WO2018104172A1 - Composés pour le traitement du syndrome de barth - Google Patents

Composés pour le traitement du syndrome de barth Download PDF

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WO2018104172A1
WO2018104172A1 PCT/EP2017/081199 EP2017081199W WO2018104172A1 WO 2018104172 A1 WO2018104172 A1 WO 2018104172A1 EP 2017081199 W EP2017081199 W EP 2017081199W WO 2018104172 A1 WO2018104172 A1 WO 2018104172A1
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protein synthesis
compound
cytosolic protein
organism
taz
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PCT/EP2017/081199
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English (en)
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Déborah TRIBOUILLARD-TANVIER
Jean-Paul LASSERRE
Jean-Paul Di Rago
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Centre National De La Recherche Scientifique (Cnrs)
Universite de Bordeaux
Association Francaise Contre Les Myopathies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/45—Non condensed piperidines, e.g. piperocaine having oxo groups directly attached to the heterocyclic ring, e.g. cycloheximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to compounds and method for treating and/or preventing Barth syndrome as well as methods for identifying such compounds.
  • Barth Syndrome is an X-linked recessive disorder that is characterized by cardiac and skeletal myopathies, growth retardation, hypocholesterolemia, increased urine levels of 3-methylglutaconic acid, increased susceptibility to bacterial infections due to cyclic neutropenia, and abnormal mitochondria and respiratory chain dysfunction. Consequences of Barth syndrome include cardiomyopathy, neutropenia, skeletal myopathy, and growth delay.
  • Mitochondria of BTHS patients have an abnormal ultrastructure and are characterized by pleiotropic respiratory defects in muscle, fibroblasts, and iPSCs- derived cardiomyocytes.
  • the Barth syndrome is very often fatal in childhood due to the cardiac failure or sepsis and there is still no effective treatment.
  • a reduced level of mature cardiolipin (CL), and an accumulation of monolysocardiolipin (MLCL), an intermediate in the CL remodeling pathway has also been shown.
  • Cardiolipin is a lipid mostly found in the inner mitochondrial membrane (IMM) of eukaryotic cells. CL has been shown to play a role in numerous processes involving the mitochondria as oxidative phosphorylation and stabilization of supercomplexes, fusion, fission, protein import, iron-sulfur biogenesis, mitophagy, apoptosis and transport of metabolites for energy metabolism. CL plays a critical role in maintaining respiratory chain enzyme function, membrane stability, and mitochondrial dynamics. CL is synthesized exclusively in the mitochondria inner membrane as immature CL and is further remodeled into mature CL. Functional mature CL corresponds to cardiolipin with unsaturated fatty acyl chains, particularly four unsaturated fatty acyl chains, particularly C18:2 chains.
  • MLCL monolysocardiolipin
  • MLCL monolysocardiolipin
  • tafazzin protein an acyl transferase located in the mitochondria, which is encoded by the nuclear TAZ gene, is located in the distal region of chromosome Xq28.12 and is crucial for the final remodelling and maturation of CL. Mutations in the TAZ gene have been shown to be responsible for Barth Syndrome.
  • Barth Syndrome is considered as a rare disease with less than 500 patients diagnosed in the world. It is very likely that this disease is highly under-diagnosed, affecting about 1 out of 300 000 children. Thanks to early diagnosis, which allows management of the various symptoms, the prognosis is strongly improved. However, in the past, children only rarely reached the age of three and often died of heart failure or infection. There is currently no definitive treatment specific to Barth syndrome and the treatment is essentially symptomatic and multidisciplinary.
  • Heart failure can be for instance treated by conventional treatments or by heart transplantation.
  • the risk of bacterial sepsis in case of intermittent neutropenia can be controlled by prophylactic antibiotic therapy or intermittent administration of granulocytes growth factor (G - CSF).
  • G - CSF granulocytes growth factor
  • EP2012800 discloses compositions comprising ketoconazole 2S, 4R enantiomer, said pharmaceutical compositions being used to increase the synthesis of cardiolipin for the treatment of Barth syndrome, diabetic myopathy and/or cardiomyopathy associated with aging, as well as mitochondrial diseases and other ailments.
  • WO2014134554 concerns methods of prevention or treatment of Barth syndrome in a mammal using a cationic peptide aromatic (D-Arg-2'6'-Dmt-Lys-Phe-NH2), so as to increase the expression of TAZ in patients in need thereof. It is thus an object of the present invention to provide means and methods for the prevention and/or treatment of Barth syndrome.
  • a cationic peptide aromatic D-Arg-2'6'-Dmt-Lys-Phe-NH2
  • the present invention concerns, in a first embodiment, a compound that inhibits the cytosolic protein synthesis for its use in the prevention and/or treatment of a disorder or disease associated to mitochondrial dysfunction, wherein the mitochondrial dysfunction is associated with a deficiency in mature cardiolipin level in mitochondria membrane.
  • the expression "disorder or disease associated to mitochondrial dysfunction” refers to disorders or diseases resulting from failures or dysfunction of the mitochondria, leading to less and less energy being generated within the cell. If this process is repeated throughout the body, whole systems begin to fail, and the life of the person in whom this is happening is severely compromised.
  • the disease primarily affects children, but adult onset is becoming more and more common.
  • Mitochondrial diseases or mitochondrial disorders may be the result of either inherited or spontaneous mutations in mtDNA or nDNA (nuclear DNA) which lead to altered functions of the proteins or RNA molecules that normally reside or play a role in mitochondria.
  • Mitochondrial diseases appear to cause the most damage to cells of the brain, heart, liver, skeletal muscles, kidneys, eyes and endocrine and respiratory systems.
  • symptoms may include loss of motor control, muscle weakness and pain, gastro-intestinal disorders and swallowing difficulties, growth delay, cardiac disease, liver disease, diabetes, respiratory complications, seizures, visual/hearing problems, lactic acidosis, developmental delays and susceptibility to infection.
  • prevention and/or treatment refers to situations where one can prevent or slow down the occurrence of the disorder or of the disease or prevent or slow down the development of the disorder or of the disease or even can treat, i.e. revert, the disorder or the disease.
  • prevention and/or treatment may also include alleviation of at least one of the symptoms of the disorder or disease.
  • mature cardiolipin refers to cardiolipin with unsaturated fatty acyl chains, particularly four unsaturated fatty acyl chains, particularly diunsaturated fatty acyl chains, more particularly C18:2 chain.
  • Mature cardiolipin thus particularly corresponds to linoleoyl-cardiolipin (TLCL), i.e. cardiolipin in which the majority, particularly all four, phosphatidyl acyl groups are specified as linoleoyi.
  • the method of direct analysis of cardiolipin and its derived lyso-compounds in intact blood white cells can represent a tool useful for the diagnosis, screening and clinical monitoring of mitochondrial diseases and disorders, such as Barth syndrome.
  • Such a ratio can be used to identify deficiency in mature cardiolipin level and such a measurement can be made according to any appropriate method described in the literature such as in J Lipid Res. 2015 Sep;56(9):1787-94 or The FASEB Journal vol. 29 no. 1 Supplement 885.27 for example.
  • the invention concerns a compound for its use according the first embodiment, characterized in that the deficiency in mature cardiolipin level in mitochondria membrane is associated with a TAZ gene mutation.
  • TAZ gene encodes a phospholipid transacylase that is localized in mitochondria and functions in cardiolipin acyl chain remodeling and maturation. While cardiac tissue usually comprises > 90 % tetra- linoleoyl cardiolipin (CL), ie mature cardiolipin, subjects with Barth syndrome show characteristic differences in cardiolipin acyl chain composition, including decreased levels of mature cardiolipin.
  • CL tetra- linoleoyl cardiolipin
  • the disorders or diseases associated to mitochondrial dysfunction that can be treated with the compound of the invention are thus preferably characterized by a mutation of TAZ gene.
  • the protein encoded by mutated TAZ gene, a phospholipid transacylase is missing and/or nonfunctional or mislocalized in cells.
  • missing it is meant that the protein is not synthetized/translated or prone to degradation and thus degraded by the cellular machinery prior to performing its function.
  • non-functional it is meant that the protein exists but is not able to perform its function, for example because of misfolding, unfolding, editing.
  • a nonfunctional protein can also be degraded by the proteolysis machinery of the cell
  • the invention concerns a compound for its use according to the second embodiment, characterized in that the mutation corresponds to an under- expression or extinction of the TAZ gene.
  • the invention concerns a compound for its use according to any preceding embodiments, characterized in that the disorder or disease is Barth syndrome.
  • the invention relates to a compound for its use according to any preceding embodiment, characterized in that the disorder or disease comprises one or more of the following pathologies : cardiomyopathy, skeletal muscle abnormalities, neutropenia, growth delay, weak muscle tone and bacterial infections.
  • cardiomyopathy is to be understood as a weak heart muscle usually associated with enlargement of the heart (usually dilated with variable myocardial hypertrophy, sometimes with left ventricular noncompaction and/or endocardial fibroelastosis).
  • Neutropenia means a reduction in neutrophils, a type of white blood cells that is most important for fighting bacterial infections. Neutropenia may predispose an individual to mouth ulcers, fevers and bacterial infections such as bacterial pneumonia and skin abscesses.
  • underdeveloped skeletal musculature and muscle weakness means that all muscles, including the heart, suffer from a cellular deficiency which limits their ability to produce energy. Muscle weakness and increased exertional fatigue are characteristic findings in Barth syndrome.
  • the invention concerns a compound for its use in the treatment of Barth syndrome, wherein such compound inhibits the cytosolic protein synthesis.
  • the invention concerns a compound for its use in the treatment of Barth syndrome, wherein the compound inhibiting the cytosolic protein synthesis is selected in the group consisting of cycloheximide, emetin, and anisomycin ; or any pharmaceutically acceptable salt or prodrug thereof.
  • prodrug relates to a compound that, after administration, is metabolized (i.e., converted within the body) into a compound that inhibits cytosolic protein synthesis.
  • the term "pharmaceutically acceptable salt” relates to any pharmaceutically acceptable salt that can be prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • the pharmaceutical salts include any salt suitable to be administered to humans or animals.
  • non-toxic pharmaceutically acceptable salts may include hydrochloride, carboxylate sodium, carboxylate disodium, sodium, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, citric acid, maleic acid, and succinic acid salts.
  • the invention deals with a compound for its use according to any preceding embodiment, characterized in that the compound is selected in the group consisting of cycloheximide, emetin, and anisomycin ; or any pharmaceutically acceptable salt or prodrug thereof.
  • the compound for its use according to the invention is selected in the group of compounds inhibiting cytosolic protein synthesis, i.e. cytosolic translation inhibitors. Suitable compounds may act at different levels of protein synthesis, and can inhibit any stage chosen among initiation, translocation, elongation and termination. As exposed above, some particular compounds are cycloheximide, emetin, and anisomycin.
  • antibiotics compounds capable of inhibiting cytosolic protein synthesis can be selected as suitable compounds according to the invention and one can cite various antibiotic inhibitors of 40S ribosome and/or 60S ribosome such as tubulosine, tylophorine, abrine, ricine or sarcine alpha for example.
  • the functionality of inhibition of cytosolic protein synthesis is a routine measurement that can easily be put into practice for screening and identifying compounds according to the invention.
  • the present description as well as abundant prior art describe usual techniques available for evaluation of protein homeostasis, thus for measuring cytosolic protein synthesis, including measuring the inhibition of cytosolic protein synthesis.
  • cytosolic protein synthesis or “inhibition of cytosolic protein synthesis” is to be understood that the compound of the invention is able to affect protein homeostasis in the cytosol to such extent that less proteins are produced in the cytosol of the cell as a result of effect at various stage of protein synthesis, be it inhibition of the transcription or inhibition of the translation, at any stage, since interaction of a large number of factors that catalyze the assembly of ribosomes, mRNA templates, and aminoacylated tRNAs may play a role in protein synthesis.
  • the functional activity defined as the present invention is the essential feature of the invention whatever the structure of the compound which can be selected in a wide variety of chemical, , particularly among antibiotics that are inhibitors of eukaryotic cytosolic protein synthesis.
  • Compounds with that functionality of inhibiting protein synthesis are part of the art and that functionality has clear technical meaning. Furthermore, this functionality can be measured/verified by tests and procedures well known or described in the present description and in any case without undue burden.
  • RNA. 2004 Mar; 10(3): 528-543 Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles
  • the invention concerns a method, preferably an in vitro method for selecting a compound capable of inhibiting the cytosolic protein synthesis and restoring respiratory function of the mitochondria comprising the steps of :
  • the deficiency of mature cardiolipin level corresponds to a ratio monolysocardiolipin (MLCL) /cardiolipin (CL) in the cell, comprised between 5 and 15.
  • the method, preferably the in vitro method, of the invention according to the seventh embodiment is characterized in that the cytosolic protein synthesis inhibition is partial.
  • the method, preferably the in vitro method, of the invention according to the eighth embodiment is characterized in that the cytosolic protein synthesis inhibition is comprised between 0.5 and 10%, particularly 0.5 and 5%, more particularly 0.5 and 2%.
  • inhibittion it is understood that the protein synthesis is decreased by 0.5 to 10%, particularly by 0.5 to 5%, more particularly by 0.5 to 2%
  • the present invention also provides a method, preferably an in vitro, method according to previous seventh to ninth embodiments, characterized in that the cytosolic protein synthesis inhibition rescues the respiratory growth of TAZ mutated organism, wherein the TAZ mutated organism is a TAZ1 mutated yeast.
  • the present invention also provides a method, preferably an in vitro method, according to previous seventh to ninth embodiments, characterized in that partial cytosolic protein synthesis inhibition allows maintenance of the mitochondrial genome in the TAZ mutated organism.
  • the present invention also provides a method, preferably an in vitro method, according to previous seventh to ninth embodiments, characterized in that partial cytosolic protein synthesis inhibition rescues the ATP production and mitochondrial membrane potential in the TAZ mutated organism.
  • the present invention also provides a method, preferably an in vitro method, according to seventh to ninth embodiments, characterized in that partial cytosolic protein synthesis inhibition rescues the OXPHOS assembly in the TAZ mutated organism.
  • the invention provides a method, preferably an in vitro method, according to any one of seventh to thirteenth embodiments, characterized in that the organism wherein the TAZ gene is mutated is a TAZ1 mutated yeast and the mutation consists in a deletion, a substitution or an insertion which leads to a null allele.
  • a null allele is herein understood as a mutant copy of a gene at a given locus that completely lacks the gene's normal function. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product.
  • the step of contacting the compound with the organism can be achieved by any technology known to the skilled person provided that the compound to be tested enters the inside of the cell(s) of the organism in order it can exert its effect.
  • the invention also relates to the use of an organism deficient in mature cardiolipin for the screening and selection of compounds capable of inhibiting the cytosolic protein synthesis and restoring respiratory mitochondrial function.
  • the present invention also provides a method for treating Barth Syndrome in a mammalian subject, the method comprising: administering to the subject a therapeutically effective amount of a compound that partially inhibits the cytosolic protein synthesis and induces at least one effect selected in the group consisting of : rescue of the ATP production and mitochondrial membrane potential ; maintenance of the mitochondrial genome ; rescue of the OXPHOS assembly; rescue the respiratory rate and decrease the reactive oxygen species (ROS) production.
  • ROS reactive oxygen species
  • Cycloheximide (CHX) and anisomycin (ANI) suppress the respiratory growth defect of the tazl mutant strain.
  • A. tazl mutant strain was spread onto respiratory ethanol-based media. Small sterile filters were placed on the agar surface and increasing concentrations of CHX and ANI were added to the filters at the indicated quantities (in nmoles). The plates were then incubated at 36°C and scanned after 5 days.
  • the indicated cultures were inoculated with precultures in respiratory media at restrictive temperature diluted to 0.5OD/ml_ in 50ml_ of complete synthetic medium CSM + 0.5% galactose + 2% ethanol in presence of the indicated concentration of DMSO (vehicule, negative controle) or cycloheximide. Cultures were diluted every day in fresh media and drugs were added in the same time. Optical densities were taken at different time over a week.
  • C Serial dilutions of the indicated strains were spotted onto ethanol and glucose media. The plates were incubated at the indicated temperature and scanned after three (glucose) or six (ethanol) days.
  • D Growth curve in liquid respiratory medium.
  • the indicated cultures were inoculated with precultures in respiratory media at restrictive temperature diluted to 0.5OD/ml_ in 50ml_ of complete synthetic medium CSM + 0.5% galactose + 2% ethanol. Optical densities were taken at different time over a week.
  • FIG. 1 Mitochondrial membrane potential. Energization of the mitochondrial inner membrane. Variations in ⁇ were monitored by the fluorescence quenching of rhodamine 123 as described in the material and methods section. 75 g of osmotically-protected mitochondria of the indicated strains were used in these experiments. (Mito: mitochondria; EtOH: ethanol; KCN: potassium cyanide; ADP: adenosine diphosphate; ATP: adenosine triphosphate; CCCP: carbonylcyanide-m- chlorophenyl hydrazone; Oligo: oligomycin). Fluorescence traces are representative of four experimental trials. Figure 3. Oxidative phosphorylation in tazIA mitochondria. A.
  • Asc Ascorbate
  • TMPD 1 .4 mM N,N,N',N'-tetramethyl-p-phenylenediamine
  • FIG. 4 OXPHOS assembly/stability/activity.
  • Mitochondrial proteins were extracted with digitonin (4g/g), separated by BN-PAGE, and transferred to membranes for western blottings with the Cox2 and Cytb antibodies.
  • D SDS-PAGE analysis. 50 g of total proteins extracts were separated in SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the Hsp60, Ccp1 and Ade13 antibodies. Data were standardized to the loading control Ade13. Data were shown as relative to the WT. Data are representive of at least 3 experiments.
  • E. ROS production Cells were grown at 36°C to the exponential phase in CSM + 0.5% galactose + 2% ethanol and the measures were done at the indicated time point as described under Experimental procedures.
  • Atazl mutant strain wild-type, WT, MATa ade2-1 ura3-1 his3-1 1 , 15 trp1 -1 Ieu2-3,1 12 can1 -100, taz1::TRP1 was used for the construction of the double mutant strains Ataz1/Arpl6b and Ataz1/Arei1.
  • Fermentable YPAD media contain 1 % (w/v) yeast extract, 2% (w/v) bacto peptone, 60 mg/L adenine and 2% (v/v) glucose;
  • Respiratory rich media YPAEthanol contain 1 % (w/v) yeast extract, 2% (w/v) bacto peptone, 60 mg/L adenine and 2% (v/v) ethanol and non-fermentable complete synthetic media
  • CSM/gal/ethanol contain 0.17% (w/v) yeast nitrogen base without aminoacids and ammoniumsulfate, 0.5% (w/v) ammonium sulfate, 0,5% (w/v) galactose, 2% ethanol and 0.8% (w/v) of a mixture of aminoacids and bases from Formedium. Ethanol was added after the sterilization.
  • the solid media contained 2% (w/v) agar.
  • Ataz1/Arpl6b and Ataz1/Arei1 strains were constructed as follows. The entire open reading frame of RPL6B or REI1 was replaced by the KanMX marker using a PCR-mediated one-step gene replacement strategy.
  • the KanMX cassette was amplified from pFA6a- Kan: Rpl6b-del-F (CTT TCT TGA ACT TGG AAG AGA AGC AAA TAT ATT CAA CGA A egg ate ccc ggg tta att aa) (SEQ ID ⁇ ) and Rpl6b-del-R (CTA TTT TAA ATC ATT TAT AAT TTT TTC AGT TCA AT gaa ttc gag etc gtt taa ac) (SEQ ID N°2), consisting of 40-nucleotide and 35-nucleotide sequences identical to the RPL6B flanking regions at the 5' ends (capital letters) and 20 nucleotides of sequence specific for the amplification of the KanMX marker gene at the 3' ends (small letters) of each primer.
  • the KanMX cassette was amplified from pFA6a-Kan: Rei1 -del-F (CAT TAG AAG TCA AGA AGA GAG CAT ATC AGT AAC AAT ACG egg ate ccc ggg tta attaa) (SEQ ID N°3) and Rei1 -del-R (GCG ACA AAA TAC TAA AAA AAG TAG TGC AAA AAG AA gaa ttc gag etc gtt taa ac) (SEQ ID N°4), consisting of 39-nucleotide and 35-nucleotide sequences identical to the REI1 flanking regions at the 5' ends (capital letters) and 20 nucleotides of sequence specific for the amplification of the KanMX marker gene at the 3' ends (small letters) of each primer.
  • the PCR product was purified and transformed into Atazl strain, and transformants were selected on agar plates added with geneticin (G418 purchased from Sigma).
  • the presence of a KanMX module instead of RPL6B or REI1 was verified by PCR analysis using primers specific for the flanking regions of RPL6B: Rpl6b-Fbis (CTG CGC TTC CGT TCA GCA TC) (SEQ ID N°5), and Rpl6b-Rbis (CGA TGA CCT GAT CTT GAA CCC) (SEQ ID N°6) or RE 11: Rei1 -Fbis (GTG GTG TAG CTA TTT GTA CAT G) (SEQ ID N°7), and Rei1 -Rbis (CAA CAT CTT CAG TCT TCA GCA GC) (SEQ ID N°8).
  • Yeast-based drug assay 125 OD of exponentially growing cell cultures was spread homogeneously with sterile glass beads on a square Petri dish (12 cm x 12cm) containing YPAEthanol solid medium. Sterile filters were placed on the agar surface. Cycloheximide (purchased from Sigma) and Anisomycin (purchased from Sigma) were applied at the indicated quantities to each filter in addition to the top left filter where DMSO, the vehicle, was added as a negative control. Plates were then incubated at 36°C for several days and scanned.
  • the mitochondria were prepared by the enzymatic method as described.
  • the protein amounts were determined by the Lowry method in the presence of 5% SDS.
  • Oxygen consumption rates were measured on 75 g of fresh mitochondria using a Clarke electrode in the respiration buffer (0.65 M mannitol, 0.36 mM ethylene glycol tetraacetic acid, 5 mM Tris-phosphate, 10 mM Tris-maleate, pH 6.8) after consecutively adding 4 mM NADH (complex I respiration) or Ascorbate (Asc) and 1 .4 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (complex IV respiration) as a substrate, 150 ⁇ ADP (state 3) and 4 ⁇ carbonyl cyanide m-chlorophenylhydrazone (CCCP) (uncoupled respiration), as described.
  • CCCP carbonyl cyanide m-chlorophenylhydrazone
  • the rates of ATP synthesis were determined in the presence of 4 mM NADH and 1 mM ADP; aliquots were withdrawn from the oxygraph cuvette every 15 seconds and the reaction was stopped with 3.5% (w/v) perchloric acid, 12.5 mM EDTA. The samples were then neutralized to pH 6.5 by addition of KOH, 0.3 M MOPS. Synthetized ATP was quantified using a luciferin/luciferase assay (ATPLite kit from Perkin Elmer) on a LKB bioluminometer. The specific ATPase activity was measured at pH 8.4 by using a previously described procedure . The participation of the FiF 0 -ATP synthase in ATP production or hydrolysis was assessed by addition of oligomycin (2 mg/ml of protein).
  • BN-PAGE & SDS-PAGE Blue native BN-PAGE and clear native CN-PAGE experiments were carried out as described previously in the art. Briefly, mitochondrial extracts solubilized with digitonin to an indicated protein ratio were separated in a native 3-12% acrylamide continuous gradient gel.
  • the gel was either stained with coomassie blue or incubated in a solution of 8 mM ATP pH7, 14 mM MgCI 2 , 0,2% lead nitrate, 35 mM Tris, 270 mM glycine pH 8.4, to detect the ATPase activity; a solution of Tris 5mM pH 7,4, Nitroblue tetrazolium (1 mg/ml), sodium succinate 20 mM, phenazine methosulfate 0,2 mM to detect the complex II activity; a solution of Tris 5 mM pH 7,4, diaminobenzidine 0,5 mg/ml, cytochrome C 0,05 mM to detect the complex IV activity or transferred to poly(vinylidene difluoride) membranes and analyzed by Western blotting.
  • Claypool 1 :1000 for Hsp60 (kind gift from Martin van der Laan) and 1 :5000 for ADE13.
  • Monoclonal antibodies against yeast porin and yeast cytochrome c oxidase COX2 (from Molecular Probes) were used at a dilution of 1 :5000.
  • Nitrocellulose membranes were incubated with peroxidase-labeled antibodies at a 1 :2500 dilution (Promega), and the blot visualization was conducted with an electrochemiluminescence reagent. Quantification was performed with the ImageJ software.
  • Lipids from isolated mitochondria were extracted with 2 ml of chloroform/methanol (2:1 , v/v). After centrifugation, the organic phase was isolated and the remaining lipids were further extracted twice by the addition of 2 ml of chloroform to the aqueous phase. After the phase separation, the organic phases were pooled and evaporated to dryness. The lipids were then suspended in chloroform/methanol (2:1 , v/v). Volumes equivalent to 50 g of acyl chains were spotted on silica plates four times for each strain.
  • Polar lipids were separated by one dimensional TLC using chloroform/methanol/1 -propanol/methyl acetate/0.25% KCI (10:4:10:10:3.6, by vol.) as a solvent. Thin layer chromatography of phospholipids. The lipids were then located by immersing the plates in a solution of 0.001 % (w/v) primuline in PBS, followed by visualization under UV light. The silica gel zones corresponding to the various lipids (PE, CL, PI and PC) were then scraped from the plates and added to 1 ml of methanol/2.5% H2SO4 containing 5 ⁇ g of heptadecanoic acid methyl ester as a standard.
  • Specimens were observed with a Hitachi 7650 (80 kV) electron microscope (Electronic Imaging Pole of Bordeaux Imaging Center). The number of mitochondria was averaged from 50 observed cells in which the nucleus and the vacuole were seen. ROS analysis by flow cytometry. 0.4 OD were taken from the indicated cultures, and the cells were pelleted in a microcentrifuge. After removal of the media, the cell pellets were resuspended in 1 ml of phosphate-buffered saline (PBS) containing 50 ⁇ dihydroethidium (DHE; Molecular Probes) and incubated at room temperature for 5 min.
  • PBS phosphate-buffered saline
  • DHE dihydroethidium
  • Flow cytometry was carried out on a Becton-Dickinson Accuri C6 model flow cytometer.
  • the DHE fluorescence indicated was the direct output of the FL2A (red fluorescence-detecting) channel without compensation. A total of 100,000 cells were analyzed for each curve.
  • Cells were harvested from exponential phase cultures by a standardized detachment procedure using 0.05% Trypsin-EDTA (Invitrogen). Flow cytometry was used to count the cells and test their viability (FSC versus propidium iodide staining). 5000 or 7500 cells in 20 ⁇ were added in each well. After leaving the plates at room temperature for 30 min to allow early cell attachment, in accordance with the manufacturer's guidelines, they were locked in the RTCA DP device in the incubator and the impedance value of each well was automatically monitored by the xCELLigence system and expressed as a Cell Index value (CI).
  • CI Cell Index value
  • Fig. 1A a halo of enhanced growth around filters was seen.
  • the advantage of this method is that, in one simple experiment, it allows compounds to be tested across a large range of concentrations due to diffusion of the drugs in the growth medium.
  • Both drugs, CHX and ANI were toxic at high concentration as seen by the halo of no growth near the filter but were very active at subtoxic concentrations.
  • CHX is active at concentrations at least 10 fold lower than active concentrations of ANI (Fig. 1A).
  • both drugs partially suppressed, in a dose-dependent manner, the respiratory growth defects of the tazl mutant strains (Fig. 1A).
  • Experiments were pursued with the most active of the drugs, CHX and confirmed its activity in liquid respiratory growth media.
  • the growth of the tazIA mutant showed a slower fermentable growth on glucose media at 28°C than the isogenic WT which correlated well with the slower fermentable growth in liquid respiratory media at 36°C (as long as galactose was present, the tazIA mutant grew by fermentation, then, the tazIA mutant stopped growing due to its respiratory deficiency that prevents it to utilize ethanol) (Fig. 1 C, 1 D).
  • the mitochondrial electrical potential ( ⁇ ) was analysed across the inner membrane via fluorescence quenching rhodamine 123. Adding ADP to WT mitochondria energized with ethanol induced a fluorescence variation that progressively disappeared due to the phosphorylating activity of the F 0 Fi-ATP synthase ( Figure 2A). The membrane potential increased when most of the ADP had been transformed into ATP. As expected, further additions of KCN and CCCP to these mitochondria led to a collapse of the membrane potential. In the mitochondria isolated from the tazIA strain, even though ethanol was able to establish an electrical membrane potential, ADP poorly induced a decrease in ⁇ ( Figure 2A).
  • the oxygen consumption activity of the indicated strains As shown previously, using either NADH or ⁇ /, ⁇ /, ⁇ /', ⁇ /'-tetramethyl-p-phenylenediamine (TMPD) / Ascorbate) in the presence of CCCP as an electron donor, the oxygen consumption was reduced by approximately 50% in tazIA mitochondria with respect to the WT (Fig. 3A). However, it was observed that REI1 or RPL6B mutations in the tazIA mutant strain allowed to restore an oxygen consumption as high as the isogenic WT (Fig. 3A). This result was consistent with the respiratory growth obtained on solid and liquid media at 36°C.
  • TMPD ⁇ /, ⁇ /, ⁇ /', ⁇ /'-tetramethyl-p-phenylenediamine
  • Hsp60 a mitochondria-specific molecular chaperone that is the marker of the mitochondrial unfolded protein response (UPRmt)
  • Ccp1 the cytochrome c peroxidase that modulate the antioxidant defense are overexpressed in the tazIA mutant in presence of CHX and in the double mutants taz1/rei1 and taz1/rpl6b (Fig. 4D).
  • Partial inhibition of cytosolic protein synthesis decreases the level of ROS and increases the number of mitochondria in the tazIA yeast mutant strain
  • a metabolic modification in the cytosolic compartment can be beneficial to restore a mitochondrial functioning in the context of a mitochondrial proteostatic stress by inducing the UPRmt that includes the generation of mitochondrial proteostasis machinery such as mitochondrial molecular chaperones and proteases as well as antioxidant genes to limit mitochondrial damages.
  • Decreasing cytosolic protein synthesis is also beneficial to tafazzin-deficient human cells.
  • the previously described HeLa cells were used, in which the TAZ gene was knocked down by RNA interference (shTazl ) and two control cell lines, shWT1 and shTazI R in which the expression of TAZ1 was not inhibited (Gonzalvez F, Schug ZT, Houtkooper RH, MacKenzie ED, Brooks DG, Wanders RJ, Petit PX, Vaz FM, Gottling E (2008) Cardiolipin provides an essential activating platform for caspase-8 on mitochondria. The Journal of cell biology 183: 681 -96).
  • ShTazl poorly accumulates Tafazzin, is defective in CL maturation, presents a destabilization of the supercomplexes, produces abnormally enlarged cells and has a higher content in mitochondria compared to shWT1 and shTazl R, as do cells from Barth syndrome patients.
  • ShTazl cells proliferate four times slower and die more rapidly, in comparison to ShWT1 and ShTazRI cells (Fig.6 A, B).
  • the inventors decided to test the capacity of CHX at counteracting the detrimental effects of a lack in CL maturation in human cells. A large beneficial effect was observed at a 50 pM concentration of CHX.
  • the drug was added 24-25 hours after inoculating 200 ⁇ of media with 5000 cells.
  • CHX induced a 48h lag phase, after which the cells grew for a long-lasting period of 170 hours, before dying and detaching from their support.
  • death was observed much more rapidly, after 72 hours of continuous growth.
  • the present description also shows that decreasing cytosolic protein synthesis is beneficial to human cells that are defective in CL remodeling.

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Abstract

La présente invention concerne des composés et une méthode pour le traitement et/ou la prévention du syndrome de Barth ainsi que des procédés d'identification de tels composés. En particulier, l'invention concerne des composés qui inhibent la synthèse des protéines cytosoliques pour utilisation dans la prévention et/ou le traitement d'un trouble ou d'une maladie associé à un dysfonctionnement des mitochondries tel que le syndrome de Barth, le dysfonctionnement des mitochondries étant associé à une insuffisance de cardiolipine mature dans la membrane mitochondriale.
PCT/EP2017/081199 2016-12-06 2017-12-01 Composés pour le traitement du syndrome de barth WO2018104172A1 (fr)

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WO2018228997A1 (fr) * 2017-06-12 2018-12-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Modulation de cellules immunitaires dans la synthèse de la cardiolipine

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