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1.
Mol Pharm ; 15(6): 2413-2422, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29763317

RESUMO

Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-blood pressure and swelling caused by congestive heart failure or kidney failure. It acts through noncovalent inhibition of the Na+-K+-2Cl- cotransporter in the thick ascending limb of Henle's loop. Chemically, EA contains a Michael acceptor group that can react covalently with nucleophilic residues in proteins; however, the proteome reactivity of EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to globally profile EA's targets in cancer cells. We discovered that EA induces impaired mitochondrial function accompanied by increased ROS production. Our profiling revealed that EA targets functional proteins on mitochondrial membranes, including adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes of action offer valuable information to repurpose EA for cancer treatment.


Assuntos
Reposicionamento de Medicamentos , Ácido Etacrínico/farmacologia , Mitocôndrias/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Linhagem Celular Tumoral , Cisteína/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/química , Translocases Mitocondriais de ADP e ATP/metabolismo , Neoplasias/patologia , Proteoma/química , Proteoma/efeitos dos fármacos , Proteômica , Espécies Reativas de Oxigênio/metabolismo
2.
Biochemistry ; 57(6): 1031-1044, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29313673

RESUMO

Through the extensive screening of our chemical library, we found epoxycyclohexenedione (ECHD)-type compounds (AMM-59 and -120) as unique inhibitors of the bovine heart mitochondrial ADP/ATP carrier (AAC). This study investigated the mechanism of inhibition of AAC by ECHDs using submitochondrial particles (SMPs). Proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH reagents showed that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs upon being incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SMP proteins, indicating that aggregation is not caused by intermolecular S-S linkages. ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration in AAC without forming intermolecular S-S linkages. When all solvent-accessible cysteines (Cys57, Cys160, and Cys257) were previously modified by N-ethylmaleimide, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. The aggregation-inducing ability of another ECHD analogue (AMM-124), which has an alkyl chain that is shorter than those of AMM-59 and -120, was significantly less efficient than that of the two compounds. On the basis of these results, the mechanism underlying the aggregation of AAC induced by ECHDs is discussed.


Assuntos
Cicloexanonas/química , Cicloexanonas/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Animais , Bovinos , Translocases Mitocondriais de ADP e ATP/metabolismo , Modelos Moleculares , Agregados Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
3.
J Med Toxicol ; 13(2): 173-179, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28105575

RESUMO

INTRODUCTION: Bongkrekic acid (BA) has a unique mechanism of toxicity among the mitochondrial toxins: it inhibits adenine nucleotide translocase (ANT) rather than the electron transport chain. Bongkrekic acid is produced by the bacterium Burkholderia gladioli pathovar cocovenenans (B. cocovenenans) which has been implicated in outbreaks of food-borne illness involving coconut- and corn-based products in Indonesia and China. Our objective was to summarize what is known about the epidemiology, exposure sources, toxicokinetics, pathophysiology, clinical presentation, and diagnosis and treatment of human BA poisoning. METHODS: We searched MEDLINE (1946 to present), EMBASE (1947 to present), SCOPUS, The Indonesia Publication Index ( http://id.portalgaruda.org/ ), ToxNet, book chapters, Google searches, Pro-MED alerts, and references from previously published journal articles. We identified a total of 109 references which were reviewed. Of those, 29 (26 %) had relevant information and were included. Bongkrekic acid is a heat-stable, highly unsaturated tricarboxylic fatty acid with a molecular weight of 486 kDa. Outbreaks have been reported from Indonesia, China, and more recently in Mozambique. Very little is known about the toxicokinetics of BA. Bongkrekic acid produces its toxic effects by inhibiting mitochondrial (ANT). ANT can also alter cellular apoptosis. Signs and symptoms in humans are similar to the clinical findings from other mitochondrial poisons, but they vary in severity and time course. Management of patients is symptomatic and supportive. CONCLUSIONS: Bongkrekic acid is a mitochondrial ANT toxin and is reported primarily in outbreaks of food-borne poisoning involving coconut and corn. It should be considered in outbreaks of food-borne illness when signs and symptoms manifest involving the liver, brain, and kidneys and when coconut- or corn-based foods are implicated.


Assuntos
Ácido Bongcréquico/envenenamento , Infecções por Burkholderia/microbiologia , Burkholderia gladioli/metabolismo , Cocos/microbiologia , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Mitocôndrias/enzimologia , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Zea mays/microbiologia , Animais , Ácido Bongcréquico/farmacocinética , Infecções por Burkholderia/enzimologia , Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/terapia , Burkholderia gladioli/patogenicidade , Doenças Transmitidas por Alimentos/enzimologia , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/terapia , Mitocôndrias/patologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Resultado do Tratamento
4.
Biochim Biophys Acta ; 1863(10): 2379-93, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27001633

RESUMO

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Membranas Mitocondriais/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Ácido Bongcréquico/farmacologia , Cardiolipinas/metabolismo , Bovinos , Sequência Consenso , Humanos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/química , Modelos Moleculares , Proteínas de Transporte de Fosfato/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
5.
J Biomol Screen ; 21(4): 381-90, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26746582

RESUMO

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.


Assuntos
Ensaios de Triagem em Larga Escala , Mitocôndrias/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Abietanos/farmacologia , Trifosfato de Adenosina/agonistas , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Proliferação de Células/efeitos dos fármacos , Humanos , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/metabolismo , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/agonistas , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/genética , Organismos Geneticamente Modificados , Retinoides/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Salicilanilidas/farmacologia , Transgenes
6.
Biochem Pharmacol ; 100: 112-32, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26616220

RESUMO

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 µM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 µM and 2.1 µM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).


Assuntos
Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/metabolismo , Simulação de Acoplamento Molecular/métodos , Sequência de Aminoácidos , Atractilosídeo/análogos & derivados , Atractilosídeo/química , Atractilosídeo/metabolismo , Atractilosídeo/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Sítios de Ligação/fisiologia , Ácido Bongcréquico/química , Ácido Bongcréquico/metabolismo , Ácido Bongcréquico/farmacologia , Relação Dose-Resposta a Droga , Humanos , Translocases Mitocondriais de ADP e ATP/química , Dados de Sequência Molecular , Transporte Proteico/fisiologia
7.
Sci Rep ; 5: 14533, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26416158

RESUMO

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.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Amidinas/química , Fármacos Antiobesidade/química , Antagonistas de Receptores de Canabinoides/química , Mitocôndrias/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Pirazóis/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Amidinas/síntese química , Amidinas/toxicidade , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/toxicidade , Antagonistas de Receptores de Canabinoides/síntese química , Antagonistas de Receptores de Canabinoides/toxicidade , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Desenho de Fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Obesidade/tratamento farmacológico , Obesidade/patologia , Consumo de Oxigênio/efeitos dos fármacos , Pirazóis/síntese química , Pirazóis/farmacologia , Pirazóis/toxicidade , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Relação Estrutura-Atividade
8.
J Gen Appl Microbiol ; 61(3): 82-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26227911

RESUMO

In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.


Assuntos
Inseticidas/isolamento & purificação , Peptaibols/química , Peptaibols/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Trichoderma/metabolismo , Peptídeos Catiônicos Antimicrobianos , Fermentação , Inseticidas/química , Testes de Sensibilidade Microbiana , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/genética , Peptaibols/farmacologia , Peptídeos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Trichoderma/classificação
9.
Chem Biol Drug Des ; 86(5): 1304-22, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26032198

RESUMO

Bongkrekic acid, isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In this study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, performed by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, KH-7, KH-16, and KH-17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of bongkrekic acid for its pH-dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated.


Assuntos
Ácido Bongcréquico/análogos & derivados , Ácido Bongcréquico/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Concentração de Íons de Hidrogênio , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Ratos
10.
J Toxicol Sci ; 40(2): 223-33, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25786526

RESUMO

Bongkrekic acid (BKA), an antibiotic isolated from Pseudomonas cocovenans, is an inhibitory molecule of adenine nucleotide translocase. Since this translocase is a core component of the mitochondrial permeability transition pore (MPTP) formed by apoptotic stimuli, BKA has been used as a tool to abrogate apoptosis. However, the other biochemical properties of BKA have not yet been resolved. Although the definition of a fatty acid is a carboxylic acid (-COOH) with a long hydrocarbon chain (tail), when focused on the chemical structure of BKA, the molecule was revealed to be a branched unsaturated tricarboxylic acid that resembled the structure of polyunsaturated fatty acids (PUFAs). Peroxisome proliferator-activated receptors (PPARs) consist of a subfamily of three isoforms: α, ß, and γ, the ligands of which include PUFAs. Using completely synthesized BKA together with simplified BKA derivatives (purity: > 98%), we herein demonstrated the utility of BKA as a selective activator of the human PPARγ isoform, which may not be associated with the anti-apoptotic nature of BKA. We also discussed the possible usefulness of BKA.


Assuntos
Antibacterianos/farmacologia , Ácido Bongcréquico/química , Ácido Bongcréquico/farmacologia , PPAR gama/metabolismo , Apoptose , Araquidonato 15-Lipoxigenase , Ácido Bongcréquico/análogos & derivados , Ácido Bongcréquico/síntese química , Humanos , Células MCF-7 , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Isoformas de Proteínas/metabolismo
11.
J Biol Chem ; 290(13): 8206-17, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25653283

RESUMO

Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here.


Assuntos
Lipídeos/química , Translocases Mitocondriais de ADP e ATP/química , Proteínas de Saccharomyces cerevisiae/química , Cardiolipinas/química , Detergentes/química , Inibidores Enzimáticos/química , Humanos , Canais Iônicos/química , Micelas , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Proteínas Mitocondriais/química , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Solubilidade , Temperatura de Transição , Proteína Desacopladora 1
12.
Biochim Biophys Acta ; 1852(5): 749-58, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25619687

RESUMO

Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca(2+) dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10µM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 µM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca(2+) leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca(2+) leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca(2+) wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca(2+) handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.


Assuntos
Cálcio/metabolismo , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Palmitoilcarnitina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Ácido Bongcréquico/farmacologia , Células Cultivadas , Depuradores de Radicais Livres/farmacologia , Immunoblotting , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/fisiologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo
13.
Food Chem Toxicol ; 70: 198-204, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24863614

RESUMO

Capsules, powders and tablets containing raw coffee extract are advertised to the consumer as antioxidant rich dietary supplements as part of a healthy diet. We isolated carboxyatractyligenin (4), 2-O-ß-d-glucopyranosyl carboxyatractyligenin (6) and 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside (8) from green coffee and found strong inhibitory effects on phosphorylating respiration in isolated mitochondria similar to the effects of the known phytotoxin carboxyatractyloside. LC-MS/MS analysis of commercial green coffee based dietary supplements revealed the occurrence of carboxyatractyligenin, 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside, and 2-O-ß-d-glucopyranosyl carboxyatractyligenin in concentrations up to 4.0, 5.7, and 41.6µmol/g, respectively. These data might help to gain first insight into potential physiological side-effects of green coffee containing dietary supplement.


Assuntos
Atractilosídeo/análogos & derivados , Café/química , Suplementos Nutricionais , Translocases Mitocondriais de ADP e ATP/metabolismo , Animais , Antioxidantes/farmacologia , Atractilosídeo/farmacologia , Cromatografia Líquida , Manipulação de Alimentos , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Fosforilação , Pós/química , Espectrometria de Massas em Tandem
14.
J Mol Graph Model ; 45: 173-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24056384

RESUMO

The protein adenine nucleotide translocase (ANT) is localized in the mitochondrial inner membrane and plays an essential role in transporting ADP into the mitochondrial matrix and ATP out from the matrix for cell utilization. In mammals there are four paralogous ANT genes, of which ANT4 is exclusively expressed in meiotic germ cells. Since ANT4 has been shown essential for spermatogenesis and male fertility in mice, inhibition of ANT4 appears to be a reasonable target for male contraceptive development. Further, in contrast to ANT1, ANT2 and ANT3 that are highly homologous to each other, ANT4 has a distinguishable amino acid sequence, which serves as a basis to develop a selective ANT4 inhibitor. In this study, we aimed to identify candidate compounds that can selectively inhibit ANT4 activity over the other ANTs. We used a structure-based method in which ANT4 was modeled then utilized as the basis for selection of compounds that interact with sites unique to ANT4. A large chemical library (>100,000 small molecules) was screened by molecular docking and effects of these compounds on ADP/ATP exchange through ANT4 were examined using yeast mitochondria expressing human ANT4. Through this, we identified one particular candidate compound, [2,2'-methanediylbis(4-nitrophenol)], which inhibits ANT4 activity with a lower IC50 than the other ANTs (5.8 µM, 4.1 µM, 5.1 µM and 1.4 µM for ANT1, 2, 3 and 4, respectively). This newly identified active lead compound and its chemical structure are expected to provide new opportunities to optimize selective ANT4 inhibitors for contraceptive purposes.


Assuntos
Inibidores Enzimáticos/química , Translocases Mitocondriais de ADP e ATP/química , Modelos Moleculares , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência
15.
Mol Cell Endocrinol ; 381(1-2): 198-209, 2013 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-23939247

RESUMO

Here, we have investigated the role of inorganic phosphate (Pi) transport in mitochondria of rat clonal ß-cells. In α-toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and Pi. In the presence of substrates, addition of Pi caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of Pi transport blocked the effects of Pi on electrochemical gradient and ATP export. Knock-down of the phosphate transporter PiC, however, neither prevented Pi-induced mitochondrial activation nor glucose-induced insulin secretion. Using (31)P NMR we observed reduction of Pi pools during nutrient stimulation of INS-1E cells. Interestingly, Pi loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial Pi pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.


Assuntos
Glicerofosfatos/metabolismo , Mitocôndrias/metabolismo , Fosfatos/metabolismo , Succinatos/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Arseniatos/farmacologia , Linhagem Celular Tumoral , Concentração de Íons de Hidrogênio , Insulinoma , Potencial da Membrana Mitocondrial , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/metabolismo , Membranas Mitocondriais/fisiologia , Ratos
16.
Phytochemistry ; 93: 124-35, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23642386

RESUMO

Atractyloside (1) and carboxyatractyloside (2) are well-known inhibitors of the adenine nucleotide translocase (ANT) in mitochondria, thus effectively blocking oxidative phosphorylation. Structurally related derivatives atractyligenin (3), 2-O-ß-D-glucopyranosyl-atractyligenin (4), 3'-O-ß-D-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-atractyligenin)-ß-D-glucopyranoside (5), and 2-O-ß-D-glucopyranosyl-carboxyatractyligenin (6) were isolated from raw beans of Coffea L. and the impact of 1-6 on ANT activity was evaluated in isolated mitochondria. Among the coffee components, 6 significantly inhibited ANT activity leading to reduced respiration. Quantitative analysis in commercial coffees, experimental roastings of coffee, and model experiments using purified compound 6 consistently revealed a complete degradation during thermal treatment. In comparison, raw coffee extracts were found to contain high levels of 6, which are therefore expected to be present in food products enriched with raw coffee extracts. This implies the necessity of analytically controlling the levels of 6 in raw coffee extracts when used as additives for food products.


Assuntos
Atractilosídeo/análogos & derivados , Coffea/química , Culinária , Inibidores Enzimáticos/farmacologia , Mitocôndrias Hepáticas/enzimologia , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Animais , Atractilosídeo/química , Atractilosídeo/isolamento & purificação , Atractilosídeo/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Conformação Molecular , Relação Estrutura-Atividade , Fatores de Tempo
17.
Dev Neurobiol ; 73(2): 127-41, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22767450

RESUMO

These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure and focused on interactions between proapoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that, following ethanol exposure, Bax proapoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least 2 h postexposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment and found such interactions were altered by ethanol treatment, but only at 2 h postexposure and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax but not by a Bax channel blocker. Therefore, we conclude that, at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex and not channel formation independent of PTP constituents.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Cerebelo/metabolismo , Etanol/farmacologia , Proteínas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Administração por Inalação , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Depressores do Sistema Nervoso Central/administração & dosagem , Depressores do Sistema Nervoso Central/sangue , Técnicas de Cocultura , Corantes , Ensaio de Imunoadsorção Enzimática , Etanol/administração & dosagem , Etanol/sangue , Feminino , Transtornos do Espectro Alcoólico Fetal/patologia , Hexoquinase/metabolismo , Masculino , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Gravidez , Conformação Proteica , Ratos , Ratos Long-Evans , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismo , Sais de Tetrazólio , Tiazóis , Canais de Ânion Dependentes de Voltagem/antagonistas & inibidores , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismo
18.
Chem Res Toxicol ; 25(10): 2253-60, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22998163

RESUMO

Bongkrekic acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Since inhibition of ANT is connected to the inhibition of cytochrome c release from mitochondria, which then results in the suppression of apoptosis, it has been used as a tool for the mechanistic investigation of apoptosis. BKA consists of a long carbon chain with two asymmetric centers, a nonconjugated olefin, two conjugated dienes, three methyl groups, a methoxyl group, and three carboxylic acids. This complicated chemical structure has caused difficulties in synthesis, supply, and biochemical mechanistic investigations. In this study, we designed and synthesized more simple tricarboxylic acids that were inspired by the molecular structure of BKA. Their cytotoxicity and apoptosis-preventing activity in HeLa cells and the effect on the mitochondrial inner membrane potential (ΔΨm) in HL-60 cells were then evaluated. All tested tricarboxylic acid derivatives including BKA showed little toxicity against HeLa cells. BKA and two of the synthesized derivatives significantly suppressed staurosporine (STS)-induced reductions in cell viability. Furthermore, STS-induced ΔΨm collapse was significantly restored by pretreatment with BKA and a tricarboxylic acid derivative. Other derivatives, in which one of three carboxylic acids was esterified, exhibited potent toxicity, especially a derivative bearing a carbon chain of the same length as that of BKA. In conclusion, we have developed a new lead compound as an apoptosis inhibitor bearing three carboxylic acids connected with the proper length of a long carbon chain.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Ácido Bongcréquico/análogos & derivados , Ácido Bongcréquico/farmacologia , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Burkholderia/química , Desenho de Fármacos , Células HL-60 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ácidos Tricarboxílicos/química , Ácidos Tricarboxílicos/farmacologia
19.
Pharmacol Res ; 65(1): 120-8, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21884796

RESUMO

The present study was aimed to provide a better understanding of the mitochondria-targeted actions of minocycline (MC), a second-generation tetracycline which has cytoprotective effects. Although the specific mechanisms underlying its activity remained elusive, considerable amounts of data indicated mitochondria as the primary pharmacological target of MC. Previous reports have shown that MC affects the oxygen-uptake rate by isolated mitochondria in different respiratory states. Here, we report on the effect of MC, in the range 50-200µM, on mitochondrial respiration. State 3 respiration titration with carboxyatractyloside revealed that MC inhibits the adenine nucleotide translocase. Furthermore, we analyze MC channel-forming capacity in the lipid membrane bilayer. Our results confirmed the crucial role of Δψ and showed a dependence on Ca(2+) for MC to have an effect on mitochondria. Our data also indicated that outer and inner mitochondrial membranes contribute differently to this effect, involving the presence of Δψ (the inner membrane) and VDAC (the outer membrane). Data from three isosmotic media indicate that MC does not increase the permeability of the inner membrane to protons or potassium. In addition, by using mitoplasts and ruthenium red, we showed that Ca(2+) uptake is not involved in the MC effect, suggesting involvement of VDAC in the MC interaction with the outer membrane. Our data contribute to unravel the mechanisms behind the mitochondria-targeted activity of the cytoprotective drug MC.


Assuntos
Respiração Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Minociclina/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Desacopladores/farmacologia , Animais , Cálcio/metabolismo , Citoproteção , Relação Dose-Resposta a Droga , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Permeabilidade , Ratos , Ratos Wistar , Fatores de Tempo , Canais de Ânion Dependentes de Voltagem/metabolismo
20.
Mech Ageing Dev ; 132(10): 488-95, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21855562

RESUMO

To find out whether and how deficit of cellular energy supply from mitochondria to cytosol occurs in aging and hypertension, we used mitochondria isolated from 5 to 72 week-old heart left ventricle of either normotensive (WKY) or spontaneous hypertensive (SH) rats as a model system. Measurements were made of the rate of ATP appearance outside mitochondria, due to externally added ADP, as an increase of NADPH absorbance which occurs when ATP is produced in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. Such a rate proved to mirror the function of the adenine nucleotide translocator (ANT) rather than other processes linked to the both oxidative and substrate level phosphorylation. The changes in both Ki for atractyloside and Km for ADP suggest the occurrence of modification of the ANT conformation during aging in which the ANT Vmax was found to decrease in normotensive but to increase under spontaneously hypertension in 24 week-old rats with a subsequent decrease in both cases. ANT function, as investigated in the ADP physiological range (20-60µM), is expected to decrease in normotensive, but to increase in hypertensive rats up to 48 weeks. Later a decrease in the ATP rate of export outside mitochondria should occur in both cases.


Assuntos
Trifosfato de Adenosina/metabolismo , Envelhecimento/metabolismo , Hipertensão/metabolismo , Mitocôndrias Cardíacas/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Atractilosídeo/farmacologia , Transporte Biológico Ativo , Metabolismo Energético , Ventrículos do Coração/metabolismo , Técnicas In Vitro , Cinética , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Translocases Mitocondriais de ADP e ATP/metabolismo , Modelos Cardiovasculares , NADP/biossíntese , Consumo de Oxigênio , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
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