KR20150041559A - A splicing regulator comprising CX-4945 as an active ingredient - Google Patents

Abstract

The present invention relates to a splicing regulator containing 5-[(3-Chlorophenyl)amino]benzo [c]-2,6-naphthyridine-8-carboxylic acid, Silmitasertib, CX-4945 or pharmaceutically acceptable salts thereof as an active ingredient. Specifically, CX-4945 of the present invention is involved in splicing regulation, and since the splicing regulation exhibits a strong inhibitory effect of Cdc-2-like kinase (Clk), independently of an inhibitory effect of casein kinase 2 (CK2), and exhibits a reduced phosphorylation level of Serin/arginine-rich proteins (SR proteins), CX-4945 or the pharmaceutically acceptable salts thereof can be useful as an active ingredient of a pharmaceutical composition for preventing or treating diseases caused by splicing defects. In addition, CX-4945 or the pharmaceutically acceptable salts thereof of the present invention can be useful in a splicing regulation method and a splicing regulation kit for biological research on splicing.

Description

A splicing regulator comprising CX-4945 as an active ingredient containing an active ingredient CX-

The present invention relates to a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid, Silmitasertib, CX-4945), or a pharmaceutically acceptable salt thereof, as an active ingredient, for the prophylaxis or treatment of diseases caused by splicing defects ≪ / RTI > The present invention also relates to a splicing control method using CX-4945 and a splicing control kit therefor for biological studies related to splicing.

Splicing removes an intron that does not have genetic information from a precursor-mRNA (Pre-mRNA), separates the exon having the genetic information only as a single polypeptide, It is an essential process for the regulation of gene expression in eukaryotes.

Alternative splicing is the process of generating mRNA from a combination of exons selected specifically from various combinations of exons of m-RNA, which allows the production of various forms of mRNA from a single gene It is known that various proteins are produced without divergence and serve to provide cell diversity. An overall study 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, an expressed gene can be translated into various proteins, each of which exhibits different functions.

Selective splicing depends on the type of development, tissue or cell type, the interaction of the cis-acting element on the pre-mRNA sequence and the trans-acting protein that binds to it Regulation (mith CW and Valcarcel J, Trends Biochem . Sci . 25 (8): 381-388,2000).

Recently, diseases related to abnormal splicing due to mutations or lacking in the factors involved in the regulation have been reported. Examples of diseases associated with splicing are shown in Table 1 below. Accordingly, there is an increasing demand for research on selective splicing and its control, and research and development of a therapeutic agent through this is also receiving attention in earnest. However, not only are very low molecular substances known as regulators of selective splicing, but their potency or selectivity is also a situation that needs to be improved.

Examples of splicing-related diseases Korean name Name of English relation
gene source  Fibrinogen deficiency  Afibrinogenemia FGB Davis RL et. al,
Hum Mutat 2009, 30: 221-227.
Propionic acidemia
Propionic acidemia PCCA Sanchez-Alcudia R et. al,
Mol Genet Metab 2011, 102: 134-138. PCCB Rincon et. al,
AmJ Hum Genet 2007, 81: 1262-1270  Neurofibromatosis  Neurofibromatosis NF1 Pros et. al,
Human Mutat 2009, 30: 454-462.  Type 1 eye albinism  Ocular albinism type 1 GRP143 Vetrini F et. al,
Hum Mutat 2006, 27: 420-426.
Alzheimer's disease /
FTDP-17
Frontal temporal dementia
Alzheimer's
disease / FTDP-17
Taupathies

MAPT Rigo F et. al,
Nat ChemBiol 2012, 8: 555-561;
Rodriguez-Martin T et. al,
HumMol Genet 2009, 18: 3266-3273;
Peacey E et. al,
Nucleic Acids Res 2012, 40: 9836-9849.

Spinal muscular atrophy
Spinal
muscular atrophy
SMN2 Rigo F et. al,
Nat ChemBiol 2012, 8: 555-561;
Fernandez Alanis E et. al,
Hum Mol Genet 2012, 21: 2389-2398;
Geib T et al, PLoSOne 2009, 4: e8204.

Serine / arginine-rich (SR protein) is a typical trans-acting protein and is very well conserved in advanced organisms. The protein contains one or two RNA-recognition motifs at the N-terminus and includes a serine and arginine-rich domain (Arginine / Serine- rich domains, RS domains) (Zahler AM et al Genes Dev. 6 (5): 837-847, 1992; Caceres JF and Krainer AR, EMBO J. 12 (12): 4715-4726, 1993). The physiological role of the SR protein has not been specifically reported, but the phosphorylation of the SR protein has been implicated in protein-protein, protein-RNA interactions, intercellular localization, intercellular trafficking or pre-mRNA Which is known to be involved in selective splicing.

Phosphorylation of SR proteins regulates the binding of pre-mRNA and SR proteins, phosphorylation facilitates the interaction of specific proteins, resulting in splicing and splicing of RNA and spliceosomes. In this process, hypofosphorylation or hyperphosphorylation of the phosphorylation level leads to abnormal splicing (Kanopka A , et al., Nature 393 (6681): 185-187. 1998).

In the splicing process, there are several kinases that phosphorylate the SR protein. Typical examples of the phosphorylation enzymes include Cdc-2-like kinase (Clk) and SR protein kinase / arginine-rich protein kinase, SRPKs). Clk is a dual-specificity kinase, known to have 44 isoforms, including Clk1, Clk2 and Clk3, and is reported to be involved in the regulation of selective splicing There is a bar (Colwill K , et al . EMBO J. 15 (2): 265-275, 1996), and other biological functions such as the signal pathway.

(Z) -1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one as a drug for controlling selective splicing (Muraki M , et & lt ; / RTI & gt ; et & lt ; RTI ID = 0.0 > al . J. Biol . Chem . 279 (23): 24246-24254, 2004; Fedorov O , et al . Chem . Biol . 18 (1): 67-76, 2001) and SRPK inhibitors including SRPIN340 (Fukuhara T , et al ., Proc . Natl . Acad . Sci . USA 103 (30): 11329-11333, 2006), the inhibitors have limited clinical application, since their efficacy and selectivity as therapeutic agents for biological and splice-related diseases have not been adequately demonstrated . Therefore, development of more selective and effective Clk and SRPK inhibitors is required.

8-carboxylic acid (5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- (CX-4945), which is a protein that is frequently found in human cancer cells and that regulates various cellular activities such as cell growth and auto-apoptosis, is known as casein kinase 2, CK2). ≪ / RTI > Adam Siddiqui-Jain et al. Have shown that CX-4945 exhibits activity as an optional inhibitor that can be bioavailable by oral administration and inhibits the proliferation of cancer cells by inhibiting the expression level of CK2?, A catalytic subunit of CK2 (Siddiqui-Jaine , et al . Cancer Res. 70 (24): 10288-10298, 2010). Currently, clinical studies are underway to use CX-4945 as a CK2 inhibitor, confirming the safety and pharmacodynamic properties of the first-line clinical trial, and identifying a randomized drug combination Additional studies are underway (Sean E, Cylene Pharmaceuticals. Accessed 22 Mar 2011). However, the effect of splicing control of CX-4945 is not known.

Accordingly, the present inventors have made efforts to develop an effective splicing control substance, and as a result, CX-4945, known as an inhibitor of casein kinase 2 (CK2), a protein overexpressed in cancer, is involved in splicing , Exhibits a strong inhibitory effect on Clk independently of the CK2 inhibitory effect and exhibits a reduced phosphorylation level of the SR protein. Thus, the CX-4945 is a pharmaceutical composition for the prevention or treatment of diseases caused by splice defects It can be used effectively as an active ingredient. Further, confirming that CX-4945 of the present invention is more effective than TG-003, which has been used as a Clk inhibitory substance, CX-4945 can be effectively used for splicing-related biological studies Thus completing the present invention.

It is an object of the present invention to provide a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-dihydroxybenzo [c] -2,6-naphthyridine-8- for the prevention or treatment of diseases caused by splicing defects containing, as an active ingredient, -naphthyridine-8-carboxylic acid, Silmitasertib, CX-4945 or a pharmaceutically acceptable salt thereof To provide a pharmaceutical composition.

In order to accomplish the above object, the present invention provides a process for producing 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- ] -2,6-naphthyridine-8-carboxylic acid, Silmitasertib, CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. Or a pharmaceutically acceptable salt thereof.

The present invention also provides a splicing regulator comprising CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health food for preventing and ameliorating 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 of splicing, comprising the step of treating isolated cells with CX-4945 or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a splicing control kit comprising CX-4945 or a pharmaceutically acceptable salt thereof.

(5 - [(3-Chlorophenyl) amino] benzo [c] -2,6-naphthyridine of the present invention is a 5 - [(3-chlorophenyl) amino] benzo [ -8-carboxylic acid, Silmitasertib, CX-4945) is involved in splicing regulation and the splicing control is associated with the inhibitory effect of casein kinase 2 (CK2) It exhibits a strong inhibitory effect on Cdc-2-like kinase (Clk) independently and exhibits a reduced phosphorylation level of the serine / arginine-rich (SR protein) , CX-4945 or a pharmaceutically acceptable salt thereof may be useful as an active ingredient of a pharmaceutical composition for treating and preventing splice-related diseases. In addition, CX-4945 or a pharmaceutically acceptable salt thereof of the present invention can be usefully used in a splicing control method and a splicing control kit therefor, for biological studies related to splicing.

Figure 1 is a schematic illustration of a method for preparing 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine Confirmation of the splicing modulating effect of CX-4945 on casein kinase 2 (CK2) mRNA according to the treatment concentration of -8-carboxylic acid, Silmitasertib, CX-4945 .
Figure 2 shows confirmation of splicing control effect of CX-4945 on CK2 mRNA according to the treatment time of CX-4945.
Figure 3 shows a schematic diagram of splicing control of CX-4945 to CK2 mRNA.
FIG. 4 shows the splicing control effect of CX-4945 on mRNA selected through an exon array.
Figure 5 shows the CK2 inhibitory effect of various CK2 inhibitors.
Figure 6 shows a comparison of the splicing pattern for CK2 mRNA of various CK2 inhibitors.
Figure 7 shows the effect of CX-4945 on SR protein phosphorylation modulation.
Figure 8 shows a quantitative analysis of the SR protein phosphorylation modulating effect of CX-4945.
FIG. 9 shows the inhibitory effect of Cdc-2-like kinase (Clk) and SR protein kinase (serine / arginine-rich protein kinase, SRPKs) of CX-4945 through kinase profiling analysis .
Figure 10 shows the inhibitory effect of CX-4945 on Clk1, Clk2, Clk3 and Clk4 and TG-003, an existing splice inhibitor.
Figure 11 shows the inhibitory effect of CX-4945 on Clk under various ATP concentrations.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid, Silmitasertib, CX-4945), or a pharmaceutically acceptable salt thereof, as an active ingredient, for the prophylaxis or treatment of diseases caused by splicing defects Lt; / RTI >

The CX-4945 preferably has a structure represented by the following formula (1), but is not limited thereto.

The CX-4945 regulates the alternative splicing of mRNA and specifically inhibits the splicing regulation kinase. However, it is not limited thereto.

Preferably, the splicing control kinase is one or both of a Cdc-2-like kinase (Clk) or a serine / arginine-rich protein kinase (SRPKs) But is not limited thereto.

The CX-4945 preferably controls the phosphorylation of a serine-arginine-rich protein (SR protein), but is not limited thereto.

The diseases are selected from the group consisting of fibrinogenemia, propionic acidemia, neurofibromatosis, Ocular albinism type 1, Alzheimer's disease / FTDP-17 Alzheimer's disease / FTDP- 17 Taupathies, and spinal muscular atrophy. However, the diseases are not limited to DMD, CFTR, FGB, PCCA, NF1, GRP143, MAPT, It is caused by a splicing defect to the SMN gene.

It is preferable that CX-4945 is contained at a concentration of 1 μM or more, but it is not limited thereto, and the concentration of CX-4945 is not effective because it does not significantly exhibit splicing control effect at a concentration of 1 μM or less.

In a specific embodiment of the present invention, the present inventors have identified CX-4945 in human embryonic kidney cell line (HEK293T) to confirm the effect that CX-4945, known as CK2 inhibitor, (Fig. 1 and Fig. 2), CK2 mRNA was spliced by CX-4945 (see Fig. 1 and Fig. 2), and CK2 ' (exon) was removed (Fig. 3).

In order to confirm whether CX-4945 affects the splicing control of other precursor-mRNA (Pre-mRNA) other than CK2? 'Within the cell, the present inventors performed an exon array analysis . The results showed that 39,001 exons were spliced by CX-4945 more than twice as much as the spliced control by CX-4945. Accordingly, the confirming the splicing modulation of CX-4945 Effect of some of the selected one exon result, due to the C X-4945 ELL2, SPAST, CTDSPL2, PRPSAP2, CPEB1, QRSL1, USP38, TRIP4, WDFY1 and ZCCHC2 gene (Fig. 4). As shown in Fig.

In addition, the present inventors have found that the effect of CX-4945 on intracellular splicing is affected by splicing by CX-4945 in order to determine whether the effect of CX-4945 is the result of inhibition of CK2. (CX-4945), 4,5,6,7-tetrabromobenzotriazole (TBB), which are known as CK2 inhibitors, have been shown to inhibit the splicing of other CK2 inhibitors. And Tetrabromocinnamic acid (TBCA) did not show dephosphorylation to S129 of AKT, which is a substrate of CK2, but significantly inhibited the activity of CK2 (see FIG. 5) It was confirmed that the splicing control effect of CX-4945 was an effect that is independent of CK2 (see FIG. 6).

In order to confirm the specific splicing control mechanism of CX-4945, the present inventors used Cdc-2-like kinase (Cdc-2) through phosphorylation and kinase profiling analysis of SR protein phosphorylated by Clk (Z) -l- (3-ethyl-4-methylpiperidin-4-yl) -propan-1-ol, which is known to be an inhibitor of Clk, Methoxy-2,3-dihydrobenzothiazol-2-ylidene) -propan-2 (see FIG. 7 and FIG. 8), and Clk1, SRSF4, SRSF6, SRSF5 and SRSF1 hyperphosphorylated by CX-4945 in the same manner as in the case of TK-propan-2- Clk2 and Clk3 phosphorylase showed an IC ₅₀ concentration of 3 to 90 nM, indicating that the phosphorylated enzyme was significantly inhibited (see FIG. 9 and Table 4).

In addition, the present inventors confirmed the inhibitory effect on Clk1, Clk2, Clk3 and Cl4k represented by CX-4945 or TG-003. As a result, CX-4945 of the present invention was compared with TG-003 which is conventionally used as a splicing inhibitor And 5 to 30 times higher Clk inhibitory activity (see FIG. 10 and Table 5). In addition, it was confirmed that CX-4945 exhibits an inhibitory activity against Clk in an ATP competitive manner (Fig. 11).

Thus, the CX-4945 of the present invention is involved in splicing control and the splicing control exhibits a strong inhibitory effect on Clk independent of the CK2 inhibitory effect and not only a reduced phosphorylation level of the SR protein CX-4945 or a pharmaceutically acceptable salt thereof of the present invention is useful for the prevention or treatment of diseases caused by splice defects, since it exhibits a remarkably increased splicing control effect when compared to conventional splicing inhibitors And can be usefully used as an active ingredient of a pharmaceutical composition.

The CX-4945 of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention may be prepared by dissolving CX-4945 in an excess amount of an acid aqueous solution and dissolving it in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile Followed by precipitation. It may be prepared by heating an equal amount of CX-4945 and an acid or alcohol in water, followed by evaporating and drying the mixture, or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, CX-4945 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which 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-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile or the like, adding an excessive amount of an organic acid, Followed by precipitation or crystallization with an aqueous acid solution. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated into various oral or parenteral dosage forms at the time of clinical administration and administered But is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing CX-4945 of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be administered parenterally, and parenteral administration can be carried out by subcutaneous injection, intravenous injection, intramuscular injection, And the like. In this case, in order to formulate the composition for parenteral administration, a pharmaceutical composition containing CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient is mixed with water together with a stabilizer or a buffer to prepare a solution or suspension, Or vial unit dosage forms. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the composition of the present invention to the human body may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient. When the patient is 60 kg body weight, And may be 0.01 to 500 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 comprising CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.

CX-4945 of the present invention is involved in splicing control, and the splicing control exhibits a strong inhibitory effect on Clk independently of the CK2 inhibitory effect and not only shows a reduced phosphorylation level of the SR protein, CX-4945 or a pharmaceutically acceptable salt thereof of the present invention can be usefully used as an active ingredient of a splicing control agent, since it exhibits a remarkably increased splicing control effect when compared with a splicing inhibitor.

In addition, the present invention provides a health food for 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, propionic acidemia, neurofibromatosis, eye albinism type 1, Alzheimer's disease / FTDP-17 frontal temporal dementia and spinal muscular atrophy.

CX-4945 of the present invention is involved in splicing control, and the splicing control exhibits a strong inhibitory effect on Clk independently of the CK2 inhibitory effect and not only shows a reduced phosphorylation level of the SR protein, CX-4945 < / RTI > or a pharmaceutically acceptable salt thereof of the present invention is useful as a medicament for preventing and ameliorating diseases caused by splice defects, as it exhibits a significantly increased splicing control effect when compared to splicing inhibitors Can be usefully used as an active ingredient.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The CX-4945 of the present invention or a pharmaceutically acceptable salt thereof may be added to the food as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients except that it contains the compound of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, CX-4945 of the present invention or a pharmaceutically acceptable salt thereof may be used as a flavoring agent, a coloring agent and a thickening agent (cheese, chocolate, etc.) such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, , A pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, CX-4945 or pharmaceutically acceptable salts thereof of the present invention may contain flesh for the production of natural fruit juice and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of CX-4945 of the present invention or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of splicing control, comprising the step of treating a separate cell with CX-4945 or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a splicing control kit comprising CX-4945 or a pharmaceutically acceptable salt thereof.

The CX-4945 preferably has a structure represented by the formula (1), but is not limited thereto.

The CX-4945 regulates the selective splicing of mRNA and specifically inhibits the splicing control kinase, but is not limited thereto.

The splicing control kinase is preferably, but not limited to, Clk or SRPKs.

The CX-4945 preferably controls the phosphorylation of the SR protein, but is not limited thereto.

The above-mentioned CX-4945 is preferably treated at a concentration of 1 μM or more, but it is not limited thereto. The concentration of CX-4945 at 1 μM or less is not effective since the effect of splicing is not significantly exhibited.

CX-4945 of the present invention is involved in splicing control, and the splicing control exhibits a strong inhibitory effect on Clk independently of the CK2 inhibitory effect and not only shows a reduced phosphorylation level of the SR protein, CX-4945 < / RTI > or a pharmaceutically acceptable salt thereof of the present invention can be used for biological studies related to splicing, as well as for splicing control methods And a splicing control kit therefor.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Casein Kinase  2( casein kinase  2, CK2 ) For the gene CX Splicing of -4945 ( splicing ) Checking the modulating effect

&Lt; 1-1 > CK2 mRNA For CX -4945 of Splicing  Confirm adjustment effect

To confirm the effect of CX-4945 (5 - [(3-Chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid, Silmitasertib, The effect of CX-4945 on the expression of the subunit gene of CK2 was confirmed in HEK293T cells.

Specifically, 5 × 10 ⁵ human embryonic kidney cell lines (HEK293T; American Type Culture Collection (ATCC)) per well were placed in a 6-well culture plate (SPL) (Dulbecco's modified Eagle's medium (Hyclon, USA) containing 1% bovine serum (FBS) and penicillin-streptomycin (1% penicillin-streptomycin) for 24 hours. When the cells grew to 90%, the culture medium of each well was removed and washed once with phosphate buffered saline (PBS) solution, followed by addition of 1 or 10 μM of CX-4945 (Celec Chemical Co., USA) And 1 ml of DMEM medium was added thereto. The cells were cultured in a CO ₂ incubator (VS-9108MS, Vision, Korea) for 3 hours at 37 ° C and 5% carbon dioxide to treat CX-4945. Then, the RNA of the cell was extracted and quantified according to the protocol of the manufacturer using a triazol reagent (Invitrogen, USA), and the RNA was extracted from the cells using an omniscript kit (Quiagen Inc., USA) And cDNA was synthesized according to the manufacturer's protocol. 25 μl of green master mix PCR (Promega, USA), 1 μl of a 10 pM forward primer (SEQ ID NO: 1), 1 μl of a reverse primer (SEQ ID NO: 2) 1 Denatured at 94 ° C for 3 minutes, and subjected to denaturation at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 45 seconds to 35 ° C. And then reacted at 72 ° C. for 7 minutes to amplify the cDNA. Then, electrophoresis was performed to confirm the expression of CK2 α 'mRNA. The sequences of the primers are shown in Table 2 below. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control group for comparison of expression levels, and the same method as above was performed using dimethyl sulfoxide (DMSO) as a negative control. mRNA was amplified and RNA expression was confirmed by the same method as above.

Primer sequence for amplification of CK2 &amp;alpha; cDNA Name of the primer order SEQ ID NO: CK2 A 'Forward 5'-CAACTCTACCAGATCCTGACAGACTTTGATA-3 ' SEQ ID NO: 1 CK2 A 'Reverse 5'-CTGATAGTCCACGAGGAGCTCTGGT-3 ' SEQ ID NO: 2

As a result, as shown in Fig. 1, when HEK293T cells were treated with 10 [mu] M of CX-4945, it was confirmed that CK2 [alpha] [Delta] band, which is an mRNA having a shorter length than that of CK2 alpha 'mRNA (FIG.

<1-2> Time dependent CX -4945 of Splicing  Confirm adjustment effect

To confirm the effect that CX-4945 regulates the splicing of CK2 alpha 'mRNA, we confirmed the effect of CX-4945 on the splicing of CK2 alpha' mRNA over time.

Specifically, HEK293T cells were cultured in the same manner as in Example <1-1>, and treated with 10 μM of CX-4945, and treated at 0, 1, 2, 3, 6, 12, Cells were obtained. Then, RNA of HEK293T cells was extracted and cDNA was prepared to confirm the expression of CK2? 'MRNA in the same manner as in the above Example <1-1>, followed by sequencing analysis to find that CK2?' The nucleotide sequence of CK2? '? Was confirmed. The same method as above was performed using DMSO as a negative control, and GAPDH mRNA was amplified as a control group for comparison of expression levels, and RNA expression was confirmed by the same method as described above.

As a result, as shown in FIG. 2 and FIG. 3, it was confirmed that the CK2? 'MRNA at the early stage of CX-4945 treatment was rapidly separated into CK2?'? MRNA and quantitatively increased the CK2? (Fig. 2), confirming that the CK2? '? Band represents a form in which the exon 6 of CK2?' MRNA is deleted due to the splicing effect of CX-4945 (Fig. 3).

< Example  2> intracellular CX -4945 of Splicing  Confirm adjustment effect

<2-1> CX By -4945 Splicing  Selection of Regulatory Gene screening )

In order to confirm whether CX-4945 affects the splicing control of other precursor-mRNAs (Precursor-mRNA, Pre-mRNA) other than CK2α 'within the cells, exon array analysis was performed to determine whether CX-4945 Were selected for the splicing control.

Specifically, HEK293T cells were cultured in the same manner as in Example <1-1>, treated with 10 μM of CX-4945, extracted and quantified, and then 1 μg of the extracted RNA was subjected to the protocol of the manufacturer And labeled with Affymetrix reagent (Affymetrix, USA). After the labeling, the labeled RNA was synthesized as a fragment-end-labeled single-stranded cDNA fragment having an end portion by performing the same method as in Example <1-1> The Affymetrix Expression Console Software program was used to perform hybridization on the Metrix-gene chip human exon 1.0 ST assay (Affymetrix-GeneChip Human Exon 1.0 ST arrays; Affymetrix, USA) ) To perform an exon array. The results of the exon array were analyzed using Gene Spring 12.6 (GeneSpring 12.6; Agilent, USA) containing GX. As a negative control for comparing the transcription level of HEK293T cells, HEK293T cells not treated with CX-4945 were cultured for 12 hours, and then exon array analysis was performed by the same method as above.

As a result, it was confirmed that when CX-4945 was treated, the level of change due to the alternative splicing was four times higher than that of the transcript indicating the stability of the mRNA. A total of 39,001 exons were found to be more than twice as spliced by CX-4945, of which 7,277 exons increase the size of the gene by the addition of exons, and 31,724 exons exon And the size of the gene was decreased.

<2-2> CX By -4945 Splicing  Identification of regulatory genes

In order to confirm the splicing control effect of CX-4945 on the selected genes in the exon array analysis, the splicing control effect of CX-4945 on some of the genes selected in Example <2-1> Respectively.

Specifically, HEK293T cells were cultured in the same manner as in Example <1-1>, and 10 μM of CX-4945 was treated to extract RNA, and cDNA was prepared. PCR using the primers was carried out in the same manner as in Example <1-1>, and splicing patterns were determined by the mRNA expression of ELL2, SPAST, CTDSPL2, PRPSAP2, CPEB1, QRSL1, USP38, TRIP4, WDFY1 and ZCCHC2 genes Respectively. The same procedure as described above was performed using DMSO as a negative control.

Primer sequences for ELL2, SPAST, CTDSPL2, PRPSAP2, CPEBl, QRSLl, USP38, TRIP4, WDFYl and ZCCHC2 cDNA amplifications Name of the primer order SEQ ID NO: ELL2
Forward 5`- CTAAATCCGTCTCAGAATGCTGCAG-3` SEQ ID NO: 3 Reverse 5`- TAATCTGGGAGTTCAATTGCTGAAGG-3` SEQ ID NO: 4 SPAST
 Forward 5`- CCCCTTAACACACACTAGTAATTCACTGC-3` SEQ ID NO: 5 Reverse 5`- CTCAGGCCTCAGAGAAGGAAGAATAAC-3` SEQ ID NO: 6 CTDSPL2
 Forward 5`- CCTTCAAAACGGAGTAGAATTGAACG-3` SEQ ID NO: 7 Reverse 5`- CTGTAAGGGGTGGGATATCACGATT-3` SEQ ID NO: 8 PRPSAP2
 Forward 5`- GGACGTGAACACCACCATCATG-3` SEQ ID NO: 9 Reverse 5`- TTCGCCGAGGCTGGAGACTT-3` SEQ ID NO: 10 CPEB1
 Forward 5`- CCTTCCTGTCTCTGTCAGGGGG-3` SEQ ID NO: 11 Reverse 5`- TAATCTGGGAGTTCAATTGCTGAAGG-3` SEQ ID NO: 12 QRSL1
 Forward 5`- CCAGGGACTCTACCACAGTACATGAA-3` SEQ ID NO: 13 Reverse 5`- GTTTCCTGAGAGAATTCTTCCTCTCACC-3` SEQ ID NO: 14 TRIP4
Forward 5`- TCGAAGGACCCAAGTCATTGATG-3` SEQ ID NO: 15 Reverse 5`- CTTTTAATCCCTCTGACAAGCAGAGAAG-3` SEQ ID NO: 16 WDFY1
Forward 5`- GCTCCTCAGTGGTTGGAAAGTGAT-3` SEQ ID NO: 17 Reverse 5`- CTTTACAATGCGGTCGGTCCC-3` SEQ ID NO: 18 USP38
Forward 5`- CAGTGTTATACAAGCCTTGTTTATGGCC-3` SEQ ID NO: 19 Reverse 5`- GCGAAAGATCTGTAAAGGCTTCCAC-3` SEQ ID NO: 20 ZCCHC2
Forward 5`- GCAGCTATTTTCAAGTTCATCACAAGCT-3` SEQ ID NO: 21 Reverse 5`-TTCACAATGGGACTTTTCCCAGTATG-3` SEQ ID NO: 22

As a result, as shown in FIG. 4, it was confirmed that the mRNA of ELL2, SPAST, CTDSPL2, PRPSAP2, CPEB1, QRSL1, USP38, TRIP4, WDFY1 and ZCCHC2 was spliced by CX- , Which showed the same pattern as the exon array analysis (Fig. 4).

< Example  3> intracellular CX -4945 of CK2 Independent Splicing  Confirm adjustment effect

<3-1> Various CK2  Inhibitor CK2  Confirmation of inhibition

In order to confirm whether the effect of CX-4945 regulating intracellular splicing was the result of CK2 inhibition of CX-4945, another inhibitor of CK2 was added to the cells and the 29th amino acid of AKT, The inhibitory effect of CK2 was confirmed by the presence of dephosphorylation of serine and S129.

In detail, 10 μM CX-4945, 100 μM 4,5,6,7-tetrabromobenzotriazole (TBB; (Sigma, USA) or 100 μM Tetrabromocinnamic acid (TBCA; Calbiochem, USA) were cultured in HEK293T cells and cultured. Cells were obtained and disrupted to obtain the whole protein of HEK293T cells Cell extracts were obtained. Then, the whole protein was developed by SDS-PAGE and transferred to a polyvinylidene fluoride transfer membrane (GE Healthcare, USA) of 0.45 μm in size, and the mixture was transferred to 5% skim milk After blocking, anti-AKT antibody (Cell Signaling Co., USA) and anti-pAKT (s129) antibody (Abcam Company, USA) were infected with primary antibody tris buffer saline Tween-20 buffered saline tween-20, TBST), and treated with the blocking membrane for overnight. Then, the cells were washed with TBST four times for 10 minutes each. Then, anti-rabbit anti-rabbit antibody (goat anti-rabbit IgG-HRP) conjugated with a horseradish peroxidase derived from a goat as a secondary antibody (Santa Cruz, USA) diluted 1: 2000 was added and reacted with anti-mouse antibody (goat anti-mouse IgG-HRP; . After the reaction, the cells were rinsed with TBST four times for 10 minutes each, and the proteins on the moving membrane were washed with WEST-ZOL plus western blotting detection system (Intron Biotechnology Co., USA) and LAS- 4,000 image analyzer (LAS-4000 Image analyzer, Fuji Film, Japan) was used to detect the phosphorylation level of the protein. The same procedure as described above was performed using DMSO as a negative control. Expression of hnRNPA1 was confirmed by the same method as above using anti-hNRNP A1 (anti-hnRNA A1, Gideon Dreyfuss, University of Pennsylvania, USA) as a primary antibody for comparison of expression levels.

As a result, it was confirmed that the activity of CK2 was significantly inhibited by CX-4945, TBB and TBCA as shown in FIG. 5, and dephosphorylation was not observed in S129 of AKT (FIG. 5).

<3-2> CX -4945 of CK2 Independent Splicing  Confirm adjustment effect

To determine whether the intracellular splicing control effect of CX-4945 was a consequence of CK2 inhibition of CX-4945, the effect of splicing control by CX-4945 on other CK2 inhibitors The effect of splicing was confirmed.

Specifically, cells were treated with CX-4945, TBB, and TBCA in the same manner as in Example <1-1>, and then treated with ELL2, CPEB1, PRPSAP2, and QRSL1 genes in the same manner as in Example < The splicing pattern was confirmed by mRNA expression. The same procedure as described above was performed using DMSO as a negative control.

As a result, as shown in FIG. 6, ELL2, CPEB1, PRPSAP2 and QRSL1 genes were regulated by CX-4945 to show a splicing pattern, whereas TBB and TBCA did not show a splicing control effect on the gene , And that splicing control effect of CX-4945 was an effect that is independent of CK2 (Fig. 6).

< Example  4> Cdc -2 similar Kinase ( Cdc -2- like kinase , Clk ) And SR  Protein kinase ( serine / arginine - rich protein kinase , SRPKs ) For CX -4945 of Splicing  Confirm control mechanism

<4-1> CX -4945 of SR  Identification of protein phosphorylation modulation effect

In order to confirm the specific splicing control mechanism of CX-4945, Clk inhibitory effect of CX-4945 was confirmed by phosphorylation of SR protein phosphorylated by Clk.

Specifically, 1 or 10 μM of CX-4945 was treated for 1 hour in HEK293T cells by performing the same method as in Example <1-1>, and then Western blotting was performed in the same manner as in Example <3-1> Was performed to use the phosphorylation level of the SR protein. Phosphorylation level of SR protein was confirmed using anti-phospho-SR protein antibody (1H4; Millipore, USA) as the primary antibody. (Anti-hnRNA A1, anti-hnRNA A1, Gideon Dreyfuss, University of Pennsylvania, USA) as a primary antibody for the comparison of the expression levels, anti-SRSF1, SRSF4, SRSF9 (Santa Cruz Bioscience, USA) Was used to confirm the expression in the same manner as described above. Based on this, the phosphorylation levels of SRSF4, SRSF6, SRSF5, SRSF1, and SRSF3 were quantified using image analysis software, ImageJ (NIA, USA). (Z) -1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (Z) -1- (TG-003; Sigma, USA) was treated with 10 or 50 [mu] M as a negative control, DMSO And the same method as described above was performed.

As a result, it was confirmed that the dephosphorylation of SSRSF4, SRSF6, SRSF5 and SRSF1 and the hyperphosphorylation of SRSF6 were shown by CX-4945 as shown in Fig. 7 and Fig. 8, indicating that TG -003, indicating that CX-4945 exhibited a Clk inhibitory effect (FIGS. 7 and 8).

<4-2> CX -4945 of Clk  And SRPKs  Confirmation of inhibition

In order to confirm the specific splicing control mechanism of CX-4945, kinase profiling analysis was performed to directly confirm the inhibitory effect of CX-4945 on Clk and SRPKs.

Specifically, various concentrations of CX-4945 were reacted with the purified purified human Clk1, Clk2, Clk3, Clk4, SRPK1 and SRPK2 proteins, followed by fluorescence-based immunoassay and radioactive- kinase profiling analysis was performed using a kinase profiler service (Invitrogen, Millipore) using a radiometric filter-binding assay to detect Clk1, Clk2, Clk3, The inhibitory effect of CX-4945 on SRPK1 and SRPK2 was assessed by measuring the CX-4945 concentration of 0.001, 0.01, 0.1, 1 and 10 μM, and the ATP concentration (ATP concentration equal to Km for ATP) Lt; RTI ID = 0.0 &gt; IC50 &lt; _{/ RTI} &gt; And the inhibitory effect of CX-4945 on each phosphorylase was compared. The inhibitory effect of CX-4945 on CK2? As a control group was confirmed in the same manner as described above.

As a result, as shown in Fig. 9 and the following Table 4, CX-4945 exhibited an IC ₅₀ concentration of 3 to 90 nM on Clk1, Clk2, Clk3 phosphorylase and showed inhibitory effect on the phosphorylated enzyme And showed an IC ₅₀ of 3.8 nM for Clk2, which was the most inhibitory effect, which was confirmed to be much more significant than when compared with the known CK2α inhibitory effect (IC ₅₀ = 14.7 nM) (FIG. 9 and Table 4).

Identification of the inhibitory effect of CX-4945 on Clk1, Clk2, Clk3, SRPK1 and SRPK2 phosphorylase Phosphorylase name IC ₅₀ (nM) Clk1   82.3 Clk2    3.8 Clk3   90.0 SRPK1 1229.0 SRPK2 1111.0 CL2 alpha   14.7

<4-3> Existing Clk  Inhibitors and CX -4945 of SR  Comparison of Protein Phosphorylation Modulating Effect

In order to compare the splicing control effect of CX-4945 of the present invention, the Clk inhibitory effects of TG-003 and CS-4945, which are drugs used as conventional Clk inhibitors, were compared.

Specifically, CX-4945 at various concentrations was reacted with purified human Clk1, Clk2, Clk3 and Clk4 proteins by the same method as in Example <1-1>, and then fluorescence-based immunoassay Kinase profiling analysis was performed using a Kinase Profiler service (Millipore) using a -based immunoassay to detect CX-4945 for Clk1, Clk2, Clk3 and Cl4k, or Inhibitory effect of TG-003 was confirmed. Then, IC ₅₀ indicates the respective kinases Were measured to compare the inhibitory effects of CX-4945 or TG-003 on the respective phosphorylated enzymes.

As a result, as shown in FIG. 10 and the following Table 5, TG-003 showed IC ₅₀ values of 18.3 nM, 91.6 nM, 2000 nM and 16.6 nM for Clk1, Clk2, Clk3 and Cl4k, respectively And [Table 5]), and CX-4945 showed an IC ₅₀ of 3.3 nM, 2.9 nM, 66.8 nM and 23.0 nM (FIG. 10 and [Table 5]). These results indicate that CX-4945 of the present invention shows 5 to 30 times higher inhibition of Clk than TG-003, which is conventionally used as a splicing inhibitor (FIG. 10 and [Table 5]).

Identification of Clk1, Clk2, Clk3 and SRPK1 and SRPK2 phosphorylase inhibitory effects of CX-4943 and TG-003 Phosphorylase name IC ₅₀ (nM) CX -4945 TG-003 Clk1  3.3    18.3 Clk2  2.9    91.6 Clk3 66.8 > 2000.0 Clk4 23.0    16.6

<4-4> CX -4945 of Clk  For the enzyme ATP  Confirming competitive inhibition

CX-4945 exhibits an inhibitory effect on CK2 in an ATP competitive manner, so that CX-4945 undergoes a competitive inhibition with Clk against ATP, The IC ₅₀ value for Clk2 was measured.

Specifically, ATP (5, 15, 45 and 135 μM) and CX-4945 were reacted with purified purified human Clk2 protein in the same manner as in Example <4-3> The inhibitory effect of CX-4945 on Clk2 was confirmed by the IC ₅₀ value in the same manner as in Example <4-3>.

As a result, as shown in FIG. 11, it was confirmed that CX-4945 inhibited Clk2 enzyme in an ATP competitive manner (FIG. 11).

<110> Korea Research Institute of Bioscience and Biotechnology <120> A splicing regulator comprising CX-4945 as an active ingredient <130> 14P-03-003 <150> 10-2013-0120022 <151> 2013-10-08 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CK2A'_F <400> 1 caactctacc agatcctgac agactttgat a 31 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CK2A'_R <400> 2 ctgatagtcc acgaggagct ctggt 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ELL2_F <400> 3 ctaaatccgt ctcagaatgc tgcag 25 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ELL2_R <400> 4 taatctggga gttcaattgc tgaagg 26 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SPAST_F <400> 5 ccccttaaca cacactagta attcactgc 29 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SPAST_R <400> 6 ctcaggcctc agagaaggaa gaataac 27 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CTDSPL2_F <400> 7 ccttcaaaac ggagtagaat tgaacg 26 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CTDSPL2_R <400> 8 ctgtaagggg tgggatatca cgatt 25 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PRPSAP2_F <400> 9 ggacgtgaac accaccatca tg 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PRPSAP2_R <400> 10 ttcgccgagg ctggagactt 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CPEB1_F <400> 11 ccttcctgtc tctgtcaggg gg 22 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CPEB1_R <400> 12 taatctggga gttcaattgc tgaagg 26 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> QRSL1_F <400> 13 ccagggactc taccacagta catgaa 26 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> QRSL1_R <400> 14 gtttcctgag agaattcttc ctctcacc 28 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TRIP4_F <400> 15 tcgaaggacc caagtcattg atg 23 <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TRIP4_R <400> 16 cttttaatcc ctctgacaag cagagaag 28 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> WDFY1_F <400> 17 gctcctcagt ggttggaaag tgat 24 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> WDFY1_R <400> 18 ctttacaatg cggtcggtcc c 21 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> USP38_F <400> 19 cagtgttata caagccttgt ttatggcc 28 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> USP38_R <400> 20 gcgaaagatc tgtaaaggct tccac 25 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> ZCCHC2_F <400> 21 gcagctattt tcaagttcat cacaagct 28 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ZCCHC2_R <400> 22 ttcacaatgg gacttttccc agtatg 26

Claims (16)

8-carboxylic acid (5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- A pharmaceutical composition for the prevention or treatment of diseases caused by splicing defects comprising, as an active ingredient, a compound of formula (I), a pharmaceutically acceptable salt thereof, a carboxylic acid, Silmitasertib, CX-4945) or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition according to claim 1, wherein the CX-4945 regulates the alternative splicing of mRNA.
The pharmaceutical composition according to claim 1, wherein the CX-4945 inhibits splicing regulation kinase. 2. The pharmaceutical composition according to claim 1, wherein the CX-4945 inhibits splicing regulation kinase.
The method according to claim 1, wherein the CX-4945 regulates the phosphorylation of a serine-arginine-rich protein (SR protein). Or a pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the disease is selected from the group consisting of Afibrinogenemia, Propionic acidemia, Neurofibromatosis, Ocular albinism type 1, Alzheimer's disease / FTDP-17 Frontal temporal dementia Alzheimer &apos; s disease / FTDP-17 Taupathies, and spinal muscular atrophy. The pharmaceutical composition for preventing or treating diseases caused by splicing defects,
A splicing regulator comprising CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
A pharmaceutical composition for preventing or treating diseases caused by splicing defects comprising CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
8. The method of claim 7, wherein the disease is selected from the group consisting of fibrinogen deficiency, propionic acidemia, neurofibromatosis, eye albinism type 1, Alzheimer's disease / FTDP-17 frontal temporal dementia and spinal cord muscle atrophy Or a pharmaceutically acceptable salt thereof, for the treatment and prevention of splice-related diseases.
Comprising treating CX-4945 or a pharmaceutically acceptable salt thereof in isolated cells, cell-free or in vitro.
10. The method of claim 9, wherein the CX-4945 regulates selective splicing of mRNA.
10. The method of claim 9, wherein the CX-4945 inhibits splicing control kinase.
10. The method of claim 9, wherein the CX-4945 regulates the phosphorylation of a serine-arginine-rich (SR) protein.
CX-4945 or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for preventing or treating diseases caused by overexpression of Cdc-2-like kinase (Clk), which comprises CX-4945 or a pharmaceutically acceptable salt thereof as an active ingredient.
15. The pharmaceutical composition according to claim 14, wherein said CX-4945 competitively inhibits the activity of Clk.
15. The pharmaceutical composition according to claim 14, wherein the disease is Alzheimer's disease. 15. A pharmaceutical composition for preventing or treating diseases caused by overexpression of Clk.

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