WO2015053452A1 - Régulateur de l'épissage contenant cx-4945 en tant qu'ingrédient actif - Google Patents

Régulateur de l'épissage contenant cx-4945 en tant qu'ingrédient actif Download PDF

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Publication number
WO2015053452A1
WO2015053452A1 PCT/KR2014/003514 KR2014003514W WO2015053452A1 WO 2015053452 A1 WO2015053452 A1 WO 2015053452A1 KR 2014003514 W KR2014003514 W KR 2014003514W WO 2015053452 A1 WO2015053452 A1 WO 2015053452A1
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Prior art keywords
splicing
pharmaceutically acceptable
acceptable salt
diseases caused
active ingredient
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PCT/KR2014/003514
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English (en)
Korean (ko)
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조성찬
김형기
최광만
강현주
임동화
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한국생명공학연구원
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Priority claimed from KR1020140035000A external-priority patent/KR101593595B1/ko
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Publication of WO2015053452A1 publication Critical patent/WO2015053452A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems

Definitions

  • the invention relates to 5-[(3-chlorophenyl) amino] benzo
  • Splicing removes introns that do not have genetic information from the precursor -mR A (Precursor-mRNA, Pre-mRNA), followed by only the exon portion that has genetic information, followed by a single polypeptide (polypeptide). It is an essential process in the regulation of gene expression in eukaryotes.
  • Selective splicing is a process of generating mRNA from a combination of axons only specifically selected from the combination of various exons of m-RNA, which allows the production of various types of mRNA from a single human It is known that a variety of proteins are produced without expansion of the genome and serve to provide cell diversity. A comprehensive survey of human transcripts predicts that 95% of genes, including introns, undergo selective splicing (Pan Q, et al, Nat. Genet. 40 (12): 1413-1415, 2008; Wang ET , et al. Nature 456 (7221): 470-476, 2008). Through selective splicing, one expressed Genes can be translated into various proteins, each representing a different function.
  • Selective splicing depends on the interaction of the cis-acting element on the pre-mRNA sequence and the trans-acting protein that binds it, depending on the development, tissue or cell type. Regulat ion (mi th CW and Valcarcel J, Trends Biochem. Sci. 25 (8): 381-388, 2000).
  • Serine / arginine-rich protein (Serin / arginine-rich, SR protein) is a representative trans-acting protein and is very well preserved in living organisms.
  • the protein contains one or two RNA-recognition motifs (13 ⁇ 4 -1- ⁇ 0 011 0111110 ⁇ ) at the amino terminus (N-terminal) and is rich in serine and arginine at the carboxyl terminus (C-terminal). Domain (Arginine / Serine-rich domain, RS domain). (Zahler AM et al Genes Dev. 6 (5): 83 G 847, 1992; Caceres JF and Krainer AR, EMBO J.
  • SR proteins The physiological role of SR proteins has not been specifically reported, but the phosphorylation of SR proteins (1 ⁇ 3 110 ⁇ 1 1011) is associated with protein-protein, protein-RNA interactions, intercellular localization, It is known to be involved in intercellular trafficking or selective splicing of pre—mRNAs.
  • SR proteins regulates the binding of pre-mRNA and SR proteins, and the phosphorylation promotes the interaction of specific proteins, resulting in the splicing of RA and splicing complexes.
  • at least at the level of the phosphorylation is induced when low phosphorylation (hypophosphorylation) 'or hyperphosphorylation Chemistry (hyperphosphorylat ion) abnormal splicing is induced (Kanopka A, et al, Nature 393 (6681):. 185- 187. 1998).
  • SR SR .
  • Clk Cdc-2-Hke kinase
  • SR protein kinases serine / arginine-rich protein kinase, SRPKs
  • Clk a dual-specific kinase, is known to exist 44 isoforms, including Clkl, Clk2 and Clk3, and has been reported to be involved in the regulation of selective splicing. (Colwill K, et al. EMBO J. 15 (2): 265-275, 1996), but little is known about other biological functions, such as signal pathways.
  • [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid, Si lmitasert ib, CX-4945) are frequently found in human cancer cells. It is known as an inhibitor of casein kinase 2 (CK2), a protein that regulates various cellular activities such as growth and self-killing.
  • CK2 casein kinase 2
  • Adam Siddiqui-Jain et al. Demonstrates that CX-4945 is a bioavailable selective inhibitor of oral administration and inhibits the expression level of CK2 ⁇ , a catalytic subunit of CK2. It has been reported to inhibit the proliferation of cancer cells (Siddiqui-Jain A, et al. Cancer Res.
  • CX-4945 is a pharmaceutical composition for the prevention or treatment of diseases caused by splicing defects. It was confirmed that it can be usefully used as an active ingredient.
  • the CX-4945 of the present invention by confirming that the overall efficacy is higher than the TG-003 that is previously used as a Clk inhibitor, by confirming that the CX-4945 can be usefully used for biological research related to splicing The present invention has been completed. ⁇ Detailed Description of the Invention ⁇ ⁇
  • the object of the present invention is 5-[(3-chlorophenyl) amino] benzo
  • the present invention provides 5-[(3-chlorophenyl) amino] benzo [(:]-2,6-naphthyridine-8-carboxylic acid (5— [(3-Ch 1 or opheny 1) am i no] benzo
  • the present invention provides a splicing regulator (spl icing regulator) containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • a splicing regulator spl icing regulator
  • the present invention also provides a health food for the prevention and improvement of diseases caused by splicing defects containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a method for controlling splice splicing comprising treating CX-4945 or a pharmaceutically acceptable salt thereof to the isolated cell.
  • the present invention also provides a kit for adjusting splicing, comprising CX-4945 or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a method for treating a disease caused by a spl i cing defect comprising administering CX-4945 or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the present invention also provides a method for preventing a disease caused by a splicing defect comprising administering CX-4945 or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the present invention also provides the use of a pharmaceutical composition for the prevention or treatment of diseases caused by splicing defects containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention is the use of splicing "modulators (spl icing regulator) containing acceptable salt CX-4945 or a pharmaceutically acceptable thereof as an active ingredient to provide.
  • the present invention also provides the use of a splicing control kit, comprising CX-4945 or a pharmaceutically acceptable salt thereof.
  • [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid, Silmitasertib, CX-4945) is involved in the regulation of splicing, the splicing Modulation has a potent inhibitory effect on Cdc-2-like kinase (Cdc-2-like kinase, Clk), independent of the casein kinase 2, C 2 inhibitory effect, and a serine / arginine-rich protein ( Serin / arginine-rich (SR protein), the reduced phosphorylation level, the CX-4945 or a pharmaceutically acceptable salt thereof is useful as an active ingredient of the pharmaceutical composition for the treatment and prevention of splicing-related diseases Can be used.
  • the CX-4945 or a pharmaceutically acceptable salt thereof of the present invention can be usefully used in a splicing control method and a kit for controlling splicing for biological research related to splicing.
  • FIG. 3 is a schematic of splicing regulation of CX-4945 for CK2 a 'mRNA Indicates.
  • Figure 4 shows the splicing regulatory effect of CX-4945 on mRNA selected via exon array.
  • FIG. 6 shows a comparison of splicing patterns for mRNA selected from axon arrays of various CK2 inhibitors.
  • Figure 7 shows the SR protein phosphorylation regulatory effect of CX-4945.
  • Figure 9 shows the CX ⁇ 4945 through kinase profiling analysis
  • FIG. 10 shows the inhibitory effect of CX-4945 and the existing splicing inhibitor TG-003 against Clkl, Clk2, Clk3 and Clk4.
  • Figure 11 shows the inhibitory effect of CX-4945 on Clk under various ATP concentrations.
  • the invention relates to 5-[(3-chlorophenyl) amino] benzo
  • the present invention provides a pharmaceutical composition for preventing or treating a disease caused by a defect.
  • the invention also provides for the treatment of a disease caused by a splicing defect comprising administering CX-4945 or a pharmaceutically acceptable salt thereof to a subject in need thereof. Provide a method.
  • the present invention also provides a method for preventing a disease caused by a splicing defect comprising administering CX-4945 or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the present invention provides a use of the pharmaceutical composition for the prevention or treatment of diseases caused by a splicing defect containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the CX-4945 preferably has a structure represented by the following [Formula 1], but is not limited thereto;
  • the CX-4945 regulates selective splicing of mRNA, and specifically, inhibits splicing regulation kinase, but is not limited thereto.
  • the splicing regulatory kinase is preferably one or both of Cdc-2 like kinase (Cdc-2 ⁇ like kinase, Clk) or serine-arginine-rich protein kinase (SRPKs).
  • Cdc-2 ⁇ like kinase Cdc-2 ⁇ like kinase, Clk
  • SRPKs serine-arginine-rich protein kinase
  • the CX-4945 preferably regulates phosphorylation of serine-arginine-rich protein (Serin-arginine-rich, SR protein), but is not limited thereto.
  • the disease may include fibrinogenemia, propionic acidemia, neurofibromatosis, neurofibromatosis, ⁇ U ocular albinism type 1, Alzheimer's disease / FTDP-17 prefrontal lobe dementia (Alzheimer's disease / FTDP-17 Taupathies) and spinal muscular atrophy
  • any one of the above diseases is specifically caused by splicing defects for DMD, CFTR, FGB, PCCA, NF1, GRP143, MAPT and SMN genes.
  • the CX-4945 is preferably included at a concentration of 1 ⁇ or more, but is not limited thereto. At the concentration of 1 ⁇ or less, the splicing control effect is not significant.
  • CX-4945 in human embryonic kidney cell line (HEK293T) in order to confirm the effect of CX-4945 known as a CK2 inhibitor modulates splicing Of the CK2 ⁇ subunit (subimit) gene expression, CK2 a 'mRNA is spliced by CX-4945 (see Fig. 1 and 2), CK2 ⁇ ' mRNA number 6 It was confirmed that the axon exhibited the removed form (FIG. 3).
  • CX-4945 caused the ELL2, SPAST, CTDSPL2, PRPSAP2, CPEB1, QRSLl, USP38, TRIP4, WDFY1 and ZCCHC2 genes. It was confirmed that the splicing of the mRNA is controlled to decrease the size of the mRNA (see FIG. 4).
  • CX-4945 to confirm whether the intracellular splicing regulation effect by CX-4945 is a result of CK2 inhibition of CX-4945.
  • CK2 inhibitors 4, 5,6, 7-tetrabromobenzotria, known as CK2 inhibitors
  • TBB 5,6, 7-tetrabromobenzotr i azole
  • TBCA tetrabromocinnamic acid
  • the inventors of the present invention through the phosphorylation and kinase profiling analysis of SR proteins phosphorylated by Clk, in order to confirm the specific splicing regulation mechanism of CX-4945, Cdc-2-like kinase (Cdc) -2-1 ike kinase, Clk) and SR protein kinase (ser ine / arginine yl r ich protein kinase,
  • CX-4945 of the present invention is involved in splicing regulation, and the splicing regulation is a potent inhibitor of Clk independently of the CK2 inhibitory effect.
  • CX-4945 or a pharmaceutically acceptable compound of the present invention because it exhibits a ⁇ 2 effect, not only reduces the phosphorylation level of SR protein, but also shows a significantly increased splicing control effect when compared to conventional splicing inhibitors.
  • Possible salts may be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating a disease caused by a splicing defect.
  • CX-4945 of the present invention can be used in the form of a pharmaceutically acceptable salt, and acid salts formed by pharmaceutically acceptable free acids are useful as salts.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and Obtained from non-toxic organic acids such as alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and Obtained from non-toxic organic acids such as alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • These pharmaceutically toxic salts include sulfate pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate monohydrogen phosphate, dihydrogen phosphate, metaphosphate pyrophosphate chloride, bromide ⁇ and iodide , Fluoride acetate, propionate, decanoate, caprylate, acrylate formate, isobutyrate, caprate, heptanoate, propiolate oxalate, malonate, succinate, suverate, sebacate, Fumarate maleate, butyne-1, 4-dioate, nucleic acid-1, 6-dioate, benzoate chlorobenzoate, methylbenzoate, dinitro benzoate hydroxybenzoate, hydroxybenzoate, phthalate, tere Phthalate Benzenesulfonane , Toluenesulfonate, chlorobenzenesulfonate
  • Methoxy invention acid addition salts are conventional methods, For example, if u i according - in the CX-4945 excess It can be prepared by dissolving in an aqueous acid solution and precipitating the salt with a hydrophobic organic solvent such as methanol, ethane, acetone or acetonitrile. The same amount of CX-4945 and acid or alcohol in water may be heated and then the mixture is evaporated to dryness or the precipitated salt may be produced by suction filtration. Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the compound salt at no cost, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).
  • a suitable negative salt eg, silver nitrate
  • CX-4945 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.
  • the addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving CX-4945 in a water-soluble organic solvent such as acetone, methanol, ethane, or acetonitrile and adding an excess of an organic acid or an inorganic acid. It can be prepared by adding an acidic aqueous solution of and then precipitating or crystallizing. This mixture can then be evaporated and dried to evaporate the solvent or excess acid to obtain additional salts or to precipitate prepared salts by suction filtration.
  • a water-soluble organic solvent such as acetone, methanol, ethane, or acetonitrile
  • composition of the present invention When the composition of the present invention is used as a medicament, a pharmaceutical composition containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated and administered in various oral or parenteral dosage forms as described below. It may be, but is not limited thereto.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations may contain diluted crabs (e.g., lactose, dex) It contains a trope, sucrose, manny, sorbetle, celrose and / or glycine, a lubricant (e.g. silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol).
  • a lubricant e.g. silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol.
  • Tablets It may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, natrium carboxymethylcellulose and / or polyvinylpyridine, and optionally with starch, agar, alginic acid or its sodium salt.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, natrium carboxymethylcellulose and / or polyvinylpyridine, and optionally with starch, agar, alginic acid or its sodium salt.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, natrium carboxymethylcellulose and / or polyvinylpyridine, and optionally with starch, agar, alginic acid or its sodium salt.
  • compositions containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration may be administered by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. It depends on how to do it.
  • the pharmaceutical composition containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient for mixing into a parenteral formulation is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, and the ampoule Or in vial unit dosage form: the composition is sterile and / or contains preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for osmotic pressure control, and other therapeutically valuable substances. It may be formulated according to a conventional method of mixing, granulating or coating.
  • the dosage of the composition of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and the degree of disease of the patient, generally based on an adult patient weighing 60 kg, 3 ⁇ 4 0.001-1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.
  • the present invention also provides a splicing regulator containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present 'invention provides the use of the splicing control agent containing acceptable salt CX-4945 or a pharmaceutically acceptable thereof as an active ingredient.
  • CX-4945 of the present invention is involved in splicing regulation, and the splicing regulation has a strong inhibitory effect on Clk independently of the CK2 inhibitory effect.
  • CB-4945 or a pharmaceutically acceptable salt thereof of the present invention indicates lb and shows a decreased phosphorylation level of SR protein, as well as a markedly increased splicing control effect when compared to existing splicing inhibitors.
  • Silver may be usefully used as an active ingredient of a splicing regulator.
  • the present invention provides a health food for the prevention and improvement of diseases caused by splicing defects containing CX-4945 as an active ingredient.
  • the disease is preferably one or more selected from the group consisting of fibrinogen deficiency, propionateemia, neurofibromatosis, ocular whiteness type 1, Alzheimer's disease / FTDP-17 prefrontal dementia and spinal muscular atrophy.
  • CX-4945 of the present invention is involved in the regulation of splicing, the splicing regulation exhibits a potent inhibitory effect on Clk independently of the CK2 inhibitory effect, not only shows a reduced phosphorylation level of SR protein, CX-4945 or a pharmaceutically acceptable salt thereof according to the present invention shows a significantly increased splicing control effect when compared to a splicing inhibitor, and is a health food for preventing and improving diseases caused by splicing defects. It can be usefully used as an active ingredient of. There is no particular limitation on the kind of food.
  • Examples of foods to which the substance may be added include drinks, meats, sausages, breads, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice creams including ice cream, various soups, Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes all the health functional foods in the usual sense.
  • CX-4945 or a pharmaceutically acceptable salt thereof of the present invention can be added to a food as it is or used with other food or food ingredients . It can be suitably used according to a conventional method.
  • the combined amount of the active ingredient can be suitably determined depending on the purpose of use (prevention or improvement).
  • the amount of the compound in the health food can be added at 0.1 to 90 ' parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • the health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound of the present invention as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as polysaccharides, for example, conventional sugars such as textine, cyclodextrin, and the like, and xylyl, sorbitol, and erythritol.
  • natural flavoring agents such as tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 compositions of the present invention.
  • CX-4945 or a pharmaceutically acceptable salt thereof of the present invention may be various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, coloring agents and neutralizing agents (such as cheese and chocolate). , Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.
  • the CX-4945 or a pharmaceutically acceptable salt thereof of the present invention may contain a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination.
  • the proportion of such additives is not so critical but is generally selected in the range of from 0.01 to about 20 parts by weight per 100 parts by weight of CX-4945 or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a method for controlling splicing, comprising treating CX-4945 or a pharmaceutically acceptable salt thereof to the isolated cell.
  • the present invention also provides a kit for controlling splicing, comprising CX-4945 or a pharmaceutically acceptable salt thereof.
  • kits for controlling splicing comprising CX-4945 or a pharmaceutically acceptable salt thereof.
  • the CX-4945 preferably has a structure represented by the above [Formula 1], but is not limited thereto.
  • the CX-4945 regulates selective splicing of mRNA, and specifically, inhibits splicing regulatory kinase, but is not limited thereto.
  • the splicing regulatory kinase is preferably one or both of Clk and SRPKs, but is not limited thereto.
  • the CX-4945 preferably regulates phosphorylation of SR protein, but is not limited thereto.
  • the CX-4945 is preferably treated at a concentration of 1 ⁇ M or more, but is not limited thereto. In the case of 1 ⁇ M or less, the splicing control effect is not significant.
  • CX-4945 of the present invention is involved in the regulation of splicing, the splicing regulation exhibits a potent inhibitory effect on Clk independently of the CK2 inhibitory effect, not only shows a reduced phosphorylation level of SR protein, CX-4945, or a pharmaceutically acceptable salt thereof, of the present invention exhibits a significantly increased splicing control effect when compared to a splicing inhibitor. It can be usefully used in the kit for adjusting the splicing for this.
  • the present invention will be described in detail by way of examples.
  • CX-4945 (5-[(3-Chlorophenyl) amino] benzo [c] -2, 6-napht hy ridi ne-8 to c ar boxy 1 ic acid, Si lmitasert ib), known as a CK2 inhibitor
  • CK2 inhibitor 5-[(3-Chlorophenyl) amino] benzo [c] -2, 6-napht hy ridi ne-8 to c ar boxy 1 ic acid, Si lmitasert ib
  • HEK293T human embryonic kidney cell line
  • ATCC American Type Culture Collection
  • SPL 6-well culture plate
  • FBS fetal bovine serum
  • penicillin-streptomycin were inoculated in DMEM medium (Dulbeccc ⁇ s modified Eaglet medhium; Hyclon, USA) and incubated for 24 hours.
  • DMEM medium Dulbeccc ⁇ s modified Eaglet medhium; Hyclon, USA
  • PBS phosphate buffered saline
  • RNA of the cell was extracted and quantified according to the manufacturer's protocol, and then the omniscript kit (Qiagen, USA) was used.
  • CDNA was synthesized according to the manufacturer's protocol. Green master mix PCR (Promega, USA) 25 ⁇ , 10 ⁇ forward primer (SEQ ID NO: 1) 1, reverse primer (SEQ ID NO: 2) 1, using the synthesized cDNA as a sample.
  • glyceraldehyde 3-phosphate dehydrogenase As a control for comparing the expression levels, glyceraldehyde 3-phosphate dehydrogenase (Glycera l dehyde 3-) was used. Phosphate dehydrogenase (GAPDH) mRNA was amplified to confirm RNA expression in the same manner as above.
  • HEK293T cells were cultured in the same manner as in Example ⁇ 1 ⁇ 1> to treat 10 ⁇ CX-4945, and at 0, 1, 2, 3, 6, 12, and 24 hours after treatment. The cells were obtained. Then, RNA was extracted from ⁇ 293 ⁇ cells in the same manner as in Example ⁇ 1-1>, cDNA was prepared to confirm the expression of CK2 ⁇ 'mRNA, and sequencing was performed by sequencing CK2 ⁇ ' and The base sequence of CK2 ⁇ ' ⁇ was confirmed. The same method was performed using DMS0 as a negative control group, and GAPDH mRNA was amplified as a control for comparing the expression levels, and RNA expression was confirmed by the same method as above.
  • FIG. 2 and FIG. 3 CK2 at the initial stage of CX-4945 treatment. It was confirmed that the ⁇ 'mRNA was very quickly separated into CK2 ⁇ ' ⁇ mRNA and the CK2 ⁇ ' ⁇ band quantitatively increased with treatment time (FIG. 2), which indicates that the CK2 ⁇ ' ⁇ band was spliced by CX-4945. Due to the effect, it was confirmed that the exon 6 of the CK2 ⁇ 'mRNA exhibited the removed form (FIG. 3).
  • CX-4945 is used to regulate splicing of other precursors -mRNAs (Precursor-mRNA, Pre-mRNA) other than CK2 ⁇ '.
  • Precursor-mRNA Pre-mRNA
  • exon array analysis was performed to select genes that are spliced by CX-4945.
  • HEK293T cells were cultured, treated with 10 ⁇ CX-4945, RNA was extracted and quantified, and the extracted RNA 1 was prepared according to the manufacturer's protocol.
  • Labeled with Affymetrix reagent Af fymetr ix reagent; Affymetrix, USA: After labeling, the labeled RNA was subjected to the same method as Example ⁇ 1-1> to obtain a single-labeled fragment of a fragment labeled with an end portion.
  • Example ⁇ 1-1> HEK293T cells were cultured to treat 10 ⁇ CX-4945, RNA was extracted, and cDNA was prepared. PCR using primers was performed in the same manner as in Example ⁇ 1-1> to determine the splicing pattern through mRNA expression of ELL2, SPAST, CTDSPL2, PRPSAP2, CPEB1, QRSLl, USP38, TRIP4, WDFY1 and ZCCHC2 genes. Each was confirmed. The same method was performed using DMS0 as a negative control.
  • PRPSAP2 forward 5 °-GGACGTGAACACCACCATCATG-3 ⁇ SEQ ID NO: 9
  • QRSL1 3 5,-CCAGGGACTCTACCACAGTACATGAA-3, SEQ ID NO: 13
  • ZCCHC2 forward 5 ′ GCAGCTA1TTTCAAGnCATCACAAGCT-3 s SEQ ID NO: 21
  • CK2 inhibitors for genes selected to undergo splicing regulation by CX-4945 to determine whether the intracellular splicing regulation effect by CX-4945 is a result of CX-4945 inhibition of CK2. The effect of splicing regulation of was confirmed.
  • Example ⁇ 1-1> 1 or 10 ⁇ CX-4945 was treated for 1 hour in HEK293T cells by performing the same method as in Example ⁇ 1-1>, and Westernble in the same manner as in Example ⁇ 3-1>. Routing was performed to use the phosphorylation level of the SR protein.
  • anti-phosphate-SR protein anti-phosphate-SR protein (ant i-phospho-SR protein ant ibody, 1H4; Millipore, USA) was used to confirm the phosphorylation level of SR protein.
  • anti-SRSFl As a control for comparing expression levels, anti-SRSFl, SRSF4, SRSF9 (Santa Cruz Biotech, USA) and anti—hnRNP AKant i-hnRNA Al, Gideon Dreyfuss, University of Pennsylvania, USA were used as primary antibodies. It was confirmed whether the expression in the same manner as above. Based on this, imaging analysis software ImageKNIA (USA) was used to quantify the phosphorylation levels of SRSF4, SRSF6, SRSF5, SRSF1 and SRSF3.
  • Positive control known as inhibitor of Clk (Z) -l- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazole-2-ylidene) propane-2-one ((Z) -1- (3-Eth 1-5 -methoxy-2 , 3 ⁇ di hydr Plumbingzo thiazol-2-y 1 i dene) pr opan-2-one, TG-003; Sigma, USA) treated with 10 or 50 ⁇ and treated with DMS0 as negative control By the same method as described above.
  • Example ⁇ 4-2> the same method as in Example ⁇ 4-2> was performed to react CX-4945 at different concentrations with purified human Clkl, Clk2, Clk3, and Clk4 proteins, followed by fluorescence-based immunoassay (fluorescence).
  • fluorescence-based immunoassay fluorescence-based immunoassay
  • kinase profiling assays using the Kinase Profiler service (provided by Millipore) using a C-based immunoassay to perform CX-4945 on Clkl, Clk2, Clk3 and C14k or The inhibitory effect of TG-003 was confirmed.
  • the IC 50 value of each kinase was measured to compare the inhibitory effect of CX-4945 or TG-003 on each kinase. As a result, as shown in FIG.
  • TG-003 showed IC 50 values of 18.3 nM, 91.6 nM, 2000 nM and 16.6 for Clkl, Clk2, Clk3 and C14k, respectively.
  • nM was shown (FIG. 10 and Table 5)
  • CX-4945 showed IC 50 of 3.3 nM, 2.9 nM, 66.8 nM, and 23.0 nM (FIG. 10 and Table 5).
  • CX-4945 The inhibitory effect of CX-4945 on CK2 is in an ATP-competitive manner. Therefore, to determine whether CX-4945 exhibits an inhibitory effect on Clk in an ATP-competitive manner, CX-4945 was tested under various ATP concentrations. The IC 50 value shown for Clk2 was measured.
  • Example ⁇ 4-3> the same method as in Example ⁇ 4-3> was performed to react ATP (5, 15, 45 and 135 ⁇ ) and CX-4945 at various concentrations with purified human Clk2 protein, and then In the same manner as in Example ⁇ 4-3>, the inhibitory effect of CX-4945 on Clk2 was confirmed by an IC 50 value.

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Abstract

La présente invention concerne un régulateur de l'épissage qui contient de l'acide 5-[(3-chlorophényl)amino]benzo[c]-2,6-naphtyridine-8-carboxylique, du silmitasertib, CX-4945 ou des sels pharmaceutiquement acceptables de ceux-ci en tant qu'ingrédient actif. Spécifiquement, CX-4945 de la présente invention est impliqué dans la régulation de l'épissage, et étant donné que la régulation de l'épissage présente un fort effet d'inhibition de la kinase de type Cdc-2 (Clk), indépendamment d'un effet inhibiteur de la caséine kinase 2 (CK2), et présente un niveau de phosphorylation réduit des protéines riches en sérine/arginine (protéines SR), CX-4945 ou les sels pharmaceutiquement acceptables de celui-ci peuvent être utiles en tant qu'ingrédient actif d'une composition pharmaceutique destinée à la prévention ou au traitement de maladies provoquées par les défauts d'épissage. En outre, CX-4945 ou les sels pharmaceutiquement acceptables de celui-ci de la présente invention peuvent être utiles dans un procédé de régulation de l'épissage et un kit de régulation de l'épissage pour la recherche biologique sur l'épissage.
PCT/KR2014/003514 2013-10-08 2014-04-22 Régulateur de l'épissage contenant cx-4945 en tant qu'ingrédient actif WO2015053452A1 (fr)

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KR1020140035000A KR101593595B1 (ko) 2013-10-08 2014-03-26 Ｃｘ－４９４５를 유효성분으로 함유하는 스플라이싱 조절제
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110212845A1 (en) * 2009-10-02 2011-09-01 Denis Drygin Biomarkers for predicting the sensitivity and response of protein kinase CK2-mediated diseases to CK2 Inhibitors
US20110305706A1 (en) * 2009-02-23 2011-12-15 Scott Thomas Brady Compositions and Methods for Treating a Disease Mediated by Soluble Oligomeric Amyloid Beta

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110305706A1 (en) * 2009-02-23 2011-12-15 Scott Thomas Brady Compositions and Methods for Treating a Disease Mediated by Soluble Oligomeric Amyloid Beta
US20110212845A1 (en) * 2009-10-02 2011-09-01 Denis Drygin Biomarkers for predicting the sensitivity and response of protein kinase CK2-mediated diseases to CK2 Inhibitors

Non-Patent Citations (3)

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Title
MYLONIS, I. ET AL.: "Protein kinase CK2 phosphorylates and activates the SR protein- specific kinase 1", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 301, no. 3, 14 February 2003 (2003-02-14), pages 650 - 656 *
PEREZ, D. I. ET AL.: "Protein kinases CK1 and CK2 as new targets for neurodegenerative diseases", MEDICINAL RESEARCH REVIEWS, vol. 31, no. 6, 1 December 2011 (2011-12-01), pages 924 - 954 *
SON, Y. H. ET AL.: "Pharmacokinetic characterization of CK2 inhibitor CX-4945", ARCHIVES OF PHARMACAL RESEARCH, vol. 36, no. 7, 31 July 2013 (2013-07-31), pages 840 - 845 *

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