KR20210021828A - A Composition for Preventing or Treating Protein Conformational Disorders Comprising Kaempferide as an Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating protein conformational disorders or cardiovascular diseases comprising kaempferide or a derivative thereof as an active component. The composition of the present invention induces efficient decomposition of denatured proteins, protein aggregates and plaques by promoting the inflow of autophages, and thus as a naturally-derived compound, the composition has fewer side effects even when administered for a long period of time, thereby being able to be usefully used as an effective therapeutic composition for protein conformational disorders and cardiovascular diseases.

Description

A Composition for Preventing or Treating Protein Conformational Disorders Comprising Kaempferide as an Active Ingredient}

The present invention relates to a composition for preventing or treating protein morphological disorders or cardiovascular diseases comprising chemperide or a derivative thereof as an active ingredient.

Protein aggregation and misfolding are a key cause of the disease group called Protein Conformational Disorder. These proteins exist even in non-disease environments, but when entering the disease stage, the tertiary structure is transformed and bonds to itself to form aggregates, and often accumulates in tissues while taking the form of toxic fibers. Therefore, it is important to inhibit or eliminate the production of modified proteins for the prevention and treatment of protein morphological disorders such as Alzheimer's disease, Parkinson's disease and type 2 diabetes.

Alzheimer's disease is a disease with a high prevalence of 35 million patients worldwide, and is expected to increase dramatically with aging, and is currently the most studied protein morphology disorder. In Alzheimer's disease, amyloid β-peptide (Aβ) aggregates to form extracellular neuritis plaques in the brain. Amyloid-forming proteins and peptides can go through a number of individual combination steps, and fibrillation of Aβ, which causes plaque accumulation, is long. It has been considered a cause of neurodegeneration in Alzheimer's disease. Accordingly, the development and clinical studies of various drugs targeting Aβ for treatment have been mainly conducted, but a lot of research has recently been focused on tau protein.

Tau is one of the microtubule-associated proteins (MAP) mainly present in the axons of normal neurons, and despite many studies, the actual role and importance of tau in cells has not been established. However, during the prodromal stage of Alzheimer's, the tau of the cerebrospinal fluid begins to increase, and the increased amount remains stable during the course of the disease, and neurofibrillary tangles (NFTs) are discovered from the beginning of the onset and the disease progresses. It gradually increases as you do. In addition to Alzheimer's, various tauopathies such as fronto-temporal dementia, progressive supranuclear palsy, corticobasal degeneration, and Pick's disease, which are the cause of tau increase and aggregation. tauopathy), but there is no development of a therapeutic agent for these diseases that effectively inhibits the increase and aggregation of tau.

On the other hand, autophagy, also called autophagy, refers to a process of decomposing intracellular proteins and organelles, including the process of moving from the cytoplasm to the lysosome. Autophagy is an essential process for cell survival, differentiation, development, and homeostasis. Abnormalities in autophagy are known to be involved in cancer and other neurodegenerative diseases, liver disease, heart disease, muscle disease, infectious disease, and aging. have. In addition, it has been reported that autophagy has a function of inhibiting accumulation in tissues by regulating and removing modified proteins that cause misfolding, aggregation, fibrosis, etc. in protein morphological disorders or tau conditions. Through this, it has been suggested that symptoms of cardiovascular disease caused by various protein morphological disorders or plaque accumulation can be improved. Accordingly, it has been expected that if a new substance that effectively induces autophagy is discovered, it can be applied as an effective therapeutic agent for these diseases.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Patent Document 1. US Patent Publication No. 2010-0087474

The present inventors have excellent therapeutic activity for protein conformational disorders and cardiovascular diseases that can be treated by removing excessively accumulated denatured proteins, plaques, lipids, etc. Efforts were made to develop an efficient natural-derived therapeutic composition suitable for the treatment of patients. As a result, it was found that camperide (Kaempferide, hereinafter “Kaem”) and its derivatives promote autophagy activation, remove tau aggregates and significantly inhibit plaque formation, and thus can be used as an effective therapeutic composition for these diseases. By doing so, the present invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating protein morphological disorders or cardiovascular diseases, and a food composition for improving or alleviating these diseases.

Another object of the present invention is to provide a composition for inducing autophagy.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention is a drug for preventing or treating protein conformational disorders or cardiovascular diseases comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient The pharmaceutical composition is provided:

Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl.

The present inventors have excellent therapeutic activity for protein conformational disorders and cardiovascular diseases that can be treated by removing excessively accumulated denatured proteins, plaques, lipids, etc. Efforts were made to develop an efficient natural-derived therapeutic composition suitable for the treatment of patients. As a result, camperide (Kaempferide, hereinafter “Kaem”) having the structure of Formula 1 and its derivatives promote autophagy activation, remove tau aggregates and significantly inhibit plaque formation, and thus an effective therapeutic composition for these diseases. Found that it can be used as.

In the present specification, the term "protein" refers to a series of macromolecules formed by bonding of amino acid residues to each other by peptide bonds. Proteins are linear molecules consisting of consecutive bonds of amino acid units, but their three-dimensional shape and state change tendency are affected by the overall size, charge and hydrophobicity of all or each constituent residue, and whether or not covalent or non-covalent bonds are formed. And if the tendency is abnormal, it may be the cause of various protein conformational disorders (PCD).

In the present specification, the term “protein morphological disorder” refers to a disease in which a structural abnormality occurs in a specific protein and causes a dysfunction of cells, tissues, organs, and individuals, and is also referred to as “proteopathy”. . Often, the three-dimensional shape change due to the folding defect of the protein leads to the aggregation of the protein, and the process of accumulation of toxic protein aggregates in the tissue proceeds.

In the present specification, the term “folding defect” means that a polypeptide cannot be folded normally so that the protein acquires a three-dimensional structure having its own function and activity. Thus, the term “folding defect” is meant to include “misfolding” and “unfolding”.

In the present specification, the term "protein aggregation" refers to the accumulation of proteins that cannot be folded normally within or outside the cell and forming agglomerates to form aggregates. Since folding defects and aggregation of proteins lead to a lack of normal proteins or increase toxicity by accumulating abnormal proteins, substances that remove these protein aggregates can reversibly reverse the progression of these diseases. It can be a fundamental treatment.

In the present specification, the term "alkyl" refers to a linear or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, and the like. C ₁ -C ₃ alkyl means an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, the number of carbon atoms of the substituent is not included.

According to a specific embodiment of the present invention, R ₁ in Formula 1 is C ₁ alkyl, that is, methyl. According to the present invention, the _{compound of formula 1 in which R 1} is methyl is campferide (Kaempferide, 3,5,7-trihydroxy-2-(4-methoxyphenyl)chromen-4-one). Chemperide is an O-methylated flavonol extracted from plants such as Kaempferia galanga , and there is no known about the effect of inducing autophagy and removing denatured proteins and plaques. In the present invention, there is little risk of side effects even when long-term administration as a pharmaceutical composition or long-term feeding as a food composition by using a natural product-derived compound or a derivative thereof.

In the present specification, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids are hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluroacetic acid, citric acid, methane And sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium and the like.

According to a specific embodiment of the present invention, Protein Conformational Disorder prevented or treated with the composition of the present invention is Alzheimer's disease, Parkinson's disease, type 2 diabetes, amyotrophic lateral sclerosis. (amyotrophic lateral sclerosis), dialysis-related amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease , Lewy body dementia, inclusion body myositis, cerebral amyloid angiopathy, traumatic brain injury, fronto-temporal dementia, progressive It is selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease.

According to a specific embodiment of the present invention, the cardiovascular disease prevented or treated with the composition of the present invention includes atherosclerosis, atherosclerosis, coronary artery disease, stable and unstable angina, stroke, aortic stenosis, aortic aneurysm, acute ischemic cardiovascular disease ( acute ischemic arteriovascular event).

In the present specification, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject that has not been diagnosed as having a disease or disease, but is likely to have such disease or disease.

As used herein, the term “treatment” refers to (a) inhibition of the development of a disease, disease or condition; (b) alleviation of the disease, disease or condition; Or (c) to eliminate the disease, disease or condition. When the composition of the present invention is administered to a subject, it directly binds to TUFM, thereby activating the influx of autophages and inducing the removal of denatured proteins or plaques, thereby inhibiting or eliminating the development of symptoms in diseases caused by excessive accumulation thereof. It plays a role of doing or reducing. Accordingly, the composition of the present invention may itself be a composition for treatment of these diseases, or may be administered together with other pharmacological components and applied as a therapeutic adjuvant for the disease. Accordingly, the term “treatment” or “therapeutic agent” in the present specification includes the meaning of “treatment aid” or “treatment aid”.

In the present specification, the term “administration” or “administer” refers to the formation of the same amount in the body of the subject by directly administering a therapeutically effective amount of the composition of the present invention to the subject.

In the present invention, the term "therapeutically effective amount" refers to the amount of the composition contained in a sufficient degree to provide a therapeutic or prophylactic effect to the individual who intends to administer the pharmaceutical composition of the present invention. It is meant to include “prophylactically effective amount”.

As used herein, the term “subject” includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

According to a specific embodiment of the present invention, the composition of the present invention induces autophagy.

In the present specification, the term “autophagy” is used to maintain normal cell function by decomposing substances and organs in cells that are old or damaged by depletion of energy sources in cells or generation of stress factors. It means the mechanism. The term “induction of autophagy” is by enhancing autophagic flux, promoting the production of autophagosomes in cells, or promoting the fusion of autophagy and lysosomes, Ultimately, it is meant to encompass all actions that increase autophagy activity that decomposes and purifies substances and organs in old or damaged cells. Therefore, the term “autophagy induction” is used in the same meaning as “autophage activation”, “autophagy inflow enhancement” and “autophage flow enhancement”.

According to the present invention, the composition of the present invention improves LC3-II accumulation even compared to rapamycin, which is a commercially available autophage inducing agent, while decomposing tau aggregates in an autophagy-lysosome system dependently, thereby effectively enhancing the influx of autophagy. Sikkim was confirmed.

As aging progresses, the intracellular autophagy activity rapidly decreases, and aging mitochondria and misfolding proteins are excessively accumulated in the cells, resulting in increased free radicals and oxidative stress, leading to cell death and aging of the individual. Therefore, by rapidly removing modified proteins, lipids, and old mitochondria by autophagy activation, not only neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, but also cardiovascular diseases such as type 2 diabetes and arteriosclerosis are treated or symptoms are relieved. You can delay its progress.

Specifically, activation of autophagy reduces the accumulation of beta-amyloid (Aβ), a major cause of Alzheimer's disease, to minimize damage to nerve cells (Hung et al. Autophagy, 5(4):502-510(2009)). , Degraded huntingtin protein, which is the main cause of Huntington's disease, is decomposed to eliminate and ameliorate its symptoms (Qi et al. PLOS one 7(10) e46834(2012)), and the accumulation of α-Syn, the cause of Parkinson's disease. In addition to alleviating neurodegeneration by suppressing it (Xilouri et al. Brain , 136: 2130-2146 (2013)), it inhibits the formation of toxic oligomers of pancreatic amyloid, thereby preventing the destruction of pancreatic beta cells. It has been reported to block the onset of diabetes (Kim et al. J Clin Invest 124(8):3311-3324 (2014)) and improve symptoms by reducing lipids and plaques in vascular diseases including atherosclerosis ( Razani, B. et al. Cell Metab 15 : 534-544 (2012)) Therefore, the composition of the present invention is useful as a fundamental treatment method that eliminates the core etiology itself, freeing from symptomatic therapy for these diseases, which are mostly intractable. Can be used.

According to a specific embodiment of the present invention, the composition of the present invention degrades tau protein aggregates.

The tau protein is a microtubule-associated protein (MAP), and its aggregation becomes another marker of Alzheimer's disease (AD) along with Aβ. As shown in the examples to be described later, the composition of the present invention can be an effective therapeutic composition for tauopathy by significantly reducing tau proteins and aggregates thereof in human tau-expressing cell lines. As used herein, the term "tauopathy" refers to a neurodegenerative disease caused by pathological aggregation of tau proteins in brain tissue, and Alzheimer's, frontotemporal dementia, progressive nuclear paralysis, cortical basal nuclear degeneration, and Pick's disease. Belongs.

According to a specific embodiment of the present invention, the composition of the present invention binds to TUFM (Tu translation elongation factor, mitochondrial).

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and specifically, may be administered orally, intravenously, subcutaneously or intraperitoneally.

A suitable dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. Can be. The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

According to another aspect of the present invention, the present invention is a food for improving or alleviating protein conformational disorders or cardiovascular diseases comprising a compound represented by the following Formula 1 or a food pharmaceutically acceptable salt thereof as an active ingredient The composition is provided:

Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl.

The compound of Formula 1 used in the present invention and the protein morphological disorder or cardiovascular disease in which symptoms are improved or alleviated using the same have been described above, and thus description thereof will be omitted to avoid excessive redundancy.

In the present specification, the term “food wise acceptable salt” refers to a salt in a form that can be used in food compositions among salts in which cations and anions are bound by electrostatic attraction, and specific examples thereof are “pharmaceutical Acceptable salts”.

When the composition of the present invention is prepared as a food composition, it may include not only the compound of the present invention as an active ingredient, but also carbohydrates, flavoring agents, and flavoring agents that are commonly added during food production. Examples of carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol are not limited thereto. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glysirhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc. are added in addition to the pine bark extract, which is the active ingredient of the present invention. Can be included as

According to another aspect of the present invention, the present invention provides a composition for inducing autophagy comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl.

Since the meaning of the compound of Formula 1 and “autophagy induction” used in the present invention has already been described above, the description thereof will be omitted to avoid excessive redundancy.

The composition for inducing autophagy of the present invention may be usefully used as a composition for preventing and treating various diseases in which etiology can be removed or improved through the promotion and activation of autophagy. It is as described above that various protein morphological disorders including neurodegenerative diseases and various cardiovascular diseases including atherosclerosis can be treated through the induction of autophagy, but in addition to infection with intracellular pathogens, various malignant tumors , Aging, various inflammatory diseases, including skin inflammatory diseases, can be effectively prevented or treated by the composition of the present invention, which significantly enhances autophagy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition and a functional food composition for the prevention or treatment of protein conformational disorders or cardiovascular diseases comprising campferide or a derivative thereof as an active ingredient.

(b) The composition of the present invention induces efficient decomposition of denatured proteins, protein aggregates and plaques through the promotion of autophagy inflow, and has fewer side effects even when administered for a long period of time as a natural compound. It can be usefully used as a therapeutic composition.

1 is a diagram showing that Kaem reduces tau protein aggregates in HEK293 expressing human tau protein. FIG. 1A shows the results of treating HEK293-tau-BiFC cells with okadaic acid for 48 hours in the presence or absence of rapamycin or Kaem to induce tau oligomers. After DAPI staining, it was observed with a fluorescence microscope (top), and VENUS fluorescence intensity was measured using the ImageJ2 program (bottom). n=10, rapamycin, 10 μM, Kaem, 20 μM. Scale bar, 20 μm. ***P<0.001. 1B shows the results of treatment with doxycycline for 48 hours in HEK293-Trex-hTau40 cells in the presence or absence of rapamycin or Kaem to induce tau protein expression. Western blot using an antibody against tau was performed on the cell extract (top). The intensities of the immunoblot bands of tau and truncated tau are plotted as a fold change normalized to β-actin (bottom). Rapamycin, 10 μM, Kaem, 5, 10, 20 μM. *P<0.05, **P<0.01 vs. You treated only doxycycline.
Figure 2 is a diagram showing that Kaem induces autophagy-dependent decomposition of tau protein aggregates. 2A shows the results of treatment with doxycycline on HEK293-Trex-hTau40 cells. Cycloheximide (CHX), GM132, chloroquine (CQ) were simultaneously treated for 48 hours in the presence or absence of Kaem. Western blot using an antibody against tau was performed on the cell extract (top). The intensity of the immunoblot band of tau is shown as a graph expressed as fold change normalized to β-actin (bottom). ns P>0.05, *P<0.05, **P<0.01. FIG. 2B shows the results of treatment of doxycycline in HEK293-Trex-hTau40 cells for 48 hours in the presence or absence of rapamycin or Kaem to induce tau protein expression. Western blots using antibodies against tau and LC3B were performed on the cell extract (top). The intensities of the immunoblot bands of tau, truncated tau, and LC3B-II are shown as graphs expressed as fold change normalized to β-actin (bottom). *P<0.05, **P<0.01 vs. You treated only doxycycline.
3 is a diagram showing that the degradation efficiency of Kaem Tau aggregates disappears when TUFM expression is suppressed. HEK293-Trex-hTau40 cells transduced with or without siRNA targeting TUFM were treated with doxycycline for 24 hours in the presence or absence of Kaem. Wiston blotting was performed using antibodies against tau and TUFM on the cell extract (top). The intensities of the immunoblot bands of tau and TUFM are shown as a graph expressed as fold change normalized to β-actin (bottom). ns P>0.05, *P<0.05
4 is a diagram showing the inhibitory activity of Kaem on plaque formation in a mouse model of atherosclerosis. Using the arteriosclerotic mouse model from which the ApoE gene was removed, a high cholesterol diet was progressed for 4 weeks and Kaem was intraperitoneally injected for 8 weeks. The artery of the mouse was isolated and stained with Oil-Red-O (left). The area of the plaque is shown as a graph expressed as fold change normalized over the total area (right).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method

Compounds and reagents

Cemperide (69545), dimethyl sulfoxide (D2650), rapamycin (553210), chloroquine (C6628), MG132 (M8699), cyclohexide (C7698), okadaic acid (O9381), Oil-Red-O (O0625) ) Was purchased from Sigma Aldrich. Hoechst33342 (H3570), Lipofectamine LTX (94756), Lipofectamine 2000 (52887), Plus reagent (10964), DMEM, Fetal bovine serum (FBS), and antibiotics were purchased from Invitrogen Thermo Fisher Scientific. For in vivo experiments, chemperide (K0057) was purchased from TCI chemicals. SiRNA targeting TUFM was purchased from Dharmacon.

Cell culture and treatment

Tau-BiFC cells and HEK293-trex-htau40 cells were cultured in DMEM supplemented with bovine serum (10% v/v) and penicillin/streptomycin (1% v/v), respectively. All cell cultures were performed in a humidified incubator at pH 7.4 while maintaining _{37° C. and 5% (v/v) CO 2.}

Immunoblotting

Soluble proteins were collected from cells using SDS lysis buffer (10% glycerol, 2% SDS, 10 mM dithiothreitol and 50 mM Tris HCl with 0.005% bromophenol blue, pH 6.8). The same volume of protein was separated using 10% or 12.5% SDS-PAGE and transferred to a fluorinated polyvinylidene membrane (EMD Millipore, Billerica, MA, USA). Afterwards, immunolabeling was performed overnight at 4°C with primary antibodies of anti-LC3B (Cell signaling technology), tau and actin (Abcam), and TUFM (Atlas antibodies). Immunolabels were visualized according to the manufacturer's instructions using an enhanced chemiluminescence kit (Amersham Life Science, Inc., Amersham, UK). Images were quantified using Image Lab (Bio-Rad, Hercules, CA, USA), and actin was used as an internal control. All band intensities are proportional to the amount of target protein on the membrane with a linear detection range.

Tau-BiFC cell analysis

Tau-BiFC cells were treated with 30 nM okadaphosphoric acid and the designated compound. Nuclei were stained with DAPI. After incubation for 20 minutes, cells and tissues were fixed with 4% formaldehyde and washed three times with PBS. An image of 400× magnification was obtained using a confocal microscope LSM880, and the fluorescence intensity of VENUS was ImageJ2 (NIH, Bethesda, MD. , USA).

Endogenous tau degradation assay using HEK293-trex-htau40 cells

HEK293-trex-htau40 cells were treated with the designated compound along with doxycycline (1 ng/mL). Soluble protein was collected from cells using SDS lysis buffer (10% glycerol, 2% SDS, 10 mM dithiothreitol and 50 mM Tris HCl, pH 6.8 containing 0.005% bromophenol blue) and immunoblotting was performed. .

Treatment of atherosclerosis-induced mouse model and analysis of atherosclerosis

Animal testing complies with the guidelines under the approval of the Yonsei University Animal Experimental Ethics Committee (IACUC), and the National Institutes of Health (NIH)'s Guide to Laboratory Animal Care and Use (The National Academies Press, 8th Edition, 2011). Performed in compliance. A 6-week-old male ApoE gene knockout C57BL/6j mouse was placed in a 12-hour light condition/12-hour dark condition, followed by a high cholesterol diet for 4 weeks. Mice were randomly divided into two groups, and chemperide (10 mg/kg) and vehicle were injected once every 2 days for 8 weeks by intraperitoneal injection. After sacrifice of the mouse, PBS perfusion was performed through the left ventricle, and heart and arteries were obtained. The heart was deeply embedded with OCT compound (Tissue tek, USA) and frozen. The artery was cut along the branch point of the iliac artery, and fibroblasts and fat outside the artery were removed. The unfolded artery was pinned and immobilized with 10% formaldehyde for 16 hours. The fixed arteries were stained with Oil-Red-O for 16 hours, washed with PBS, and then images were obtained using a microscope (NS-6T, China).

Statistical analysis

All data were expressed as mean ± standard error using GraphPad Prism (Windows ver. 5.00; GraphPad Software, Inc., San Diego, CA, USA). Data were obtained through at least 3 independent experiments. Statistical analysis was performed using the independent sample two-sided distribution Student t-test. P-values less than 0.05 were considered statistically significant (* p <0.05, ** p <0.01, *** p <0.001).

Experiment result

The natural compound kaem reduces tau protein in human cellular systems capable of inducing tau protein

Pathologically, aggregation of tau proteins is a marker of Alzheimer's disease (AD). Therefore, the discovery of a therapeutic composition capable of decomposing tau aggregates is very important for the development of a therapeutic agent for neurodegenerative diseases ¹ . The present inventors investigated whether the natural compound Kaem can regulate tau protein using a human tau expression cell line system. In HEK293-htau40-BiFC cells, the N-terminal and C-terminal portions of the Venus protein were independently fused to htau40. When this tau protein is hyper-phosphorylated by a chemical inducing agent such as okadaic acid, the fluorescence intensity of Venus increases ² . In particular, tau-BiFC cells treated with Kaem significantly reduced tau aggregates compared to the control group treated with rapamycin ³ ( FIG. 1A ). Next, the present inventors investigated the protein level by performing immunoblotting using the HEK293-htau40-Trex system, which is a tau-inducing cell line treated with doxycycline ⁴ . Doxycycline-induced Tau accumulation was dose-dependently reduced by treatment with Kaem. ^{Furthermore, not only all forms of tau, but also truncated forms of tau 5,6} , which are considered major toxic molecules and initiate and promote neurofibrillary tangle (NFT) formation, were also significantly reduced following Kaem treatment. From these results, it can be seen that Kaem inhibits aggregates or overexpressed tau proteins.

The breakdown of Tau aggregates promoted by Kaem is dependent on the autophage-lysosome system.

To investigate whether the regulation of tau protein by Kaem is due to inhibition of protein synthesis, promotion of proteolysis such as UPS (ubiquitin-proteasomal system), or by autophagy-lysosome system (ALS), We treated Kaem in tau-trex cells with or without the following various inhibitors: cycloheximide (CHX, protein synthesis inhibitor), MG132 (proteosome inhibitor) and chloroquine (CQ, lysosome inhibitor). (Fig. 2a). By confirming that Kaem reduced the Tau protein even under the condition that protein synthesis was inhibited, it was found that it regulates the degradation of the tau protein rather than the synthesis. Furthermore, by observing that tau protein degradation is repaired by CQ treatment rather than MG132, it can be seen that the degradation of tau aggregates is promoted dependently on the autophage-lysosome system. In order to demonstrate the autophagy activity of Kaem in the situation where tau protein is overexpressed, Western blot analysis was performed. Kaem also improved LC3-II accumulation compared to ^{rapamycin 7} , a well-known autophagy inducer (FIG. 2B). It is unusual tau to promote so vice versa can cause auto phage defective when there are ^8,9, tau aggregates automatic gripping inlet is important. From the above results, it can be seen that Kaem promotes tau decomposition by inducing autophagy influx.

Kaem improves tauopathy in the presence of the target protein TUFM

In order to investigate the mechanism by which Kaem induces autophagy, the present inventors have investigated the biophysical interaction between Kaem and the mitochondrial protein TUFM. Thus, it was investigated whether the decomposition of tau aggregates by Kaem depends on the presence or absence of the target protein, TUFM. Upon transduction of the silencing RNA for TUFM, the degradation efficiency of tau disappeared (Fig. 3). In conclusion, Kaem can degrade tau protein by using an autophagy-lysosome system in the presence of TUFM.

Kaem Improves Atherosclerosis in ApoE Gene Abled Mice

Atherosclerosis has been reported that low-density lipoprotein (LDL) accumulates in the arterial intima, and the disease is plaque formation ^10, a lack of recent automatic grasping the deteriorated atherosclerosis ^11. Accordingly, the present inventors hypothesized that Kaem, an autophagy influx enhancer, would inhibit plaque formation in atherosclerosis. ApoE gene knockout mice fed high-cholesterol-diet (HCD) were intraperitoneally (IP) injected with 10 mg/kg of Kaem once every two days for 8 weeks, and the content of triglycerides stained with oil-red-o was significantly reduced. Was observed (Fig. 4). Through this, it was found that Kaem improved cholesterol-related cardiovascular disease.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

references

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Claims (13)

A pharmaceutical composition for preventing or treating protein conformational disorders or cardiovascular diseases comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl.
The composition of claim 1, wherein R ₁ in Formula 1 is C ₁ alkyl.
The method of claim 1, wherein the Protein Conformational Disorder is Alzheimer's disease, Parkinson's disease, type 2 diabetes, amyotrophic lateral sclerosis, and dialysis-related amyloidosis. -related amyloidosis), cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease, Lewy body dementia, including Inclusion body myositis, cerebral amyloid angiopathy, traumatic brain injury, fronto-temporal dementia, progressive supranuclear palsy, cortical basal nuclear degeneration A composition, characterized in that it is selected from the group consisting of (corticobasal degeneration), Pick's disease, and argyrophilic grain disease.
The method of claim 1, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, aortic stenosis, aortic aneurysm, acute ischemic arteriovascular event. Composition, characterized in that.
The composition of claim 1, wherein the composition induces autophagy.
The composition of claim 1, wherein the composition degrades tau protein aggregates.
The composition of claim 1, wherein the composition binds to TUFM (Tu translation elongation factor, mitochondrial).
A food composition for improving or alleviating protein conformational disorders or cardiovascular diseases comprising a compound represented by the following Formula 1 or a food pharmaceutically acceptable salt thereof as an active ingredient:
Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl.
The composition of claim 8, wherein R ₁ in Formula 1 is C ₁ alkyl.
The method of claim 8, wherein the Protein Conformational Disorder is Alzheimer's disease, Parkinson's disease, type 2 diabetes, amyotrophic lateral sclerosis, and dialysis-related amyloidosis. -related amyloidosis), cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease, Lewy body dementia, including Inclusion body myositis, cerebral amyloid angiopathy, traumatic brain injury, fronto-temporal dementia, progressive supranuclear palsy, cortical basal nuclear degeneration A composition, characterized in that it is selected from the group consisting of (corticobasal degeneration), Pick's disease, and argyrophilic grain disease.
The method of claim 8, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina, stroke, aortic stenosis, aortic aneurysm, acute ischemic arteriovascular event. Composition, characterized in that.
A composition for inducing autophagy comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
Formula 1

In Formula 1, R ₁ is C ₁ -C ₃ alkyl.
13. The composition of claim 12, wherein R ₁ in Formula 1 is C ₁ alkyl.

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