WO2018172588A1 - Use of mok modulating agents for the treatment of inflammatory diseases and neurodegenerative diseases - Google Patents

Use of mok modulating agents for the treatment of inflammatory diseases and neurodegenerative diseases Download PDF

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WO2018172588A1
WO2018172588A1 PCT/ES2018/070216 ES2018070216W WO2018172588A1 WO 2018172588 A1 WO2018172588 A1 WO 2018172588A1 ES 2018070216 W ES2018070216 W ES 2018070216W WO 2018172588 A1 WO2018172588 A1 WO 2018172588A1
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mok
disease
treatment
compound
tdp
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PCT/ES2018/070216
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French (fr)
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Cintia ROODVELDT CATELLANI
María Magdalena LEAL LASARTE
Jaime MUÑOZ FRANCO
David POZO PÉREZ
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Fundación Pública Andaluza Progreso Y Salud
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  • the present invention is within the field of molecular biology and medicine, and specifically refers to compounds and compositions capable of modulating the activity of MOK (MAPK / MAK / MRK overlapping kinase) or any of its biologically active isoforms and variants , for the treatment of diseases with inflammatory component, among others, neurodegerative conformational diseases (protein misfolding diseases), and more specifically for the treatment of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobe degeneration (Frontotemporal lobar degeneration or FTLD ).
  • MOK MOK
  • MAK / MAK / MRK overlapping kinase any of its biologically active isoforms and variants
  • diseases with inflammatory component among others, neurodegerative conformational diseases (protein misfolding diseases), and more specifically for the treatment of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobe degeneration (Frontotemporal lobar degeneration or FTLD ).
  • neuroinflammatory processes are a critical factor in triggering the pathological cascade that leads to neuronal degeneration.
  • Signs of external or internal damage activate microglial cells, favoring the production of cytotoxic factors that induce neuronal degeneration. These factors activate protein kinases, which lead to hyperphosphorylation of aggregating proteins, and their consequent oligomerization. These aggregates released into the extracellular environment, upon activation of the microglial cell, would cause a positive feedback mechanism favoring neurodegeneration.
  • ALS / ALS amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • Parkinson's disease Alzheimer's disease
  • Huntington's disease Huntington's disease
  • spongiform encephalopathies multiple tauopathies
  • amyloid polyneuropathy family etc.
  • Amyotrophic lateral sclerosis (ALS / ALS) is a fatal neurodegenerative disorder currently incurable, mainly because the molecular mechanisms of this and related diseases (e.g. FTLD) remain enigmatic (Fujisawa et al., Hum. Mol. Gen. 2016).
  • FTLD molecular mechanisms of this and related diseases
  • Currently, the only drug approved for treatment is 'riluzole', which increases the life expectancy of patients in only 2-3 months.
  • Agents modulating the activity of protein kinases are known in the state of the art.
  • Garske AL et al., PNAS 201 1 describe a chemical-genetic approach based on the covalent complementarity between a gatekeeper cysteine and an electrophilic inhibitor.
  • MAPK mitogen-activated protein kinases
  • FTLD is a clinical syndrome caused by the degeneration of the frontal lobe of the human brain, which can spread to the temporal lobe. It is one of three syndromes caused by frontotemporal lobular degeneration, and the second most common cause of early onset dementia after Alzheimer's disease
  • the identification of a key therapeutic target in this case the MOK protein kinase or some of its isoforms, and its possible modulation, could represent an important advance in the development of new therapeutic approaches for the treatment of neurodegerative conformational diseases, and mainly where the aggregating protein is TDP-43, such as ALS and FTLD.
  • the aggregating protein is TDP-43, such as ALS and FTLD.
  • the authors of the invention demonstrate that the modulation of the expression and / or activity of the human protein kinase MOK (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07) or any of its isoforms or biologically active variants, whose expression and phosphorylation status (required for its activity as MAPK) are strongly affected in a cellular model of ALS / FTLD, which would serve to modulate the neuroinflammatory-neurodegenerative profile associated with these conformational diseases 'neurodegenerative.
  • MOK human protein kinase MOK
  • a first aspect of the invention relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of inflammatory diseases, more preferably neurodegenerative diseases. mediated by neuroinflammatory processes.
  • MOK modulating agent of the invention in the preparation of a medicament for the prevention, improvement and / or treatment of inflammatory diseases, more preferably of neurodegenerative diseases mediated by inflammation.
  • neurodegenerative disease is a neurodegerative conformational disease.
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the disease is ALS or FTLD. Therefore, MOK inhibitors would be useful for the treatment of nurodegenerative conformational diseases, and more specifically, where the aggregating protein is TDP-43.
  • MOK activity modulating agents are selected from a list consisting of:
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (I):
  • n 0 or 1
  • R is a methyl (Me), isopropyl ( ⁇ Pr), tere-Butyl (tBu), ciciopeniadienil ⁇ (Cp)
  • X is H or NH 2
  • Y is H
  • NHCOCHCH2, NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, and Z is H
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (II):
  • n is equal to 1
  • R is an isopropyl ( ⁇ Pr),
  • X is NH 2
  • the MOK modulating agent of the invention comprises an antisense oligonucleotide and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • the MOK modulating agent of the invention comprises an antibody and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • the MOK modulating agent of the invention comprises a ribozyme and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • a second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a MOK modulating agent of the invention.
  • a composition of the invention comprising a MOK modulating agent of the invention.
  • it further comprises a pharmaceutically acceptable carrier or excipient.
  • the composition of the invention is a pharmaceutical composition.
  • it also comprises another active ingredient.
  • a third aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • Hirayama disease or monomelic atrophy Spinal muscular atrophy.
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • a fifth aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • a method for the collection of data useful in the diagnosis and / or prognosis of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD. DESCRIPTION OF THE FIGURES
  • Fig. 1 The aggregates of TDP-43 are internalized and modified by microglial cells, mobilizing the endogenous TDP-43 protein.
  • Fused images include DAPI fluorescence (blue).
  • IF images show ubiquitin labeling (green), TDP-43 labeling (red), and fusion images, including DAPI fluorescence (D).
  • F IF analysis with TDP-43 immunostaining (red) of N13 cells treated with 5 ⁇ g / ml of TDP43 or simulation for 4 h.
  • TDP-43 nuclear protein levels (TDP-43 nuclear clearance) in cells exposed to TDP43 treatment.
  • G) IF confocal microscopy analysis of CD11 b (white), His-tag (red) and TDP-43 (green) immunotyped cultures of spinal cord organotypic cultures after 24 h of treatment with 5 ⁇ g / mL of TDP43 (or control mock) showing the presence of inclusions containing His-tag / TDP-43 in the microglial cell cytoplasm and the elimination of nuclear TDP-43, in the sample treated with exogenous TDP-43 aggregates.
  • Cell nuclei were contrasted with DAPI (blue).
  • the bars are the average of at least 3 independent experiments (N ⁇ 3), each performed with duplicate samples.
  • E) Western blot analysis of IL-18 cleaved in supernatants of primary microglial cells collected 24 h after treatment with 5 ⁇ g / ml of TDP43 or simulation (mock). Recombinant murine IL-18 was used as a control in the same gel (right end). Lower: quantification of the band density (relative values) of three independent experiments (N 3).
  • Fig. 3 The aggregates of TDP-43 induce a neuroinflammatory response that involves caspase-3.
  • Fused images include DAPI fluorescence. graduated bars: 25 ⁇ .
  • B, C Quantitative evaluation of the levels of activated caspase-3 of IF and confocal microscopy images (as shown in A), based on total activated caspase 3 (B) or granules of activated caspase-3 in the compartments Cytoplasmic and nuclear cells of the primary microglia (C).
  • the values shown were normalized by the untreated control (Ctrl) within each of the three experiments, and subsequently referred to the average granules / cell of the three Ctrl samples.
  • Fig. 4 Aggregates of extracellular TDP-43 are targets of the MOK microglia signaling pathway.
  • FIG. 5 (Supplementary Figure 1). Alternative processing of IL-18 in ca. 16 kDa in primary microglia culture. Expression of caspase-1 and secretion of IL-18 in cultures of the spinal cord organotypic section.
  • Fig. 6 (Supplementary Figure 2). IF analysis of activated caspase-3 in cells 4 h and 24 h after treatment.
  • Fig 7 (Supplementary Figure 3).
  • B Quantitative analysis of His6-tag / MOK colocalization (over total MOK) based on co-IF and confocal microscopy analysis.
  • the white arrow indicates a differential band / s and the regions in the gel (for both lanes) that were cleaved and analyzed by MALDI / MS.
  • the object of the invention is to modulate the expression and / or activity of the human protein MOK (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms).
  • MAPK / MAK / MRK overlapping kinase 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms.
  • said protein kinase - a member of the MAPK superfamily that expresses itself in the brain and needs to be phosphorylated to phosphorylate other substrates - is strongly affected in terms of its expression and phosphorylation status, in a model ELA / FTD cell.
  • a first aspect of the invention relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of inflammatory diseases, more preferably neurodegenerative diseases. mediated by inflammation.
  • MOK modulating agent of the invention in the preparation of a medicament for the prevention, improvement and / or treatment of inflammatory diseases, more preferably of neurodegenerative diseases mediated by inflammation.
  • the invention also relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of neurodegenerative conformational diseases, or alternatively, a MOK modulating agent of the invention, for use in the prevention, improvement and / or treatment of neurodegenerative conformational diseases.
  • MOK modulating agent of the invention for the prevention, improvement and / or treatment of neurodegenerative conformational diseases
  • MOK modulating agent of the invention for use in the prevention, improvement and / or treatment of neurodegenerative conformational diseases.
  • This report refers to an inflammatory disease and / or that has an inflammatory component or is mediated by inflammation, which refers to a condition in a subject characterized by inflammation, for example, chronic inflammation.
  • Non-limiting, inflammatory disorders include, but are not limited to, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (for example, idiopathic pulmonary fibrosis), inflammatory vacuhtides (for example, polyarteritisnode, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosis), post-traumatic vascular angioplasty (for example, restenosis after of angioplasty), inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacterial infections. Also included in this report are inflammatory diseases,
  • neurodegerative conformational disease is understood as the kind of pathology in which one (or more) proteins specific ones become structurally abnormal, and therefore interfere with the function of cells, tissues and organs in the body. Often proteins fail to fold in their normal conformation; In this poorly folded state, the proteins form aggregates and become toxic in some way (eg, causing neuroinflammation) can lose their normal function.
  • PD Parkinson's disease
  • DLB Lewy body dementia
  • MSA multiple system atrophy
  • FAP amyotrophic lateral sclerosis
  • ALS fronto-temporal dementia
  • Gaucher disease Down syndrome
  • Huntington's disease prion disease
  • Creutzfeldt-Jakob Jakob disease and other transmissible spongiform encephalopathies
  • motor neuron Gerstmann-Stráussler-Scheinker syndrome
  • Kuru fatal familial insomnia
  • type II diabetes cerebrovascular amyloidosis
  • glaucoma age-related macular degeneration
  • psychiatric syndromes multiple tauopathies
  • familial amyloid polyneuropathy and neurodegeneration due to aggregation of age-related proteins.
  • ALS amyotrophic lateral sclerosis
  • primary lateral sclerosis progressive muscular atrophy
  • Kennedy disease or progressive spinobulbar muscular atrophy Hirayama disease or monomelic atrophy
  • spinal muscular atrophy frontotemporal lobe degeneration (FTLD), or Any of your combinations.
  • FTLD frontotemporal lobe degeneration
  • neurodegenerative disease is a neurodegerative conformational disease, that is, the invention relates to the use of the MOK modulating agent of the invention in the preparation of a medicament for prevention, improvement or treatment. of a neurodegerative conformational disease.
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the aggregating neurodegenrative disease is ALS or FTLD.
  • MOK MAK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms
  • This gene belongs to the MAP kinase superfamily.
  • the gene is regulated by the flow rate transcription factor 2 (Cdx2).
  • the encoded protein which is located in the epithelial cells in the intestinal crypt, can play a role in the growth arrest and differentiation of the cells of the upper crypt and regions of lower villi. Multiple transcription variants alternately linked encoding different isoforms have been observed for this gene.
  • MOK refers to both the gene and the human MOK protein.
  • MOK is also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the MOK protein coding sequence, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,
  • nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) are nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
  • nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MOK protein.
  • SEQ I D NO: 2 is, but not limited to, SEQ I D NO: 2.
  • the activity of MOK can be modulated by the modification of the levels and / or activity of the MOK protein, or by the modification of the levels at which the MOK gene is transcribed such that the levels of activity of the MOK protein in The cell is modulated.
  • Modulating agents may also be agonists (substances that are capable of binding to a receptor and eliciting a response in the cell, specifically an increase in MOK activity), as antagonists (substances that not only do not activate the receptor, but also in actually blocks its activation by agonists). In the context of the present invention, inhibition is the preferred form of modulation.
  • TDP-43 also TARDBP or ALS10
  • TAR DNA binding protein a protein that is a transcriptional repressor that binds to chromosomally integrated DNA TAR and represses HIV-1 transcription.
  • this protein regulates the alternative splicing of the CFTR gene. It is located on chromosome 1, but a similar pseudogen is present on chromosome 20.
  • TDP-43 protein was identified as the main disease protein in most sporadic and familial FTLD cases. Subsequently, it was also found that ubiquitinated compact inclusions, characteristic of amyotrophic lateral sclerosis (ALS), were composed of TDP-43, thus providing evidence that both conditions are mechanically linked and are part of the spectrum of diseases that can be classified as proteopathies. TDP-43, (See, for example, Neumann et al., Science 314 (2006), 130-133).
  • TDP-43 accumulates in the nerve cells and glial cells of the dementia complex of ALS-Parkinsonism of Guam, corticobasal degeneration, dementia with Lewy bodies, Huntington's disease, body disease Lewy, motor neuron disease, frontotemporal dementia, frontotemporal lobar degeneration with positive inclusions to ubiquitin, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease, Parkinson's disease dementia, Parkinson's complex- dementia on the Kii peninsula and Pick's disease and the like, among other pathologies; See, for example, Lagier-Tourenne et al. , Hum. Mol. Gen. 19 (2010), R46-64, which is incorporated herein by reference in its entirety.
  • TDP-43 refers to both the gene and the human MOK protein.
  • TDP-43 is also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the MOK protein coding sequence, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
  • nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MOK protein.
  • nucleotide sequences encoding MOK is, but not limited to, SEQ ID NO: 4.
  • MOK activity modulating agents are selected from a list consisting of:
  • the organic molecules will preferably have a weight of 100 to 20,000 daltons, more preferably 500 to 15,000 daltons, and more preferably 1000 to 10,000 daltons. Bookstores of organic molecules are commercially available.
  • the route of administration may be, without limitation, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (I):
  • n 0 or 1
  • R is a methyl (Me), isopropyl ( ⁇ Pr), tere-Butyl (tBu), cyclopentadienyl (Cp)
  • X is H or NH 2
  • Y is H, NHCOCHCH 2 , NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, and Z is H,
  • the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (II):
  • n is equal to 1
  • R is an isopropyl ( ⁇ Pr),
  • X is NH 2
  • the compounds of the present invention represented by formula (I) and / or (II) may include isomers, depending on the presence of multiple bonds, including optical isomers or enantiomers, depending on the presence of chiral centers.
  • the individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to any mixture of isomers, such as diastereomers, racemic, etc., even their optically isomers. assets or mixtures in different proportions thereof.
  • prodrugs of the compounds of formula (I) and / or (II) include any derivative of a compound of formula (I) and / or (II) - for example and not limited to: esters (including esters of carboxylic acids, amino acid esters, phosphate esters, sulphonate esters of metal salts, etc.), carbamates, amides, etc- that when administered to an individual can be transformed directly or indirectly into said compound of formula (I) and / or (II) in the mentioned individual.
  • said derivative is a compound that increases the bioavailability of the compound of formula (I) and / or (II) when administered to an individual or that enhances the release of the compound of formula (I) and / or (II) in a biological compartment
  • said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I) and / or (II) in a biological compartment of an individual.
  • the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • the term "derivative” includes both pharmaceutically acceptable compounds, that is, derivatives of the compound of formula (I) and / or (II) that can be used in the manufacture of a medicament or food compositions, such as Pharmaceutically acceptable derivatives, since these may be useful in the preparation of pharmaceutically acceptable derivatives.
  • the compounds of the invention may be in crystalline form as free compounds or as solvates.
  • the term "solvate”, as used herein includes both pharmaceutically acceptable solvates, that is, solvates of the compound of formula (I) and / or (II) that can be used in the manufacture of a medicament, as pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically acceptable solvates or salts.
  • the nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable.
  • the solvate is a hydrate. Solvates can be obtained by conventional solvation methods known to those skilled in the art.
  • the compounds of formula (I) and / or (II), their salts, prodrugs or solvates will preferably be in a pharmaceutically acceptable or substantially pure form, that is, having a level of purity pharmaceutically acceptable excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels.
  • the purity levels for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and still more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or of its salts, solvates or prodrugs.
  • nucleotide sequences specifically complementary to a particular DNA or RNA sequence could form complexes and block transcription or translation.
  • interfering RNA interfering RNA
  • tools have been developed that allow specific inhibition of gene expression.
  • the inhibition of the expression of MOK would therefore constitute the inhibition of its biological activity, and in particular, of the activity that is contributing to the aggravation of the neurodegerative conformational disease.
  • antisense polynucleotides are understood ribonucleotide or deoxyribonucleotide chains that can inhibit the production of the MOK protein by one of these three mechanisms:
  • Antisense oligonucleotides capable of modulating the activity of MOK are known in the state of the art. For example, and without limiting our, it could be a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the MOK protein.
  • siRNA small interfering RNA
  • silencing RNA small interfering RNA
  • siRNA small interfering RNA
  • siRNA small interfering RNA
  • silencing RNA small interfering RNA
  • this specific gene is MOK. It could also be any siRNA capable of hybridizing a nucleic acid molecule encoding the human MOK protein (or any of its isoforms or transcriptional variant) whose sequence (transcriptional variant 1; Acc. No. NM_014226.2) is collected in SEQ ID NO: 5.
  • RNA constructs that contains at least any one of the possible nucleotide sequences of siRNA capable of inhibiting MOK expression, and notwithstanding that any of the RNA sequences and constructs of RNA are additionally part of the present invention.
  • the invention described above that are subject to modifications, preferably chemical, that lead to greater stability against the action of ribonucleases and thereby greater efficiency. Without these modifications involving the alteration of its mechanism of action, which is the specific binding to the RISC complex (RNA-induced silencing complex), activating it and manifesting a helicase activity that separates the two strands leaving only the antisense strand associated with the complex.
  • the resulting ribonucleoproteic complex binds to the target mRNA (MOK messenger RNA, which is collected in SEQ ID NO: 2 or in SEQ ID NO: 5).
  • MOK messenger RNA which is collected in SEQ ID NO: 2 or in SEQ ID NO: 5.
  • RISC is associated with the messenger and the translation is attenuated. But if it is perfect, RISC acts as RNasa, cutting the messenger and being free to repeat the process.
  • siRNA capable of inhibiting the translation of these mRNAs also forms part of the invention.
  • siRNA sequence of the invention or of the RNA construct of the invention would be apparent to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications in both different nucleotides of the product such as the incorporation of other chemical compounds at any of the ends.
  • the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitor products or RNAs.
  • the nucleotide sequence design of the siRNA of the invention would also be apparent to one skilled in the art. Thus, it could be done through a random design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or positional information it has in the transcript.
  • Another non-limiting alternative of the present invention would be the conventional design by simple parameters developed by the pioneers of the art (Calipel, A. et al., 2003. J Biol Chem. 278 (43): 42409-42418) completed with an analysis BLAST of nucleotides.
  • Another possibility could be a rational design, in which a computer procedure is used to identify the optimal siRNA targets in an mRNA. The target sequences are analyzed in groups of 19 nucleotides at a time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters.
  • a genetic DNA construct could also be part of the composition of the invention, which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and comprising at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, comprising at least the sequence encoding the siRNA of the invention or the RNA construct of the invention for transcription, or, b) nucleotide sequence of DNA, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the RNA sequence of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, signals of start and end, cut sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.
  • compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro. Transfection could be carried out, but not limited to, direct transfection or vectors that facilitate the access of siRNA into the cell.
  • these vectors are, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates.
  • the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs can be conjugated with release peptides or other compounds to favor the transport of these siRNAs into the cell. Therefore, in another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises an antisense oligonucleotide and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • the MOK modulating agent of the invention comprises an antibody and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) the protein.
  • portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.
  • Antibodies capable of binding to the MOK protein can be used to inhibit the activity of said protein.
  • Such antibodies are commercially available, as per example, but not limited to, Anti-MOK antibodies marketed by Sigma-Aldrich HPA027282, HPA027292, SAB4502568, SAB1300406, SAB1305100, SAB4504627, SAB2105049.
  • the antibodies, or fragments thereof might be able to inhibit the activity of the MOK protein that contributes to the acquisition of the characteristics characteristic of neurodegerative conformational disease.
  • the antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen).
  • the monoclonal antibody can be altered biochemically, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in whole or in parts, of portions that are not necessary for the recognition of the MOK and being replaced by others that communicate to the antibody additional advantageous properties.
  • the antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.
  • a "recombinant antibody or polypeptide” is one that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.
  • rAc can be expressed and directed to specific cellular sub-compartments when the appropriate sequences for intracellular traffic are incorporated.
  • These antibodies are called intrabodies, and have proven effective not only to divert proteins from their usual compartment or block interactions between proteins involved in signaling pathways, but also to activate intracellular proteins.
  • Also part of the invention are genetic DNA constructs capable of transcribing to a peptide, antibody or antibody fragment, for use in the treatment of ALS.
  • Said genetic construction of DNA would direct the in vitro or intracellular transcription of the antibody sequence or fragment thereof, and comprises at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, which it comprises, at least, the coding sequence of the antibody of the invention or of the antibody fragment of the invention for transcription in vitro, or intracellular,
  • DNA nucleotide sequence preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the antibody sequence or antibody fragment of the invention operatively linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, start and end signals, sites of section, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts related to neurodegerative conformational disease.
  • ribozyme refers to a catalytic polynucleotide (typically RNA), which can be constructed to specifically recognize, by hybridization, an mRNA and fragment it or eliminate its expression. Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules.
  • RNA a catalytic polynucleotide
  • Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules.
  • the MOK modulating agent of the invention comprises a ribozyme and is used for the prevention, improvement and / or treatment of a neurodegerative conformational disease.
  • a second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a MOK modulating agent of the invention.
  • a composition of the invention comprising a MOK modulating agent of the invention.
  • it further comprises a pharmaceutically acceptable carrier or excipient.
  • the composition of the invention is a pharmaceutical composition.
  • it also comprises another active ingredient.
  • the pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.
  • the term "therapeutically effective amount” refers to the amount of the agent or compound capable of developing the therapeutic action determined by its pharmacological properties, calculated to produce the desired effect and, in general, will be determined, among other causes, due to the characteristics of the compounds, including the age, condition of the patient, the severity of the alteration or disorder, and the route and frequency of administration.
  • the compounds described in the present invention, their salts, prodrugs and / or solvates as well as the pharmaceutical compositions containing them can be used together with other drugs, or active ingredients, additional to provide a combination therapy.
  • Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising a compound of formula (I) and / or (II ), or a salt, prodrug or solvate thereof.
  • the pharmaceutical composition further comprises another active ingredient.
  • active substance means any component that potentially provides a pharmacological activity or other different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • the term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
  • Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising the compound of the invention or the composition of the invention.
  • compositions and pharmaceutical forms of the invention are suitable for oral administration, in solid or liquid form.
  • Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone), fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinyl pyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate.
  • Other pharmaceutical forms may be colloidal systems, which include nanoemulsions, nanocapsules and polymeric nanoparticles.
  • compositions for oral administration can be prepared by conventional methods of Galenic Pharmacy, as mixing and dispersion.
  • the tablets can be coated following methods known in the pharmaceutical industry.
  • the compositions and pharmaceutical forms can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dose.
  • Suitable excipients such as pH buffering agents or surfactants, can be used.
  • the aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
  • medication refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
  • the administration of the compounds, compositions or pharmaceutical forms of the present invention can be performed by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred for the convenience of patients and for the chronic nature of the diseases to be treated.
  • the amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / kg / day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.
  • the compounds and compositions of the present invention can be used together with other medicaments in combination therapies.
  • the other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.
  • a third aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • test compounds to be tested used in the method of selection of therapeutic agents are not limited to low molecular weight organic molecules, proteins (including antibodies), peptides, oliogonucleotides, etc. They can be natural and / or synthetic.
  • antibodies capable of binding to an MOK epitope can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in The state of the art.
  • MOK can be used to immunize an animal, to obtain polyclonal antibodies.
  • Monoclonal antibodies can also be prepared by techniques that allow the production of antibodies by cultured cell lines, including, but not limited to, hybridomas, human B-cell hybridomas. Techniques for producing chimeric, humanized or synthetic antibodies are known.
  • the therapeutic agents identified by the selection method described herein can be used in an animal or other model to determine the mechanism of action of said agent. Moreover, the therapeutic agents selected by the method described herein would be used in the treatment of diseases that occur with the alteration of MOK and, in particular, a neurodegerative conformational disease.
  • a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • a fifth aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR).
  • Procedures for detecting the expression of the MOK protein are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
  • a method for the collection of data useful in the diagnosis and / or prognosis of a neurodegerative conformational disease comprising:
  • the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
  • ALS Amyotrophic lateral sclerosis
  • FTLD frontotemporal lobe degeneration
  • the neurodegerative conformational disease is ALS or FTLD.
  • the ELA and FTLD model used recapitulates the effects observed for TDP-43 in patient samples and animal models of ELA and FTLD
  • microglia support the results described above in neurons, and recapitulate the effects observed in samples of patients and animal models of ALS and FTLD (Boeynaems et al., 2016. Acta Neuropathol. 132 (2): 159- 73).
  • TDP43 ' aggregates of TDP-43
  • 'mock' immunofluorescence
  • IL-18 activated and secreted by Western blot has been observed (Fig. 2E, Fig. SI B), as well as total intracellular IL-18 by IF (Fig. 2F) and a kind of IL-18 alternative processing product, 16 kDa, by Western blot (Fig. 2G).
  • Non-cell autonomic neurodegeneration 'non-cell autonomous' mechanism
  • IL-18 and caspase-3 Non-cell autonomic neurodegeneration ('non-cell autonomous' mechanism) involving IL-18 and caspase-3
  • 'conformational' neurodegenerative diseases / 'misfolding diseases' in particular, ELA and FTLD
  • others related Alzheimer, Parkinson, Huntington, prionosis, etc.
  • aging also characterized by the increasing presence of aberrantly added protein species in the intra and / or extracellular environment
  • diseases with an inflammatory component including Cancer.
  • TDP-43 The aggregates of TDP-43 affect the signaling pathway of the MAPK / MAK / MRK kinase overlapping kinase (MOK) in microglia. Implications for 'conformational', inflammatory and cancer neurodegenerative diseases
  • MOK is a serine / threonine kinase belonging to the MAPK kinase superfamily and which is expressed in different tissues (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248). MOK is capable of phosphorylating a variety of substrates and for its activity requires the phosphorylation of two residues within the TEY motif, one of which is autophosphorylated (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248) .
  • MOK sequence at the RNA level contains in its 3 'terminal region a segment identical to the RAGE family (renal cell carcinoma antigen) proteins, which are overexpressed in various cancers (renal, melanoma, leukemia, etc.) (Eichmuller et al., 2002. Exp. Dermatol. 1 1 (4): 292-301). This extends the possible role of MOK to the pathogenesis and / or development of cancer, considering also that there is a proven relationship between cancer and inflammation.

Abstract

The invention relates to compounds and compositions that can modulate the activity of MOK (MAPK/MAK/MRK) or any of the biologically active variants and isoforms thereof, and to a method for selecting same for the treatment of diseases with an inflammatory component and/or conformational neurodegenerative diseases and, more specifically, for the treatment of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

Description

USO DE AGENTES MODULADORES DE MOK PARA EL TRATAMIENTO DE ENFERMEDADES INFLAMATORIAS Y ENFERMEDADES NEURODEGENERATIVAS  USE OF MOK MODULATING AGENTS FOR THE TREATMENT OF INFLAMMATORY DISEASES AND NEURODEGENERATIVE DISEASES
La presente invención se encuentra dentro del campo de la biología molecular y de la medicina, y específicamente se refiere a compuestos y composiciones capaces de modular la actividad de MOK (MAPK/MAK/MRK overlapping kinase) o cualquiera de sus isoformas y variantes biológicamente activas, para el tratamiento de enfermedades con componente inflamatorio, entre otras, las enfermedades conformacionales neurodegerativas (protein misfolding diseases), y más concretamente para el tratamiento de la Esclerosis Lateral Amiotrófica (ELA) y de la degeneración del lóbulo frontotemporal (Frontotemporal lobar degeneration o FTLD). The present invention is within the field of molecular biology and medicine, and specifically refers to compounds and compositions capable of modulating the activity of MOK (MAPK / MAK / MRK overlapping kinase) or any of its biologically active isoforms and variants , for the treatment of diseases with inflammatory component, among others, neurodegerative conformational diseases (protein misfolding diseases), and more specifically for the treatment of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobe degeneration (Frontotemporal lobar degeneration or FTLD ).
ESTADO DE LA TÉCNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
Varias enfermedades neurodegenerativas se asocian a mutaciones en los genes que codifican para las proteínas que se agregan, indicando que la agregación proteica es causa y no consecuencia de la muerte celular. Sin embargo, este concepto se aplica también a los casos 'esporádicos' de dichas enfermedades. Se postulan dos modelos conceptuales del mecanismo de la citotoxicidad de los agregados. El primero plantea que las proteínas mutantes agregadas pierden su funcionalidad y no pueden desarrollar sus funciones normales esenciales. El segundo es que los agregados se hacen tóxicos al interferir con los procesos celulares normales y generan estrés al gatillar respuestas de daño e inflamación, o porque los agregados atrapan otras proteínas esenciales, interfiriendo con procesos celulares cruciales. Several neurodegenerative diseases are associated with mutations in the genes that code for the proteins that are added, indicating that protein aggregation is a cause and not a consequence of cell death. However, this concept also applies to 'sporadic' cases of such diseases. Two conceptual models of the mechanism of cytotoxicity of aggregates are postulated. The first states that aggregate mutant proteins lose their functionality and cannot develop their essential normal functions. The second is that the aggregates become toxic by interfering with normal cellular processes and generate stress by triggering damage and inflammation responses, or because the aggregates trap other essential proteins, interfering with crucial cellular processes.
Se sabe que los agregados proteicos aberrantes desencadenan respuestas pro- inflamatorias y que las enfermedades conformacionales neurodegerativas están asociadas a un aumento progresivo y exacerbado de neuroinflamación mediado por microglia. It is known that aberrant protein aggregates trigger pro-inflammatory responses and that neurodegerative conformational diseases are associated with a progressive and exacerbated increase in microglia-mediated neuroinflammation.
Por tanto, los procesos neuroinflamatorios son un factor crítico al desencadenar la cascada patológica que conduce a la degeneración neuronal. Señales de daños externos o internos activan las células microgliales, favoreciendo la producción de factores citotóxicos que inducen la degeneración neuronal. Estos factores activan la proteína-quinasas, que conducen a la hiperfosforilación de proteínas agregantes, y su consecuente oligomerización. Estos agregados liberados al medio extracelular, al activar a la célula microglial, provocarían un mecanismo de retroalimentación positiva favoreciendo la neurodegeneración. Entre lo que podíamos denominar enfermedades conformacionales neurodegerativas se encuentran, pero sin limitarse, esclerosis lateral amiotrófica (ELA/ALS), degeneración del lóbulo frontotemporal (FTLD), Enfermedad de Parkinson, Enfermedad de Alzheimer, Enfermedad de Huntington, encefalopatías espongiformes, tauopatías múltiples, polineuropatía amiloidótica familiar, etc. Therefore, neuroinflammatory processes are a critical factor in triggering the pathological cascade that leads to neuronal degeneration. Signs of external or internal damage activate microglial cells, favoring the production of cytotoxic factors that induce neuronal degeneration. These factors activate protein kinases, which lead to hyperphosphorylation of aggregating proteins, and their consequent oligomerization. These aggregates released into the extracellular environment, upon activation of the microglial cell, would cause a positive feedback mechanism favoring neurodegeneration. Between What we could call neurodegerative conformational diseases are, but not limited to, amyotrophic lateral sclerosis (ALS / ALS), frontotemporal lobe degeneration (FTLD), Parkinson's disease, Alzheimer's disease, Huntington's disease, spongiform encephalopathies, multiple tauopathies, amyloid polyneuropathy family, etc.
La esclerosis lateral amiotrófica (ELA/ALS) es un trastorno neurodegenerativo fatal actualmente incurable, principalmente porque los mecanismos moleculares de esta enfermedad y otras relacionadas (e.g. FTLD) permanecen enigmáticos (Fujisawa et al., Hum. Mol. Gen. 2016). Actualmente, la única droga aprobada para su tratamiento es el 'riluzol', que aumenta la esperanza de vida de los pacientes en sólo 2-3 meses. Amyotrophic lateral sclerosis (ALS / ALS) is a fatal neurodegenerative disorder currently incurable, mainly because the molecular mechanisms of this and related diseases (e.g. FTLD) remain enigmatic (Fujisawa et al., Hum. Mol. Gen. 2016). Currently, the only drug approved for treatment is 'riluzole', which increases the life expectancy of patients in only 2-3 months.
Agentes moduladores de la actividad de las proteín kinasas son conocidos en el estado del arte. Por ejemplo, Garske AL et al., PNAS 201 1 describen una aproximación químico- genética basado en la complementariedad covalente entre una cisteína gatekeeper y un inhibidor electrofílico. Agents modulating the activity of protein kinases are known in the state of the art. For example, Garske AL et al., PNAS 201 1 describe a chemical-genetic approach based on the covalent complementarity between a gatekeeper cysteine and an electrophilic inhibitor.
Está demostrado que la alteración de las rutas de señalización de las MAPK (mitogen- activated protein kinases) en los mamíferos, contribuye a la patología de diversas enfermedades humanas incluyendo el cáncer y los trastornos neurodegenerativos como Alzheimer, Parkinson, y ELA (Kim EK & Choi EJ, Arch. Toxicol. 2015). Por ejemplo, se conoce que la MAPK p38 y sus sustratos juegan un papel importante en la patología del Alzheimer, y actualmente se están evaluando en ensayos clínicos de fase II una serie de inhibidores de la p38 MAPK como agentes terapéuticos para la depresión y otras enfermedades neuronales (Ramesh G, Inflamm. Cell Signal. 2014). Si bien se conoce la participación de ciertos miembros de la superfamilia de las MAPK en el Alzheimer, la identidad de las proteínas MAPK involucradas en la neuroinflamación exacerbada en ELA y otras enfermedades relacionadas, no se conoce aún. El FTLD es un síndrome clínico causado por la degeneración del lóbulo frontal del cerebro humano, que puede extenderse al lóbulo temporal. Es uno de los tres síndromes provocados por la degeneración lobular frontotemporal, y la segunda causa más común de la demencia de inicio temprano tras la enfermedad de Alzheimer It is shown that the alteration of the signaling pathways of MAPK (mitogen-activated protein kinases) in mammals, contributes to the pathology of various human diseases including cancer and neurodegenerative disorders such as Alzheimer's, Parkinson's, and ELA (Kim EK & Choi EJ, Arch. Toxicol. 2015). For example, it is known that p38 MAPK and its substrates play an important role in Alzheimer's disease, and a series of p38 MAPK inhibitors as therapeutic agents for depression and other diseases are currently being evaluated in phase II clinical trials. neuronal (Ramesh G, Inflamm. Cell Signal. 2014). While the participation of certain members of the MAPK superfamily in Alzheimer's disease is known, the identity of MAPK proteins involved in exacerbated neuroinflammation in ALS and other related diseases is not yet known. FTLD is a clinical syndrome caused by the degeneration of the frontal lobe of the human brain, which can spread to the temporal lobe. It is one of three syndromes caused by frontotemporal lobular degeneration, and the second most common cause of early onset dementia after Alzheimer's disease
Por tanto, la identificación de una diana terapéutica clave, en este caso la proteína kinasa MOK o alguna de sus isoformas, y su posible modulación, podrían representar un importante avance en el desarrollo de nuevas aproximaciones terapéuticas para el tratamiento de enfermedades conformacionales neurodegerativas, y principalmente donde la proteína agregante es la TDP-43, como la ELA y la FTLD. Therefore, the identification of a key therapeutic target, in this case the MOK protein kinase or some of its isoforms, and its possible modulation, could represent an important advance in the development of new therapeutic approaches for the treatment of neurodegerative conformational diseases, and mainly where the aggregating protein is TDP-43, such as ALS and FTLD.
BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
Los autores de la invención, en los ejemplos, demuestran que la modulación de la expresión y/o actividad de la proteína kinasa humana MOK (MAPK/MAK/MRK overlapping kinase; 419 aa; Acc. Number BAA81688 o Q9UQ07) o de cualquiera de sus isoformas o variantes biológicamente activas, cuya expresión y estado de fosforilación (requerido para su actividad como MAPK) se ven fuertemente afectados en un modelo celular de ELA/FTLD, lo que serviría para modular el perfil neuroinflamatorio-neurodegenerativo asociado a estas enfermedades 'conformacionales' neurodegenerativas. En particular, demuestran que el inhibidor específico de MOK 'compuesto 13' (Garske AL et al., PNAS 201 1) o algún análogo o derivado del mismo, podría utilizarse para modular la actividad de MOK como tratamiento terapéutico para la ELA/FTLD en sus variantes genéticas o esporádicas, estando su alteración -de acuerdo a nuestros nuevos resultados- asociada a los agregados/inclusiones de TDP-43, presentes en la casi totalidad de los casos de ELA y FTLD (genéticos y esporádicos). The authors of the invention, in the examples, demonstrate that the modulation of the expression and / or activity of the human protein kinase MOK (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07) or any of its isoforms or biologically active variants, whose expression and phosphorylation status (required for its activity as MAPK) are strongly affected in a cellular model of ALS / FTLD, which would serve to modulate the neuroinflammatory-neurodegenerative profile associated with these conformational diseases 'neurodegenerative. In particular, they demonstrate that the specific 'compound 13' MOK inhibitor (Garske AL et al., PNAS 201 1) or some analogue or derivative thereof, could be used to modulate MOK activity as a therapeutic treatment for ALS / FTLD in its genetic or sporadic variants, being its alteration -according to our new results- associated with the aggregates / inclusions of TDP-43, present in almost all cases of ALS and FTLD (genetic and sporadic).
Por tanto, un primer aspecto de la invención se refiere un agente modulador de la actividad de MOK, de ahora en adelante agente modulador de MOK de la invención, para la prevención, mejora y/o tratamiento de enfermedades inflamatorias, más preferiblemente de enfermedades neurodegenerativas mediadas por procesos neuroinflamatorios. Therefore, a first aspect of the invention relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of inflammatory diseases, more preferably neurodegenerative diseases. mediated by neuroinflammatory processes.
Alternativamente también se refiere al uso de un agente modulador de la actividad de MOK (agente modulador de MOK de la invención) en la elaboración de un medicamento para la prevención, mejora y/o tratamiento de enfermedades inflamatorias, más preferiblemente de enfermedades neurodegenerativas mediadas por inflamación. Alternatively it also refers to the use of a MOK activity modulating agent (MOK modulating agent of the invention) in the preparation of a medicament for the prevention, improvement and / or treatment of inflammatory diseases, more preferably of neurodegenerative diseases mediated by inflammation.
En una realización preferida de este aspecto de la invención, la enfermedad neurodegenerativa es una enfermedad conformacional neurodegerativa. En otra realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment of this aspect of the invention, neurodegenerative disease is a neurodegerative conformational disease. In another preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA), Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Atrofia muscular progresiva, Primary lateral sclerosis Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal. Spinal muscular atrophy.
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad es la ELA o el FTLD. Por tanto, los inhibidores de MOK serían útiles para el tratamiento de enfermedades conformacionales nurodegenerativas, y más concretamente, donde la proteína agregante es la TDP-43. More preferably, the disease is ALS or FTLD. Therefore, MOK inhibitors would be useful for the treatment of nurodegenerative conformational diseases, and more specifically, where the aggregating protein is TDP-43.
En una realización preferida de este aspecto de la invención, los agentes moduladores de la actividad de MOK se seleccionan de una lista que consiste en: In a preferred embodiment of this aspect of the invention, MOK activity modulating agents are selected from a list consisting of:
a) una molécula o compuesto orgánico, a) an organic molecule or compound,
b) una molécula de ARN, b) an RNA molecule,
c) un oligonucleótido antisentido, c) an antisense oligonucleotide,
d) un anticuerpo, o d) an antibody, or
e) una ribozima. e) a ribozyme.
En una realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención es una molécula orgánica, y más preferiblemente, la molécula orgánica de fórmula (I): In a preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (I):
Figure imgf000005_0001
Figure imgf000005_0001
Fórmula (I) Formula (I)
Donde Where
n es igual a 0 ó 1 , n is equal to 0 or 1,
R es un metilo (Me), isopropilo (¡Pr), tere-Butilo (tBu), ciciopeniadienil© (Cp)  R is a methyl (Me), isopropyl (¡Pr), tere-Butyl (tBu), ciciopeniadienil © (Cp)
X es H ó NH2, Y es H, NHCOCHCH2, NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, y Z es H, X is H or NH 2 , Y is H, NHCOCHCH2, NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, and Z is H,
o cualquiera de sus sales, preferiblemente cualquier sal farmacéuticamente aceptable, ésteres, tautómeros, polimorfos, hidratos farmacéuticamente aceptables, o un isómero, profármacos, derivados, solvatos o análogos, o cualquiera de sus combinaciones. or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
En una realización más preferida, de este aspecto de la invención, el agente modulador de MOK de la invención es una molécula orgánica, y más preferiblemente, la molécula orgánica de fórmula (II): In a more preferred embodiment, of this aspect of the invention, the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (II):
Figure imgf000006_0001
Figure imgf000006_0001
Fórmula (I) Formula (I)
Donde Where
n es igual a 1 , n is equal to 1,
R es un isopropilo (¡Pr), R is an isopropyl (¡Pr),
X es NH2, X is NH 2 ,
Y es COCH2F And it is COCH2F
Z es H, Z is H,
o cualquiera de sus sales, preferiblemente cualquier sal farmacéuticamente aceptable, ésteres, tautómeros, polimorfos, hidratos farmacéuticamente aceptables, o un isómero, profármacos, derivados, solvatos o análogos, o cualquiera de sus combinaciones. or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
Recientemente, con el desarrollo de la tecnología antisentido, secuencias de nucleótidos específicamente complementarios a una determinada secuencia de ADN o ARN, podrían formar complejos y bloquear la transcripción o traducción. Así, con el progreso del silenciamiento génico post-transcripcional, y en particular del ARN de interferencia (RNA interferente o RNAi), se han desarrollado herramientas que permiten la inhibición específica de la expresión de un gen. La inhibición de la expresión de MOK constituiría por ende la inhibición de su actividad biológica, y en concreto, de la actividad que está contribuyendo al agravamiento de la enfermedad conformacional neurodegerativa. Por tanto, en otra realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención comprende un oligonucleotido antisentido y se usa para la prevención, mejora y/o tratamiento de la enfermedad conformacional neurodegerativa. Recently, with the development of antisense technology, nucleotide sequences specifically complementary to a particular DNA or RNA sequence could form complexes and block transcription or translation. Thus, with the progress of post-transcriptional gene silencing, and in particular of interfering RNA (interfering RNA or RNAi), tools have been developed that allow specific inhibition of gene expression. The inhibition of the expression of MOK would therefore constitute the inhibition of its biological activity, and in particular, of the activity that is contributing to the aggravation of the neurodegerative conformational disease. Therefore, in another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises an antisense oligonucleotide and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
En otra realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención comprende un anticuerpo y se usa para la prevención, mejora y/o tratamiento de la enfermedad conformacional neurodegerativa. En otra realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención comprende una ribozima y se usa para la prevención, mejora y/o tratamiento de la enfermedad conformacional neurodegerativa. In another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises an antibody and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease. In another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises a ribozyme and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
Un segundo aspecto de la invención se refiere a una composición, de ahora en adelante composición de la invención, que comprende un agente modulador de MOK de la invención. En una realización preferida, además comprende un vehículo o excipiente farmacéuticamente aceptable. En otra realización preferida la composición de la invención es una composición farmacéutica. En otra realización preferida, además comprende otro principio activo. A second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a MOK modulating agent of the invention. In a preferred embodiment, it further comprises a pharmaceutically acceptable carrier or excipient. In another preferred embodiment the composition of the invention is a pharmaceutical composition. In another preferred embodiment, it also comprises another active ingredient.
Un tercer aspecto de la invención consiste en un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad conformacional neurodegerativa que comprende: A third aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
a) poner en contacto el compuesto a analizar con el polipéptido MOK, a) contacting the compound to be analyzed with the MOK polypeptide,
b) detectar la unión de dicho compuesto a analizar con el polipéptido MOK. b) detect the binding of said compound to be analyzed with the MOK polypeptide.
Los compuestos que se unen al polipéptido MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad conformacional neurodegerativa. En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: Compounds that bind to the MOK polypeptide would be identified as potential therapeutic agents against neurodegerative conformational disease. In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA),  Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria  Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Atrofia muscular espinal. Hirayama disease or monomelic atrophy Spinal muscular atrophy.
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. or any of its combinations. More preferably, the neurodegerative conformational disease is ALS or FTLD.
En un cuarto aspecto de la invención se describe un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad conformacional neurodegerativa que comprende: In a fourth aspect of the invention a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
a) determinar la actividad de MOK a una concentración establecida del compuesto a analizar o en ausencia de dicho compuesto, a) determine the activity of MOK at an established concentration of the compound to be analyzed or in the absence of said compound,
b) determinar la actividad de MOK a una concentración del compuesto a analizar diferente de la de a). Compuestos que den lugar a una actividad diferente de MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad conformacional neurodegerativa. b) determine the activity of MOK at a concentration of the compound to be analyzed different from that of a). Compounds that give rise to a different activity of MOK would be identified as potential therapeutic agents against neurodegerative conformational disease.
En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA),  Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal, Spinal muscular atrophy,
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. Un quinto aspecto de la invención consiste en un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad conformacional neurodegerativa que comprende: or any of its combinations. More preferably, the neurodegerative conformational disease is ALS or FTLD. A fifth aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
a) poner en contacto el compuesto a analizar con el polinucleótido MOK, a) contacting the compound to be analyzed with the MOK polynucleotide,
b) detectar la unión de dicho compuesto a analizar con el polinucleótido MOK, Los compuestos que se unen al polinucleótido MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad conformacional neurodegerativa. b) detecting the binding of said compound to be analyzed with the MOK polynucleotide, Compounds that bind to the MOK polynucleotide would be identified as potential therapeutic agents against neurodegerative conformational disease.
En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA), Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal, Spinal muscular atrophy,
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. More preferably, the neurodegerative conformational disease is ALS or FTLD.
En un sexto aspecto de la invención se describe un método para la recolección de datos útiles en el diagnóstico y/o pronóstico de una enfermedad conformacional neurodegerativa que comprende: In a sixth aspect of the invention a method is described for the collection of data useful in the diagnosis and / or prognosis of a neurodegerative conformational disease comprising:
a) determinar la expresión de MOK en una muestra extraída de un mamífero, a) determine the expression of MOK in a sample taken from a mammal,
b) comparar los valores de la expresión de MOK obtenidos en a) con los valores estándar en mamíferos sanos o enfermos. En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: b) compare the values of the MOK expression obtained in a) with the standard values in healthy or diseased mammals. In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA),  Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria  Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica  Hirayama disease or monomelic atrophy
Atrofia muscular espinal.  Spinal muscular atrophy.
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. DESCRIPCIÓN DE LAS FIGURAS More preferably, the neurodegerative conformational disease is ALS or FTLD. DESCRIPTION OF THE FIGURES
Fig. 1. Los agregados de TDP-43 son internalizados y modificados por células microgliales, movilizando la proteína TDP-43 endógena. A) Caracterización por SDS- PAGE al 12% de cuerpos de inclusión que contienen TDP-43 purificados (TDP43) y cuerpos de inclusión simulados (simulado), 10 de proteína cargada por muestra, mostrando un contenido de proteína TDP-43 de aprox. 50%. B) Inmunofluorescencia (IF) con His-tag (verde) y TDP-43 (rojo) inmunomarcación en células microgliales primarias después de 4 h de tratamiento con 2,5 μg / mL de TDP43 (o control simulado) mostrando TDP-43- conteniendo puncta internalizada en el citoplasma. Las imágenes fusionadas incluyen fluorescencia DAPI (azul). Barras graduadas: 25 μηι. C) Western blot de lisados celulares de células tratadas usando inmunomarcaje anti-TDP-43. Las células microgliales primarias habían sido expuestas a 5 μg / mi de TDP43 o mock (control), durante 24 h. Un número de bandas adicionales, especialmente de masa molecular ca. 20-25 kDa, podría verse en las células expuestas a TDP43. D-E) La ubiquitinación de proteínas intracelulares y el TDP-43 ubiquitinado en células microgliales primarias tratadas con TDP43 o mock durante 24 h, se determinaron mediante estudios de IF (2,5 μg / mi de proteína total). IF imágenes muestran el etiquetado de ubiquitina (verde), TDP-43 etiquetado (rojo), y las imágenes de fusión, incluyendo DAPI fluorescencia (D). Western blot de lisados celulares (5 μ g / mi) (E), con ubiquitina específica immunolabeling. La flecha muestra la presencia de una banda adicional de ca. 51 kDa en células expuestas a TDP43. Se usaron células tratadas con MG132 5 μΜ durante 90 minutos como control positivo para la ubiquitina IF (D). Barras de escala E: 20 μηι. F) Análisis de IF con inmunomarcación TDP-43 (rojo) de células N13 tratadas con 5 \ mu g / mi de TDP43 o simulación durante 4 h. Los resultados muestran una reducción significativa en los niveles de proteína nuclear TDP-43 (aclaramiento nuclear de TDP-43) en células expuestas al tratamiento con TDP43. Barras graduadas: 20 μηι. G) IF análisis por microscopía confocal de CD11 b (blanco), His-tag (rojo) y TDP-43 (verde) immunotipado de los cultivos de corte organotípico de la médula espinal después de 24 h de tratamiento con 5 μ g / mL de TDP43 (o mock de control) mostrando la presencia de inclusiones que contenían His-tag / TDP-43 en el citoplasma de células microgliales y la eliminación de TDP-43 nuclear, en la muestra tratada con agregados TDP-43 exógenos. Los núcleos celulares se contrastaron con DAPI (azul). Barras graduadas: 25 μηι. Se utilizaron cultivos sin tratamiento y cultivos tratados simuladamente (mock) como controles en todos los experimentos. Las imágenes y los resultados mostrados son representativos de al menos tres experimentos independientes en todos los casos. Fig. 2. Los agregados de TDP-43 activan las respuestas neuroinflamatorias independientes dependientes de NLRP3. A) Determinación de la citotoxicidad con células BV-2 células microgliales expuestas durante 24 h y 48 h a TDP43 o mock a 5 μ g / mi o inferior, se realizaron mediante el ensayo de MTT (ensayo de viabilidad celular). B) La expresión de Pro-caspasa-1 en células tratadas se analizó mediante transferencia Western con lisados celulares de células microgliales primarias tratadas durante 24 h con 2,5 \ μg / mi de TDP43 o simulación (mock). La mancha es representativa de 5 experimentos independientes (N = 5). C) Determinación cuantitativa por ELISA de los niveles de citoquinas de I L-1 β y TNF-α en sobrenadantes de N13 (TNFa) o células microgliales primarias (IL-1 β), 6 h o 24 h después del tratamiento con 5, 2,5 ó 1 ,25 μg / mL de TDP43 o simulación (mock), con o sin pretratamiento con Bay 11-7082. Las barras son la media de al menos 3 experimentos independientes (N≥3), cada uno realizado con muestras duplicadas. D) Determinación cuantitativa por ELISA de IL-18 activada secretada en sobrenadantes de cultivo de microglia primaria 24 h después del tratamiento con 2,5 ó 5 μg / mL de TDP43 o simulación (mock). Los niveles medidos absolutos de IL-18 para células tratadas con TDP43 oscilaron entre 60 y 90 pg/ml entre experimentos independientes. Las barras son la media de seis experimentos independientes (N = 6), cada uno realizado con muestras duplicadas. E) El análisis Western blot de IL - 18 escindida en sobrenadantes de células microgliales primarias recogidas 24 h después del tratamiento con 5 μg / mi de TDP43 o simulación (mock). La IL-18 murina recombinante se utilizó como control en el mismo gel (extremo derecho). Inferior: cuantificación de la densidad de banda (valores relativos) de tres experimentos independientes (N = 3). F) Análisis IF de IL-18 intracelular en células N13, 24 h después del tratamiento con 5 μg / mi de TDP43 o simulación. Barras graduadas: 20 μηι. G) Análisis Western blot de IL-18 escindida en lisados celulares de células microgliales primarias tratadas con 5 μg /mi de TDP43 o simulación (mock), durante 24 h. Análisis cuantitativo de ca. 16 kDa IL-18 (valores relativos) a partir de cuatro experimentos independientes (N = 4). Las células sin tratamiento y las células tratadas simuladamente se usaron como controles en todos los experimentos. A menos que se indique lo contrario, las barras de error corresponden a ± SEM de al menos 3 experimentos independientes. En todos los casos, p <0,05, ** p <0,01 , *** p <0,001 indican diferencias significativas para los resultados de N≥3 experimentos independientes. Fig. 1. The aggregates of TDP-43 are internalized and modified by microglial cells, mobilizing the endogenous TDP-43 protein. A) Characterization by 12% SDS-PAGE of inclusion bodies containing purified TDP-43 (TDP43) and simulated inclusion bodies (simulated), 10 of protein loaded per sample, showing a TDP-43 protein content of approx. fifty%. B) Immunofluorescence (IF) with His-tag (green) and TDP-43 (red) immunostaining in primary microglial cells after 4 h of treatment with 2.5 μg / mL of TDP43 (or simulated control) showing TDP-43- containing internalized puncta in the cytoplasm. Fused images include DAPI fluorescence (blue). Graduated bars: 25 μηι. C) Western blotting of cell lysates from cells treated using anti-TDP-43 immunomarking. The primary microglial cells had been exposed to 5 μg / ml of TDP43 or mock (control), for 24 h. A number of additional bands, especially of molecular mass ca. 20-25 kDa, could be seen in cells exposed to TDP43. DE) The ubiquitination of intracellular proteins and the ubiquitinated TDP-43 in primary microglial cells treated with TDP43 or mock for 24 h, were determined by IF studies (2.5 μg / mi total protein). IF images show ubiquitin labeling (green), TDP-43 labeling (red), and fusion images, including DAPI fluorescence (D). Western blot of cell lysates (5 μg / mi) (E), with specific ubiquitin immunolabeling. The arrow shows the presence of an additional band of ca. 51 kDa in cells exposed to TDP43. Cells treated with 5 mg MG132 were used for 90 minutes as a positive control for ubiquitin IF (D). Scale bars E: 20 μηι. F) IF analysis with TDP-43 immunostaining (red) of N13 cells treated with 5 µg / ml of TDP43 or simulation for 4 h. The results show a significant reduction in TDP-43 nuclear protein levels (TDP-43 nuclear clearance) in cells exposed to TDP43 treatment. Graduated bars: 20 μηι. G) IF confocal microscopy analysis of CD11 b (white), His-tag (red) and TDP-43 (green) immunotyped cultures of spinal cord organotypic cultures after 24 h of treatment with 5 μg / mL of TDP43 (or control mock) showing the presence of inclusions containing His-tag / TDP-43 in the microglial cell cytoplasm and the elimination of nuclear TDP-43, in the sample treated with exogenous TDP-43 aggregates. Cell nuclei were contrasted with DAPI (blue). Graduated bars: 25 μηι. Untreated cultures and mock treated cultures were used as controls in all experiments. The images and the results shown are representative of at least three independent experiments in all cases. Fig. 2. The aggregates of TDP-43 activate NLRP3-dependent independent neuroinflammatory responses. A) Determination of cytotoxicity with BV-2 cells microglial cells exposed for 24 h and 48 h TDP43 or mock at 5 μg / ml or less, were performed by MTT test (cell viability test). B) Pro-caspase-1 expression in treated cells was analyzed by Western blotting with cell lysates of primary microglial cells treated for 24 h with 2.5 µg / ml of TDP43 or simulation (mock). The stain is representative of 5 independent experiments (N = 5). C) Quantitative determination by ELISA of cytokine levels of I L-1β and TNF-α in supernatants of N13 (TNFa) or primary microglial cells (IL-1β), 6 h or 24 h after treatment with 5, 2 , 5 or 1, 25 μg / mL of TDP43 or simulation (mock), with or without pretreatment with Bay 11-7082. The bars are the average of at least 3 independent experiments (N≥3), each performed with duplicate samples. D) Quantitative determination by ELISA of activated IL-18 secreted in primary microglia culture supernatants 24 h after treatment with 2.5 or 5 μg / mL of TDP43 or simulation (mock). The absolute measured levels of IL-18 for cells treated with TDP43 ranged from 60 to 90 pg / ml between independent experiments. The bars are the average of six independent experiments (N = 6), each performed with duplicate samples. E) Western blot analysis of IL-18 cleaved in supernatants of primary microglial cells collected 24 h after treatment with 5 μg / ml of TDP43 or simulation (mock). Recombinant murine IL-18 was used as a control in the same gel (right end). Lower: quantification of the band density (relative values) of three independent experiments (N = 3). F) IF analysis of intracellular IL-18 in N13 cells, 24 h after treatment with 5 μg / ml of TDP43 or simulation. Graduated bars: 20 μηι. G) Western blot analysis of IL-18 cleaved in cell lysates of primary microglial cells treated with 5 μg / ml of TDP43 or simulation (mock), for 24 h. Quantitative analysis of ca. 16 kDa IL-18 (relative values) from four independent experiments (N = 4). Untreated cells and simulated cells were used as controls in all experiments. Unless otherwise indicated, the error bars correspond to ± SEM of at least 3 independent experiments. In all cases, p <0.05, ** p <0.01, *** p <0.001 indicate significant differences for the results of N≥3 independent experiments.
Fig. 3. Los agregados de TDP-43 inducen una respuesta neuroinflamatoria que involucra caspasa-3. A) Análisis por IF y microscopía confocal de niveles de caspasa-3 activada en células de microglia primaria 4 h después del tratamiento con 5 μg / mL de TDP43 o simulación (mock). El tratamiento con 1 μΜ de estaurosporina (STS) durante 4 hy 1 μg / mi de LPS durante 24 horas sirvió como controles de "apoptosis" e "inflamación", respectivamente. Las imágenes fusionadas incluyen fluorescencia DAPI. Barras graduadas: 25 μηι. B, C) Evaluación cuantitativa de los niveles de caspasa-3 activada de IF y de las imágenes de microscopía confocal (como se muestra en A), basadas en caspasa activada total 3 (B) o gránulos de caspasa-3 activada en los compartimientos citoplásmicos y nucleares de células de la microglia primaria (C). Los valores mostrados se normalizaron mediante el control sin tratar (Ctrl) dentro de cada uno de los tres experimentos, y posteriormente se refirieron al promedio de gránulos / célula de las tres muestras Ctrl. D) Estudio de neurotoxicidad mediada por microglia realizado mediante ensayo LDH de células cultivadas con neurona motora NSC-34 después de incubación durante 24 h o 48 h con medio acondicionado libre de agregados de cultivos microgliales primarios tratados con 5 μg / mi de TDP43 o simulación (mock). El diagrama es representativo de tres experimentos independientes cada uno realizado por muestras duplicadas. E) IF análisis por microscopía confocal de CD11 b (rojo) y caspasa-3 (verde) immunotipado de los cultivos de corte organotípico de la médula espinal después de 24 h de tratamiento con 5 μ g / ml_ de TDP43 (o simulacro de control), mostrando la presencia de gránulos de caspasa-3 en el citoplasma de células microgliales de la muestra tratada con agregados TDP-43 exógenos. Los núcleos celulares se contrastaron con DAPI (azul). Barras graduadas: 25 μηι. Las células sin tratamiento y las células tratadas simuladamente se usaron como controles en todos los experimentos. Las imágenes y los resultados mostrados son representativos de al menos tres experimentos independientes en todos los casos. A menos que se indique lo contrario, las barras de error corresponden a ± SEM de al menos 3 experimentos independientes. En todos los casos, p <0,05, ** p <0,01 , *** p <0,001 indican diferencias significativas para los resultados de N≥3 experimentos independientes. Fig. 3. The aggregates of TDP-43 induce a neuroinflammatory response that involves caspase-3. A) Analysis by IF and confocal microscopy of activated caspase-3 levels in primary microglia cells 4 h after treatment with 5 μg / mL of TDP43 or simulation (mock). Treatment with 1 μΜ of staurosporin (STS) for 4 h and 1 μg / ml of LPS for 24 hours served as controls for "apoptosis" and "inflammation", respectively. Fused images include DAPI fluorescence. Graduated bars: 25 μηι. B, C) Quantitative evaluation of the levels of activated caspase-3 of IF and confocal microscopy images (as shown in A), based on total activated caspase 3 (B) or granules of activated caspase-3 in the compartments Cytoplasmic and nuclear cells of the primary microglia (C). The values shown were normalized by the untreated control (Ctrl) within each of the three experiments, and subsequently referred to the average granules / cell of the three Ctrl samples. D) Microglia-mediated neurotoxicity study conducted by means of LDH assay of cells cultured with NSC-34 motor neuron after incubation for 24 hours or 48 hours with aggregate-free medium of aggregates of primary microglial cultures treated with 5 μg / ml of TDP43 or simulation ( mock) The diagram is representative of three independent experiments each performed by duplicate samples. E) IF confocal microscopy analysis of CD11 b (red) and caspase-3 (green) immunotyped from organotypic cultures of the spinal cord after 24 h of treatment with 5 μg / ml_ of TDP43 (or control simulation) ), showing the presence of caspase-3 granules in the cytoplasm of microglial cells of the sample treated with exogenous TDP-43 aggregates. Cell nuclei were contrasted with DAPI (blue). Graduated bars: 25 μηι. Untreated cells and simulated cells were used as controls in all experiments. The images and the results shown are representative of at least three independent experiments in all cases. Unless otherwise indicated, the error bars correspond to ± SEM of at least 3 independent experiments. In all cases, p <0.05, ** p <0.01, *** p <0.001 indicate significant differences for the results of N≥3 independent experiments.
Fig. 4. Agregados de TDP-43 extracelular son blanco de la vía MOK de señalización en microglia. A) Análisis de Co-IF por microscopía confocal de cultivos inmunotipados CD1 1 b (blanco), His-tag (verde) y MOK (rojo) de cortes organotípicos de ratones de 4 semanas de edad después de 24 h de tratamiento con 2,5 μg / mL de TDP43 (o control simulado) mostrando la co-localización (amarilla) en el citoplasma de MOK con especies TDP43 internalizadas (que contienen His6), en la muestra tratada con agregados TDP-43 exógenos. Los núcleos celulares se contrastaron con DAPI (azul). Barras graduadas: 25 μηι. B) Western blot del SDS-PAGE de la fracción eluida después del ensayo de Co-IP, con anticuerpo anti-MOK, confirma la interacción entre los agregados TDP-43 y MOK microglial. C) Estudios de Co-IF mediante microscopía confocal con proteínas His-tag y MOK- inmunomarcadas de células microgliales primarias expuestas a 5 μg / mL de TDP43 o de correlación simulada entre especies TDP-43 internalizadas y MOK en células expuestas a agregados de TDP- 43. Las imágenes fusionadas incluyen fluorescencia DAPI. Barras graduadas: 25 μηι. D) Análisis cuantitativo de His6-tag / MOK co-localización basada en co- IF y microscopía confocal análisis. E) Transferencia de Western de lisado total a partir de células microgliales primarias, 30 minutos después del tratamiento con 5 / mi de TDP43 o simulación, usando anticuerpo específico de fosfo-Tyr MOK (izquierda). Análisis cuantitativo por densitometría de banda de los niveles de MOK fosforilados con Tyr en el ensayo Western Blot (derecha). La normalización se realizó contra GAPDH, y el simulacro de tratamiento se consideró como 100%. Los valores son media ± SD y derivan de dos experimentos independientes (N = 2), cada uno realizado en triplicado. En todos los demás casos, las imágenes y los resultados mostrados son representativos de tres experimentos independientes. Fig. 4. Aggregates of extracellular TDP-43 are targets of the MOK microglia signaling pathway. A) Co-IF analysis by confocal microscopy of CD1 1b (white), His-tag (green) and MOK (red) immunotyped cultures of organotypic sections of 4-week-old mice after 24 h of treatment with 2, 5 μg / mL of TDP43 (or simulated control) showing co-localization (yellow) in the MOK cytoplasm with internalized TDP43 species (containing His6), in the sample treated with exogenous TDP-43 aggregates. Cell nuclei were contrasted with DAPI (blue). Graduated bars: 25 μηι. B) Western blot of the SDS-PAGE of the fraction eluted after the Co-IP assay, with anti-MOK antibody, confirms the interaction between the TDP-43 aggregates and microglial MOK. C) Co-IF studies using confocal microscopy with His-tag and MOK-immunostained proteins of primary microglial cells exposed to 5 μg / mL of TDP43 or simulated correlation between internalized TDP-43 and MOK species in cells exposed to TDP aggregates - 43. Merged images include DAPI fluorescence. Graduated bars: 25 μηι. D) Quantitative analysis of His6-tag / MOK co-location based on co- IF and confocal microscopy analysis. E) Western blot of total lysate from primary microglial cells, 30 minutes after treatment with 5 µm of TDP43 or simulation, using phospho-Tyr MOK specific antibody (left). Quantitative analysis by band densitometry of phosphorylated MOK levels with Tyr in the Western Blot assay (right). Normalization was performed against GAPDH, and the mock treatment was considered as 100%. The values are mean ± SD and derive from two independent experiments (N = 2), each performed in triplicate. In all other cases, the images and results shown are representative of three independent experiments.
Fig. 5 (Figura Suplementaria 1 ). Procesamiento alternativo de IL-18 en especies ca. 16 kDa en cultivo primario de microglia. Expresión de la caspasa-1 y secreción de IL-18 en cultivos del corte organotípico de médula espinal. A) Western blot del lisado total de microglia primaria tratada durante 24 h con 2,5 μg /mi de TDP43 o simular, y cotratamiento con inhibidor de inflammasoma NLRP3 Bay1 1 - 7082, usando un anticuerpo anti-IL-18 (izquierda). El análisis cuantitativo por densitometría de banda de aprox. 16 kDa IL-18 (derecha). Los valores mostrados (media ± SEM) son relativos al control no tratado (Ctrl) en cada experimento, y derivan de cuatro experimentos independientes (N = 2). B) Western blot usando anticuerpo anti-IL-18, del lisado total de células microgliales primarias tratadas durante 24 h con 2,5 μg/ml de TDP43 o simular, con o sin cotratamiento con Hsp27 o Hsp70 (izquierda). El análisis cuantitativo por densitometría de banda de aprox. 16 kDa IL-18 (derecha). Los valores mostrados (media ± SEM) son relativos al control no tratado (Ctrl) en cada experimento, y derivan de tres experimentos independientes (N = 3). C) Western blot del lisado total de cultivos de corte organotípico de médula espinal de ratones de 4 semanas de edad, después de 24 h de tratamiento con 2,5 μg/ml de TDP43 (o control similar), usando anticuerpo anti-capase-1 (izquierda). Análisis cuantitativo mostrado (niveles relativos) derivados de dos experimentos independientes (N = 2). Los valores son la media ± DE. D) Western blot con anticuerpo anti-IL-18 de sobrenadantes de cultivos de corte organotípico tratados, después de IP con anticuerpo anti-IL-18 con fines de concentración. La imagen mostrada es representativa de dos experimentos independientes. Fig. 5 (Supplementary Figure 1). Alternative processing of IL-18 in ca. 16 kDa in primary microglia culture. Expression of caspase-1 and secretion of IL-18 in cultures of the spinal cord organotypic section. A) Western blot of the total lysate of primary microglia treated for 24 h with 2.5 μg / ml of TDP43 or simulate, and co-treatment with NLRP3 Bay1 1 - 7082 inflammasome inhibitor, using an anti-IL-18 antibody (left). The quantitative analysis by band densitometry of approx. 16 kDa IL-18 (right). The values shown (mean ± SEM) are relative to the untreated control (Ctrl) in each experiment, and derive from four independent experiments (N = 2). B) Western blot using anti-IL-18 antibody, of the total lysate of primary microglial cells treated for 24 h with 2.5 μg / ml of TDP43 or simulated, with or without cotrareatment with Hsp27 or Hsp70 (left). The quantitative analysis by band densitometry of approx. 16 kDa IL-18 (right). The values shown (mean ± SEM) are relative to the untreated control (Ctrl) in each experiment, and are derived from three independent experiments (N = 3). C) Western blot of the total lysate of organotypic spinal cord cultures of 4-week-old mice, after 24 h of treatment with 2.5 μg / ml of TDP43 (or similar control), using anti-layer antibody. 1 (left). Quantitative analysis shown (relative levels) derived from two independent experiments (N = 2). Values are the mean ± SD. D) Western blot with anti-IL-18 antibody from supernatants of treated organotypic cultures, after IP with anti-IL-18 antibody for concentration purposes. The image shown is representative of two independent experiments.
Fig. 6 (Figura Suplementaria 2). Análisis IF de la caspasa-3 activada en las células 4 h y 24 h después del tratamiento. A) Análisis por IF y microscopía confocal de niveles de caspasa-3 activada en células microgliales primarias 4 h y 24 h después del tratamiento con 5 μg/mL de TDP43 o similar, con o sin cotratamiento con Hsp27 exógena o Hsp70. Las imágenes son de un experimento, representativo de tres experimentos independientes. Barra de escala: 25 μηι. B) El gráfico corresponde a una evaluación cuantitativa por análisis de microscopía confocal de "granulos de caspasa-3 activados" en el compartimiento citoplasmático, después de 24 h de tratamiento. Fig. 6 (Supplementary Figure 2). IF analysis of activated caspase-3 in cells 4 h and 24 h after treatment. A) Analysis by IF and confocal microscopy of activated caspase-3 levels in primary microglial cells 4 h and 24 h after treatment with 5 μg / mL of TDP43 or similar, with or without co-treatment with exogenous Hsp27 or Hsp70. The images are from one experiment, representative of three independent experiments. Scale bar: 25 μηι. B) The graph corresponds to a quantitative evaluation by analysis of confocal microscopy of "activated caspase-3 granules" in the cytoplasmic compartment, after 24 h of treatment.
Fig 7 (Figura Suplementaria 3). A) Estudios de Co-IF con inmunotipado His-tag (rojo) y MOK (verde) mediante microscopía confocal de células BV-2 expuestas a 5 μg/mL de TDP43 o similar muestran colocalización entre especies TDP-43 internalizadas y proteínas MOK en células expuestas a agregados TDP-43 exógenos. Las imágenes fusionadas incluyen fluorescencia DAPI. Barras de escala: 25 μηι. B) Análisis cuantitativo de la colocalización His6-tag/MOK (sobre MOK total) basado en co-IF y análisis microscopía confocal. C) Análisis SDS-PAGE teñido con plata de la fracción eluida en el ensayo de Co-IP realizado después de incubar TDP43 (o control similar) con lisados primarios de microglia, usando anticuerpo de captura anti-TDP-43. La flecha blanca indica una/s banda/s diferenciales y las regiones en el gel (para ambos carriles) que fueron escindidas y analizadas por MALDI/MS. D) Western blot con anticuerpos anti-MOK y anti-fosfo-Tyr MOK específicos de lisado total de BV-2 y microglia primaria, 30 minutos después del tratamiento con 1 μg/mL de LPS o 5 μg/mL de TDP43. Las células sin tratamiento se usaron como control negativo. E) Western blot de lisado total de células BV-2, 30 min después del tratamiento con 5 μg/ml de TDP43 o similar, usando anticuerpo específico de fosfo - Tyr MOK (izquierda). Análisis cuantitativo por densitometría de banda de los niveles de MOK fosforilados con Tyr en el ensayo de Western Blot (derecha). La normalización se realizó contra a-tubulina, y el tratamiento simulación se consideró como la referencia (100%). Fig 7 (Supplementary Figure 3). A) Co-IF studies with His-tag immunotyping (red) and MOK (green) by confocal microscopy of BV-2 cells exposed to 5 μg / mL of TDP43 or similar show colocalization between internalized TDP-43 species and MOK proteins in cells exposed to exogenous TDP-43 aggregates. Fused images include DAPI fluorescence. Scale bars: 25 μηι. B) Quantitative analysis of His6-tag / MOK colocalization (over total MOK) based on co-IF and confocal microscopy analysis. C) Silver-stained SDS-PAGE analysis of the fraction eluted in the Co-IP assay performed after incubating TDP43 (or similar control) with primary microglia lysates, using anti-TDP-43 capture antibody. The white arrow indicates a differential band / s and the regions in the gel (for both lanes) that were cleaved and analyzed by MALDI / MS. D) Western blotting with specific anti-MOK and anti-phospho-Tyr MOK antibodies for total lysate of BV-2 and primary microglia, 30 minutes after treatment with 1 μg / mL of LPS or 5 µg / mL of TDP43. Untreated cells were used as a negative control. E) Western blot of total lysate of BV-2 cells, 30 min after treatment with 5 μg / ml of TDP43 or similar, using phospho-Tyr MOK specific antibody (left). Quantitative analysis by band densitometry of phosphorylated MOK levels with Tyr in the Western Blot assay (right). Normalization was performed against a-tubulin, and the simulation treatment was considered as the reference (100%).
DESCRIPCIÓN DE LA INVENCIÓN El objeto de la invención consiste en modular la expresión y/o actividad de la proteína humana MOK (MAPK/MAK/MRK overlapping kinase; 419 aa; Acc. Number BAA81688 o Q9UQ07, o cualquiera de sus isoformas). De acuerdo a nuestros resultados, dicha proteína kinasa -un miembro de la superfamilia de las MAPK que se expresa en cerebro y que necesita estar fosforilada para fosforilar otros sustratos- se ve fuertemente afectada en cuanto a su expresión y estado de fosforilación, en un modelo celular de ELA/FTD. El interés en modular la expresión de esta proteína o su actividad, por ejemplo mediante la tecnología de siRNA o mediante un inhibidor específico (como la molécula pequeña e inhibidor específico 'compuesto 13', Garske AL et al., PNAS 201 1 , o alguno de sus derivados), consiste en modular la ruta de señalización celular involucrada en la respuesta neuroinflamatoria asoociada a neurodegeneración en la que, de acuerdo a nuestros resultados en modelo celular de ELA/FTD, tendría una participación clave dicha proteína. USOS MÉDICOS DE LA INVENCIÓN DESCRIPTION OF THE INVENTION The object of the invention is to modulate the expression and / or activity of the human protein MOK (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms). According to our results, said protein kinase - a member of the MAPK superfamily that expresses itself in the brain and needs to be phosphorylated to phosphorylate other substrates - is strongly affected in terms of its expression and phosphorylation status, in a model ELA / FTD cell. The interest in modulating the expression of this protein or its activity, for example by means of siRNA technology or by a specific inhibitor (such as the small molecule and specific inhibitor 'compound 13', Garske AL et al., PNAS 201 1, or some of its derivatives), consists in modulating the cellular signaling pathway involved in the neuroinflammatory response associated with neurodegeneration in which, according to our results in the cellular model of ELA / FTD, said protein would have a key participation. MEDICAL USES OF THE INVENTION
Por tanto, un primer aspecto de la invención se refiere un agente modulador de la actividad de MOK, de ahora en adelante agente modulador de MOK de la invención, para la prevención, mejora y/o tratamiento de enfermedades inflamatorias, más preferiblemente de enfermedades neurodegenerativas mediadas por inflamación. Therefore, a first aspect of the invention relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of inflammatory diseases, more preferably neurodegenerative diseases. mediated by inflammation.
Alternativamente también se refiere al uso de un agente modulador de la actividad de MOK (agente modulador de MOK de la invención) en la elaboración de un medicamento para la prevención, mejora y/o tratamiento de enfermedades inflamatorias, más preferiblemente de enfermedades neurodegenerativas mediadas por inflamación.  Alternatively it also refers to the use of a MOK activity modulating agent (MOK modulating agent of the invention) in the preparation of a medicament for the prevention, improvement and / or treatment of inflammatory diseases, more preferably of neurodegenerative diseases mediated by inflammation.
La invención se refiere también a un agente modulador de la actividad de MOK, de ahora en adelante agente modulador de MOK de la invención, para la prevención, mejora y/o tratamiento de enfermedades conformacionales neurodegenerativoas, o alternativamente, a un agente modulador de MOK de la invención, para su uso en la prevención, mejora y/o tratamiento de enfermedades conformacionales neurodegenerativoas. The invention also relates to a MOK activity modulating agent, hereinafter MOK modulating agent of the invention, for the prevention, improvement and / or treatment of neurodegenerative conformational diseases, or alternatively, a MOK modulating agent of the invention, for use in the prevention, improvement and / or treatment of neurodegenerative conformational diseases.
En esta memoria se entiende por una enfermedad inflamatoria y/o que tienen un componente inflamatorio o están mediadas por inflamación, las que se refieren a una afección en un sujeto caracterizada por la inflamación, por ejemplo, inflamación crónica. Ejemplos ilustrativ This report refers to an inflammatory disease and / or that has an inflammatory component or is mediated by inflammation, which refers to a condition in a subject characterized by inflammation, for example, chronic inflammation. Illustrative examples
os, no limitativos, de trastornos inflamatorios incluyen, pero no se limitan a, la artritis reumatoide (RA), enfermedad inflamatoria intestinal (IBD), el asma, la encefalitis, enfermedad pulmonar obstructiva crónica (EPOC), osteólisis inflamatoria, trastornos alérgicos, shock séptico, fibrosis pulmonar (por ejemplo, fibrosis pulmonar idiopática), vacuhtides inflamatorias (por ejemplo, polyarteritisnodosa, granulomatosis de Wegner, arteritis de Takayasu, arteritis temporal, y granulomatosis linfomatoide), angioplastia vascular post-traumático (por ejemplo, la restenosis después de la angioplastia), enfermedades inflamatorias intestinales como la enfermedad de Crohn y la colitis ulcerosa, la espondiloartropatía indiferenciada, artropatía indiferenciada, la artritis, la osteólisis inflamatoria, hepatitis crónica, y la inflamación crónica resultante de infecciones virales o bacterianas crónicas. También se incluyen en esta memoria, dentro del concepto enfermedades inflamatorias, el cáncer, y particularmente el cáncer renal, el melanoma y las leucemias. Non-limiting, inflammatory disorders include, but are not limited to, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (for example, idiopathic pulmonary fibrosis), inflammatory vacuhtides (for example, polyarteritisnode, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosis), post-traumatic vascular angioplasty (for example, restenosis after of angioplasty), inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacterial infections. Also included in this report are inflammatory diseases, cancer, and particularly renal cancer, melanoma and leukemia.
En esta memoria se entiende por enfermedad conformacional neurodegerativa (enfermedad conformacional o proteinopatía) la clase de patología en la cual una (o más) proteínas específicas se tornan estructuralmente anormales, y por lo tanto interfieren con la función de las células, tejidos y órganos en el cuerpo. A menudo las proteínas no consiguen plegarse en su conformación normal; en este estado mal plegado, las proteíonas forman agregados y se tornan tóxicas de alguna manera (por ej., provocando neuroinflamación) pueden perder su función normal. Entre otras se incluyen, pero sin limitarnos, enfermedad de Parkinson (EP), demencia con cuerpos de Lewy (DLB), la variante de cuerpos de Lewy de la enfermedad de Alzheimer, atrofia de sistemas múltiples (MSA), enfermedad de Alzheimer, fallo autonómico puro (FAP), esclerosis lateral amiotrófica (ELA), demencia fronto-temporal, enfermedad de Gaucher, síndrome de Down, enfermedad de Huntington, la enfermedad de priones, la enfermedad de Creutzfeldt-Jakob Jakob y otras encefalopatías espongiformes transmisibles, enfermedad de la motoneurona, síndrome de Gerstmann-Stráussler- Scheinker, Kuru, el insomnio familiar fatal, diabetes de tipo II, amiloidosis cerebrovascular, glaucoma, degeneración macular relacionada con la edad, síndromes psiquiátricos, tauopatías múltiples, polineuropatía amiloidótica familiar, y neurodegeneración debida a agregación de proteínas relacionado con la edad. Más preferiblemente se refiere a la esclerosis lateral amiotrófica (ELA), esclerosis lateral primaria, atrofia muscular progresiva, enfermedad de Kennedy o atrofia muscular progresiva espinobulbar, enfermedad de Hirayama o atrofia monomélica, atrofia muscular espinal, degeneración del lóbulo frontotemporal (FTLD), o cualquiera de sus combinaciones. In this report, neurodegerative conformational disease (conformational disease or proteinopathy) is understood as the kind of pathology in which one (or more) proteins specific ones become structurally abnormal, and therefore interfere with the function of cells, tissues and organs in the body. Often proteins fail to fold in their normal conformation; In this poorly folded state, the proteins form aggregates and become toxic in some way (eg, causing neuroinflammation) can lose their normal function. Others include, but are not limited to, Parkinson's disease (PD), Lewy body dementia (DLB), Lewy body variant of Alzheimer's disease, multiple system atrophy (MSA), Alzheimer's disease, failure Autonomic pure (FAP), amyotrophic lateral sclerosis (ALS), fronto-temporal dementia, Gaucher disease, Down syndrome, Huntington's disease, prion disease, Creutzfeldt-Jakob Jakob disease and other transmissible spongiform encephalopathies, motor neuron, Gerstmann-Stráussler-Scheinker syndrome, Kuru, fatal familial insomnia, type II diabetes, cerebrovascular amyloidosis, glaucoma, age-related macular degeneration, psychiatric syndromes, multiple tauopathies, familial amyloid polyneuropathy, and neurodegeneration due to aggregation of age-related proteins. More preferably it refers to amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, Kennedy disease or progressive spinobulbar muscular atrophy, Hirayama disease or monomelic atrophy, spinal muscular atrophy, frontotemporal lobe degeneration (FTLD), or Any of your combinations.
En una realización preferida de este aspecto de la invención, la enfermedad neurodegenerativa es una enfermedad conformacional neurodegerativa, es decir, la invención se refiere al uso del agente modulador de MOK de la invención en la elaboración de un medicamento para la prevención, mejora o tratamiento de una enfermedad conformacional neurodegerativa. En otra realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment of this aspect of the invention, neurodegenerative disease is a neurodegerative conformational disease, that is, the invention relates to the use of the MOK modulating agent of the invention in the preparation of a medicament for prevention, improvement or treatment. of a neurodegerative conformational disease. In another preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA),  Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria  Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica  Hirayama disease or monomelic atrophy
Atrofia muscular espinal.  Spinal muscular atrophy.
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad neurodegenrativa agregante es la ELA o el FTLD. El término "MOK" (MAPK/MAK/MRK overlapping kinase; 419 aa; Acc. Number BAA81688 o Q9UQ07, o cualquiera de sus isoformas), también llamada RAGE; RAGE1 ; STK30; RAGE- 1 . Este gen pertenece a la superfamilia MAP quinasa. El gen esta regulado por el factor de transcripción de tipo caudal 2 (Cdx2). La proteína codificada, que se localiza en las células epiteliales en la cripta intestinal, puede desempeñar un papel en la detención del crecimiento y la diferenciación de las células de la cripta superior y las regiones de vellosidades inferiores. Se han observado múltiples variantes de transcripción unidas alternativamente codificando diferentes isoformas para este gen. More preferably, the aggregating neurodegenrative disease is ALS or FTLD. The term "MOK" (MAPK / MAK / MRK overlapping kinase; 419 aa; Acc. Number BAA81688 or Q9UQ07, or any of its isoforms), also called RAGE; RAGE1; STK30; RAGE- 1. This gene belongs to the MAP kinase superfamily. The gene is regulated by the flow rate transcription factor 2 (Cdx2). The encoded protein, which is located in the epithelial cells in the intestinal crypt, can play a role in the growth arrest and differentiation of the cells of the upper crypt and regions of lower villi. Multiple transcription variants alternately linked encoding different isoforms have been observed for this gene.
A no ser que se indique lo contrario, cuando se utiliza en el presente documento el término "MOK", se refiere tanto al gen como a la proteína MOK humana. En el contexto de la presente invención, MOK se define también, pero sin limitarse, por una secuencia de nucleótidos o polinucleótido, que constituye la secuencia codificante de la proteína MOK, y que comprendería diversas variantes procedentes de: a) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica de la SEQ I D NO: 1 , Unless otherwise indicated, when the term "MOK" is used herein, it refers to both the gene and the human MOK protein. In the context of the present invention, MOK is also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the MOK protein coding sequence, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,
b) moléculas de ácido nucleico cuya cadena complementaria híbrida con la secuencia polinucleotídica de a),  b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
c) moléculas de ácido nucleico cuya secuencia difiere de a) y/o b) debido a la degeneración del código genético, c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,
d) moléculas de ácido nucleico que codifican un polipétptido que comprende la secuencia aminoacídica con una identidad de al menos un 50%, un 60%, un 70%, un 80%, un 90%, un 95%, un 98% o un 99% con la SEQ I D NO: 1 . en las que el polipéptido codificado por dichos ácidos nucleicos posee la actividad y las características estructurales de la proteína MOK. Entre otras posibles secuencias nucleotídicas que codifican MOK se encuentra, pero sin limitarse, la SEQ I D NO:2. d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MOK protein. Among other possible nucleotide sequences encoding MOK is, but not limited to, SEQ I D NO: 2.
SEQ I D NO: 1 SEQ I D NO: 1
MKNYKAIGKIGEGTFSEVMKMQSLRDGNYYACKQMKQRFESI EQVNNLREIQALRRLNPHP NI LMLHEWFDRKSGSLALICELM DMNIYELI RGRRYPLSEKKIMHYMYQLCKSLDHIH RNGI F HRDVKPENI LI KQDVLKLGDFGSCRSVYSKQPYTEYISTRWYRAPECLLTDGFYTYKMDLWS AGCVFYEIASLQPLFPGVN ELDQISKI HDVIGTPAQKI LTKFKQSRAM NFDFPFKKGSGI PLLT TNLSPQCLSLLHAMVAYDPDERIAAHQALQHPYFQEQRKTEKRALGSHRKAGFPEHPVAPE PLSNSCQISKEGRKQKQSLKQEEDRPKRRGPAYVMELPKLKLSGVVRLSSYSSPTLQSVLG SGTNGRVPVLRPLKCIPASKKTDPQKDLKPAPQQCRLPTIVRKGGR SEQ ID NO:2 atcttgcgcatgcgcaggcgacagctcatggcgttcagggcctgacggttgctagggtgacagggacacaacatggcggcggg atctctaacgctctccttcgagggaccaccacggagatcctagtgcgggaccccgcctcagggaagtggaaagcagggggac aaccttcctgcttctttcttttccgtccagtgtcggcaaggggttgtcaccggcttccgcatccaagatgaagaactataaagcaattg gcaaaataggagagggaacgttttctgaagttatgaagatgcaaagcctgagagatggaaactactatgcatgtaaacaaatga agcagcgctttgaaagtattgagcaagtcaacaacctacgagagatccaagcactgaggcgcctgaatccgcacccaaacatt cttatgttgcatgaagtggtttttgacagaaaatctggttctcttgcactaatatgtgaacttatggacatgaatatttatgagctaatacg agggagaagatacccattatcagaaaaaaaaattatgcactatatgtaccagttatgtaagtccctggatcatattcacagaaatg gaatatttcacagagatgtaaaaccagaaaatatactaataaagcaggatgtcctgaaattaggggactttggctcctgccggagt gtctattccaagcagccgtacacggaatacatctccacccgctggtaccgggccccggagtgtctcctcactgatgggttctacac gtacaagatggacctgtggagcgccggctgtgtgttctacgagatcgccagtctgcagcccctctttcctggagtaaatgaactgg accaaatctcaaaaatccacgatgtcatcggcacacccgctcagaagatcctcaccaagttcaaacagtcgagagctatgaattt tgattttccttttaaaaagggatcaggaatacctctactaacaaccaatttgtccccacaatgcctctccctcctgcacgcaatggtgg cctatgatcccgatgagagaatcgccgcccaccaggccctgcagcacccctacttccaagaacagaggaaaacagagaagc gggctctgggcagccacagaaaagctggctttccggagcaccctgtggcaccggaaccactcagtaacagctgccagatttcc aaggagggcagaaagcagaaacagtccctaaagcaagaggaggaccgtcccaagagacgaggaccggcctatgtcatgg aactgcccaaactaaagctttcgggagtggtcagactgtcgtcttactccagccccacgctgcagtccgtgcttggatctggaaca aatggaagagtgccggtgctgagacccttgaagtgcatccctgcgagcaagaagacagatccgcagaaggaccttaagcctg ccccgcagcagtgtcgcctgcccaccatagtgcggaaaggcggaagataactgagcagcaccgtcgtctcgacttcggaggc aacaccaagcccgaccgggccaggcctgggtgatctgctgctgagacgccacggagggctggggatgcgcctgcgtccgtttc gcgctggccggggctctgggtgctgccctgcgccctgccgcacccgcggcccgcgcagctgcctaggatgttctgggctaatata cttgtaaaaccaccgcattctagggttttctttcattttcgttaagaatttggggcaggaaatactttgtaactttgtatatgaatcaaaac aaacgagcaggcatttctgtgatgtgttgggcgtggttggaaggtgggttctgcgtgtcccttcccagcgctgctggtcagtcgtgga gcgccatcatgtcttaccagtgacgctgctgacacccctgacttttattaaagaataagctgtcgttacagtattgcattttaatgttaaa aaaaaaaaaaaaaaa MKNYKAIGKIGEGTFSEVMKMQSLRDGNYYACKQMKQRFESI EQVNNLREIQALRRLNPHP NI LMLHEWFDRKSGSLALICELM DMNIYELI RGRRYPLSEKKIMHYMYQLCKSLDHIH RNGI F HRDVKPENI LI KQDVLKLGDFGSCRSVYSKQPYTEYISTRWYRAPECLLTDGFYTYKMDLWS AGCVFYEIASLQPLFPGVN ELDQISKI HDVIGTPAQKI LTKFKQSRAM NFDFPFKKGSGI PLLT TNLSPQCLSLLHAMVAYDPDERIAAHQALQHPYFQEQRKTEKRALGSHRKAGFPEHPVAPE PLSNSCQISKEGRKQKQSLKQEEDRPKRRGPAYVMELPKLKLSGVVRLSSYSSPTLQSVLG SGTNGRVPVLRPLKCIPASKKTDPQKDLKPAPQQCRLPTIVRKGGR SEQ ID NO: 2 atcttgcgcatgcgcaggcgacagctcatggcgttcagggcctgacggttgctagggtgacagggacacaacatggcggcggg atctctaacgctctccttcgagggaccaccacggagatcctagtgcgggaccccgcctcagggaagtggaaagcagggggac aaccttcctgcttctttcttttccgtccagtgtcggcaaggggttgtcaccggcttccgcatccaagatgaagaactataaagcaattg gcaaaataggagagggaacgttttctgaagttatgaagatgcaaagcctgagagatggaaactactatgcatgtaaacaaatga agcagcgctttgaaagtattgagcaagtcaacaacctacgagagatccaagcactgaggcgcctgaatccgcacccaaacatt cttatgttgcatgaagtggtttttgacagaaaatctggttctcttgcactaatatgtgaacttatggacatgaatatttatgagctaatacg agggagaagatacccattatcagaaaaaaaaattatgcactatatgtaccagttatgtaagtccctggatcatattcacagaaatg gaatatttcacagagatgtaaaaccagaaaatatactaataaagcaggatgtcctgaaattaggggactttggctcctgccggagt gtctattccaagcagccgtacacggaatacatctccacccgctggtaccgggccccggagtgtctcctcactgatgggttctacac gtacaagatggacctgtggagcgccggctgtgtgttctacgagatcgccagtctgcagcccctctttcctggagtaaatgaactgg accaaatctcaaaaatccacgatgtcatcggcacacccgctcagaagatcctcaccaagttcaaacagtcgagagctatgaattt tgattttccttttaaaaagggatcaggaatacctc tactaacaaccaatttgtccccacaatgcctctccctcctgcacgcaatggtgg cctatgatcccgatgagagaatcgccgcccaccaggccctgcagcacccctacttccaagaacagaggaaaacagagaagc gggctctgggcagccacagaaaagctggctttccggagcaccctgtggcaccggaaccactcagtaacagctgccagatttcc aaggagggcagaaagcagaaacagtccctaaagcaagaggaggaccgtcccaagagacgaggaccggcctatgtcatgg aactgcccaaactaaagctttcgggagtggtcagactgtcgtcttactccagccccacgctgcagtccgtgcttggatctggaaca aatggaagagtgccggtgctgagacccttgaagtgcatccctgcgagcaagaagacagatccgcagaaggaccttaagcctg ccccgcagcagtgtcgcctgcccaccatagtgcggaaaggcggaagataactgagcagcaccgtcgtctcgacttcggaggc aacaccaagcccgaccgggccaggcctgggtgatctgctgctgagacgccacggagggctggggatgcgcctgcgtccgtttc gcgctggccggggctctgggtgctgccctgcgccctgccgcacccgcggcccgcgcagctgcctaggatgttctgggctaatata cttgtaaaaccaccgcattctagggttttctttcattttcgttaagaatttggggcaggaaatactttgtaactttgtatatgaatcaaaac aaacgagcaggcatttctgtgatgtgttgggcgtggttggaaggtgggttctgcgtgtcccttcccagcgctgctggtcagtcgtgga aa gcgccatcatgtcttaccagtgacgctgctgacacccctgacttttattaaagaataagctgtcgttacagtattgcattttaatgttaaa aaaaaaaaaaaaa
El término "que modula la actividad" como se usa aquí, se refiere tanto a que inhibe (disminuye) o estimula (incrementa) el nivel de actividad de MOK en una célula. La actividad de MOK puede ser modulada por la modificación de los niveles y/o de la actividad de la proteína MOK, o por la modificación de los niveles a los que se transcribe el gen MOK tal que los niveles de actividad de la proteína MOK en la célula es modulada. Los agentes moduladores pueden ser también agonistas (sustancias que son capaces de unirse a un receptor y provocar una respuesta en la célula, en concreto un aumento de la actividad de MOK), como antagonistas (sustancias que no solamente no activan el receptor, sino que en realidad bloquea su activación por los agonistas). En el contexto de la presente invención, la inhibición es la forma preferida de modulación. The term "which modulates activity" as used herein, refers to both that it inhibits (decreases) or stimulates (increases) the level of MOK activity in a cell. The activity of MOK can be modulated by the modification of the levels and / or activity of the MOK protein, or by the modification of the levels at which the MOK gene is transcribed such that the levels of activity of the MOK protein in The cell is modulated. Modulating agents may also be agonists (substances that are capable of binding to a receptor and eliciting a response in the cell, specifically an increase in MOK activity), as antagonists (substances that not only do not activate the receptor, but also in actually blocks its activation by agonists). In the context of the present invention, inhibition is the preferred form of modulation.
En esta memoria se entiende por TDP-43 (también TARDBP o ALS10), o TAR DNA binding protein, una proteína que es un represor trasncripcional que se une al TAR DNA cromosómicamente integrado y reprime la transcripción de HIV-1. Además, esta proteína regula el empalme alternativo del gen CFTR. Se localiza en el cromosoma 1 , pero un pseudogen similar está presente en el cromosoma 20. In this specification, TDP-43 (also TARDBP or ALS10), or TAR DNA binding protein, a protein that is a transcriptional repressor that binds to chromosomally integrated DNA TAR and represses HIV-1 transcription. In addition, this protein regulates the alternative splicing of the CFTR gene. It is located on chromosome 1, but a similar pseudogen is present on chromosome 20.
La proteína TDP-43 fue identificada como la principal proteína de la enfermedad en la mayoría de los casos FTLD esporádicos y familiares. Posteriormente, también se encontró que las inclusiones compactas ubiquitinadas, características de la esclerosis lateral amiotrófica (ALS), estaban compuestas por TDP-43, proporcionando así evidencia de que ambas condiciones están mecánicamente ligadas y forman parte del espectro de enfermedades que pueden clasificarse como proteopatías TDP-43, (Véase, por ejemplo, Neumann et al. , Science 314 (2006), 130 - 133). The TDP-43 protein was identified as the main disease protein in most sporadic and familial FTLD cases. Subsequently, it was also found that ubiquitinated compact inclusions, characteristic of amyotrophic lateral sclerosis (ALS), were composed of TDP-43, thus providing evidence that both conditions are mechanically linked and are part of the spectrum of diseases that can be classified as proteopathies. TDP-43, (See, for example, Neumann et al., Science 314 (2006), 130-133).
Además de FTLD y ALS, también se sabe que TDP-43 se acumula en las células nerviosas y células gliales del complejo de demencia de ALS-Parkinsonismo de Guam, degeneración corticobasal, demencia con cuerpos de Lewy, enfermedad de Huntington, enfermedad de cuerpos de Lewy, enfermedad de la motoneurona, demencia frontotemporal, degeneración lobar frontotemporal con inclusiones positivas a la ubiquitina, esclerosis del hipocampo, miopatía del cuerpo de inclusión, miositis del cuerpo de inclusión, enfermedad de Parkinson, demencia de la enfermedad de Parkinson, complejo Parkinson-demencia en la península Kii y enfermedad de Pick y similares, entre otras patologías; Véase, por ejemplo, Lagier - Tourenne et al. , Hum. Mol. Gen. 19 (2010), R46-64, que se incorpora aquí como referencia en su totalidad. In addition to FTLD and ALS, it is also known that TDP-43 accumulates in the nerve cells and glial cells of the dementia complex of ALS-Parkinsonism of Guam, corticobasal degeneration, dementia with Lewy bodies, Huntington's disease, body disease Lewy, motor neuron disease, frontotemporal dementia, frontotemporal lobar degeneration with positive inclusions to ubiquitin, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease, Parkinson's disease dementia, Parkinson's complex- dementia on the Kii peninsula and Pick's disease and the like, among other pathologies; See, for example, Lagier-Tourenne et al. , Hum. Mol. Gen. 19 (2010), R46-64, which is incorporated herein by reference in its entirety.
A no ser que se indique lo contrario, cuando se utiliza en el presente documento el término "TDP-43", se refiere tanto al gen como a la proteína MOK humana. En el contexto de la presente invención, TDP-43 se define también, pero sin limitarse, por una secuencia de nucleótidos o polinucleótido, que constituye la secuencia codificante de la proteína MOK, y que comprendería diversas variantes procedentes de: a) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica de la SEQ ID NO: 3, b) moléculas de ácido nucleico cuya cadena complementaria híbrida con la secuencia polinucleotídica de a), Unless otherwise indicated, when the term "TDP-43" is used herein, it refers to both the gene and the human MOK protein. In the context of the present invention, TDP-43 is also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the MOK protein coding sequence, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
c) moléculas de ácido nucleico cuya secuencia difiere de a) y/o b) debido a la degeneración del código genético, c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,
d) moléculas de ácido nucleico que codifican un polipétptido que comprende la secuencia aminoacídica con una identidad de al menos un 50%, un 60%, un 70%, un 80%, un 90%, un 95%, un 98% o un 99% con la SEQ ID NO: 1. en las que el polipéptido codificado por dichos ácidos nucleicos posee la actividad y las características estructurales de la proteína MOK. Entre otras posibles secuencias nucleotídicas que codifican MOK se encuentra, pero sin limitarse, la SEQ ID NO: 4. d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or a 99% with SEQ ID NO: 1. in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MOK protein. Among other possible nucleotide sequences encoding MOK is, but not limited to, SEQ ID NO: 4.
SEQ ID NO: 3 SEQ ID NO: 3
MSEYI RVTEDEN DEPI El PSEDDGTVLLSTVTAQFPGACGLRYRN PVSQCM RGVRLVEGI LH APDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKE YFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQ SQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSL CGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQ GSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMM MLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGS GFNGGFGSSMDSKSSGWGM MSEYI RVTEDEN DEPI The PSEDDGTVLLSTVTAQFPGACGLRYRN PVSQCM RGVRLVEGI LH APDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKE YFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQ SQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSL CGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQ GSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMM MLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGS GFNGGFGSSMDSKSSGWGM
SEQ ID NO:4 atgtctgaatatattcgggtaaccgaagatgagaacgatgagcccattgaaataccatcggaagacgatgggacggtgctgctct ccacggttacagcccagtttccaggggcgtgtgggcttcgctacaggaatccagtgtctcagtgtatgagaggtgtccggctggta gaaggaattctgcatgccccagatgctggctggggaaatctggtgtatgttgtcaactatccaaaagataacaaaagaaaaatgg atgagacagatgcttcatcagcagtgaaagtgaaaagagcagtccagaaaacatccgatttaatagtgttgggtctcccatggaa aacaaccgaacaggacctgaaagagtattttagtacctttggagaagttcttatggtgcaggtcaagaaagatcttaagactggtc attcaaaggggtttggctttgttcgttttacggaatatgaaacacaagtgaaagtaatgtcacagcgacatatgatagatggacgat ggtgtgactgcaaacttcctaattctaagcaaagccaagatgagcctttgagaagcagaaaagtgtttgtggggcgctgtacaga ggacatgactgaggatgagctgcgggagttcttctctcagtacggggatgtgatggatgtcttcatccccaagccattcagggccttt gcctttgttacatttgcagatgatcagattgcgcagtctctttgtggagaggacttgatcattaaaggaatcagcgttcatatatccaat gccgaacctaagcacaatagcaatagacagttagaaagaagtggaagatttggtggtaatccaggtggctttgggaatcagggt ggatttggtaatagcagagggggtggagctggtttgggaaacaatcaaggtagtaatatgggtggtgggatgaactttggtgcgtt cagcattaatccagccatgatggctgccgcccaggcagcactacagagcagttggggtatgatgggcatgttagccagccagc agaaccagtcaggcccatcgggtaataaccaaaaccaaggcaacatgcagagggagccaaaccaggccttcggttctggaa ataactcttatagtggctctaattctggtgcagcaattggttggggatcagcatccaatgcagggtcgggcagtggttttaatggagg ctttggctcaagcatggattctaagtcttctggctggggaatgaatcactag SEQ ID NO: 4 ccacggttacagcccagtttccaggggcgtgtgggcttcgctacaggaatccagtgtctcagtgtatgagaggtgtccggctggta atgtctgaatatattcgggtaaccgaagatgagaacgatgagcccattgaaataccatcggaagacgatgggacggtgctgctct gaaggaattctgcatgccccagatgctggctggggaaatctggtgtatgttgtcaactatccaaaagataacaaaagaaaaatgg atgagacagatgcttcatcagcagtgaaagtgaaaagagcagtccagaaaacatccgatttaatagtgttgggtctcccatggaa aacaaccgaacaggacctgaaagagtattttagtacctttggagaagttcttatggtgcaggtcaagaaagatcttaagactggtc attcaaaggggtttggctttgttcgttttacggaatatgaaacacaagtgaaagtaatgtcacagcgacatatgatagatggacgat ggtgtgactgcaaacttcctaattctaagcaaagccaagatgagcctttgagaagcagaaaagtgtttgtggggcgctgtacaga ggacatgactgaggatgagctgcgggagttcttctctcagtacggggatgtgatggatgtcttcatccccaagccattcagggccttt gcctttgttacatttgcagatgatcagattgcgcagtctctttgtggagaggacttgatcattaaaggaatcagcgttcatatatccaat gccgaacctaagcacaatagcaatagacagttagaaagaagtggaagatttggtggtaatccaggtggctttgggaatcagggt ggatttggtaatagcagagggggtggagctggtttgggaaacaatcaaggtagtaatatgggtggtgggatgaactttggtgcgtt cagcattaatccagccatgatggctgccgc ccaggcagcactacagagcagttggggtatgatgggcatgttagccagccagc agaaccagtcaggcccatcgggtaataaccaaaaccaaggcaacatgcagagggagccaaaccaggccttcggttctggaa ataactcttatagtggctctaattctggtgcagcaattggttggggatcagcatccaatgcagggtcgggcagtggttttaatggagg ctttggctcaagcatggattctaagtcttctggctggggaatgaatcactag
En una realización preferida de este aspecto de la invención, los agentes moduladores de la actividad de MOK se seleccionan de una lista que consiste en: In a preferred embodiment of this aspect of the invention, MOK activity modulating agents are selected from a list consisting of:
a) una molécula orgánica, a) an organic molecule,
b) una molécula de ARN, b) an RNA molecule,
c) un oligonucleótido antisentido, c) an antisense oligonucleotide,
d) un anticuerpo, o d) an antibody, or
e) una ribozima. e) a ribozyme.
Un experto en la materia podría preparar moléculas orgánicas que pueden unirse específicamente a MOK sin unirse a otros polipéptidos o proteínas. Las moléculas orgánicas tendrán preferiblemente un peso de 100 a 20.000 daltons, más preferiblemente 500 a 15.000 daltons, y más preferiblemente 1000 a 10.000 daltons. Librerías de moléculas orgánicas se encuentran disponibles comercialmente. La vía de administración puede ser, sin limitarse a estas, intraperitoneal, intratecal, intravenosa, intramuscular, subcutánea, intraventricular, oral, enteral, parenteral, intranasal o dérmica. En una realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención es una molécula orgánica, y más preferiblemente, la molécula orgánica de fórmula (I):
Figure imgf000022_0001
One skilled in the art could prepare organic molecules that can specifically bind MOK without binding to other polypeptides or proteins. The organic molecules will preferably have a weight of 100 to 20,000 daltons, more preferably 500 to 15,000 daltons, and more preferably 1000 to 10,000 daltons. Bookstores of organic molecules are commercially available. The route of administration may be, without limitation, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal. In a preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (I):
Figure imgf000022_0001
Fórmula (I) Formula (I)
Donde Where
n es igual a 0 ó 1 , n is equal to 0 or 1,
R es un metilo (Me), isopropilo (¡Pr), tere-Butilo (tBu), ciclopentadienilo (Cp) R is a methyl (Me), isopropyl (¡Pr), tere-Butyl (tBu), cyclopentadienyl (Cp)
X es H ó NH2, X is H or NH 2 ,
Y es H, NHCOCHCH2, NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, y Z es H, Y is H, NHCOCHCH 2 , NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, 0 COCH3, and Z is H,
o cualquiera de sus sales, preferiblemente cualquier sal farmacéuticamente aceptable, ésteres, tautómeros, polimorfos, hidratos farmacéuticamente aceptables, o un isómero, profármacos, derivados, solvatos o análogos, o cualquiera de sus combinaciones. or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
En una realización más preferida, de este aspecto de la invención, el agente modulador de MOK de la invención es una molécula orgánica, y más preferiblemente, la molécula orgánica de fórmula (II): In a more preferred embodiment, of this aspect of the invention, the MOK modulating agent of the invention is an organic molecule, and more preferably, the organic molecule of formula (II):
Figure imgf000022_0002
Figure imgf000022_0002
Fórmula (I) Formula (I)
Donde Where
n es igual a 1 , n is equal to 1,
R es un isopropilo (¡Pr),  R is an isopropyl (¡Pr),
X es NH2, X is NH 2 ,
Y es COCH2F Z es H, And it is COCH2F Z is H,
o cualquiera de sus sales, preferiblemente cualquier sal farmacéuticamente aceptable, ésteres, tautómeros, polimorfos, hidratos farmacéuticamente aceptables, o un isómero, profármacos, derivados, solvatos o análogos, o cualquiera de sus combinaciones. or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
Los compuestos de la presente invención representados por la fórmula (I) y/o (II) pueden incluir isómeros, dependiendo de la presencia de enlaces múltiples, incluyendo isómeros ópticos o enantiómeros, dependiendo de la presencia de centros quirales. Los isómeros, enantiómeros o diastereoisómeros individuales y las mezclas de los mismos caen dentro del alcance de la presente invención, es decir, el término isómero también se refiere a cualquier mezcla de isómeros, como diastereómeros, racémicos, etc., incluso a sus isómeros ópticamente activos o las mezclas en distintas proporciones de los mismos. Los enantiómeros o diastereoisómeros individuales, así The compounds of the present invention represented by formula (I) and / or (II) may include isomers, depending on the presence of multiple bonds, including optical isomers or enantiomers, depending on the presence of chiral centers. The individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to any mixture of isomers, such as diastereomers, racemic, etc., even their optically isomers. assets or mixtures in different proportions thereof. The individual enantiomers or diastereoisomers, as well
como sus mezclas, pueden separarse mediante técnicas convencionales. Like their mixtures, they can be separated by conventional techniques.
Asimismo, dentro del alcance de esta invención se encuentran los profármacos de los compuestos de fórmula (I) y/o (II). El término "prodroga" o "profármaco" tal como aquí se utiliza incluye cualquier derivado de un compuesto de fórmula (I) y/o (II) -por ejemplo y no limitativamente: ésteres (incluyendo ésteres de ácidos carboxílicos, ésteres de aminoácidos, ésteres de fosfato, ésteres de sulfonato de sales metálicas, etc.), carbamatos, amidas, etc- que al ser administrado a un individuo puede ser transformado directa o indirectamente en dicho compuesto de fórmula (I) y/o (II) en el mencionado individuo. Ventajosamente, dicho derivado es un compuesto que aumenta la biodisponibilidad del compuesto de fórmula (I) y/o (II) cuando se administra a un individuo o que potencia la liberación del compuesto de fórmula (I) y/o (II) en un compartimento biológico. Also, within the scope of this invention are the prodrugs of the compounds of formula (I) and / or (II). The term "prodrug" or "prodrug" as used herein includes any derivative of a compound of formula (I) and / or (II) - for example and not limited to: esters (including esters of carboxylic acids, amino acid esters, phosphate esters, sulphonate esters of metal salts, etc.), carbamates, amides, etc- that when administered to an individual can be transformed directly or indirectly into said compound of formula (I) and / or (II) in the mentioned individual. Advantageously, said derivative is a compound that increases the bioavailability of the compound of formula (I) and / or (II) when administered to an individual or that enhances the release of the compound of formula (I) and / or (II) in a biological compartment
La naturaleza de dicho derivado no es crítica siempre y cuando pueda ser administrado a un individuo y proporcione el compuesto de fórmula (I) y/o (II) en un compartimento biológico de un individuo. La preparación de dicho profármaco puede llevarse a cabo mediante métodos convencionales conocidos por los expertos en la materia. The nature of said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I) and / or (II) in a biological compartment of an individual. The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
Tal como aquí se utiliza, el término "derivado" incluye tanto a compuestos farmacéuticamente aceptables, es decir, derivados del compuesto de fórmula (I) y/o (II) que pueden ser utilizados en la elaboración de un medicamento o composiciones alimentarias, como derivados farmacéuticamente no aceptables, ya que éstos pueden ser útiles en la preparación de derivados farmacéuticamente aceptables. Los compuestos de la invención pueden estar en forma cristalina como compuestos libres o como solvatos. En este sentido, el término "solvato", tal como aquí se utiliza, incluye tanto solvatos farmacéuticamente aceptables, es decir, solvatos del compuesto de fórmula (I) y/o (II) que pueden ser utilizados en la elaboración de un medicamento, como solvatos farmacéuticamente no aceptables, los cuales pueden ser útiles en la preparación de solvatos o sales farmacéuticamente aceptables. La naturaleza del solvato farmacéuticamente aceptable no es crítica siempre y cuando sea farmacéuticamente aceptable. En una realización particular, el solvato es un hidrato. Los solvatos pueden obtenerse por métodos convencionales de solvatacion conocidos por los expertos en la materia. As used herein, the term "derivative" includes both pharmaceutically acceptable compounds, that is, derivatives of the compound of formula (I) and / or (II) that can be used in the manufacture of a medicament or food compositions, such as Pharmaceutically acceptable derivatives, since these may be useful in the preparation of pharmaceutically acceptable derivatives. The compounds of the invention may be in crystalline form as free compounds or as solvates. In this sense, the term "solvate", as used herein, includes both pharmaceutically acceptable solvates, that is, solvates of the compound of formula (I) and / or (II) that can be used in the manufacture of a medicament, as pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically acceptable solvates or salts. The nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. Solvates can be obtained by conventional solvation methods known to those skilled in the art.
Para su aplicación en terapia, los compuestos de fórmula (I) y/o (II), sus sales, profármacos o solvatos, se encontrarán, preferentemente, en una forma farmacéuticamente aceptable o sustancialmente pura, es decir, que tiene un nivel de pureza farmacéuticamente aceptable excluyendo los aditivos farmacéuticos normales tales como diluyentes y portadores, y no incluyendo material considerado tóxico a niveles de dosificación normales. Los niveles de pureza para el principio activo son preferiblemente superiores al 50%, más preferiblemente superiores al 70%, y todavía más preferiblemente superiores al 90%. En una realización preferida, son superiores al 95% de compuesto de fórmula (I), o de sus sales, solvatos o profármacos. For their application in therapy, the compounds of formula (I) and / or (II), their salts, prodrugs or solvates, will preferably be in a pharmaceutically acceptable or substantially pure form, that is, having a level of purity pharmaceutically acceptable excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels. The purity levels for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and still more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or of its salts, solvates or prodrugs.
Recientemente, con el desarrollo de la tecnología antisentido, secuencias de nucleótidos específicamente complementarios a una determinada secuencia de ADN o ARN, podrían formar complejos y bloquear la transcripción o traducción. Así, con el progreso del silenciamiento génico post-transcripcional, y en particular del ARN de interferencia (RNA interferente o RNAi), se han desarrollado herramientas que permiten la inhibición específica de la expresión de un gen. La inhibición de la expresión de MOK constituiría por ende la inhibición de su actividad biológica, y en concreto, de la actividad que está contribuyendo al agravamiento de la enfermedad conformacional neurodegerativa. Recently, with the development of antisense technology, nucleotide sequences specifically complementary to a particular DNA or RNA sequence could form complexes and block transcription or translation. Thus, with the progress of post-transcriptional gene silencing, and in particular of interfering RNA (interfering RNA or RNAi), tools have been developed that allow specific inhibition of gene expression. The inhibition of the expression of MOK would therefore constitute the inhibition of its biological activity, and in particular, of the activity that is contributing to the aggravation of the neurodegerative conformational disease.
Por "polinucleótidos antisentido" se entienden cadenas de ribonucleótidos o desoxirribonucleóitidos que pueden inhibir la producción de la proteína MOK por uno de estos tres mecanismos: By "antisense polynucleotides" are understood ribonucleotide or deoxyribonucleotide chains that can inhibit the production of the MOK protein by one of these three mechanisms:
1- Interfiriendo la transcripción, al hibridar en el gen estructural o en una región regulatoria del gen que codifica para MOK. Puesto que la transcripción o expresión es bloqueada de manera efectiva por la hibridación del oligonucleótido antisentido con el ADN, disminuye la producción de MOK. 1- Interfering transcription by hybridizing in the structural gene or in a regulatory region of the gene encoding MOK. Since the transcript or expression is blocked from Effectively by hybridizing the antisense oligonucleotide with DNA, decreases the production of MOK.
2- La unión del oligonucleótido antisentido en el citoplasma con el ARNm, interfiriendo con la formación de la construcción de traducción propiamente dicha, inhibiendo la traducción de ARNm a la proteína.  2- The binding of the antisense oligonucleotide in the cytoplasm with the mRNA, interfering with the formation of the translation construct itself, inhibiting the translation of mRNA into the protein.
3- La formación de un ARNm - antisentido dúplex que permite una rápida degradación del ARNm dúplex por ARNasas (como ARNasa H). Esto da lugar a una menor producción de MOK.  3- The formation of a duplex antisense mRNA that allows rapid degradation of the duplex mRNA by RNases (such as RNase H). This results in lower production of MOK.
Oligonucleótidos antisentido capaces de modular la actividad de la MOK son conocidas en el estado de la técnica. Por ejemplo, y sin limitarnos, podría ser una secuencia de ribonucleótidos o ARN que pertenece al denominado siRNA (small interferíng RNA), ARN pequeño de interferencia o ARN de silenciamiento, capaz de inhibir la expresión genética de la proteína MOK. En el contexto de la presente memoria se entiende como "siRNA" (small interferíng RNA ó ARN pequeño de interferencia) una clase de ARN de doble cadena de 19 a 25 nucleótidos de largo, y más preferentemente entre 21 y 23 nucleótidos, que está involucrado en la ruta de la interferencia de ARN, donde el siRNA interfiere la expresión de un gen específico. En la presente invención, este gen específico es el MOK. También podría ser cualquier siRNA capaz de hibridar una molécula de ácido nucleico que codifique la proteína MOK humana (o cualquiera de sus isoformas o variante transcripcional) cuya secuencia (variante transcripcional 1 ; Acc. No. NM_014226.2) se recoge en la SEQ ID NO: 5. Antisense oligonucleotides capable of modulating the activity of MOK are known in the state of the art. For example, and without limiting ourselves, it could be a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the MOK protein. In the context of the present specification, "siRNA" (small interfering RNA or small interfering RNA) is understood to be a class of double stranded RNA 19 to 25 nucleotides long, and more preferably between 21 and 23 nucleotides, which is involved in the route of RNA interference, where siRNA interferes with the expression of a specific gene. In the present invention, this specific gene is MOK. It could also be any siRNA capable of hybridizing a nucleic acid molecule encoding the human MOK protein (or any of its isoforms or transcriptional variant) whose sequence (transcriptional variant 1; Acc. No. NM_014226.2) is collected in SEQ ID NO: 5.
ATCTTGCGCATGCGCAGGCGACAGCTCATGGCGTTCAGGGCCTGACGGTTGCTAGGGT GACAGGGACACAACATGGCGGCGGGATCTCTAACGCTCTCCTTCGAGGGACCACCACG GAGATCCTAGTGCGGGACCCCGCCTCAGGGAAGTGGAAAGCAGGGGGACAACCTTCC TGCTTCTTTCTTTTCCGTCCAGTGTCGGCAAGGGGTTGTCACCGGCTTCCGCATCCAAG ATGAAGAACTATAAAGCAATTGGCAAAATAGGAGAGGGAACGTTTTCTGAAGTTATGAA GATGCAAAGCCTGAGAGATGGAAACTACTATGCATGTAAACAAATGAAGCAGCGCTTTG AAAGTATTGAGCAAGTCAACAACCTACGAGAGATCCAAGCACTGAGGCGCCTGAATCCG CACCCAAACATTCTTATGTTGCATGAAGTGGTTTTTGACAGAAAATCTGGTTCTCTTGCA CTAATATGTGAACTTATGGACATGAATATTTATGAGCTAATACGAGGGAGAAGATACCCA TTATCAGAAAAAAAAATTATGCACTATATGTACCAGTTATGTAAGTCCCTGGATCATATTC ACAGAAATGGAATATTTCACAGAGATGTAAAACCAGAAAATATACTAATAAAGCAGGATG TCCTGAAATTAGGGGACTTTGGCTCCTGCCGGAGTGTCTATTCCAAGCAGCCGTACACG GAATACATCTCCACCCGCTGGTACCGGGCCCCGGAGTGTCTCCTCACTGATGGGTTCT ACA CGTACAAGATGGACCTGTGGAGCGCCGGCTGTGTGTTCTACGAGATCGCCAGTCTGCA GCCCCTCTTTCCTGGAGTAAATGAACTGGACCAAATCTCAAAAATCCACGATGTCATCG GCACACCCGCTCAGAAGATCCTCACCAAGTTCAAACAGTCGAGAGCTATGAATTTTGAT TTTCCTTTTAAAAAGGGATCAGGAATACCTCTACTAACAACCAATTTGTCCCCACAATGC CTCTCCCTCCTGCACGCAATGGTGGCCTATGATCCCGATGAGAGAATCGCCGCCCACC AGGCCCTGCAGCACCCCTACTTCCAAGAACAGAGGAAAACAGAGAAGCGGGCTCTGGG CAGCCACAGAAAAGCTGGCTTTCCGGAGCACCCTGTGGCACCGGAACCACTCAGTAAC AGCTGCCAGATTTCCAAGGAGGGCAGAAAGCAGAAACAGTCCCTAAAGCAAGAGGAGG ACCGTCCCAAGAGACGAGGACCGGCCTATGTCATGGAACTGCCCAAACTAAAGCTTTC GGGAGTGGTCAGACTGTCGTCTTACTCCAGCCCCACGCTGCAGTCCGTGCTTGGATCT GGAACAAATGGAAGAGTGCCGGTGCTGAGACCCTTGAAGTGCATCCCTGCGAGCAAGA AGACAGATCCGCAGAAGGACCTTAAGCCTGCCCCGCAGCAGTGTCGCCTGCCCACCAT AGTGCGGAAAGGCGGAAGATAACTGAGCAGCACCGTCGTCTCGACTTCGGAGGCAACA CCAAGCCCGACCGGGCCAGGCCTGGGTGATCTGCTGCTGAGACGCCACGGAGGGCTG GGGATGCGCCTGCGTCCGTTTCGCGCTGGCCGGGGCTCTGGGTGCTGCCCTGCGCCC TGCCGCACCCGCGGCCCGCGCAGCTGCCTAGGATGTTCTGGGCTAATATACTTGTAAA ACCACCGCATTCTAGGGTTTTCTTTCATTTTCGTTAAGAATTTGGGGCAG GAAATACTTTGTAACTTTGTATATGAATCAAAACAAACGAGCAGGCATTTCTGTGATGTG TTGGGCGTGGTTGGAAGGTGGGTTCTGCGTGTCCCTTCCCAGCGCTGCTGGTCAGTCG TGGAGCGCCATCATGTCTTACCAGTGACGCTGCTGACACCCCTGACTTTTATTAAAGAA TAAGCTGTCGTTACAGTATTGCATTTTAATGTTAAAAAAAAAAAAAAAAAA ATCTTGCGCATGCGCAGGCGACAGCTCATGGCGTTCAGGGCCTGACGGTTGCTAGGGT GACAGGGACACAACATGGCGGCGGGATCTCTAACGCTCTCCTTCGAGGGACCACCACG GAGATCCTAGTGCGGGACCCCGCCTCAGGGAAGTGGAAAGCAGGGGGACAACCTTCC TGCTTCTTTCTTTTCCGTCCAGTGTCGGCAAGGGGTTGTCACCGGCTTCCGCATCCAAG ATGAAGAACTATAAAGCAATTGGCAAAATAGGAGAGGGAACGTTTTCTGAAGTTATGAA GATGCAAAGCCTGAGAGATGGAAACTACTATGCATGTAAACAAATGAAGCAGCGCTTTG AAAGTATTGAGCAAGTCAACAACCTACGAGAGATCCAAGCACTGAGGCGCCTGAATCCG CACCCAAACATTCTTATGTTGCATGAAGTGGTTTTTGACAGAAAATCTGGTTCTCTTGCA CTAATATGTGAACTTATGGACATGAATATTTATGAGCTAATACGAGGGAGAAGATACCCA TTATCAGAAAAAAAAATTATGCACTATATGTACCAGTTATGTAAGTCCCTGGATCATATTC ACAGAAATGGAATATTTCACAGAGATGTAAAACCAGAAAATATACTAATAAAGCAGGATG TCCTGAAATTAGGGGACTTTGGCTCCTGCCGGAGTGTCTATTCCAAGCAGCCGTACACG GAATACATCTCCACCCGCTGGTACCGGGCCCCGGAGTGTCTCCTCACTGATGGGTTCT ACA CGTACAAGATGGACCTGTGGAGCGCCGGCTGTGTGTTCTACGAGATCGCCAGTCTGCA GCCCCTCTTTCCTGGAGTAAATGAACTGGACCAAATCTCAAAAATCCACGATGTCATCG GCACACCCGCTCAGAAGATCCTCACCAAGTTCAAACAGTCGAGAGCTATGAATTTTGAT TTTCCTTTTAAAAAGGGATCAGGAATACCTCTACTAACAACCAATTTGTCCCCACAATGC CTCTCCCTCCTGCACGCAATGGTGGCCTATGATCCCGATGAGAGAATCGCCGCCCACC AGGCCCTGCAGCACCCCTACTTCCAAGAACAGAGGAAAACAGAGAAGCGGGCTCTGGG CAGCCACAGAAAAGCTGGCTTTCCGGAGCACCCTGTGGCACCGGAACCACTCAGTAAC AGCTGCCAGATTTCCAAGGAGGGCAGAAAGCAGAAACAGTCCCTAAAGCAAGAGGAGG ACCGTCCCAAGAGACGAGGACCGGCCTATGTCATGGAACTGCCCAAACTAAAGCTTTC GGGAGTGGTCAGACTGTCGTCTTACTCCAGCCCCACGCTGCAGTCCGTGCTTGGATCT GGAACAAATGGAAGAGTGCCGGTGCTGAGACCCTTGAAGTGCATCCCTGCGAGCAAGA AGACAGATCCGCAGAAGGACCTTAAGCCTGCCCCGCAGCAGTGTCGCCTGCCCACCAT AGTGCGGAAAGGCGGAAGATAACTGAGCAGCACCGTCGTCTCGACTTCGGAGGCAACA CCAAGCCCGACCGGGCCAGGCCTGGGTGATCTGCTGCTGAGACGCCACGGAGGGCTG GGGATGCGCCTGCGTCCGTTTCGCGCTGGCCGGGGCTCTGGGTGCTGCCCTGCGCCC TGCCGCACCCGCGGCCCGCGCAGCTGCCTAGGATGTTCTGGGCTAATATACTTGTAAA GAA ACCACCGCATTCTAGGGTTTTCTTTCATTTTCGTTAAGAATTTGGGGCAG ATACTTTGTAACTTTGTATATGAATCAAAACAAACGAGCAGGCATTTCTGTGATGTG TTGGGCGTGGTTGGAAGGTGGGTTCTGCGTGTCCCTTCCCAGCGCTGCTGGTCAGTCG TGGAGCGCCATCATGTCTTACCAGTGACGCTGCTGACACCCCTGACTTTTATTAAAGAA TAAGCTGTCGTTACAGTATTGCATTTTAATGTTAAAAAAAAAAAAAAAAAA
También podrían ser una construcción de ARN que al menos contenga una cualquiera de las secuencias de nucleótidos posibles de siRNA capaces de inhibir la expresión de MOK, y sin perjuicio de que adicionalmente formen parte de la presente invención cualquiera de las secuencias y construcciones de RNA de la invención anteriormente descritas que sean objeto de modificaciones, preferentemente químicas, que conduzcan a una mayor estabilidad frente a la acción de ribonucleasas y con ello a una mayor eficiencia. Sin que dichas modificaciones supongan la alteración de su mecanismo de acción, que es la unión específica al complejo RISC (RNA-induced silencing complex), activándolo y manifestando una actividad helicasa que separa las dos hebras dejando solo la hebra antisentido asociada al complejo. El complejo ribonucleoprotéico resultante se une al mRNA diana (ARN mensajero de MOK, que se recoge en la SEQ ID NO: 2 o en la SEQ ID NO: 5). Si la complementariedad no es perfecta, RISC queda asociado al mensajero y se atenúa la traducción. Pero si es perfecta, RISC actúa como RNasa, cortando al mensajero y quedando libre para repetir el proceso. Adicionalmente resulta evidente para un experto en la materia que una gran cantidad de polinucleótidos de mARN pueden traducirse a MOK como consecuencia, por ejemplo, de que el código genético es degenerado. Cualquier siRNA capaz de inhibir la traducción de estos mARN también forman parte de la invención. They could also be an RNA construct that contains at least any one of the possible nucleotide sequences of siRNA capable of inhibiting MOK expression, and notwithstanding that any of the RNA sequences and constructs of RNA are additionally part of the present invention. the invention described above that are subject to modifications, preferably chemical, that lead to greater stability against the action of ribonucleases and thereby greater efficiency. Without these modifications involving the alteration of its mechanism of action, which is the specific binding to the RISC complex (RNA-induced silencing complex), activating it and manifesting a helicase activity that separates the two strands leaving only the antisense strand associated with the complex. The resulting ribonucleoproteic complex binds to the target mRNA (MOK messenger RNA, which is collected in SEQ ID NO: 2 or in SEQ ID NO: 5). If the complementarity is not perfect, RISC is associated with the messenger and the translation is attenuated. But if it is perfect, RISC acts as RNasa, cutting the messenger and being free to repeat the process. Additionally, it is apparent to one skilled in the art that a large number of mRNA polynucleotides can be translated into MOK as a consequence, for example, that the genetic code is degenerated. Any siRNA capable of inhibiting the translation of these mRNAs also forms part of the invention.
La preparación de la secuencia de siRNA de la invención o de la construcción de RNA de la invención sería evidente para un experto en la materia, y se podría llevar a cabo por síntesis química, lo cual permite además la incorporación de modificaciones químicas tanto en los distintos nucleotidos del producto como la incorporación de otros compuestos químicos en cualquiera de los extremos. Por otro lado, la síntesis también podría realizarse enzimáticamente utilizando cualquiera de las RNA polimerasas disponibles. La síntesis enzimática también permite alguna modificación química de los productos o RNAs inhibidores. The preparation of the siRNA sequence of the invention or of the RNA construct of the invention would be apparent to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications in both different nucleotides of the product such as the incorporation of other chemical compounds at any of the ends. On the other hand, the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitor products or RNAs.
El diseño de la secuencia de nucleotidos del siRNA de la invención también sería evidente para un experto en la materia. Así, se podría realizar mediante un diseño aleatorio en el que se seleccionen 19-25 bases del ARNm diana sin tener en cuenta la secuencia o la información posicional que tiene en el transcrito. Otra alternativa no limitativa de la presente invención sería el diseño convencional mediante parámetros simples desarrollados por los pioneros de la técnica (Calipel, A. et al., 2003. J Biol Chem. 278(43): 42409-42418) completados con un análisis BLAST de nucleotidos. Otra posibilidad podría ser un diseño racional, en el que se emplee un procedimiento informático dirigido a identificar las dianas óptimas de siRNA en un ARNm. Las secuencias diana se analizan en grupos de 19 nucleotidos a la vez y se identifican las que tienen mejores características en función de un algoritmo que incorpora un gran número de parámetros termodinámicos y de secuencia. The nucleotide sequence design of the siRNA of the invention would also be apparent to one skilled in the art. Thus, it could be done through a random design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or positional information it has in the transcript. Another non-limiting alternative of the present invention would be the conventional design by simple parameters developed by the pioneers of the art (Calipel, A. et al., 2003. J Biol Chem. 278 (43): 42409-42418) completed with an analysis BLAST of nucleotides. Another possibility could be a rational design, in which a computer procedure is used to identify the optimal siRNA targets in an mRNA. The target sequences are analyzed in groups of 19 nucleotides at a time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters.
También podría formar parte de la composición de la invención una construcción genética de ADN, la cual dirigiría la transcripción in vitro o intracelular de la secuencia siRNA o construcción de ARN de la invención, y que comprende, al menos, uno de los siguientes tipos de secuencias: a) secuencia de nucleotidos de ADN, preferentemente de doble cadena, que comprende, al menos, la secuencia codificante del siRNA de la invención o de la construcción de ARN de la invención para su transcripción, o, b) secuencia de nucleotidos de ADN, preferentemente de doble cadena, correspondiente a un sistema o vector de expresión génica que comprende la secuencia codificante de la secuencia de ARN de la invención operativamente enlazada con, al menos, un promotor que dirija la transcripción de dicha secuencia de nucleotidos de interés, y con otras secuencias necesarias o apropiadas para la transcripción y su regulación adecuada en tiempo y lugar, por ejemplo, señales de inicio y terminación, sitios de corte, señal de poliadenilación, origen de replicación, activadores transcripcionales (enhancers), silenciadores transcripcionales (silencers), etc.. para su uso en aquellos contextos patológicos en los que MOK está contribuyendo al agravamiento de las características tumorales de las células cancerosas y/o a la aparición de acantolisis. Múltiples de estas construcciones, sistemas o vectores de expresión pueden ser obtenidos por métodos convencionales conocidos por los expertos en la materia (Sambrook et al. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York). A genetic DNA construct could also be part of the composition of the invention, which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and comprising at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, comprising at least the sequence encoding the siRNA of the invention or the RNA construct of the invention for transcription, or, b) nucleotide sequence of DNA, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the RNA sequence of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, signals of start and end, cut sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts in which MOK is contributing to the worsening of tumor characteristics of cancer cells and / or the onset of acolysis. Multiple of these constructs, systems or expression vectors can be obtained by conventional methods known to those skilled in the art (Sambrook et al. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York).
Las composiciones de la presente invención permiten la transfección del siRNA de la invención al interior de una célula, in vivo o in vitro. La transfección se podría llevar a cabo, pero sin limitarnos a, transfección directa o vectores que faciliten el acceso del siRNA al interior de la célula. Así, ejemplos de estos vectores son, sin limitarse a, retrovirus, lentivirus, adenovirus, virus adeno-asociados, virus del Herpes simplex, plásmidos de DNA no virales, liposomas catiónicos y conjugados moleculares. Así, por ejemplo, los siRNA de la presente invención, así como ARN o ADN precursores de estos siRNA, pueden conjugarse con péptidos de liberación u otros compuestos para favorecer el transporte de estos siRNA al interior de la célula. Por tanto, en otra realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención comprende un oligonucleotido antisentido y se usa para la prevención, mejora y/o tratamiento de la enfermedad conformacional neurodegerativa. The compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro. Transfection could be carried out, but not limited to, direct transfection or vectors that facilitate the access of siRNA into the cell. Thus, examples of these vectors are, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates. Thus, for example, the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs, can be conjugated with release peptides or other compounds to favor the transport of these siRNAs into the cell. Therefore, in another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises an antisense oligonucleotide and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
En otra realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención comprende un anticuerpo y se usa para la prevención, mejora y/o tratamiento de la enfermedad conformacional neurodegerativa. In another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises an antibody and is used for the prevention, improvement and / or treatment of neurodegerative conformational disease.
El término "anticuerpo" tal como se emplea en esta memoria, se refiere a moléculas de inmunoglobulinas y porciones inmunológicamente activas de moléculas de inmunoglobulinas, es decir, moléculas que contienen un sitio de fijación de antígeno que se une específicamente (inmunorreacciona con) la proteína MOK. Ejemplos de porciones de moléculas de inmunoglobulinas inmunológicamente activas, incluyen fragmentos F(ab) y F(ab')2 que pueden ser generados tratando el anticuerpo con una enzima tal como la pepsina. Puede ser un anticuerpo monoclonal o policlonal. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) the protein. MOK Examples of portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.
Los anticuerpos capaces de unirse a la proteína MOK pueden ser empleados para inhibir la actividad de dicha proteína. Tales anticuerpos están disponibles comercialmente, como por ejemplo, pero sin limitarse a ellos, los anticuerpos Anti-MOK comercializados por Sigma- Aldrich HPA027282, HPA027292, SAB4502568, SAB1300406, SAB1305100, SAB4504627, SAB2105049. Los anticuerpos, o fragmentos de los mismos, podrían ser capaces de inhibir la actividad de la proteína MOK que contribuye a la adquisición de las características propias de la enfermedad conformacional neurodegerativa. Los anticuerpos pueden ser policlonales (incluyen típicamente anticuerpos distintos dirigidos contra determinantes o epitopos distintos) o monoclonales (dirigidos contra un único determinante en el antígeno). El anticuerpo monoclonal puede ser alterado bioquímicamente, por manipulación genética, o puede ser sintético, careciendo, posiblemente, el anticuerpo en su totalidad o en partes, de porciones que no son necesarias para el reconocimiento de la MOK y estando sustituidas por otras que comunican al anticuerpo propiedades ventajosas adicionales. El anticuerpo puede ser también recombinante, quimérico, humanizado, sintético o una combinación de cualquiera de los anteriores. Un "anticuerpo o polipéptido recombinante" (rAC) es uno que ha sido producido en una célula hospedadora que ha sido transformada o transfectada con el ácido nucleico codificante del polipéptido, o produce el polipéptido como resultado de la recombinación homologa. Antibodies capable of binding to the MOK protein can be used to inhibit the activity of said protein. Such antibodies are commercially available, as per example, but not limited to, Anti-MOK antibodies marketed by Sigma-Aldrich HPA027282, HPA027292, SAB4502568, SAB1300406, SAB1305100, SAB4504627, SAB2105049. The antibodies, or fragments thereof, might be able to inhibit the activity of the MOK protein that contributes to the acquisition of the characteristics characteristic of neurodegerative conformational disease. The antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen). The monoclonal antibody can be altered biochemically, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in whole or in parts, of portions that are not necessary for the recognition of the MOK and being replaced by others that communicate to the antibody additional advantageous properties. The antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing. A "recombinant antibody or polypeptide" (rAC) is one that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.
Estos rAc se pueden expresar y dirigir hacia subcompartimentos celulares específicos cuando se les incorpora las secuencias apropiadas para el tráfico intracelular. Estos anticuerpos se denominan intrabodies, y han demostrado su eficacia no sólo para desviar proteínas de su compartimento habitual o bloquear interacciones entre proteínas implicadas en vías de señalización, sino también para activar proteínas intracelulares. These rAc can be expressed and directed to specific cellular sub-compartments when the appropriate sequences for intracellular traffic are incorporated. These antibodies are called intrabodies, and have proven effective not only to divert proteins from their usual compartment or block interactions between proteins involved in signaling pathways, but also to activate intracellular proteins.
También forman parte de la invención las construcciones genéticas de DNA capaces de transcribirse a un péptido, anticuerpo o fragmento de anticuerpo, para su uso en el tratamiento de la ELA. Dicha construcción genética de DNA dirigiría la transcripción in vitro o intracelular de la secuencia del anticuerpo o fragmento del mismo, y comprende, al menos, uno de los siguientes tipos de secuencias: a) secuencia de nucleótidos de DNA, preferentemente de doble cadena, que comprende, al menos, la secuencia codificante del anticuerpo de la invención o del fragmento de anticuerpo de la invención para su transcripción in vitro, o intracelular, Also part of the invention are genetic DNA constructs capable of transcribing to a peptide, antibody or antibody fragment, for use in the treatment of ALS. Said genetic construction of DNA would direct the in vitro or intracellular transcription of the antibody sequence or fragment thereof, and comprises at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, which it comprises, at least, the coding sequence of the antibody of the invention or of the antibody fragment of the invention for transcription in vitro, or intracellular,
b) secuencia de nucleótidos de DNA, preferentemente de doble cadena, correspondiente a un sistema o vector de expresión génica que comprende la secuencia codificante de la secuencia de anticuerpo o fragmento de anticuerpo de la invención operativamente enlazada con, al menos, un promotor que dirija la transcripción de dicha secuencia de nucleótidos de interés, y con otras secuencias necesarias o apropiadas para la transcripción y su regulación adecuada en tiempo y lugar, por ejemplo, señales de inicio y terminación, sitios de corte, señal de poliadenilación, origen de replicación, activadores transcripcionales (enhancers), silenciadores transcripcionales (silencers), etc.. para su uso en aquellos contextos patológicos relacionados con la enfermedad conformacional neurodegerativa. b) DNA nucleotide sequence, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the antibody sequence or antibody fragment of the invention operatively linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its appropriate regulation in time and place, for example, start and end signals, sites of section, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts related to neurodegerative conformational disease.
Un "ribozima" tal y como se entiende en la presente invención, se refiere a un polinucleótido catalítico (típicamente RNA), que puede construirse para reconocer específicamente, por hibridación, un mRNA y fragmentarlo o eliminar su expresión. Las ribozimas pueden introducirse en la célula como moléculas de RNA catalíticas o como construcciones genéticas que se expresan a moléculas catalíticas de RNA. A "ribozyme" as understood in the present invention, refers to a catalytic polynucleotide (typically RNA), which can be constructed to specifically recognize, by hybridization, an mRNA and fragment it or eliminate its expression. Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed to RNA catalytic molecules.
En otra realización preferida de este aspecto de la invención, el agente modulador de MOK de la invención comprende una ribozima y se usa para la prevención, mejora y/o tratamiento de una enfermedad conformacional neurodegerativa. In another preferred embodiment of this aspect of the invention, the MOK modulating agent of the invention comprises a ribozyme and is used for the prevention, improvement and / or treatment of a neurodegerative conformational disease.
Un segundo aspecto de la invención se refiere a una composición, de ahora en adelante composición de la invención, que comprende un agente modulador de MOK de la invención. En una realización preferida, además comprende un vehículo o excipiente farmacéuticamente aceptable. En otra realización preferida la composición de la invención es una composición farmacéutica. En otra realización preferida, además comprende otro principio activo. Los adyuvantes y vehículos farmacéuticamente aceptables que pueden ser utilizados en dichas composiciones son los adyuvantes y vehículos conocidos por los técnicos en la materia y utilizados habitualmente en la elaboración de composiciones terapéuticas. A second aspect of the invention relates to a composition, hereinafter composition of the invention, comprising a MOK modulating agent of the invention. In a preferred embodiment, it further comprises a pharmaceutically acceptable carrier or excipient. In another preferred embodiment the composition of the invention is a pharmaceutical composition. In another preferred embodiment, it also comprises another active ingredient. The pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.
En el sentido utilizado en esta descripción, la expresión "cantidad terapéuticamente efectiva" se refiere a la cantidad del agente o compuesto capaz de desarrollar la acción terapéutica determinada por sus propiedades farmacológicas, calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de los compuestos, incluyendo la edad, estado del paciente, la severidad de la alteración o trastorno, y de la ruta y frecuencia de administración. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the agent or compound capable of developing the therapeutic action determined by its pharmacological properties, calculated to produce the desired effect and, in general, will be determined, among other causes, due to the characteristics of the compounds, including the age, condition of the patient, the severity of the alteration or disorder, and the route and frequency of administration.
Los compuestos descritos en la presente invención, sus sales, profármacos y/o solvatos así como las composiciones farmacéuticas que los contienen pueden ser utilizados junto con otros fármacos, o principios activos, adicionales para proporcionar una terapia de combinación. Dichos fármacos adicionales pueden formar parte de la misma composición farmacéutica o, alternativamente, pueden ser proporcionados en forma de una composición separada para su administración simultánea o no a la de la composición farmacéutica que comprende un compuesto de fórmula (I) y/o (II), o una sal, profármaco o solvato del mismo. The compounds described in the present invention, their salts, prodrugs and / or solvates as well as the pharmaceutical compositions containing them can be used together with other drugs, or active ingredients, additional to provide a combination therapy. Said additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising a compound of formula (I) and / or (II ), or a salt, prodrug or solvate thereof.
Por tanto, en otra realización preferida, la composición farmacéutica además comprende otro principio activo. Como se emplea aquí, el término "principio activo", "substancia activa", "substancia farmacéuticamente activa", "ingrediente activo" ó "ingrediente farmacéuticamente activo" significa cualquier componente que potencialmente proporcione una actividad farmacológica u otro efecto diferente en el diagnóstico, cura, mitigación, tratamiento, o prevención de una enfermedad, o que afecta a la estructura o función del cuerpo del hombre u otros animales. El término incluye aquellos componentes que promueven un cambio químico en la elaboración del fármaco y están presentes en el mismo de una forma modificada prevista que proporciona la actividad específica o el efecto. Therefore, in another preferred embodiment, the pharmaceutical composition further comprises another active ingredient. As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
Otro aspecto de la invención se refiere a una forma farmacéutica, de ahora en adelante forma farmacéutica de la invención, que comprende el compuesto de la invención o la composición de la invención. Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising the compound of the invention or the composition of the invention.
En esta memoria se entiende por "forma farmacéutica" la mezcla de uno o más principios activos con o sin aditivos que presentan características físicas para su adecuada dosificación, conservación, administración y biodisponibilidad. En otra realización preferida de la presente invención, las composiciones y formas farmacéuticas de la invención son adecuadas para la administración oral, en forma sólida o líquida. Las posibles formas para la administración oral son tabletas, cápsulas, siropes o soluciones y pueden contener excipientes convencionales conocidos en el ámbito farmacéutico, como agentes agregantes (p.e. sirope, acacia, gelatina, sorbitol, tragacanto o polivinil pirrolidona), rellenos (p.e. lactosa, azúcar, almidón de maíz, fosfato de calcio, sorbitol o glicina), disgregantes (p.e. almidón, polivinil pirrolidona o celulosa microcristalina) o un surfactante farmacéuticamente aceptable como el lauril sulfato de sodio. Otras formas farmacéuticas pueden ser los sistemas coloidales, dentro de los cuales se incluyen nanoemulsiones, nanocápsulas y nanopartículas poliméricas. Las composiciones para administración oral pueden ser preparadas por métodos los convencionales de Farmacia Galénica, como mezcla y dispersión. Las tabletas se pueden recubrir siguiendo métodos conocidos en la industria farmacéutica. Las composiciones y formas farmacéuticas se pueden adaptar para la administración parenteral, como soluciones estériles, suspensiones, o liofilizados de los productos de la invención, empleando la dosis adecuada. Se pueden emplear excipientes adecuados, como agentes tamponadores del pH o surfactantes. In this specification, "pharmaceutical form" means the mixture of one or more active ingredients with or without additives that have physical characteristics for proper dosage, preservation, administration and bioavailability. In another preferred embodiment of the present invention, the compositions and pharmaceutical forms of the invention are suitable for oral administration, in solid or liquid form. Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, as aggregating agents (eg syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone), fillers (eg lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), disintegrants (eg starch, polyvinyl pyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate. Other pharmaceutical forms may be colloidal systems, which include nanoemulsions, nanocapsules and polymeric nanoparticles. Compositions for oral administration can be prepared by conventional methods of Galenic Pharmacy, as mixing and dispersion. The tablets can be coated following methods known in the pharmaceutical industry. The compositions and pharmaceutical forms can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dose. Suitable excipients, such as pH buffering agents or surfactants, can be used.
Las formulaciones anteriormente mencionadas pueden ser preparadas usando métodos convencionales, como los descritos en las Farmacopeas de diferentes países y en otros textos de referencia. The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.
El término "medicamento", tal y como se usa en esta memoria, hace referencia a cualquier sustancia usada para prevención, diagnóstico, alivio, tratamiento o curación de enfermedades en el hombre y los animales. The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
La administración de los compuestos, composiciones o formas farmacéuticas de la presente invención puede ser realizada mediante cualquier método adecuado, como la infusión intravenosa y las vías oral, tópica o parenteral. La administración oral es la preferida por la conveniencia de los pacientes y por el carácter crónico de las enfermedades a tratar. The administration of the compounds, compositions or pharmaceutical forms of the present invention can be performed by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred for the convenience of patients and for the chronic nature of the diseases to be treated.
La cantidad administrada de un compuesto de la presente invención dependerá de la relativa eficacia del compuesto elegido, la severidad de la enfermedad a tratar y el peso del paciente. Sin embargo, los compuestos de esta invención serán administrados una o más veces al día, por ejemplo 1 , 2, 3 ó 4 veces diarias, con una dosis total entre 0.1 y 1000 mg/Kg/día. Es importante tener en cuenta que puede ser necesario introducir variaciones en la dosis, dependiendo de la edad y de la condición del paciente, así como modificaciones en la vía de administración. Los compuestos y composiciones de la presente invención pueden ser empleados junto con otros medicamentos en terapias combinadas. Los otros fármacos pueden formar parte de la misma composición o de otra composición diferente, para su administración al mismo tiempo o en tiempos diferentes. The amount administered of a compound of the present invention will depend on the relative efficacy of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg / kg / day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration. The compounds and compositions of the present invention can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.
MÉTODOS DE SCREENING Un tercer aspecto de la invención consiste en un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad conformacional neurodegerativaque comprende: SCREENING METHODS A third aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
a) poner en contacto el compuesto a analizar con el polipéptido MOK, a) contacting the compound to be analyzed with the MOK polypeptide,
b) detectar la unión de dicho compuesto a analizar con el polipéptido MOK. b) detect the binding of said compound to be analyzed with the MOK polypeptide.
En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA), Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal. Spinal muscular atrophy.
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. More preferably, the neurodegerative conformational disease is ALS or FTLD.
Los compuestos que se unen al polipéptido MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad conformacional neurodegerativa. Compounds that bind to the MOK polypeptide would be identified as potential therapeutic agents against neurodegerative conformational disease.
Como se ha dicho, estos ensayos pueden implicar el polipéptido completo MOK, un fragmento biológicamente activo del mismo, o una proteína de fusión que implique toda o una porción del polipéptido MOK. Determinar la capacidad de un compuesto para modular la actividad de MOK puede realizarse, por ejemplo, determinando la capacidad de MOK de unirse o interaccionar con una molécula diana de dicho compuesto, de manera directa o indirecta. Pueden ser también ensayos de actividad, midiendo de manera directa o indirecta la actividad de MOK. También puede ser un ensayo de expresión, determinando de manera directa o indirecta la expresión del mARN de MOK o de la proteína MOK. Estos ensayos también pueden combinarse con un ensayo in vivo midiendo el efecto de un compuesto test sobre los síntomas de enfermedades relacionadas con MOK, y en concreto una enfermedad conformacional neurodegerativa (por ejemplo, pero sin limitarse, sobre modelos animales u otros sistemas modelo conocidos en la técnica). Los compuestos a testar empleados en el método de selección de agentes terapéuticos no se limitan a moléculas orgánicas de bajo peso molecular, proteínas (incluyendo anticuerpos), péptidos, oliogonucleótidos, etc. Pueden ser naturales y/o sintéticos. Por ejemplo, anticuerpos capaces de unirse a un epitopo de MOK, que pueden ser empleados terapéuticamente, como se ha expuesto anteriormente, pueden emplearse también en ensayos inmunohistoquímicos, como Western blots, ELISAs, radioinmunoensayos, ensayos de inmunoprecipitación, o otros ensayos inmunohistoquímicos conocidos en el estado de la técnica. MOK puede emplearse para inmunizar a un animal, para obtener anticuerpos policlonales. También se pueden preparar anticuerpos monoclonales mediante técnicas que permiten la producción de anticuerpos por líneas celulares en cultivo, entre las que se incluyen, pero sin limitarse, hibridomas, hibridomas de células B humanas. Técnicas para producir anticuerpos quiméricos, humanizados o sintéticos son conocidas. As stated, these assays may involve the complete MOK polypeptide, a biologically active fragment thereof, or a fusion protein that involves all or a portion of the MOK polypeptide. Determining the ability of a compound to modulate the activity of MOK can be performed, for example, by determining the ability of MOK to bind or interact with a target molecule of said compound, directly or indirectly. They can also be activity tests, directly or indirectly measuring the activity of MOK. It can also be an expression assay, directly or indirectly determining the expression of the MOK mRNA or MOK protein. These tests can also be combined with an in vivo test by measuring the effect of a test compound on the symptoms of MOK-related diseases, and in particular a neurodegerative conformational disease (for example, but not limited to, on animal models or other model systems known in The technique). The compounds to be tested used in the method of selection of therapeutic agents are not limited to low molecular weight organic molecules, proteins (including antibodies), peptides, oliogonucleotides, etc. They can be natural and / or synthetic. For example, antibodies capable of binding to an MOK epitope, which can be used therapeutically, as discussed above, can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in The state of the art. MOK can be used to immunize an animal, to obtain polyclonal antibodies. Monoclonal antibodies can also be prepared by techniques that allow the production of antibodies by cultured cell lines, including, but not limited to, hybridomas, human B-cell hybridomas. Techniques for producing chimeric, humanized or synthetic antibodies are known.
Los agentes terapéuticos identificados por el método de selección aquí descrito pueden ser usados en un modelo animal o de otro tipo para determinar el mecanismo de acción de dicho agente. Más aún, los agentes terapéuticos seleccionados por el método aquí descrito se emplearían en el tratamiento de enfermedades que cursen con la alteración de MOK y, en concreto, una enfermedad conformacional neurodegerativa. The therapeutic agents identified by the selection method described herein can be used in an animal or other model to determine the mechanism of action of said agent. Moreover, the therapeutic agents selected by the method described herein would be used in the treatment of diseases that occur with the alteration of MOK and, in particular, a neurodegerative conformational disease.
En un cuarto aspecto de la invención se describe un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad conformacional neurodegerativa que comprende:  In a fourth aspect of the invention a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
a) determinar la actividad de MOK a una concentración establecida del compuesto a analizar o en ausencia de dicho compuesto, a) determine the activity of MOK at an established concentration of the compound to be analyzed or in the absence of said compound,
b) determinar la actividad de MOK a una concentración del compuesto a analizar diferente de la de a). b) determine the activity of MOK at a concentration of the compound to be analyzed different from that of a).
Compuestos que den lugar a una actividad diferente de MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad conformacional neurodegerativa. Compounds that give rise to a different activity of MOK would be identified as potential therapeutic agents against neurodegerative conformational disease.
En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA), Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Primary lateral sclerosis
Atrofia muscular progresiva, Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar Progressive muscular atrophy, Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal, Spinal muscular atrophy,
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. More preferably, the neurodegerative conformational disease is ALS or FTLD.
Un quinto aspecto de la invención consiste en un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad conformacional neurodegerativa que comprende: A fifth aspect of the invention consists in a method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a neurodegerative conformational disease comprising:
a) poner en contacto el compuesto a analizar con el polinucleótido MOK, a) contacting the compound to be analyzed with the MOK polynucleotide,
b) detectar la unión de dicho compuesto a analizar con el polinucleótido MOK, Los compuestos que se unen al polinucleótido MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad conformacional neurodegerativa. b) detecting the binding of said compound to be analyzed with the MOK polynucleotide. Compounds that bind to the MOK polynucleotide would be identified as potential therapeutic agents against neurodegerative conformational disease.
En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en: In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA),  Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal, Spinal muscular atrophy,
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. or any of its combinations. More preferably, the neurodegerative conformational disease is ALS or FTLD.
MÉTODOS DE OBTENCIÓN DE DATOS ÚTILES PARA EL DIAGNÓSTICO METHODS OF OBTAINING USEFUL DATA FOR DIAGNOSIS
Las enfermedades en las que la alteración de la actividad de MOK puede ser diagnóstica, y en concreto las enfermedades conformacionales neurodegerativas, más concretamente donde la proteína agregante es la TDP-43, y aún más concretamente la ELA, pueden ser detectadas midiendo la cantidad de ácidos nucleicos (ADN y/o ARN y/o mARN) que codifican para MOK, o la cantidad de proteína MOK que se expresa, en comparación con células normales. La detección de los oligonucleótidos puede hacerse por métodos bien conocidos en el estado de la técnica (como por ejemplo, pero sin limitarse, sondas con nucleótidos marcados, hibridación ADN-ADN ó ADN-ARN, amplificación por PCR empleando nucleótidos marcados, la RT-PCR). Diseases in which the alteration of MOK activity can be diagnostic, and in particular neurodegerative conformational diseases, more specifically where the aggregating protein is TDP-43, and even more specifically ALS, can be detected by measuring the amount of nucleic acids (DNA and / or RNA and / or mRNA) that encode for MOK, or the amount of MOK protein that is expressed, compared to normal cells. The detection of oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR).
Procedimientos para detectar la expresión de la proteína MOK también son bien conocidos en el estado de la técnica, como por ejemplo anticuerpos poli o monoclonales, ELISA, radioinmunoensayo (RIA), y FACS (fluorescence activated cell sorting). Procedures for detecting the expression of the MOK protein are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
En un sexto aspecto de la invención se describe un método para la recolección de datos útiles en el diagnóstico y/o pronóstico de una enfermedad conformacional neurodegerativa que comprende: In a sixth aspect of the invention a method is described for the collection of data useful in the diagnosis and / or prognosis of a neurodegerative conformational disease comprising:
a) determinar la expresión de MOK en una muestra extraída de un mamífero, a) determine the expression of MOK in a sample taken from a mammal,
b) comparar los valores de la expresión de MOK obtenidos en a) con los valores estándar en mamíferos sanos o enfermos. b) compare the values of the MOK expression obtained in a) with the standard values in healthy or diseased mammals.
En una realización preferida, la principal proteína agregante de la enfermedad conformacional neurodegerativa es la TDP-43, y aún más preferiblemente se selecciona de la lista que consiste en:  In a preferred embodiment, the main aggregative protein of neurodegerative conformational disease is TDP-43, and even more preferably it is selected from the list consisting of:
Esclerosis lateral amiotrófica (ELA), Amyotrophic lateral sclerosis (ALS),
Esclerosis lateral primaria Primary lateral sclerosis
Atrofia muscular progresiva, Progressive muscular atrophy,
Enfermedad de Kennedy o atrofia muscular progresiva espinobulbar  Kennedy disease or progressive spinobulbar muscle atrophy
Enfermedad de Hirayama o atrofia monomélica Hirayama disease or monomelic atrophy
Atrofia muscular espinal. Spinal muscular atrophy.
degeneración del lóbulo frontotemporal (FTLD), frontotemporal lobe degeneration (FTLD),
o cualquiera de sus combinaciones. or any of its combinations.
Más preferiblemente, la enfermedad conformacional neurodegerativa es la ELA o el FTLD. More preferably, the neurodegerative conformational disease is ALS or FTLD.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. EJEMPLOS DE LA INVENCIÓN Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES OF THE INVENTION
El modelo de ELA y FTLD utilizado recapitula los efectos observados para TDP-43 en muestras de pacientes y modelos animales de ELA y FTLD The ELA and FTLD model used recapitulates the effects observed for TDP-43 in patient samples and animal models of ELA and FTLD
Se utilizó un modelo experimental basado en agregados de proteína recombinante His-tag- TDP-43 humana, previamente caracterizados y validados (Capitini et al., 2014. PLoS One 9, e86720; Cascella et al., 2016. J. Biol. Chem. 291 (37): 19437-48; Fig.lA) en cultivo celular primario de microglia murina (Roodveldt et al., 2013. PLoS One Nov 13;8(11):e79160; Leal- Lasarte et al., FASEB J. 2017, en prensa) y cultivo organotípico de espina dorsal de ratón de 4 semanas (Guzmán-Lenis et al., 2008. Neuroscience 155(1): 104-13). Los resultados de la presente invención en microglia apoyan los resultados anteriormente descriptos en neuronas, y recapitulan los efectos observados en muestras de pacientes y modelos animales de ELA y FTLD (Boeynaems et al., 2016. Acta Neuropathol. 132(2): 159-73). En particular, hemos observado que la exposición de células de microglia a al tratamiento con agregados de TDP-43 (llamado aquí TDP43') vs. el tratamiento control con agregados sin TDP-43 (llamado aquí 'mock') producen una serie de efectos diferenciales: internalización de los agregados exógenos de TDP-43 y formación de inclusiones en citoplasma, mediante inmunofluorescencia (IF) (Fig.l B), modificación proteolítica de la TDP-43 internalizada por Western blot (Fig. 1 C), ubiquitinación de especies TDP-43 por IF y Western blot (Fig. 1 D, E), retro-translocación aberrante de TDP-43 nuclear (endógena) al citoplasma por IF ('nuclear clearing', Fig. 1 F). Además, estas observaciones han sido apoyadas por los resultados obtenidos en análisis por IF de cultivo organotípico de espina dorsal murina (Fig. 1 G). An experimental model based on human His-tag-TDP-43 recombinant protein aggregates, previously characterized and validated was used (Capitini et al., 2014. PLoS One 9, e86720; Cascella et al., 2016. J. Biol. Chem 291 (37): 19437-48; Fig.lA) in primary cell culture of murine microglia (Roodveldt et al., 2013. PLoS One Nov 13; 8 (11): e79160; Leal-Lasarte et al., PHASEB J 2017, in press) and organotypic culture of 4-week-old mouse spine (Guzmán-Lenis et al., 2008. Neuroscience 155 (1): 104-13). The results of the present invention in microglia support the results described above in neurons, and recapitulate the effects observed in samples of patients and animal models of ALS and FTLD (Boeynaems et al., 2016. Acta Neuropathol. 132 (2): 159- 73). In particular, we have observed that the exposure of microglia cells to treatment with aggregates of TDP-43 (here called TDP43 ') vs. Control treatment with aggregates without TDP-43 (called 'mock' here) produces a series of differential effects: internalization of exogenous aggregates of TDP-43 and formation of cytoplasm inclusions, by immunofluorescence (IF) (Fig. 1 B) , proteolytic modification of TDP-43 internalized by Western blot (Fig. 1 C), ubiquitination of TDP-43 species by IF and Western blot (Fig. 1 D, E), aberrant nuclear retro-translocation of TDP-43 (endogenous) ) to the cytoplasm by IF ('nuclear clearing', Fig. 1 F). In addition, these observations have been supported by the results obtained in IF analysis of murine spine organotypic culture (Fig. 1 G).
Los agregados de TDP-43 inducen respuesta inflamatoria dependiente de NLRP3/vía clásica y alternativa sobre la microglia Aggregates of TDP-43 induce NLRP3-dependent inflammatory response / classical and alternative pathway on microglia
El hecho de que, a las concentraciones analizadas, los agregados de TDP-43 no resultan citotóxicos sobre las células de microglia (Fig. 2A), hizo que se estudiara el efecto sobre la respuesta inflamatoria mediado por este tipo de células. En concreto, mediante Western blot de lisados celulares de cultivos de microglia y organotípicos de espinal dorsal tratados, observamos una fuerte inducción de la expresión de caspasa-1 (clave en la respuesta mediada por el inflamasoma) en su forma de pro-caspasa-1 (Fig. 2B, Fig.SIA), así como un aumento en la secreción de citoquinas pro-inflamatorias claves (TNF-α, I L- β, IL-18), medido por ELISA (Figs. 2C, D). Además, se ha observado un aumento de IL-18 activada y secretada por Western blot (Fig. 2E, Fig.SI B), así como de IL-18 total intracelular por IF (Fig. 2F) y de una especie de IL-18 producto de procesamiento alternativo, de 16 kDa, por Western blot (Fig. 2G). The fact that, at the concentrations analyzed, the TDP-43 aggregates are not cytotoxic on the microglia cells (Fig. 2A), the effect on the inflammatory response mediated by this type of cells was studied. Specifically, by Western blotting of cell lysates of treated microglia and organotypic cultures of the dorsal spinal column, we observed a strong induction of caspase-1 expression (key in the response mediated by inflamasome) in its pro-caspase-1 form (Fig. 2B, Fig. SIA), as well as an increase in the secretion of key pro-inflammatory cytokines (TNF-α, I L-β, IL-18), measured by ELISA (Figs. 2C, D). In addition, an increase in IL-18 activated and secreted by Western blot has been observed (Fig. 2E, Fig. SI B), as well as total intracellular IL-18 by IF (Fig. 2F) and a kind of IL-18 alternative processing product, 16 kDa, by Western blot (Fig. 2G).
También estudiamos el efecto sobre la activación de caspasa-3 en microglia, que recientemente ha sido relacionada con un perfil neuroinflamatorio y neurotóxico en el contexto del Alzheimer y el Parkinson (Burguillos et al., 201 1. Nature 472(7343): 319-24; Kavanagh et al., 2014. Cell Death Dis. 5:e1565), dos enfermedades 'conformacionales' neurodegenerativas relacionadas con ELA y FTLD (Cooper-Knock et al., 2012. Nat. Rev. Neurol. 518-30). Los resultados de la invención demuestran que los agregados de TDP-43 producen un aumento de los niveles de caspasa-3 activada en cultivo de microglia primaria (Fig. 3A, B, C, Fig.S2) y en cultivo organotípico de cortes de espina dorsal (Fig. 1 E). Además, la respuesta de la microglia a los agregados de TDP-43 resultó ser citotóxica sobre motoneuronas (línea NSC-34) en cultivo (Fig. 3D), lo que demuestra que la microglia activada puede producir respuesta neuroinflamatoria neurotóxica y proporciona un mecanismo de neurodegeneración no autónomo celularmente ('non-cell autonomous' mechanism) que involucre a IL-18 y caspasa-3, en el contexto de: enfermedades 'conformacionales' neurodegenerativas/'misfolding diseases' (en particular, ELA y FTLD), y otras relacionadas (Alzheimer, Parkinson, Huntington, las prionosis, etc.), el envejecimiento (caracterizado también por la presencia creciente de especies proteicas agregadas de forma aberrante en el medio intra y/o extracelular), y enfermedades con un componente inflamatorio, incluido el cáncer. We also studied the effect on the activation of caspase-3 in microglia, which has recently been related to a neuroinflammatory and neurotoxic profile in the context of Alzheimer's and Parkinson's (Burguillos et al., 201 1. Nature 472 (7343): 319- 24; Kavanagh et al., 2014. Cell Death Dis. 5: e1565), two neurodegenerative 'conformational' diseases related to ALS and FTLD (Cooper-Knock et al., 2012. Nat. Rev. Neurol. 518-30). The results of the invention demonstrate that TDP-43 aggregates produce an increase in activated caspase-3 levels in primary microglia culture (Fig. 3A, B, C, Fig.S2) and in organotypic culture of spine cuts dorsal (Fig. 1 E). In addition, the microglia response to TDP-43 aggregates was found to be cytotoxic on motor neurons (NSC-34 line) in culture (Fig. 3D), demonstrating that activated microglia can produce neurotoxic neuroinflammatory response and provide a mechanism of Non-cell autonomic neurodegeneration ('non-cell autonomous' mechanism) involving IL-18 and caspase-3, in the context of: 'conformational' neurodegenerative diseases / 'misfolding diseases' (in particular, ELA and FTLD), and others related (Alzheimer, Parkinson, Huntington, prionosis, etc.), aging (also characterized by the increasing presence of aberrantly added protein species in the intra and / or extracellular environment), and diseases with an inflammatory component, including Cancer.
Los agregados de TDP-43 afectan la ruta de señalización de la kinasa MAPK/MAK/MRK overlapping kinase (MOK) en microglia. Implicaciones para las enfermedades neurodegenerativas 'conformacionales', inflamatorias y el cáncer The aggregates of TDP-43 affect the signaling pathway of the MAPK / MAK / MRK kinase overlapping kinase (MOK) in microglia. Implications for 'conformational', inflammatory and cancer neurodegenerative diseases
Para identificar en las células de microglia posibles proteínas interactoras con los agregados aberrantes de TDP-43 asociados a ELA y FLTD, los autores de la presente invención han realizado una inmunoprecipitación (IP) luego de incubar agregados de TDP-43 (o su control mock) con lisados celulares de microglia y capturando con anticupero anti-TDP-43. El resultado del análisis de la fracción eluida por SDS_PAGE (Fig. S3) mostró una banda diferencial de aprox. 46-50 kDa para la muestra TDP43 cuyo análisis por MALDI-TOF/MS identificó a la proteína murina 'MAPK/MAK/MRK overlapping kinase (MOK) protein' (Acc. No. BAA81689) para la muestra TDP43 pero no para su control mock. Este resultado fue confirmado por Wetern blot con el producto de la IP y utilizando un anticuerpo anti-MOK específico (Fig. 4B), que dio una banda de 46-50 kDa, que es consistente con el tamaño predicho para MOK (48 kDa). Habiendo identificado MOK como interactor con los agregados aberrantes de TDP-43, realizamos estudios de co-localización entre los agregados internalizados de TDP-43 y MOK, por IF con anticuerpos específicos y microscopía confocal. Los resultados obtenidos en la línea de microglia BV-2 (Fig. S3A, B), cultivo de microglia primaria (Fig. 4C, D) y en cultivo organotípico de espina dorsal (Fig. 4A) demuestran, además de un significativo aumento de los niveles de MOK como resultado del tratamiento con TDP43, la co-localización His-tag/MOK e indican una interacción específica y estable entre los agregados aberrantes de TDP-43 internalizados y la proteína MOK en microglia. To identify possible interactor proteins in the microglia cells with aberrant aggregates of TDP-43 associated with ELA and FLTD, the authors of the present invention have performed an immunoprecipitation (IP) after incubating aggregates of TDP-43 (or their mock control ) with microglia cell lysates and capturing with anti-TDP-43 anticupero. The result of the analysis of the fraction eluted by SDS_PAGE (Fig. S3) showed a differential band of approx. 46-50 kDa for the TDP43 sample whose MALDI-TOF / MS analysis identified the murine protein 'MAPK / MAK / MRK overlapping kinase (MOK) protein' (Acc. No. BAA81689) for the TDP43 sample but not for its control mock This result was confirmed by Wetern blot with the IP product and using a specific anti-MOK antibody (Fig. 4B), which gave a band of 46-50 kDa, which is consistent with the predicted size for MOK (48 kDa) . Having identified MOK as an interactor with the aggregates aberrant of TDP-43, we conducted co-localization studies between the internalized aggregates of TDP-43 and MOK, by IF with specific antibodies and confocal microscopy. The results obtained in the BV-2 microglia line (Fig. S3A, B), primary microglia culture (Fig. 4C, D) and in organotypic spine culture (Fig. 4A) demonstrate, in addition to a significant increase in MOK levels as a result of treatment with TDP43, His-tag / MOK co-localization and indicate a specific and stable interaction between the aberrant aggregates of internalized TDP-43 and the MOK protein in microglia.
MOK es una serin/treonin kinasa perteneciente a la superfamilia de las MAPK kinasas y que se expresa en diferentes tejidos (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229- 248). MOK es capaz de fosforilar una variedad de sustratos y para su actividad requiere de la fosforilación de dos residuos dentro del motivo TEY, uno de los cuales es autofosforilado (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248). En base a esto, los autores de la presente invención han estudiado el posible efecto de esta interacción aberrante entre TDP-43 y MOK— asociada a una respuesta inflamatoria— sobre la actividad de MOK, para lo cual han analizamos el grado de fosforilación del residuo de Tyr, con un anticuerpo anti- pTyr-MOK, luego del tratamiento con los agregados de TDP-43 o mock durante 30 minutos, por Western blot de lisados celulares. Los resultados mostraron una banda tenue y aparentemente constante a la altura de 48 kDa, y otra inducible con lipopolisacárido (LPS) — un inductor de respuesta pro-inflamatoria/modelo de inflamación— correspondiente a aprox. 46 kDa, consistente con el tamaño predicho para las isoformas '2' y '3' de MOK (Fig. S3D). Sin embargo, se observó una reducción relativa en la intensidad de esta banda del 30%, luego del tratamiento con TDP43 vs. mock, tanto en células BV-2 como de microglia primaria (Fig. 4E y S3E). Sabiendo que la fosforilación de este residuo de MOK es crítico para su actividad de kinasa, estos resultados indican que los agregados de TDP-43 afectan tanto los niveles como el estado y localización proteica de MOK (aglutinamiento en torno a los agregados de TDP-43 internalizados) y al estado de fosforilación/actividad y función en la señalización celular, en la activación pro-inflamatoria de la microglia. MOK is a serine / threonine kinase belonging to the MAPK kinase superfamily and which is expressed in different tissues (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248). MOK is capable of phosphorylating a variety of substrates and for its activity requires the phosphorylation of two residues within the TEY motif, one of which is autophosphorylated (Fu JZ, 2012. In Protein Kinases, Intech Open, pp: 229-248) . Based on this, the authors of the present invention have studied the possible effect of this aberrant interaction between TDP-43 and MOK - associated with an inflammatory response - on the activity of MOK, for which we have analyzed the degree of phosphorylation of the residue Tyr, with an anti-pTyr-MOK antibody, after treatment with the TDP-43 aggregates or mock for 30 minutes, by Western blotting of cell lysates. The results showed a faint and apparently constant band at the height of 48 kDa, and another inducible with lipopolysaccharide (LPS) - a pro-inflammatory response inducer / inflammation model - corresponding to approx. 46 kDa, consistent with the predicted size for the '2' and '3' isoforms of MOK (Fig. S3D). However, a relative reduction in the intensity of this band of 30% was observed, after treatment with TDP43 vs. mock, both in BV-2 and primary microglia cells (Fig. 4E and S3E). Knowing that phosphorylation of this MOK residue is critical for its kinase activity, these results indicate that the TDP-43 aggregates affect both the levels and the protein status and location of MOK (agglutination around the TDP-43 aggregates internalized) and the state of phosphorylation / activity and function in cell signaling, in the pro-inflammatory activation of microglia.
Esto tendría importantes consecuencias en el contexto de las enfermedades 'conformacionales' neurodegenerativas, así como en un contexto más amplio de 'inflamación' asociada a diversas patologías. Es de resaltar que la secuencia de MOK a nivel de RNA contiene en su región 3' terminal un segmento idéntico a las proteínas de la familia RAGE (renal cell carcinoma antigen), que se encuentran sobre-expresadas en diversos cánceres (renal, melanoma, leucemias, etc.) (Eichmuller et al., 2002. Exp. Dermatol. 1 1 (4):292-301). Esto amplía el posible rol de MOK al a la patogénesis y/o desarrollo del cáncer, considerando además que hay una relación demostrada entre cáncer e inflamación. This would have important consequences in the context of neurodegenerative 'conformational' diseases, as well as in a broader context of 'inflammation' associated with various pathologies. It is noteworthy that the MOK sequence at the RNA level contains in its 3 'terminal region a segment identical to the RAGE family (renal cell carcinoma antigen) proteins, which are overexpressed in various cancers (renal, melanoma, leukemia, etc.) (Eichmuller et al., 2002. Exp. Dermatol. 1 1 (4): 292-301). This extends the possible role of MOK to the pathogenesis and / or development of cancer, considering also that there is a proven relationship between cancer and inflammation.

Claims

REIVINDICACIONES
1.- El agente modulador de MOK de fórmula (I): 1.- The MOK modulating agent of formula (I):
Figure imgf000040_0001
Fórmula (I)
Figure imgf000040_0001
Formula (I)
donde where
n es igual a 0 ó 1 , n is equal to 0 or 1,
R es un metilo (Me), isopropilo (¡Pr), tere-Butilo (tBu), ciclopentadienilo (Cp).  R is a methyl (Me), isopropyl (¡Pr), tere-Butyl (tBu), cyclopentadienyl (Cp).
X es H ó NH2, X is H or NH 2 ,
Y es H, NHCOCHCH2, NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, ó COCH3, y Z es H, Y is H, NHCOCHCH 2 , NHSO2CHCH2, NHCOCH2CI, NHSO2CH2CH3, COCH2F, or COCH3, and Z is H,
o cualquiera de sus sales, preferiblemente cualquier sal farmacéuticamente aceptable, ésteres, tautómeros, polimorfos, hidratos farmacéuticamente aceptables, o un isómero, profármacos, derivados, solvatos o análogos, o cualquiera de sus combinaciones para su uso en la prevención, mejora y/o tratamiento de una enfermedad inflamatoria y/o una enfermedad neurodegenerativa. or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof for use in prevention, improvement and / or treatment of an inflammatory disease and / or a neurodegenerative disease.
2. - El uso del agente modulador de MOK según la reivindicación 1 , donde 2. - The use of the MOK modulating agent according to claim 1, wherein
n es igual a 1 , n is equal to 1,
R es un isopropilo (¡Pr),  R is an isopropyl (¡Pr),
X es NH2, X is NH 2 ,
Y es COCH2F And it is COCH2F
Z es H, Z is H,
o cualquiera de sus sales, preferiblemente cualquier sal farmacéuticamente aceptable, ésteres, tautómeros, polimorfos, hidratos farmacéuticamente aceptables, o un isómero, profármacos, derivados, solvatos o análogos, o cualquiera de sus combinaciones. or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or the like, or any combination thereof.
3. - El uso del agente modulador de MOK según cualquiera de las reivindicaciones 1-2, donde enfermedad neurodegenerativa es una enfermedad conformacional neurodegerativa. 3. - The use of the MOK modulating agent according to any of claims 1-2, wherein neurodegenerative disease is a neurodegerative conformational disease.
4.- El uso del agente modulador de MOK según la reivindicación 3, donde la proteína agregante es la TDP-43. 4. The use of the MOK modulating agent according to claim 3, wherein the aggregating protein is TDP-43.
5.- El uso del agente modulador de MOK según cualquiera de las reivindicaciones 1-4, donde la enfermedad conformacional neurodegerativa se selecciona de la lista que consiste en: esclerosis lateral amiotrófica (ELA), esclerosis lateral primaria, atrofia muscular progresiva, enfermedad de Kennedy o atrofia muscular progresiva espinobulbar, enfermedad de Hirayama o atrofia monomélica, atrofia muscular espinal, degeneración del lóbulo frontotemporal (FTLD), o cualquiera de sus combinaciones. 5. The use of the MOK modulating agent according to any of claims 1-4, wherein the neurodegerative conformational disease is selected from the list consisting of: amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, disease of Kennedy or progressive spinobulbar muscular atrophy, Hirayama disease or monomelic atrophy, spinal muscular atrophy, frontotemporal lobe degeneration (FTLD), or any combination thereof.
6.- El uso del agente modulador de MOK según cualquiera de las reivindicaciones 1-5, donde la enfermedad es la ELA o la FLDT. 6. The use of the MOK modulating agent according to any of claims 1-5, wherein the disease is ALS or FLDT.
7.- El uso de una composición que comprende un agente modulador de MOK según cualquiera de las reivindicaciones 1-6, en la elaboración de un medicamento para la prevención, mejora y/o tratamiento de una enfermedad según se describe en cualquiera de las reivindicaciones 1-6. 7. The use of a composition comprising an MOK modulating agent according to any of claims 1-6, in the preparation of a medicament for the prevention, improvement and / or treatment of a disease as described in any of the claims 1-6.
8.- El uso de una composición según la reivindicación anterior, donde la composición además comprende un vehículo o excipiente farmacéuticamente aceptable. 8. The use of a composition according to the preceding claim, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
9. - El uso de una composición según cualquiera de las reivindicaciones 7-8, donde la composición es una composición farmacéutica. 9. - The use of a composition according to any of claims 7-8, wherein the composition is a pharmaceutical composition.
10. - El uso de una composición según cualquiera de las reivindicaciones 7-9, donde la composición además comprende otro principio activo. 10. - The use of a composition according to any of claims 7-9, wherein the composition further comprises another active ingredient.
1 1. - Un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad según cualquiera de las reivindicaciones 1-6 que comprende: 1 1. - A method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a disease according to any of claims 1-6 comprising:
a) poner en contacto el compuesto a analizar con el polipéptido MOK, a) contacting the compound to be analyzed with the MOK polypeptide,
b) detectar la unión de dicho compuesto a analizar con el polipéptido MOK. b) detect the binding of said compound to be analyzed with the MOK polypeptide.
donde los compuestos que se unen al polipéptido MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad según se describe en cualquiera de las reivindicaciones 1-6. wherein compounds that bind to the MOK polypeptide would be identified as potential therapeutic agents against the disease as described in any of claims 1-6.
12. - Un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad según cualquiera de las reivindicaciones 1-6 que comprende: 12. - A method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a disease according to any of claims 1-6 comprising:
a) determinar la actividad de MOK a una concentración establecida del compuesto a analizar o en ausencia de dicho compuesto, a) determine the activity of MOK at an established concentration of the compound to be analyzed or in the absence of said compound,
b) determinar la actividad de MOK a una concentración del compuesto a analizar diferente de la de a). b) determine the activity of MOK at a concentration of the compound to be analyzed different from that of a).
donde los compuestos que den lugar a una actividad diferente de MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad según se describe en cualquiera de las reivindicaciones 1-6. wherein the compounds that give rise to a different activity of MOK would be identified as potential therapeutic agents against the disease as described in any of claims 1-6.
13. - Un método de selección de agentes terapéuticos útiles en la prevención, mejora y/o el tratamiento de una enfermedad según cualquiera de las reivindicaciones 1-6 que comprende: 13. - A method of selecting therapeutic agents useful in the prevention, improvement and / or treatment of a disease according to any of claims 1-6 comprising:
a) poner en contacto el compuesto a analizar con el polinucleotido MOK, a) contacting the compound to be analyzed with the MOK polynucleotide,
b) detectar la unión de dicho compuesto a analizar con el polinucleotido MOK, b) detecting the binding of said compound to be analyzed with the MOK polynucleotide,
donde los compuestos que se unen al polinucleotido MOK se identificarían como agentes terapéuticos potenciales frente a la enfermedad según se describe en cualquiera de las reivindicaciones 1-6. wherein the compounds that bind to the MOK polynucleotide would be identified as potential therapeutic agents against the disease as described in any of claims 1-6.
14. - Un método para la obtención de datos útiles en el diagnóstico y/o pronóstico de una enfermedad según cualquiera de las reivindicaciones 1-6 que comprende: 14. - A method for obtaining useful data in the diagnosis and / or prognosis of a disease according to any of claims 1-6 comprising:
a) determinar la expresión de MOK en una muestra biológica extraída de un mamífero, b) comparar los valores de la expresión de MOK obtenidos en a) con los valores estándar en mamíferos sanos o enfermos. a) determine the expression of MOK in a biological sample extracted from a mammal, b) compare the values of the MOK expression obtained in a) with the standard values in healthy or diseased mammals.
PCT/ES2018/070216 2017-03-21 2018-03-21 Use of mok modulating agents for the treatment of inflammatory diseases and neurodegenerative diseases WO2018172588A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011153553A2 (en) * 2010-06-04 2011-12-08 The Regents Of The University Of California Methods and compositions for kinase inhibition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011153553A2 (en) * 2010-06-04 2011-12-08 The Regents Of The University Of California Methods and compositions for kinase inhibition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEAL-LASARTE, M.M. ET AL.: "Extracellular TDP-43 aggregates target MAPK/MAK/MRK overlapping kinase (MOK) and trigger caspase-3/ IL -18 signaling in microglia", THE FASEB JOURNAL, vol. 31, no. 7, 23 March 2017 (2017-03-23), pages 2797 - 2816, XP055538709 *

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