KR20200052191A - Ligand Specific for Olfactory Receptor and Use thereof - Google Patents

Abstract

The present invention relates to an olfactory receptor-specific ligand and uses thereof. More particularly, the present invention relates to a stilbene compound that specifically binds to an olfactory receptor, and a pharmaceutical composition and a functional health food composition for the treatment or prevention of a disease, the composition comprising the stilbene compound as an active ingredient.

Description

Oligoceptor specific ligands and uses thereof {Ligand Specific for Olfactory Receptor and Use thereof}

The present invention relates to olfactory receptor specific ligands and uses thereof, and more specifically, a stilbene compound that specifically binds olfactory receptors and pharmaceutical compositions for the treatment or prevention of diseases comprising it as an active ingredient, health functional food It relates to a composition.

It is known that the most unknown area of the human sense is the olfactory field due to its complexity, and in particular, the cognition of the olfactory is made by a combination of nerve signals generated by numerous olfactory neurons, and the mechanism is known to be very complex.

In particular, the olfactory receptor, which is known to be composed of membrane proteins expressed in cell membranes, has a very complex structure and is extremely difficult to express in xenogeneic cells, is a protein that selectively binds odor substances and is expressed on olfactory neuron membranes. have. Humans have about 390 olfactory receptors, and olfactory nerve signals are generated in olfactory neurons by selective binding between odor substances and olfactory receptors. The generated olfactory nerve signals are collected in the olfactory bulb, which is a part of the human brain, and a combination of signals occurs, and the smell is recognized by recognizing the combined olfactory neurons in the cerebrum.

Recently, research on mechanisms and diseases of these olfactory receptors has been started, and as new physiological roles of these receptors have been identified, interest in developing new drugs using the receptors has been generated.

On the other hand, pterostilbene (pterostilbene) is one of the antioxidants extracted from the blueberry has an antioxidant activity and anti-cholesterol effect is known, but other physiological mechanisms have not been studied yet.

Korean Registered Patent 10-1795495

It is an object of the present invention to provide an olfactory receptor-specific ligand stilbene compound, a composition comprising the same, and its pharmaceutical, food, and cosmetic uses.

In order to solve the above-mentioned problems, the present invention provides a stilbene compound which is an olfactory receptor specific ligand.

More specifically, the present invention provides scentbene compounds which are olfactory receptor specific ligands, as well as pharmaceutically acceptable salts, hydrates, hydrated salts, polymorphic crystal structures, racemates, diastereomers or enantiomers thereof. Includes.

In one example, the present invention includes pterostilbene having the structure of Formula 1 among several stilbene compounds as olfactory receptor specific ligands:

[Formula 1]

The present invention is also a composition comprising the stilbene compound, a pharmaceutically acceptable salt, hydrate, hydrated salt, polymorphic crystal structure, racemate, diastereomer or enantiomer thereof, for the treatment and prevention of diseases A composition, a food composition, and a cosmetic composition are provided.

The stilbene compound, which is an olfactory receptor specific ligand according to the present invention, can be used in various industrial applications as an active ingredient in health functional foods, for the treatment or prevention of various diseases associated with olfactory receptors.

1 shows a cleavage map of a recombinant vector used for constructing olfactory receptor-expressing cell lines according to the present invention (pIRES-RTP1-IRES-GST-OR5V1-IRES-puroR).
2 shows a cleavage map of a recombinant vector used for constructing olfactory receptor-expressing cell lines according to the present invention (pIRES-RTP1-IRES-GST-OR6M1-IRES-puroR).
Figure 3 is an image confirming the protein expression results in olfactory receptor-expressing cells obtained according to the present invention.
4 is a view schematically showing the surface modification process in the SPR sensor in the present invention.
5 is a result showing the RU value of pterostilbene in the SPR sensor according to the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Hereinafter, the present invention will be described in detail.

The present invention, in one aspect, the olfactory receptor specific ligand stilbene compounds, pharmaceutically acceptable salts, hydrates, hydrated salts, polymorphic crystal structures, racemates, diastereomers or enantiomers, containing the compounds It relates to a composition.

In the present invention, the stilbene compound is a resveratrol derivative, which collectively refers to compounds obtained by methylation of a resveratrol hydroxyl group. As a preferred example, pterostilbene having the structure of Formula 1 is a newly disclosed compound as a ligand specifically binding to the olfactory receptor, and has the structure of Formula 1 below:

[Formula 1]

In the present invention, the olfactory receptor is OR6M1, but is not limited thereto.

In the present invention, for use according to such olfactory receptor specific binding, the stilbene compound of the present invention can be utilized for the treatment and / or prevention of various diseases associated with olfactory receptors. Alternatively, pharmaceutically acceptable salts, hydrates, hydrated salts, polymorphic crystal structures, racemates, diastereomers or enantiomers thereof may also be utilized.

For example, such diseases include various metabolic diseases such as fatty liver and cholesterol, digestive system diseases, liver-related diseases, various cardiovascular diseases based on antioxidant activity, neurological diseases, and various skin-related diseases. In addition, olfactory receptors All diseases related to are included.

The term “compound” herein refers to compounds represented by the corresponding formulas, as well as their corresponding clathrates, hydrates, solvates, crystalline and non-crystalline forms, isoforms, and polymorphs of interest. It is defined to include all forms of. In addition, the term "compound" of the present invention is meant to include a pharmaceutically acceptable salt of a compound of the present invention, unless a pharmaceutically acceptable salt thereof is mentioned. It is also understood that the compounds and salts may form solvates or may exist in substantially uncomplexed forms, such as anhydrous forms.

The term "solvate" herein refers to a molecular complex in which a solvent molecule, such as a crystallization solvent, has been incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is referred to as a hydrate. Pharmaceutically acceptable solvates include hydrates, alkoxylates such as methanolate and ethanolate, acetonitrile, and the like. Such compounds may also exist in polymorphic form.

The term "salt" in the context of the present invention refers to those salts that retain the biological effect and properties of the parent compound and are not biologically or otherwise harmful at the doses administered, and "pharmaceutically acceptable salts" It is meant to include. Salts of the compounds of the present invention can be prepared from inorganic or organic acids or bases. The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids or bases. The term 'pharmaceutically acceptable salt' is suitable for use in contact with tissues of human and lower animals without excessive toxicity, irritation, allergic reactions and the like, within the scope of proper medical judgment and corresponds to a reasonable benefit / risk ratio Refers to these salts, and non-toxic acid addition salts derived from inorganic and organic acids. For example, S. M. Berge et al. Details pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 1977, 66: 1 et seq.

Suitable salt derivatives are, for example, halide, tosylate, mesylate, thiocyanate, sulfate, bisulfate, sulfite, bisulfite, arylsulfonate, alkylsulfate, fumarate, oxalate, phosphonate, phosphate , Monohydrogen-phosphate, dihydrogenphosphate, metaphosphate, pyrophosphonate, alkanoate, cycloalkylalkanoate, arylalkonate, adipate, alginate, aspartate, benzoate, fumarate, Glucoheptanoate, glycerophosphate, lactate, malate, nicotinate, palmitate, pectinate, picrate, pivalate, succinate, tartrate, citrate, camphorate, camphorsulfonate, digluco Nate, trifluoroacetate, hydrochloride, hydrobromide, or salicylate It should be, but is not limited thereto. Acid addition salts can be prepared by dissolving the compound in a conventional method such as an aqueous solution of excess acid and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol in water may be heated, and then the mixture may be evaporated to dryness or the precipitated salt may be suction filtered.

On the other hand, when the stilbene compound according to the present invention has a prophylactic and therapeutic effect against diseases, such as olfactory receptors, the salts, solvates and polymorphs also have the same effect. It's self-explanatory.

According to an aspect of the present invention, there is provided a composition for preventing, improving, alleviating or treating a disease comprising the stilbene compound or a salt thereof as an active ingredient. The composition may be variously commercialized, for example, a pharmaceutical composition, a food composition including a dietary supplement, a quasi-drug composition, and the like.

The term “prevention” herein refers to blocking the appearance of a disease or symptom associated with the disease, in asymptomatic subjects at risk of developing the disease, eg, by exposure to the cause of the disease.

The term "treatment" herein refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after onset. “Improvement” and “mitigation” in relation to signs or symptoms of the disease or pathological condition refer to the observable beneficial effects of treatment. The beneficial effects are delayed onset of the clinical symptoms of the disease in susceptible subjects, reduced severity of some or all clinical symptoms of the disease, slower progression of the disease, improvement of the subject's overall health or well-being, or specific to a specific disease It can be demonstrated by other parameters well known in the art.

In the present invention, the stilbene compound in the composition may be included as an active ingredient, wherein the term "comprising as an active ingredient" includes the prevention, reduction, alleviation or elimination of signs or symptoms of a disease or disorder, but is not limited to It is meant to include an amount sufficient to affect the desired biological effect, such as beneficial results, and this amount is referred to as an “effective amount”. Thus, the total amount of each active ingredient in the composition or method is sufficient to represent a significant individual benefit. Thus, the effective amount will vary depending on the situation being administered. An effective amount can be administered upon one or more prophylactic or therapeutic administrations.

According to an aspect of the present invention, there is provided a food composition for preventing, improving or alleviating a disease comprising the stilbene compound or a salt thereof as an active ingredient.

According to an aspect of the present invention, there is provided a quasi-drug composition for preventing, improving or alleviating a disease comprising the stilbene compound or a salt thereof as an active ingredient.

The composition according to the invention can be used for the prevention, alleviation, improvement and treatment of various olfactory receptor-related diseases.

In the composition according to the present invention, the stilbene compound or a salt thereof is at least about 0.0001% to 90%, 0.001% to 90%, 0.01% to 90%, 0.1% to 90%, 0.1% to 90%, 0.1% in the final form. Can be included in the range of ~ 80%, 0.1% ~ 70%, 0.1% ~ 60%, 0.1% ~ 50%, 0.1% ~ 40%, 0.1% ~ 30%, 0.1% ~ 20% or 0.1% ~ 10% have. The above percentage can be calculated based on the total weight of the composition relative to the weight or the total volume, and the concentration can be adjusted according to the desired effect of the composition or the product into which the composition is incorporated.

The composition according to the invention can be applied systemically or topically.

The composition of the present invention may further contain physiologically acceptable additives. The additives include excipients, diluents, fillers, solubilizers, lubricants, auxiliaries, binders, disintegrants, coating agents, encapsulating agents, ointment bases, suppository bases, aerosolizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, tonicity agents. , Buffers, sedatives, preservatives, antioxidants, corrective agents, flavoring agents, colorants, functional substances such as cyclodextrins and biodegradable polymers, stabilizers, and the like. The additives are well known in the art and can be selected from those described in general pharmaceutical literature.

The composition of the present invention may be various formulations, oral or parenteral. When formulating the composition, one or more buffers (e.g. saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g. Aluminum hydroxide), suspending agents, thickener wetting agents, disintegrating agents or surfactants, diluents or excipients.

Examples of solid compositions for oral administration include tablets, oral tablets, sublingual tablets, capsules, pills, powders, granules, and the like. Solid compositions can be prepared by mixing one or more active ingredients with one or more inert diluents. The composition may further contain additives other than inert diluents, such as lubricants, disintegrants and stabilizers. Tablets and pills may be coated with an enteric or gastrointestinal film as needed. These can be coated with two or more layers. They can also be adsorbed to sustained release materials, or can be microencapsulated. Moreover, the composition can be encapsulated by easily degradable materials such as gelatin. They can also be dissolved in suitable solvents such as fatty acids or mono, di or triglycerides thereof to form soft capsules. Sublingual tablets can be used when rapid action properties are required.

Examples of liquid compositions for oral administration include emulsions, solutions, suspensions, syrups and elixirs, and the like. The composition may further contain a commonly used inert diluent, for example purified water or ethyl alcohol. The composition may contain, in addition to inert diluents, adjuvants, such as wetting and suspending agents, sweeteners, flavoring agents, flavoring agents and additives such as preservatives.

The composition of the present invention may be in the form of a composition for injection for parenteral administration containing one or more active ingredients, and examples of the composition for injection include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Diluents for aqueous solutions or suspensions may include, for example, distilled water for injection, physiological saline and Ringer's solution. Non-aqueous diluents for solutions and suspensions can include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol and polysorbates. The composition may further include additives such as preservatives, wetting agents, emulsifying agents, dispersing agents, and the like. They can be sterilized, for example, by filtration with a bacteria-retaining filter, blending with a sterilizer, or by gas or radioisotope irradiation sterilization. Injectable compositions can also be provided as sterile powder compositions to dissolve in sterile injectable solvents prior to use.

The pharmaceutical composition of the present invention may contain a single active ingredient or a combination of two or more active ingredients. In the combination of multiple active ingredients, their respective contents can be appropriately increased or decreased in consideration of their therapeutic effect and safety.

In addition, the formulations of the present invention may contain other pharmacologically active ingredients as appropriate, as long as they are not contrary to the object of the present invention.

In the present invention, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6727 on the Health Functional Food, and the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful effects for health purposes such as physiological action.

When the composition of the present invention is used as a food additive, the composition may be added as it is or used with other foods or ingredients, and may be suitably used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the intended use. The food additive usually refers to a small amount intentionally added to the food for the purpose of improving the appearance, flavor, texture, or storage, and improves the quality of the food, improves the preservation or palatability, as well as the nutritional value and the practicality of the food. It is meant to be used for the purpose of enhancing value. This may be a substance used in addition, mixing, infiltrating, or other methods to food in manufacturing or preserving food, as defined in Article 2, No. 2 of the Food Sanitation Act.

There are no particular restrictions on the type of food of the present invention. Examples of foods to which the composition of the present invention can be added are meat, sausage, bread, chocolate, candy, snack, confectionery, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages and vitamin complexes, and the like, may include all foods in a conventional sense, and include foods used as feed for animals.

The health functional food of the present invention may include a normal food additive, and whether or not it is suitable as a food additive is related to the item according to the general rules and general test methods of food additives approved by the Food and Drug Administration, unless otherwise specified. Judging by standards and standards.

In addition, the food composition of the present invention is a variety of nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol , Carbonic acid used in carbonated beverages, and the like. In addition, it may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

In addition, the food may be prepared in the form of tablets, granules, powders, capsules, liquid solutions and pills, etc. according to a known manufacturing method. There are no particular limitations on other components except for including the compound of the present invention as an active ingredient, and may include various conventional flavoring agents or natural carbohydrates as additional components.

In the present invention, it may also be provided in the form of quasi-drug composition containing the stilbene compound.

Here, "quasi-drug" is a fiber, rubber product or the like used for the purpose of treating, alleviating, treating or preventing a human or animal disease, and has a weak effect on the human body or does not directly affect the human body. This is a product that is one of the products that are not similar to the other, or used for sterilization, pesticide, and similar purposes to prevent infection, and are used for the purpose of diagnosing, treating, alleviating, treating or preventing human or animal diseases. Means that are not devices, machines, or devices, and items that are used for the purpose of pharmacologically affecting the structure and function of a person or animal, excluding items that are not devices, machines, or devices, and include external preparations for skin and personal hygiene products do. The external preparation for skin is not particularly limited thereto, but is preferably used in the form of ointment, lotion, spray, patch, cream, powder, suspension, gel, or gel. The personal hygiene products are not particularly limited, but preferably soap, cosmetics, wipes, tissues, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, ball touch, mascara, eye shadow, sun cream lotion , Hair care products, air freshener gel or cleaning gel. In addition, the quasi-drug composition of the present invention is not particularly limited thereto, but is preferably manufactured in the form of a product selected from the group consisting of disinfectant cleaners, shower foams, eye wash liquids, wipes, hand washes, humidifier cleaners, ointments, and filter cleaners. Can be used.

When the composition of the present invention is added to a quasi-drug composition, the composition may be added as it is or used with other quasi-drug components, and may be suitably used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the intended use.

Further, the stilbene compound according to the present invention or a pharmaceutically acceptable salt thereof may also be provided as a cosmetic composition for preventing, improving or alleviating a disease.

The cosmetic composition according to the present invention includes, but is not limited to, lotions, ointments, gels, creams, patches or sprays, and the like. In addition, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, Skin such as fillers, metal ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles, or any other ingredient commonly used in skin external composition. It may contain adjuvants commonly used in science. In addition, the ingredients may be introduced in an amount commonly used in the field of skin science.

The stilbene compounds of the present invention can be added to feed compositions for the purpose of preventing, improving or alleviating disease.

In the present invention, the term, “feed additive” is a substance added to feed for various effects such as supplementing nutrients and preventing weight loss, improving digestibility of fiber in feed, improving oil quality, preventing reproductive disorder and improving conception rate, and preventing high temperature stress in summer. It includes. The feed additive of the present invention corresponds to the supplementary feed under the feed management method, mineral preparations such as sodium hydrogen carbonate, bentonite, magnesium oxide, and composite minerals, mineral preparations such as zinc, copper, cobalt, and selenium, mineral preparations and kerosene , Vitamin AD, E, nicotinic acid, vitamins such as vitamin B complex, protective amino acids such as methionine and lysine, protective fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria), live cultures such as yeast cultures, mold fermentation products, Yeast agents and the like may be further included.

In the present invention, the term "feed" may be any natural or artificial diet, a meal, or the like, or a component of the meal, for animals to eat, ingest, and digest.

The feed additive according to the present invention can be applied without limitation as long as it is an object aimed at preventing or improving a disease. For example, it can be applied to any individual such as non-human animals such as dogs, cats, monkeys, rabbits, mormots, rats, mice, cows, sheep, pigs, goats, birds and fish.

In addition, the present invention provides a method for preventing, improving, alleviating or treating a disease, comprising administering a therapeutically effective amount of the stilbene compound or a salt thereof.

Suitable individuals to be treated according to the present invention include mammalian individuals. The mammal according to the invention is, but is not limited to, humans, dogs (canine), feline, bovine, caprine, equine, ovine, pig (porcine), rodents, lagomorphs, primates, etc., and in utero mammals. The individual can be both sex and can be any stage of development.

The administration route of the treatment method according to the present invention is intravenous, intraperitoneal, subcutaneous, intracranial, intradermal, intracellular, intraocular, intrathecal, intracranial, intranasal, through muscle, by inhalation, orally, rectal As, but not limited to, by a drop, a patch and an implant.

Suitable dosages and dosing regimens of the present invention can be determined by conventional range-measurement techniques known to those skilled in the art. Generally, treatment is initiated with a smaller dose that is less than the optimal dose of the compound. Subsequently, the dosage is increased in small increments until the optimum effect is reached under the environment. The methods of the invention will typically involve the administration of about 0.1 to about 300 mg of one or more of the above compounds per kg of body weight of an animal or mammal.

Hereinafter, examples and the like will be described in detail to help understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be interpreted as being limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example 1. Identification of specific ligands of olfactory receptor OR6M1 according to the present invention

(1) Recombinant olfactory receptor expression cell line construction for fixation on SPR chip

To build a cell line stably expressing the olfactory receptor, it is cloned into a mammalian expression vector, pIRES vector (purchased by Clontech), and a chaperone protein RTP1 (receptor transporter protein1, NP_714919.2 origene purchased) for expression of human olfactory receptor protein. To prepare pIRES-RTP1.

After subcloning the human olfactory receptor OR6M1 (NP_001005325, purchased from origene), OR5V1 (NP_110503, purchased from origene) clone to the GST tagging vector pGEX4T1 (GE), followed by GST-tag It was subcloned into the prepared pIRES-RTP1. Through this, pIRES-RTP1-IRES-GST-OR5V1 (or OR6M1) was constructed.

In order to establish a stable expression cell line, screening was performed using puromycine as a selective drug marker, and for this purpose, the expression vector must have a puromycin resistance gene, and for this, a pRIES-puro3 vector is used. Was used.

In the previously prepared pIRES-RTP1-IRES-GST-OR5V1, the RTP1-IRES-GST-OR5V1 portion was subcloned into the pIRES-puro3 vector (clonetech) through restriction enzyme treatment (refer to the vector cleavage map of FIGS. 1 and 2). ).

(2) Stable olfactory receptor expression cell line construction

① transformation

Transient transfection (temporary infusion) Using the lipofectamine 3000 kit (Invitrogen) in the HEK293t cell line (ATCC®CRL-11268 ™, purchased from ATCC), which is a human embryonic kidney cell with high efficiency, the expression according to the protocol supplied to the kit Dragon clones were transformed.

② Screening of transformed cells to establish stable cell weight

Transfected cells were selected by treatment with puromycin (10 ug / ml) 2 days after the temporary injection for screening of the expressing cell lines only. Expression cells were diluted to a concentration of 1 cell / well, and cultured at the level of 96 wells, and a single clone was selected and cultured.

③ Stable expression of target protein in cell line

To confirm the stable expression cell line, the olfactory receptor GST tag was immunostained using a gst antibody (purchased from ab chem) to confirm the expression level and expression frequency, and as a result, uniform expression could be confirmed.

In detail, in order to confirm the stable expression of the GST-tagged olfactory receptor through the following process, the cells were immunostained using GST antibodies to confirm expression and expression patterns.

For immunostaining, first, cells cultured for 2 days on Coverslip were immobilized with 4% paraformaldehyde in PBS for 20 minutes. After fixation, to remove the remaining paraformaldehyde, it was wiped three times for 5 minutes using PBS (phosphate buffered saline). For stable access of the antibody, the solution dissolved in PBS at a concentration of 5% BSA (Bovine Serum Albumin, Sigma Aldrich), and 0.3% Triton X-100 (Sigma Aldrich) is treated for 30 minutes to perform cell osmosis. did. Cells were treated with GST antibody diluted to a concentration of 1: 200 at a concentration of 1: 200 using a solution in which 0.1% Tween 20 (Sigma Aldrich) and 5% BSA were diluted in PBS. To remove excess GST antibody that was not bound, wipe the cells 5 times for 10 minutes with a solution of 0.1% Tween 20 (Sigma Aldrich) and 5% BSA diluted in PBS, and then add FITC (Fluorescein isothiocyanate) to the same solution. ) Was tagged with a secondary antibody (anti-mouse IgG-FITC, Sigma-Aldrich) diluted 1: 1000. When FITC is used as a fluorescent substance and cells are observed using a fluorescent microscope, a green fluorescent signal is displayed. Cells were wiped 5 times for 10 minutes using a solution of excess antibody in 0.1% Tween 20 (Sigma Aldrich) and 5% BSA diluted in PBS. After washing with PBS for 3 more 10 minutes, the cells were treated with 300 uM DAPI (4 ′, 6-diamidino-2-phenylindole) in 1: 1000 diluted in PBS for 20 minutes. Since DAPI can bind to DNA, the DAPI signal that can be observed with a fluorescence microscope tells the location of the nucleus. After wiping with PBS three times for 5 minutes, samples were prepared by mounting coverslips on slide glass. The prepared cell sample was observed for a fluorescence signal at a specific wavelength using a fluorescence microscope (Nikon Corporation).

As a result, as shown in FIG. 3, cells expressing the olfactory receptor can observe a green fluorescence signal, and it is confirmed that all the cells are expressed, so that the olfactory receptor is stably expressed in all cells. In particular, since the green signal is observed in the cytoplasm and the cell membrane, it can be seen that the expression of the olfactory receptor is normally performed. On the other hand, in the case of non-transformed Hek293t cells used as a negative control, although staining was performed through the same immunostaining method, only the blue DAPI signal representing the nucleus could be identified, and no green fluorescent signal could be identified. Through this, stable expression of the olfactory receptor was confirmed.

(3) Cell-fixed SPR chip fabrication

To make a large number of aldehyde groups on the surface of the obtained recombinant olfactory receptor cells, the 2ⅹ10 ⁶ cells were resuspensioned in 1mL of 1mM sodium metaperiodate solution in sodium acetate buffer (pH5.5), reacted with ice for 20 minutes, and reacted with 1mL of sodium. Acetate buffer pH4.0 was sequentially applied.

Meanwhile, the SPR chip used for cell immobilization used a CM5 sensor chip (Biocore, GE healthcare) with dextran treated on the surface. As in the process shown in FIG. 3, the dextran-treated CM5 sensor chip is an SPR device. Phosphorus Biacore T200, where 4 flow cells (FC) are blank (control surface without cells) in 4 flow cells (FC), FC2 is a cell that does not express olfactory receptors, and FC3 is a cell that stably expresses olfactory receptors Ordered. Specifically, in order to modify the carbonyl group on the surface of the flow cell, a crosslinking agent of ethylene dichloride (EDC) 0.4M and imide-based compound N-hydrosuccinimide (NHS) 0.1M using a 1: 1 mixture is used to flow at 10 ug / min. Was activated for 3 minutes. Then, hydrazine was treated with 5 mM, and finally, ethanolamine was treated to inactivate the ester functional group of the imide compound that did not react. Olfactory receptor expressing cells were injected into flow cell 3 at a rate of 10 ul / min for 12 minutes, and exhibited a surface density of 9,000 to 10,000 reaction units (response uit, RU).

(4) Discovering ligands that bind directly to olfactory receptors

Various substances were injected into the produced SPR chip to confirm whether the olfactory receptor was bound. Among them, as a result of injecting 50 uM of pterostilbene for 60 seconds, the RU value began to increase from the time of injection, and reactivity returned to 0 after 1 minute after the injection was completed.

In order to confirm that it is an OR6M1-specific reaction, the reaction of the cell not expressing the olfactory receptor was removed from the OR6M1 expressing cell line reaction to pterostilbene. As a result, as shown in FIG. 5, it was confirmed that direct interaction with the OR6M1 was finally performed, and pterostilbene was excavated as a ligand. In contrast, in the case of Irinotecan, it was confirmed that the RU value is less than or equal to 0, so that it does not specifically bind to OR6M1.

In the above, the applicant has described preferred embodiments of the present invention, but these embodiments are only one embodiment for implementing the technical concept of the present invention, and any modification or modification is possible as long as the technical concept of the present invention is implemented. It should be interpreted as being within the scope of.

Claims (9)

Stilbene compounds that specifically bind to olfactory receptors.
According to claim 1,
A stilbene compound characterized by being pterostilbene having the structure of Formula 1:
[Formula 1]

A composition for specific binding of olfactory receptors containing a stilbene compound as an active ingredient.
According to claim 3,
The stilbene compound is a composition characterized in that pterostilbene having the structure of Formula 1:
[Formula 1]

According to claim 3,
The olfactory receptor is characterized in that OR6M1.
According to claim 3,
The stilbene compound is a composition characterized in that the olfactory receptor ligand.
A pharmaceutical composition for treating or preventing a disease, comprising the stilbene compound according to claim 1.
A health functional food composition comprising the stilbene compound according to claim 1.
A cosmetic composition comprising the stilbene compound according to claim 1.

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