KR20210060362A - COMPOSITION FOR THE TREATMENT OF INFLAMMATORY DISEASES USING CPTH2, TAK-242 AND GCN5 siRNA - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases or neurodegenerative diseases. More particularly, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or neurodegenerative diseases, comprising a compound represented by a formula 1 below, a compound represented by a formula 2 below, a pharmaceutically acceptable salt thereof, and siRNA of GCN5 protein as an active ingredient.

Description

Composition for the treatment of inflammatory diseases using siRNA of CPTH2, TAK-242 and GCN5 {COMPOSITION FOR THE TREATMENT OF INFLAMMATORY DISEASES USING CPTH2, TAK-242 AND GCN5 siRNA}

The present invention relates to a composition for the treatment of inflammatory diseases comprising a GCN5 inhibitor and a use thereof, and more particularly, to provide a therapeutic composition specific for neurological diseases including TK242, CPTH2 compound, or siRNA of GCN5 protein.

Microglial is present in the central nervous system (CNS) and brain and is involved in the protection and recovery of primary neurons. However, many studies have been reported that activated glial cells induce neuroinflammatory reactions, and neuroinflammatory reactions lead to various neurodegenerative diseases. Activated glial cells include nitrite (NO), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). ) And induces the activities of pro-inflammatory cytokines such as IL-1b, IL-6, and TNF-a (Graeberand Streit, 2010).

These substances are reported to be involved in inflammatory diseases such as sepsis, inflammatory bowel disease, encephalitis, and atopy, and are involved in the pathological mechanisms of neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease (Kim and Joh, 2006; Koning et al., 2007; Krause and Muller, 2010; Moller, 2010). Therefore, the control of neuroinflammation can play an important role in the prevention of neurodegenerative diseases, and is also important in preventing inflammatory diseases.

On the other hand, GCN5 is a histone acetyltransferase (HAT), which mainly acts as a transcriptional activator. GCN5 also acts as a repressor of NF-kappa-B by promoting the ubiquitination of the NF-kappa-B subunit RELA in a HAT-independent manner.

Under this background, the present inventors were searching for novel biomarkers capable of diagnosing inflammatory diseases and neurodegenerative diseases, and when GCN5 was inhibited in BV-2 glial cells stimulated with lipopolysaccharide (LPS), a neuroinflammation mediator It was confirmed that the gene expression of BV-2 glial cells was decreased, and the change pattern of GCN5 by LPS in BV-2 glial cells was analyzed, and through this, the possibility of GCN5 as a novel biomarker capable of diagnosing neurodegenerative diseases was confirmed.

However, research on inhibitors of GCN5 is insufficient, and pharmaceutical compositions for preventing or treating inflammatory diseases, neuroinflammatory diseases and neurodegenerative diseases containing GCN5 inhibitors as active ingredients and/or prevention or improvement of inflammatory diseases or neurodegenerative diseases There is a need for research on the composition of health functional food for use.

The present invention has been developed to overcome the limitations and problems of the prior art as described above, and specifically aims to provide a biomarker capable of diagnosing inflammatory diseases using GCN5.

An object of the present invention is to provide a composition for preventing or treating inflammatory diseases or neurodegenerative diseases that inhibits GCN5 by using a compound represented by the following formula (1), a compound represented by the following formula (2), or siRNA of the GCN5 protein.

Another object of the present invention is to provide a health functional food composition for the prevention or improvement of inflammatory diseases or neurodegenerative diseases that inhibit GCN5 using a compound represented by the following Formula 1, a compound represented by the following Formula 2, or siRNA of the GCN5 protein It is to do.

[Chemical Formula 1] CPTH2

[Chemical Formula 2] TAK-242

In order to solve the above-described problems, the present invention provides a kit for diagnosing inflammatory diseases comprising a biomarker for diagnosing inflammatory diseases including GCN5 protein and a detection reagent specifically binding to the nucleic acid sequence of the biomarker or a gene encoding the biomarker. do.

The present invention also provides a composition for diagnosing or predicting prognosis of inflammatory diseases, including a substance for measuring the mRNA expression level of a GCN5 protein or a gene encoding the protein.

In the present invention, the substance for measuring the mRNA level is a composition for diagnosing or predicting prognosis of an inflammatory disease comprising a primer, a probe or an antisense nucleotide that specifically binds to the gene, and an inflammatory disease diagnosis or prognosis comprising the composition Kits are provided.

According to another embodiment of the present invention, for the prevention, treatment, or improvement of inflammatory diseases and neurodegenerative diseases including a compound represented by the following formula (1), a compound represented by the following formula (2), or siRNA (small interfering RNA) of GCN5 protein It provides a pharmaceutical composition and a health functional food composition.

[Formula 1]

[Formula 2]

In addition, the pharmaceutical composition or health functional food composition may alleviate inflammation by inhibiting phosphorylation of the tyrosine residue of the GCN5 protein.

According to another embodiment of the present invention, (a) measuring the expression level of a biomarker containing the GCN5 protein in a biological sample isolated from the individual, (b) the expression level of the biomarker in the step (a) is measured in a normal control group. Diagnosing a patient with an inflammatory disease or neurodegenerative disease when the expression level is higher than that of the normal control group, and (c) a compound represented by the following formula (1) in a biological sample isolated from a patient with the inflammatory disease or neurodegenerative disease; or There is provided a method of providing customized treatment information for each patient of inflammatory disease or neurodegenerative disease comprising the step of selecting a composition having a high gene expression inhibition degree of GCN5 by treating a composition containing a compound represented by the following Formula 2, respectively.

[Formula 1]

[Formula 2]

In the present invention, the neurodegenerative diseases are multiple sclerosis, Parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and fronto-temporal dementia. temporal dementia), cortical-basal ganglia degeneration, and progressive supranuclear palsy.

In the present invention, the inflammatory disease includes sepsis, inflammatory bowel disease, brain nerve inflammation, and atopic dermatitis, and preferably refers to brain nerve inflammation. The brain nerve inflammation includes inflammation occurring in brain tissues of the hippocampus, striatum, or substantia nigra compacta.

According to the present invention, inflammatory diseases can be diagnosed using a biomarker containing GCN5.

In addition, there is an advantage in that inflammatory diseases can be easily diagnosed with a diagnostic kit using a biomarker containing GCN5.

The present invention includes siRNAs of CPTH2 or TAK-242 and GCN5, which are compounds represented by Chemical Formula 1, as active ingredients, and can be usefully used for inhibiting GCN5 or TLR4 receptors.

The present invention can effectively prevent, improve and treat neurodegenerative diseases by inhibiting the GCN5 or TLR4 receptor.

According to an embodiment of the present invention, there is an advantage of increasing the treatment effect of inflammatory diseases by providing customized treatment information for each patient.

1 is a graph showing the effect of CPTH-2 on cell viability in mouse BV-2 glial cells stimulated with LPS and the effect of reducing the amount of nitric oxide (NO) released.
FIG. 2 shows the effect of inhibiting protein expression of iNOS and COX-2 according to CPTH2 concentration in mouse BV-2 glial cells stimulated with LPS by Western Blot.
3 is a Western blot confirming the interaction between GCN5 and RelA by neuroinflammatory stimulation in mouse BV-2 glial cells stimulated with LPS.
4 is a Western blot confirming the protein expression pattern of iNOS, NF-kappaB pathway-related protein expression pattern, and GCN5 protein expression pattern in the brain tissues of mice stimulated with LPS.
5 is a graph showing the effect of inhibiting gene expression of iNOS and GCN5 by TAK-242 in the brain tissue of stimulated mice.
6 is an image showing the expression level of GCN5 mRNA by tissue in C57BL/6 mice treated with LPS.
7 is an image showing changes in the expression levels of GCN5 and iNOS mRNA for each brain tissue site due to neuroinflammation in C57BL/6 mice treated with LPS.
8 is an image comparing changes in the mRNA expression levels of GCN5 and pro-inflammatory mediators (iNOS, TNF-a and IL-6) due to neuroinflammation in glial cells (microglia) and astrocytes (astrocyte).
9 is an image comparing changes in the expression levels of GCN5, ac-p65, and p-p65 proteins due to neuroinflammation in glial cells (microglia) and astrocytes (astrocyte).
10 is an image confirming changes in phosphorylation and acetylation of p65 in BV-2 glial cells overexpressing GCN5.
11 is an image showing changes in the expression of inflammatory mediators in BV-2 glial cells overexpressing GCN5.
12 is an image confirming changes in phosphorylation and acetylation of p65 in HEK293 (TLR4) cells overexpressing GCN5.
13 is an image confirming changes in phosphorylation and acetylation of p65 in HEK293 (TLR4) cells in which GCN5 expression was suppressed.
14 is an image comparing the results of expression of inflammatory mediators in HEK293 (TLR4) cells overexpressing GCN5.
15 is an image showing the results of expression of inflammatory mediators in HEK293 (TLR4) cells that inhibited the expression of GCN5.
16 is an image confirming the PTM change of GCN5 in BV-2 glial cells stimulated with LPS.

It should be noted that the technical terms used in the present specification are only used to describe specific embodiments, and are not intended to limit the present invention. In addition, the technical terms used in the present specification should be interpreted as generally understood by those of ordinary skill in the technical field to which the present invention belongs, unless otherwise defined in this specification. It should not be construed as a human meaning or an excessively reduced meaning. In addition, when a technical term used in the present specification is an incorrect technical term that does not accurately express the spirit of the present invention, it should be understood by being replaced with a technical term that can be correctly understood by those skilled in the art. General terms used in the present invention should be interpreted as defined in the dictionary or according to the context before and after, and should not be interpreted as an excessively reduced meaning.

In this specification, “GCN5” means General control non-derepressible 5. The GCN5 of the present invention may include the gene sequence of SEQ ID NO: 1 or a partial sequence thereof.

GCN5 used in the present invention is NCBI Accession No. May mean NP_066564.2.

In addition, the GCN5 overexpression vector used in the present invention is as follows.

GCN5 Lentiviral vector (human, BC032743) (CMV)(pLenti-GIII-CMV-C-term-HA)　: ABMGOOD, CAT. NO 2529406

GCN5 Lentivral vector (Moues, NM020004) (CMV) (pLenti-GIII-CMV-C-term-HA)?; ABMGOOD, CAT. NO 2529406

In the present specification, “siRNA of GCN5” refers to an RNA having a gene sequence complementary to the gene sequence of GCN5 or a partial sequence thereof, which is a small interfering RNA in a conventional sense. The siRNA of GCN5 according to the present invention may have a gene sequence of SEQ ID NOs: 3 to 6, but is not limited thereto.

Gene sequence information Sequence number Gene sequence/ amino acid sequence SEQ ID NO: 1
(Homo sapiens K(lysine) acetyltransferase 2A, mRNA (cDNA clone MGC:45022 IMAGE:5575574), complete CDs (BC032743) 2514 out of 3100 bp)

MAEPSQAPTPAPAAQPRPLQSPAPAPTPTPAPSPASAPIPTPTPAPAPAPAAAPAGSTGTGGPGVGSGGAGSGGDPARPGLSQQQRASQRKAQVRGLPRAKKLEKLGVFSACKANETCKCNGWKNPKPPTAPRMDLQQPAANLSELCRSCEHPLADHVSHLENVSEDEINRLLGMVVDVENLFMSVHKEEDTDTKQVYFYLFKLLRKCILQMTRPVVEGSLGSPPFEKPNIEQGVLNFVQYKFSHLAPRERQTMFELSKMFLLCLNYWKLETPAQFRQRSQAEDVATYKVNYTRWLCYCHVPQSCDSLPRYETTHVFGRSLLRSIFTVTRRQLLEKFRVEKDKLVPEKRTLILTHFPKFLSMLEEEIYGANSPIWESGFTMPPSEGTQLVPRPASVSAAVVPSTPIFSPSMGGGSNSSLSLDSAGAEPMPGEKRTLPENLTLEDAKRLRVMGDIPMELVNEVMLTITDPAAMLGPETSLLSANAARDETARLEERRGIIEFHVIGNSLTPKANRRVLLWLVGLQNVFSHQLPRMPKEYIARLVFDPKHKTLALIKDGRVIGGICFRMFPTQGFTEIVFCAVTSNEQVKGYGTHLMNHLKEYHIKHNILYFLTYADEYAIGYFKKQGFSKDIKVPKSRYLGYIKDYEGATLMECELNPRIPYTELSHIIKKQKEIIKKLIERKQAQIRKVYPGLSCFKEGVRQIPVESVPGIRETGWKPLGKEKGKELKDPDQLYTTLKNLLAQIKSHPSAWPFMEPVKKSEAPDYYEVIRFPIDLKTMTERLRSRYYVTRKLFVADLQRVIANCREYNPPDSEYCRCASALEKFFYFKLKEGGLIDK SEQ ID NO: 2
(Mus musculus K(lysine) acetyltransferase 2A (Kat2a), transcript variant 1, mRNA (NM_020004) 2493 out of 3048 bp) MAEPSQAPNPVPAAQPRPLHSPAPAPTSTPAPSPASASTPAPTPAPAPAPAAAPAGSTGSGGAGVGSGGDPARPGLSQQQRASQRKAQVRGLPRAKKLEKLGVFSACKANETCKCNGWKNPKPPTAPRMDLQQPAANLSELCRSCEHPLADHVSHLENVSEDEINRLLGMVVDVENLFMSVHKEEDTDTKQVYFYLFKLLRKCILQMTRPVVEGSLGSPPFEKPNIEQGVLNFVQYKFSHLAPRERQTMFELSKMFLLCLNYWKLETPAQFRQRSQSEDVATYKVNYTRWLCYCHVPQSCDSLPRYETTHVFGRSLLRSIFTVTRRQLLEKFRVEKDKLVPEKRTLILTHFPKFLSMLEEEIYGANSPIWESGFTMPPSEGTQLVPRPATVSATVVPSFSPSMGGGSNSSLSLDSAGTEPMPAGEKRKLPENLTLEDAKRLRVMGDIPMELVNEVMLTITDPAAMLGPETSLLSANAARDETARLEERRGIIEFHVIGNSLTPKANRRVLLWLVGLQNVFSHQLPRMPKEYIARLVFDPKHKTLALIKDGRVIGGICFRMFPTQGFTEIVFCAVTSNEQVKGYGTHLMNHLKEYHIKHSILYFLTYADEYAIGYFKKQGFSKDIKVPKSRYLGYIKDYEGATLMECELNPRIPYTELSHIIKKQKEIIKKLIERKQAQIRKVYPGLSCFKEGVRQIPVESVPGIRETGWKPLGKEKGKELKDPDQLYTTLKNLLAQIKSHPSAWPFMEPVKKSEAPDYYEVIRFPIDLKTMTERLRSRYYVTRKLFVADLQRVIANCREYNPPDSEYCRCASALEKFFYFKLKEGGLIDK SEQ ID NO: 3
(ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-17) UCCUGUUUAACCACGGGCU
(Target sequence: GUUCCUGGCAUUCGAGAGA)

SEQ ID NO: 4
(ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-18) CAAGGACCGUAAGCUCUCU
(Target sequence: GUUCCUGGCAUUCGAGAGA) SEQ ID NO: 5
(ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-19) CGAUGAUGCACUGGGCCUU
(Target sequence: GCUACUACGUGACCCGGAA)
SEQ ID NO: 6
(ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-20) UGAGUACAGAAACCCGCUU
(Target sequence: ACUCAUGUCUUUGGGCGAA)

In the present specification, the term "diagnosis" refers to confirming the existence or characteristics of a pathological condition, and in this specification, "prognosis" refers to a prospect for a future symptom or course determined by diagnosing a disease. The prognosis prediction also includes prediction of the patient's response to the disease treatment and the course of treatment. Preferably, in the present invention, the prognosis may mean overall survival or disease free survival rate, but is not limited thereto.

The term "biomarker" used in the present invention is a substance that can be determined by classifying whether or not inflammation occurs or the degree of inflammation progression, and is expressed in cells or tissues in which inflammation has occurred compared to cells or tissues in which inflammation has not occurred. It includes proteins or mRNAs that show differences by increasing or decreasing. For the purposes of the present invention, a biomarker for diagnosing inflammation or predicting prognosis is a GCN 5 protein in a sample, or a gene encoding it, and diagnostic efficiency can be improved by using a set containing one or two or more of these biomarkers.

Hereinafter, a biomarker for diagnosing inflammatory diseases, a composition for treating inflammatory diseases, and a composition for diagnosing inflammatory diseases or predicting prognosis including the present invention GCN5 will be described in detail with reference to the accompanying drawings.

Microglial is present in the central nervous system (CNS) and brain and is involved in the protection and recovery of primary neurons. However, many studies have been reported that activated glial cells induce neuroinflammatory reactions, and neuroinflammatory reactions lead to various neurodegenerative diseases. Activated glial cells include nitrite (NO), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). ) And induces the activities of pro-inflammatory cytokines such as IL-1b, IL-6, and TNF-a (Graeberand Streit, 2010).

These substances have been reported to be involved in the pathologic mechanisms of neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease (Kim and Joh, 2006; Koning et al., 2007; Krause and Muller, 2010; Moller, 2010). Therefore, the control of neuroinflammation can play an important role in the prevention of neurodegenerative diseases.

On the other hand, GCN5 is a histone acetyltransferase (HAT), which mainly acts as a transcriptional activator. GCN5 also acts as a repressor of NF-kappa-B by promoting the ubiquitination of the NF-kappa-B subunit RELA in a HAT-independent manner.

Under this background, the present inventors were searching for a novel biomarker capable of diagnosing neurodegenerative diseases, and when GCN5 was inhibited in BV-2 glial cells stimulated with lipopolysaccharide (LPS), gene expression of neuroinflammation mediators It was confirmed that this decrease, and the change pattern of GCN5 by LPS in BV-2 glial cells was analyzed, and through this, the possibility of GCN5 as a novel biomarker capable of diagnosing inflammatory diseases and neurodegenerative diseases was confirmed.

The present invention provides a biomarker for diagnosing inflammatory diseases including GCN5 protein. The marker according to the present invention can be used for diagnosis of inflammatory diseases by detecting the nucleic acid or protein level (concentration).

In addition, the biomarker in the present invention including GCN5 can be used to diagnose neurodegenerative diseases, and the neurodegenerative diseases are multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Lou Gehrig. Any selected from the group consisting of amyotrophic lateral sclerosis, huntingington's disease, fronto-temporal dementia, cortical-basal degeneration, and progressive supranuclear palsy. There may be one or more, and particularly preferably Parkinson's disease, but any neurodegenerative disease that can be diagnosed through detection of the GCN5 nucleic acid or protein level may be included without limitation. In addition, the inflammatory disease may be any one or more selected from the group consisting of sepsis, inflammatory bowel disease (Crohn's disease, etc.), brain nerve inflammation, and atopy.

According to a preferred embodiment of the present invention, the brain nerve inflammation refers to inflammation occurring in the hippocampus (Hippocampus), striatum (STRIATUM), or substantia nigra compacta brain tissue, but is not limited thereto.

In addition, it may be provided as a kit for diagnosing inflammatory diseases including a detection reagent that specifically binds to the biomarker of the present invention or a nucleic acid sequence of a gene encoding the biomarker.

The detection reagent that specifically binds to the biomarker is at least one selected from the group consisting of antibodies, antibody fragments, and aptamers, and the detection reagent that specifically binds to the nucleic acid sequence of the gene encoding the biomarker is a primer ( primer), probes, and anti-sense nucleotides.

According to an embodiment of the present invention, a binding agent or an array containing a binding agent that specifically binds to the biomarker of the present invention or an mRNA derived from a gene encoding the same is used.

In another embodiment, a sandwich type immunoassay such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay), etc. may be used. Such a method is applied to a first antibody bound to a solid substrate such as beads, membranes, slides or microtiterplates made of a solid substrate such as glass, plastic (e.g. polystyrene), polysaccharide, nylon or nitrocellulose. After adding, the labeling material that can be directly or indirectly detected, for example, a radioactive material such as 3H or 125I, a fluorescent material, a chemiluminescent material, a hapten, biotin, digoxigenin, etc. Alternatively, the protein can be qualitatively or quantitatively detected through binding of an antibody conjugated with an enzyme such as horseradish peroxidase, alkaline phosphatase, and maleate dehydrogenase capable of emitting light.

Other immune response-based methods can be used, and in other embodiments, immunoelectricity such as Ouchterlony plate, Western blot, Crossed IE, Rocket IE, Fused Rocket IE, and Affinity IE, which can simply detect a marker through antigen-antibody binding. Immuno Electrophoresis can be used. Reagents or substances used in this method are known, for example, antigen-antibody reactions, substrates that specifically bind to the markers, nucleic acids or peptide aptamers, receptors or ligands that specifically interact with the markers, or It can be detected through reaction with a cofactor, or a mass spectrometer can be used. The reagent or material that specifically interacts or binds to the marker of the present application may be used in a chip method or in combination with nanoparticles. By analyzing the intensity of the final signal by the above-described immunoassay process, that is, performing signal contrast with a normal sample, it is possible to diagnose whether a disease has occurred.

Each of the markers herein can be used in the diagnosis of inflammatory diseases or neurodegenerative diseases through quantitative and/or qualitative detection at the nucleic acid level, particularly at the mRNA level. A variety of known methods can be used. For example, reverse transcription polymerase chain reaction (RT-PCR)/polymerase chain reaction, competitive RT-PCR, real-time RT-PCR, nuclease protection assay (NPA) for detection at the RNA level, expression level or pattern. For example, RNase, S1 nuclease analysis, in situ hybridization, DNA microarray or a method using a chip or Northern blot may be used, and such an analysis method is known and can be performed using a commercially available kit, and those skilled in the art If so, it will be possible to select one suitable for the practice of the present application. For example, Northern blot can know the size of transcripts present in cells, has the advantage of using a variety of probes, NPA is useful for multi-marker analysis, and in situ hybridization is used for cells of transcripts such as mRNA. Alternatively, it is easy to determine the location in the tissue, and the reverse transcription polymerase chain reaction is useful for detecting a small amount of samples.

In a method for measuring the presence or absence of the mRNA and its amount or pattern by RT-PCR, as a detection reagent, for example, a probe and/or primer pair specific to the mRNA of the present marker is included. “Primer” or “probe” means a nucleic acid sequence that can bind complementarily to a template and has a free 3'hydroxyl group that allows reverse transcriptase or DNA polymerase to initiate replication of the template. do. The detection reagent used herein may be labeled with a color, luminescence, or fluorescent substance as described above for signal detection. In one embodiment, Northern blot or reverse transcription PCR (polymerase chain reaction) is used to detect mRNA. In the latter case, a specific gene in the sample is detected using a specific primer or a combination of primers and probes after separating the RNA of the sample, particularly mRNA, and then synthesizing cDNA therefrom. Or it is a method that can determine the expression level.

As described above, when GCN5 is suppressed in BV-2 glial cells stimulated with Lipopolysaccharide (LPS), the expression of genes and proteins of neuroinflammation mediators is reduced. In BV-2 glial cells, it was confirmed that the binding of GCN5 to p65, a subunit of NF-kappaB, which is one of the inflammatory signaling mechanisms, is regulated. It can be seen that GCN5 can be used as a biomarker capable of diagnosing degenerative diseases.

In an embodiment of the present invention, the GCN5 inhibitor may be CPTH2 represented by Formula 1 below. Therefore, it can be seen that the composition comprising the GCN5 inhibitor of the present invention, specifically CPTH2 or a pharmaceutically acceptable salt thereof, as an active ingredient, is effective in preventing, treating or improving inflammatory diseases or neurodegenerative diseases.

In the present invention, a compound of Formula 1 and a compound of Formula 2 may be used. Hereinafter, the compound of Formula 1 is referred to as CPTH2, and the compound of Formula 2 is referred to as TAK-242.

[Chemical Formula 1] CPTH2

[Chemical Formula 2] TAK-242

In another embodiment of the present invention, TAK-242 may be used as an inhibitor of TLR4 receptor or GCN5 to which LPS binds. Therefore, it can be seen that the composition comprising TAK-242 or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient is effective in preventing, treating or improving inflammatory diseases or neurodegenerative diseases.

According to another embodiment of the present invention, a health functional food composition comprising CPTH2, TAK-242 or a pharmaceutically acceptable salt thereof as an active ingredient exhibits an effect of improving memory.

[Experimental Example 1]

Cell culture

Inactivated 5% FBS and 100 units/ml 1% penicilline/streptomycin were incubated in DMEM with 5% CO₂ and 95% O₂ incubator maintaining humidity.

In all experiments, the number of cells was 1.25 x 10 cells/ml. LPS was treated with 200 ng/ml in BV-2 glial cells, respectively, and CPTH2 was diluted in medium and pretreated with 1, 5 and 15 μM concentrations for 1 hour in BV-2 glial cells.

[Experimental Example 2]

Experimental animals

Male C57BL/6J mice (22-25 g, 8 weeks old) were purchased from Daehan Biolink. They were divided into 3 groups and purified for 1 week under standard conditions, and then used for experiments (temperature 22±2 ℃, humidity 55±5%, 12 h-light/dark cycle, food/water free diet).

All experiments were conducted in accordance with the guidelines of NIH publication No. 85-23, revised 1985 and Konkuk University IACUC (Institutional Animal Care and Use Committee).

[Experimental Example 3]

Animal group used in the experiment and drug administration method

Mice were divided into 3 groups of 5 mice each (1. vehicle, 2. LPS 5 mg/kg, 3. LPS+TAK 242 3 mg/kg) and then put into the experiment, and TAK-242 and LPS were dissolved in physiological saline and used. I did. TAK-242 was administered to mice via oral administration 3 hours before LPS treatment, and 5 mg/kg of LPS was administered via intraperitoneal injection.

According to an embodiment of the present invention, a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising as an active ingredient any one or more selected from the group consisting of CPTH2 and TAK-242 or a pharmaceutically acceptable salt thereof may be provided. .

In addition, in the pharmaceutical composition for preventing or treating neurodegenerative diseases, the neurodegenerative diseases are multiple sclerosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. (huntington's disease), fronto-temporal dementia, cortical-basal ganglia degeneration, and progressive supranuclear palsy.

The kind of neurodegenerative disease is as described above, and the pharmaceutical composition and/or health functional food composition of the present invention is particularly useful for the prevention, improvement or treatment of Parkinson's disease.

As used herein, the term "prevention" refers to any action of inhibiting or delaying the onset of the disease by administration of a composition comprising a GCN5 inhibitor or a pharmaceutically acceptable salt thereof according to the present invention, and the term, " "Treatment" refers to any action in which the symptoms of the disease are improved or beneficially changed by the administration of the composition according to the present invention.

According to another embodiment of the present invention, a health functional food for preventing or improving inflammatory diseases or neurodegenerative diseases comprising any one or more selected from the group consisting of CPTH2 and TAK-242 or a pharmaceutically acceptable salt thereof as an active ingredient Compositions can be provided.

In the health functional food composition for preventing or improving neurodegenerative diseases of the present invention, the neurodegenerative diseases are, multiple sclerosis, Parkinson's disease, Alzheimer's disease, ALS (amyotrophic lateral sclerosis). , Huntington's disease, fronto-temporal dementia, cortical-basal degeneration, and progressive supranuclear palsy. In addition, the inflammatory disease may be any one or more selected from the group consisting of sepsis, inflammatory bowel disease (Crohn's disease, etc.), encephalitis, and atopy.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are provided so that the present invention may be sufficiently understood by those of ordinary skill in the art, and may be modified in various other forms, and the scope of the present invention is limited to the examples described below. It is not.

According to another embodiment of the present invention, (a) measuring the expression level of a biomarker containing GCN5 protein in a biological sample isolated from an individual, (b) normalizing the expression level of the biomarker in the step (a). Diagnosing a patient with an inflammatory disease or neurodegenerative disease when it is higher than the expression level of the normal control compared to the expression level of the control group, and (c) a compound represented by the following formula (1) in a biological sample isolated from the inflammatory disease patient, the following formula: A method of providing customized treatment information for each patient with inflammatory disease or neurodegenerative disease comprising the step of selecting a composition having a high degree of inhibition of gene expression of GCN5 by treating a composition containing the compound represented by 2 may be provided, Accordingly, it is possible to select an advantageous treatment method for each patient.

In the method of providing customized treatment information for each patient of the inflammatory disease or neurodegenerative disease, the inflammatory disease may be any one or more selected from the group consisting of sepsis, inflammatory bowel disease (Crohn's disease, etc.), encephalitis, and atopy.

According to an embodiment of the present invention, the present invention may use a biomarker for diagnosing inflammatory diseases and neurodegenerative diseases including GCN5 protein, and includes a detection reagent that specifically binds to the nucleic acid sequence of the biomarker or a gene encoding the biomarker. A kit for diagnosing an inflammatory disease or a neurodegenerative disease may be used.

According to a preferred embodiment of the present invention, the detection reagent specifically binding to the biomarker is at least one selected from the group consisting of antibodies, antibody fragments, and aptamers, and the nucleic acid sequence of the gene encoding the biomarker is The detection reagent that specifically binds may be any one or more selected from the group consisting of a primer, a probe, and an antisense nucleotide.

The present invention also includes the steps of: (a) measuring the expression level of the biomarker of claim 1 in a biological sample isolated from an individual; (b) comparing the expression level of the biomarker in step (a) with the expression level of a normal control group, and diagnosing a patient with a neurodegenerative disease if it is higher than that of the normal control group; (c) treating a composition containing CPTH2 and TAK-242 in a biological sample isolated from the neurodegenerative disease patient, respectively, to select a composition having a high degree of inhibition of gene expression of GCN5; customized for each patient with a neurodegenerative disease comprising Provides a way to provide treatment information.

As used herein, detection includes quantitative and/or qualitative analysis, and includes detection of presence and absence and detection of expression levels, and such methods are known in the art, and those skilled in the art may select an appropriate method for the practice of the present application. I will be able to.

As used herein, a biological sample refers to a substance or a mixture of substances containing one or more components capable of detecting a biomarker, and includes cells, tissues or body fluids derived from living organisms, particularly humans, such as whole blood, urine, plasma, and serum. However, it is not limited to this. It also includes cells or tissues cultured in vitro as well as directly derived from an organism. Various samples may be used for the detection of the neurodegenerative disease marker according to the present application, but are not limited thereto.

The marker according to the present application can be detected at the level of detection of the presence or absence of a nucleic acid, particularly an mRNA and/or a protein, and/or its expression level itself, a change in the expression level, and a difference in the expression level through quantitative or qualitative analysis.

Among the present inventions, the diagnostic markers for inflammatory diseases or neurodegenerative diseases are based on their functional characteristics and/or antigenic characteristics. This allows detection using nucleic acids encoding proteins, in particular agents that interact specifically at the mRNA and/or protein level.

1-1 Nitric Oxide Assay (NO Assay)

In order to confirm the inhibitory effect of CPTHH2 on inflammatory diseases (especially neuroinflammation), the inhibitory effect on the amount of nitric oxide (NO) released in BV-2 glial cells was confirmed through the NO assay. BV-2 glial cells were cultured to 1.25 X 10 ⁵ cells/well in a 24 well plate, and CPTH2 was pretreated for 1 hour at each concentration (1, 5 or 15 μM). And the cells were stimulated with LPS (200 ng/ml). After 24 hours, 100 μl of the cell supernatant was added to 96 wells and reacted with Griess reagent (1% sulfanylamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, 2.5% H3PO4). After that, the value was measured using a Vmax 96-well microplate spectrophotometer with a wavelength of 550 nm. As a result, as shown in Fig. 1A, it was confirmed that the nitric oxide inhibitory effect was shown in a concentration-dependent manner of CPTH2, an inhibitor of GCN5, in BV-2 glial cells.

1-2 MTT assay

BV-2 glial cells were cultured to 1.25 X 10 ⁵ cells/well in a 24-well plate, and CPTH2 was pretreated for 1 hour at each concentration (1, 5 or 15 μM). And the cells were stimulated with LPS (200 ng/ml). After the MTT reagent was added to the supernatant, the values obtained by measuring the surviving cells were expressed as a percentage. As a result, as shown in FIG. 1B, it was confirmed that cytotoxicity by treatment by LPS and CPTH2 concentration was not observed when compared to the control group that was not treated with anything.

Through these results, it was confirmed that CPTHH2, an inhibitor of GCN5, can effectively treat inflammatory diseases and neurodegenerative diseases by inhibiting inflammatory reactions, particularly neuroinflammatory reactions.

Inhibitory Effect of iNOS and COX-2 on Protein Expression by CPTH2 Treatment

In order to confirm the change in the protein expression pattern of iNOS and COX-2 (cyclooxygenase type 2), which are enzymes involved in inflammation production, in this example, LPS and CPTH2 were added to BV-2 glial cells by concentration (1, 5 or 15 μM) and cultured. After 20 hours, proteins were identified and Western blot was performed to confirm the expression patterns of iNOS and COX-2 proteins.

Specifically, for the identification of proteins in BV-2 glial cells, the cells were identified using a sample buffer [Glycerol, 10% SDS, β-mercaptomethanol, bromo-phenol blue, 0.5M Tris-Hcl]. The same amount of protein was electrophoresed on 10% SDS-PAGE, and then transferred from the gel to the membrane using 0.45 μm polyvinylidene fluoride (PVDF, Millipore). Dilute anti-β-actin, anti-COX-2 (1:2000; Santa Cruz), and anti-iNOS (1:2000; Cell Signaling) as a primary antibody and perform O/N (over-night) at 4°C. After reacting an antibody with HRP (Horseradish peroxidase) as a secondary antibody for 1 hour at room temperature, react with an ECL kit (pierce, CA), and confirm the result using Davinch Cas-400SM (Davinch-K, Korea) I did. β-actin was used as a control for protein expression analysis.

As a result, as shown in FIG. 2, the expression levels of iNOS and COX-2 in the group treated with only LPS increased compared to the control group, but the level of expression in the group treated with CPTH2 by concentration was CPTH2 compared to the group treated with only LPS. It was confirmed that the expression levels of iNOS and COX-2 proteins were decreased in a concentration-dependent manner.

Through these results, it was confirmed that CPTHH2, an inhibitor of GCN5, can effectively treat inflammatory diseases and neurodegenerative diseases by inhibiting inflammatory reactions, particularly neuroinflammatory reactions.

Confirmation of the interaction between GCN5 and RelA in LPS-stimulated BV-2 microglia

Neuroglial cells are stimulated by an external stimulus to activate the NF-κB mechanism to express inflammatory genes, thereby expressing inflammatory cytokines such as TNF-α, IL-1β, and IL-6. The present inventors tried to confirm the role and mechanism analysis of GCN5 in the NF-κB mechanism.

In this example, BV-2 glial cells were treated with LPS and cultured for each time period. After 30 minutes, the protein was identified and a specific binding protein was obtained by adding GCN5 antibody or p65 antibody. Western blot was performed to confirm the expression pattern according to the mutual binding of GCN5, ac-p65, and p65 proteins.

The same amount of protein was electrophoresed on 10% SDS-PAGE, and then transferred from the gel to the membrane using 0.45 μm polyvinylidene fluoride (PVDF, Millipore). Dilute anti-GCN5 (1;1000, Sigma Aldrich), anti-β-actin (1:5000; Sigma Aldrich), anti-ac-p65, anti-p65 (1:2000; Cell Signaling) with a primary antibody. O/N (over-night) at 4°C and an antibody having HRP (Horseradish peroxidase) as a secondary antibody was reacted at room temperature for 1 hour, and then reacted with an ECL kit (pierce, CA), and Davinch Cas-400SM ( Davinch-K, Korea) was used to confirm the results. β-actin was used as a control for protein expression analysis.

In this example, it was possible to confirm the progression of the NF-κB mechanism through the mutual coupling between GCN5 and RelA due to LPS treatment. In order to confirm the activation of NF-κB, the amount of change in p-p65 and ac-p65 was confirmed through BV-2 glial cells. As a result, as shown in FIG. 3, when LPS treatment in BV-2 glial cells, it was confirmed that the amount of protein increased due to the binding of GCN5 and RelA.

Through these results, it was confirmed that CPTHH2, an inhibitor of GCN5, can effectively treat inflammatory diseases and neurodegenerative diseases by inhibiting inflammatory reactions, particularly neuroinflammatory reactions.

Identification of protein expression mechanisms of iNOS, p-p65, ac-p65 and GCN5 by neuroinflammation in LPS-stimulated C57BL/6J mice

In order to confirm the change in the protein expression pattern of iNOS and COX-2 (cyclooxygenase type 2), which are enzymes involved in inflammation production, in this example, C57BL/5J mice were treated with 5 mg/kg of LPS. After 6 hours, brain tissue was obtained to identify proteins, and Western blot was performed to confirm the expression patterns of iNOS protein and GCN5.

Specifically, for the identification of proteins in C57BL/6J mouse brain tissue, cells were identified using Sample buffer [Glycerol, 10% SDS, β-mercaptomethanol, bromo-phenol blue, 0.5M Tris-Hcl]. The same amount of protein was electrophoresed on 10% SDS-PAGE, and then transferred from the gel to the membrane using 0.45 μm polyvinylidene fluoride (PVDF, Millipore). Anti-β-actin(1:5000, Sigma Aldrich), anti-iNOS, anti-p65, anti-p-p65, anti-ac-p65 (1:2000; Cell Signaling) anti-GCN5 (1 :1000; Sigma Aldrich) was diluted, O/N (over-night) at 4°C, and an antibody having HRP (Horseradish peroxidase) as a secondary antibody was reacted at room temperature for 1 hour, and then ECL kit (pierce, CA) And confirmed the results using Davinch Cas-400SM (Davinch-K, Korea). β-actin was used as a control for protein expression analysis.

As a result, as shown in FIG. 4, it was confirmed that the expression levels of iNOS, p-p65, ac-p65, and GCN5 in the group treated with only LPS were increased compared to the control group.

Effect of TAK-242 Treatment on Gene Expression Inhibition of iNOS and GCN5

In order to confirm the changes in the expression patterns of the iNOS and GCN5 genes, which are representative inflammatory mediators, in this example, the expression patterns of the inflammatory mediator genes were evaluated in C57BL/6J mice with 5 mg/kg of LPS and 3 mg/kg of TAK-242. After 6 hours of administration, RNA was identified for each part of brain tissue, and RT-PCR was performed to analyze the expression pattern of the gene through a quantitative method.

Isolation and RT-PCR of all RNAs were performed as follows. All RNA identification of C57BL/6J mouse brain tissue was isolated using Trizaol reagent according to the instructions suggested by the manufacturer. Add 200µl of Trizol reagent to C57BL/6J brain tissue, add 50µl of chloroform, mix well, react at room temperature for 5 minutes, and centrifuge (4℃, 13,000rpm, 15 minutes). 200 μl of the supernatant is transferred to a new tube. 200µl of isopropanol in the same amount was added, reacted at room temperature for 10 minutes, and centrifuged (4℃, 16000rpm, 15 minutes). Remove the supernatant, wash twice with 75% EtOH, dry the pellet well, and dissolve it with DEPC-treated distilled water. RT (real-time) qPCR was adjusted to the concentration of 2.5 μg/μl of the prepared RNA, and the final volume was equal to 10 μl. After pre-heating with 1 μl of Oligo(dT) 15 primer and 1 μl of random primers, 5 X Reaction Buffer 4µl, MgCl ₂ 2µl, dNTP 1µl under the reaction conditions 25℃ for 5 minutes, 42℃ for 60 minutes, 70℃ for 15 minutes, 4℃ for more than 5 minutes, total 20 µl was synthesized, and the concentration of the template used was 0.5 Dyne qPCR 2X premix (DyneBio, Korea) was used as a reaction composition, 2 µl of template, 10 µl of PreMix, 1 µl of primer-F, 1 µl of primer-R, and DEPC 6 µl of reaction conditions 94°C for 5 minutes, 94°C for 30 seconds-58°C for 1 minute-72°C for 1 minute 70 times, 72°C for 5 minutes, 4°C storage for a total of 20µl was reacted.

As a result, as shown in FIG. 5, it was confirmed that the expression of iNOS gene and GCN5 gene expression, which are inflammatory mediators, were inhibited when TAK-242 was administered.

Through these results, it was confirmed that TAK-242, an inhibitor of GCN5, can effectively treat inflammatory diseases and neurodegenerative diseases by inhibiting inflammatory reactions, especially neuroinflammatory reactions.

Confirmation of changes in the expression level of GCN5 mRNA by tissue in C57BL/6 mice treated with LPS

8-week-old C57BL/6 mice (average 23~25g) were received at Daehan Biolink and stabilized in the SPF room for a week. After that, LPS (lipopolysaccharide) was injected intraperitoneally at a concentration of 5mg/kg, and after 6 hours, brain, liver and heart were obtained. RNA samples were obtained from the obtained tissue using the chloroform-phenol method, and cDNA was synthesized using reverse transcriptase, and the expression of GCN5 mRNA was compared through the RT-PCR method using DNA polymerase.

All RNA isolation and RT-PCR were performed using the following method. All RNA identification was isolated using Trizol reagent according to the instructions suggested by the manufacturer. C57BL/6 mouse brain, liver, and heart tissues were crushed by adding 500 ul of Trizol reagent to the tissues, mixed well with 100 ul of chloroform, reacted for 5 minutes at room temperature, and then centrifuged (4 degrees, 12,000 rpm, 10 minutes). Transfer 250 ul of the supernatant to a new tube. Add the same amount of isopropanol, react at room temperature for 15 minutes, and centrifuge (4 degrees, 12,000 rpm, 15 minutes). Remove the supernatant, wash twice with 75% ethanol, dry the pellet well, and dissolve it with DEPC-treated distilled water. The resulting RNA concentration was adjusted to 2.5 ug/ul, and the final volume was equal to 10 ul.After pre-heating with Oligo(dT) 15 primers 1 ul and Random primers 1 ul, 5X reaction buffer 4ul, MgC12 2ul, 1 ul of dNTP was synthesized for a total of 20 ul over 25 degrees 5 minutes, 42 degrees 60 minutes, 70 degrees 15 minutes, and 4 degrees 5 minutes under reaction conditions, and the concentration of the template used was diluted to 0.5 ug/ul.

As a result, referring to FIG. 6, it was confirmed that when LPS was injected into C57BL/6 mice, the expression of GCN5 mRNA in brain, liver and heart was increased when compared to the control group not treated with LPS, especially in brain tissue. It was confirmed that the expression of GCN5 mRNA was further increased. That is, it can be seen that GCN5 is more involved in neuroinflammation in brain tissue than in other tissues.

Changes in GCN5 and iNOS mRNA expression levels by neuroinflammation in C57BL/6 mice treated with LPS

8-week-old C57BL/6 mice (average 23~25g) were received at Daehan Biolink and stabilized in the SPF room for a week. Thereafter, LPS (lipopolysaccharide) was injected intraperitoneally at a concentration of 5 mg/kg, and after 6 hours, brain tissue was separated and obtained. RNA samples were obtained from the obtained tissue using the chloroform-phenol method, and cDNA was synthesized using reverse transcriptase, and the expression of GCN5 mRNA was compared through the RT-PCR method using DNA polymerase.

All RNA isolation and RT-PCR were performed using the following method. All RNA identification was isolated using Trizol reagent according to the instructions suggested by the manufacturer. C57BL/6 mouse brain, liver, and heart tissues were crushed by adding 500 ul of Trizol reagent to the tissues, mixed well with 100 ul of chloroform, reacted for 5 minutes at room temperature, and then centrifuged (4 degrees, 12,000 rpm, 10 minutes). Transfer 250 ul of the supernatant to a new tube. Add the same amount of isopropanol, react at room temperature for 15 minutes, and centrifuge (4 degrees, 12,000 rpm, 15 minutes). Remove the supernatant, wash twice with 75% ethanol, dry the pellet well, and dissolve it with DEPC-treated distilled water. The resulting RNA concentration was adjusted to 2.5 ug/ul, and the final volume was equal to 10 ul.After pre-heating with Oligo(dT) 15 primers 1 ul and Random primers 1 ul, 5X reaction buffer 4ul, MgC12 2ul, A total of 20 ul of dNTP was synthesized at 25 degrees 5 minutes, 42 degrees 60 minutes, 70 degrees 15 minutes, 4 degrees 5 minutes or more under reaction conditions, and the concentration of the template used was diluted to 0.5 ug/ul.

As a result, referring to FIG. 7, it was confirmed that when PS was injected into C57BL/6 mice, the expression of GCN5 mRNA in brain SNpc, Hippocampus, and Cortex was increased when compared to the control group not treated with LPS, and increased. It was confirmed that the expression of GCN5 mRNA was inhibited when TAK-242, a TLR4-specific inhibitor, was treated with LPS. That is, it can be seen that GCN5 may play an important role in neuroinflammation in brain tissues, especially in the hippocampus, striatum, and SNpc.

Changes in mRNA expression levels of GCN5 and pro-inflammatory mediators (iNOS, TNF-a and IL-6) due to neuroinflammation in glial cells (microglia) and astrocytes (astrocyte)

C57BL/6 mice of 2 days of age) were received at Daehan Biolink, and brain tissue was obtained, and blood was removed and homogenized using sterile filter paper. The brain tissue using homogenization is filtered through a nylone mesh, and the medium is added to centrifuge for 5 minutes, and the supernatant is removed. After seeding with the medium, cultured in a cell incubator for 1 day, replaced with fresh medium to remove cell debris, and then changed the medium every 5 days. Cells cultured for 3 weeks were washed with Serum Free medium and incubated for 15 minutes with 0.05% TE for mild trypsinization, and then gently shaken to obtain astrocytes. Incubated cells with 0.25% TE for 10 minutes for mild trypsinization. Obtain glial cells. The obtained glial cells and astrocytes are seeded and stored in a cell incubator for one day. After that, LPS (lipopolysaccharide) was treated at a concentration of 200 ng/ml and cells were obtained after 6 hours. The obtained cells were obtained by obtaining RNA samples using the chloroform-phenol method, and cDNA was synthesized using reverse transcriptase, and RT using DNA polymerase. -The expression of GCN5 mRNA was compared through the PCR method.

All RNA isolation and RT-PCR were performed using the following method. All RNA identification was isolated using Trizol reagent according to the instructions suggested by the manufacturer. C57BL/6 mice brain, liver, and heart tissues were crushed by adding 500 ul of Trizol reagent, and 100 ul of chloroform was mixed well, reacted for 5 minutes at room temperature, and then centrifuged (4 degrees, 12,000 rpm, 10 minutes). Transfer 250 ul of the supernatant to a new tube. Add the same amount of isopropanol, react at room temperature for 15 minutes, and centrifuge (4 degrees, 12,000 rpm, 15 minutes). Remove the supernatant, wash twice with 75% ethanol, dry the pellet well, and dissolve it with DEPC-treated distilled water. The resulting RNA concentration was adjusted to 2.5 ug/ul, and the final volume was equal to 10 ul.After pre-heating with Oligo(dT) 15 primers 1 ul and Random primers 1 ul, 5X reaction buffer 4ul, MgC1 _{2 2ul} , 1 ul of dNTP was synthesized for a total of 20 ul over 25 degrees 5 minutes, 42 degrees 60 minutes, 70 degrees 15 minutes, and 4 degrees 5 minutes under reaction conditions, and the concentration of the used template was diluted to 0.5 ug/ul.

As a result, referring to FIG. 8, the expression of the inflammatory mediators iNOS, TNF-a, and IL-6, including GCN5, in glial cells treated with LPS increased, but the expression of GCN5 mRNA was not increased in astrocytes. It was confirmed that the mRNA expression of the inflammatory mediator was increased.

Changes in the expression levels of GCN5, ac-p65, and p-p65 proteins due to neuroinflammation in glial cells (microglia) and astrocytes (astrocyte)

C57BL/6 mice of 2 days of age) were received at Daehan Biolink, and brain tissue was obtained, and blood was removed and homogenized using sterile filter paper. The brain tissue using homogenization is filtered through a nylone mesh, and the medium is added to centrifuge for 5 minutes, and the supernatant is removed. After seeding with the medium, cultured in a cell incubator for 1 day, replaced with fresh medium to remove cell debris, and then changed the medium every 5 days. Cells cultured for 3 weeks were washed with Serum Free medium and incubated for 15 minutes with 0.05% TE for mild trypsinization, and then gently shaken to obtain astrocytes. Incubated cells with 0.25% TE for 10 minutes for mild trypsinization. Obtain glial cells. The obtained glial cells and astrocytes are seeded and stored in a cell incubator for one day. Thereafter, LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 30 minutes. Proteins were identified in the obtained cells, and expressions of GCN5, ac-p65, and p-p65 proteins were confirmed using Western blot.

Specifically, cells were identified using a sample buffer [Glycerol, 10% SDS, beta-mercaptomethanil, bromo-phenol blue, 0.5M Tris-HCL]. The same amount of protein was electrophoresed on 10% SDS-PAGE and then transferred from the gel to the membraine using 0.45 um polyvinylidene fluoride (PVDF, Millipore). After diluting each antibody and over-night at 4°C, reacting the antibody with HRP as a secondary antibody at room temperature for 1 hour, then reacted with the ECL kit, and the result using Davinch Cas-400SM (Davinch-K, Korea). Was confirmed. Beta-actin was used as a control for protein expression analysis. As a result, the expression of ac-p65 and p-p65 proteins including GCN5 was increased in glial cells treated with LPS, but the expression of GCN5 protein was not increased in astrocytes, but the expression of ac-p65 and p-p65 proteins was increased. Was confirmed. From the above results, it can be seen that glial cells among brain cells are involved in the regulation by GCN5 in neuroinflammation (see FIG. 9).

Changes in phosphorylation and acetylation of p65 in BV-2 glial cells overexpressing GCN5

To increase the expression of GCN5, BV-2 glial cells were treated with Lipofectamine LTX (Thermo Fisher) according to the instructions suggested by the manufacturer. The GCN5 Lentivirus vector was purchased through abmgood, and the purchased lentivirus vector was transformed and cultured, followed by DNA isolation. DNA identification is performed using the PureLink Hipure Plasmid Midiprep Kit (Thermo, K210004), and the method of use is separated by the method provided by the manufacturer. The separated DNA isolation is injected into BV-2 glial cells and replaced with growth medium after 4 hours. Lipofectamine LTX (Thermo) is used to inject into cells, and the method of use is injected through the method recommended by the manufacturer. The injected cells were proliferated for 1 day, and LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 30 minutes. Proteins were identified in the obtained cells, and expressions of GCN5, ac-p65, and p-p65 proteins were confirmed using Western blot.

As a result, it was confirmed that BV-2 glial cells overexpressing GCN5 increased the expression of ac-p65 and p-p65 proteins compared to the non-overexpressed control group. In addition, when the LPS was treated, it was confirmed that it increased more than when it was not treated (see FIG. 10).

Changes in the expression of inflammatory mediators in BV-2 glial cells overexpressing GCN5

To increase the expression of GCN5, BV-2 glial cells were treated with Lipofectamine LTX (Thermo Fisher) according to the instructions suggested by the manufacturer. The GCN5 Lentivirus vector was purchased through abmgood, and the purchased lentivirus vector was transformed and cultured, followed by DNA isolation. DNA identification is performed using the PureLink Hipure Plasmid Midiprep Kit (Thermo, K210004), and the method of use is separated by the method provided by the manufacturer. The separated DNA isolation is injected into BV-2 glial cells and replaced with growth medium after 4 hours. Lipofectamine LTX (Thermo) is used to inject into cells, and the method of use is injected through the method recommended by the manufacturer. The injected cells were proliferated for 1 day and LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 6 hours. RNA was identified in the obtained cells, and expression of GCN5, iNOS, TNA-a, and IL-6 mRNA was confirmed using RT-PCR.

As a result, referring to FIG. 11, it was confirmed that BV-2 glial cells overexpressing GCN5 increased the expression of inflammatory mediator mRNA compared to the control group not overexpressed. In addition, when LPS was treated, it was confirmed that the expression of inflammatory mediators was further increased.

Changes in phosphorylation and acetylation of p65 in HEK293 (TLR4) cells overexpressing GCN5

To increase the expression of GCN5, HEK293 (TLR4) cells were treated with Lipofectamine LTX (Thermo Fisher) according to the instructions suggested by the manufacturer. The GCN5 Lentivirus vector was purchased through abmgood, and the purchased lentivirus vector was transformed and cultured, followed by DNA isolation. DNA identification is performed using the PureLink Hipure Plasmid Midiprep Kit (Thermo, K210004), and the method of use is separated by the method provided by the manufacturer. The isolated DNA isolation is injected into HEK293 (TLR4) cells and replaced with growth medium after 6 hours. Lipofectamine LTX (Thermo) is used to inject into cells, and the method of use is injected through the method recommended by the manufacturer. The injected cells were proliferated for 1 day and LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 30 minutes. Proteins were identified in the obtained cells, and expressions of GCN5, ac-p65, and p-p65 proteins were confirmed using Western blot.

As a result, referring to FIG. 12, it was confirmed that HEK293 (TLR4) cells overexpressing GCN5 increased the expression of ac-p65 and p-p65 proteins compared to the control group not overexpressed. In addition, it was confirmed that when LPS was treated, it increased more than when it was not treated.

Changes in phosphorylation and acetylation of p65 in HEK293 (TLR4) cells in which GCN5 expression was inhibited

To inhibit the expression of GCN5, HEK293 (TLR4) cells were treated with Lipofectamine LTX (Thermo Fisher) according to the instructions suggested by the manufacturer. GCN5 siRNA was purchased through Dharmacon, and the concentration was set and stored separately. Separately stored siRNA is injected into HEK293 (TLR4) cells and replaced with growth medium after 6 hours. Lipofectamine LTX (Thermo) is used to inject into cells, and the method of use is injected through the method recommended by the manufacturer. The injected cells were proliferated for 1 day, and LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 30 minutes. Proteins were identified in the obtained cells, and expressions of GCN5, ac-p65, and p-p65 proteins were confirmed using Western blot.

As a result, referring to FIG. 13, it was confirmed that HEK293 (TLR4) cells overexpressing GCN5 increased the expression of ac-p65 and p-p65 proteins compared to the control group not overexpressed. In addition, it was confirmed that when LPS was treated, it increased more than when it was not treated. On the other hand, it was confirmed that the experimental group in which the expression of GCN5 was suppressed did not increase even after treatment with LPS.

Confirmation of changes in the expression of inflammatory mediators in HEK293 (TLR4) cells overexpressing GCN5

To inhibit the expression of GCN5, HEK293 (TLR4) cells were treated with Lipofectamine LTX (Thermo Fisher) according to the instructions suggested by the manufacturer. GCN5 siRNA was purchased through Dharmacon, and the concentration was set and stored separately. Separately stored siRNA is injected into HEK293 (TLR4) cells and replaced with growth medium after 6 hours. Lipofectamine LTX (Thermo) is used to inject into cells, and the method of use is injected through the method recommended by the manufacturer. The injected cells were proliferated for 1 day, and LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 6 hours. RNA was identified in the obtained cells, and expression of GCN5, iNOS, TNA-a and IL-6 mRNA was confirmed using RT-PCR.

As a result, referring to FIG. 14, it was confirmed that HEK293 (TLR4) cells overexpressing GCN5 increased the expression of inflammatory mediator mRNA compared to the control group not overexpressed. In addition, when LPS was treated, it was confirmed that the expression of inflammatory mediators was further increased.

Confirmation of changes in the expression of inflammatory mediators in HEK293 (TLR4) cells that inhibited the expression of GCN5

To inhibit the expression of GCN5, HEK293 (TLR4) cells were treated with Lipofectamine LTX (Thermo Fisher) according to the instructions suggested by the manufacturer. GCN5 siRNA was purchased through Dharmacon, and the concentration was set and stored separately. Separately stored siRNA is injected into HEK293 (TLR4) cells and replaced with growth medium after 6 hours. Lipofectamine LTX (Thermo) is used to inject into cells, and the method of use is injected through the method recommended by the manufacturer. The injected cells were proliferated for 1 day, and LPS was treated at a concentration of 200 ng/ml, and cells were obtained after 6 hours. RNA was identified in the obtained cells, and expression of GCN5, iNOS, TNA-a and IL-6 mRNA was confirmed using RT-PCR.

As a result, referring to FIG. 15, it was confirmed that HEK293 (TLR4) cells overexpressing GCN5 increased the expression of inflammatory mediator mRNA compared to the control group not overexpressed. In addition, when LPS was treated, it was confirmed that the expression of inflammatory mediators was further increased. On the other hand, it was confirmed that the experimental group in which the expression of GCN5 was suppressed did not increase even after treatment with LPS. From the above results, it can be seen that the regulation of GCN5 expression can play an important role in regulating neuroinflammation or inflammation.

Confirmation of changes in PTM of GCN5 in BV-2 glial cells stimulated with LPS

BV-2 glial cells were treated with LPS, and 30 minutes later, proteins were identified, and p65 antibody was added to obtain specific binding proteins. -serine, acetyl-lysine and the expression pattern according to the mutual binding was confirmed. As a result, it was confirmed that phsophso-tyrosine was increased at the site identified as the location of GCN5 due to LPS treatment, but phospho-thereonine, phosphoe-serine, and acetyl-lysine were not changed. From the above results, it can be seen that phosphorylation of tyrosine residues takes place in the regulation of neuroinflammation of GCN5 (see FIG. 16).

Statistical analysis

In order to confirm the reproducibility of the experimental results, all experimental data of the examples of the present invention were expressed as mean values ± standard deviations, and each experiment was performed three times. For statistical analysis of the experimental results, Graphpad Prism V5.01. One-way ANOVA was performed according to the Tukey method using software.

<110> GLOCAL Industry-Academic Cooperation Foundation Konkuk University <120> COMPOSITION FOR THE TREATMENT OF INFLAMMATORY DISEASES USING CPTH2, TAK-242 AND GCN5 siRNA <130> 1068485 <150> KR 10-2019-0147540 <151> 2019-11-18 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 837 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens K(lysine) acetyltransferase 2A, mRNA (cDNA clone MGC:45022 IMAGE:5575574), complete CDs (BC032743) <400> 1 Met Ala Glu Pro Ser Gln Ala Pro Thr Pro Ala Pro Ala Ala Gln Pro 1 5 10 15 Arg Pro Leu Gln Ser Pro Ala Pro Ala Pro Thr Pro Thr Pro Ala Pro 20 25 30 Ser Pro Ala Ser Ala Pro Ile Pro Thr Pro Thr Pro Ala Pro Ala Pro 35 40 45 Ala Pro Ala Ala Ala Pro Ala Gly Ser Thr Gly Thr Gly Gly Pro Gly 50 55 60 Val Gly Ser Gly Gly Ala Gly Ser Gly Gly Asp Pro Ala Arg Pro Gly 65 70 75 80 Leu Ser Gln Gln Gln Arg Ala Ser Gln Arg Lys Ala Gln Val Arg Gly 85 90 95 Leu Pro Arg Ala Lys Lys Leu Glu Lys Leu Gly Val Phe Ser Ala Cys 100 105 110 Lys Ala Asn Glu Thr Cys Lys Cys Asn Gly Trp Lys Asn Pro Lys Pro 115 120 125 Pro Thr Ala Pro Arg Met Asp Leu Gln Gln Pro Ala Ala Asn Leu Ser 130 135 140 Glu Leu Cys Arg Ser Cys Glu His Pro Leu Ala Asp His Val Ser His 145 150 155 160 Leu Glu Asn Val Ser Glu Asp Glu Ile Asn Arg Leu Leu Gly Met Val 165 170 175 Val Asp Val Glu Asn Leu Phe Met Ser Val His Lys Glu Glu Asp Thr 180 185 190 Asp Thr Lys Gln Val Tyr Phe Tyr Leu Phe Lys Leu Leu Arg Lys Cys 195 200 205 Ile Leu Gln Met Thr Arg Pro Val Val Glu Gly Ser Leu Gly Ser Pro 210 215 220 Pro Phe Glu Lys Pro Asn Ile Glu Gln Gly Val Leu Asn Phe Val Gln 225 230 235 240 Tyr Lys Phe Ser His Leu Ala Pro Arg Glu Arg Gln Thr Met Phe Glu 245 250 255 Leu Ser Lys Met Phe Leu Leu Cys Leu Asn Tyr Trp Lys Leu Glu Thr 260 265 270 Pro Ala Gln Phe Arg Gln Arg Ser Gln Ala Glu Asp Val Ala Thr Tyr 275 280 285 Lys Val Asn Tyr Thr Arg Trp Leu Cys Tyr Cys His Val Pro Gln Ser 290 295 300 Cys Asp Ser Leu Pro Arg Tyr Glu Thr Thr His Val Phe Gly Arg Ser 305 310 315 320 Leu Leu Arg Ser Ile Phe Thr Val Thr Arg Arg Gln Leu Leu Glu Lys 325 330 335 Phe Arg Val Glu Lys Asp Lys Leu Val Pro Glu Lys Arg Thr Leu Ile 340 345 350 Leu Thr His Phe Pro Lys Phe Leu Ser Met Leu Glu Glu Glu Ile Tyr 355 360 365 Gly Ala Asn Ser Pro Ile Trp Glu Ser Gly Phe Thr Met Pro Pro Ser 370 375 380 Glu Gly Thr Gln Leu Val Pro Arg Pro Ala Ser Val Ser Ala Ala Val 385 390 395 400 Val Pro Ser Thr Pro Ile Phe Ser Pro Ser Met Gly Gly Gly Ser Asn 405 410 415 Ser Ser Leu Ser Leu Asp Ser Ala Gly Ala Glu Pro Met Pro Gly Glu 420 425 430 Lys Arg Thr Leu Pro Glu Asn Leu Thr Leu Glu Asp Ala Lys Arg Leu 435 440 445 Arg Val Met Gly Asp Ile Pro Met Glu Leu Val Asn Glu Val Met Leu 450 455 460 Thr Ile Thr Asp Pro Ala Ala Met Leu Gly Pro Glu Thr Ser Leu Leu 465 470 475 480 Ser Ala Asn Ala Ala Arg Asp Glu Thr Ala Arg Leu Glu Glu Arg Arg 485 490 495 Gly Ile Ile Glu Phe His Val Ile Gly Asn Ser Leu Thr Pro Lys Ala 500 505 510 Asn Arg Arg Val Leu Leu Trp Leu Val Gly Leu Gln Asn Val Phe Ser 515 520 525 His Gln Leu Pro Arg Met Pro Lys Glu Tyr Ile Ala Arg Leu Val Phe 530 535 540 Asp Pro Lys His Lys Thr Leu Ala Leu Ile Lys Asp Gly Arg Val Ile 545 550 555 560 Gly Gly Ile Cys Phe Arg Met Phe Pro Thr Gln Gly Phe Thr Glu Ile 565 570 575 Val Phe Cys Ala Val Thr Ser Asn Glu Gln Val Lys Gly Tyr Gly Thr 580 585 590 His Leu Met Asn His Leu Lys Glu Tyr His Ile Lys His Asn Ile Leu 595 600 605 Tyr Phe Leu Thr Tyr Ala Asp Glu Tyr Ala Ile Gly Tyr Phe Lys Lys 610 615 620 Gln Gly Phe Ser Lys Asp Ile Lys Val Pro Lys Ser Arg Tyr Leu Gly 625 630 635 640 Tyr Ile Lys Asp Tyr Glu Gly Ala Thr Leu Met Glu Cys Glu Leu Asn 645 650 655 Pro Arg Ile Pro Tyr Thr Glu Leu Ser His Ile Ile Lys Lys Gln Lys 660 665 670 Glu Ile Ile Lys Lys Leu Ile Glu Arg Lys Gln Ala Gln Ile Arg Lys 675 680 685 Val Tyr Pro Gly Leu Ser Cys Phe Lys Glu Gly Val Arg Gln Ile Pro 690 695 700 Val Glu Ser Val Pro Gly Ile Arg Glu Thr Gly Trp Lys Pro Leu Gly 705 710 715 720 Lys Glu Lys Gly Lys Glu Leu Lys Asp Pro Asp Gln Leu Tyr Thr Thr 725 730 735 Leu Lys Asn Leu Leu Ala Gln Ile Lys Ser His Pro Ser Ala Trp Pro 740 745 750 Phe Met Glu Pro Val Lys Lys Ser Glu Ala Pro Asp Tyr Tyr Glu Val 755 760 765 Ile Arg Phe Pro Ile Asp Leu Lys Thr Met Thr Glu Arg Leu Arg Ser 770 775 780 Arg Tyr Tyr Val Thr Arg Lys Leu Phe Val Ala Asp Leu Gln Arg Val 785 790 795 800 Ile Ala Asn Cys Arg Glu Tyr Asn Pro Pro Asp Ser Glu Tyr Cys Arg 805 810 815 Cys Ala Ser Ala Leu Glu Lys Phe Phe Tyr Phe Lys Leu Lys Glu Gly 820 825 830 Gly Leu Ile Asp Lys 835 <210> 2 <211> 830 <212> PRT <213> Artificial Sequence <220> <223> Mus musculus K(lysine) acetyltransferase 2A (Kat2a), transcript variant 1, mRNA (NM_020004) <400> 2 Met Ala Glu Pro Ser Gln Ala Pro Asn Pro Val Pro Ala Ala Gln Pro 1 5 10 15 Arg Pro Leu His Ser Pro Ala Pro Ala Pro Thr Ser Thr Pro Ala Pro 20 25 30 Ser Pro Ala Ser Ala Ser Thr Pro Ala Pro Thr Pro Ala Pro Ala Pro 35 40 45 Ala Pro Ala Ala Ala Pro Ala Gly Ser Thr Gly Ser Gly Gly Ala Gly 50 55 60 Val Gly Ser Gly Gly Asp Pro Ala Arg Pro Gly Leu Ser Gln Gln Gln 65 70 75 80 Arg Ala Ser Gln Arg Lys Ala Gln Val Arg Gly Leu Pro Arg Ala Lys 85 90 95 Lys Leu Glu Lys Leu Gly Val Phe Ser Ala Cys Lys Ala Asn Glu Thr 100 105 110 Cys Lys Cys Asn Gly Trp Lys Asn Pro Lys Pro Pro Thr Ala Pro Arg 115 120 125 Met Asp Leu Gln Gln Pro Ala Ala Asn Leu Ser Glu Leu Cys Arg Ser 130 135 140 Cys Glu His Pro Leu Ala Asp His Val Ser His Leu Glu Asn Val Ser 145 150 155 160 Glu Asp Glu Ile Asn Arg Leu Leu Gly Met Val Val Asp Val Glu Asn 165 170 175 Leu Phe Met Ser Val His Lys Glu Glu Asp Thr Asp Thr Lys Gln Val 180 185 190 Tyr Phe Tyr Leu Phe Lys Leu Leu Arg Lys Cys Ile Leu Gln Met Thr 195 200 205 Arg Pro Val Val Glu Gly Ser Leu Gly Ser Pro Pro Phe Glu Lys Pro 210 215 220 Asn Ile Glu Gln Gly Val Leu Asn Phe Val Gln Tyr Lys Phe Ser His 225 230 235 240 Leu Ala Pro Arg Glu Arg Gln Thr Met Phe Glu Leu Ser Lys Met Phe 245 250 255 Leu Leu Cys Leu Asn Tyr Trp Lys Leu Glu Thr Pro Ala Gln Phe Arg 260 265 270 Gln Arg Ser Gln Ser Glu Asp Val Ala Thr Tyr Lys Val Asn Tyr Thr 275 280 285 Arg Trp Leu Cys Tyr Cys His Val Pro Gln Ser Cys Asp Ser Leu Pro 290 295 300 Arg Tyr Glu Thr Thr His Val Phe Gly Arg Ser Leu Leu Arg Ser Ile 305 310 315 320 Phe Thr Val Thr Arg Arg Gln Leu Leu Glu Lys Phe Arg Val Glu Lys 325 330 335 Asp Lys Leu Val Pro Glu Lys Arg Thr Leu Ile Leu Thr His Phe Pro 340 345 350 Lys Phe Leu Ser Met Leu Glu Glu Glu Ile Tyr Gly Ala Asn Ser Pro 355 360 365 Ile Trp Glu Ser Gly Phe Thr Met Pro Pro Ser Glu Gly Thr Gln Leu 370 375 380 Val Pro Arg Pro Ala Thr Val Ser Ala Thr Val Val Pro Ser Phe Ser 385 390 395 400 Pro Ser Met Gly Gly Gly Ser Asn Ser Ser Leu Ser Leu Asp Ser Ala 405 410 415 Gly Thr Glu Pro Met Pro Ala Gly Glu Lys Arg Lys Leu Pro Glu Asn 420 425 430 Leu Thr Leu Glu Asp Ala Lys Arg Leu Arg Val Met Gly Asp Ile Pro 435 440 445 Met Glu Leu Val Asn Glu Val Met Leu Thr Ile Thr Asp Pro Ala Ala 450 455 460 Met Leu Gly Pro Glu Thr Ser Leu Leu Ser Ala Asn Ala Ala Arg Asp 465 470 475 480 Glu Thr Ala Arg Leu Glu Glu Arg Arg Gly Ile Ile Glu Phe His Val 485 490 495 Ile Gly Asn Ser Leu Thr Pro Lys Ala Asn Arg Arg Val Leu Leu Trp 500 505 510 Leu Val Gly Leu Gln Asn Val Phe Ser His Gln Leu Pro Arg Met Pro 515 520 525 Lys Glu Tyr Ile Ala Arg Leu Val Phe Asp Pro Lys His Lys Thr Leu 530 535 540 Ala Leu Ile Lys Asp Gly Arg Val Ile Gly Gly Ile Cys Phe Arg Met 545 550 555 560 Phe Pro Thr Gln Gly Phe Thr Glu Ile Val Phe Cys Ala Val Thr Ser 565 570 575 Asn Glu Gln Val Lys Gly Tyr Gly Thr His Leu Met Asn His Leu Lys 580 585 590 Glu Tyr His Ile Lys His Ser Ile Leu Tyr Phe Leu Thr Tyr Ala Asp 595 600 605 Glu Tyr Ala Ile Gly Tyr Phe Lys Lys Gln Gly Phe Ser Lys Asp Ile 610 615 620 Lys Val Pro Lys Ser Arg Tyr Leu Gly Tyr Ile Lys Asp Tyr Glu Gly 625 630 635 640 Ala Thr Leu Met Glu Cys Glu Leu Asn Pro Arg Ile Pro Tyr Thr Glu 645 650 655 Leu Ser His Ile Ile Lys Lys Gln Lys Glu Ile Ile Lys Lys Leu Ile 660 665 670 Glu Arg Lys Gln Ala Gln Ile Arg Lys Val Tyr Pro Gly Leu Ser Cys 675 680 685 Phe Lys Glu Gly Val Arg Gln Ile Pro Val Glu Ser Val Pro Gly Ile 690 695 700 Arg Glu Thr Gly Trp Lys Pro Leu Gly Lys Glu Lys Gly Lys Glu Leu 705 710 715 720 Lys Asp Pro Asp Gln Leu Tyr Thr Thr Leu Lys Asn Leu Leu Ala Gln 725 730 735 Ile Lys Ser His Pro Ser Ala Trp Pro Phe Met Glu Pro Val Lys Lys 740 745 750 Ser Glu Ala Pro Asp Tyr Tyr Glu Val Ile Arg Phe Pro Ile Asp Leu 755 760 765 Lys Thr Met Thr Glu Arg Leu Arg Ser Arg Tyr Tyr Val Thr Arg Lys 770 775 780 Leu Phe Val Ala Asp Leu Gln Arg Val Ile Ala Asn Cys Arg Glu Tyr 785 790 795 800 Asn Pro Pro Asp Ser Glu Tyr Cys Arg Cys Ala Ser Ala Leu Glu Lys 805 810 815 Phe Phe Tyr Phe Lys Leu Lys Glu Gly Gly Leu Ile Asp Lys 820 825 830 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-17 <400> 3 uccuguuuaa ccacgggcu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-18 <400> 4 caaggaccgu aagcucucu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-19 <400> 5 cgaugaugca cugggccuu 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ON-TARGETplus Human KAT2A (2648) siRNA-SMARTpool siRNA: Dharmacon, L-009722-02-0010: J-009722-20 <400> 6 ugaguacaga aacccgcuu 19

Claims (18)

Biomarkers for diagnosing inflammatory diseases or predicting prognosis containing GCN5 protein. A kit for diagnosing or predicting prognosis of an inflammatory disease comprising a detection reagent that specifically binds to the biomarker of claim 1 or a nucleic acid sequence of a gene encoding the same. A composition for diagnosing or predicting prognosis of an inflammatory disease, comprising a substance for measuring the mRNA expression level of the GCN5 protein or the gene encoding the protein. The method of claim 3,
The inflammatory disease is caused by nerve inflammation of the brain, the composition for diagnosing inflammatory disease or predicting the prognosis. The method of claim 4,
The brain nerve inflammation is inflammation occurring in at least one brain tissue selected from the group consisting of hippocampus, striatum, and substantia nigra compacta, a composition for diagnosing inflammatory diseases or predicting prognosis. The method of claim 3,
The substance for measuring the mRNA level is a composition for diagnosing or predicting prognosis of an inflammatory disease, characterized in that it comprises a primer, a probe, or an antisense nucleotide that specifically binds to the gene. A kit for diagnosing inflammatory diseases or predicting prognosis comprising the composition of claim 3. A compound represented by the following formula (1);
A compound represented by the following formula (2); or
Pharmaceutical composition for the prevention or treatment of inflammatory diseases or neurodegenerative diseases containing siRNA (small interfering RNA) of GCN5 protein:
[Formula 1]

[Formula 2]

The method of claim 8, wherein the inflammatory disease,
Sepsis, inflammatory bowel disease, cerebral nerve inflammation, a pharmaceutical composition for preventing or treating inflammatory disease or neurodegenerative disease, characterized in that any one or more selected from the group consisting of atopy. The method of claim 9,
The brain nerve inflammation is inflammation occurring in at least one brain tissue selected from the group consisting of hippocampus, striatum, and substantia nigra compacta, preventing or treating inflammatory diseases or neurodegenerative diseases Pharmaceutical composition for use. The method of claim 8,
The composition is a pharmaceutical composition for preventing or treating inflammatory diseases or neurodegenerative diseases, characterized in that it inhibits the phosphorylation of tyrosine residues in the GCN5 protein. A compound represented by the following formula (1);
A compound represented by the following formula (2); or
Health functional food composition for preventing or improving inflammatory diseases or neurodegenerative diseases comprising siRNA (small interfering RNA) of GCN5 protein as an active ingredient:
[Formula 1]

[Formula 2]

The method of claim 12, wherein the inflammatory disease,
Sepsis, inflammatory bowel disease, brain nerve inflammation, atopy, characterized in that any one or more selected from the group consisting of inflammatory disease or neurodegenerative disease prevention or improvement health functional food composition. The method of claim 12,
The brain nerve inflammation is inflammation occurring in at least one brain tissue selected from the group consisting of hippocampus, striatum, and substantia nigra compacta, health functional food composition for improving memory. (a) measuring the expression level of a biomarker containing GCN5 protein in a biological sample isolated from the individual;
(b) comparing the expression level of the biomarker in step (a) with the expression level of a normal control group, and diagnosing a patient with an inflammatory disease or a neurodegenerative disease when it is higher than that of the normal control group; And
(c) A composition containing a compound represented by the following formula (1) or a composition containing a compound represented by the following formula (2) is treated in a biological sample isolated from a patient with the inflammatory disease or neurodegenerative disease to obtain a composition having a high degree of inhibition of gene expression of GCN5 A method of providing personalized treatment information for each patient with an inflammatory disease or neurodegenerative disease comprising the step of selecting:
[Formula 1]

[Formula 2]

The method of claim 15, wherein the neurodegenerative disease,
Multiple sclerosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, fronto-temporal dementia, cortical-basal ganglia degeneration (cortico basal degeneration), and progressive supranuclear palsy (progressive supranuclear palsy), characterized in that any one or more selected from the group consisting of inflammatory disease or neurodegenerative disease method for providing customized treatment information for each patient. The method of claim 16, wherein the inflammatory disease,
Sepsis, inflammatory bowel disease, brain nerve inflammation, method for providing customized treatment information for each patient of inflammatory disease or neurodegenerative disease, characterized in that any one or more selected from the group consisting of atopy. The method of claim 17,
The brain neuroinflammation is inflammation occurring in at least one brain tissue selected from the group consisting of hippocampus, striatum, and substantia nigra compacta. How to provide information.

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