WO2016103223A1 - Procédés de traitement de l'ataxie de friedreich au moyen d'inhibiteurs de src - Google Patents

Procédés de traitement de l'ataxie de friedreich au moyen d'inhibiteurs de src Download PDF

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WO2016103223A1
WO2016103223A1 PCT/IB2015/059963 IB2015059963W WO2016103223A1 WO 2016103223 A1 WO2016103223 A1 WO 2016103223A1 IB 2015059963 W IB2015059963 W IB 2015059963W WO 2016103223 A1 WO2016103223 A1 WO 2016103223A1
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src inhibitor
frataxin
administered
subject
src
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PCT/IB2015/059963
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Roberto Testi
Florence MALISAN
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Fratagene Therapeutics Ltd.
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Publication of WO2016103223A1 publication Critical patent/WO2016103223A1/fr
Priority to US15/630,328 priority Critical patent/US20170296540A1/en

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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • FRDA Friedreich's ataxia
  • FRDA is caused by homozygous hyperexpansion of GAA triplets in intron 1 of the frataxin (FXN) gene on chromosome 9ql3.
  • FXN frataxin
  • Frataxin is an extremely conserved mitochondrial protein synthesized as a cytosolic 210 amino acid precursor, which is then imported into mitochondria following a two-step proteolytic processing by a mitochondrial processing peptidase.
  • frataxin deficiency in humans critically affects survival of large primary neurons of the dorsal root ganglia, cardiomyocytes, and pancreatic ⁇ -cells.
  • frataxin-defective cells have reduced activity of iron sulfur cluster (ISC)-containing enzymes, a general imbalance in intracellular iron distribution, reduced ATP content, and increased sensitivity to oxidative stress, with increased ROS generation.
  • ISC iron sulfur cluster
  • Src tyrosine kinases can be regulated by a variety of important mitochondrial signals such as ATP levels, redox state, which are dysregulated in FRDA. Hebert-Chatelain, E. (2013), Int J Biochem Cell Biol, 45, 90-98.
  • the present invention shows that blocking Src activity with Src inhibitors increases frataxin levels.
  • Src inhibitors induce frataxin expression in cells derived from FRDA patients, though Src inhibitors failed to upregulate frataxin in human cells in which a frataxin-Yl 18F mutant was expressed. Therefore, Src inhibitors can promote frataxin accumulation in living cells, thus mitigating or alleviating the damaging effects of FRDA.
  • the present invention sets forth a method of preventing ubiquitination on K 147 could grant frataxin an increased stability and a prolonged half-life.
  • the invention provides a method of treating Friedreich's ataxia that includes administering to the subject a therapeutically effective amount of an Src inhibitor or a pharmaceutically acceptable salt.
  • the invention provides a method of modulating the phosphorylation of frataxin on Yl 18 by Src kinase. In one aspect, the invention provides a method of increasing frataxin levels in living cells, which may be in a subject suffering from FRDA or from low levels of frataxin. In one aspect, the method slows or otherwise palliates one or more clinical effects associated with FRDA or with low levels of frataxin.
  • FIG. 1A Src kinase phosphorylates frataxin.
  • FIG. 1A HEK293 cells were transiently transfected with frataxin (FXN), and either constitutively active Src (Y527F) or its kinase inactive counterpart (Y527F-Kin " ).
  • Total protein extracts (TOT) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin and tubulin (TUB) as loading control. Data are representative of ten independent experiments.
  • HEK293 cells were transiently transfected with wild type frataxin (FXN), or non- phosphorylable frataxin mutants Y175F, Yl 18F, Y123F, and either constitutively active Src (Y527F) or its kinase inactive counterpart (Y527F-Kin " ).
  • Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin, phosphorylated tyrosine (pY) and tubulin (TUB) as loading control.
  • WB Western protein extracts
  • WB immunoblotted
  • pY phosphorylated tyrosine
  • TAB tubulin
  • HEK293 cells were transiently transfected with wild type frataxin (FXN), or non- phosphorylable mutants Yl 18F, Y166F, Y175F, and hemaglutinin (HA)-tagged ubiquitin (HA-Ub).
  • FXN wild type frataxin
  • HA-Ub hemaglutinin-tagged ubiquitin
  • TOT total protein extracts
  • IP oc- HA immunoprecipitated ubiquitinated frataxin
  • the graph illustrates the relative ubiquitination level quantitated as the ratio between mono-ubiquitinated frataxin (Mono-Ub) level versus the frataxin precursor expression in the MG132-treated lanes.
  • the precursor (P), intermediate (I) and mature (M) frataxin forms are indicated.
  • FIG. 4 Src inhibitors upregulate wild type frataxin but not the non- phosphorylable Yl 18F frataxin mutant.
  • HEK293 FXN cells stably expressing single copy of wild type frataxin (WT) or non-phosphorylable Y 118F frataxin mutant (Yl 18F) were treated for 24 h with 1, 3, and 10 ⁇ of either Src inhibitor SU6656, PP2, dasatinib, bosutinib, saracatinib, or vehicle (-).
  • Left panels Frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated.
  • Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (-) set to one. Data represent the mean ⁇ 1 S.E.M. from three different independent experiments performed for each inhibitor. P- values were calculated with the Student's /-test and were statistically significant (TO.05; **P ⁇ 0.0 ⁇ ) for each treatment compared to non- treated conditions. [0018] Figure 5. Src inhibitors promote endogenous frataxin accumulation in HEK293 cells. Human HEK293 cells were treated with 1, 3 and 10 ⁇ of SU6656, PP2, and dasatinib Src inhibitor or vehicle (-) for 24 h. Frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot.
  • FRDA patient-derived B cells were treated with 10 ⁇ of either Src inhibitor SU6656, PP2, dasatinib, or vehicle (-) for the time indicated.
  • Left panels mature frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments.
  • Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (NT) set to one. Data represent the mean ⁇ 1 S.E.M. from three different independent experiments performed for each inhibitor in the left panels. -values were calculated with the Student's /-test and were statistically significant (TO.05; * ⁇ 0.01) for each treatment compared to non-treated conditions.
  • Figures 7A-7B show a western blot of the frataxin precursor (FXN) and tubulin expression (TUB) for cells treated with the Src inhibitor dasatinib (Dasa, 100 nM), the ubiquitin competing molecule F166 (1 uM), and a combination. Data are representative of four independent experiments.
  • Figure 7B show a densitometric
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • Antibodies can be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which are recognized or specifically bound by the antibody.
  • the epitope(s) or polypeptide portion(s) can be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues.
  • composition comprising A must include at least the component A, but it may also include one or more other components (e.g. , B; B and C; B, C, and D; and the like).
  • compositions comprising A or B would typically present an aspect with a composition comprising both A and B.
  • Or should, however, be construed to exclude those aspects presented that cannot be combined without contradiction (e.g. , a composition pH that is between 9 and 10 or between 7 and 8).
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the invention which is made with counterions understood in the art to be generally acceptable for pharmaceutical uses and which possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid,
  • cyclopentanepropionic acid glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
  • an alkali metal ion an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine,
  • Specific binding refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment.
  • “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold.
  • the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant is about 10 "7 M or stronger (e.g., about 10 "8 M, 10 "9 M or even stronger) .
  • the invention presents a method of treating Friedreich's ataxia in a subject in need thereof, including administering to the subject a therapeutically effective amount of a Src inhibitor or a pharmaceutically acceptable salt thereof.
  • the subject is a mammal. In one aspect, the mammal is a human. [0035] In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor inhibits Yl 18 phosphorylation of frataxin.
  • the invention presents a method as set forth herein, wherein the Src inhibitor is a SrcY527F antagonist.
  • the invention presents a method as set forth herein, wherein the Src inhibitor increases endogenous frataxin levels by at least 5% over an inactive control.
  • the Src inhibitor increases endogenous frataxin levels by at least 10% over an inactive control (e.g., 10%, 15%, or 20%).
  • the Src inhibitor increases endogenous frataxin levels by at least 25% over an inactive control (e.g., 25%, 30%, 35%, 40%, or 45%).
  • the Src inhibitor increases endogenous frataxin levels by at least 50% over an inactive control (e.g., 50%, 55%, 60%, 65%, or 70%).
  • the Src inhibitor increases endogenous frataxin levels by at least 75% over an inactive control (e.g. , 75%, 80%, 85%, 90%, or 95%). In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 100% over an inactive control (e.g., 100%, 105%, 1 10%, 1 15%, or 120%). In one aspect, the Src inhibitor increases endogenous frataxin levels by at least 125% over an inactive control (e.g., 125%, 130% 135%, 140%, 145%, 150%, 155%, 160% 165%, 170%, 175%, 180%, 185%, 190%, 200%, 225%, or 250%).
  • the invention presents a method as set forth herein, wherein the Src inhibitor is selected from the group consisting of saracatinib (AZD-530), dasatinib, bafetinib, KX01, XL228, DCC-2036, ponatinib (AP24534), and TG100435.
  • the Src inhibitor is AZD-530.
  • the Src inhibitor is dasatinib.
  • the Src inhibitor is bafetinib.
  • the Src inhibitor is KX01 (a.k.a. KX2-391).
  • the Src inhibitor is XL228.
  • the Src inhibitor is DCC-2036.
  • the Src inhibitor is ponatinib.
  • the Src inhibitor is TGI 00435.
  • the invention presents a method as set forth herein, wherein the Src inhibitor is selected from the group consisting of SU6656, PP2, dasatinib, bafetinib, saracatinib, XL999, KX01, XL228, and bosutinib.
  • the Src inhibitor is SU6656.
  • the Src inhibitor is PP2.
  • the Src inhibitor is XL999.
  • the Src inhibitor is KX01 (a.k.a. KX2-391).
  • the Src inhibitor is XL228.
  • the Src inhibitor is bosutinib.
  • the invention presents a method as set forth herein, wherein the Src inhibitor is administered at a daily dose of from about 2 to 25 mg of compound per kg of the subject's body weight (mg/kg) (e.g. , 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28).
  • the Src inhibitor is administered at a daily dose of from about 2 to 20 mg of compound per kg of the subject's body weight (mg/kg).
  • the Src inhibitor is administered at a daily dose of from about 1 to 15 mg of compound per kg of the subject's body weight (mg/kg).
  • the Src inhibitor is administered at a daily dose of from about 1 to 10 mg of compound per kg of the subject's body weight (mg/kg) (e.g. , 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 1 1). In one aspect, the Src inhibitor is administered at a daily dose of from about 500 to 5000 meg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a dose of from about 100 to 1000 meg of compound per kg of the subject's body weight (mcg/kg).
  • the Src inhibitor is administered at a daily dose of from about 50 to 500 meg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 25 to 250 meg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 10 to 100 meg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 50 meg of compound per kg of the subject's body weight (mcg/kg).
  • the Src inhibitor is administered at a daily dose of from about 1 to 25 mg of compound per kg of the subject's body weight (mcg/kg). In one aspect, the Src inhibitor is administered at a daily dose of from about 1 to 10 meg of compound per kg of the subject's body weight (mcg/kg).
  • the invention presents a method as set forth herein, wherein the Src inhibitor is administered by controlled release. In one aspect, the Src inhibitor is administered topically. In one aspect, the Src inhibitor is administered intraveneously. In one aspect, the Src inhibitor is administered orally. [0042] In one aspect, the invention presents a method as set forth herein, wherein the Src inhibitor is administered once weekly. In one aspect, the Src inhibitor is administered twice weekly. In one aspect, the Src inhibitor is administered three time weekly. In one aspect, the Src inhibitor is administered once daily. In one aspect, the Src inhibitor is administered twice daily. In one aspect, the Src inhibitor is administered three times daily. [0043] In one aspect, the invention presents a method as set forth herein, wherein the method of treating Friedreich's ataxia comprises inhibiting ubiquitination of frataxin.
  • the invention presents a method of inhibiting ubiquitination of frataxin in a subject comprising administering to a subject a therapeutically effective amount of a Src inhibitor or a pharmaceutically acceptable salt thereof. In one aspect, the method is as otherwise set forth herein. [0045] In one aspect, the invention presents a method as set forth herein, further comprising administering to the subject one or more agents selected from the group consisting of a ubiquitination inhibitor, an antioxidant, and a siderophore.
  • the invention presents a method as set forth herein, further comprising administering to the subject one or more agents selected from the group consisting of an interferon and a ubiquitin-competing molecule.
  • the agent is an interferon.
  • the interferon is gamma interferon, for example, using a method as set forth in U.S. Patent No. 8,815,230.
  • the invention presents the use of an Src inhibitor in a method of treating Friedreich's ataxia in a subject in need thereof, comprising the method as set forth herein.
  • HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HEK-293 cells were transfected with the calcium/phosphate precipitation method, using 20 ⁇ g of total DNA (5 or 10 ⁇ g of pIRES2frataxin, 10 ⁇ g of Tyrosine mutated non-phosphorylable frataxin mutants, 10 ⁇ g of Src or Abl constructs and 10 ⁇ g of HA-Ub, or the corresponding empty vectors) on 10 cm dishes.
  • HEK-293 Flp-In cells are HEK-293 variants allowing the stable and isogenic integration and expression of a transfected gene.
  • Y118F expressing frataxin (Condo et al. 2007) and the clone stably expressing frataxin were generated by mutagenesis as described below.
  • HEK-293 Flp-In cells were maintained in DMEM supplemented with 10% FBS.
  • Immortalized GM16214 lymphoblasts, from a clinically affected FRDA patient were obtained from NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research (Camden, NJ, USA) and cultured in RPMI 1640 supplemented with 15% fetal bovine serum.
  • the construct pIRES2-frataxin 1 210 contains human frataxin cDNA cloned into the bicistronic expression vector pIRES2-EGFP (BD Clontech) and was previously generated in this laboratory. Condo, I.; Ventura, N.; Malisan, F.; Tomassini, B.; Testi, R. (2006), J Biol Chem, 281, 16750-56. All the tyrosine mutant constructs were generated using the Quick-Change site-directed mutagenesis kit (Stratagene) with specific primers using pIRES2-frataxin 1 ⁇ 210 as template. The HA-Ub construct was generated by M. Treier.
  • CIP dephosphorylation assay (New England BioLabs ® Inc.) was used to release phosphate groups from residues of tyrosine. 50 units of CIP (Alkaline Phosphatase, Calf Intestinal) were added to total cell extracts resuspended in NE3 buffer pH 7.9 (1 M NaCl, 0.5 M Tris-HCl, 100 mM MgCl 2 , 10 mM dithiothreitol), and incubated for 60 minutes at 37 °C. Sodium orthovanadate lOmM and EDTA 50 mM were used to inhibit CIP activity.
  • Cell lysates (100 ⁇ g) were resolved by SDS-PAGE and analyzed by immunoblot with specific mAb anti-frataxin clone 1G2 and STR-23 (Immunological Sciences, Rome, Italy) mAb anti-tubulin (Sigma), mAb anti-actin (Sigma), mAb anti-phosphotyrosine (Millipore), pAb anti-phospho-Src (Life Technologies), mAb anti-GFP (Takara), secondary antibody horseradish peroxidase-conjugated goat anti-mouse (Pierce), secondary antibody horseradish peroxidase-conjugated mouse anti-rabbit (Pierce), secondary antibody horseradish peroxidase-conjugated goat anti-Fc anti-mouse (Thermo Scientific) using ECL system detection (GE Healthcare Europe GmbH, Milan, Italy).
  • peptide mixture was analyzed by nanoflow- reversed-phase liquid chromatography tandem mass spectrometry (RP-LC -MS/MS) using an HPLC Ultimate 3000 (DIONEX, Sunnyvale, CA U.S.A.) connected on line with a linear Ion Trap (LTQ, ThermoElectron, San Jose, CA).
  • RP-LC -MS/MS nanoflow- reversed-phase liquid chromatography tandem mass spectrometry
  • Peptides were desalted in a trap-column (AcclaimPepMaplOO CI 8, LC Packings, DIONEX) and then separated in a reverse phase column, a 10-cm-long fused-silica capillary (SilicaTipsFS 360-75-8, New Objective, Woburn, MA, USA), slurry-packed in-house with 5 ⁇ , 200 A pore size CI 8 resin (Michrom BioResources, CA). Peptides were eluted using a linear gradient from 96% aqueous phase (H 2 0 with 5% ACN, 0.1% formic acid) to 60% organic buffer (ACN with 5% H 2 0 with 0.1% formic acid) in 30 min, at 300 nl/min flow rate.
  • Tandem mass spectra were matched against Swiss-Prot protein database and through SEQUEST algorithm (Y ates et al , 1995) incorporated in Bioworks software (version3.3, Thermo Electron) using no enzyme constrain, static cysteine alkylation by iodoacetamide, variable modification by oxidation on methionine and phosphorylation on tyrosine residues (Am: + 80 Da).
  • a peptide has been considered legitimately identified when it achieved cross correlation scores of 1.8 for [M+H] 1+ , 2.5 for[M+2H] 2+ , 3 for[M+3H] 3+ , and a probability cut-off for randomized identification of p ⁇ 0.001.
  • frataxin was transiently transfected in human embryo kidney (HEK) 293 cells, together with several constructs encoding different forms of Src and Abl kinases.
  • HEK human embryo kidney
  • the mutants were analyzed in cotransfection assays with constitutively active SrcY527F and its kinase inactive counterpart SrcY527FKin-.
  • HEK293 cells were transiently transfected with frataxin (FXN), and either constitutively active Src (Y527F) or its kinase inactive counterpart (Y527F-Kin " ) (Fig. 1A).
  • the total protein extracts (TOT) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin and tubulin (TUB) as loading control.
  • WB immunoblotted
  • TAB tubulin
  • HEK293 cells were transiently transfected with frataxin, and either constitutively active Src (Y527F), its kinase inactive counterpart (Y527F-Kin " ) and constitutively active Abl (Abl-PP).
  • Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS-Page and immunoblotted (WB) with specific antibodies against frataxin (FXN), phosphorylated tyrosine (pY) and tubulin (TUB) as loading controls (Figure 1C). The data shown are representative of three independent experiments.
  • Figure 2A shows three representative mutants, out of the eight analyzed, while the associated table summarizes the results for all mutants. Only mutation of Yl 18 abrogated tyrosine phosphorylation of frataxin precursor, indicating that Yl 18 is the main Src phosphorylation site. [0071] To further confirm that Yl 18 was the main phosphorylation site, mass spectrometry was performed (Fig. 2B). In vitro phosphorylation reaction using recombinant frataxin 1-210 and recombinant Src was performed, recovered on SDS-PAGE gel after visualization by Coomassie staining, digested with chymotrypsin, and analyzed by MALDI-TOF mass spectrometry. One phosphopeptide was isolated, corresponding to amino acids 96-123, identifying the phosphorylated residue as Yl 18.
  • Example 4 Non-phosphorylable Y118F frataxin mutant is less ubiquitinated
  • HEK-293 cells were transfected with wild type frataxin or non-phosphorylable frataxin mutants Y 118F, Yl 16F, and Y175F together with hemaglutinin (HA)-tagged ubiquitin (HA-Ub) in the absence or presence of proteasome inhibitor MG132.
  • HA hemaglutinin
  • HA-Ub hemaglutinin-tagged ubiquitin
  • frataxin ubiquitination status was evaluated by anti-frataxin monoclonal antibodies on total lysates or after immunoprecipitation of ubiquitinated forms with anti-HA antibody only in the presence of MG132. Frataxin monoubiquitinated forms can be detected as slower migrating bands above the frataxin precursor.
  • Figure 3 illustrates that accumulation of ubiquitinated frataxin forms was reduced when non-phosphorylable Yl 18F mutant, but not other non-phosphorylable mutants such as Yl 16F and Y175F, was transfected. Relative ubiquitination level was quantitated as the ratio between mono-ubiquitinated frataxin level versus the frataxin precursor expression in the MG132-treated lanes. Non-phosphorylable Yl 18F frataxin mutant is >60% less
  • Dasatinib is delivered orally as a solution in citric acid buffer, with a regimen that has shown no toxicity in the nude mice. Lombardo et al. (2004), J. Med. Chem. , 47, 6658- 61. A daily dose of 50 mcg/kg, administered on a 5 days on and 2 days off schedule, will be tested.
  • Bosutinib is delivered orally at a daily dose of 100 mcg/kg according to a previously described therapeutic regimen in the nude mouse. Golas et al. (2005), Cancer Res., 65, 5358-64.
  • mice are subjected to a number of behavioural tests at regular intervals in time. These include body weight analysis, rotarod, beam-walk, hang wire, grip strength, and footprint tests. Moreover, at the end of the treatment, mice are sacrificed, and the amount of frataxin within the dorsal root ganglia neurons is quantitated by SDS-PAGE and Western blot analysis. These results clarify whether selected src inhibitors treatment has an impact on the progression of the disease in the mouse and whether this is associated with an increase of frataxin in critical neuronal compartments.
  • Src inhibitors and ubiquitin competing molecules can enhance each other's ability to promote frataxin precursor accumulation, as they impinge on the same molecular pathway.
  • the Src inhibitor dasatinib further enhances the ability of the ubiquitin competing molecule F 166 to accumulate frataxin precursor in living cells ( Figure 7).
  • HEK293 FXN cells stably expressing a single copy of wild-type frataxin were treated for 24 h with Src inhibitor dasatinib (Dasa, 100 nM), the ubiquitin competing molecule F166 (1 uM) or a combination thereof.
  • the frataxin precursor (FXN) and tubulin expression (TUB) were analyzed by western blot (Fig. 7A). The data shown are representative of four independent experiments.

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  • Organic Chemistry (AREA)
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Abstract

Selon un aspect, la présente invention concerne un procédé de traitement de l'ataxie de Friedreich (FRDA) par administration d'une quantité thérapeutiquement efficace d'un inhibiteur de Src.
PCT/IB2015/059963 2014-12-23 2015-12-23 Procédés de traitement de l'ataxie de friedreich au moyen d'inhibiteurs de src WO2016103223A1 (fr)

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US11266635B2 (en) 2019-08-12 2022-03-08 Deciphera Pharmaceuticals, Llc Methods of treating gastrointestinal stromal tumors
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US11779572B1 (en) 2022-09-02 2023-10-10 Deciphera Pharmaceuticals, Llc Methods of treating gastrointestinal stromal tumors
US11986463B2 (en) 2018-01-31 2024-05-21 Deciphera Pharmaceuticals, Llc Combination therapy for the treatment of gastrointestinal stromal tumor

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US10442779B2 (en) 2014-09-22 2019-10-15 Fratagene Therapeutics S.R.L. Compositions and methods for treating Friedreich's ataxia
WO2018217683A1 (fr) * 2017-05-23 2018-11-29 Children's Hospital Medical Center Méthodes de traitement de la mucoviscidose
US10426775B2 (en) 2017-09-11 2019-10-01 Fratagene Therapeutics Srl Methods for treating Friedreich's ataxia with etravirine
US11986463B2 (en) 2018-01-31 2024-05-21 Deciphera Pharmaceuticals, Llc Combination therapy for the treatment of gastrointestinal stromal tumor
US11344536B1 (en) 2019-08-12 2022-05-31 Deciphera Pharmaceuticals, Llc Methods of treating gastrointestinal stromal tumors
US11266635B2 (en) 2019-08-12 2022-03-08 Deciphera Pharmaceuticals, Llc Methods of treating gastrointestinal stromal tumors
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US11801237B2 (en) 2019-12-30 2023-10-31 Deciphera Pharmaceuticals, Llc Amorphous kinase inhibitor formulations and methods of use thereof
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US11185535B2 (en) 2019-12-30 2021-11-30 Deciphera Pharmaceuticals, Llc Amorphous kinase inhibitor formulations and methods of use thereof
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