RESUMEN
PURPOSE: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide. METHODS AND RESULTS: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice. The myocardium of Akita mice had a decreased ATP/ADP ratio, elevated mitochondrial oxidative stress and increased organelle density, compared with that of wild-type mice. MitoGamide, a mitochondria-targeted 1,2-dicarbonyl scavenger, exhibited good stability in vivo, uptake into cells and mitochondria and reactivity with dicarbonyls. Treatment of Akita mice with MitoGamide for 12 weeks significantly improved the E/A ratio compared with the vehicle-treated group. CONCLUSION: Our work confirms that the Akita mouse model of diabetes replicates key clinical features of diabetic HFpEF, including cardiac and mitochondrial dysfunction. Furthermore, in this independent study, MitoGamide treatment improved diastolic function in Akita mice.
Asunto(s)
Benzamidas/farmacología , Fármacos Cardiovasculares/farmacología , Cardiomiopatías Diabéticas/prevención & control , Insuficiencia Cardíaca/prevención & control , Volumen Sistólico/efectos de los fármacos , Disfunción Ventricular Izquierda/prevención & control , Función Ventricular Izquierda/efectos de los fármacos , Animales , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Productos Finales de Glicación Avanzada/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria- targeted cyclooctyne (MitoOct) that accumulates several- hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondria-targeted therapies.
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Química Clic/métodos , Sistemas de Liberación de Medicamentos/métodos , Mitocondrias/metabolismo , Azidas/análisis , Azidas/química , Azidas/farmacocinética , Ciclooctanos/química , Ciclooctanos/farmacocinética , Portadores de Fármacos/química , Humanos , Espectrometría de Masas , Membranas Mitocondriales/metabolismo , Terapia Molecular Dirigida/métodosRESUMEN
BACKGROUND: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. SCOPE OF REVIEW: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
Asunto(s)
Biomarcadores/análisis , Mitocondrias/metabolismo , Modelos Biológicos , Sondas Moleculares , Especies Reactivas de Oxígeno/análisis , Animales , Ratones , Estrés OxidativoRESUMEN
A series of mitochondria-targeted antioxidants comprising a lipophilic triphenylphosphonium cation attached to the antioxidant chroman moiety of vitamin E by an alkyl linker have been prepared. The synthesis of a series of mitochondria-targeted vitamin E derivatives with a range of alkyl linkers gave compounds of different hydrophobicities. This work will enable the dependence of antioxidant defence on hydrophobicity to be determined in vivo.
RESUMEN
BACKGROUND: Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs. METHODS: To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp. RESULTS: There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls. CONCLUSION: Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver. GENERAL SIGNIFICANCE: These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Barrera Hematoencefálica/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Compuestos de Organoselenio/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Compuestos Heterocíclicos/farmacocinética , Compuestos Heterocíclicos/farmacología , Hígado/metabolismo , Ratones , Ratones Noqueados , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/genética , Compuestos Organofosforados/farmacocinética , Compuestos Organofosforados/farmacología , Compuestos de Organoselenio/farmacología , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. The need for improved therapies is required to kill different M. tuberculosis populations present during infection and to kill drug resistant strains. Protein complexes associated with energy generation, required for the survival of all M. tuberculosis populations, have shown promise as targets for novel therapies (e.g., phenothiazines that target type II NADH dehydrogenase (NDH-2) in the electron transport chain). However, the low efficacy of these compounds and their off-target effects has made the development of phenothiazines as a therapeutic agent for TB limited. This study reports that a series of alkyltriphenylphosphonium (alkylTPP) cations, a known intracellular delivery functionality, improves the localization and effective concentration of phenothiazines at the mycobacterial membrane. AlkylTPP cations were shown to accumulate at biological membranes in a range of bacteria and lipophilicity was revealed as an important feature of the structure-function relationship. Incorporation of the alkylTPP cationic function significantly increased the concentration and potency of a series of phenothiazine derivatives at the mycobacterial membrane (the site of NDH-2), where the lead compound 3a showed inhibition of M. tuberculosis growth at 0.5µg/mL. Compound 3a was shown to act in a similar manner to that previously published for other active phenothiazines by targeting energetic processes (i.e., NADH oxidation and oxygen consumption), occurring in the mycobacterial membrane. This shows the enormous potential of alkylTPP cations to improve the delivery and therefore efficacy of bioactive agents targeting oxidative phosphorylation in the mycobacterial membrane.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Compuestos Organofosforados/farmacología , Fenotiazinas/química , Fenotiazinas/farmacología , Antibacterianos/síntesis química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Compuestos Organofosforados/química , Fenotiazinas/síntesis química , Relación Estructura-ActividadRESUMEN
Protein kinase G type I (PKGI) plays a critical role in survival signaling of pre- and postconditioning downstream of cardiac cGMP. However, it is unclear whether PKGI exerts its protective effects in the cardiomyocyte or if other cardiac cell types are involved, and whether nitric oxide (NO) metabolism can target cardiomyocyte mitochondria independently of cGMP/PKGI. We tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation and inhibition, adenosine A(2B) receptor (A(2B)AR) agonist, phosphodiesterase type-5 (PDE-5) inhibitor, or mitochondria-targeted S-nitrosothiol (MitoSNO) was affected by a cardiomyocyte-specific ablation of the PKGI gene in the mouse (CMG-KO). In situ hearts underwent 30 min of regional ischemia followed by 2 h of reperfusion. As expected, in CMG-CTRs all interventions at early reperfusion lead to profound infarct size reduction: IPost (six cycles of 10-s reperfusion and 10-s coronary occlusion) with or without treatment with the sGC inhibitor ODQ, treatment with the specific sGC activator BAY58-2667 (BAY58), the selective A(2B)AR agonist BAY60-6583 (BAY60), PDE-5 inhibitor sildenafil, and MitoSNO. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO· and S-nitrosates thiol proteins. In contrast, the hearts of CMG-KO animals were not protected by BAY58 and sildenafil, whereas the protective effects of IPost, IPost with ODQ, BAY60, and MitoSNO were unaffected by the lack of PKGI. Taken together, PKGI is important for the protection against ischemia reperfusion injury afforded by sGC activation or PDE-5 inhibition. However, the beneficial effects of IPost, activation of the A(2B)AR, as well as the direct effects via mitochondrial S-nitrosation do not depend on PKGI in cardiomyocytes.
Asunto(s)
Benzoatos/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Poscondicionamiento Isquémico/métodos , Daño por Reperfusión Miocárdica/prevención & control , Piperazinas/farmacología , S-Nitrosotioles/farmacología , Sulfonas/farmacología , Animales , Benzoatos/metabolismo , Western Blotting , Corazón/efectos de los fármacos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Inhibidores de Fosfodiesterasa 5/metabolismo , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/metabolismo , Purinas/metabolismo , Purinas/farmacología , S-Nitrosotioles/metabolismo , Citrato de Sildenafil , Sulfonas/metabolismoRESUMEN
Mitochondria play key roles in a broad range of biomedical situations, consequently there is a need to direct bioactive compounds to mitochondria as both therapies and probes. A successful approach has been to target compounds to mitochondria by conjugation to lipophilic cations, such as triphenylphosphonium (TPP), which utilize the large mitochondrial membrane potential (Δψ(m), negative inside) to drive accumulation. This has proven effective both in vitro and in vivo for a range of bioactive compounds and probes. However so far only neutral appendages have been targeted to mitochondria in this way. Many bioactive functional moieties that we would like to send to mitochondria contain ionisable groups with pK (a) in the range that creates an assortment of charged species under physiological conditions. To see if such ionisable compounds can also be taken up by mitochondria, we determined the general requirements for the accumulation within mitochondria of a TPP cation conjugated to a carboxylic acid or an amine. Both were taken up by energised mitochondria in response to the protonmotive force. A lipophilic TPP cation attached to a carboxylic acid was accumulated to a greater extent than a simple TPP cation due to the interaction of the weakly acidic group with the pH gradient (ΔpH). In contrast, a lipophilic TPP cation attached to an amine was accumulated less than the simple cation due to exclusion of the weakly basic group by the ΔpH. From these data we derived a simple equation that describes the uptake of lipophilic cations containing ionisable groups as a function of Δψ(m), ΔpH and pK(a). These findings may facilitate the rational design of additional mitochondrial targeted probes and therapies.
Asunto(s)
Diseño de Fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Hepáticas/química , Sondas Moleculares , Fuerza Protón-Motriz/efectos de los fármacos , Animales , Femenino , Mitocondrias Hepáticas/metabolismo , Sondas Moleculares/química , Sondas Moleculares/farmacología , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Ratas , Ratas WistarRESUMEN
OBJECTIVE: We have previously found abrogated ischemia-induced coronary collateral growth in Zucker obese fatty (ZOF) rats compared with Zucker lean (ZLN) rats. Because ZOF rats have structural abnormalities in their mitochondria suggesting dysfunction and also show increased production of O(2), we hypothesized that mitochondrial dysfunction caused by oxidative stress impairs coronary collateral growth in ZOF. METHODS AND RESULTS: Increased levels of reactive oxygen species were observed in aortic endothelium and smooth muscle cells in ZOF rats compared with ZLN rats. Reactive oxygen species levels were decreased by the mitochondria-targeted antioxidants MitoQuinone (MQ) and MitoTempol (MT) as assessed by MitoSox Red and dihydroethidine staining. Lipid peroxides (a marker of oxidized lipids) were increased in ZOF by ≈47% compared with ZLN rats. The elevation in oxidative stress was accompanied by increased antioxidant enzymes, except glutathione peroxidase-1, and by increased uncoupling protein-2 in ZOF versus ZLN rats. In addition, elevated respiration rates were also observed in the obese compared with lean rats. Administration of MQ significantly normalized the metabolic profiles and reduced lipid peroxides in ZOF rats to the same level observed in lean rats. The protective effect of MQ also suppressed the induction of uncoupling protein-2 in the obese rats. Resolution of mitochondrial oxidative stress by MQ or MT restored coronary collateral growth to the same magnitude observed in ZLN rats in response to repetitive ischemia. CONCLUSIONS: We conclude that mitochondrial oxidative stress and dysfunction play a key role in disrupting coronary collateral growth in obesity and the metabolic syndrome, and elimination of the mitochondrial oxidative stress with MQ or MT rescues collateral growth.
Asunto(s)
Antioxidantes/farmacología , Circulación Colateral/efectos de los fármacos , Vasos Coronarios/crecimiento & desarrollo , Síndrome Metabólico/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Obesidad/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Circulación Colateral/fisiología , Vasos Coronarios/efectos de los fármacos , Modelos Animales de Enfermedad , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Peróxidos Lipídicos/metabolismo , Masculino , Síndrome Metabólico/fisiopatología , Mitocondrias Cardíacas/fisiología , Proteínas Mitocondriales/metabolismo , Obesidad/fisiopatología , Compuestos Organofosforados/farmacología , Estrés Oxidativo/fisiología , Piperidinas/farmacología , Ratas , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismo , Ubiquinona/farmacologíaRESUMEN
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 IC(50) of 80 µM, whereas the electron transfer from CII to CIII was inhibited with IC(50) 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 Ser(68) 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.
Asunto(s)
Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Complejo II de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Vitamina E/administración & dosificación , Animales , Antineoplásicos/farmacología , Bovinos , Transporte de Electrón , Humanos , Concentración 50 Inhibidora , Células Jurkat , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa , Vitamina E/farmacologíaRESUMEN
Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.
Asunto(s)
Mitocondrias/metabolismo , Daño por Reperfusión/prevención & control , S-Nitrosotioles/farmacología , Compuestos de Sulfhidrilo/metabolismo , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Línea Celular , Complejo I de Transporte de Electrón/metabolismo , Células HeLa , Corazón/efectos de los fármacos , Corazón/fisiopatología , Humanos , Técnicas In Vitro , Masculino , Espectrometría de Masas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/fisiología , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/fisiología , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Óxido Nítrico/metabolismo , Nitrosación/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , S-Nitrosotioles/síntesis química , S-Nitrosotioles/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential. CONCLUSIONS: Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function. METHODS: To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration. RESULTS: AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10-30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration. GENERAL SIGNIFICANCE: These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.
Asunto(s)
Antioxidantes/farmacocinética , Sistemas de Liberación de Medicamentos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/tratamiento farmacológico , Compuestos Organofosforados/farmacocinética , Animales , Antioxidantes/farmacología , Cationes , Femenino , Inyecciones Intravenosas , Ratones , Mitocondrias/patología , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Compuestos Organofosforados/farmacologíaRESUMEN
The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox-active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild-type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild-type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1-mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Benzoquinonas/farmacología , Estrés Fisiológico , Benzoquinonas/síntesis química , Línea Celular Tumoral , Medio de Cultivo Libre de Suero , Transporte de Electrón , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/genética , Oxidación-Reducción , Interferencia de ARN , Sirtuina 1/genética , Sirtuina 1/metabolismo , Superóxidos/metabolismo , Sales de Tetrazolio/química , Sales de Tetrazolio/metabolismo , Tiazoles/química , Tiazoles/metabolismoRESUMEN
The majority of kidneys used for transplantation are obtained from deceased donors. These kidneys must undergo cold preservation/storage before transplantation to preserve tissue quality and allow time for recipient selection and transport. However, cold storage (CS) can result in tissue injury, kidney discardment, or long-term renal dysfunction after transplantation. We have previously determined mitochondrial superoxide and other downstream oxidants to be important signaling molecules that contribute to CS plus rewarming (RW) injury of rat renal proximal tubular cells. Thus, this study's purpose was to determine whether adding mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to University of Wisconsin (UW) preservation solution could offer protection against CS injury. CS was initiated by placing renal cells or isolated rat kidneys in UW solution alone (4 h at 4°C) or UW solution containing MitoQ or its control compound, decyltriphenylphosphonium bromide (DecylTPP) (1 µM in vitro; 100 µM ex vivo). Oxidant production, mitochondrial function, cell viability, and alterations in renal morphology were assessed after CS exposure. CS induced a 2- to 3-fold increase in mitochondrial superoxide generation and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or renal damage. MitoQ treatment decreased oxidant production ~2-fold, completely prevented mitochondrial dysfunction, and significantly improved cell viability and/or renal morphology, whereas DecylTPP treatment did not offer any protection. These findings implicate that MitoQ could potentially be of therapeutic use for reducing organ preservation damage and kidney discardment and/or possibly improving renal function after transplantation.
Asunto(s)
Antioxidantes/farmacología , Frío/efectos adversos , Túbulos Renales Proximales/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Preservación de Órganos/efectos adversos , Compuestos Organofosforados/farmacología , Ubiquinona/farmacología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Sustancias Protectoras/farmacología , Ratas , Ratas Endogámicas F344RESUMEN
The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and alpha-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.
Asunto(s)
Proteínas Mitocondriales/metabolismo , S-Nitrosotioles/metabolismo , Animales , Electroforesis en Gel Bidimensional , Técnicas In Vitro , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/fisiología , Proteínas Mitocondriales/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Miocardio/patología , Oxidación-Reducción , Proteómica , Ratas , Superóxidos/metabolismoRESUMEN
Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.
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Citocinas/metabolismo , Isotiocianatos/química , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Inflamación , Células Jurkat , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
Multiple lines of evidence point to mitochondrial oxidative stress as a potential pathogenic cause for Parkinson's disease (PD). MitoQ is a powerful mitochondrial antioxidant. It is absorbed orally and concentrates within mitochondria where it has been shown to protect against oxidative damage. We enrolled 128 newly diagnosed untreated patients with PD in a double-blind study of two doses of MitoQ compared with placebo to explore the hypothesis that, over 12 months, MitoQ would slow the progression of PD as measured by clinical scores, particularly the Unified Parkinson Disease Rating Scale. We showed no difference between MitoQ and placebo on any measure of PD progression. MitoQ does not slow the progression of PD, and this finding should be taken into account when considering the oxidative stress hypothesis for the pathogenesis of PD.
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Antioxidantes/uso terapéutico , Compuestos Organofosforados/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Ubiquinona/análogos & derivados , Adulto , Anciano , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del Tratamiento , Ubiquinona/uso terapéuticoRESUMEN
BACKGROUND: Increased oxidative stress and subsequent mitochondrial damage are important pathways for liver damage in chronic hepatitis C virus (HCV) infection; consequently, therapies that decrease mitochondrial oxidative damage may improve outcome. The mitochondria-targeted anti-oxidant mitoquinone combines a potent anti-oxidant with a lipophilic cation that causes it to accumulate several-hundred fold within mitochondria in vivo. AIMS: In this phase II study, we investigated the effect of oral mitoquinone on serum aminotransferases and HCV RNA levels in HCV-infected patients. METHODS: Thirty HCV patients who were either non-responders or unsuitable candidates for standard-of-care (pegylated interferon plus ribavirin) were randomized to receive mitoquinone (40 or 80 mg) or placebo once daily for 28 days, and serum aminotransferases and HCV RNA levels were measured. RESULTS: Both treatment groups showed significant decreases in absolute and percentage changes in serum alanine transaminase (ALT) from baseline to treatment day 28 (P<0.05). There was also a significant difference between incremental area under the curve for ALT between baseline and day 28 for the 40 mg treatment group against placebo (P<0.05). The differences in plasma ALT activity from baseline to day 28 in both mitoquinone groups compared with placebo did not reach significance (P>0.05). There was no change in HCV load on mitoquinone treatment. CONCLUSIONS: Administration of the mitochondria-targeted anti-oxidant mitoquinone significantly decreased plasma ALT and aspartate aminotransferase in patients with chronic HCV infection, and this suggests that mitoquinone may decrease necroinflammation in the liver in these patients. As mitochondrial oxidative damage contributes to many other chronic liver diseases, such as steatohepatitis, further studies using mitochondria-targeted anti-oxidants in HCV and other liver diseases are warranted.
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Antioxidantes/uso terapéutico , Antivirales/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Compuestos Organofosforados/uso terapéutico , Ubiquinona/uso terapéutico , Administración Oral , Adulto , Alanina Transaminasa/sangre , Antioxidantes/administración & dosificación , Aspartato Aminotransferasas/sangre , Biomarcadores/sangre , Método Doble Ciego , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/diagnóstico , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/patología , Humanos , Interferones/uso terapéutico , Hígado/metabolismo , Hígado/patología , Hígado/virología , Masculino , Persona de Mediana Edad , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Mitocondrias Hepáticas/virología , Compuestos Organofosforados/administración & dosificación , ARN Viral/sangre , Ribavirina/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Ubiquinona/administración & dosificación , Carga ViralRESUMEN
Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was approximately 5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with approximately 8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.
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Membrana Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Mitocondrias Hepáticas/metabolismo , Compuestos Onio/metabolismo , Compuestos de Tritilo/metabolismo , Animales , Transporte Biológico , Cationes/química , Cationes/metabolismo , Fibroblastos , Humanos , Células Jurkat , Estructura Molecular , Compuestos Onio/química , Oxidación-Reducción , Ratas , Factores de Tiempo , Compuestos de Tritilo/químicaRESUMEN
Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H() from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.