite their significance, there is no method to experimentally disrupt and interrogate the essentiality of individual endogenous PPIs. The ability to computationally predict or infer PPI essentiality would help prioritize PPIs for drug discovery and help advance understanding of cancer biology.

Computational drug discovery

PubMed Central

Ou-Yang, Si-sheng; Lu, Jun-yan; Kong, Xiang-qian; Liang, Zhong-jie; Luo, Cheng; Jiang, Hualiang

2012-01-01

Computational drug discovery is an effective strategy for accelerating and economizing drug discovery and development process. Because of the dramatic increase in the availability of biological macromolecule and small molecule information, the applicability of computational drug discovery has been extended and broadly applied to nearly every stage in the drug discovery and development workflow, including target identification and validation, lead discovery and optimization and preclinical tests. Over the past decades, computational drug discovery methods such as molecular docking, pharmacophore modeling and mapping, de novo design, molecular similarity calculation and sequence-based virtual screening have been greatly improved. In this review, we present an overview of these important computational methods, platforms and successful applications in this field. PMID:22922346

Mitochondrial control by DRP1 in brain tumor initiating cells.

PubMed

Xie, Qi; Wu, Qiulian; Horbinski, Craig M; Flavahan, William A; Yang, Kailin; Zhou, Wenchao; Dombrowski, Stephen M; Huang, Zhi; Fang, Xiaoguang; Shi, Yu; Ferguson, Ashley N; Kashatus, David F; Bao, Shideng; Rich, Jeremy N

2015-04-01

Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca(2+)-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

Mitochondrial Cyclophilin D in Vascular Oxidative Stress and Hypertension.

PubMed

Itani, Hana A; Dikalova, Anna E; McMaster, William G; Nazarewicz, Rafal R; Bikineyeva, Alfiya T; Harrison, David G; Dikalov, Sergey I

2016-06-01

Vascular superoxide (O˙2 (-)) and inflammation contribute to hypertension. The mitochondria are an important source of O˙2 (-); however, the regulation of mitochondrial O˙2 (-) and the antihypertensive potential of targeting the mitochondria remain poorly defined. Angiotensin II and inflammatory cytokines, such as interleukin 17A and tumor necrosis factor-α (TNFα) significantly contribute to hypertension. We hypothesized that angiotensin II and cytokines co-operatively induce cyclophilin D (CypD)-dependent mitochondrial O˙2 (-) production in hypertension. We tested whether CypD inhibition attenuates endothelial oxidative stress and reduces hypertension. CypD depletion in CypD(-/-) mice prevents overproduction of mitochondrial O˙2 (-) in angiotensin II-infused mice, attenuates hypertension by 20 mm Hg, and improves vascular relaxation compared with wild-type C57Bl/6J mice. Treatment of hypertensive mice with the specific CypD inhibitor Sanglifehrin A reduces blood pressure by 28 mm Hg, inhibits production of mitochondrial O˙2 (-) by 40%, and improves vascular relaxation. Angiotensin II-induced hypertension was associated with CypD redox activation by S-glutathionylation, and expression of the mitochondria-targeted H2O2 scavenger, catalase, abolished CypD S-glutathionylation, prevented stimulation mitochondrial O˙2 (-), and attenuated hypertension. The functional role of cytokine-angiotensin II interplay was confirmed by co-operative stimulation of mitochondrial O˙2 (-) by 3-fold in cultured endothelial cells and impairment of aortic relaxation incubated with combination of angiotensin II, interleukin 17A, and tumor necrosis factor-α which was prevented by CypD depletion or expression of mitochondria-targeted SOD2 and catalase. These data support a novel role of CypD in hypertension and demonstrate that targeting CypD decreases mitochondrial O˙2 (-), improves vascular relaxation, and reduces hypertension. © 2016 American Heart Association, Inc.

Clinical benefit of drugs targeting mitochondrial function as an adjunct to reperfusion in ST-segment elevation myocardial infarction: A meta-analysis of randomized clinical trials.

PubMed

Campo, Gianluca; Pavasini, Rita; Morciano, Giampaolo; Lincoff, A Michael; Gibson, C Michael; Kitakaze, Masafumi; Lonborg, Jacob; Ahluwalia, Amrita; Ishii, Hideki; Frenneaux, Michael; Ovize, Michel; Galvani, Marcello; Atar, Dan; Ibanez, Borja; Cerisano, Giampaolo; Biscaglia, Simone; Neil, Brandon J; Asakura, Masanori; Engstrom, Thomas; Jones, Daniel A; Dawson, Dana; Ferrari, Roberto; Pinton, Paolo; Ottani, Filippo

2017-10-01

To perform a systematic review and meta-analysis of randomized clinical trials (RCT) comparing the effectiveness of drugs targeting mitochondrial function vs. placebo in patients with ST-segment elevation myocardial infarction (STEMI) undergoing mechanical coronary reperfusion. Inclusion criteria: RCTs enrolling STEMI patients treated with primary percutaneous coronary intervention (PCI) and comparing drugs targeting mitochondrial function vs. placebo. Odds ratios (OR) were computed from individual studies and pooled with random-effect meta-analysis. Fifteen studies were identified involving 5680 patients. When compared with placebo, drugs targeting mitochondrial component/pathway were not associated with significant reduction of cardiovascular and all-cause mortality (OR 0.9, 95% CI 0.7-1.17 and OR 0.92, 95% CI 0.69-1.23, respectively). However, these agents significantly reduced hospital admission for heart failure (HF) (OR 0.64; 95% CI 0.45-0.92) and increased left ventricular ejection fraction (LVEF) (OR 1.44; 95% CI 1.15-1.82). After analysis for subgroups according to the mechanism of action, drugs with direct/selective action did not reduce any outcome. Conversely, those with indirect/unspecific action showed a significant effect on cardiovascular mortality (0.65, 95% CI 0.46-0.92), all-cause mortality (OR 0.69, 95% CI 0.52-0.92), hospital readmission for HF (OR 0.41, 95% CI 0.28-0.6) and LVEF (OR 1.49, 95% CI 1.09-2.05). Administration of drugs targeting mitochondrial function in STEMI patients undergoing primary PCI appear to have no effect on mortality, but may reduce hospital readmission for HF. The drugs with a broad-spectrum mechanism of action seem to be more effective in reducing adverse events. Copyright © 2017 Elsevier B.V. All rights reserved.

Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function

PubMed Central

Romanino, Klaas; Mazelin, Laetitia; Albert, Verena; Conjard-Duplany, Agnès; Lin, Shuo; Bentzinger, C. Florian; Handschin, Christoph; Puigserver, Pere; Zorzato, Francesco; Schaeffer, Laurent; Gangloff, Yann-Gaël; Rüegg, Markus A.

2011-01-01

Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy. PMID:22143799

Mitochondrial Dysfunction in Retinal Diseases

PubMed Central

Barot, Megha; Gokulgandhi, Mitan R.; Mitra, Ashim K.

2015-01-01

The mitochondrion is a vital intracellular organelle for retinal cell function and survival. There is growing confirmation to support an association between mitochondrial dysfunction and a number of retinal degenerations. Investigations have also unveiled mitochondrial genomic instability as one of the contributing factors for age-related retinal pathophysiology. This review highlights the role of mitochondrial dysfunction originating from oxidative stress in the etiology of retinal diseases including diabetic retinopathy, glaucoma and age-related macular degeneration (AMD). Moreover, mitochondrial DNA (mtDNA) damage associated with AMD due to susceptibility of mtDNA to oxidative damage and failure of mtDNA repair pathways is also highlighted in this review. The susceptibility of neural retina and retinal pigment epithelium (RPE) mitochondria to oxidative damage with ageing appears to be a major factor in retinal degeneration. It thus appears that the mitochondrion is a weak link in the antioxidant defenses of retinal cells. In addition, failure of mtDNA repair pathways can also specifically contribute towards pathogenesis of AMD. This review will further summarize the prospective role of mitochondria targeting therapeutic agents for the treatment of retinal disease. Mitochondria based drug targeting to diminish oxidative stress or promote repair of mtDNA damage may offer potential alternatives for the treatment of various retinal degenerative diseases. PMID:21978133

Mitochondrial dysfunction in retinal diseases.

PubMed

Barot, Megha; Gokulgandhi, Mitan R; Mitra, Ashim K

2011-12-01

The mitochondrion is a vital intracellular organelle for retinal cell function and survival. There is growing confirmation to support an association between mitochondrial dysfunction and a number of retinal degenerations. Investigations have also unveiled mitochondrial genomic instability as one of the contributing factors for age-related retinal pathophysiology. This review highlights the role of mitochondrial dysfunction originating from oxidative stress in the etiology of retinal diseases including diabetic retinopathy, glaucoma and age-related macular degeneration (AMD). Moreover, mitochondrial DNA (mtDNA) damage associated with AMD due to susceptibility of mtDNA to oxidative damage and failure of mtDNA repair pathways is also highlighted in this review. The susceptibility of neural retina and retinal pigment epithelium (RPE) mitochondria to oxidative damage with ageing appears to be a major factor in retinal degeneration. It thus appears that the mitochondrion is a weak link in the antioxidant defenses of retinal cells. In addition, failure of mtDNA repair pathways can also specifically contribute towards pathogenesis of AMD. This review will further summarize the prospective role of mitochondria targeting therapeutic agents for the treatment of retinal disease. Mitochondria based drug targeting to diminish oxidative stress or promote repair of mtDNA damage may offer potential alternatives for the treatment of various retinal degenerative diseases.

Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets.

PubMed

Kimple, Adam J; Bosch, Dustin E; Giguère, Patrick M; Siderovski, David P

2011-09-01

Because G-protein coupled receptors (GPCRs) continue to represent excellent targets for the discovery and development of small-molecule therapeutics, it is posited that additional protein components of the signal transduction pathways emanating from activated GPCRs themselves are attractive as drug discovery targets. This review considers the drug discovery potential of two such components: members of the "regulators of G-protein signaling" (RGS protein) superfamily, as well as their substrates, the heterotrimeric G-protein α subunits. Highlighted are recent advances, stemming from mouse knockout studies and the use of "RGS-insensitivity" and fast-hydrolysis mutations to Gα, in our understanding of how RGS proteins selectively act in (patho)physiologic conditions controlled by GPCR signaling and how they act on the nucleotide cycling of heterotrimeric G-proteins in shaping the kinetics and sensitivity of GPCR signaling. Progress is documented regarding recent activities along the path to devising screening assays and chemical probes for the RGS protein target, not only in pursuits of inhibitors of RGS domain-mediated acceleration of Gα GTP hydrolysis but also to embrace the potential of finding allosteric activators of this RGS protein action. The review concludes in considering the Gα subunit itself as a drug target, as brought to focus by recent reports of activating mutations to GNAQ and GNA11 in ocular (uveal) melanoma. We consider the likelihood of several strategies for antagonizing the function of these oncogene alleles and their gene products, including the use of RGS proteins with Gα(q) selectivity.

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessedmore » by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. �

A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

PubMed

Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

2008-10-01

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

PubMed

Panozzo, C; Laleve, A; Tribouillard-Tanvier, D; Ostojić, J; Sellem, C H; Friocourt, G; Bourand-Plantefol, A; Burg, A; Delahodde, A; Blondel, M; Dujardin, G

2017-12-01

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III 2 /IV in yeast or I/III 2 /IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes. Copyright © 2017. Published by Elsevier B.V.

Computer-based prediction of mitochondria-targeting peptides.

PubMed

Martelli, Pier Luigi; Savojardo, Castrense; Fariselli, Piero; Tasco, Gianluca; Casadio, Rita

2015-01-01

Computational methods are invaluable when protein sequences, directly derived from genomic data, need functional and structural annotation. Subcellular localization is a feature necessary for understanding the protein role and the compartment where the mature protein is active and very difficult to characterize experimentally. Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in the precursor sequence from where it is possible to recognize a peptide targeting the protein to its final destination. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequences and benchmark our and other methods on the human mitochondrial proteins endowed with experimentally characterized targeting peptides. Furthermore, we illustrate our newly implemented web server and its usage on the whole human proteome in order to infer mitochondrial targeting peptides, their cleavage sites, and whether the targeting peptide regions contain or not arginine-rich recurrent motifs. By this, we add some other 2,800 human proteins to the 124 ones already experimentally annotated with a mitochondrial targeting peptide.

Early Probe and Drug Discovery in Academia: A Minireview.

PubMed

Roy, Anuradha

2018-02-09

Drug discovery encompasses processes ranging from target selection and validation to the selection of a development candidate. While comprehensive drug discovery work flows are implemented predominantly in the big pharma domain, early discovery focus in academia serves to identify probe molecules that can serve as tools to study targets or pathways. Despite differences in the ultimate goals of the private and academic sectors, the same basic principles define the best practices in early discovery research. A successful early discovery program is built on strong target definition and validation using a diverse set of biochemical and cell-based assays with functional relevance to the biological system being studied. The chemicals identified as hits undergo extensive scaffold optimization and are characterized for their target specificity and off-target effects in in vitro and in animal models. While the active compounds from screening campaigns pass through highly stringent chemical and Absorption, Distribution, Metabolism, and Excretion (ADME) filters for lead identification, the probe discovery involves limited medicinal chemistry optimization. The goal of probe discovery is identification of a compound with sub-µM activity and reasonable selectivity in the context of the target being studied. The compounds identified from probe discovery can also serve as starting scaffolds for lead optimization studies.

A Novel Methodology for Bioenergetic Analysis of Plasmodium falciparum Reveals a Glucose-Regulated Metabolic Shift and Enables Mode of Action Analyses of Mitochondrial Inhibitors.

PubMed

Sakata-Kato, Tomoyo; Wirth, Dyann F

2016-12-09

Given that resistance to all drugs in clinical use has arisen, discovery of new antimalarial drug targets is eagerly anticipated. The Plasmodium mitochondrion has been considered a promising drug target largely based on its significant divergence from the host organelle as well as its involvement in ATP production and pyrimidine biosynthesis. However, the functions of Plasmodium mitochondrial protein complexes and associated metabolic pathways are not fully characterized. Here, we report the development of novel and robust bioenergetic assay protocols for Plasmodium falciparum asexual parasites utilizing a Seahorse Bioscience XFe24 Extracellular Flux Analyzer. These protocols allowed us to simultaneously assess the direct effects of metabolites and inhibitors on mitochondrial respiration and glycolytic activity in real-time with the readout of oxygen consumption rate and extracellular acidification rate. Using saponin-freed parasites at the schizont stage, we found that succinate, malate, glycerol-3-phosphate, and glutamate, but not pyruvate, were able to increase the oxygen consumption rate and that glycerol-3-phosphate dehydrogenase had the largest potential as an electron donor among tested mitochondrial dehydrogenases. Furthermore, we revealed the presence of a glucose-regulated metabolic shift between oxidative phosphorylation and glycolysis. We measured proton leak and reserve capacity and found bioenergetic evidence for oxidative phosphorylation in erythrocytic stage parasites but at a level much lower than that observed in mammalian cells. Lastly, we developed an assay platform for target identification and mode of action studies of mitochondria-targeting antimalarials. This study provides new insights into the bioenergetics and metabolomics of the Plasmodium mitochondria.

Mitochondrial Metabolism in Aging Heart

PubMed Central

Lesnefsky, Edward J.; Chen, Qun; Hoppel, Charles L.

2016-01-01

Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area there is an approximate 50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction. PMID:27174952

Stem cell signaling as a target for novel drug discovery: recent progress in the WNT and Hedgehog pathways.

PubMed

An, Songzhu Michael; Ding, Qiang Peter; Li, Ling-song

2013-06-01

One of the most exciting fields in biomedical research over the past few years is stem cell biology, and therapeutic application of stem cells to replace the diseased or damaged tissues is also an active area in development. Although stem cell therapy has a number of technical challenges and regulatory hurdles to overcome, the use of stem cells as tools in drug discovery supported by mature technologies and established regulatory paths is expected to generate more immediate returns. In particular, the targeting of stem cell signaling pathways is opening up a new avenue for drug discovery. Aberrations in these pathways result in various diseases, including cancer, fibrosis and degenerative diseases. A number of drug targets in stem cell signaling pathways have been identified. Among them, WNT and Hedgehog are two most important signaling pathways, which are the focus of this review. A hedgehog pathway inhibitor, vismodegib (Erivedge), has recently been approved by the US FDA for the treatment of skin cancer, while several drug candidates for the WNT pathway are entering clinical trials. We have discovered that the stem cell signaling pathways respond to traditional Chinese medicines. Substances isolated from herbal medicine may act specifically on components of stem cell signaling pathways with high affinities. As many of these events can be explained through molecular interactions, these phenomena suggest that discovery of stem cell-targeting drugs from natural products may prove to be highly successful.

Targeting the human genome-microbiome axis for drug discovery: inspirations from global systems biology and traditional Chinese medicine.

PubMed

Zhao, Liping; Nicholson, Jeremy K; Lu, Aiping; Wang, Zhengtao; Tang, Huiru; Holmes, Elaine; Shen, Jian; Zhang, Xu; Li, Jia V; Lindon, John C

2012-07-06

Most chronic diseases impairing current human public health involve not only the human genome but also gene-environment interactions, and in the latter case the gut microbiome is an important factor. This makes the classical single drug-receptor target drug discovery paradigm much less applicable. There is widespread and increasing international interest in understanding the properties of traditional Chinese medicines (TCMs) for their potential utilization as a source of new drugs for Western markets as emerging evidence indicates that most TCM drugs are actually targeting both the host and its symbiotic microbes. In this review, we explore the challenges of and opportunities for harmonizing Eastern-Western drug discovery paradigms by focusing on emergent functions at the whole body level of humans as superorganisms. This could lead to new drug candidate compounds for chronic diseases targeting receptors outside the currently accepted "druggable genome" and shed light on current high interest issues in Western medicine such as drug-drug and drug-diet-gut microbial interactions that will be crucial in the development and delivery of future therapeutic regimes optimized for the individual patient.

Mitochondrial Dysfunction in Parkinson's Disease.

PubMed

Moon, Hyo Eun; Paek, Sun Ha

2015-06-01

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) with motor and nonmotor symptoms. Defective mitochondrial function and increased oxidative stress (OS) have been demonstrated as having an important role in PD pathogenesis, although the underlying mechanism is not clear. The etiopathogenesis of sporadic PD is complex with variable contributions of environmental factors and genetic susceptibility. Both these factors influence various mitochondrial aspects, including their life cycle, bioenergetic capacity, quality control, dynamic changes of morphology and connectivity (fusion, fission), subcellular distribution (transport), and the regulation of cell death pathways. Mitochondrial dysfunction has mainly been reported in various non-dopaminergic cells and tissue samples from human patients as well as transgenic mouse and fruit fly models of PD. Thus, the mitochondria represent a highly promising target for the development of PD biomarkers. However, the limited amount of dopaminergic neurons prevented investigation of their detailed study. For the first time, we established human telomerase reverse transcriptase (hTERT)-immortalized wild type, idiopathic and Parkin deficient mesenchymal stromal cells (MSCs) isolated from the adipose tissues of PD patients, which could be used as a good cellular model to evaluate mitochondrial dysfunction for the better understanding of PD pathology and for the development of early diagnostic markers and effective therapy targets of PD. In this review, we examine evidence for the roles of mitochondrial dysfunction and increased OS in the neuronal loss that leads to PD and discuss how this knowledge further improve the treatment for patients with PD.

Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins.

PubMed

Hansel, Alfred; Kuschel, Lioba; Hehl, Solveig; Lemke, Cornelius; Agricola, Hans-Jürgen; Hoshi, Toshinori; Heinemann, Stefan H

2002-06-01

Peptide methionine sulfoxide reductase (MSRA) catalyzes the reduction of methionine sulfoxide to methionine. This widely expressed enzyme constitutes an important repair mechanism for oxidatively damaged proteins, which accumulate during the manifestation of certain degenerative diseases and aging processes. In addition, it is discussed to be involved in regulatory processes. Here we address the question of how the enzyme's diverse functions are reflected in its subcellular localization. Using fusions of the human version of MSRA with the enhanced green fluorescence protein expressed in various mammalian cell lines, we show a distinct localization at mitochondria. The N-terminal 23 amino acid residues contain the signal for this mitochondrial targeting. Activity tests showed that they are not required for enzyme function. Mitochondrial localization of native MSRA in mouse and rat liver slices was verified with an MSRA-specific antibody by using immunohistochemical methods. The protein was located in the mitochondrial matrix, as demonstrated by using pre-embedding immunostaining and electron microscopy. Mitochondria are the major source of reactive oxygen species (ROS). Therefore, MSRA has to be considered an important means for the general reduction of ROS release from mitochondria.

Repurposing atovaquone: Targeting mitochondrial complex III and OXPHOS to eradicate cancer stem cells

PubMed Central

Fiorillo, Marco; Lamb, Rebecca; Tanowitz, Herbert B.; Mutti, Luciano; Krstic-Demonacos, Marija; Cappello, Anna Rita; Martinez-Outschoorn, Ubaldo E.; Sotgia, Federica; Lisanti, Michael P.

2016-01-01

Atovaquone is an FDA-approved anti-malarial drug, which first became clinically available in the year 2000. Currently, its main usage is for the treatment of pneumocystis pneumonia (PCP) and/or toxoplasmosis in immune-compromised patients. Atovaquone is a hydroxy-1,4-naphthoquinone analogue of ubiquinone, also known as Co-enzyme Q10 (CoQ10). It is a well-tolerated drug that does not cause myelo-suppression. Mechanistically, it is thought to act as a potent and selective OXPHOS inhibitor, by targeting the CoQ10-dependence of mitochondrial complex III. Here, we show for the first time that atovaquone also has anti-cancer activity, directed against Cancer Stem-like Cells (CSCs). More specifically, we demonstrate that atovaquone treatment of MCF7 breast cancer cells inhibits oxygen-consumption and metabolically induces aerobic glycolysis (the Warburg effect), as well as oxidative stress. Remarkably, atovaquone potently inhibits the propagation of MCF7-derived CSCs, with an IC-50 of 1 μM, as measured using the mammosphere assay. Atovaquone also maintains this selectivity and potency in mixed populations of CSCs and non-CSCs. Importantly, these results indicate that glycolysis itself is not sufficient to maintain the proliferation of CSCs, which is instead strictly dependent on mitochondrial function. In addition to targeting the proliferation of CSCs, atovaquone also induces apoptosis in both CD44+/CD24low/− CSC and ALDH+ CSC populations, during exposure to anchorage-independent conditions for 12 hours. However, it has no effect on oxygen consumption in normal human fibroblasts and, in this cellular context, behaves as an anti-inflammatory, consistent with the fact that it is well-tolerated in patients treated for infections. Future studies in xenograft models and human clinical trials may be warranted, as the IC-50 of atovaquone's action on CSCs (1 μM) is >50 times less than its average serum concentration in humans. PMID:27136895

Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

Joubert, Fourie; Harrison, Claudia M; Koegelenberg, Riaan J; Odendaal, Christiaan J; de Beer, Tjaart AP

2009-01-01

Background Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties. Methods A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available. Results An in silico system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from Plasmodium falciparum. Conclusion The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties. PMID:19642978

Mitochondrial Dynamics in Diabetic Cardiomyopathy

PubMed Central

Galloway, Chad A.

2015-01-01

Abstract Significance: Cardiac function is energetically demanding, reliant on efficient well-coupled mitochondria to generate adenosine triphosphate and fulfill the cardiac demand. Predictably then, mitochondrial dysfunction is associated with cardiac pathologies, often related to metabolic disease, most commonly diabetes. Diabetic cardiomyopathy (DCM), characterized by decreased left ventricular function, arises independently of coronary artery disease and atherosclerosis. Dysregulation of Ca2+ handling, metabolic changes, and oxidative stress are observed in DCM, abnormalities reflected in alterations in mitochondrial energetics. Cardiac tissue from DCM patients also presents with altered mitochondrial morphology, suggesting a possible role of mitochondrial dynamics in its pathological progression. Recent Advances: Abnormal mitochondrial morphology is associated with pathologies across diverse tissues, suggesting that this highly regulated process is essential for proper cell maintenance and physiological homeostasis. Highly structured cardiac myofibers were hypothesized to limit alterations in mitochondrial morphology; however, recent work has identified morphological changes in cardiac tissue, specifically in DCM. Critical Issues: Mitochondrial dysfunction has been reported independently from observations of altered mitochondrial morphology in DCM. The temporal relationship and causative nature between functional and morphological changes of mitochondria in the establishment/progression of DCM is unclear. Future Directions: Altered mitochondrial energetics and morphology are not only causal for but also consequential to reactive oxygen species production, hence exacerbating oxidative damage through reciprocal amplification, which is integral to the progression of DCM. Therefore, targeting mitochondria for DCM will require better mechanistic characterization of morphological distortion and bioenergetic dysfunction. Antioxid. Redox Signal. 22, 1545–1562. PMID

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

Targeted mitochondrial therapy using MitoQ shows equivalent renoprotection to angiotensin converting enzyme inhibition but no combined synergy in diabetes.

PubMed

Ward, Micheal S; Flemming, Nicole B; Gallo, Linda A; Fotheringham, Amelia K; McCarthy, Domenica A; Zhuang, Aowen; Tang, Peter H; Borg, Danielle J; Shaw, Hannah; Harvie, Benjamin; Briskey, David R; Roberts, Llion A; Plan, Manuel R; Murphy, Michael P; Hodson, Mark P; Forbes, Josephine M

2017-11-09

Mitochondrial dysfunction is a pathological mediator of diabetic kidney disease (DKD). Our objective was to test the mitochondrially targeted agent, MitoQ, alone and in combination with first line therapy for DKD. Intervention therapies (i) vehicle (D); (ii) MitoQ (DMitoQ;0.6 mg/kg/day); (iii) Ramipril (DRam;3 mg/kg/day) or (iv) combination (DCoAd) were administered to male diabetic db/db mice for 12 weeks (n = 11-13/group). Non-diabetic (C) db/m mice were followed concurrently. No therapy altered glycaemic control or body weight. By the study end, both monotherapies improved renal function, decreasing glomerular hyperfiltration and albuminuria. All therapies prevented tubulointerstitial collagen deposition, but glomerular mesangial expansion was unaffected. Renal cortical concentrations of ATP, ADP, AMP, cAMP, creatinine phosphate and ATP:AMP ratio were increased by diabetes and mostly decreased with therapy. A higher creatine phosphate:ATP ratio in diabetic kidney cortices, suggested a decrease in ATP consumption. Diabetes elevated glucose 6-phosphate, fructose 6-phosphate and oxidised (NAD+ and NADP+) and reduced (NADH) nicotinamide dinucleotides, which therapy decreased generally. Diabetes increased mitochondrial oxygen consumption (OCR) at complex II-IV. MitoQ further increased OCR but decreased ATP, suggesting mitochondrial uncoupling as its mechanism of action. MitoQ showed renoprotection equivalent to ramipril but no synergistic benefits of combining these agents were shown.

Motor neuron mitochondrial dysfunction in spinal muscular atrophy

PubMed Central

Miller, Nimrod; Shi, Han; Zelikovich, Aaron S.; Ma, Yong-Chao

2016-01-01

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease. PMID:27488123

Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

PubMed

Raveh, Avi; Delekta, Phillip C; Dobry, Craig J; Peng, Weiping; Schultz, Pamela J; Blakely, Pennelope K; Tai, Andrew W; Matainaho, Teatulohi; Irani, David N; Sherman, David H; Miller, David J

2013-01-01

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the

Discovery of Potent Broad Spectrum Antivirals Derived from Marine Actinobacteria

PubMed Central

Raveh, Avi; Delekta, Phillip C.; Dobry, Craig J.; Peng, Weiping; Schultz, Pamela J.; Blakely, Pennelope K.; Tai, Andrew W.; Matainaho, Teatulohi; Irani, David N.; Sherman, David H.; Miller, David J.

2013-01-01

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the

Mitochondrial proteome remodelling in pressure overload-induced heart failure: the role of mitochondrial oxidative stress

PubMed Central

Dai, Dao-Fu; Hsieh, Edward J.; Liu, Yonggang; Chen, Tony; Beyer, Richard P.; Chin, Michael T.; MacCoss, Michael J.; Rabinovitch, Peter S.

2012-01-01

Aims We investigate the role of mitochondrial oxidative stress in mitochondrial proteome remodelling using mouse models of heart failure induced by pressure overload. Methods and results We demonstrate that mice overexpressing catalase targeted to mitochondria (mCAT) attenuate pressure overload-induced heart failure. An improved method of label-free unbiased analysis of the mitochondrial proteome was applied to the mouse model of heart failure induced by transverse aortic constriction (TAC). A total of 425 mitochondrial proteins were compared between wild-type and mCAT mice receiving TAC or sham surgery. The changes in the mitochondrial proteome in heart failure included decreased abundance of proteins involved in fatty acid metabolism, an increased abundance of proteins in glycolysis, apoptosis, mitochondrial unfolded protein response and proteolysis, transcription and translational control, and developmental processes as well as responses to stimuli. Overexpression of mCAT better preserved proteins involved in fatty acid metabolism and attenuated the increases in apoptotic and proteolytic enzymes. Interestingly, gene ontology analysis also showed that monosaccharide metabolic processes and protein folding/proteolysis were only overrepresented in mCAT but not in wild-type mice in response to TAC. Conclusion This is the first study to demonstrate that scavenging mitochondrial reactive oxygen species (ROS) by mCAT not only attenuates most of the mitochondrial proteome changes in heart failure, but also induces a subset of unique alterations. These changes represent processes that are adaptive to the increased work and metabolic requirements of pressure overload, but which are normally inhibited by overproduction of mitochondrial ROS. PMID:22012956

Brain mitochondrial iron accumulates in Huntington's disease, mediates mitochondrial dysfunction, and can be removed pharmacologically.

PubMed

Agrawal, Sonal; Fox, Julia; Thyagarajan, Baskaran; Fox, Jonathan H

2018-05-20

Mitochondrial bioenergetic dysfunction is involved in neurodegeneration in Huntington's disease (HD). Iron is critical for normal mitochondrial bioenergetics but can also contribute to pathogenic oxidation. The accumulation of iron in the brain occurs in mouse models and in human HD. Yet the role of mitochondria-related iron dysregulation as a contributor to bioenergetic pathophysiology in HD is unclear. We demonstrate here that human HD and mouse model HD (12-week R6/2 and 12-month YAC128) brains accumulated mitochondrial iron and showed increased expression of iron uptake protein mitoferrin 2 and decreased iron-sulfur cluster synthesis protein frataxin. Mitochondria-enriched fractions from mouse HD brains had deficits in membrane potential and oxygen uptake and increased lipid peroxidation. In addition, the membrane-permeable iron-selective chelator deferiprone (1 μM) rescued these effects ex-vivo, whereas hydrophilic iron and copper chelators did not. A 10-day oral deferiprone treatment in 9-week R6/2 HD mice indicated that deferiprone removed mitochondrial iron, restored mitochondrial potentials, decreased lipid peroxidation, and improved motor endurance. Neonatal iron supplementation potentiates neurodegeneration in mouse models of HD by unknown mechanisms. We found that neonatal iron supplementation increased brain mitochondrial iron accumulation and potentiated markers of mitochondrial dysfunction in HD mice. Therefore, bi-directional manipulation of mitochondrial iron can potentiate and protect against markers of mouse HD. Our findings thus demonstrate the significance of iron as a mediator of mitochondrial dysfunction and injury in mouse models of human HD and suggest that targeting the iron-mitochondrial pathway may be protective. Copyright © 2018 Elsevier Inc. All rights reserved.

Screening the receptorome to discover the molecular targets for plant-derived psychoactive compounds: a novel approach for CNS drug discovery.

PubMed

Roth, Bryan L; Lopez, Estela; Beischel, Scott; Westkaemper, Richard B; Evans, Jon M

2004-05-01

Because psychoactive plants exert profound effects on human perception, emotion, and cognition, discovering the molecular mechanisms responsible for psychoactive plant actions will likely yield insights into the molecular underpinnings of human consciousness. Additionally, it is likely that elucidation of the molecular targets responsible for psychoactive drug actions will yield validated targets for CNS drug discovery. This review article focuses on an unbiased, discovery-based approach aimed at uncovering the molecular targets responsible for psychoactive drug actions wherein the main active ingredients of psychoactive plants are screened at the "receptorome" (that portion of the proteome encoding receptors). An overview of the receptorome is given and various in silico, public-domain resources are described. Newly developed tools for the in silico mining of data derived from the National Institute of Mental Health Psychoactive Drug Screening Program's (NIMH-PDSP) K(i) Database (K(i) DB) are described in detail. Additionally, three case studies aimed at discovering the molecular targets responsible for Hypericum perforatum, Salvia divinorum, and Ephedra sinica actions are presented. Finally, recommendations are made for future studies.

A computational approach for predicting off-target toxicity of antiviral ribonucleoside analogues to mitochondrial RNA polymerase.

PubMed

Freedman, Holly; Winter, Philip; Tuszynski, Jack; Tyrrell, D Lorne; Houghton, Michael

2018-06-22

In the development of antiviral drugs that target viral RNA-dependent RNA polymerases, off-target toxicity caused by the inhibition of the human mitochondrial RNA polymerase (POLRMT) is a major liability. Therefore, it is essential that all new ribonucleoside analogue drugs be accurately screened for POLRMT inhibition. A computational tool that can accurately predict NTP binding to POLRMT could assist in evaluating any potential toxicity and in designing possible salvaging strategies. Using the available crystal structure of POLRMT bound to an RNA transcript, here we created a model of POLRMT with an NTP molecule bound in the active site. Furthermore, we implemented a computational screening procedure that determines the relative binding free energy of an NTP analogue to POLRMT by free energy perturbation (FEP), i.e. a simulation in which the natural NTP molecule is slowly transformed into the analogue and back. In each direction, the transformation was performed over 40 ns of simulation on our IBM Blue Gene Q supercomputer. This procedure was validated across a panel of drugs for which experimental dissociation constants were available, showing that NTP relative binding free energies could be predicted to within 0.97 kcal/mol of the experimental values on average. These results demonstrate for the first time that free-energy simulation can be a useful tool for predicting binding affinities of NTP analogues to a polymerase. We expect that our model, together with similar models of viral polymerases, will be very useful in the screening and future design of NTP inhibitors of viral polymerases that have no mitochondrial toxicity. © 2018 Freedman et al.

Yeast mitochondrial glutathione is an essential antioxidant with mitochondrial thioredoxin providing a back-up system

PubMed Central

Gostimskaya, Irina; Grant, Chris M.

2016-01-01

Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146

Oxidative Stress and Mitochondrial Dysfunction across Broad-Ranging Pathologies: Toward Mitochondria-Targeted Clinical Strategies

PubMed Central

d'Ischia, Marco; Gadaleta, Maria Nicola; Pallardó, Federico V.; Petrović, Sandra; Tiano, Luca; Zatterale, Adriana

2014-01-01

Beyond the disorders recognized as mitochondrial diseases, abnormalities in function and/or ultrastructure of mitochondria have been reported in several unrelated pathologies. These encompass ageing, malformations, and a number of genetic or acquired diseases, as diabetes and cardiologic, haematologic, organ-specific (e.g., eye or liver), neurologic and psychiatric, autoimmune, and dermatologic disorders. The mechanistic grounds for mitochondrial dysfunction (MDF) along with the occurrence of oxidative stress (OS) have been investigated within the pathogenesis of individual disorders or in groups of interrelated disorders. We attempt to review broad-ranging pathologies that involve mitochondrial-specific deficiencies or rely on cytosol-derived prooxidant states or on autoimmune-induced mitochondrial damage. The established knowledge in these subjects warrants studies aimed at elucidating several open questions that are highlighted in the present review. The relevance of OS and MDF in different pathologies may establish the grounds for chemoprevention trials aimed at compensating OS/MDF by means of antioxidants and mitochondrial nutrients. PMID:24876913

Discovery and molecular characterization of a Bcl-2-regulated cell death pathway in schistosomes.

PubMed

Lee, Erinna F; Clarke, Oliver B; Evangelista, Marco; Feng, Zhiping; Speed, Terence P; Tchoubrieva, Elissaveta B; Strasser, Andreas; Kalinna, Bernd H; Colman, Peter M; Fairlie, W Douglas

2011-04-26

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2-regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with "BH3 mimetic" drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.

Mitochondrial Energy and Redox Signaling in Plants

PubMed Central

Schwarzländer, Markus

2013-01-01

Abstract Significance: For a plant to grow and develop, energy and appropriate building blocks are a fundamental requirement. Mitochondrial respiration is a vital source for both. The delicate redox processes that make up respiration are affected by the plant's changing environment. Therefore, mitochondrial regulation is critically important to maintain cellular homeostasis. This involves sensing signals from changes in mitochondrial physiology, transducing this information, and mounting tailored responses, by either adjusting mitochondrial and cellular functions directly or reprogramming gene expression. Recent Advances: Retrograde (RTG) signaling, by which mitochondrial signals control nuclear gene expression, has been a field of very active research in recent years. Nevertheless, no mitochondrial RTG-signaling pathway is yet understood in plants. This review summarizes recent advances toward elucidating redox processes and other bioenergetic factors as a part of RTG signaling of plant mitochondria. Critical Issues: Novel insights into mitochondrial physiology and redox-regulation provide a framework of upstream signaling. On the other end, downstream responses to modified mitochondrial function have become available, including transcriptomic data and mitochondrial phenotypes, revealing processes in the plant that are under mitochondrial control. Future Directions: Drawing parallels to chloroplast signaling and mitochondrial signaling in animal systems allows to bridge gaps in the current understanding and to deduce promising directions for future research. It is proposed that targeted usage of new technical approaches, such as quantitative in vivo imaging, will provide novel leverage to the dissection of plant mitochondrial signaling. Antioxid. Redox Signal. 18, 2122–2144. PMID:23234467

A Molecular Approach to Mitophagy and Mitochondrial Dynamics

PubMed Central

Yoo, Seung-Min; Jung, Yong-Keun

2018-01-01

Mitochondrial quality control systems are essential for the maintenance of functional mitochondria. At the organelle level, they include mitochondrial biogenesis, fusion and fission, to compensate for mitochondrial function, and mitophagy, for degrading damaged mitochondria. Specifically, in mitophagy, the target mitochondria are recognized by the autophagosomes and delivered to the lysosome for degradation. In this review, we describe the mechanisms of mitophagy and the factors that play an important role in this process. In particular, we focus on the roles of mitophagy adapters and receptors in the recognition of damaged mitochondria by autophagosomes. In addition, we also address a functional association of mitophagy with mitochondrial dynamics through the interaction of mitophagy adaptor and receptor proteins with mitochondrial fusion and fission proteins. PMID:29370689

Mitochondrial oxidative stress in aging and healthspan

PubMed Central

2014-01-01

The free radical theory of aging proposes that reactive oxygen species (ROS)-induced accumulation of damage to cellular macromolecules is a primary driving force of aging and a major determinant of lifespan. Although this theory is one of the most popular explanations for the cause of aging, several experimental rodent models of antioxidant manipulation have failed to affect lifespan. Moreover, antioxidant supplementation clinical trials have been largely disappointing. The mitochondrial theory of aging specifies more particularly that mitochondria are both the primary sources of ROS and the primary targets of ROS damage. In addition to effects on lifespan and aging, mitochondrial ROS have been shown to play a central role in healthspan of many vital organ systems. In this article we review the evidence supporting the role of mitochondrial oxidative stress, mitochondrial damage and dysfunction in aging and healthspan, including cardiac aging, age-dependent cardiovascular diseases, skeletal muscle aging, neurodegenerative diseases, insulin resistance and diabetes as well as age-related cancers. The crosstalk of mitochondrial ROS, redox, and other cellular signaling is briefly presented. Potential therapeutic strategies to improve mitochondrial function in aging and healthspan are reviewed, with a focus on mitochondrial protective drugs, such as the mitochondrial antioxidants MitoQ, SkQ1, and the mitochondrial protective peptide SS-31. PMID:24860647

Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage.

PubMed

Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P; Morales-Nebreda, Luisa; Cheng, Yuan; Hogan, Erin; Yeldandi, Anjana; Chi, Monica; Piseaux, Raul; Ridge, Karen; Michael Hart, C; Chandel, Navdeep; Scott Budinger, G R; Kamp, David W

2016-12-01

Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. Crocidolite asbestos (100µg/50µL), TiO 2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role

Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage

PubMed Central

Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P.; Morales-Nebreda, Luisa; Cheng, Yuan; Hogan, Erin; Yeldandi, Anjana; Chi, Monica; Piseaux, Raul; Ridge, Karen; Hart, C. Michael; Chandel, Navdeep; Budinger, G.R. Scott; Kamp, David W.

2018-01-01

Rationale Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. Objective To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. Methods Crocidolite asbestos (100 μg/50 μL), TiO2 (negative control), bleomycin (0.025 units/50 μL), or PBS was instilled intratracheally in 8–10 week-old wild-type (WT - C57Bl/6 J) or MCAT mice. The lungs were harvested at 21 d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. Results Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. Conclusions Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

A Mitochondrial-Targeted Coenzyme Q Analog Prevents Weight Gain and Ameliorates Hepatic Dysfunction in High-Fat–Fed Mice

PubMed Central

Fink, Brian D.; Herlein, Judith A.; Guo, Deng Fu; Kulkarni, Chaitanya; Weidemann, Benjamin J.; Yu, Liping; Grobe, Justin L.; Rahmouni, Kamal; Kerns, Robert J.

2014-01-01

We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 µM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF- and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics. PMID:25301169

Insulin Resistance and Mitochondrial Dysfunction.

PubMed

Gonzalez-Franquesa, Alba; Patti, Mary-Elizabeth

2017-01-01

Insulin resistance precedes and predicts the onset of type 2 diabetes (T2D) in susceptible humans, underscoring its important role in the complex pathogenesis of this disease. Insulin resistance contributes to multiple tissue defects characteristic of T2D, including reduced insulin-stimulated glucose uptake in insulin-sensitive tissues, increased hepatic glucose production, increased lipolysis in adipose tissue, and altered insulin secretion. Studies of individuals with insulin resistance, both with established T2D and high-risk individuals, have consistently demonstrated a diverse array of defects in mitochondrial function (i.e., bioenergetics, biogenesis and dynamics). However, it remains uncertain whether mitochondrial dysfunction is primary (critical initiating defect) or secondary to the subtle derangements in glucose metabolism, insulin resistance, and defective insulin secretion present early in the course of disease development. In this chapter, we will present the evidence linking mitochondrial dysfunction and insulin resistance, and review the potential for mitochondrial targets as a therapeutic approach for T2D.

Mitochondrial pathogenic mutations are population-specific.

PubMed

Breen, Michael S; Kondrashov, Fyodor A

2010-12-31

Surveying deleterious variation in human populations is crucial for our understanding, diagnosis and potential treatment of human genetic pathologies. A number of recent genome-wide analyses focused on the prevalence of segregating deleterious alleles in the nuclear genome. However, such studies have not been conducted for the mitochondrial genome. We present a systematic survey of polymorphisms in the human mitochondrial genome, including those predicted to be deleterious and those that correspond to known pathogenic mutations. Analyzing 4458 completely sequenced mitochondrial genomes we characterize the genetic diversity of different types of single nucleotide polymorphisms (SNPs) in African (L haplotypes) and non-African (M and N haplotypes) populations. We find that the overall level of polymorphism is higher in the mitochondrial compared to the nuclear genome, although the mitochondrial genome appears to be under stronger selection as indicated by proportionally fewer nonsynonymous than synonymous substitutions. The African mitochondrial genomes show higher heterozygosity, a greater number of polymorphic sites and higher frequencies of polymorphisms for synonymous, benign and damaging polymorphism than non-African genomes. However, African genomes carry significantly fewer SNPs that have been previously characterized as pathogenic compared to non-African genomes. Finding SNPs classified as pathogenic to be the only category of polymorphisms that are more abundant in non-African genomes is best explained by a systematic ascertainment bias that favours the discovery of pathogenic polymorphisms segregating in non-African populations. This further suggests that, contrary to the common disease-common variant hypothesis, pathogenic mutations are largely population-specific and different SNPs may be associated with the same disease in different populations. Therefore, to obtain a comprehensive picture of the deleterious variability in the human population, as well as

A review of the basics of mitochondrial bioenergetics, metabolism, and related signaling pathways in cancer cells: Therapeutic targeting of tumor mitochondria with lipophilic cationic compounds.

PubMed

Kalyanaraman, Balaraman; Cheng, Gang; Hardy, Micael; Ouari, Olivier; Lopez, Marcos; Joseph, Joy; Zielonka, Jacek; Dwinell, Michael B

2018-04-01

The present review is a sequel to the previous review on cancer metabolism published in this journal. This review focuses on the selective antiproliferative and cytotoxic effects of mitochondria-targeted therapeutics (MTTs) in cancer cells. Emerging research reveals a key role of mitochondrial respiration on tumor proliferation. Previously, a mitochondria-targeted nitroxide was shown to selectively inhibit colon cancer cell proliferation at submicromolar levels. This review is centered on the therapeutic use of MTTs and their bioenergetic profiling in cancer cells. Triphenylphosphonium cation conjugated to a parent molecule (e.g., vitamin-E or chromanol, ubiquinone, and metformin) via a linker alkyl chain is considered an MTT. MTTs selectively and potently inhibit proliferation of cancer cells and, in some cases, induce cytotoxicity. MTTs inhibit mitochondrial complex I activity and induce mitochondrial stress in cancer cells through generation of reactive oxygen species. MTTs in combination with glycolytic inhibitors synergistically inhibit tumor cell proliferation. This review discusses how signaling molecules traditionally linked to tumor cell proliferation affect tumor metabolism and bioenergetics (glycolysis, TCA cycle, and glutaminolysis). Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma

PubMed Central

Tsoli, Maria; Liu, Jie; Franshaw, Laura; Shen, Han; Cheng, Cecilia; Jung, MoonSun; Joshi, Swapna; Ehteda, Anahid; Khan, Aaminah; Montero-Carcabosso, Angel; Dilda, Pierre J.; Hogg, Philip; Ziegler, David S.

2018-01-01

Diffuse Intrinsic Pontine Gliomas (DIPG) are the most devastating of all pediatric brain tumors. They mostly affect young children and, as there are no effective treatments, almost all patients with DIPG will die of their tumor within 12 months of diagnosis. A key feature of this devastating tumor is its intrinsic resistance to all clinically available therapies. It has been shown that glioma development is associated with metabolic reprogramming, redox state disruption and resistance to apoptotic pathways. The mitochondrion is an attractive target as a key organelle that facilitates these critical processes. PENAO is a novel anti-cancer compound that targets mitochondrial function by inhibiting adenine nucleotide translocase (ANT). Here we found that DIPG neurosphere cultures express high levels of ANT2 protein and are sensitive to the mitochondrial inhibitor PENAO through oxidative stress, while its apoptotic effects were found to be further enhanced upon co-treatment with mTOR inhibitor temsirolimus. This combination therapy was found to act through inhibition of PI3K/AKT/mTOR pathway, HSP90 and activation of AMPK. In vivo experiments employing an orthotopic model of DIPG showed a marginal anti-tumour effect likely due to poor penetration of the inhibitors into the brain. Further testing of this anti-DIPG strategy with compounds that penetrate the BBB is warranted. PMID:29484131

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death

PubMed Central

Wallace, Martina; Buren, Caodu; Martyniuk, Kelly; Andreyev, Alexander Y.; Li, Edward; Fields, Jerel A.; Cordes, Thekla; Reynolds, Ian J.; Bloodgood, Brenda L.; Metallo, Christian M.

2017-01-01

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity. PMID:28254829

Postgenomic strategies in antibacterial drug discovery.

PubMed

Brötz-Oesterhelt, Heike; Sass, Peter

2010-10-01

During the last decade the field of antibacterial drug discovery has changed in many aspects including bacterial organisms of primary interest, discovery strategies applied and pharmaceutical companies involved. Target-based high-throughput screening had been disappointingly unsuccessful for antibiotic research. Understanding of this lack of success has increased substantially and the lessons learned refer to characteristics of targets, screening libraries and screening strategies. The 'genomics' approach was replaced by a diverse array of discovery strategies, for example, searching for new natural product leads among previously abandoned compounds or new microbial sources, screening for synthetic inhibitors by targeted approaches including structure-based design and analyses of focused libraries and designing resistance-breaking properties into antibiotics of established classes. Furthermore, alternative treatment options are being pursued including anti-virulence strategies and immunotherapeutic approaches. This article summarizes the lessons learned from the genomics era and describes discovery strategies resulting from that knowledge.

Systems Pharmacology-Based Discovery of Natural Products for Precision Oncology Through Targeting Cancer Mutated Genes.

PubMed

Fang, J; Cai, C; Wang, Q; Lin, P; Zhao, Z; Cheng, F

2017-03-01

Massive cancer genomics data have facilitated the rapid revolution of a novel oncology drug discovery paradigm through targeting clinically relevant driver genes or mutations for the development of precision oncology. Natural products with polypharmacological profiles have been demonstrated as promising agents for the development of novel cancer therapies. In this study, we developed an integrated systems pharmacology framework that facilitated identifying potential natural products that target mutated genes across 15 cancer types or subtypes in the realm of precision medicine. High performance was achieved for our systems pharmacology framework. In case studies, we computationally identified novel anticancer indications for several US Food and Drug Administration-approved or clinically investigational natural products (e.g., resveratrol, quercetin, genistein, and fisetin) through targeting significantly mutated genes in multiple cancer types. In summary, this study provides a powerful tool for the development of molecularly targeted cancer therapies through targeting the clinically actionable alterations by exploiting the systems pharmacology of natural products. © 2017 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Mitochondrial quality control: Easy come, easy go

PubMed Central

Stotland, Aleksandr; Gottlieb, Roberta A.

2015-01-01

“Friends come and go but enemies accumulate.”Arthur Bloch Mitochondrial networks in eukaryotic cells are maintained via regular cycles of degradation and biogenesis. These complex processes function in concert with one another to eliminate dysfunctional mitochondria in a specific and targeted manner and coordinate the biogenesis of new organelles. This review covers the two aspects of mitochondrial turnover, focusing on the main pathways and mechanisms involved. The review also summarizes the current methods and techniques for analyzing mitochondrial turnover in vivo and in vitro, from the whole animal proteome level to the level of single organelle. PMID:25596427

The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

PubMed Central

Liao, Yajin; Dong, Yuan; Cheng, Jinbo

2017-01-01

The mitochondrial calcium uniporter (MCU)—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP); however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders. PMID:28208618

Disposable Amperometric Polymerase Chain Reaction-Free Biosensor for Direct Detection of Adulteration with Horsemeat in Raw Lysates Targeting Mitochondrial DNA.

PubMed

Ruiz-Valdepeñas Montiel, Víctor; Gutiérrez, María L; Torrente-Rodríguez, Rebeca M; Povedano, Eloy; Vargas, Eva; Reviejo, Á Julio; Linacero, Rosario; Gallego, Francisco J; Campuzano, Susana; Pingarrón, José M

2017-09-05

A novel electrochemical disposable nucleic acid biosensor for simple, rapid, and specific detection of adulterations with horsemeat is reported in this work. The biosensing platform involves immobilization of a 40-mer RNA probe specific for a characteristic fragment of the mitochondrial DNA D-loop region of horse onto the surface of magnetic microcarriers. In addition, signal amplification was accomplished by using a commercial antibody specific to RNA/DNA duplexes and a bacterial protein conjugated with a horseradish peroxidase homopolymer (ProtA-HRP40). Amperometric detection at -0.20 V vs Ag pseudoreference electrode was carried out at disposable screen-printed carbon electrodes. The methodology achieved a limit of detection (LOD) of 0.12 pM (3.0 attomoles) for the synthetic target and showed ability to discriminate between raw beef and horsemeat using just 50 ng of total extracted mitochondrial DNA (∼16 660 bp in length) without previous fragmentation. The biosensor also allowed discrimination between 100% raw beef and beef meat samples spiked with only 0.5% (w/w) horse meat (levels established by the European Commission) using raw mitochondrial lysates without DNA extraction or polymerase chain reaction (PCR) amplification in just 75 min. These interesting features made the developed methodology an extremely interesting tool for beef meat screening, and it can be easily adapted to the determination of other meat adulterations by selection of the appropriate specific fragments of the mitochondrial DNA region and capture probes.

Applications of chemogenomic library screening in drug discovery.

PubMed

Jones, Lyn H; Bunnage, Mark E

2017-04-01

The allure of phenotypic screening, combined with the industry preference for target-based approaches, has prompted the development of innovative chemical biology technologies that facilitate the identification of new therapeutic targets for accelerated drug discovery. A chemogenomic library is a collection of selective small-molecule pharmacological agents, and a hit from such a set in a phenotypic screen suggests that the annotated target or targets of that pharmacological agent may be involved in perturbing the observable phenotype. In this Review, we describe opportunities for chemogenomic screening to considerably expedite the conversion of phenotypic screening projects into target-based drug discovery approaches. Other applications are explored, including drug repositioning, predictive toxicology and the discovery of novel pharmacological modalities.

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Discovery and characterization of olokizumab: a humanized antibody targeting interleukin-6 and neutralizing gp130-signaling.

PubMed

Shaw, Stevan; Bourne, Tim; Meier, Chris; Carrington, Bruce; Gelinas, Rich; Henry, Alistair; Popplewell, Andrew; Adams, Ralph; Baker, Terry; Rapecki, Steve; Marshall, Diane; Moore, Adrian; Neale, Helen; Lawson, Alastair

2014-01-01

Interleukin-6 (IL-6) is a critical regulator of the immune system and has been widely implicated in autoimmune disease. Here, we describe the discovery and characterization of olokizumab, a humanized antibody to IL-6. Data from structural biology, cell biology and primate pharmacology demonstrate the therapeutic potential of targeting IL-6 at "Site 3", blocking the interaction with the signaling co-receptor gp130.

Discovery and molecular characterization of a Bcl-2–regulated cell death pathway in schistosomes

PubMed Central

Lee, Erinna F.; Clarke, Oliver B.; Evangelista, Marco; Feng, Zhiping; Speed, Terence P.; Tchoubrieva, Elissaveta B.; Strasser, Andreas; Kalinna, Bernd H.; Colman, Peter M.; Fairlie, W. Douglas

2011-01-01

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2–regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2–like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with “BH3 mimetic” drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment. PMID:21444803

Overcoming Multidrug Resistance through the GLUT1-Mediated and Enzyme-Triggered Mitochondrial Targeting Conjugate with Redox-Sensitive Paclitaxel Release.

PubMed

Ma, Pengkai; Chen, Jianhua; Bi, Xinning; Li, Zhihui; Gao, Xing; Li, Hongpin; Zhu, Hongyu; Huang, Yunfang; Qi, Jing; Zhang, Yujie

2018-04-18

Multidrug resistance (MDR) is thought to be the major obstacle leading to the failure of paclitaxel (PTX) chemotherapy. To solve this problem, a glucose transporter-mediated and matrix metalloproteinase 2 (MMP2)-triggered mitochondrion-targeting conjugate [glucose-polyethylene glycol (PEG)-peptide-triphenylphosponium-polyamidoamine (PAMAM)-PTX] composed of a PAMAM dendrimer and enzymatic detachable glucose-PEG was constructed for mitochondrial delivery of PTX. The conjugate was characterized by a 30 nm sphere particle, MMP2-sensitive PEG outer layer detachment from PAMAM, and glutathione (GSH)-sensitive PTX release. It showed higher cellular uptake both in glucose transporter 1 (GLUT1) overexpressing MCF-7/MDR monolayer cell (2D) and multicellular tumor spheroids (3D). The subcellular location study showed that it could specifically accumulate in the mitochondria. Moreover, it exhibited higher cytotoxicity against MCF-7/MDR cells, which significantly reverse the MDR of MCF-7/MDR cells. The MDR reverse might be caused by reducing the ATP content through destroying the mitochondrial membrane as well as by down-regulating P-gp expression. In vivo imaging and tissue distribution indicated more conjugate accumulated in the tumor of the tumor-bearing mice model. Consequently, the conjugate showed better tumor inhibition rate and lower body weight loss, which demonstrated that it possessed high efficiency and low toxicity. This study provides glucose-mediated GLUT targeting, MMP2-responsive PEG detachment, triphenylphosponium-mediated mitochondria targeting, and a GSH-sensitive intracellular drug release conjugate that has the potential to be exploited for overcoming MDR of PTX.

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death.

PubMed

Divakaruni, Ajit S; Wallace, Martina; Buren, Caodu; Martyniuk, Kelly; Andreyev, Alexander Y; Li, Edward; Fields, Jerel A; Cordes, Thekla; Reynolds, Ian J; Bloodgood, Brenda L; Raymond, Lynn A; Metallo, Christian M; Murphy, Anne N

2017-04-03

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity. © 2017 Divakaruni et al.

Parkin suppresses Drp1-independent mitochondrial division.

PubMed

Roy, Madhuparna; Itoh, Kie; Iijima, Miho; Sesaki, Hiromi

2016-07-01

The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson's disease-associated protein-parkin, which biochemically and genetically interacts with Drp1-in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division. Copyright © 2016 Elsevier Inc. All rights reserved.

Small mitochondria-targeting molecules as anti-cancer agents

PubMed Central

Wang, Feng; Ogasawara, Marcia A.; Huang, Peng

2009-01-01

Alterations in mitochondrial structure and functions have long been observed in cancer cells. Targeting mitochondria as a cancer therapeutic strategy has gained momentum in the recent years. The signaling pathways that govern mitochondrial function, apoptosis and molecules that affect mitochondrial integrity and cell viability have been important topics of the recent review in the literature. In this article, we first briefly summarize the rationale and biological basis for developing mitochondrial-targeted compounds as potential anticancer agents, and then provide key examples of small molecules that either directly impact mitochondria or functionally affect the metabolic alterations in cancer cells with mitochondrial dysfunction. The main focus is on the small molecular weight compounds with potential applications in cancer treatment. We also summarize information on the drug developmental stages of the key mitochondria-targeted compounds and their clinical trial status. The advantages and potential shortcomings of targeting the mitochondria for cancer treatment are also discussed. PMID:19995573

The Origin of Mitochondrial Cristae from Alphaproteobacteria.

PubMed

Muñoz-Gómez, Sergio A; Wideman, Jeremy G; Roger, Andrew J; Slamovits, Claudio H

2017-04-01

Mitochondria are the respiratory organelles of eukaryotes and their evolutionary history is deeply intertwined with that of eukaryotes. The compartmentalization of respiration in mitochondria occurs within cristae, whose evolutionary origin has remained unclear. Recent discoveries, however, have revived the old notion that mitochondrial cristae could have had a pre-endosymbiotic origin. Mitochondrial cristae are likely homologous to the intracytoplasmic membranes (ICMs) used by diverse alphaproteobacteria for harnessing energy. Because the Mitochondrial Contact site and Cristae Organizing System (MICOS) that controls the development of cristae evolved from a simplified version that is phylogenetically restricted to Alphaproteobacteria (alphaMICOS), ICMs most probably transformed into cristae during the endosymbiotic origin of mitochondria. This inference is supported by the sequence and structural similarities between MICOS and alphaMICOS, and the expression pattern and cellular localization of alphaMICOS. Given that cristae and ICMs develop similarly, alphaMICOS likely functions analogously to mitochondrial MICOS by culminating ICM development with the creation of tubular connections and membrane contact sites at the alphaproteobacterial envelope. Mitochondria thus inherited a pre-existing ultrastructure adapted to efficient energy transduction from their alphaproteobacterial ancestors. The widespread nature of purple bacteria among alphaproteobacteria raises the possibility that cristae evolved from photosynthetic ICMs. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

PubMed

Bahaji, Abdellatif; Ovecka, Miroslav; Bárány, Ivett; Risueño, María Carmen; Muñoz, Francisco José; Baroja-Fernández, Edurne; Montero, Manuel; Li, Jun; Hidalgo, Maite; Sesma, María Teresa; Ezquer, Ignacio; Testillano, Pilar S; Pozueta-Romero, Javier

2011-04-01

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.

Arrayed antibody library technology for therapeutic biologic discovery.

PubMed

Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

2013-03-15

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

PubMed

Favaro, Elena; Ramachandran, Anassuya; McCormick, Robert; Gee, Harriet; Blancher, Christine; Crosby, Meredith; Devlin, Cecilia; Blick, Christopher; Buffa, Francesca; Li, Ji-Liang; Vojnovic, Borivoj; Pires das Neves, Ricardo; Glazer, Peter; Iborra, Francisco; Ivan, Mircea; Ragoussis, Jiannis; Harris, Adrian L

2010-04-26

Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

A mitochondrial-targeted coenzyme q analog prevents weight gain and ameliorates hepatic dysfunction in high-fat-fed mice.

PubMed

Fink, Brian D; Herlein, Judith A; Guo, Deng Fu; Kulkarni, Chaitanya; Weidemann, Benjamin J; Yu, Liping; Grobe, Justin L; Rahmouni, Kamal; Kerns, Robert J; Sivitz, William I

2014-12-01

We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 µM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF- and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics. U.S. Government work not protected by U.S. copyright.

Bioinformatics in translational drug discovery.

PubMed

Wooller, Sarah K; Benstead-Hume, Graeme; Chen, Xiangrong; Ali, Yusuf; Pearl, Frances M G

2017-08-31

Bioinformatics approaches are becoming ever more essential in translational drug discovery both in academia and within the pharmaceutical industry. Computational exploitation of the increasing volumes of data generated during all phases of drug discovery is enabling key challenges of the process to be addressed. Here, we highlight some of the areas in which bioinformatics resources and methods are being developed to support the drug discovery pipeline. These include the creation of large data warehouses, bioinformatics algorithms to analyse 'big data' that identify novel drug targets and/or biomarkers, programs to assess the tractability of targets, and prediction of repositioning opportunities that use licensed drugs to treat additional indications. © 2017 The Author(s).

Role of Mitochondrial Homeostasis and Dynamics in Alzheimer’s Disease

PubMed Central

Selfridge, J. Eva; Lezi, E; Lu, Jianghua; Swerdlow, Russell H.

2012-01-01

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that affects a staggering percentage of the aging population and causes memory loss and cognitive decline. Mitochondrial abnormalities can be observed systemically and in brains of patients suffering from AD, and may account for part of the disease phenotype. In this review, we summarize some of the key findings that indicate mitochondrial dysfunction is present in AD-affected subjects, including cytochrome oxidase deficiency, endophenotype data, and altered mitochondrial morphology. Special attention is given to recently described perturbations in mitochondrial autophagy, fission-fusion dynamics, and biogenesis. We also briefly discuss how mitochondrial dysfunction may influence amyloidosis in Alzheimer’s disease, why mitochondria are a valid therapeutic target, and strategies for addressing AD-specific mitochondrial dysfunction. PMID:22266017

MitoBreak: the mitochondrial DNA breakpoints database.

PubMed

Damas, Joana; Carneiro, João; Amorim, António; Pereira, Filipe

2014-01-01

Mitochondrial DNA (mtDNA) rearrangements are key events in the development of many diseases. Investigations of mtDNA regions affected by rearrangements (i.e. breakpoints) can lead to important discoveries about rearrangement mechanisms and can offer important clues about the causes of mitochondrial diseases. Here, we present the mitochondrial DNA breakpoints database (MitoBreak; http://mitobreak.portugene.com), a free, web-accessible comprehensive list of breakpoints from three classes of somatic mtDNA rearrangements: circular deleted (deletions), circular partially duplicated (duplications) and linear mtDNAs. Currently, MitoBreak contains >1400 mtDNA rearrangements from seven species (Homo sapiens, Mus musculus, Rattus norvegicus, Macaca mulatta, Drosophila melanogaster, Caenorhabditis elegans and Podospora anserina) and their associated phenotypic information collected from nearly 400 publications. The database allows researchers to perform multiple types of data analyses through user-friendly interfaces with full or partial datasets. It also permits the download of curated data and the submission of new mtDNA rearrangements. For each reported case, MitoBreak also documents the precise breakpoint positions, junction sequences, disease or associated symptoms and links to the related publications, providing a useful resource to study the causes and consequences of mtDNA structural alterations.

Mitochondria and Mitochondrial Cascades in Alzheimer’s Disease

PubMed Central

Swerdlow, Russell H.

2017-01-01

Decades of research indicate mitochondria from Alzheimer’s disease (AD) patients differ from those of non-AD individuals. Initial studies revealed structural differences, and subsequent studies showed functional deficits. Observations of structure and function changes prompted investigators to consider the consequences, significance, and causes of AD-related mitochondrial dysfunction. Currently, extensive research argues mitochondria may mediate, drive, or contribute to a variety of AD pathologies. The perceived significance of these mitochondrial changes continues to grow, and many currently believe AD mitochondrial dysfunction represents a reasonable therapeutic target. Debate continues over the origin of AD mitochondrial changes. Some argue amyloid-β (Aβ) induces AD mitochondrial dysfunction, a view that does not challenge the amyloid cascade hypothesis and that may in fact help explain that hypothesis. Alternatively, data indicate mitochondrial dysfunction exists independent of Aβ, potentially lies upstream of Aβ deposition, and suggest a primary mitochondrial cascade hypothesis that assumes mitochondrial pathology hierarchically supersedes Aβ pathology. Mitochondria, therefore, appear at least to mediate or possibly even initiate pathologic molecular cascades in AD. This review considers studies and data that inform this area of AD research. PMID:29036828

Novel role of NOX in supporting aerobic glycolysis in cancer cells with mitochondrial dysfunction and as a potential target for cancer therapy.

PubMed

Lu, Weiqin; Hu, Yumin; Chen, Gang; Chen, Zhao; Zhang, Hui; Wang, Feng; Feng, Li; Pelicano, Helene; Wang, Hua; Keating, Michael J; Liu, Jinsong; McKeehan, Wallace; Wang, Huamin; Luo, Yongde; Huang, Peng

2012-01-01

Elevated aerobic glycolysis in cancer cells (the Warburg effect) may be attributed to respiration injury or mitochondrial dysfunction, but the underlying mechanisms and therapeutic significance remain elusive. Here we report that induction of mitochondrial respiratory defect by tetracycline-controlled expression of a dominant negative form of DNA polymerase γ causes a metabolic shift from oxidative phosphorylation to glycolysis and increases ROS generation. We show that upregulation of NOX is critical to support the elevated glycolysis by providing additional NAD+. The upregulation of NOX is also consistently observed in cancer cells with compromised mitochondria due to the activation of oncogenic Ras or loss of p53, and in primary pancreatic cancer tissues. Suppression of NOX by chemical inhibition or genetic knockdown of gene expression selectively impacts cancer cells with mitochondrial dysfunction, leading to a decrease in cellular glycolysis, a loss of cell viability, and inhibition of cancer growth in vivo. Our study reveals a previously unrecognized function of NOX in cancer metabolism and suggests that NOX is a potential novel target for cancer treatment.

Rolling Circle Amplification of Complete Nematode Mitochondrial Genomes

PubMed Central

Tang, Sha; Hyman, Bradley C.

2005-01-01

To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle replication strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis of the rhabditid nematode Caenorhabditis elegans and a mermithid nematode, Thaumamermis cosgrovei. These taxa, representing the two major nematode classes Chromodorea and Enoplea, maintain mitochondrial genomes of 13.8 kb and 20.0 kb, respectively. Efficient amplifications were conducted on template DNA isolated from individual or pooled nematodes that were alive or stored at -80°C. Unexpectedly, these experiments revealed that multiple T. cosgrovei mitochondrial DNA haplotypes are maintained in our local population. Rolling circle amplification products can be used as templates for standard PCR reactions with specific primers that target mitochondrial genes or for direct DNA sequencing. PMID:19262866

Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia.

PubMed

Crouser, Elliott D; Julian, Mark W; Huff, Jennifer E; Joshi, Mandar S; Bauer, John A; Gadd, Martha E; Wewers, Mark D; Pfeiffer, Douglas R

2004-02-01

concentrations of Bax and ceramide. These studies confirm that liver mitochondria are early targets of injury during endotoxemia and that inner and outer mitochondrial membrane damage occurs through different mechanisms. Inner mitochondrial membrane damage appears to relate to the mitochondrial permeability transition, whereas outer mitochondrial membrane damage can occur independent of the mitochondrial permeability transition. Preliminary evidence suggests that Bax may participate in lipopolysaccharide-induced outer mitochondrial membrane damage, but further investigations are needed to confirm this.

Mind the gap! The mitochondrial control region and its power as a phylogenetic marker in echinoids.

PubMed

Bronstein, Omri; Kroh, Andreas; Haring, Elisabeth

2018-05-30

In Metazoa, mitochondrial markers are the most commonly used targets for inferring species-level molecular phylogenies due to their extremely low rate of recombination, maternal inheritance, ease of use and fast substitution rate in comparison to nuclear DNA. The mitochondrial control region (CR) is the main non-coding area of the mitochondrial genome and contains the mitochondrial origin of replication and transcription. While sequences of the cytochrome oxidase subunit 1 (COI) and 16S rRNA genes are the prime mitochondrial markers in phylogenetic studies, the highly variable CR is typically ignored and not targeted in such analyses. However, the higher substitution rate of the CR can be harnessed to infer the phylogeny of closely related species, and the use of a non-coding region alleviates biases resulting from both directional and purifying selection. Additionally, complete mitochondrial genome assemblies utilizing next generation sequencing (NGS) data often show exceptionally low coverage at specific regions, including the CR. This can only be resolved by targeted sequencing of this region. Here we provide novel sequence data for the echinoid mitochondrial control region in over 40 species across the echinoid phylogenetic tree. We demonstrate the advantages of directly targeting the CR and adjacent tRNAs to facilitate complementing low coverage NGS data from complete mitochondrial genome assemblies. Finally, we test the performance of this region as a phylogenetic marker both in the lab and in phylogenetic analyses, and demonstrate its superior performance over the other available mitochondrial markers in echinoids. Our target region of the mitochondrial CR (1) facilitates the first thorough investigation of this region across a wide range of echinoid taxa, (2) provides a tool for complementing missing data in NGS experiments, and (3) identifies the CR as a powerful, novel marker for phylogenetic inference in echinoids due to its high variability, lack of

Modulation of Epigenetic Targets for Anticancer Therapy: Clinicopathological Relevance, Structural Data and Drug Discovery Perspectives

PubMed Central

Andreol, Federico; Barbosa, Arménio Jorge Moura; Daniele Parenti, Marco; Rio, Alberto Del

2013-01-01

Research on cancer epigenetics has flourished in the last decade. Nevertheless growing evidence point on the importance to understand the mechanisms by which epigenetic changes regulate the genesis and progression of cancer growth. Several epigenetic targets have been discovered and are currently under validation for new anticancer therapies. Drug discovery approaches aiming to target these epigenetic enzymes with small-molecules inhibitors have produced the first pre-clinical and clinical outcomes and many other compounds are now entering the pipeline as new candidate epidrugs. The most studied targets can be ascribed to histone deacetylases and DNA methyltransferases, although several other classes of enzymes are able to operate post-translational modifications to histone tails are also likely to represent new frontiers for therapeutic interventions. By acknowledging that the field of cancer epigenetics is evolving with an impressive rate of new findings, with this review we aim to provide a current overview of pre-clinical applications of small-molecules for cancer pathologies, combining them with the current knowledge of epigenetic targets in terms of available structural data and drug design perspectives. PMID:23016851

A Single Missense Mutation in 77% of Prostate Cancer Bone Metastases: Novel Opportunity for Genetic Biomarker and Novel Therapeutic Mitochondrial Target

DTIC Science & Technology

2017-10-01

Biomarker and Novel Therapeutic Mitochondrial Target PRINCIPAL INVESTIGATOR: John A. Petros, MD RECIPIENT: Emory University Atlanta GA 30322 REPORT ...DATE: October 2017 TYPE OF REPORT : Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012...and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

PubMed Central

Sideris, Dionisia P.; Petrakis, Nikos; Katrakili, Nitsa; Mikropoulou, Despina; Gallo, Angelo; Ciofi-Baffoni, Simone; Banci, Lucia; Bertini, Ivano

2009-01-01

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. PMID:20026652

Targeting mitochondria with small molecules: the preparation of MitoB and MitoP as exomarkers of mitochondrial hydrogen peroxide.

PubMed

Cairns, Andrew G; McQuaker, Stephen J; Murphy, Michael P; Hartley, Richard C

2015-01-01

Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP-conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow temporal control, and be applied in a reversible dose-dependent fashion. An example is the detection and quantification of hydrogen peroxide in mitochondria of whole living organisms by MitoB. Hydrogen peroxide produced within the mitochondrial matrix is involved in signalling and implicated in the oxidative damage associated with aging and a wide range of age-associated conditions including cardiovascular disease, neurodegeneration, and cancer. MitoB accumulates in mitochondria and is converted into the exomarker, MitoP, by hydrogen peroxide in the mitochondrial matrix. The hydrogen peroxide concentration is determined from the ratio of MitoP to MitoB after a period of incubation, and this ratio is determined by mass spectrometry using d15-MitoP and d15-MitoB as standard. Here we describe the synthesis of MitoB and MitoP and the deuterated standards necessary for this method of quantification.

Mitochondrial Redox Signaling and Tumor Progression.

PubMed

Chen, Yuxin; Zhang, Haiqing; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

2016-03-25

Cancer cell can reprogram their energy production by switching mitochondrial oxidative phosphorylation to glycolysis. However, mitochondria play multiple roles in cancer cells, including redox regulation, reactive oxygen species (ROS) generation, and apoptotic signaling. Moreover, these mitochondrial roles are integrated via multiple interconnected metabolic and redox sensitive pathways. Interestingly, mitochondrial redox proteins biphasically regulate tumor progression depending on cellular ROS levels. Low level of ROS functions as signaling messengers promoting cancer cell proliferation and cancer invasion. However, anti-cancer drug-initiated stress signaling could induce excessive ROS, which is detrimental to cancer cells. Mitochondrial redox proteins could scavenger basal ROS and function as "tumor suppressors" or prevent excessive ROS to act as "tumor promoter". Paradoxically, excessive ROS often also induce DNA mutations and/or promotes tumor metastasis at various stages of cancer progression. Targeting redox-sensitive pathways and transcriptional factors in the appropriate context offers great promise for cancer prevention and therapy. However, the therapeutics should be cancer-type and stage-dependent.

False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform.

PubMed

Strickland, Erin C; Geer, M Ariel; Hong, Jiyong; Fitzgerald, Michael C

2014-01-01

Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2% and <0.8% are calculated for SPROX experiments using Q-TOF and Orbitrap mass spectrometer systems, respectively. Our results indicate that the false-positive rate is largely determined by random errors associated with the mass spectral analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

Exercise training improves vascular mitochondrial function

PubMed Central

Park, Song-Young; Rossman, Matthew J.; Gifford, Jayson R.; Bharath, Leena P.; Bauersachs, Johann; Richardson, Russell S.; Abel, E. Dale; Symons, J. David

2016-01-01

Exercise training is recognized to improve cardiac and skeletal muscle mitochondrial respiratory capacity; however, the impact of chronic exercise on vascular mitochondrial respiratory function is unknown. We hypothesized that exercise training concomitantly increases both vascular mitochondrial respiratory capacity and vascular function. Arteries from both sedentary (SED) and swim-trained (EX, 5 wk) mice were compared in terms of mitochondrial respiratory function, mitochondrial content, markers of mitochondrial biogenesis, redox balance, nitric oxide (NO) signaling, and vessel function. Mitochondrial complex I and complex I + II state 3 respiration and the respiratory control ratio (complex I + II state 3 respiration/complex I state 2 respiration) were greater in vessels from EX relative to SED mice, despite similar levels of arterial citrate synthase activity and mitochondrial DNA content. Furthermore, compared with the SED mice, arteries from EX mice displayed elevated transcript levels of peroxisome proliferative activated receptor-γ coactivator-1α and the downstream targets cytochrome c oxidase subunit IV isoform 1, isocitrate dehydrogenase (Idh) 2, and Idh3a, increased manganese superoxide dismutase protein expression, increased endothelial NO synthase phosphorylation (Ser1177), and suppressed reactive oxygen species generation (all P < 0.05). Although there were no differences in EX and SED mice concerning endothelium-dependent and endothelium-independent vasorelaxation, phenylephrine-induced vasocontraction was blunted in vessels from EX compared with SED mice, and this effect was normalized by NOS inhibition. These training-induced increases in vascular mitochondrial respiratory capacity and evidence of improved redox balance, which may, at least in part, be attributable to elevated NO bioavailability, have the potential to protect against age- and disease-related challenges to arterial function. PMID:26825520

Mitochondrial fragmentation in excitotoxicity requires ROCK activation.

PubMed

Martorell-Riera, Alejandro; Segarra-Mondejar, Marc; Reina, Manuel; Martínez-Estrada, Ofelia M; Soriano, Francesc X

2015-01-01

Mitochondria morphology constantly changes through fission and fusion processes that regulate mitochondrial function, and it therefore plays a prominent role in cellular homeostasis. Cell death progression is associated with mitochondrial fission. Fission is mediated by the mainly cytoplasmic Drp1, which is activated by different post-translational modifications and recruited to mitochondria to perform its function. Our research and other studies have shown that in the early moments of excitotoxic insult Drp1 must be nitrosylated to mediate mitochondrial fragmentation in neurons. Nonetheless, mitochondrial fission is a multistep process in which filamentous actin assembly/disassembly and myosin-mediated mitochondrial constriction play prominent roles. Here we establish that in addition to nitric oxide production, excitotoxicity-induced mitochondrial fragmentation also requires activation of the actomyosin regulator ROCK. Although ROCK1 has been shown to phosphorylate and activate Drp1, experiments using phosphor-mutant forms of Drp1 in primary cortical neurons indicate that in excitotoxic conditions, ROCK does not act directly on Drp1 to mediate fission, but may act on the actomyosin complex. Thus, these data indicate that a wider range of signaling pathways than those that target Drp1 are amenable to be inhibited to prevent mitochondrial fragmentation as therapeutic option.

A Framework of Knowledge Integration and Discovery for Supporting Pharmacogenomics Target Predication of Adverse Drug Events: A Case Study of Drug-Induced Long QT Syndrome.

PubMed

Jiang, Guoqian; Wang, Chen; Zhu, Qian; Chute, Christopher G

2013-01-01

Knowledge-driven text mining is becoming an important research area for identifying pharmacogenomics target genes. However, few of such studies have been focused on the pharmacogenomics targets of adverse drug events (ADEs). The objective of the present study is to build a framework of knowledge integration and discovery that aims to support pharmacogenomics target predication of ADEs. We integrate a semantically annotated literature corpus Semantic MEDLINE with a semantically coded ADE knowledgebase known as ADEpedia using a semantic web based framework. We developed a knowledge discovery approach combining a network analysis of a protein-protein interaction (PPI) network and a gene functional classification approach. We performed a case study of drug-induced long QT syndrome for demonstrating the usefulness of the framework in predicting potential pharmacogenomics targets of ADEs.

Mitochondrial dysfunctions in Parkinson's disease.

PubMed

Gautier, C A; Corti, O; Brice, A

2014-05-01

Neurodegenerative disorders (ND) include a wide spectrum of diseases characterized by progressive neuronal dysfunctions or degeneration. With an estimated cost of 135 billion € in 2010 in the European Union (Olesen et al., 2012), they put an enormous economic as well as social burden on modern societies. Hence, they have been the subject of a huge amount of research for the last fifty years. For many of these diseases, our understanding of their profound causes is incomplete and this hinders the discovery of efficient therapies. ND form a highly heterogeneous group of diseases affecting various neuronal subpopulations reflecting different origins and different pathological mechanisms. However, some common themes in the physiopathology of these disorders are emerging. There is growing evidence that mitochondrial dysfunctions play a pivotal role at some point in the course of neurodegeneration. In some cases (e.g. Alzheimer's disease, amyotrophic lateral sclerosis), impairment of mitochondrial functions probably occurs late in the course of the disease. In a subset of ND, current evidence suggests that mitochondrial dysfunctions play a more seminal role in neuronal demise. Parkinson's disease (PD) presents one of the strongest cases based in part on post-mortem studies that have shown mitochondrial impairment (e.g. reduced complex I activity) and oxidative damage in idiopathic PD brains. The occurrence of PD is largely sporadic, but clinical syndromes resembling sporadic PD have been linked to specific environmental insults or to mutations in at least 5 distinct genes (α-synuclein, parkin, DJ-1, PINK1 and LRRK2). It is postulated that the elucidation of the pathogenic mechanisms underlying the selective dopaminergic degeneration in familial and environmental Parkinsonism should provide important clues to the pathogenic mechanisms responsible for idiopathic PD. Hence, numerous cellular and animal models of the disease have been generated that mimic these

Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting.

PubMed

Monfregola, Jlenia; Cevenini, Armando; Terracciano, Antonio; van Vlies, Naomi; Arbucci, Salvatore; Wanders, Ronald J A; D'Urso, Michele; Vaz, Frédéric M; Ursini, Matilde Valeria

2005-09-01

epsilon-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Fe(++) and 2-oxoglutarate (2OG) dependent, catalyzing the first of four enzymatic reactions of the highly conserved carnitine biosynthetic pathway. Otherwise from all the other enzymes of carnitine biosynthesis, TMLH was found to be associated to the mitochondrial fraction. We here report molecular cloning of two alternative spliced forms of TMLH, which appear ubiquitously expressed in human adult and fetal tissues. The deduced proteins are designated TMLH-a and TMLH-b, and contain 421 and 399 amino acids, respectively. They share the first N-terminal 332 amino acids, including a mitochondrial targeting signal, but diverge at the C-terminal end. TMLH-a and TMLH-b exogenous expression in COS-1 cells shows that the first 15 amino acids are necessary and sufficient for mitochondrial import. Furthermore, comparative evolutionary analysis of the C-terminal portion of TMLH-a identifies a conserved domain characterized by a key triad of residues, His242-Glu244-His389 predicted to bind 2OG end. This sequence is conserved in the TMLH enzyme from all species but is partially substituted by a unique sequence in the TMLH-b variant. Indeed, TMLH-b is not functional by itself as well as a TMLH-H389L mutant produced by site directed mutagenesis. As great interest, we found that TMLH-b and TMLH-H389L, individually co-expressed with TMLH-a in COS-1 cells, negatively affect TMLH activity. Therefore, our studies on the TMLH alternative form provide relevant novel information, first that the C-terminal region of TMLH contains the main determinants for its enzymatic activity including a key H389 residue, and second that TMLH-b could act as a crucial physiological negative regulator of TMLH. Copyright 2005 Wiley-Liss, Inc.

Antisense antibiotics: a brief review of novel target discovery and delivery.

PubMed

Bai, Hui; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Meng, Jingru; Luo, Xiaoxing

2010-06-01

The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.

Toxoplasma gondii infection confers resistance against BimS-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic Bax.

PubMed

Hippe, Diana; Weber, Arnim; Zhou, Liying; Chang, Donald C; Häcker, Georg; Lüder, Carsten G K

2009-10-01

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein Bim(S) to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated Bim(S)-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of Bim(S) was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of Bim(S)-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.

Mitochondrial DNA disease—molecular insights and potential routes to a cure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, Oliver; Turnbull, Doug, E-mail: doug.turnbull@newcastle.ac.uk

2014-07-01

Mitochondrial DNA diseases are common neurological conditions caused by mutations in the mitochondrial genome or nuclear genes responsible for its maintenance. Current treatments for these disorders are focussed on the management of the symptoms, rather than the correction of biochemical defects caused by the mutation. This review focuses on the molecular effects of mutations, the symptoms they cause and current work focusing on the development of targeted treatments for mitochondrial DNA disease. - Highlights: • We discuss several common disease causing mtDNA mutations. • We highlight recent work linking pathogenicity to deletion size and heteroplasmy. • We discuss recent advancesmore » in the development of targeted mtDNA disease treatments.« less

Hyperuricemia induces endothelial dysfunction via mitochondrial Na+/Ca2+ exchanger-mediated mitochondrial calcium overload.

PubMed

Hong, Quan; Qi, Ka; Feng, Zhe; Huang, Zhiyong; Cui, Shaoyuan; Wang, Liyuan; Fu, Bo; Ding, Rui; Yang, Jurong; Chen, Xiangmei; Wu, Di

2012-05-01

Uric acid (UA) has proven to be a causal agent in endothelial dysfunction in which ROS production plays an important role. Calcium overload in mitochondria can promote the mitochondrial production of ROS. We hypothesize that calcium transduction in mitochondria contributes to UA-induced endothelial dysfunction. We first demonstrated that high concentrations of UA cause endothelial dysfunction, marked by a reduction in eNOS protein expression and NO release in vitro. We further found that a high concentration of UA increased levels of [Ca2+]mito, total intracellular ROS, H2O2, and mitochondrial O2·-, and Δψmito but not the [Ca2+]cyt level. When the mitochondrial calcium channels NCXmito and MCU were blocked by CGP-37157 and Ru360, respectively, the UA-induced increases in the levels of [Ca2+]mito and total intracellular ROS were significantly reduced. Mitochondrial levels of O2·- and Δψmito were reduced by inhibition of NCXmito but not of MCU. Moreover, inhibition of NCXmito, but not of MCU, blocked the UA-induced reductions in eNOS protein expression and NO release. The increased generation of mitochondrial O2·- induced by a high concentration of UA is triggered by mitochondrial calcium overload and ultimately leads to endothelial dysfunction. In this process, the activation of NCXmito is the major cause of the influx of calcium into mitochondria. Our results provide a new pathophysiological mechanism for UA-induced endothelial dysfunction and may offer a new therapeutic target for clinicians. Copyright © 2012 Elsevier Ltd. All rights reserved.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

PubMed

Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

2017-07-14

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Discovery of Anthelmintic Drug Targets and Drugs Using Chokepoints in Nematode Metabolic Pathways

PubMed Central

Taylor, Christina M.; Wang, Qi; Rosa, Bruce A.; Huang, Stanley Ching-Cheng; Powell, Kerrie; Schedl, Tim; Pearce, Edward J.; Abubucker, Sahar; Mitreva, Makedonka

2013-01-01

Parasitic roundworm infections plague more than 2 billion people (1/3 of humanity) and cause drastic losses in crops and livestock. New anthelmintic drugs are urgently needed as new drug resistance and environmental concerns arise. A “chokepoint reaction” is defined as a reaction that either consumes a unique substrate or produces a unique product. A chokepoint analysis provides a systematic method of identifying novel potential drug targets. Chokepoint enzymes were identified in the genomes of 10 nematode species, and the intersection and union of all chokepoint enzymes were found. By studying and experimentally testing available compounds known to target proteins orthologous to nematode chokepoint proteins in public databases, this study uncovers features of chokepoints that make them successful drug targets. Chemogenomic screening was performed on drug-like compounds from public drug databases to find existing compounds that target homologs of nematode chokepoints. The compounds were prioritized based on chemical properties frequently found in successful drugs and were experimentally tested using Caenorhabditis elegans. Several drugs that are already known anthelmintic drugs and novel candidate targets were identified. Seven of the compounds were tested in Caenorhabditis elegans and three yielded a detrimental phenotype. One of these three drug-like compounds, Perhexiline, also yielded a deleterious effect in Haemonchus contortus and Onchocerca lienalis, two nematodes with divergent forms of parasitism. Perhexiline, known to affect the fatty acid oxidation pathway in mammals, caused a reduction in oxygen consumption rates in C. elegans and genome-wide gene expression profiles provided an additional confirmation of its mode of action. Computational modeling of Perhexiline and its target provided structural insights regarding its binding mode and specificity. Our lists of prioritized drug targets and drug-like compounds have potential to expedite the discovery

Prevalence of somatic mitochondrial mutations and spatial distribution of mitochondria in non-small cell lung cancer.

PubMed

Kazdal, Daniel; Harms, Alexander; Endris, Volker; Penzel, Roland; Kriegsmann, Mark; Eichhorn, Florian; Muley, Thomas; Stenzinger, Albrecht; Pfarr, Nicole; Weichert, Wilko; Warth, Arne

2017-07-11

Mitochondria are considered relevant players in many tumour entities and first data indicate beneficial effects of mitochondria-targeted antioxidants in both cancer prevention and anticancer therapies. To further dissect the potential roles of mitochondria in NSCLC we comprehensively analysed somatic mitochondrial mutations, determined the spatial distribution of mitochondrial DNA within complete tumour sections and investigated the mitochondrial load in a large-scale approach. Whole mitochondrial genome sequencing of 26 matched tumour and non-neoplastic tissue samples extended by reviewing published data of 326 cases. Systematical stepwise real-time PCR quantification of mitochondrial DNA covering 16 whole surgical tumour sections. Immunohistochemical determination of the mitochondrial load in 171 adenocarcinoma and 145 squamous cell carcinoma. Our results demonstrate very low recurrences (max. 1.7%) and a broad distribution of 456 different somatic mitochondrial mutations. Large inter- and intra-tumour heterogeneity were seen for mitochondrial DNA copy numbers in conjunction with a correlation to the predominant histological growth pattern. Furthermore, tumour cells had significantly higher mitochondrial level compared to adjacent stroma, whereas differences between tumour entities were negligible. Non-evident somatic mitochondrial mutations and highly varying mitochondrial DNA level delineate challenges for the approach of mitochondria-targeted anticancer therapies in NSCLC.

Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase.

PubMed

Bridges, Hannah R; Sirviö, Ville A; Agip, Ahmed-Noor A; Hirst, Judy

2016-08-09

The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known. Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments. Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.

Application of multi-target phytotherapeutic concept in malaria drug discovery: a systems biology approach in biomarker identification.

PubMed

Tarkang, Protus Arrey; Appiah-Opong, Regina; Ofori, Michael F; Ayong, Lawrence S; Nyarko, Alexander K

2016-01-01

There is an urgent need for new anti-malaria drugs with broad therapeutic potential and novel mode of action, for effective treatment and to overcome emerging drug resistance. Plant-derived anti-malarials remain a significant source of bioactive molecules in this regard. The multicomponent formulation forms the basis of phytotherapy. Mechanistic reasons for the poly-pharmacological effects of plants constitute increased bioavailability, interference with cellular transport processes, activation of pro-drugs/deactivation of active compounds to inactive metabolites and action of synergistic partners at different points of the same signaling cascade. These effects are known as the multi-target concept. However, due to the intrinsic complexity of natural products-based drug discovery, there is need to rethink the approaches toward understanding their therapeutic effect. This review discusses the multi-target phytotherapeutic concept and its application in biomarker identification using the modified reverse pharmacology - systems biology approach. Considerations include the generation of a product library, high throughput screening (HTS) techniques for efficacy and interaction assessment, High Performance Liquid Chromatography (HPLC)-based anti-malarial profiling and animal pharmacology. This approach is an integrated interdisciplinary implementation of tailored technology platforms coupled to miniaturized biological assays, to track and characterize the multi-target bioactive components of botanicals as well as identify potential biomarkers. While preserving biodiversity, this will serve as a primary step towards the development of standardized phytomedicines, as well as facilitate lead discovery for chemical prioritization and downstream clinical development.

Targeted Modification of Mitochondrial ROS Production Converts High Glucose-Induced Cytotoxicity to Cytoprotection: Effects on Anesthetic Preconditioning.

PubMed

Sedlic, Filip; Muravyeva, Maria Y; Sepac, Ana; Sedlic, Marija; Williams, Anna Marie; Yang, Meiying; Bai, Xiaowen; Bosnjak, Zeljko J

2017-01-01

Contradictory reports on the effects of diabetes and hyperglycemia on myocardial infarction range from cytotoxicity to cytoprotection. The study was designed to investigate acute effects of high glucose-driven changes in mitochondrial metabolism and osmolarity on adaptive mechanisms and resistance to oxidative stress of isolated rat cardiomyocytes. We examined the effects of high glucose on several parameters of mitochondrial bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential, and NAD(P)H fluorometry. Effects of high glucose on the endogenous cytoprotective mechanisms elicited by anesthetic preconditioning (APC) and the mediators of cell injury were also tested. These experiments included real-time measurements of reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening in single cells by laser scanning fluorescence confocal microscopy, and cell survival assay. High glucose rapidly enhanced mitochondrial energy metabolism, observed by increase in NAD(P)H fluorescence intensity, oxygen consumption, and mitochondrial membrane potential. This substantially elevated production of ROS, accelerated opening of the mPTP, and decreased survival of cells exposed to oxidative stress. Abrogation of high glucose-induced mitochondrial hyperpolarization with 2,4 dinitrophenol (DNP) significantly, but not completely, attenuated ROS production to a level similar to hyperosmotic mannitol control. DNP treatment reversed high glucose-induced cytotoxicity to cytoprotection. Hyperosmotic mannitol treatment also induced cytoprotection. High glucose abrogated APC-induced mitochondrial depolarization, delay in mPTP opening and cytoprotection. In conclusion, high glucose-induced mitochondrial hyperpolarization abolishes APC and augments cell injury. Attenuation of high glucose-induced ROS production by eliminating mitochondrial hyperpolarization protects cardiomyocytes. J. Cell. Physiol. 232: 216-224, 2017

Mitochondrial Aging: Is There a Mitochondrial Clock?

PubMed

Zorov, Dmitry B; Popkov, Vasily A; Zorova, Ljubava D; Vorobjev, Ivan A; Pevzner, Irina B; Silachev, Denis N; Zorov, Savva D; Jankauskas, Stanislovas S; Babenko, Valentina A; Plotnikov, Egor Y

2017-09-01

Fragmentation (fission) of mitochondria, occurring in response to oxidative challenge, leads to heterogeneity in the mitochondrial population. It is assumed that fission provides a way to segregate mitochondrial content between the "young" and "old" phenotype, with the formation of mitochondrial "garbage," which later will be disposed. Fidelity of this process is the basis of mitochondrial homeostasis, which is disrupted in pathological conditions and aging. The asymmetry of the mitochondrial fission is similar to that of their evolutionary ancestors, bacteria, which also undergo an aging process. It is assumed that mitochondrial markers of aging are recognized by the mitochondrial quality control system, preventing the accumulation of dysfunctional mitochondria, which normally are subjected to disposal. Possibly, oncocytoma, with its abnormal proliferation of mitochondria occupying the entire cytoplasm, represents the case when segregation of damaged mitochondria is impaired during mitochondrial division. It is plausible that mitochondria contain a "clock" which counts the degree of mitochondrial senescence as the extent of flagging (by ubiquitination) of damaged mitochondria. Mitochondrial aging captures the essence of the systemic aging which must be analyzed. We assume that the mitochondrial aging mechanism is similar to the mechanism of aging of the immune system which we discuss in detail. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Changing paradigm from one target one ligand towards multi target directed ligand design for key drug targets of Alzheimer disease: An important role of Insilco methods in multi target directed ligands design.

PubMed

Kumar, Akhil; Tiwari, Ashish; Sharma, Ashok

2018-03-15

Alzheimer disease (AD) is now considered as a multifactorial neurodegenerative disorder and rapidly increasing to an alarming situation and causing higher death rate. One target one ligand hypothesis is not able to provide complete solution of AD due to multifactorial nature of disease and one target one drug seems to fail to provide better treatment against AD. Moreover, current available treatments are limited and most of the upcoming treatments under clinical trials are based on modulating single target. So the current AD drug discovery research shifting towards new approach for better solution that simultaneously modulate more than one targets in the neurodegenerative cascade. This can be achieved by network pharmacology, multi-modal therapies, multifaceted, and/or the more recently proposed term "multi-targeted designed drugs. Drug discovery project is tedious, costly and long term project. Moreover, multi target AD drug discovery added extra challenges such as good binding affinity of ligands for multiple targets, optimal ADME/T properties, no/less off target side effect and crossing of the blood brain barrier. These hurdles may be addressed by insilico methods for efficient solution in less time and cost as computational methods successfully applied to single target drug discovery project. Here we are summarizing some of the most prominent and computationally explored single target against AD and further we discussed successful example of dual or multiple inhibitors for same targets. Moreover we focused on ligand and structure based computational approach to design MTDL against AD. However is not an easy task to balance dual activity in a single molecule but computational approach such as virtual screening docking, QSAR, simulation and free energy are useful in future MTDLs drug discovery alone or in combination with fragment based method. However, rational and logical implementations of computational drug designing methods are capable of assisting AD drug

Cyclin D1 Determines Mitochondrial Function In Vivo†

PubMed Central

Sakamaki, Toshiyuki; Casimiro, Mathew C.; Ju, Xiaoming; Quong, Andrew A.; Katiyar, Sanjay; Liu, Manran; Jiao, Xuanmao; Li, Anping; Zhang, Xueping; Lu, Yinan; Wang, Chenguang; Byers, Stephen; Nicholson, Robert; Link, Todd; Shemluck, Melvin; Yang, Jianguo; Fricke, Stanley T.; Novikoff, Phyllis M.; Papanikolaou, Alexandros; Arnold, Andrew; Albanese, Christopher; Pestell, Richard

2006-01-01

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function. PMID:16809779

A critique of the molecular target-based drug discovery paradigm based on principles of metabolic control: advantages of pathway-based discovery.

PubMed

Hellerstein, Marc K

2008-01-01

Contemporary drug discovery and development (DDD) is dominated by a molecular target-based paradigm. Molecular targets that are potentially important in disease are physically characterized; chemical entities that interact with these targets are identified by ex vivo high-throughput screening assays, and optimized lead compounds enter testing as drugs. Contrary to highly publicized claims, the ascendance of this approach has in fact resulted in the lowest rate of new drug approvals in a generation. The primary explanation for low rates of new drugs is attrition, or the failure of candidates identified by molecular target-based methods to advance successfully through the DDD process. In this essay, I advance the thesis that this failure was predictable, based on modern principles of metabolic control that have emerged and been applied most forcefully in the field of metabolic engineering. These principles, such as the robustness of flux distributions, address connectivity relationships in complex metabolic networks and make it unlikely a priori that modulating most molecular targets will have predictable, beneficial functional outcomes. These same principles also suggest, however, that unexpected therapeutic actions will be common for agents that have any effect (i.e., that complexity can be exploited therapeutically). A potential operational solution (pathway-based DDD), based on observability rather than predictability, is described, focusing on emergent properties of key metabolic pathways in vivo. Recent examples of pathway-based DDD are described. In summary, the molecular target-based DDD paradigm is built on a naïve and misleading model of biologic control and is not heuristically adequate for advancing the mission of modern therapeutics. New approaches that take account of and are built on principles described by metabolic engineers are needed for the next generation of DDD.

Dual-acting of Hybrid Compounds - A New Dawn in the Discovery of Multi-target Drugs: Lead Generation Approaches.

PubMed

Abdolmaleki, Azizeh; Ghasemi, Jahan B

2017-01-01

Finding high quality beginning compounds is a critical job at the start of the lead generation stage for multi-target drug discovery (MTDD). Designing hybrid compounds as selective multitarget chemical entity is a challenge, opportunity, and new idea to better act against specific multiple targets. One hybrid molecule is formed by two (or more) pharmacophore group's participation. So, these new compounds often exhibit two or more activities going about as multi-target drugs (mtdrugs) and may have superior safety or efficacy. Application of integrating a range of information and sophisticated new in silico, bioinformatics, structural biology, pharmacogenomics methods may be useful to discover/design, and synthesis of the new hybrid molecules. In this regard, many rational and screening approaches have followed by medicinal chemists for the lead generation in MTDD. Here, we review some popular lead generation approaches that have been used for designing multiple ligands (DMLs). This paper focuses on dual- acting chemical entities that incorporate a part of two drugs or bioactive compounds to compose hybrid molecules. Also, it presents some of key concepts and limitations/strengths of lead generation methods by comparing combination framework method with screening approaches. Besides, a number of examples to represent applications of hybrid molecules in the drug discovery are included. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

CDK1 enhances mitochondrial bioenergetics for radiation-induced DNA repair

DOE PAGES

Qin, Lili; Fan, Ming; Candas, Demet; ...

2015-12-06

Nuclear DNA repair capacity is a critical determinant of cell fate under genotoxic stress conditions. DNA repair is a well-defined energy-consuming process. However, it is unclear how DNA repair is fueled and whether mitochondrial energy production contributes to nuclear DNA repair. Here, we report a dynamic enhancement of oxygen consumption and mitochondrial ATP generation in irradiated normal cells, paralleled with increased mitochondrial relocation of the cell-cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is reduced significantly in cells harboring CDK1 phosphorylation-deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair aremore » also compromised severely in cells harboring mitochondrially targeted, kinase-deficient CDK1. These findings demonstrate a mechanism governing the communication between mitochondria and the nucleus by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress conditions.« less

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

PubMed

Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

2015-08-01

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE

PubMed Central

Caza, Tiffany N; Fernandez, David R; Talaber, Gergely; Oaks, Zachary; Haas, Mark; Madaio, Michael P; Lai, Zhi-wei; Miklossy, Gabriella; Singh, Ram R; Chudakov, Dmitriy M; Malorni, Walter; Middleton, Frank; Banki, Katalin; Perl, Andras

2014-01-01

Objective Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice. Methods Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology. Results Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (−49%; p=0.01) and mouse lymphocytes (−41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (−86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001). Conclusions These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE. PMID:23897774

Discovery on Pad 39a

NASA Image and Video Library

2010-10-31

The space shuttle Discovery is seen on launch pad 39a early in the morning of Sunday, Oct. 31, 2010 at the NASA Kennedy Space Center in Cape Canaveral, Fla. During Space Shuttle Discovery's final spaceflight, the STS-133 crew members will take important spare parts to the International Space Station along with the Express Logistics Carrier-4. Discovery is targeted for launch at 3:52 p.m. Wednesday, Nov. 3, 2010. Photo Credit: (NASA/Bill Ingalls)

Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules

PubMed Central

Bakajsova, Diana; Samarel, Allen M.

2011-01-01

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC. PMID:21289057

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery.

PubMed

Prieto, DaRue A; Chan, King C; Johann, Donald J; Ye, Xiaoying; Whitely, Gordon; Blonder, Josip

2017-01-01

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein*

PubMed Central

Vest, Katherine E.; Leary, Scot C.; Winge, Dennis R.; Cobine, Paul A.

2013-01-01

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria. PMID:23846699

Copper import into the mitochondrial matrix in Saccharomyces cerevisiae is mediated by Pic2, a mitochondrial carrier family protein.

PubMed

Vest, Katherine E; Leary, Scot C; Winge, Dennis R; Cobine, Paul A

2013-08-16

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis