KR102569276B1 - Composition for preventing or treating neurodegerative diseases comprising flavanone derivatives - Google Patents

Abstract

The present invention relates to a composition for preventing or treating neurodegenerative diseases comprising a flavanone derivative as an active ingredient, wherein the flavanone derivative is AChE (acetylcholinesterase), BChE (butyrylcholinesterase) or BACE1 (β-site amyloid precursor protein cleaving enzyme 1) can be usefully used for the treatment of neurodegenerative diseases caused by overexpression or overactivity.

Description

Composition for preventing or treating neurodegenerative diseases comprising flavanone derivatives

The present invention relates to a composition for preventing or treating neurodegenerative diseases containing a flavanone derivative.

Alzheimer's disease is one of the degenerative neurological diseases that mainly occurs in the elderly. It has been reported that 13% of the elderly over 65 years old suffer from Alzheimer's disease, and the proportion increases to 45% over the age of 85 years (Clin. Chim. Acta. 456 (2016) 107-114). It is known that the main mechanisms of Alzheimer's disease include abnormal amyloid plaque deposition, tau protein aggregation, excessive metal ions, oxidative stress, reduced inflammatory processes, and reduced acetylcholine (ACh) levels (Indian J. Psychiatry. 51 (2009) 55-61). Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are reported to play an important role in the pathophysiology of Alzheimer's disease by hydrolyzing acetylcholine, a neurotransmitter, and interfering with synaptic transmission (JAMA. 273 (1995) 1354-1359).

Cholinesterase inhibitors can improve cholinergic function by inhibiting the enzyme that breaks down acetylcholine, increasing acetylcholine, which can activate nicotinic and muscarinic receptors in the brain (Curr. Neuropharmacol. 11 (2013) 315-335). Currently, approved Alzheimer's disease therapies include the AChE inhibitors rivastigmine, tacrine, donepezil, galantamine, huperzine A and heptylphysostigmine and the N-methyl-D-aspartate receptor antagonist (Curr. Neuropharmacol. 11 (2013) 315-335; and Curr. Neuropharmacol. 11 (2013) 315-335). However, these therapeutic agents are effective only in mild or moderately ill patients, and thus there is a need to develop more fundamental therapeutic agents that are effective even in severely ill patients.

A major histopathological feature of Alzheimer's disease is the presence of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) derived from APP (amyloid precursor protein) (Physiol. Rev. 81 (2001 ) 741-766 and Cell 120 (2005) 545-555). Amyloid beta is produced by cleavage of APP by two aspartic proteases, β-secretase and γ-secretase (Science 275 (1997) 630-631). In particular, β-secretase cleaves APP at the β-site and is referred to as BACE1 (β-site APP cleaving enzyme 1). In the amyloidogenic pathway, β-secretase cleaves APP to produce a soluble N-terminal fragment (sAPPβ) and a 12-kDa C-terminal fragment (C99) . In addition, C99 can be cleaved by γ-secretase to generate amyloid beta (Proc. Natl. Acad. Sci. USA 96 (1999) 11049-11053).

The fragment containing residues 25-35 of Aβ _1-42 , ie Aβ _25-35 , is the shortest fragment of Aβ _1-42 that remains active (PLOS ON 9 (2014) 1-18). Aβ _25-35 has a large β-sheet structure with the same physical, biological and toxicological properties as full-length peptides (PLOS ON 9 (2014) 1-18; and J. Biosci. 34 (2009) 293-303). In addition, Aβ _25-35 has very fast aggregation and improved neurotoxicity, so the peptide was used as a neurotoxin in the present invention.

Flavonoids are phenolic compounds widely distributed in the plant kingdom. Flavonoids are reactive secondary metabolites abundant in foods of plant origin, especially fruits, seeds and vegetables (Ann.N.Y. Acad. Sci. 854 (1998) 435-442; and Curr. Med. Chem. 18 (2011) 5289-5302), natural flavonoids are mostly present in the form of O-glycoside or C-glycoside in plants. Flavonoids structurally contain a C6-C3-C6 backbone derived from phenylpropanoids.

Korean Patent Publication No. 10-2018-0115916

An object of the present invention is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a flavanone derivative as an active ingredient.

Another object of the present invention is to provide a health functional food for the prevention or improvement of neurodegenerative diseases comprising a flavanone derivative as an active ingredient.

Another object of the present invention is to provide a composition for protecting nerve cells comprising a flavanone derivative as an active ingredient.

Another object of the present invention is AChE (acetylcholinesterase), BChE (butyrylcholinesterase) or BACE1 (β-site amyloid) comprising the step of treating a sample with a flavanone derivative in vitro or ex-vivo. It is to provide a method of inhibiting the expression or activity of precursor protein cleaving enzyme 1).

Another object of the present invention is to provide a method for protecting neurons from neurotoxicity induced by amyloid beta, comprising administering a flavanone derivative to a subject.

Another object of the present invention is to provide binding agents for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) containing flavanone derivatives.

Another object of the present invention is to provide antagonists for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), including flavanone derivatives.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a flavanone derivative as an active ingredient.

In one embodiment of the present invention, the flavanone derivative may be selected from the group consisting of didymin, poncirin, and naringenin.

In one embodiment of the present invention, the neurodegenerative disease is caused by overexpression or overactivity of one or more enzymes from the group consisting of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). It may be caused by a degenerative neurological disease.

In one embodiment of the present invention, the neurodegenerative disease may be a neurodegenerative disease caused by the β-sheet structure of amyloid protein, and the amyloid protein may be Aβ _25-35 .

In one embodiment of the present invention, the neurodegenerative disease may be a neurodegenerative disease caused by overexpression of sAPPβ (soluble N-terminal fragment) and C99 (12-kDa C-terminal fragment).

In one embodiment of the present invention, the neurodegenerative disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, senile dementia, diabetic dementia, alcoholic dementia, and vascular dementia.

In addition, the present invention provides a health functional food for preventing or improving neurodegenerative diseases comprising a flavanone derivative as an active ingredient.

In addition, the present invention provides a composition for protecting nerve cells comprising a flavanone derivative as an active ingredient.

In one embodiment of the present invention, the nerve cell protection may be to protect nerve cells from toxicity caused by the β-sheet structure of amyloid protein, and the amyloid protein may be Aβ _25-35 . there is.

In addition, the present invention relates to AChE (acetylcholinesterase), BChE (butyrylcholinesterase) or BACE1 (β-site amyloid precursor protein) comprising the step of treating a sample with a flavanone derivative in vitro or ex vivo. A method for inhibiting the expression or activity of cleaving enzyme 1) is provided.

In addition, the present invention provides a method for protecting nerve cells from neurotoxicity induced by amyloid beta, comprising administering a flavanone derivative to a subject.

In addition, the present invention provides binding agents for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) containing flavanone derivatives.

In addition, the present invention provides antagonists for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) including flavanone derivatives.

The flavanone derivative according to the present invention can be usefully used in the treatment of neurodegenerative diseases caused by overexpression or overactivity of AChE (acetylcholinesterase), BChE (butyrylcholinesterase) or BACE1 (β-site amyloid precursor protein cleaving enzyme 1). .

1 shows the chemical structure of flavanone derivatives.
Figure 2 shows the inhibitory activities of AChE (A), BChE (B) and BACE1 (C) by flavanone derivatives (berberine, galantamine and quercetin were used as positive controls).
Figures 3A and 3B show Dixon plots of AChE inhibition by didimine (A) and ponyrin (B) at different concentrations of substrate conditions (0.6 mM (●), 0.3 mM (○) and 0.1 mM (▼)). 3C is a Lineweaver-Burk plot of AChE inhibition by different concentrations of didimine (0 μM (Δ), 0.8 μM (▼), 4 μM (○) and 20 μM (●)), and FIG. 3D Shows the Lineweaver-Burk plot of AChE inhibition by different concentrations of ponsillin (0 μM (Δ), 4 μM (▼), 20 μM (○) and 100 μM (●)).
Figures 4A and 4B show Dixon plots of BChE inhibition by didimine (A) and poncerin (B) at different concentrations of substrate conditions (0.6 mM (●), 0.3 mM (○) and 0.1 mM (▼)). 4C shows a Lineweaver-Burk plot of BChE inhibition by different concentrations of didimine (0 μM (Δ), 4 μM (▼), 20 μM (○) and 100 μM (●)), and FIG. 4D Shows the Lineweaver-Burk plot of BChE inhibition by different concentrations of Ponsillin (0 μM (Δ), 4 μM (▼), 20 μM (○) and 100 μM (●)).
Figures 5A and 5B show Dixon plots of BACE1 inhibition by didimine (A) and fonsillin (B) at different concentrations of substrate conditions (150 nM (●), 250 nM (○), and 750 nM (▼)). Figure 5C is a Lineweaver-Burk plot of BACE1 inhibition by different concentrations of didimine (0 μM (■), 0.5 μM (Δ), 2.5 μM (▼), 12.5 μM (○) and 62.6 μM (●))) , and Figure 5D shows the lineweaver-line of BACE1 inhibition by different concentrations of poncerin (0 μM (■), 0.5 μM (Δ), 2.5 μM (▼), 12.5 μM (○) and 62.6 μM (●))). It shows the Burk plot.
Figure 6 shows the interaction of flavanone derivatives with 2WJO at the docked position, didimine (A), ponyrin (B), naringenin (C), QUD (D) and 2WJO-QUD complex docking protocol ( E) is shown as a pink stick, hydrogen bonds formed with 2WJO are shown as green sticks, and residues of 2WJO related to hydrophobic interactions (van der Waals interaction) are shown as green lines.
7A and 7B show molecular docking between flavanone derivatives (didimine (A), fonsillin (B)) and AChE, and FIGS. 7C and 7D show flavanone derivatives (didimine (C), fonsillin (D) )) and molecular docking between BChE.
Figure 8 shows the results of measuring cell viability through an MTT assay after pre-treating PC12 cells with a flavanone derivative for 24 hours and treating them with Aβ _25-35 (10 μM) for 2 hours (data are shown in three experiments). expressed as mean ± SEM for ; and letters ^aj indicate statistical differences relative to the mean).
Figure 9A is the result of measuring cell viability after pre-treatment with didimine and treatment with Aβ _25-35 in PC12 cells, and 9B is the expression of BACE1 after treatment with didimine in PC12 cells treated with Aβ _25-35 . 9C is the result of measuring the expression levels of APP, sAPPsβ and C99 through Western blotting (results were normalized to β-actin and expressed as the mean ± SEM of three independent experiments; and letters ^af indicate statistical differences about the mean).

The flavanone derivatives of the present invention are didymin, poncirin, and naringenin, which are compounds represented by Chemical Formulas 1 to 3, respectively. The flavanone derivative of the present invention may be chemically synthesized by a method known in the art or a commercially available material may be used.

[Formula 1]

[Formula 2]

[Formula 3]

In the present invention, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, senile dementia, diabetic dementia, alcoholic dementia and vascular dementia, but are not limited thereto.

Neurodegenerative diseases according to an embodiment of the present invention are caused by overexpression or overactivity of one or more enzymes from the group consisting of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) In particular, the flavanone derivative of the present invention can triple the inhibition of the three enzymes, and furthermore, sAPPβ (soluble N-terminal fragment) and C99 (12-kDa C-terminal fragment) It may be a degenerative neurological disease caused by overexpression of .

Neurodegenerative diseases according to an embodiment of the present invention may be neurodegenerative diseases caused by the β-sheet structure of amyloid protein, and the amyloid protein may be Aβ _25-35 .

According to one embodiment of the present invention, flavanone derivatives can be used for neuronal cell protection, and the neuronal cell protection can be toxic caused by the β-sheet structure of amyloid protein.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means that it exhibits non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like.

In addition, the pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Furthermore, it may be used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or skin external preparations in the form of gels. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, but not limited thereto.

The term "individual" refers to all animals such as rats, mice, livestock, and the like, including humans. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's gender, age, Factors including body weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, drugs used in combination or concurrently, and other medical disciplines. It can be easily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dose, or may be administered in several divided doses.

The food composition of the present invention may include all food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food and health functional food.

The term of the present invention, "functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body according to the Act on Health Functional Foods No. 6727, and "functional" means It refers to intake for the purpose of obtaining useful effects for health purposes, such as regulating nutrients with respect to structure and function or physiological action.

The term of the present invention, "health functional food" refers to a food manufactured and processed by methods such as extracting, concentrating, refining, mixing, etc., using specific ingredients as raw materials or specific ingredients contained in food raw materials for the purpose of health supplementation. It refers to a food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. It can perform related functions, etc.

There are no restrictions on the types of foods in which the composition of the present invention can be used. In addition, the composition of the present invention may be prepared by mixing suitable other auxiliary ingredients that may be included in food and known additives according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and Vitamin complexes, etc., can be prepared by adding the extract and its fractions according to the present invention to juices, teas, jellies, juices, etc. prepared as main components.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formula milk, infant, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplement food , seasonings (eg soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.) ), beverages (eg fruit, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional components, like conventional beverages. In addition to the above, the health functional food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, and glycerin. , alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages.

As used herein, the term "antagonist" refers to a molecule that partially or completely inhibits the effects of other molecules such as receptors or intracellular mediators by any mechanism, and in the present invention, the antagonist is AChE ( acetylcholinesterase), BChE (butyrylcholinesterase), and BACE1 (β-site amyloid precursor protein cleaving enzyme 1), thereby preventing agonists from binding to receptors, and substances that do not themselves exhibit physiological actions through receptors. it means. Therefore, the antagonists of AChE (acetylcholinesterase), BChE (butyrylcholinesterase), and BACE1 (β-site amyloid precursor protein cleaving enzyme 1) of the present invention have strong binding affinity to the enzymes, thereby partially inhibiting the enzyme-related signaling at the micromolecular level. However, it may work without completely abolishing it.

As used herein, the term "AChE (acetylcholinesterase), BChE (butyrylcholinesterase) and BACE1 (β-site amyloid precursor protein cleaving enzyme 1) antagonist" refers to AChE (acetylcholinesterase), BChE (butyrylcholinesterase) and BACE1 (β-site amyloid precursor protein cleaving enzyme 1) antagonist. A substance that can directly, indirectly, or substantially interfere with, reduce, or inhibit the biological activity of protein cleaving enzyme 1).

As used herein, the term "inhibition" refers to a phenomenon in which biological activity or activity is lowered due to deficiency, incompatibility, or many other causes, and partially or completely blocks, reduces, or prevents the activity / expression of an enzyme or by delaying, inactivating, or down-regulating activation.

In one aspect, the present invention relates to acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) binding agents including flavanone derivatives.

The binding positions of each of the flavanone derivatives to acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) will be described in detail in the Examples below.

As used herein, the term "sample" may include a biological sample, and the biological sample may include samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Experimental Materials and Methods

1.1. experimental material

Electric eel-derived AChE (acetylcholinesterase, EC3.1.1.7), horse serum-derived BChE (butyrylcholinesterase, EC 3.1.1.8), ACh (acetylthiocholine iodide), BCh (butyrylthiocholine chloride), DTNB (5,5′- dithiobis (2-nitrobenzoic acid)), didymin (>98% purity), poncirin (>95% purity), naringenin, quercetin (>95% purity), galantamine ( galanthamine) and berberine were purchased from Merck, Fluka or Sigma-Aldrich unless otherwise noted. BACE1 FRET (fluorescence resonance energy transfer) assay kits (β-secretase, human recombinant) were purchased from Pan Vera Co. (Madison, WI, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and DMSO (N, N-dimethyl sulfoxide) were purchased from Sigma (St. Louis, MO, USA). Amyloid beta (Aβ 25-35) is a product of Bachem California Inc. (Torrance, CA, USA). RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, and horse serum were purchased from Gibco (Logan, UT, USA). Anti-BACE1 (ab2077; Abcam), anti-C99 (M066-3; MBL), human anti-sAPPβ Swedish (sAPPβ-sw), APP, and monoclonal β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Purchased. The chemical structure of flavanone derivatives is shown in FIG. 1.

1.2. In vitro ChE (Cholinesterase) enzyme assay

The inhibitory activity of flavonoids against ChE was investigated by Ellman et al. (Biochem. Pharmacol. 7 (1961) 88-95) was measured using a spectrophotometric method. Acetylcholine (ACh) and butyrylcholine (BCh) are used as substrates to measure inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively. The reaction consisted of 140 μL of sodium phosphate buffer (pH 8.0); 20 μL of flavonoid sample (final concentration 100 μM); and 20 μL of AChE or BChE solution (0.36 U/mL), mixed, and reacted at room temperature for 10 minutes. All samples and positive controls (berberine, galantamine) were dissolved in 10% DMSO. Reactions were each treated with 10 μL DTNB; and 10 μL of ACh or BCh. Hydrolysis of ACh or BCh resulted in the formation of yellow 5-thio-2-nitrobenzoate anion resulting from the reaction of DTNB with thiocholine released by enzymatic hydrolysis of ACh or BCh at 412 nm for 5 min. It was observed by tracing the formation. All reactions were performed and recorded in triplicate in a 96-well microplate using a spectrophotometer (VERSAmax, Molecular Devices, Sunnyvale, CA, USA). Percent (%) inhibition was calculated with the following formula: % inhibition = (1 - S / E) × 100 (S is the enzyme activity when the sample is treated and E is the enzyme activity when the sample is not treated) . The ChE inhibitory activity of each sample was expressed as an IC ₅₀ value (concentration required to inhibit hydrolysis of ACh or BCh as a substrate by 50%, μM) calculated from a log-dose inhibition curve.

1.3. In vitro BACE1 (β-site amyloid precursor protein cleaving enzyme 1) enzyme assay

For the BACE1 enzyme assay, BACE1 FRET (fluorescence resonance energy transfer) assay kits (β-secretase, human recombinant, PanVera Co., Madison, WI, USA) were used. The assay was performed with minor modifications to the provided manual (Asian. Pac. J. Trop. Med. 9 (2016) 103-111). As a positive control, quercetin was used.

1.4. Kinetic parameters of flavanone derivatives that inhibit AChE, BChE and BACE1

The present inventors used two complementary kinetic methods, Lineweaver-Burk plot and Dixon plot, to identify mechanisms by which flavanone derivatives inhibit AChE, BChE and BACE1 (J. Am. Chem. Soc. 56 (1934) 658-666; Biochem. J. 137 (1974) 143-144; and Biochem. J. 55 (1953) 170-171). The mechanism of AChE and BChE inhibition was confirmed by observing the effects of different concentrations of the substrate (0.6 to 0.1 mM for flavanone derivatives). AChE and BChE inhibition assays were performed with various substrate concentrations. Briefly, 20 μL of the flavanone derivative sample dissolved in 10% DMSO was mixed with 140 μL of buffer and 20 μL of AChE or BChE (0.36 U/mL) and reacted at room temperature for 10 minutes. Then, 10 μL of DTNB (0.5 mM) and 10 μL of ACh or BCh (0.6 mM) were added, and the final mixture was reacted at room temperature for 5 minutes. The absorbance of the mixture was measured at 412 nm using a microplate reader. Dixon plots for BACE1 inhibition by flavanone derivatives were obtained in the presence of 375, 250 and 150 nM substrate. Experimental concentrations of each flavanone derivative for the inhibition kinetic assay were 0 μM (■), 0.5 μM (Δ), 2.5 μM (▼), 12.5 μM (○) and 62.6 μM (●). The inhibition constant (K _i ) was determined by taking the x-axis intercept value as ^_ K _i in the analysis of the Dixon plot.

1.5. Molecular docking simulation of AChE and BChE

Docking studies were performed with the advanced docking program AutoDock 4.2 (J. Comput. Chem. 30 (2009) 2785-2791). The X-ray crystallographic structures of AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) were retrieved from the Brookhaven protein data bank (http://www.rcsb.org/). For the preparation of template target proteins (PDB codes: 4ey7 & 1p0i), the cognate ligand and all crystallographic water molecules were omitted from the initial acceptor structure, and all docking simulations were performed on chain A. The ADT program was used to incorporate the non-polar hydrogen into the relevant carbon atom of the acceptor and assigned a Kollman charge. Non-polar hydrogen was added to the docked ligands, Gasteiger charges were assigned, and torsional degrees of freedom were assigned by the ADT program. The Lamarckian genetic algorithm (LGA) was applied to model the interaction/binding at the active site. For each ligand, 100 preferred independent genetic algorithms were considered. 27,000 maximum generations for the Lamarckian genetic algorithm; a gene mutation rate of 0.02; and a crossover ratio of 0.8 was used. A grid map characterizing proteins during the actual docking process was calculated with AutoGrid. The size of the grid was set in such a way that it covered not only the active site but also a significant portion of the surrounding surface. For this purpose, a grid of 60 * 60 * 60 points in the x, y and z directions was designed with a spacing of 0.375 Å from the center of mass of the catalytic sites of AChE and BChE. Cluster analysis was performed using a RMSD tolerance of 3 Å on the docked results.

1.6. Molecular docking simulation of BACE1

The BACE1 structure (PDB ID-2WJO) was used for docking studies. Missing residues 162-171 were modeled using the SWISS-MODEL (Bioinformatics. 22 (2006)195-201) web-server, and the quality of the generated models was judged using ANOLEA and QMEAN analyses. Afterwards, the selected model was run in a docking study. Non-polar charges are combined and Gasteiger charges are assigned to all acceptor atoms. All ligands were drawn and optimized using MM4+ implemented in Hyper Chem 8 for an energy convergence of 0.003 kcal/Å mol ^-1 with the Polak Ribiere bond gradient algorithm. Gasteiger charges are assigned to atoms of each ligand and non-polar hydrogens are combined. The used Lamarckian genetic algorithm implemented with Autodock 4 (J. Mol. Recogn. 9 (1996) 1-5) was used as a search method. A basic grid spacing of 0.375 Å was maintained, and the grid was chosen to accommodate the active site residues of the protein. Parameters for docking included 150 initial populations with a maximum energy rating of 2,500,000 and a maximum of 27,000 generations as final criteria. An Elitism value of 1, mutation probability of 0.02, and crossover rate of 0.8 were used. 200 docking runs were performed for each ligand.

1.7. cell culture

PC12 cells, pheochromocytoma cells, were cultured and maintained as previously described (Mol. Cells 13 (2002) 113-117). The PC12 cell line was derived from a transplantable rat pheochromocytoma (ACCT, Manassas, VA, USA). Cells respond reversibly to NGF (nerve growth factor) by inducing a neuronal phenotype. The growth medium consisted of RPMI 1640, 10% heat-inactivated horse serum, 5% fetal bovine serum (FBS), 50 units/mL penicillin and 100 μg/mL streptomycin. . Cultures were maintained in an incubator at 5% CO ₂ and 37 °C. PC12 cells were detached from the surface of a culture dish (100 mm) when they reached 80-90% confluency, and the medium was repeatedly and strongly dispensed directly to the cells to disperse them into single cells and subcultured.

1.8. Amyloid beta (Aβ _25-35 ) Preparation of stock solution

Amyloid beta (hereinafter referred to as "Aβ") is a potential cause of Alzheimer's (Nature 382 (1997) 685-691; and Cell 7 (1993) 1039-1042), and Aβ oligomers are monomeric (soluble) or It has been reported that it is more toxic to neurons than fibril (J. Biol. Chem. 281 (2006) 28079-28089). Accordingly, Aβ _25-35 used in the present invention was pre-aggregated before use. Briefly, Aβ _25-35 (1 mg) was dissolved in 1 ml of RPMI 1640 medium and placed in a 37° C. water bath for 3 days to induce aggregation. Thereafter, the aggregated Aβ _25-35 was diluted to 100 μg/ml (100 μM) and stored at -20°C until use.

1.9. Cell viability measurement

Whether or not the flavanone derivatives have an effect of protecting PC12 cells from Aβ _25-35 was determined by measuring the cell's ability to reduce MTT to MTT formazan, that is, cell viability. Briefly, PC12 cells were seeded at 4×10 ⁴ cells/well in a 96-well plate, and pretreated with a given concentration of flavanone derivatives for 24 hours. Then, the cells were treated with Aβ _25-35 (10 μM) for an additional 2 hours. Thereafter, MTT solution (20 μl/well, 1 mg/ml stock solution in PBS) was added and treated at 37° C. for 3 hours. Then, the cells were lysed overnight at 37 °C with 100 μl of lysis buffer (10%, w/v of SDS in 0.01N HCl). Then, OD (optical density) was measured at 570 nm using a microplate reader (Molecular Devices, MN, USA).

1.10. Western blot analysis

Cells were extracted 24 hours after transfection. Total protein was extracted with RIPA lysis buffer, and its concentration was quantified by bicinchoninic acid assay (Pierce, USA). Then, 5X SDS (sodium dodecyl sulfate) loading buffer was added to 20 μg of protein and denatured at 95 °C for 5 minutes. Then, the total protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm NC (nitrocellulose) membrane (Beyotime). Afterwards, the membrane was blocked with 5% skim milk, BACE1, APP, C99 and sAPPβ antibodies (1:1,000) and β-actin antibody (1:1,000) (both from Cell Signaling Technology, USA) were added and overnight at 4 °C. reacted with After washing, the cells were reacted with a specific secondary antibody for 1 hour, and then washed three times with PBST. Protein bands were detected using an odyssey scanning system (LI-COR, Lincoln, NE, USA). The experiment was performed three times in triplicate, and representative figures are shown. Densitometric analysis of the data was performed using a cooled CCD camera system EZ-Capture II and CS analyzer version 3.00 software (ATTO Co., Tokyo, Japan).

1.11. statistical analysis

All experiments are presented as mean ± SEM for triplicate experiments. Statistically significant values were analyzed using ANOVA (analysis of variance) and Duncan's test (Systat Inc., Evanston, IL, USA), and a P value <0.05 was considered statistically significant.

Example 2. Inhibitory effect of flavanone derivatives on AChE, BChE and BACE1

The present inventors investigated the inhibitory effects of flavanone derivatives on AChE, BChE and BACE1 in order to confirm the therapeutic effect of flavanone derivatives on Alzheimer's disease. As a result, AChE, BChE and BACE1 were inhibited concentration-dependently by the flavanone derivative (Table 1 and FIG. 2). All of the tested flavanone derivatives showed remarkable AChE inhibitory activity. In particular, the IC ₅₀ value of didimine was 2.13 ± 0.16 µM, which was higher than that of ponsillin (IC ₅₀ : 12.96 ± 0.69 µM) and naringenin (IC ₅₀ : 36.20 µM). ± 0.10 μM), showed a potent inhibitory effect of AChE (Fig. 2A).

Similar to the results of the AChE inhibition assay, didimine and foncerin had IC ₅₀ values of 8.12, respectively, compared to the positive controls (IC ₅₀ of berberine: 1.69 ± 0.14 μM; and IC ₅₀ of galantamine: 1.23 ± 0.11 μM). showed inhibitory activity of BChE as ± 0.42 and 6.29 ± 0.52 μM (Fig. 2B).

In addition, in the case of BACE1, didimine, poncerin, and naringenin inhibited BACE1 by showing IC ₅₀ values of 2.34 ± 0.07, 3.96 ± 0.09, and 38.06 ± 3.23 μM, respectively, compared to the positive control, quercetin (21.71 ± 1.42 μM). It was confirmed that there was activity (FIG. 2C).

^a,b,c 50% inhibitory concentrations (IC ₅₀ , μM) are presented as mean ± SEM for triplicate experiments;

^d SI: selection index (BChE/AChE); and

^e Berberine, ^f Galanthamine and ^g Quercetin were used as positive controls for ChE and BACE1 assays, respectively.

Example 3. Kinetic analysis of inhibition of flavanone derivatives against AChE, BChE and BACE1

In order to determine the mechanism of inhibition of BACE1, AChE and BChE induced by flavanone derivatives, the present inventors investigated the inhibition kinetics using Lineweaver-Burk plot and Dixon plot at different substrate and inhibitor concentrations. The inhibition constant (K _i ) was determined by taking the x-axis intercept value as ^_ K _i in the analysis of the Dixon plot.

As a result, AChE inhibition by Ponsillin was confirmed as a non-competitive inhibitor with a Ki value of 20.43 μM, and didimine was confirmed to be a mixed type AChE inhibitor with a Ki value of 6.86 μM (FIG. 3 and Table 2). In the inhibition of BChE, didimine was found to be a mixed-type inhibitor with a Ki value of 9.11 μM, and foncerin was identified as a competitive inhibitor with a Ki value of 10.33 μM (FIG. 4 and Table 2). In addition, in the inhibition of BACE1, didimine and poncerin were identified as mixed type inhibitors with Ki values of 4.99 and 7.61 µM, respectively (FIG. 5 and Table 2).

determined by ^a Dixon plot; and ^b determined by Dixon and Lineweaver plot.

Example 4. Molecular Docking Analysis of Flavanone Derivatives to BACE1

Molecular docking models of the flavanone derivative and QUD (2-amino-3-{(1R)-1-cyclohexyl-2-[(cyclohexylcarbonyl) amino] ethyl}-6-phenoxyquinazolin-3-Ium) are shown in Figure 6. . According to the Protein Data Bank, QUD is the most potent BACE1 inhibitor reported to date. Docking studies were performed with the protein structure 2WJO. The 2WJO residues in the results are usually numbered differently compared to BACE1. For example, in BACE1, the catalytic pair includes Asp32 and Asp228, whereas in the 2WJO structure they correspond to Asp36 and Asp232. In the ligand-enzyme complex generated by Autodock 4.2, the flavanone derivative and QUD were found to be stably located in the same pocket of BACE1.

The interaction of didimine with the corresponding ligand at the active site of BACE1 was achieved through hydrogen bonding between the sugar moiety of didimine and Asp36 and Lys111 residues of BACE1 (FIG. 6A and Table 3). In addition, Phe112, Gly78, Lys79, Tyr75, Trp80, Val73, Arg132, Ile130, and Tyr202 showed a hydrophobic interaction (Van der Waals interaction), thereby enhancing the interaction between BACE1 and didimine.

Poncerin formed two hydrogen bonds with BACE1, and showed a binding affinity of -8.2 kcal/mol (FIG. 6B and Table 3). Residues Ile130 and Asp232 of BACE1 form two hydrogen bonds with the sugar moiety of Ponsirin. Strong hydrophobic interactions were formed between Ponsyrin and the residues Phe112, Tyr75, Trp119, Gln16, Leu34, Thr236, Gly234, Gly38 and Tyr202 of BACE1.

Naringenin formed a hydrogen bond with BACE1 and showed a binding affinity of -6.98 kcal/mol (FIG. 6C and Table 3). Ile130 of BACE1 formed a single hydrogen bond with naringenin. Strong hydrogen interactions were formed between naringenin and the residues Arg132, Asn41, Ser39, Ile122, Phe112, Gln77, Trp80, Val73 and Lys79 of BACE1.

In contrast, residues Gly234, Asp232 and Asp36 of BACE1 formed hydrogen bonds with QUD and hydrophobic interactions with residues Leu34, Phe112, Lys79, Gly78, Tyr75, Tyr202 and Arg239 (Fig. 6D). In addition, the docking protocol of the 2WJO-QUD complex was evaluated (Fig. 6E).

Example 5. Molecular Docking Analysis of Flavanone Derivatives to AChE and BChE

The present inventors performed molecular docking studies to confirm that flavanone derivatives bind to AChE and BChE. According to the results of the Autodock 4.2 simulation, didimine and ponyrin were found to have binding energies of -11.7 and -10.1 kcal/mol with AChE, respectively (FIG. 7 and Table 3).

Didimine formed five hydrogen bonds with Arg296, Asp74, Tyr341, Asn87, and Gln71 residues present in the active site of AChE (FIG. 7A and Table 3). Two residues, Asn87 and Gln71, form hydrogen bonds with the methoxy group of didimine, and residues Arg296, Asp74, and Tyr341 form three hydrogen bonds with the sugar moiety of didimine. Residues Leu289, Trp286, Leu76, Val73, Phe338, Thr75 and Tyr337 of AChE formed hydrophobic interactions with didimine.

Ponsillin formed three hydrogen bonds with AChE, and showed a binding energy of -10.1 kcal/mol (FIG. 7B and Table 3). Residue Asp74 of AChE formed a hydrogen bond with the hydroxyl group of fonnsilin, residue Arg296 formed a hydrogen bond with the oxygen atom of fonsilin, and residue Ser293 formed a hydrogen bond with the sugar moiety of fonsilin. In addition, residues Gln291, Glu292, Val294, Phe295, Phe297, Tyr124, Tyr337, Trp286, Tyr341, Phe338 and Leu289 of AChE formed hydrophobic interactions with ponyrin.

Donepezil, a positive control, showed a binding energy of -11.06 kcal/mol with AChE and formed a hydrogen bond with the Phe295 residue of AChE (Table 3). Tyr72, Trp286, Tyr 337, Trp86, His447, Gly448, Glu202, Ser203, Gly121, Gly120, Phe338, Tyr124, Phe297, Tyr341, Val294 and Ser293 residues of AChE formed hydrophobic interactions with donepezil.

Didimine exhibited a binding affinity of -7.8 kcal/mol with BChE, and formed five hydrogen bonds with residues Gln517, Gln518, Asp391, Glu422, and Lys513 present in the active site of BChE (FIG. 7C and Table 3). Lys513, Gln517 and Gln518 residues formed hydrogen bonds with didimine's methoxy group, hydroxyl group and oxygen atom, respectively. In addition, Asp391 and Glu422 residues formed a hydrogen bond with the sugar moiety of didimine. Thr512, Met511, Tyr500, Tyr420, Thr502, Arg515 and Ala516 residues of BChE form strong hydrophobic interactions with didimine.

Ponsyrin interacts with 16 residues of Ala516, Asp375, Glu422, Met511, Gln518, Tyr373, Thr374, Val377, Lys513, Arg515, Gln517, Leu514, Tyr500, Tyr420, Thr502 and His372 at the active site of BChE ( Figure 7D and Table 3). Residues Asp375 and Gln518 of BChE form two hydrogen bonds with the sugar moiety of fonnsilin, Glu422 and Met511 form two hydrogen bonds with the methoxy group of fonnsilin, and residue Ala516 forms hydrogen with the oxygen atom of fonnsilin. bond was formed.

In contrast, the positive control, donepezil, formed interactions with 14 residues of Asp74, Arg296, Ser293, Gln291, Glu292, Val294, Phe295, Phe297, Tyr124, Tyr337, Trp286, Tyr341, Phe338 and Leu289 in the active site of BChE. , of which three hydrogen bonds were formed (Table 3).

Example 6. Aβ _25-35 -Protective effect of flavanone derivatives against induced neuronal cell death

The present inventors performed experiments to measure cell viability by pretreatment with flavanone derivatives at different concentrations in order to confirm whether flavanone derivatives have an effect of protecting cells from Aβ _25-35 -induced neuronal cell death. As a result, it was confirmed that when PC12 cells were treated with 10 μM of Aβ _25-35 , cell viability was reduced by 46%, but when PC12 cells were pretreated with flavanone derivatives, cell viability was remarkably increased (FIG. 8). . This means that the flavanone derivative has a cytoprotective effect against the death of nerve cells induced by Aβ _25-35 .

In particular, when 10 and 20 μM of didimine were pretreated, the cell viability of PC12 cells was significantly increased to 82 ± 0.3% and 94 ± 0.3%, respectively. In addition, pre-treatment with 15 and 30 μM of naringenin increased cell viability to 66 ± 0.7% and 82 ± 0.5%, respectively, and pre-treatment with 50 and 100 μM of naringenin increased cell viability to 61 ± 0.6% and increased to 69 ± 1.5%. Positive controls, curcumin and resveratrol, increased cell viability to 86 ± 0.9% and 67 ± 1.2%, respectively, at a concentration of 25 µM.

From the above results, it was confirmed that the flavanone derivatives had a remarkable cytoprotective effect against the cytotoxicity of neurons induced by Aβ _25-35 .

Example 7. Aβ _25-35 -Effect of didimine on induction of neurotoxicity

The present inventors performed an experiment to confirm whether didimine, among flavanone derivatives, has a cytoprotective effect against neurotoxicity induced by Aβ _25-35 . As a result, when PC12 cells were treated with Aβ _25-35 , the cell viability was significantly reduced to 43%, but when pre-treated with 5 to 40 μM of didimine, the cytotoxicity induced by Aβ _25-35 was significantly reduced. It was confirmed that there was an effect of recovering cell viability (FIG. 9A).

In addition, the present inventors performed experiments to determine whether didimine affects the expression of BACE1, amyloid precursor protein (APP), soluble N-terminal fragment (sAPPβ), and 12-kDa C-terminal fragment (C99) proteins. As a result, it was confirmed that the expression of BACE1, sAPPβ, and C99 was markedly reduced in didimine-treated cells compared to the control group in a concentration-dependent manner (FIGS. 9B and 9C). However, there was no significant change in the expression of APP.

From the above results, didimine protects cells from cytotoxicity induced by Aβ _25-35 and significantly reduces the expression of BACE1, sAPPβ and C99 to block Aβ aggregation, thereby preventing or treating Alzheimer's disease. Confirmed.

Claims (7)

delete delete delete delete delete delete In vitro (in vitro), the step of treating the cells with didymin represented by the following formula (1); inhibiting the overexpression or overactivity of AChE, BChE and BACE1 enzymes, including; As a method of suppressing the overexpression of sAPPβ (soluble N-terminal fragment) and C99 (12-kDa C-terminal fragment),
The didimine hydrogen bonds with residues Arg296, Asp74, Tyr341, Asn87, and Gln71 present in the active site of AChE, and forms hydrophobic interactions with residues Leu289, Trp286, Leu76, Val73, Phe338, Thr75, and Tyr337 to form AChE inhibit activity,
The didimine hydrogen bonds with residues Gln517, Gln518, Asp391, Glu422 and Lys513 present in the active site of BChE, and forms hydrophobic interactions with residues Thr512, Met511, Tyr500, Tyr420, Thr502, Arg515 and Ala516 to form BChE inhibit activity,
The didimine hydrogen bonds with Asp36 and Lys111 residues of BACE1 and forms hydrophobic interactions with Phe112, Gly78, Lys79, Tyr75, Trp80, Val73, Arg132, Ile130 and Tyr202 to inhibit the activity of BACE1,
The didimine inhibits the expression of sAPPβ (soluble N-terminal fragment) and C99 (12-kDa C-terminal fragment), thereby inhibiting the formation of β-sheet structure of amyloid protein, method
[Formula 1]
.