WO2013186422A1 - Method for screening and/or evaluating the efficacy of medicaments for the treatment of mitochondrial diseases and melas syndrome - Google Patents

Method for screening and/or evaluating the efficacy of medicaments for the treatment of mitochondrial diseases and melas syndrome Download PDF

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WO2013186422A1
WO2013186422A1 PCT/ES2013/070388 ES2013070388W WO2013186422A1 WO 2013186422 A1 WO2013186422 A1 WO 2013186422A1 ES 2013070388 W ES2013070388 W ES 2013070388W WO 2013186422 A1 WO2013186422 A1 WO 2013186422A1
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mitochondrial
melas
yeasts
drug
melas syndrome
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French (fr)
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José A SÁNCHEZ ALCÁZAR
Mario CORDERO MORALES
Mario DE LA MATA
David COTÁN MARÍN
Manuel OROPESA ÁVILA
Juan GARRIDO MARAVER
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Universidad Pablo De Olavide
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/395—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Saccharomyces

Definitions

  • the present invention falls within the general field of biomedicine and in particular refers to a method for screening and / or evaluation of the efficacy of a treatment for mitochondrial diseases and MELAS syndrome.
  • Mitochondrial diseases cover a broad spectrum of neurodegenerative, chronic and progressive disorders, with phenotypic manifestations and varying degrees of affection, as a consequence of alterations in mitochondrial oxidative metabolism [Zeviani M, Carelli V. Mitochondrial disorders. Curr Opin Neurol 2007; 20: 564-571].
  • the pathogenesis of these disorders has its origin in a chronic state of energy insufficiency, due to the inability of the affected mitochondria to generate enough ATP through the OXPHOS system (oxidative phosphorylation).
  • OXPHOS system oxidative phosphorylation
  • a conversion of pyruvate to lactate occurs, which systemically manifests itself as a chronic lactic acidosis.
  • mitochondrial cytopathies have a multisystemic pattern, with tissues with a strong energy demand such as the brain and muscle, the organs that are most frequently affected.
  • the 37 mitochondrial DNA (mtDNA) genes are essential for oxidative phosphorylation. Of these, 13 encode subunits of respiratory chain complexes: seven subunits of complex I, one subunit of complex III, three subunits of complex IV and two subunits of complex V. Mutations of these genes cause various mitochondrial alterations and generally present maternal inheritance In addition, 22 tRNA and 2 ribosomal RNAs (rRNA) are required for mitochondrial protein synthesis. In the last decade, clinical researchers have also been interested in mitochondrial alterations with Mendelian inheritance.
  • nDNA Nuclear DNA
  • nDNA nuclear DNA
  • genes necessary for oxidative phosphorylation including 72 polypeptide subunits, as well as all the factors required for the correct assembly of the respiratory chain and the machinery necessary for integrity, replication, repair and expression of mtDNA.
  • mutations in the factors required for the translation of proteins in the mitochondria, the importation of proteins, and the fusion / fission of mitochondria also cause mitochondrial abnormalities [Debray FG, Lambert M, Mitchell GA. Disorders of mitochondrial function. Curr Opin Pediatr 2008; 20: 471-482].
  • Mitochondrial diseases are clinically heterogeneous due to the uneven distribution of mutations in the different tissues, the degree of heteroplasmia of the affected tissues, the mitotic segregation and the variability of penetrance and threshold effect of the different mutations.
  • the prevalence of mitochondrial diseases is approximately 1: 5000 among the population worldwide.
  • MELAS syndrome owes its name to the English acronym of Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (mitochondrial encephalomyopathy, lactic acidosis and episodes similar to strokes). It was first described by Pavlakis et al., In 1984. Patients present with clinical manifestations that include the triad of symptoms that give name to the disease. Stroke mainly affects the parieto-occipital region of the brain which leads to defects in the visual field. Seizures are common in these patients associated with stroke episodes or as an isolated phenomenon.
  • MELAS syndrome is a polygenic disorder, associated with at least 29 specific point mutations in mtDNA.
  • the most common mutation related to this syndrome is the transition from an adenine to a guanine in the position 3243 of the mitochondrial genome (A3243G), in the gene that codes for tRNA (UUR), with a prevalence of 0.06% of the general population [Sproule DM, Kaufmann P. Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences 2008; 1 142: 133-158].
  • the A3243G mutation makes it difficult to modify the base U of balancing, hindering the translation of the UUA and UUG codons, resulting in an altered incorporation of the amino acids to the proteins synthesized in the mitochondria [Kirino Y, Yasukawa T, Ohta S, Akira S, Ishihara K, Watanabe K et al. Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease. Proceedings of the National Academy of Sciences of the United States of America 2004; 101: 15070-15075].
  • Other proposed factors that also influence the altered synthesis of mitochondrial proteins are: disorders in the processing of mRNAs, incorrect aminoacylation kinetics of tRNALeu (UUR) or incorrect conjugation of amino acids to tRNALeu (UUR).
  • mitochondria are not capable of producing sufficient amounts of ATP. This leads to a chronic state of energy deficiency, due to an imbalance between energy requirements and available energy. Finally, this energy imbalance causes cellular and tissue damage.
  • the A3243G mutation causes a higher glycolytic rate, increased lactate production, reduced glucose oxidation, an altered response to NADH, low ⁇ ⁇ , decreased ATP production, increased ROS and intracellular calcium homeostasis altered decrease in insulin secretion, premature aging and a deregulation of amino acid metabolism and urea synthesis.
  • mtDNA mutations cause cell damage and the compensatory mechanisms that the cell activates to survive.
  • MELAS fibroblasts showed reduced respiratory enzymatic activities, CoQ deficiency and mitochondrial depolarization.
  • Mitochondrial dysfunction was associated with an increase in ROS production, activation of the mitochondrial permeability transition (MPT) and the elimination of mitochondria altered by mitophagy.
  • MPT mitochondrial permeability transition
  • Saccharomyces cerevisiae yeast is a useful tool, since specific mutations can be introduced into your mtDNA by biobalistics, such as base substitution in mitochondrial tRNA (mt tRNA) genes equivalent to those that originate human neurodegenerative diseases. This is possible because mitochondrial yeast and human tRNAs are similar in sequence and structure, except for the presence of a longer loop in yeasts than in humans.
  • mt tRNA mitochondrial tRNA
  • the advantages of the use of S. cerevisiae as a model organism are diverse: high growth rate, economic maintenance, classification as GRAS microorganism (generally recognized as safe), fully sequenced genome, suitable for the expression of heterologous proteins, contains a multitude of selective markers including auxotrophic and resistance markers.
  • Yeasts are also particularly useful for the study of human mitochondrial diseases thanks to their ability to survive in a medium with a fermentable carbon source, even though their respiratory chain is not functional.
  • concentration of glucose When the concentration of glucose is reduced, the mutant yeasts deficient in respiration grow slowly, giving rise to small colonies (petite).
  • These petite mutants have abnormalities in mtDNA in the form of multiple rearrangements (petites rho-) or loss of mtDNA (petites rho 5 ).
  • the The causative mutation of 80% of MELAS syndrome cases is the transition from an adenine to a guanine at position 3243 of the mitochondrial genome (m.3243A> G), in the gene encoding the tRNA (UUR).
  • This mutation is found in a highly conserved region between the human mitochondrial genome and the yeast genome ( Figure 1).
  • the A14 nucleotide of the tRNA participates in a canonical tertiary interaction with the nucleotidouridine in position 8 ( Figure 2), which stabilizes the secondary structure of the tRNA and determines its functionality. Therefore, the A14G mutation in yeast results in a conformational rearrangement of the D arm of the tRNA and a decrease in the efficiency of aminoacylation [Montanari A, Besagni C, De Luca C, Morea V, Olive R, Tramontane A et al. Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects. RNA (New York, NY 2008; 14: 275-283].
  • the A3243G mutation like other mutations related to MELAS syndrome in humans, has been shown to prevent the modification of uridine with a taurine residue (5-taurinomethyl uridine, xm 5 U) in the staggered position of the anticodon, and The lack of this modification has been proposed as responsible for the pathological effect. It seems that the enzyme responsible for carrying out this modification recognizes the tertiary structure of the complete tRNA, which is affected as a result of this mutation.
  • the alteration of mitochondrial tRNA (UUR) shows a reduced translation of UUG, while there is no decrease in the translation of UUA ( Figure 3).
  • the main molecular cause of MELAS syndrome is the poor translation of the UUG codon as a result of the defect in the modification of taurine in the staggering position of the anticodon, which translates into a reduction in the activity of the Complex I, which is one of the characteristic symptoms that has been found in patients.
  • the A14G mutation in tRNA (Leu) (UUR) equivalent to that produced in humans, causes severe respiratory deficiencies with a high production of mtDNA-deficient mutants (rho 5 ). The percentage of rho 5 colonies is a good indicator of the severity of the respiratory phenotype.
  • yeast strains carrying the A14G mutation can grow in fermentation medium (with glucose or galactose as a carbon source), but rapidly lose mtDNA, indicating that they have a serious defect in the synthesis of mitochondrial proteins. Instead, these yeasts are unable to grow in between respiratory (with glycerol as a carbon source).
  • the use of yeasts for the study of mitochondrial diseases due to alterations in the mitochondrial tRNA presents as a limitation that they are homoplasmic unlike human cells, which are heteroplastic. Therefore, the yeast models of these pathologies do not allow the threshold effect to be evaluated. In spite of this, they constitute a very useful tool for the simplification of a complex system.
  • Yeasts are not exclusively useful for understanding the effects of mutations of mitochondrial diseases, they can also be used for mass screening of drugs capable of reversing the defects in the respiration-dependent growth characteristic of mitochondrial mutant yeasts. This is a fast and sensitive trial that allows the screening of thousands of drugs in a robotic and economical way. However, and despite the fact that dozens of yeast models of mitochondrial diseases are available, there are few studies of their usefulness for mass drug screening.
  • Drugs that modify the function of the respiratory chain or prevent oxidative stress CoQ, idebenone, succinate, vitamin C, vitamin K3, riboflavin-B2, thiamine-B1, cytochrome c, creatine monohydrate, copper, uridine.
  • the present invention relates to a method for the identification and evaluation of the efficacy of drugs for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome characterized in that it comprises the following steps: a) drug screening in a model of Saccharomices cerevisiae mutant A14G, by exposing said yeasts to at least one drug and determining whether said drug produces cellular growth of the A14G mutant yeasts.
  • the cell growth of the A14G mutant yeasts of step a) can be performed by any prior art method known to a person skilled in the art.
  • the cell growth of the mutant yeasts is determined by optical density.
  • step b) evaluation of the efficacy of the drug that produces cellular growth of the A14G mutant yeasts of step a) in cellular models derived from patients with MELAS syndrome and to determine if the drug is effective by means of the ability of said drug to restore pathophysiological alterations said cellular models,
  • the cellular models derived from patients with MELAS syndrome in step b) of the method of the present invention are fibroblasts derived from patients with MELAS syndrome and / or in MELAS transmitochondrial cybrids.
  • the pathophysiological alterations restored in the cellular models of step b) of the method of the present invention are an increase in cell proliferation, an increase in ATP levels, a decrease in ROS, a decrease in mitophageal activity, an increase in mitochondrial protein expression and / or increased mitochondrial activity.
  • mitochondrial dysfunction refers to mitochondrial and other adult diseases such as diabetes, Parkinson's disease, arteriosclerosis, cerebrovascular disease, Alzheimer's disease, and cancer in which mitochondrial dysfunction plays a determining role in the course of the disease.
  • the present invention relates to a drug identified by the method of the present invention for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome. Description of the figures
  • Figure 1 shows the structure of the Leu tRNA (UUR) of yeasts (A) and humans (B), showing the position of the A14G and A3243G mutation.
  • UUR Leu tRNA
  • Figure 2 shows the three-dimensional structure of tRNA (A) and the base pairing of A14 and U8 (B).
  • Figure 3 shows the chemical structure of 5-Taurinomethyl uridine (xm 5 U) (A) and taurine (B).
  • C Punctual mutation of the tRNA Leu (UUR) , which prevents the modification of a uridine to taurine in the staggering position of the anticodon, resulting in an abnormal pattern of codon recognition.
  • Figure 4 shows the results obtained with the method described in the present invention: A) CoQ, B) riboflavin, C) carnitine, D) creatinine, E) vitamin E, F) Lithium, G) menadione, H) lipoic acid, I) thiamine, J) uridine, K) vitamin C, L) resveratrol.
  • FIG. 5 Immunofluorescence images that show that treatment with CoQ and riboflavin dramatically decreases the mitophagy present in MELAS fibroblasts (panel A). The mitophagy is quantified as the number of discrete points where the mitochondrial cytochrome c marker and the autophagic marker LC3 panel B and C are placed.
  • FIG. 6 A: wild yeasts (WT) grown in YPD medium, B: mutant yeasts (MUT) A14G in YPD medium, C: WT yeasts in YPG medium, D: A14G MUT yeasts in YPG medium.
  • Yeasts were grown in complete medium containing 2% glucose (YPD) or 3% glycerol (YPG). The media were solidified with 1 , 5% agar. For the experiments, the yeasts were collected from colonies on YPG plates and transferred to 5ml of YPG medium to obtain an optical density of 0.5.
  • each well was replicated 4 times in YPG. The average of the 4 replicates was normalized. The control wells were also placed on each plate.
  • the drugs tested at different concentrations were: Creatine, Thiamine, Vitamin E, Vitamin C (ascorbic acid), Menadione, Lipoic acid, L-Arginine, Carnitine, Riboflavin, Resveratrol, Lithium, Uridine and CoQ.
  • Example 2 Evaluation of selected drugs in fibroblast models derived from MELAS patients and in MELAS transmitochondrial cybrids.
  • the active ingredients selected in the initial screening in yeasts were subsequently evaluated for their ability to improve pathophysiological alterations in fibroblasts and MELAS cybrids.
  • the optimal concentration of the two drugs (CoQ and riboflavin) was determined, by means of the yeast growth test, the fibroblasts and cybrids were treated for two weeks to determine their performance on the pathophysiological alterations previously detected.
  • the proliferation of the cells was determined by microscopic counting with Neubauer chamber.
  • the enzymatic activities of the respiratory chain and oxygen consumption in the cultured cells were first determined. Specifically, the activities of NADH dehydrogenase (Complex I), succinate dehydrogenase (complex II), NADH-cytochrome c reductase sensitive to Rotenone (complex l + lll), succinate-cytochrome c reductase (complex ll + lll) and cytochrome c oxidase (complex IV).
  • citrate synthase an enzyme of mitochondrial hue
  • Cytochemical stains for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) completed the biochemical studies.
  • the respiratory capacity of the cells was analyzed by polarographic techniques that measure the rate of oxygen consumption in intact fibroblasts with a Clark electrode (Yellow Spring).
  • the levels of lactic acid production in the culture medium were measured as a measure of the degree of mitochondrial dysfunction and compensation for the glycolytic pathway and the intracellular ATP levels as a guideline measure in the degree of bioenergetic alteration.
  • Oxygen consumption was determined using a Clark electrode (Yellow Springs Instruments Co, Yellow Springs, OH).
  • Lactic acid levels in the culture medium were measured using the Boehringer-Mannhein L-lactic acid commercial kit.
  • ATP levels were determined using the commercial ATP bioluminescence assay kit HS II (Roche Applied Science) according to the manufacturer's instructions. Samples were measured using a luminometer equipped with an injector (Lumat LB 9505, Berthold).
  • the synthesis of mitochondrial proteins in cultures of control fibroblasts and MELAS was essentially evaluated by measuring the incorporation of [ 35 S] -methionine in the presence of emetine (an inhibitor of cytoplasmic protein synthesis). Radioactively labeled proteins corresponding to mitochondrial synthesis were precipitated with trichloroacetic acid (10%) and analyzed by a liquid scintillation counter.
  • ATP is synthesized from ADP and phosphate by mitochondrial oxidative phosphorylation.
  • the three redox centers of the mitochondrial respiratory chain pump hydrogenions from the mitochondrial matrix to the intermembrane space, developing a protonmotrial force or mitochondrial membrane potential of the order of - 180 mV.
  • This protonmotrial force allows the synthesis of ATP by means of ATPsintetase.
  • the alterations of the mitochondrial membrane potential reflect functional alterations of the mitochondria.
  • the mitochondrial membrane potential was measured using JC-1 fluorochrome.
  • the 2'-7 'dichlorofluorescein diacetate (DCFDA) probe is preferably oxidized by H 2 0 2 generating a green fluorescence. Fluorescence was measured in a flow cytometer.
  • isolated mitochondria were resuspended in PBS and lipoperoxides were measured using an LPO-560 kit (OxisResearch, Portland, OR).
  • the mitophagy was characterized by the following techniques: 1) Autophagosome observation by electron microscopy; 2) Increased lysosomal activity: beta-galactosidase assay; 3) Colocalization of mitochondrial markers such as cytochrome c with lysosomal markers such as cathepsin or Lysotracker (invitrogen); 4) Mitochondrial localization of autophagy markers such as LC3, ATG12; 5) Expression of genes (ATG genes) and proteins that participate in the mitophagy by Western Blotting and real-time PCR. Autophageal flow integrity was measured by incubating the MELAS fibroblasts and cybrids in the presence of bafilomycin and subsequently determining the increase in LC3-II by Western blotting and real-time PCR. Autophageal flow integrity was measured by incubating the MELAS fibroblasts and cybrids in the presence of bafilomycin and subsequently determining the increase in LC3-II
  • Mitochondrial biogenesis was determined by mitochondrial mass (citrate synthase activity) and expression levels of mitochondrial biogenesis factors as a compensatory mechanism before eliminating dysfunctional mitochondria.
  • the expression and activation of factors related to mitochondrial biogenesis such as mitochondrial transcription factor Tfam, nuclear respiratory factor NRF-1 and NFR-2, and activated peroxisome proliferation receptor (PGC-1-alpha) were studied.
  • the number of mitochondrial DNA copies was determined by real-time PCR.
  • apoptosis was studied by treating cells with camptothecin (a topoisomerase I inhibitor) and serum withdrawal or treatment with staurosporine (protein kinase C inhibitor). Apoptosis was assessed by flow cytometry and immunofluorescence. Cells with the activation of caspases, cytochrome c release, cell condensation and fragmentation and other characteristic parameters of apoptotic cells were detected in fibroblast cultures.
  • camptothecin a topoisomerase I inhibitor
  • staurosporine protein kinase C inhibitor

Abstract

The invention relates to a method for identifying and evaluating the efficacy of drugs for the treatment of diseases that present mitochondrial dysfunction and/or MELAS syndrome in an A14G mutant Saccharomyces cerevisiae model, in fibroblasts derived from patients suffering from MELAS syndrome and in MELAS transmitochondrial cybrids.

Description

MÉTODO PARA EL CRIBADO Y/O EVALUACIÓN DE LA EFICACIA DE MEDICAMENTOS PARA EL TRATAMIENTO DE ENFERMEDADES MITOCONDRIALES Y SÍNDROME MELAS  METHOD FOR THE SCREENING AND / OR EVALUATION OF THE EFFECTIVENESS OF MEDICINES FOR THE TREATMENT OF MITOCONDRIAL DISEASES AND HONEY SYNDROME
Campo de la invención Field of the Invention
La presente invención se encuadra en el campo general de la biomedicina y en particular se refiere a un método para el cribado y/o evaluación de la eficacia de un tratamiento para las enfermedades mitocondriales y síndrome MELAS. The present invention falls within the general field of biomedicine and in particular refers to a method for screening and / or evaluation of the efficacy of a treatment for mitochondrial diseases and MELAS syndrome.
Estado de la técnica State of the art
Las enfermedades mitocondriales abarcan un amplio espectro de trastornos neurodegenerativos, crónicos y progresivos, con manifestaciones fenotípicas y grados de afección variables, como consecuencia de alteraciones en el metabolismo oxidativo mitocondrial [Zeviani M, Carelli V. Mitochondrial disorders. Curr Opin Neurol 2007;20:564-571 ]. A nivel celular, la patogénesis de estos desórdenes tiene su origen en un estado crónico de insuficiencia energética, debido a la incapacidad de las mitocondrias afectadas de generar suficiente ATP mediante el sistema OXPHOS (fosforilación oxidativa). Como consecuencia, se produce una conversión de piruvato a lactato, que sistémicamente se manifiesta como una acidosis láctica crónica. A menudo, las citopatías mitocondriales presentan un patrón multisistémico, siendo los tejidos con una fuerte demanda energética como el cerebro y el músculo, los órganos que aparecen afectados con mayor frecuencia. Mitochondrial diseases cover a broad spectrum of neurodegenerative, chronic and progressive disorders, with phenotypic manifestations and varying degrees of affection, as a consequence of alterations in mitochondrial oxidative metabolism [Zeviani M, Carelli V. Mitochondrial disorders. Curr Opin Neurol 2007; 20: 564-571]. At the cellular level, the pathogenesis of these disorders has its origin in a chronic state of energy insufficiency, due to the inability of the affected mitochondria to generate enough ATP through the OXPHOS system (oxidative phosphorylation). As a consequence, a conversion of pyruvate to lactate occurs, which systemically manifests itself as a chronic lactic acidosis. Often, mitochondrial cytopathies have a multisystemic pattern, with tissues with a strong energy demand such as the brain and muscle, the organs that are most frequently affected.
Los 37 genes del DNA mitocondrial (mtDNA) son imprescindibles para la fosforilación oxidativa. De éstos, 13 codifican subunidades de los complejos de la cadena respiratoria: siete subunidades del complejo I, una subunidad del complejo III, tres subunidades del complejo IV y dos subunidades del complejo V. Las mutaciones de estos genes causan diversas alteraciones mitocondriales y generalmente presentan herencia materna. Además, se requieren 22 tRNA y 2 RNA ribosomales (rRNA) para la síntesis proteica mitocondrial. En la última década, los investigadores clínicos también se han interesado por las alteraciones de la mitocondria con herencia mendeliana. El DNA nuclear (nDNA) codifica un amplio número de genes necesarios para la fosforilación oxidativa, incluyendo 72 subunidades polipeptídicas, así como todos los factores requeridos para el ensamblaje correcto de la cadena respiratoria y la maquinaria necesaria para la integridad, replicación, reparación y expresión del mtDNA. Del mismo modo, las mutaciones en los factores que se requieren para la traducción de proteínas en la mitocondria, la importación de proteínas, y la fusión/fisión de mitocondrias también causan alteraciones mitocondriales [Debray FG, Lambert M, Mitchell GA. Disorders of mitochondrial function. Curr Opin Pediatr 2008;20:471 -482]. Las enfermedades mitocondriales son clínicamente heterogéneas debido a la distribución desigual de las mutaciones en los distintos tejidos, al grado de heteroplasmia de los tejidos afectados, a la segregación mitótica y a la variabilidad de penetrancia y de efecto umbral de las distintas mutaciones. La prevalencia de las enfermedades mitocondriales es de aproximadamente 1 :5000 entre la población a nivel mundial. The 37 mitochondrial DNA (mtDNA) genes are essential for oxidative phosphorylation. Of these, 13 encode subunits of respiratory chain complexes: seven subunits of complex I, one subunit of complex III, three subunits of complex IV and two subunits of complex V. Mutations of these genes cause various mitochondrial alterations and generally present maternal inheritance In addition, 22 tRNA and 2 ribosomal RNAs (rRNA) are required for mitochondrial protein synthesis. In the last decade, clinical researchers have also been interested in mitochondrial alterations with Mendelian inheritance. Nuclear DNA (nDNA) encodes a large number of genes necessary for oxidative phosphorylation, including 72 polypeptide subunits, as well as all the factors required for the correct assembly of the respiratory chain and the machinery necessary for integrity, replication, repair and expression of mtDNA. Similarly, mutations in the factors required for the translation of proteins in the mitochondria, the importation of proteins, and the fusion / fission of mitochondria also cause mitochondrial abnormalities [Debray FG, Lambert M, Mitchell GA. Disorders of mitochondrial function. Curr Opin Pediatr 2008; 20: 471-482]. Mitochondrial diseases are clinically heterogeneous due to the uneven distribution of mutations in the different tissues, the degree of heteroplasmia of the affected tissues, the mitotic segregation and the variability of penetrance and threshold effect of the different mutations. The prevalence of mitochondrial diseases is approximately 1: 5000 among the population worldwide.
Actualmente no se dispone de tratamientos eficaces para la mayor parte de ellas [Stacpoole PW. Why are there no proven therapies for genetic mitochondrial diseases? Mitochondrion 201 1 ], limitándose éstos a medidas paliativas, generales y farmacológicas. Por lo general, se trata de procesos degenerativos, pero pueden tener un curso crónico estacionario, en forma de manifestaciones crónicas recurrentes, pudiendo mostrar en ocasiones una mejoría espontánea hasta su recuperación. Currently there are no effective treatments available for most of them [Stacpoole PW. Why are there no proven therapies for genetic mitochondrial diseases? Mitochondrion 201 1], limiting these to palliative, general and pharmacological measures. Usually, these are degenerative processes, but they can have a chronic stationary course, in the form of recurrent chronic manifestations, and can sometimes show spontaneous improvement until recovery.
El síndrome MELAS debe su nombre al acrónimo en inglés de Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (encefalomiopatía mitocondrial, acidosis láctica y episodios semejantes a los accidentes cerebrovasculares). Fue descrito por primera vez por Pavlakis et al., en 1984. Los pacientes presentan unas manifestaciones clínicas que incluyen la tríada de síntomas que dan nombre a la enfermedad. Los accidentes cerebrovasculares afectan principalmente la región parieto-occipital del cerebro lo que conduce a defectos en el campo visual. Las convulsiones son comunes en estos pacientes asociadas a episodios de ictus o como un fenómeno aislado. Otras manifestaciones incluyen migrañas intermitentes, vómitos, depresión, ataxia, trastornos cognitivos, baja estatura, sordera, intolerancia al ejercicio, cardiomiopatía y diabetes mellitas [Sproule DM, Kaufmann P. Mitochondrial encephalopathy, lactic acidosis, and strokelikeepisodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences 2008;1 142:133-158]. La edad de aparición de esta enfermedad se ha descrito inicialmente dentro del rango entre los 2 y los 60 años, aunque casi el 70% de los pacientes presentan los síntomas iniciales entre los 2 y los 20 años. La progresión de la enfermedad es frecuentemente dramática y los pacientes experimentan un progresivo deterioro neurológico y neuromuscular que resulta en demencia, severa invalidez y muerte repentina, a menudo antes de los 20 años. La supervivencia media después del diagnóstico es de 6,5 años. Dada la alta morbilidad y mortalidad del síndrome MELAS es necesaria la búsqueda de nuevos tratamientos capaces mejorar el pronóstico de la enfermedad. MELAS syndrome owes its name to the English acronym of Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (mitochondrial encephalomyopathy, lactic acidosis and episodes similar to strokes). It was first described by Pavlakis et al., In 1984. Patients present with clinical manifestations that include the triad of symptoms that give name to the disease. Stroke mainly affects the parieto-occipital region of the brain which leads to defects in the visual field. Seizures are common in these patients associated with stroke episodes or as an isolated phenomenon. Other manifestations include intermittent migraines, vomiting, depression, ataxia, cognitive disorders, short stature, deafness, exercise intolerance, cardiomyopathy and diabetes mellites [Sproule DM, Kaufmann P. Mitochondrial encephalopathy, lactic acidosis, and strokelikeepisodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences 2008; 1 142: 133-158]. The age of onset of this disease has been initially described within the range between 2 and 60 years, although almost 70% of patients have initial symptoms between 2 and 20 years. The progression of the disease is often dramatic and patients experience progressive neurological and neuromuscular deterioration resulting in dementia, severe disability and sudden death, often before the age of 20. The average survival after diagnosis is 6.5 years. Given the high morbidity and mortality of MELAS syndrome, the search for new treatments capable of improving the prognosis of the disease is necessary.
El síndrome MELAS se trata de un desorden poligénico, asociado con al menos 29 mutaciones puntuales específicas en el mtDNA. La mutación más común relacionada con este síndrome, que supone un 80% de los casos, es la transición de una adenina a una guanina en la posición 3243 del genoma mitocondrial (A3243G), en el gen que codifica para el ARNtLeu (UUR), con una prevalencia de 0,06% de la población general [Sproule DM, Kaufmann P. Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences 2008;1 142:133-158]. Por otra parte, se han asociado a esta enfermedad al menos otras siete mutaciones puntuales en dicho gen, mutaciones en otros genes de ARNt (His, Lys, Gln y Glu) y en genes que codifican proteínas (MT-ND1 , MT-C03, MTND4, MT-ND5, MT-ND6 y MT-CYB) [Wong LJ. Pathogenic mitochondrial DNA mutations in protein-coding genes. Muscle & nerve 2007;36:279-293]. Muchas de estas mutaciones, particularmente las que afectan a subunidades proteicas, están implicadas en otros síndromes mitocondriales (Neuropatía Óptica Hereditaria de Leber (LHON), Enfermedad de Leigh, Epilepsia Mioclónica asociada a Fibras Rojo-Rasgadas (MERRF)). MELAS syndrome is a polygenic disorder, associated with at least 29 specific point mutations in mtDNA. The most common mutation related to this syndrome, which accounts for 80% of cases, is the transition from an adenine to a guanine in the position 3243 of the mitochondrial genome (A3243G), in the gene that codes for tRNA (UUR), with a prevalence of 0.06% of the general population [Sproule DM, Kaufmann P. Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes: basic concepts, clinical phenotype, and therapeutic management of MELAS syndrome. Annals of the New York Academy of Sciences 2008; 1 142: 133-158]. On the other hand, at least seven other point mutations have been associated with this disease, mutations in other tRNA genes (His, Lys, Gln and Glu) and in genes encoding proteins (MT-ND1, MT-C03, MTND4, MT-ND5, MT-ND6 and MT-CYB) [Wong LJ. Pathogenic mitochondrial DNA mutations in protein-coding genes. Muscle & nerve 2007; 36: 279-293]. Many of these mutations, particularly those affecting protein subunits, are involved in other mitochondrial syndromes (Leber Hereditary Optic Neuropathy (LHON), Leigh Disease, Myoclonic Epilepsy Associated with Red-Ripped Fibers (MERRF)).
La mutación A3243G dificulta la modificación de la base U de balanceo, entorpeciendo la traducción de los codones UUA y UUG, lo que resulta en una incorporación alterada de los aminoácidos a las proteínas sintetizadas en la mitocondria [Kirino Y, Yasukawa T, Ohta S, Akira S, Ishihara K, Watanabe K et al. Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease. Proceedings of the National Academy of Sciences of the United States of America 2004;101 :15070-15075]. Otros factores propuestos que también influyen en la síntesis alterada de proteínas mitocondriales son: trastornos en el procesamiento de los mRNA, cinética de aminoacilación incorrecta de los tRNALeu(UUR) o conjugación incorrecta de los aminoácidos a tRNALeu(UUR). The A3243G mutation makes it difficult to modify the base U of balancing, hindering the translation of the UUA and UUG codons, resulting in an altered incorporation of the amino acids to the proteins synthesized in the mitochondria [Kirino Y, Yasukawa T, Ohta S, Akira S, Ishihara K, Watanabe K et al. Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease. Proceedings of the National Academy of Sciences of the United States of America 2004; 101: 15070-15075]. Other proposed factors that also influence the altered synthesis of mitochondrial proteins are: disorders in the processing of mRNAs, incorrect aminoacylation kinetics of tRNALeu (UUR) or incorrect conjugation of amino acids to tRNALeu (UUR).
En esta enfermedad existe una deficiencia generalizada en la síntesis de proteínas mitocondriales, una disminución de la actividad de las enzimas de la cadena respiratoria mitocondrial y graves defectos respiratorios. Al menos el 42% de los pacientes con MELAS muestran una disminución de la actividad del complejo I, seguido de un 29% con disminución del complejo III y un 23% en el complejo IV [Santa KM. Treatment options for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Pharmacotherapy 2010;30:1 179- 1 196.] In this disease there is a generalized deficiency in the synthesis of mitochondrial proteins, a decrease in the activity of mitochondrial respiratory chain enzymes and serious respiratory defects. At least 42% of patients with MELAS show a decrease in the activity of complex I, followed by 29% with a decrease in complex III and 23% in complex IV [Santa KM. Treatment options for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Pharmacotherapy 2010; 30: 1 179-1 196.]
En presencia de una cadena respiratoria disfuncional las mitocondrias no son capaces de producir suficientes cantidades de ATP. Esto conduce a un estado crónico de deficiencia energética, debido a un desequilibrio entre los requerimientos energéticos y la energía disponible. Finalmente, este desequilibrio energético causa daño celular y tisular. En general, la mutación A3243G provoca una mayor tasa glicolítica, aumento de la producción de lactato, oxidación reducida de la glucosa, una respuesta alterada a NADH, baja ΔΨη, una producción disminuida de ATP, aumento de ROS y una homeostasis del calcio intracelular alterada, disminución en la secreción de insulina, envejecimiento prematuro y una desregulación del metabolismo de los aminoácidos y la síntesis de la urea. Sin embargo, no se conoce con claridad como las mutaciones del mtDNA causan el daño celular y los mecanismos compensatorios que activa la célula para sobrevivir. En trabajos previos, nuestro grupo ha demostrado como la mutación afecta la función mitocondrial en fibroblastos cultivados derivados de dos pacientes MELAS con la mutación A3243G [David Cotán MDC, Juan Garrido-Maraver, Manuel Oropesa-Ávila, Ángeles Rodríguez- Hernández, Lourdes Gómez Izquierdo, Mario De la Mata, Manuel De Miguel, Juan Bautista Lorite, Eloy Rivas Infante, Sandra Jackson, Plácido Navas, and José A. Sánchez-Alcázar. Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts. FASEB J 201 1 ]. Los fibroblastos MELAS mostraron actividades enzimáticas respiratorias reducidas, deficiencia en CoQ y despolarización mitocondrial. La disfunción mitocondria se asoció a un aumento de la producción de ROS, la activación de la transición de permeabilidad mitocondrial (MPT) y la eliminación de las mitocondrias alteradas por mitofagia. Para el estudio de las bases moleculares y celulares de las enfermedades mitocondriales, así como para la identificación y evaluación de la acción de agentes terapéuticos, son necesarios modelos sencillos de investigación. Hay diferentes organismos modelos para el estudio de los defectos mitocondriales codificados por el genoma nuclear. Sin embargo éstos no son válidos en el caso de los defectos codificados por el genoma mitocondrial, ante la imposibilidad, en general, de manipular el mtDNA. Por ello, la levadura Saccharomyces cerevisiae constituye una herramienta útil, ya que se pueden introducir mutaciones puntuales en su ADNmt por biobalística, tales como sustitución de bases en genes del ARNt mitocondrial (ARNt mt) equivalentes a las que originan enfermedades neurodegenerativas humanas. Esto es posible porque los ARNt mitocondriales de levaduras y de humanos son similares en secuencia y estructura, excepto por la presencia de un loop más largo en levaduras que en humanos. Son diversas las ventajas del empleo de S. cerevisiae como organismo modelo: elevada tasa de crecimiento, mantenimiento económico, clasificación como microorganismo GRAS (generalmente reconocido como seguro), genoma totalmente secuenciado, adecuado para la expresión de proteínas heterólogas, contiene multitud de marcadores selectivos incluyendo marcadores de auxotrofías y de resistencia. Las levaduras son además particularmente útiles para el estudio de enfermedades mitocondriales humanas gracias a su capacidad de sobrevivir en un medio con una fuente de carbono fermentable, a pesar de que su cadena respiratoria no sea funcional. Cuando la concentración de glucosa es reducida, las levaduras mutantes deficientes en respiración crecen lentamente, dando lugar a pequeñas colonias (petité). Estos mutantes petite presentan anormalidades en el ADNmt en forma de reordenamientos múltiples (petites rho-) o de pérdida de ADNmt (petites rho5). Como ya se ha indicado anteriormente, la mutación causante del 80% de los casos de síndrome MELAS es la transición de una adenina a una guanina en la posición 3243 del genoma mitocondrial (m.3243A>G), en el gen que codifica el ARNtLeu (UUR). Esta mutación se encuentra en una región muy conservada entre el genoma mitocondrial humano y el de levaduras (Figura 1 ). El nucleótido A14 del ARNt participa en una interacción terciaria canónica con el nucleótidouridina en posición 8 (Figura 2), lo que estabiliza la estructura secundaria del ARNt y condiciona su funcionalidad. Por tanto, la mutación A14G en levaduras da lugar a un reordenamiento conformacional del brazo D del ARNt y una disminución en la eficiencia de la aminoacilación [Montanari A, Besagni C, De Luca C, Morea V, Oliva R, Tramontano A et al. Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects. RNA (New York, NY 2008;14:275-283]. In the presence of a dysfunctional respiratory chain, mitochondria are not capable of producing sufficient amounts of ATP. This leads to a chronic state of energy deficiency, due to an imbalance between energy requirements and available energy. Finally, this energy imbalance causes cellular and tissue damage. In general, the A3243G mutation causes a higher glycolytic rate, increased lactate production, reduced glucose oxidation, an altered response to NADH, low ΔΨ η , decreased ATP production, increased ROS and intracellular calcium homeostasis altered decrease in insulin secretion, premature aging and a deregulation of amino acid metabolism and urea synthesis. However, it is not clearly known how mtDNA mutations cause cell damage and the compensatory mechanisms that the cell activates to survive. In previous work, our group has demonstrated how the mutation affects mitochondrial function in cultured fibroblasts derived from two MELAS patients with the A3243G mutation [David Cotán MDC, Juan Garrido-Maraver, Manuel Oropesa-Ávila, Ángeles Rodríguez- Hernández, Lourdes Gómez Izquierdo , Mario De la Mata, Manuel De Miguel, Juan Bautista Lorite, Eloy Rivas Infante, Sandra Jackson, Plácido Navas, and José A. Sánchez-Alcázar. Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts. PHASEB J 201 1]. MELAS fibroblasts showed reduced respiratory enzymatic activities, CoQ deficiency and mitochondrial depolarization. Mitochondrial dysfunction was associated with an increase in ROS production, activation of the mitochondrial permeability transition (MPT) and the elimination of mitochondria altered by mitophagy. For the study of the molecular and cellular bases of mitochondrial diseases, as well as for the identification and evaluation of the action of therapeutic agents, simple research models are necessary. There are different model organisms for the study of mitochondrial defects encoded by the nuclear genome. However, these are not valid in the case of defects encoded by the mitochondrial genome, given the impossibility, in general, of manipulating mtDNA. Therefore, Saccharomyces cerevisiae yeast is a useful tool, since specific mutations can be introduced into your mtDNA by biobalistics, such as base substitution in mitochondrial tRNA (mt tRNA) genes equivalent to those that originate human neurodegenerative diseases. This is possible because mitochondrial yeast and human tRNAs are similar in sequence and structure, except for the presence of a longer loop in yeasts than in humans. The advantages of the use of S. cerevisiae as a model organism are diverse: high growth rate, economic maintenance, classification as GRAS microorganism (generally recognized as safe), fully sequenced genome, suitable for the expression of heterologous proteins, contains a multitude of selective markers including auxotrophic and resistance markers. Yeasts are also particularly useful for the study of human mitochondrial diseases thanks to their ability to survive in a medium with a fermentable carbon source, even though their respiratory chain is not functional. When the concentration of glucose is reduced, the mutant yeasts deficient in respiration grow slowly, giving rise to small colonies (petite). These petite mutants have abnormalities in mtDNA in the form of multiple rearrangements (petites rho-) or loss of mtDNA (petites rho 5 ). As indicated above, the The causative mutation of 80% of MELAS syndrome cases is the transition from an adenine to a guanine at position 3243 of the mitochondrial genome (m.3243A> G), in the gene encoding the tRNA (UUR). This mutation is found in a highly conserved region between the human mitochondrial genome and the yeast genome (Figure 1). The A14 nucleotide of the tRNA participates in a canonical tertiary interaction with the nucleotidouridine in position 8 (Figure 2), which stabilizes the secondary structure of the tRNA and determines its functionality. Therefore, the A14G mutation in yeast results in a conformational rearrangement of the D arm of the tRNA and a decrease in the efficiency of aminoacylation [Montanari A, Besagni C, De Luca C, Morea V, Olive R, Tramontane A et al. Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects. RNA (New York, NY 2008; 14: 275-283].
La mutación A3243G, al igual que otras mutaciones relacionadas con el síndrome MELAS en humanos, se ha comprobado que impide la modificación de uridina con un residuo de taurina (5-taurinometil uridina, xm5U) en la posición de tambaleo del anticodón, y la falta de esta modificación ha sido propuesta como la responsable del efecto patológico. Parece ser que la enzima responsable de llevar a cabo esta modificación reconoce la estructura terciaria del ARNt completo, la cual se ve afectada como consecuencia de esta mutación. La alteración del ARNtLeu (UUR) mitocondrial muestra una traducción reducida de UUG, mientras que no se produce una disminución en la traducción de UUA (Figura 3). Este defecto en la traducción específica del codón UUG se debe a la incapacidad de formar el apareamiento de bases entre el codón y el anticodón en el sitio A del ribosoma, lo cual sugiere que la modificación xm5U en este ARNt juega un papel importante en la estabilización del apareamiento de las bases U:G en la posición de tambaleo. Esto podría explicar el defecto en la traducción del gen que codifica el componente ND6 del Complejo I de la cadena respiratoria y que es rico en codones UUG. Por todo ello, se piensa que la principal causa molecular del síndrome MELAS es la traducción deficiente del codón UUG como consecuencia del defecto en la modificación de la taurina en la posición de tambaleo del anticodón, que se traduce en una reducción de la actividad del Complejo I, que es uno de los síntomas característicos que se ha encontrado en los pacientes. En levaduras, la mutación A14G en el ARNt(Leu) (UUR), equivalente a la que se produce en humanos, origina deficiencias respiratorias graves con una alta producción de mutantes deficientes en ADNmt (rho5). El porcentaje de colonias rho5 supone un buen indicador de la gravedad del fenotipo respiratorio. Las cepas de levaduras portadoras de la mutación A14G pueden crecer en medio fermentativo (con glucosa o galactosa como fuente de carbono), pero pierden rápidamente el ADNmt, lo que indica que tienen un defecto grave en la síntesis de proteínas mitocondriales. En cambio, estas levaduras son incapaces de crecer en medio respiratorio (con glicerol como fuente de carbono). La utilización de levaduras para el estudio de enfermedades mitocondriales debidas a alteraciones en el ARNt mitocondrial presenta como limitación que éstas son homoplásmicas a diferencia de las células humanas, que son heteroplásmicas. Por tanto, los modelos de levaduras de estas patologías no permiten evaluar el efecto umbral. A pesar de esto, constituyen una herramienta muy útil para la simplificación de un sistema complejo. Las levaduras no son útiles exclusivamente para comprender los efectos de las mutaciones de las enfermedades mitocondriales, pueden utilizarse también para el cribado masivo de fármacos capaces de revertir los defectos en el crecimiento dependiente de la respiración característico de las levaduras mutantes mitocondriales. Éste es un ensayo rápido y sensible que permite el cribado de miles de fármacos de forma robotizada y económica. Sin embargo, y a pesar de que decenas de modelos de levaduras de enfermedades mitocondriales están disponibles, hay escasos estudios de su utilidad para el cribado masivo de fármacos. The A3243G mutation, like other mutations related to MELAS syndrome in humans, has been shown to prevent the modification of uridine with a taurine residue (5-taurinomethyl uridine, xm 5 U) in the staggered position of the anticodon, and The lack of this modification has been proposed as responsible for the pathological effect. It seems that the enzyme responsible for carrying out this modification recognizes the tertiary structure of the complete tRNA, which is affected as a result of this mutation. The alteration of mitochondrial tRNA (UUR) shows a reduced translation of UUG, while there is no decrease in the translation of UUA (Figure 3). This defect in the specific translation of the UUG codon is due to the inability to form the base pairing between the codon and the anticodon at the A site of the ribosome, which suggests that the xm 5 U modification in this tRNA plays an important role in the pairing stabilization of the U: G bases in the wobble position. This could explain the defect in the translation of the gene that encodes the ND6 component of Complex I of the respiratory chain and that is rich in UUG codons. Therefore, it is thought that the main molecular cause of MELAS syndrome is the poor translation of the UUG codon as a result of the defect in the modification of taurine in the staggering position of the anticodon, which translates into a reduction in the activity of the Complex I, which is one of the characteristic symptoms that has been found in patients. In yeasts, the A14G mutation in tRNA (Leu) (UUR), equivalent to that produced in humans, causes severe respiratory deficiencies with a high production of mtDNA-deficient mutants (rho 5 ). The percentage of rho 5 colonies is a good indicator of the severity of the respiratory phenotype. The yeast strains carrying the A14G mutation can grow in fermentation medium (with glucose or galactose as a carbon source), but rapidly lose mtDNA, indicating that they have a serious defect in the synthesis of mitochondrial proteins. Instead, these yeasts are unable to grow in between respiratory (with glycerol as a carbon source). The use of yeasts for the study of mitochondrial diseases due to alterations in the mitochondrial tRNA presents as a limitation that they are homoplasmic unlike human cells, which are heteroplastic. Therefore, the yeast models of these pathologies do not allow the threshold effect to be evaluated. In spite of this, they constitute a very useful tool for the simplification of a complex system. Yeasts are not exclusively useful for understanding the effects of mutations of mitochondrial diseases, they can also be used for mass screening of drugs capable of reversing the defects in the respiration-dependent growth characteristic of mitochondrial mutant yeasts. This is a fast and sensitive trial that allows the screening of thousands of drugs in a robotic and economical way. However, and despite the fact that dozens of yeast models of mitochondrial diseases are available, there are few studies of their usefulness for mass drug screening.
Los estudios bioquímicos de los fibroblastos derivados de pacientes mitocondriales han suministrado una gran cantidad de información para comprender las alteraciones fisiopatologicas presentes en esta enfermedad. Por su parte, las líneas celulares transmitocondriales o cíbridos son una de las herramientas fundamentales en la investigación mitocondrial. Los cíbridos son generados cuando los contenidos citoplasmáticos de dos líneas celulares diferentes coexisten dentro de una misma membrana plasmática. Específicamente, esta aproximación experimental se diseña para que las mitocondrias que residen en una célula se incorporen permanentemente al citoplasma de otra célula a la que previamente se ha desprovisto de mtDNA y que por tanto no tienen mitocondrias funcionales [Khan SM, Smigrodzki RM, Swerdlow RH. Cell and animal models of mtDNA biology: progress and prospects. American journal of physiology 2007;292:C658-669]. De esta forma las alteraciones fisiopatologicas detectadas en los cíbridos se deberán a las mitocondrias disfuncionales independientemente del contexto nuclear. Ambos modelos, fibroblastos y cíbridos, resultan pues muy eficaces para conocer los mecanismos moleculares de la enfermedad mitocondrial y el cribado de diferentes tratamientos que supriman o mejoren las alteraciones fisiopatologicas detectadas. Actualmente, las opciones terapéuticas para el tratamiento del síndrome MELAS y para otras enfermedades mitocondriales son muy limitadas. En general, el tratamiento recibido por los pacientes con citopatías mitocondriales está orientado a tratar los síntomas (por ejemplo, alteraciones cardíacas, renales, nutricionales, tratamiento de la epilepsia, etc), junto con el suplemento con un cóctel mitocondrial. Existen en la actualidad diferentes fármacos dirigidos a tratar de corregir los mecanismos patogénicos de las enfermedades mitocondriales, siendo tres los principales mecanismos sobre los que puede actuar los fármacos de forma específica: Biochemical studies of fibroblasts derived from mitochondrial patients have provided a great deal of information to understand the pathophysiological alterations present in this disease. On the other hand, transmitochondrial or cybrid cell lines are one of the fundamental tools in mitochondrial research. Cybrids are generated when the cytoplasmic contents of two different cell lines coexist within the same plasma membrane. Specifically, this experimental approach is designed so that mitochondria that reside in one cell are permanently incorporated into the cytoplasm of another cell that has previously been devoid of mtDNA and therefore does not have functional mitochondria [Khan SM, Smigrodzki RM, Swerdlow RH . Cell and animal models of mtDNA biology: progress and prospects. American journal of physiology 2007; 292: C658-669]. In this way the pathophysiological alterations detected in cybrids will be due to dysfunctional mitochondria regardless of the nuclear context. Both models, fibroblasts and cybrids, are therefore very effective in knowing the molecular mechanisms of mitochondrial disease and the screening of different treatments that suppress or improve the pathophysiological alterations detected. Currently, the therapeutic options for the treatment of MELAS syndrome and other mitochondrial diseases are very limited. In general, the treatment received by patients with mitochondrial cytopathies is aimed at treating the symptoms (for example, cardiac, renal, nutritional disorders, epilepsy treatment, etc.), together with the supplement with a mitochondrial cocktail. There are currently different drugs aimed at trying to correct the pathogenic mechanisms of mitochondrial diseases, with three being the main mechanisms on which drugs can act specifically:
1 ) Fármacos que modifican la función de la cadena respiratoria o previenen el estrés oxidativo: CoQ, idebenona, succinato, vitamina C, vitamina K3, riboflavina-B2, tiamina- B1 , citocromo c, monohidrato de creatina, cobre, uridina. 1) Drugs that modify the function of the respiratory chain or prevent oxidative stress: CoQ, idebenone, succinate, vitamin C, vitamin K3, riboflavin-B2, thiamine-B1, cytochrome c, creatine monohydrate, copper, uridine.
2) Fármacos que reducen el acumulo de metabolitos tóxicos para las células: Carnitina, dicloroacetato, inhibidores del flujo del calcio mitocondrial (CGP37157). 2) Drugs that reduce the accumulation of toxic metabolites for cells: Carnitine, dichloroacetate, mitochondrial calcium flow inhibitors (CGP37157).
3) Fármacos que actúan como antioxidantes: CoQ, vitamina E. 4) Otros tratamientos farmacológicos: Ácido fólico, cuerpos cetónicos. 3) Drugs that act as antioxidants: CoQ, vitamin E. 4) Other pharmacological treatments: Folic acid, ketone bodies.
Desafortunadamente, los estudios que demuestran la eficacia de los diversos tratamientos farmacológicos actuales no son concluyentes. Por otro lado, la evaluación de la eficacia de estos tratamientos es complicada por la relativa rareza de la enfermedad, la presencia de síntomas diversos, y el curso impredecible de la enfermedad. La ausencia de pruebas concluyentes a favor de las terapias o combinación de las terapias actuales y la naturaleza progresiva del síndrome hace extremadamente difícil el tratamiento y la búsqueda de nuevas opciones terapéuticas. Unfortunately, studies that demonstrate the efficacy of the various current pharmacological treatments are inconclusive. On the other hand, the evaluation of the efficacy of these treatments is complicated by the relative rarity of the disease, the presence of various symptoms, and the unpredictable course of the disease. The absence of conclusive evidence in favor of therapies or combination of current therapies and the progressive nature of the syndrome makes treatment and the search for new therapeutic options extremely difficult.
La baja prevalencia de la enfermedad dificulta el diseño de ensayos clínicos y estudios de nuevos fármacos a gran escala, dejando sólo los casos clínicos como la primera fuente de conocimiento y guía para los médicos clínicos. Además, muchos de los ensayos clínicos disponibles incluyen pacientes con múltiples tipos de enfermedades mitocondriales, haciendo difícil la extrapolación de los resultados a los pacientes MELAS. The low prevalence of the disease makes it difficult to design clinical trials and studies of new large-scale drugs, leaving only clinical cases as the first source of knowledge and guidance for clinical doctors. In addition, many of the available clinical trials include patients with multiple types of mitochondrial diseases, making it difficult to extrapolate the results to MELAS patients.
Existe pues la necesidad de encontrar y clarificar distintas terapias para el síndrome MELAS, que permitan verificar la eficacia de los principios activos ganando con ello en especificidad y eficacia en el tratamiento. De esta manera además de proporcionar terapias eficaces en el síndrome MELAS se pueden proporcionar terapias relevantes para otras enfermedades mitocondriales y otras enfermedades del adulto como la diabetes, la enfermedad de Parkinson, arteriosclerosis, la enfermedad cerebrovascular, la enfermedad de Alzheimer, y el cáncer en las que también juega un papel determinante la disfunción mitocondrial. Además, muchas drogas utilizadas en la práctica clínica (como los antivirales, antibióticos, etc ..) causan daño mitocondrial que podría aliviarse con los nuevos tratamientos. Descripción de la invención There is therefore a need to find and clarify different therapies for MELAS syndrome, which allow verifying the efficacy of the active ingredients, thereby gaining specificity and efficacy in treatment. In this way, in addition to providing effective therapies in MELAS syndrome, relevant therapies for other mitochondrial diseases and other adult diseases such as diabetes, Parkinson's disease, arteriosclerosis, cerebrovascular disease, Alzheimer's disease, and cancer can be provided. which also plays a determining role mitochondrial dysfunction. In addition, many drugs used in clinical practice (such as antivirals, antibiotics, etc.) cause mitochondrial damage that could be relieved by new treatments. Description of the invention
Así pues la presente invención se refiere a un procedimiento para la identificación y evaluación de la eficacia de fármacos para el tratamiento de enfermedades que cursan con disfunción mitocondrial y/o síndrome de MELAS caracterizado porque comprende los siguientes pasos: a) cribado de fármacos en un modelo de Saccharomices cerevisiae mutante A14G, mediante la exposición de dichas levaduras a al menos un fármaco y determinar si dicho fármaco produce crecimiento celular de las levaduras mutantes A14G. En un aspecto más en particular el crecimiento celular de las levaduras mutantes A14G del paso a) se puede realizar por cualquier método del estado de la técnica conocido por un experto en la materia. En una realización preferida, en el procedimiento de la presente invención el crecimiento celular de las levaduras mutantes se determina mediante densidad óptica. b) evaluación de la eficacia del fármaco que produce crecimiento celular de las levaduras mutantes A14G del paso a) en modelos celulares derivados de pacientes con síndrome MELAS y determinar si el fármaco es eficaz mediante la capacidad que tiene dicho fármaco para restaurar las alteraciones fisiopatológicas de dichos modelos celulares, Thus, the present invention relates to a method for the identification and evaluation of the efficacy of drugs for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome characterized in that it comprises the following steps: a) drug screening in a model of Saccharomices cerevisiae mutant A14G, by exposing said yeasts to at least one drug and determining whether said drug produces cellular growth of the A14G mutant yeasts. In a more particular aspect the cell growth of the A14G mutant yeasts of step a) can be performed by any prior art method known to a person skilled in the art. In a preferred embodiment, in the process of the present invention the cell growth of the mutant yeasts is determined by optical density. b) evaluation of the efficacy of the drug that produces cellular growth of the A14G mutant yeasts of step a) in cellular models derived from patients with MELAS syndrome and to determine if the drug is effective by means of the ability of said drug to restore pathophysiological alterations said cellular models,
En un aspecto más en particular, los modelos celulares derivados de pacientes con síndrome MELAS del paso b) del procedimiento de la presente invención, son fibroblastos derivados de pacientes con síndrome MELAS y/o en cíbridos transmitocondriales MELAS. In a more particular aspect, the cellular models derived from patients with MELAS syndrome in step b) of the method of the present invention are fibroblasts derived from patients with MELAS syndrome and / or in MELAS transmitochondrial cybrids.
En un aspecto más en particular las alteraciones fisiopatológicas restauradas en los modelos celulares del paso b) del procedimiento de la presente invención, son un aumento de la proliferación celular, un aumento de los niveles de ATP, una disminución de ROS, una disminución de la actividad mitofágica, un aumento de la expresión de proteínas mitocondriales y/o aumento de la actividad mitocondrial. In a more particular aspect, the pathophysiological alterations restored in the cellular models of step b) of the method of the present invention are an increase in cell proliferation, an increase in ATP levels, a decrease in ROS, a decrease in mitophageal activity, an increase in mitochondrial protein expression and / or increased mitochondrial activity.
En la presente invención por enfermedades que cursan con disfunción mitocondrial se refiere a enfermedades mitocondriales y otras enfermedades del adulto como la diabetes, la enfermedad de Parkinson, arteriosclerosis, la enfermedad cerebrovascular, la enfermedad de Alzheimer, y el cáncer en las que la disfunción mitocondrial juega un papel determinante en el curso de la enfermedad. In the present invention, for diseases that occur with mitochondrial dysfunction, it refers to mitochondrial and other adult diseases such as diabetes, Parkinson's disease, arteriosclerosis, cerebrovascular disease, Alzheimer's disease, and cancer in which mitochondrial dysfunction plays a determining role in the course of the disease.
En un segundo aspecto, la presente invención se refiere a un fármaco identificado mediante el procedimiento de la presente invención para el tratamiento de enfermedades que cursan con disfunción mitocondrial y/o síndrome de MELAS. Descripción de las figuras In a second aspect, the present invention relates to a drug identified by the method of the present invention for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome. Description of the figures
La figura 1 muestra la estructura del ARNtLeu(UUR) de levaduras (A) y de humanos (B), mostrando la posición de la mutación A14G y A3243G. Figure 1 shows the structure of the Leu tRNA (UUR) of yeasts (A) and humans (B), showing the position of the A14G and A3243G mutation.
La figura 2 muestra la estructura tridimensional del ARNt (A) y el apareamiento de bases de A14 y U8 (B). Figure 2 shows the three-dimensional structure of tRNA (A) and the base pairing of A14 and U8 (B).
La figura 3 muestra la estructura química de la 5-Taurinometil uridina (xm5U) (A) y de la taurina (B). (C) Mutación puntual del ARNtLeu(UUR), que evita la modificación de una uridina a taurina en la posición de tambaleo del anticodón, dando lugar a un patrón anormal de reconocimiento del codón. La figura 4 muestra los resultados obtenidos con el método descrito en la presente invención: A) CoQ, B) riboflavina, C) carnitina, D) creatinina, E) vitamina E, F) Litio, G) menadiona, H) ácido lipoico, I) tiamina, J) uridina, K) vitamina C, L) resveratrol. Figure 3 shows the chemical structure of 5-Taurinomethyl uridine (xm 5 U) (A) and taurine (B). (C) Punctual mutation of the tRNA Leu (UUR) , which prevents the modification of a uridine to taurine in the staggering position of the anticodon, resulting in an abnormal pattern of codon recognition. Figure 4 shows the results obtained with the method described in the present invention: A) CoQ, B) riboflavin, C) carnitine, D) creatinine, E) vitamin E, F) Lithium, G) menadione, H) lipoic acid, I) thiamine, J) uridine, K) vitamin C, L) resveratrol.
La figura 5: imágenes de inmunofluorescencia que demuestran que el tratamiento con CoQ y riboflavina disminuye drásticamente la mitofagia presente en los fibroblastos MELAS (panel A). La mitofagia es cuantificada como el número de puntos discretos donde se colocalizan el marcador mitocondrial citocromo c y el marcador autofágico LC3 panel B y C. Figure 5: Immunofluorescence images that show that treatment with CoQ and riboflavin dramatically decreases the mitophagy present in MELAS fibroblasts (panel A). The mitophagy is quantified as the number of discrete points where the mitochondrial cytochrome c marker and the autophagic marker LC3 panel B and C are placed.
La figura 6: A: levaduras silvestres (WT) crecidas en medio YPD, B: levaduras mutantes (MUT) A14G en medio YPD, C: levaduras WT en medio YPG, D: levaduras MUT A14G en medio YPG. Descripción detallada de la invención Figure 6: A: wild yeasts (WT) grown in YPD medium, B: mutant yeasts (MUT) A14G in YPD medium, C: WT yeasts in YPG medium, D: A14G MUT yeasts in YPG medium. Detailed description of the invention
Ejemplo 1: Cribado de fármacos en modelo de Saccharomices cerevisiae Example 1: Screening of drugs in Saccharomices cerevisiae model
Para demostrar la validez de los modelos celulares en la búsqueda de nuevos tratamientos farmacológicos para el síndrome MELAS se realizaron una serie de ensayos piloto para comprobar la validez de los tratamientos utilizados más habitualmente en la práctica clínica en los modelos de levaduras, fibroblastos y cíbridos MELAS. To demonstrate the validity of cellular models in the search for new pharmacological treatments for MELAS syndrome, a series of pilot trials were carried out to verify the validity of the treatments most commonly used in clinical practice in the models of yeasts, fibroblasts and MELAS cybrids. .
Para el cribado de fármacos, se utilizaron dos estirpes de S. cerevisiae: la estirpe silvestre MCC123 (Mat a, ade2-1 , ura3-52, kar1 -1 , (WT) que se utiliza como estirpe control y la estirpe mutante A14G equivalente a la mutación A3243G en el gen mitocondrial tRNALeu(UUR) responsable del síndrome MELAS en humanos. Las levaduras fueron crecidas en medio completo que contenía 2% de glucosa (YPD) o 3% de glicerol (YPG). Los medios fueron solidificados con 1 ,5% de agar. Para la realización los experimentos las levaduras se recogieron de colonias en placas YPG y fueron transferidas a 5ml de medio YPG para obtener una densidad óptica de 0,5. Tras 3 h de incubación a 285C en medio líquido, se controló la presencia de mtDNA por tinción con DAPI y el experimento se continuó cuando el cultivo tuvo un 70-80% de células con mtDNA. Posteriormente, los cultivos de levaduras fueron distribuidos en placas de 96 pocilios y fueron expuestas a las diferentes concentraciones de los tratamientos. For the screening of drugs, two strains of S. cerevisiae were used: the wild strain MCC123 (Mat a, ade2-1, ura3-52, kar1 -1, (WT) which is used as a control strain and the equivalent A14G mutant strain to the A3243G mutation in the mitochondrial gene tRNALeu (UUR) responsible for MELAS syndrome in humans. Yeasts were grown in complete medium containing 2% glucose (YPD) or 3% glycerol (YPG). The media were solidified with 1 , 5% agar. For the experiments, the yeasts were collected from colonies on YPG plates and transferred to 5ml of YPG medium to obtain an optical density of 0.5. After 3 h of incubation at 28 5 C in liquid medium, the presence of mtDNA was monitored by DAPI staining and the experiment was continued when the culture had 70-80% of cells with mtDNA. Subsequently, the yeast cultures were distributed in 96-well plates and were exposed to the different concentrations of the treatments.
Tras 24 horas de incubación, se midió la densidad óptica a 660 nm de cada pocilio como indicador de crecimiento celular. Cada placa fue replicada 4 veces en YPG. La media de los 4 replicados se normalizó. Los pocilios control fueron colocados igualmente en cada placa. Los fármacos ensayados a diferentes concentraciones fueron: Creatina, Tiamina, Vitamina E, Vitamina C (Ácido ascórbico), Menadiona, Ácido lipoico, L- Arginina, Carnitina, Riboflavina, Resveratrol, Litio, Uridina y CoQ. After 24 hours of incubation, the optical density at 660 nm of each well was measured as an indicator of cell growth. Each plate was replicated 4 times in YPG. The average of the 4 replicates was normalized. The control wells were also placed on each plate. The drugs tested at different concentrations were: Creatine, Thiamine, Vitamin E, Vitamin C (ascorbic acid), Menadione, Lipoic acid, L-Arginine, Carnitine, Riboflavin, Resveratrol, Lithium, Uridine and CoQ.
En el cribado inicial en levaduras mutantes MELAS sólo se consiguieron resultados positivos con dos fármacos, CoQ y riboflavina como muestra la figura 4, dos de los fármacos más utilizados y con mejor respuesta terapéutica en el tratamiento del síndrome MELAS en la práctica clínica. In the initial screening in MELAS mutant yeasts, only positive results were achieved with two drugs, CoQ and riboflavin as shown in Figure 4, two of the most used drugs and with the best therapeutic response in the treatment of MELAS syndrome in clinical practice.
Ejemplo 2: Evaluación de los fármacos seleccionados en modelos fibroblastos derivados de pacientes MELAS y en cíbridos transmitocondriales MELAS. Example 2: Evaluation of selected drugs in fibroblast models derived from MELAS patients and in MELAS transmitochondrial cybrids.
Los principios activos seleccionados en el cribado inicial en levaduras fueron posteriormente evaluados por su capacidad de mejorar las alteraciones fisiopatologicas en fibroblastos y cíbridos MELAS. The active ingredients selected in the initial screening in yeasts were subsequently evaluated for their ability to improve pathophysiological alterations in fibroblasts and MELAS cybrids.
Una vez determinada la concentración óptima de los dos fármacos (CoQ y riboflavina), mediante el ensayo de crecimiento en levaduras, se procedió a tratar durante dos semanas con dichas concentraciones a los fibroblastos y cíbridos para determinar su actuación sobre las alteraciones fisiopatologicas detectadas previamente. Once the optimal concentration of the two drugs (CoQ and riboflavin) was determined, by means of the yeast growth test, the fibroblasts and cybrids were treated for two weeks to determine their performance on the pathophysiological alterations previously detected.
La proliferación de las células fue determinada mediante el recuento microscópico con cámara de Neubauer. Las alteraciones mitocondriales que afectaban al nivel energético celular y promovían la acumulación de metabolitos tóxicos repercutieron directa y negativamente en el correcto crecimiento y proliferación de las células. Para el análisis de la cadena respiratoria mitocondrial en primer lugar se determinaron las actividades enzimáticas de la cadena respiratoria y el consumo de oxígeno en las células en cultivo. Específicamente, se determinaron las actividades de la NADH deshidrogenasa (Complejo I), succinato deshidrogenasa (complejo II), NADH-citocromo c reductasa sensible a rotenona (complejo l+lll), succinato-citocromo c reductasa (complejo ll+lll) y citocromo c oxidasa (complejo IV). La actividad de la citrato sintasa, un enzima de matiz mitocondrial, se midió para normalizar las actividades a la cantidad relativa de mitocondrias. Las tinciones citoquímicas para la citocromo c oxidasa (COX) y la succinato deshidrogenasa (SDH) completaron los estudios bioquímicos. A continuación, se analizó la capacidad respiratoria de las células mediante técnicas polarográficas que miden la velocidad de consumo de oxígeno en los fibroblastos intactos con un electrodo de Clark (Yellow Spring). Igualmente se midieron los niveles de producción de ácido láctico en el medio de cultivo como medida del grado de disfunción mitocondrial y compensación por la ruta glicolítica y los niveles de ATP intracelulares como medida que orientativa en el grado de alteración bioenergética. The proliferation of the cells was determined by microscopic counting with Neubauer chamber. The mitochondrial alterations that affected the cellular energy level and promoted the accumulation of toxic metabolites directly and negatively affected the correct growth and proliferation of the cells. For the analysis of the mitochondrial respiratory chain, the enzymatic activities of the respiratory chain and oxygen consumption in the cultured cells were first determined. Specifically, the activities of NADH dehydrogenase (Complex I), succinate dehydrogenase (complex II), NADH-cytochrome c reductase sensitive to Rotenone (complex l + lll), succinate-cytochrome c reductase (complex ll + lll) and cytochrome c oxidase (complex IV). The activity of citrate synthase, an enzyme of mitochondrial hue, was measured to normalize activities to the relative amount of mitochondria. Cytochemical stains for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) completed the biochemical studies. Next, the respiratory capacity of the cells was analyzed by polarographic techniques that measure the rate of oxygen consumption in intact fibroblasts with a Clark electrode (Yellow Spring). Likewise, the levels of lactic acid production in the culture medium were measured as a measure of the degree of mitochondrial dysfunction and compensation for the glycolytic pathway and the intracellular ATP levels as a guideline measure in the degree of bioenergetic alteration.
El consumo de oxígeno se determinó utilizando un electrodo de Clark (Yellow Springs Instruments Co, Yellow Springs, OH). Oxygen consumption was determined using a Clark electrode (Yellow Springs Instruments Co, Yellow Springs, OH).
Los niveles de ácido láctico en el medio de cultivo se midieron utilizando el kit comercial L-lactic acid de Boehringer-Mannhein. Los niveles de ATP se determinaron mediante el kit comercial ATP bioluminescence assay kit HS II (Roche Applied Science) de acuerdo a las instrucciones del fabricante. Las muestras se midieron utilizando un luminómetro equipado con inyector (Lumat LB 9505, Berthold). Lactic acid levels in the culture medium were measured using the Boehringer-Mannhein L-lactic acid commercial kit. ATP levels were determined using the commercial ATP bioluminescence assay kit HS II (Roche Applied Science) according to the manufacturer's instructions. Samples were measured using a luminometer equipped with an injector (Lumat LB 9505, Berthold).
La biosíntesis del CoQ en las células en cultivo se estudió con el mareaje metabólico con precursores radiactivos. Para confirmar el defecto bioquímico en la síntesis del CoQ, se utilizó como precursor radiactivo, el ácido 4-hidroxi[U-14C]benzoico (4-[U-14C]HB) un precursor del anillo quinónico del CoQ, y [5-3H]mevalonolactona ([3H]MVL) como precursor de la cadena lateral isoprenoide (American Radiolabeled Chemicals, St. Louis, MO), de acuerdo al protocolo seguido por Nambudiri y colaboradores [Nambudiri, A. M., Ranganathan, S., and Rudney, H. (1980) The role of 3-hydroxy-3-methylglutaryl coenzyme A reducíase activity in the regulation of ubiquinone synthesis in human fibroblasts. The Journal of Biológica! Chemistry 255, 5894- 5899]. The biosynthesis of CoQ in the cultured cells was studied with the metabolic tide with radioactive precursors. To confirm the biochemical defect in the synthesis of CoQ, 4-hydroxy [U-14C] benzoic acid (4- [U-14C] HB), a precursor of the quinonic ring of CoQ, was used as a radioactive precursor, and [5- 3H] mevalonolactone ([3H] MVL) as a precursor to the isoprenoid side chain (American Radiolabeled Chemicals, St. Louis, MO), according to the protocol followed by Nambudiri et al [Nambudiri, AM, Ranganathan, S., and Rudney, H. (1980) The role of 3-hydroxy-3-methylglutaryl coenzyme A reduce activity in the regulation of ubiquinone synthesis in human fibroblasts. The Journal of Biological! Chemistry 255, 5894-5899].
La síntesis de proteínas mitocondriales en cultivos de fibroblastos control y MELAS se evaluó esencialmente midiendo la incorporación de [35S]- metionina en presencia de emetina (un inhibidor de la síntesis de proteínas citoplasmáticas). Las proteínas marcadas radioactivamente que corresponden a la síntesis mitocondrial fueron precipitadas con ácido tricloroacético (10%) y analizadas mediante un contador de centelleo líquido. The synthesis of mitochondrial proteins in cultures of control fibroblasts and MELAS was essentially evaluated by measuring the incorporation of [ 35 S] -methionine in the presence of emetine (an inhibitor of cytoplasmic protein synthesis). Radioactively labeled proteins corresponding to mitochondrial synthesis were precipitated with trichloroacetic acid (10%) and analyzed by a liquid scintillation counter.
La electrophoresis nativa en geles de poliacrilamida fue utilizada para la separación de los complejos respiratorios mitocondriales. Posteriormente las proteínas fueron electro-transferidas a una membrana de celulosa. Los diferentes complejos respiratorios fueron visualizados utilizando anticuerpos contra el complejo I, complejol I, complejo III, complejo IV y complejo V y son revelados por quimioluminiscencia. Native electrophoresis in polyacrylamide gels was used for the separation of mitochondrial respiratory complexes. Subsequently the proteins were electro-transferred to a cellulose membrane. The different respiratory complexes were visualized using antibodies against complex I, complexol I, complex III, complex IV and complex V and are revealed by chemiluminescence.
Los niveles de expresión de las proteínas mitocondriales fueron determinados por Western Blotting e Imnunofluorescencia. Muchas de las alteraciones mitocondriales cursan con niveles de expresión disminuidos de las proteínas mitocondriales. Sin embargo, en ocasiones se observan aumentos compensatorios de otras proteínas mitocondriales. La monitorización de los niveles de expresión de estas proteínas mediante las técnicas de Western Blotting e inmunofluorescencia sirvió para valorar la severidad de la enfermedad mitocondrial y comprobar la eficacia de los tratamientos ensayados. Expression levels of mitochondrial proteins were determined by Western Blotting and Imnunofluorescence. Many of the mitochondrial alterations present with decreased expression levels of mitochondrial proteins. However, sometimes compensatory increases in other mitochondrial proteins are observed. The monitoring of the expression levels of these proteins by Western Blotting and immunofluorescence techniques served to assess the severity of mitochondrial disease and verify the efficacy of the treatments tested.
En una célula animal típica, la mayor parte del ATP se sintetiza a partir de ADP y fosfato mediante la fosforilación oxidativa mitocondrial. En este proceso los tres centros redox de la cadena respiratoria mitocondrial bombean hidrogeniones desde la matriz mitocondrial al espacio intermembranas, desarrollando una fuerza protonmotriz o potencial de membrana mitocondrial del orden de - 180 mV. Esa fuerza protonmotriz permite la síntesis de ATP mediante la ATPsintetasa. Las alteraciones del potencial de membrana mitocondrial reflejan alteraciones funcionales de las mitocondrias. El potencial de membrana mitocondrial fue medido utilizando el fluorocromo JC-1 . In a typical animal cell, most of the ATP is synthesized from ADP and phosphate by mitochondrial oxidative phosphorylation. In this process, the three redox centers of the mitochondrial respiratory chain pump hydrogenions from the mitochondrial matrix to the intermembrane space, developing a protonmotrial force or mitochondrial membrane potential of the order of - 180 mV. This protonmotrial force allows the synthesis of ATP by means of ATPsintetase. The alterations of the mitochondrial membrane potential reflect functional alterations of the mitochondria. The mitochondrial membrane potential was measured using JC-1 fluorochrome.
Para determinar la producción de ROS, se utilizaron distintos fluorocromos sensibles a dichas ROS. La sonda 2'-7' diclorofluoresceina diacetato (DCFDA) es oxidada preferentemente por el H202 generando una fluorescencia verde. La fluorescencia se midió en un citómetro de flujo. To determine the production of ROS, different fluorochromes sensitive to said ROS were used. The 2'-7 'dichlorofluorescein diacetate (DCFDA) probe is preferably oxidized by H 2 0 2 generating a green fluorescence. Fluorescence was measured in a flow cytometer.
Para analizar la peroxidación lipídica, las mitocondrias aisladas se resuspendieron en PBS y se midieron los lipoperóxidos utilizando un kit LPO-560 (OxisResearch, Portland, OR). To analyze lipid peroxidation, isolated mitochondria were resuspended in PBS and lipoperoxides were measured using an LPO-560 kit (OxisResearch, Portland, OR).
Para determinar los niveles de daño oxidativo a las proteínas de las mitocondrias se midieron las proteínas mitocondriales carboniladas utilizando el kit OxyBlot (Intergen, Purchase, NY). To determine the levels of oxidative damage to mitochondrial proteins, carbonylated mitochondrial proteins were measured using the OxyBlot kit (Intergen, Purchase, NY).
En las enfermedades mitocondriales la alteración de la función mitocondrial induciría un aumento del estrés oxidativo mitocondrial, la activación de la transición de permeabilidad mitocondrial, y la activación de un programa de degradación selectiva de las mitocondrias malfuncionantes por mitofagia. La mitofagia fue caracterizada mediante las siguientes técnicas: 1 ) Observación de los autofagosomas por microscopía electrónica; 2) Aumento de la actividad lisosomal: ensayo de la beta-galactosidasa; 3) Colocalización de marcadores mitocondriales como el citocromo c con marcadores lisosomales como la catepsina o Lysotracker (invitrogen); 4) Localización mitocondrial de marcadores de autofagia como LC3, ATG12; 5) Expresión de los genes (genes ATG) y proteínas que participan en la mitofagia por Western Blotting y PCR a tiempo real. La integridad del flujo autofágico se midió mediante la incubación de los fibroblastos y cíbridos MELAS en presencia de bafilomicina y posteriormente determinando el aumento de LC3-II mediante Western blotting. In mitochondrial diseases, the alteration of mitochondrial function would induce an increase in mitochondrial oxidative stress, the activation of the mitochondrial permeability transition, and the activation of a program of selective degradation of malfunctioning mitochondria by mitophagy. The mitophagy was characterized by the following techniques: 1) Autophagosome observation by electron microscopy; 2) Increased lysosomal activity: beta-galactosidase assay; 3) Colocalization of mitochondrial markers such as cytochrome c with lysosomal markers such as cathepsin or Lysotracker (invitrogen); 4) Mitochondrial localization of autophagy markers such as LC3, ATG12; 5) Expression of genes (ATG genes) and proteins that participate in the mitophagy by Western Blotting and real-time PCR. Autophageal flow integrity was measured by incubating the MELAS fibroblasts and cybrids in the presence of bafilomycin and subsequently determining the increase in LC3-II by Western blotting.
Biogénesis mitocondrial se determinó mediante la masa mitocondrial (actividad citrato sintasa) y los niveles de expresión de los factores de biogénesis mitocondrial como mecanismo compensatorio ante la eliminación de las mitocondrias disfuncionales. Se estudió la expresión y activación de factores relacionados con la biogénesis mitocondrial como el factor de transcripción mitocondrial Tfam, el factor respiratorio nuclear NRF-1 y NFR-2, y el receptor activado de proliferación de peroxisomas (PGC-1 -alfa). Además se determinó el número de copias de ADN mitocondrial por PCR en tiempo real. Mitochondrial biogenesis was determined by mitochondrial mass (citrate synthase activity) and expression levels of mitochondrial biogenesis factors as a compensatory mechanism before eliminating dysfunctional mitochondria. The expression and activation of factors related to mitochondrial biogenesis such as mitochondrial transcription factor Tfam, nuclear respiratory factor NRF-1 and NFR-2, and activated peroxisome proliferation receptor (PGC-1-alpha) were studied. In addition, the number of mitochondrial DNA copies was determined by real-time PCR.
La apoptosis inducida fue estudiada tratando las células con camptotecina (un inhibidor de la topoisomerasa I) y retirada de suero o tratamiento con estaurosporina (inhibidor de la proteína quinasa C). La apoptosis se valoró mediante citometría de flujo e inmunofluorescencia. Se detectó en los cultivos de fibroblastos las células con la activación de las caspasas, la liberación de citocromo c, la condensación y fragmentación celular y demás parámetros característicos de las células apoptóticas. Induced apoptosis was studied by treating cells with camptothecin (a topoisomerase I inhibitor) and serum withdrawal or treatment with staurosporine (protein kinase C inhibitor). Apoptosis was assessed by flow cytometry and immunofluorescence. Cells with the activation of caspases, cytochrome c release, cell condensation and fragmentation and other characteristic parameters of apoptotic cells were detected in fibroblast cultures.
Los resultados demostraron que el tratamiento con CoQ y riboflavina aumentó la proliferación celular, aumentó los niveles de ATP, redujo la producción de ROS, disminuyó la actividad mitofágica y aumentó la expresión de proteínas y actividades enzimáticas mitocondriales en fibroblastos y cíbridos MELAS (Figura 5). Estos resultados indicaron que aquellos fármacos capaces de suprimir el defecto respiratorio en el cribado inicial en levaduras fueron igualmente capaces de revertir las alteraciones fisiopatológicas en fibroblastos y cíbridos MELAS. The results showed that treatment with CoQ and riboflavin increased cell proliferation, increased ATP levels, reduced ROS production, decreased mitophageal activity and increased protein expression and mitochondrial enzymatic activities in MELAS fibroblasts and cybrids (Figure 5) . These results indicated that those drugs capable of suppressing the respiratory defect in the initial screening in yeasts were also able to reverse the pathophysiological alterations in fibroblasts and MELAS cybrids.

Claims

REIVINDICACIONES
1 . Procedimiento para la identificación y evaluación de la eficacia de fármacos para el tratamiento de enfermedades que cursan con disfunción mitocondrial y/o síndrome de MELAS caracterizado porque comprende los siguientes pasos: a) cribado de fármacos en un modelo de Saccharomices cerevisiae mutante A14G, mediante la exposición de dichas levaduras a al menos un fármaco y determinar si dicho fármaco produce crecimiento celular de las levaduras mutantes A14G, b) evaluación de la eficacia del fármaco que produce crecimiento celular de las levaduras mutantes A14G del paso a) en modelos celulares derivados de pacientes con síndrome MELAS y determinar si el fármaco es eficaz mediante la capacidad que tiene dicho fármaco para restaurar las alteraciones fisiopatológicas de dichos modelos celulares, one . Procedure for the identification and evaluation of the efficacy of drugs for the treatment of diseases that occur with mitochondrial dysfunction and / or MELAS syndrome characterized in that it comprises the following steps: a) drug screening in a model of Saccharomices cerevisiae mutant A14G, by means of exposure of said yeasts to at least one drug and determine if said drug produces cellular growth of the A14G mutant yeasts, b) evaluation of the efficacy of the drug that produces cellular growth of the A14G mutant yeasts of step a) in cellular models derived from patients with MELAS syndrome and determine if the drug is effective through the ability of said drug to restore the pathophysiological alterations of said cellular models,
2. Procedimiento según la reivindicación 1 , caracterizado porque los modelos celulares derivados de pacientes con síndrome MELAS del paso b), son fibroblastos derivados de pacientes con síndrome MELAS y/o en cíbridos transmitocondriales MELAS. 2. Method according to claim 1, characterized in that the cellular models derived from patients with MELAS syndrome in step b) are fibroblasts derived from patients with MELAS syndrome and / or in MELAS transmitochondrial cybrids.
3. Procedimiento según cualquiera de las reivindicaciones anteriores, caracterizado porque las alteraciones fisiopatológicas restauradas en los modelos celulares del paso b) son un aumento de la proliferación celular, un aumento de los niveles de ATP, una disminución de ROS, una disminución de la actividad mitofágica, un aumento de la expresión de proteínas y/o aumento de la actividad mitocondrial. 3. Method according to any of the preceding claims, characterized in that the pathophysiological alterations restored in the cellular models of step b) are an increase in cell proliferation, an increase in ATP levels, a decrease in ROS, a decrease in activity mitophageal, an increase in protein expression and / or increased mitochondrial activity.
4. Procedimiento según cualquiera de las reivindicaciones anteriores, caracterizado porque el crecimiento celular de las levaduras mutantes A14G del paso a) se realiza mediante densidad óptica. 4. Method according to any of the preceding claims, characterized in that the cell growth of the A14G mutant yeasts of step a) is performed by optical density.
PCT/ES2013/070388 2012-06-15 2013-06-14 Method for screening and/or evaluating the efficacy of medicaments for the treatment of mitochondrial diseases and melas syndrome WO2013186422A1 (en)

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Citations (2)

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WO2003087768A2 (en) * 2002-04-12 2003-10-23 Mitokor Targets for therapeutic intervention identified in the mitochondrial proteome

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