KR101146010B1 - COMPOSITION FOR PREVENTING OR TREATING TNF-alpha MEDIATED DISEASES - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of TNF-α (tumor necrosis factor-α) mediated diseases comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, Compounds inhibit the secretion of TNF-α by inhibiting the binding of HuR protein to the ARE (AU-rich element) region present in TNF-α mRNA, thereby reducing mRNA half-life, thereby treating TNF-α-mediated diseases and It can be usefully used as a prophylactic agent.

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Tumor necrosis factor-α (TNF-α), HuR protein, AU-rich element (ARE)

Description

Composition for the prevention and treatment of TNP-α mediated diseases {COMPOSITION FOR PREVENTING OR TREATING TNF-α MEDIATED DISEASES}

The present invention relates to a composition for the prevention and treatment of TNF-α mediated diseases comprising the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

Hu family (Hu family of RNA-binding proteins) binds to the AU-rich element (ARE) in the 3'-UTR of mRNA to regulate its stability, nuclear release, and translation As a protein related to, HuR (HuA), HuB (Hel-N1), HuC, and HuD, etc., these proteins have homology with the Drosophila ELV (embryonic lethal abnormal vision) protein. Unlike members of other ELVA families (HuB, HuC and HuD) found only in nervous tissues, HuR is expressed in several tissues.

HuR proteins are mostly in the nucleus, but migrate to the cytoplasm by certain events. Stabilization of specific mRNA by HuR is associated with extracellular stimulation. For example, if there is LPS administration or chronic ethanol exposure to macrophage cells, HuR responds to induce stability of TNF-α mRNA. HuR is also known to regulate p21 mRNA stability by UV light and VEGF (vascular endothelial growth factor) mRNA stability by hypoxia. In addition, HuR may bind to the 3'-UTR of p53 mRNA to stabilize the transcript in response to stress adversely affecting the genetic material. When exposed to UV light (UVC), the level of p53 protein increased, and overexpression of HuR in RKO cells resulted in an increase in p53 level using Western blot analysis. The level also showed a decrease. In addition, HuR has been shown to promote transcription by binding to p53 mRNA, and HuR is known to act as an adapter protein for nuclear release of various mRNAs, including ARE (Brennan, CM, and Steitz, JA (2001). HuR and mRNA stability.Cell Mol Life Sci 58 , 266-277.

TNF-α, on the other hand, initiates an inflammatory response with important inflammatory cytokines. TNF-α mRNA generally contains an ARE sequence in the 3'-UTR. Under normal conditions, the ARE blocks translation or quickly turns over a transcript.

T-cell intracellular antigen-1 (TIA-1) is one of the RNA recognition motif (RRM) family proteins and is a translation inhibitor of TNF-α mRNA. Tristetraprolin (TTP) is also commonly known as a destabilizer of TNF-α mRNA. In contrast, Hu proteins are known to increase the half-life of target mRNAs (Seko, Y., Cole, S., Kasprzak, W., Shapiro, BA, and Ragheb, JA (2006) .The role of cytokine mRNA stability in the pathogenesis of autoimmune disease.Autoimmun Rev 5 , 299-305.).

TNF-α has become a major target for many pharmaceutical schemes. Typically, etanercept (Enbrel ™), infliximab (Remicade ™), adalimumab (Humira ™) function to neutralize antibodies of TNF-α. However, Grattendick and co-workers studied the in vitro effects of three anti-TNF-α drugs on the levels of secreted TNF-α and the levels of TNF-α associated with cells. Anti-TNF-α drugs based on proteins that partially neutralize the bioactivity of -α have demonstrated many side effects. Therefore, the development of drugs that regulate stabilization of TNF-α mRNA is expected to provide a better therapeutic strategy in the treatment of chronic inflammatory diseases.

In addition, compounds such as SB-242235 and RWJ-67657 are known as anti-inflammatory actives that inhibit the production of TNF-α, IL-1, IL-6, but the mechanism of action of these inhibitors in the post-transcriptional regulation of inflammatory mediators It has not been investigated yet. In addition, we have demonstrated in the previous report that flavonoids interfere with the binding of HuC, a stabilizing factor, and TNF-α mRNA, a major inflammatory cytokine (Kwak, H., Jeong, KC, Chae). , MJ, Kim, SY, and Park, WY (2009) .Flavonoids inhibit the AU-rich element binding of Hu C. BMB Rep 42 , 41-46.

The inventors have identified chemical inhibitors that can inhibit the interaction of the mRNA of the ARE-containing protein with the HuR protein. Specifically, the present inventors screened compounds that could interfere with HuR interaction with TNF-α mRNA, one of the ARE-containing proteins, and found a compound that strongly inhibits the binding of the mRNA and HuR of the ARE-containing protein. These results can be effectively used in post-transcriptional regulation and in the development of new anti-inflammatory drugs. In addition, the use of compound inhibitors that act on the stability of TNF-α mRNA is expected to be an alternative to current methods of treatment using anti-TNF-α antibodies to treat chronic inflammatory diseases.

An object of the present invention is to provide a composition for the prevention and treatment of TNF-α mediated diseases comprising the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the object of the present invention,

In one aspect, the present invention provides a composition for the prevention and treatment of TNF-α-mediated diseases comprising the compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

Formula I

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The compound also includes prodrugs, and the term “prodrug compound” herein refers to, after administration, being converted to the desired compound form through several chemical or physiological processes, for example by reaching physiological pH or by enzymatic action. It refers to an active or inactive compound. Suitability and the techniques for the preparation and use of the precursor compounds are known to those skilled in the art.

In addition, the compounds of the present invention can also be prepared in a variety of ways using methods well known to those skilled in the art.

As used herein, the term “pharmaceutically acceptable salts” refers to salts prepared by conventional methods and known to those skilled in the art. "Pharmaceutically acceptable salts" include, but are not limited to, basic salts of the following inorganic and organic acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid , Lactic acid, fumaric acid, succinic acid, maleic acid, silicic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. Other examples of “pharmaceutically acceptable salts” are described below and in J. Pharm. Sci. 66, 1 (1977), by reference.

The composition of the present invention induces degradation of TNF-α mRNA by reducing the mRNA half-life by inhibiting HuR protein binding to ARE (AU-rich element) mRNA, more specifically TNF-α mRNA. And ultimately TNF-α mRNA

Inhibits secretion of the encoding protein.

As used herein, the term “ARE (AU-rich element)” refers to a region of high A and U bases in mRNA and is a target of mRNA degradation and is generally present in the 3 ′ UTR. It is estimated that 5-8% of human mRNAs will contain AREs, in particular AREs in mRNAs of cytokines and transcription factors. One of the characteristics of the ARE is that U has the AUUUA central sequence (eg, WWWU (AUUUA) UUUW, where W is A or U) in many sequences. Repetition of the central AUUUA element is required to function. Many proteins bind to ARE to stabilize or destabilize mRNA.

In eukaryotic cells, mRNA undergoes several types of post-transcriptional regulation, including splicing, nuclear export, stabilization, and localization. Half-life is determined by the stability of the mRNA in response to various stimuli. Stability of mRNA plays an important role in homeostasis, differentiation, development, and growth of differentiated cells in vivo. mRNA half-life ranges from approximately 15 minutes to more than 24 hours, varying the stability of the mRNA. In particular, the synthesis of inflammatory cytokines such as TNF-α, IL-2, IL-6, and IFN-γ are regulated according to these mRNA stability levels. The cis- element of mRNA (eg ARE) and trans-acting RNA-binding protein (eg Hu protein) regulate mRNA half-life. In steady state, cytokine mRNAs are unstable due to the presence of ARE in the 3'-untranslated region (3'-UTR) containing various copies of the typical AUUUA pentamer motif.

The HuR protein consists of two N-terminal RRMs with strong affinity for ARE and a C-terminal RRM that recognizes the poly (A) tail. HuR proteins bind to and form complexes with mRNAs comprising ARE (eg, TNF-α), thereby stabilizing these mRNAs and ultimately increasing their expression.

The compound herein is preferably 2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl) -2-oxoethylthio) pyridine-3,5 Dicarbonitrile (2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl) -2-oxoethylthio) pyridine-3,5-dicarbonitrile (hereinafter b-40) )) Or 2- (6-amino-3,5-dicyano-4- (4-methoxyphenyl) pyridin-2-ylthio) -N- (4-chlorophenyl) acetamide (2- (6 -amino-3,5-dicyano-4- (4-methoxyphenyl) pyridin-2-ylthio) -N- (4-chlorophenyl) acetamide (hereinafter b-41)).

More specifically, the chemical formulas of b-40 and b-41 are as follows.

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2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl) -2-oxoethylthio) pyridine-3,5-dicarbonitrile (hereinafter, b-40) and 2- (6-amino-3,5-dicyano-4- (4-methoxyphenyl) pyridin-2-ylthio) -N- (4-chlorophenyl) acetamide (hereinafter, b-41) inhibits the stabilization of TNF-α mRNA by inhibiting the binding of the HuR protein to the ARE region of TNF-α mRNA, and ultimately may be useful for the prevention and treatment of TNF-α mediated diseases. .

According to a specific experimental example of the present invention, the IC ₅₀ (The half-maximal inhibitory concentration) of the HuR: ARE complex stability of b-40 and b-41 was measured, and b-40 is 0.38 μM and b-41 is 6.21. It was shown that μM, even at low concentrations effectively inhibited the formation of HuR: ARE complex (Table 1).

Also, most preferably the compound is 2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl) -2-oxoethylthio) pyridine-3 , 5-dicarbonitrile (b-40).

According to a specific experimental example of the present invention, as a result of adding b-40 to LPS-treated RAW264.7 cells (LPS-treated RAW264.7 cells), degradation of IL-6 mRNA compared to the case without addition The speed increased (FIG. 3B). In other words, b-40 inhibits the formation of HuR: ARE (IL-6) complex, thereby reducing the stability of IL-6 mRNA. In addition, the treatment of 0.25 μM b-40 effectively inhibited the formation of the complexes HuR: ARE (TNF-α) and HuR: ARE (cFos) (FIG. 4).

In addition, the concentration of the preferred compound inhibitor for inhibiting the secretion of TNF-α is 2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl)- In the case of 2-oxoethylthio) pyridine-3,5-dicarbonitrile (b-40), it is preferably 4 µM or less, more preferably 1-4 µM.

According to a specific experimental example of the present invention, after measuring the level of the TNF-α secreted in the medium (medium) of RAW264.7 cells after treatment of b-40 b-40 TNF when treated at 4 μM or less The synthesis of -α was effectively inhibited (FIG. 3D).

As used herein, the term “TNF-α” is a cytokine produced by macrophages in animals, including humans, in response to various immune stimulants, including endotoxins that are bacterial toxins, and play an important role in the immune system. It is a substance. Administration of TNF-α to animals or humans results in inflammation, fever, cardiovascular activity, bleeding, agglutination and acute phase reactions similar to those in acute infections and shock conditions. If not, several diseases are known to cause.

"TNF-α mediated disease" means a local and / or systemic physiological disorder in which TNF-α is the primary mediator of inducing signs of the disease. More specifically, examples of TNF-α mediated diseases are as follows.

A. Sepsis

TNF-α plays an established role in the pathophysiology of sepsis in that it has biological effects, including hypotension, myocardial repression, vascular leak syndrome, organ necrosis, stimulation of toxic secondary mediator release, and activation of coagulation chain amplification. do. Thus, the compositions of the present invention can be used to treat sepsis in any clinical setting, including septic shock, endotoxin shock, gram negative sepsis and toxin shock syndrome.

In addition, to treat sepsis, the compositions of the present invention are co-administered with one or more additional therapeutic agents that can further alleviate sepsis, such as interleukin-1 inhibitors, antagonists of cytokine interleukin-6 or platelet activating factors. Can be.

B. Autoimmune Diseases

TNF-α is involved in playing a role in the pathophysiology of various autoimmune diseases. For example, it is associated with activating tissue inflammation and causing joint destruction in rheumatoid arthritis. TNF-α is also involved in promoting death of islet cells and mediating insulin resistance in diabetes. TNF-α also mediates cytotoxicity to oligodendrocytes in multiple sclerosis and is associated with inflammatory plaque induction.

The compositions of the present invention treat autoimmune diseases, in particular autoimmune diseases with inflammation, such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome It can be used to Typically, the composition is administered systemically, but for certain diseases it may be advantageous to topically administer the composition to the site of inflammation. For example, topical administration to the joints for rheumatoid arthritis or topical application alone or with cyclohexane-ylidene derivatives for diabetic ulcers.

C. Infectious Diseases

TNF-α is associated with the mediation of biological effects observed in various infectious diseases. For example, TNF-α causes brain inflammation, capillary thrombosis and infarction in malaria. TNF-α also mediates brain inflammation, induces disruption of the blood brain barrier, triggers septic shock and activates venous infarction in meningitis. TNF-α also induces cachexia, stimulates viral proliferation and mediates central nervous system damage in AIDS.

Thus, the compositions of the present invention can be used for the treatment of infectious diseases such as bacterial meningitis, brain malaria, AIDS and AIDS-related syndromes (ARCs), and cytomegalovirus infections concomitant with transplantation. The compositions of the present invention can be used to relieve symptoms associated with an infectious disease, such as fever and myalgia due to infection (eg influenza) and cachexia concomitant with infection (eg, accompanying AIDS or ARC).

D. Transplant Rejection

TNF-α is a major mediator of allograft rejection and graft-versus-host disease (GVHD), which is observed when the rat antibody OKT3 induced against the T cell receptor CD3 complex is used to inhibit rejection of kidney transplants. It is involved in mediating side effects.

Thus, the compositions of the present invention can be used to inhibit transplant rejection, such as allograft and rejection of xenografts, and to inhibit GVHD. Although the composition may be used alone, more preferably it is used in combination with one or more agents that inhibit an immune response to allograft or inhibit GVDH. For example, the compositions of the present invention can be used with OKT3 to inhibit OKT3-induced reactions. In addition, other targets involved in modulating the immune response, such as cell surface molecules CD25 (interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 It may be used with one or more antibodies directed to (B7-1) and / or CD86 (B7-2). It may also be used in combination with one or more immunosuppressive agents, for example cyclosporin A or FK506.

E. Malignant Tumors

TNF-α induces cachexia in malignancies, stimulates tumor growth, increases metastasis potential and mediates cytotoxicity. Thus, the compositions of the present invention can be used to treat malignant tumors, inhibit tumor growth or metastasis and / or mitigate cachexia concomitant with malignancies. The composition may be administered systemically or topically to the tumor site.

F. Lung Disease

TNF-α is involved in the pathophysiology of adult respiratory distress syndrome, such as stimulating leukocyte-endothelial activation, inducing cytotoxicity to alveolar cells, and inducing vascular leak syndrome. Accordingly, the compositions of the present invention can be used to treat various lung diseases, such as adult respiratory distress syndrome, respiratory shock lung, chronic lung inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The composition can be administered systemically, for example, as an aerosol or topically to the lung surface.

G. Intestinal Disease

TNF-α is involved in the pathophysiology of inflammatory bowel disease. The human composition of the invention can be used to treat intestinal diseases, including idiopathic inflammatory bowel disease, including two syndromes, Crohn's disease and ulcerative colitis.

H. Heart Disease

The compositions of the present invention can also be used to treat various heart diseases, such as ischemia and heart failure (weakness of the heart muscle) of the heart.

I. Other

The compositions of the present invention can also be used to treat a variety of other diseases mediated by TNF-α. Since TNF-α activity relates to pathophysiology, examples of other diseases and disorders that can be treated using the compositions of the present invention include inflammatory bone disease and bone resorption disease, hepatitis such as alcoholic hepatitis and viral hepatitis, coagulation Disorders, burns, reperfusion injury, keloid formation, scar tissue formation and fever.

Therefore, the TNF-α mediated diseases to which the composition of the present invention is applicable include sepsis, autoimmune disease, infectious disease, allograft rejection, transplant-versus-host disease (GVHD), malignancy, lung disease, Crohn's disease, Ulcerative colitis, heart disease, bone disease, bone resorption disease, hepatitis, alcoholic hepatitis, viral hepatitis, coagulation disorders, burns, reperfusion injury, keloid formation, scar tissue formation and fever, but are not limited thereto. .

As used herein, the term “treatment and prevention” refers to treating, curing, healing, alleviating, altering, or treating a disease, a symptom of a disease, or a predisposition to a disease. administering a composition comprising a compound to a patient having one of the above-mentioned diseases, symptoms of symptoms or predisposition to the disease for the purpose of improving, ameliorating, improving or effecting Say that.

Compositions of the present invention may be prepared in pharmaceutical formulations using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of the formulation, it is preferred that the active ingredient is mixed or diluted with the carrier or enclosed in a carrier in the form of a container. When the carrier is used as a diluent, it may be a solid, semisolid or liquid substance which acts as a carrier, excipient or medium for the active ingredient. Thus, the formulations may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydride Oxybenzoate, talc, magnesium stearate, mineral oil and the like. In some cases, the formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

The dosage of the active ingredient, such as the composition or pharmaceutically acceptable salt thereof, used in the present invention depends on the subject being treated, the severity of the disease or condition, the rate of administration and the judgment of the prescribing physician. As used herein, the term "effective amount" is the amount of compound required to produce the desired effect. Effective amounts may vary depending on the route of administration, the use of excipients, and the possibility of use with other agents, as will be appreciated by those skilled in the art.

In order to carry out the treatment method of the present invention, it may be administered (treated) orally, rectally, parenterally, by inhalation spray, or through a planted reservoir or the like.

Parenteral includes subcutaneous, subcutaneous, intravenous, intramuscular, intraarticular, arterial, synovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.

Compositions for oral administration may be in any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions and aqueous suspensions, dispersants and solutions. Commonly used tablet carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added to the tablets. Diluents useful for oral administration in a capsule form include lactose and dried corn starch. When an aqueous suspension or emulsion is administered orally, the active moiety can be suspended or dissolved in the oil phase in combination with an emulsifying or suspending agent. If necessary, some sweetening, flavoring or coloring may be added. Sterile injectable compositions (eg, water-soluble or oleaginous suspensions) can be prepared by known techniques using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions, such as nontoxic parenterally acceptable diluents or solvents, for example 1,3-butanediol solution. Among the acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are traditionally used as a solvent or dispersion medium (eg, synthetic mono- or di-glycerides). Fatty acids such as oleic acid and its glyceride derivatives are particularly useful for the preparation of injectables as natural pharmaceutically acceptable oils such as olive or castor oil in their polyoxyethylated form. Such oil solutions or suspensions may also include long chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants.

Inhalation compositions may be prepared according to known techniques of pharmaceutical formulations, and may be salts using benzyl alcohol or other suitable preservatives, absorption enhancers that enhance bioavailability, fluorocarbons, and / or other known solubilizers or dispersants. It can be prepared as a solution of.

One or more active compounds may be administered rectally. One example is a suppository comprising the active compound with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides or paraffin hydrocarbons. Another example is a gelatin rectal capsule comprising an active compound and a base. Base materials that can be used include, for example, liquid triglycerides, polyethylene glycols or paraffin hydrocarbons.

Compositions applied to the skin can be prepared in oils, creams, lotions, ointments and other forms. Suitable carriers for the composition include vegetable or mineral oils, white petrolatum ointment (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohols (higher than C12). Preferred carriers are those in which the active ingredient is soluble. Emulsifiers, stabilizers, wetting agents and antioxidants also include color-expressing or fragrances, if necessary. In addition, skin penetration enhancers can be used in such topical formulations.

The cream is preferably prepared from a mixture of mineral oil, self-emulsifying beeswax and water mixed with a mixture of active ingredients dissolved in a small amount of oil such as almond oil. One example of such a cream comprises a ratio of about 40 water, about 20 wax, about 40 mineral oil and about 1 almond oil.

Ointments are prepared by mixing the active ingredient solution with vegetable oils such as almond oil, along with cooling the warm soft paraffin and the mixture. One example of such ointment includes 30% by weight almond oil and 70% by weight white soft paraffin.

In another aspect, the present invention provides a method of inhibiting the secretion of TNF-α by treating a compound represented by the formula (I).

The compounds of the present invention effectively inhibit TNF-α secretion by inhibiting the binding of HuR protein to TNF-α mRNA and reducing mRNA half-life, and thus can be usefully used as a treatment and prevention agent for diseases mediated by TNF-α. Can be.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Example  1. Experiment Preparation and Methods

1-1. compound

Representative compounds used by the present inventors were provided by the Korea Compound Bank of the Korea Institute of Chemical Research (Yoo Sung-gu, Daejeon, Korea).

1-2. DNA  Produce

Full-length mouse recombinant HuR and TTP genes were cloned into the pGEX4T1 vector. Escherichia as described in Kang et al., 2002. coli ( E. coli ) BL21 strain was transformed to express the protein. For in vitro synthesis of TNF-α-ARE transcripts, sense oligonucleotides were used as templates for PCR reactions. All oligonucleotides contain the 5'-GGGAGATACGGTCCACTACC-3 'sequence at 5' and the 5'-CGAAGTCTGAGTGATGCATG-3 'sequence at 3'. PCR templates were amplified using the forward primer 5'-GCTAATACGACTCACTATAGGGAGATACGGTCCACTACC-3 'and the reverse primer 3'-GCTTCAGACTCACTACGTAC-5'.

1-3. Recombination HuR  And TTP  Preparation of Protein

Colonies containing mouse recombinant HuR and TTP genes were cultured in LB culture medium (10 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) with stirring at 37 ° C. until the A600 value was 0.4. It was. Thereafter, IPTG was added to the culture solution to a concentration of 0.5 mM based on the final culture solution, followed by further incubation for 4 hours. The cells were then collected and resuspended in cold 5 ml of lysis buffer, 50 mM Tris-HCl (pH 8.0), 120 mM NaCl and 5 mM DTT, and then the cells were sonicated (30 W, 90 cells were destroyed via s). The lysate was incubated at 4 ° C. for 30 minutes and then centrifuged at 12,000 × g and 4 ° C. for 30 minutes. After mixing the supernatant obtained here and glutathione Sepharose 4B (Glutathione Sepharose 4B, GE Healthcare, Sweden), and incubated with gentle stirring at room temperature for 90 minutes. Sepharose medium was washed three times with TBS (50 mM Tris-HCl (pH 8.0), 120 mM sodium chloride) and the fusion protein was eluted with elution buffer (50 mM Tris-HCl pH 8.0, 20 mM reduced glutathione). Eluted using.

1-4. RNA Of the probe In vitro  Warrior ( In in vitro transcription )

RNA probes were constructed from PCR templates containing the T7 RNA transcriptase promoter site. The template was transcribed using [α- ³² P] UTP (GE Healthcare, Sweden) and T7 MEGAscript kit (Ambion, TX, USA) according to the manufacturer's instructions. The transferred probe was treated with DNaseI at 37 ° C. for 15 minutes, extracted with phenol: chloroform, and then isopropanol precipitated.

1-5. RNA EMSA  ( RNA electrophoretic mobility gel shift assay  )

The labeled RNA probe and the GST-HuR protein were mixed in a binding buffer (50 mM Tris-HCl, pH 8.0), 120 mM NaCl, and then each compound dissolved in DMSO solution was added. The mixture was reacted at room temperature for 20 minutes. Protein-RNA conjugates were separated from dissociated RNA on 6% polyacrylamide gels. Pre-electrophoresis was performed in 0.5x TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA). Electrophoresis was performed for 1 h at 8 mA in 0.5x TBE buffer (Jung, KC, Park, CH, Hwang, YH, Rhee, HS, Lee, JH, Kim, HK, and Yang, CH (2006). Fatty acids, inhibitors for the DNA binding of c-Myc / Max dimer, suppress proliferation and induce apoptosis of differentiated HL-60 human leukemia cell.Leukemia 20 , 122-127.). Bands were visualized using the BAS2500 automated detector system (Fujifilm, Tokyo, Japan), and the intensity of each band was image analysis software (Multi-Gauge V3.0, Fujifilm, Tokyo). , Japan).

1-6. protein- RNA  Filter combination analysis ( Protein - RNA filter binding assay )

³² P-labeled RNA and GST-HuR or GST-TTP protein were allowed to react for 20 minutes at room temperature with a total volume of 100 μl of reaction in the binding buffer. After the reaction, the reaction solution previously boiled in binding buffer for 30 minutes was filtered through a 0.45 μm nitrocellulose disc filter (Millipore, MA, USA). The filter was washed twice with binding buffer and analyzed with a Tri-Carb 2900TR liquid scintillation counter (Perkin Elmer, CT, USA).

1-7. Cell culture

RAW264.7, a mouse macrophage cell line, contains DMEM medium (Welgene, Daegu, USA) containing 10% fetal bovine serum (Hyclone Laboratories, UT, USA) and 1% penicillin / streptomycin (Invitrogen, CA, USA) South Korea) was cultured in 5% CO ₂ , 37 ℃ culture conditions.

1-8. mRNA  Stability studies

For RNA stability analysis, actinomycin D (5 μg / ml) was added to RAW264.7 cells to inhibit transcription. Actinomycin D and / or quercetin (0, 5, 20 M) or compound b-40 (0, 1 and 4 μM) were treated with time difference before total RNA was extracted from the cells. Total RNA (200 ng) was prepared using TRIZOL, and cDNA was prepared by reverse transcription of the RNA using reverse transcriptase and oligo-dT primers. PCR was performed using 1 or 2 μl of cDNA and specific primers.

1-9. RT - PCR

Reverse transcriptase involving oligo-dT primers (Invitrogen, Calif., USA) has been used to make 0.1 μg total RNA to cDNA. PCR with special primers was run using 0.1 μl of cDNA.

Primer sets were as follows: TNF-α: forward 5'-CCAGGCAGTCAGATCATCTTC-3 'and reverse 5'-TTGATGGCAGAGAGGAGGTT-3'; IL-6: forward 5'-AACGATGATGCACTTGCAGA-3 'and reverse 5'-GGAAATTGGGGTAGGAAGGA-3'; And glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward 5′-TTCACCACCATGGAGAAGGC-3 ′ and reverse 5′-GGCATGGACTGTGGTCATGA-3 ′.

The conditions of PCR were as follows: deaturation at 95 ° C. for 30 seconds followed by annealing at 55 ° C. for 30 seconds and primer synthesis at 55 ° C. for 30 seconds and extension at 72 ° C. for 30 seconds. Cycle 30 times; The last step is followed at 72 ° C for 10 minutes.

PCR products were separated by electrophoresis on a 1.5% agarose gel containing 0.1 μg / ml EthiBr (ethidium bromide) and UV light with a bioimaging system. Visually recognized (Syngene, Cambridge, United Kingdom).

Experimental Example  One. TNF -α mRNA of ARE To HuR  And tristetraprolin  ( TTP Binding affinity analysis

Recombinant GST fusion proteins were made with HuR and TTP (FIG. 1A), and RNA EMSA was implemented using radiolabeled RNA comprising these fusion proteins and ARE sequences derived from TNF-α (FIG. 1A). 1b). HuR: ARE complexes with two different molecular weights were formed in a concentration-dependent manner. As observed through RNA EMSA, HuR was more strongly bound to ARE sequence than TTP. The binding efficiency of both proteins was also quantified by filter binding assay using the same recombinant protein and RNA probe. HuR's TNF-α mRNA binding affinity was higher than TTP. In addition, the total amount of protein: RNA complex was higher in HuR: ARE.

Experimental Example  2. HuR : ARE  Inhibitors that inhibit complex formation ( inhibitor ) analysis

2-1. HuR : ARE  Inhibitors that inhibit complex formation ( inhibitor Screening Screening )

Chemical inhibitors for HuR: ARE binding were screened based on RNA EMSA and filter binding assays. We obtained a subset of a readily available compound library from the Korea Research Institute of Chemical Technology, and we used 179 electrophoretic mobility gel shifts using a genetically modified HuR. Other compounds were screened. At the initial screening, we Compounds were applied to HuR: ARE complexes at 100-μM concentration. Of the compounds, nine candidates A strong inhibitory effect (cut-off = 25% inhibition) on the binding of HuR to ARE.

2-2. Of candidate compounds IC ₅₀  result

In order to measure the IC ₅₀ of each compound according to the stability of the HuR: ARE complex, the amount of the compound was adjusted to determine the degree of formation of the RNA-protein complex through filter binding analysis. The half-maximal inhibitory concentration (IC ₅₀ ) is determined using GraphPadprism software (FIG. 2). As summarized in Table 1, the IC ₅₀ values were 0.38 μM for b-40 and 6.21 μM for b-41.

Experimental Example  3. Quercetin  And by b-40 mRNA  Control of stability

Since quercetin and b-40 can inhibit HuR: ARE (TNF-α) complex formation, these two compounds contribute to the stability of TNF-α mRNA. TNF-α and IL-6 mRNAs were tested in LPS-treated RAW264.7 (LPS-treated RAW264.7) mouse macrophage macrophages after the addition of Actinomycin D. Without quercetin, the levels of TNF-α and IL-6 mRNA are reduced with the addition of actinomycin. Quercetin was added to LPS-treated RAW264.7 cells (LPS-treated RAW264.7 cells), the degradation rate of TNF-α and IL-6 mRNA compared with the case without addition (Fig. 3a). b-40 induced rapid degradation of IL-6 mRNA (FIG. 3B).

In order to confirm the effects of quercetin and b-40, the level of TNF-α protein secreted in the medium of LPS treated RAW264.7 cells was measured. As shown in FIG. 3C, the amount of TNF-α protein in quercetin treated cells decreased in proportion to the amount of quercetin. b-40 effectively inhibited the synthesis of TNF-α when treated with 4 μM or less (FIG. 3D) .

Experimental Example  4. HuR : ARE  Specificity of compound inhibitors of the complex

Although screening compounds using AREs of HuR and TNF-α mRNA, HuR can bind to other ARE sequences, and AREs of TNF-α can also be bound by TTP. Therefore, in order to evaluate the specificity of the inhibitory activity of b-40 and quercetin (inhibitory activity) to the RNA binding protein, the following experiment was performed.

4-1. Of compound inhibitors TNF -α mRNA ARE To HuR  And tristetraprolin  ( TTP Analysis of binding inhibition

The inhibitory effect of the compound inhibitor was analyzed in the state of substituting with TTP instead of HuR and forming a complex with TNF-α ARE [ARE (TNF-α)] (FIG. 4). As shown in Figure 1B, TTP: ARE (Kd = 2.194 x 10-7 M) binding is lower than HuR: ARE (Kd = 0.677 x 10-7 M), and TTP: ARE binding is either quercetin or b-40. None was suppressed.

4-2. Diverse Targets of Compound Inhibitors ARE  Effect of Inhibitory Activity on Sequences

COX2, IL-6 and cFOS were also used to test the properties of inhibitory activity on the target ARE sequence (FIG. 4). Treatment with 0.5 μM quercetin did not effectively inhibit any RNA-protein interaction except HuR: ARE (TNF-α). Treatment with 0.25 μM of b-40 inhibited the formation of the complexes HuR: ARE (TNF-α) and HuR: ARE (cFos).

1, TNF -α mRNA of ARE  For sequence HuR Wow TTP Combination of Affinity  It is shown.

Specifically, FIG. 1A shows the structure of a fusion protein of huR (NP_034615) or TTP (NP_035886) and GST as used herein, and the ARE sequence of TNF-α, COX-2, IL-6, cFos mRNA (RRM, RNA -recognition motif; ZnF, Zn2 + -finger binding motif).

FIG. 1B shows the results of confirming that HuR (top) and TTP (bottom) bind to ARE sequences of TNF-α mRNA through RNA EMSA analysis.

Figure 1c is a result of confirming the affinity of HuR, TTP, GST to TNF-α through a filter binding assay (filter binding assay).

Figure 2 shows the results confirming the inhibitory activity of b-40 and b-41 binding to the ARE sequence of TNF- α mRNA through the filter binding analysis. Dotted lines represent IC ₅₀ values for each compound.

3 is LPS -process RAW264 .7 TNF-α and IL For -6 expression Quercetin  And b-40.

Specifically, when RAW264.7 cells were pretreated with 1 μg / ml LPS for 1 hour and then treated with actinomycin D (5 mg / ml) and quercetin (a) or b-40 (b), TNF- Degradation rate of α and IL-6 mRNA, and also when treated with quercetin (c) or b-40 (d) in LPS-activated RAW264.7 cells , Levels of TNF-α and IL-6 proteins are shown.

4 is Quercetin The specificity of the inhibitory activity of (A) and b-40 (B) is shown.

Claims (6)

Sepsis, autoimmune disease, infectious disease, allograft rejection, graft-versus-host disease (GVHD), Crohn's disease, ulcer, comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient: A composition for preventing and treating one or more diseases selected from the group consisting of colitis, bone disease, bone absorption disease, hepatitis, alcoholic hepatitis, viral hepatitis, coagulation disorders, burns, keloid formation, scar tissue formation and fever: Formula I

Where R1 is

, R2 is

to be. The composition of claim 1, wherein the composition inhibits the binding of HuR protein to the AU-rich element (ARE-region) region present in TNF-α mRNA to reduce TNF-α mRNA half-life. The compound of claim 1, wherein the compound is 2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl) -2-oxoethylthio) pyridine-3 , 5-dicarbonitrile or 2- (6-amino-3,5-dicyano-4- (4-methoxyphenyl) pyridin-2-ylthio) -N- (4-chlorophenyl) acetamide A composition, characterized in that. The method of claim 3, wherein the 2-amino-4- (4- (benzyloxy) phenyl) -6- (2- (3,4-dihydroxyphenyl) -2-oxoethylthio) pyridine-3,5 The concentration of dicarbonitrile is 1 μM-4 μM. delete delete

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