KR20170108967A - A method for screening for the prophylactic or therapeutic agent for female menopausal disease using PDK2 and a pharmaceutical composition for preventing or treating female menopausal disease comprising the therapeutic agent screened by the above method as an active ingredient - Google Patents

Abstract

The present invention relates to a method for screening a substance that can be used as a preventive or therapeutic agent for female menopausal disease by inhibiting the expression or activity of PDK2, and a pharmaceutical composition for preventing or treating female menopausal disease comprising PDK2 inhibitor as an active ingredient.

Description

A method for screening for the prophylactic or therapeutic agent for female menopausal disease using PDK2 and a pharmaceutical composition for preventing or treating female menopausal disease comprising the therapeutic agent screened by the above method as an active ingredient

The present invention relates to a method for screening for a prophylactic or therapeutic agent for female menopausal disease using PDK2 and a pharmaceutical composition for the prevention or treatment of female menopausal disease comprising the therapeutic agent screened by the above method as an active ingredient.

With the development of science and civilization, the average life expectancy of men has been prolonged. As the elderly population has increased, the life after middle age has been lengthened, and one third of the lives of modern women have been sent to menopausal menopause. Efforts are being made to improve quality.

Menopause is a transitional period in which the overall and gradual decrease of ovarian function occurs and the physiological function and sexual function are diminished or lost. Menopause, which is a permanent stoppage of menstruation after ovarian function is stopped, is a process of menopause. When menopause comes, a variety of diseases arise due to changes in hormones such as estrogen production, follicular stimulating hormone (FSH), and elevated luteininzing hormone (LH).

The disease associated with the hormone change is also called climacteric disorder, postmenopausal syndrome or menopausal syndrome, and is mainly seen in women in their 40s to 50s. In other words, menopausal disease is caused by decreased secretion of estrogen (female hormone) due to aging of the ovary, and it occurs over about 2 to 10 years before and after menopause.

Menopause is a type of endocrine syndrome and it is a transitional period in which general and gradual decrease of ovarian function occurs and physiological function and sexual function is reduced or lost. In menopausal disease, five symptoms mainly occur: i) ii) symptoms due to musculoskeletal changes, iii) symptoms due to genitourinary changes, iv) symptoms due to cranial nervous system changes, and v) general changes. Symptoms due to the change in vascularity are facial flushing, tachycardia, sweating or headache. Symptoms due to the musculoskeletal changes include muscle pain, arthralgia or back pain, and symptoms due to the change in genitourinary period include frequent urination or urinary incontinence Symptoms due to the cranial nervous system change include memory loss, depression, loss of concentration, and dizziness. The symptoms due to the general change include visual loss or change in skin and hair.

Although these symptoms vary in degree, about 80% of pre- and postmenopausal women are reported to experience it. Above all, it is noteworthy that the above-mentioned menopausal diseases have a long-term effect on important changes in the life and health of women, thereby increasing the risk of women's health-related diseases, such as osteoporosis and cardiovascular disease.

Therefore, development of a therapeutic agent for the above-mentioned menopausal disease is required for improving the physical and mental health of middle-aged women and improving the quality of life.

For the treatment of these menopausal disorders, hormonal replacement therapies and non-steroidal drugs have been developed. Hitherto, the most effective method has been hormone replacement therapy. However, it is required to develop a treatment that can replace the hormone replacement therapy because of the incessant side effects such as controversy about cancer occurrence, side effects such as headache, weight increase, and resumption of menstruation.

On the other hand, PDK (Pyruvate dehydrogenase kinase) is an enzyme that inactivates its function by phosphorylating pyruvate dehydrogenase using ATP and is distributed in eukaryotic mitochondria. The human PDK family consists of four types of alleles, PDK1, PKD2, PDK3 and PDK4, which have been shown to be important targets in the treatment of tumors, type 2 diabetes, and fatty liver. However, it is not known in the art that PDK2 is effective for the prevention or treatment of menopausal female diseases.

The inventors of the present invention found that osteoblast differentiation in osteoblast differentiated with PDK2 is lower than osteoblast differentiation in osteoblast differentiation, and bone loss is defended when PDK2 is deficient in ovariectomized animal model, Or osteoporosis, which is one type of osteoporosis. Accordingly, the present invention has been completed based on the finding that a substance that reduces the expression or activity of PDK2 can be useful for the prevention or treatment of menopausal female diseases.

Accordingly, an object of the present invention is to provide a method for screening a preventive or therapeutic agent for female menopausal disease which inhibits the expression or activity of PDK2.

It is another object of the present invention to provide a method for screening a prophylactic or therapeutic agent for female menopausal disease using cells expressing PKD2 or cells having increased PDK2 expression.

It is another object of the present invention to provide a method for the treatment and prophylaxis of osteoporosis, osteoporosis, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, memory loss, headache, neuropsychiatric disorder, dizziness, palpitations, tachycardia, nausea, And weight gain. The method of claim 1,

It is another object of the present invention to provide a composition for promoting osteoblast differentiation comprising a PDK2 inhibitor as an active ingredient.

Another object of the present invention is to provide a method for screening a substance that inhibits the expression or activity of PDK2 to promote osteoclast differentiation.

Another object of the present invention is to provide a composition for the prevention or treatment of female menopausal disease comprising PDK2 inhibitor as an active ingredient.

Another object of the present invention is to provide a composition characterized in that the PDK2 inhibitor is siRNA or antisense RNA for PDK2.

Another object of the present invention is to provide a composition wherein said PDK2 has an amino acid sequence represented by SEQ ID NO: 1.

It is another object of the present invention to provide a method for the treatment and prophylaxis of osteoporosis, osteoporosis, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, memory loss, headache, neuropsychiatric disorder, dizziness, palpitations, tachycardia, nausea, And weight gain. The present invention also provides a composition comprising the same.

In order to achieve the above object, the present invention provides a method for screening a test substance, comprising: (a) contacting a test substance to a cell; and (b) selecting a test substance that decreases the expression or activity of PDK2 protein The present invention provides a method for screening for a preventive or therapeutic agent for female menopausal disease.

In order to accomplish another object of the present invention, the present invention provides a method characterized in that the cell of step (a) is a PDK2-expressing cell or an PDK2-expressing cell.

In order to achieve the other object of the present invention, the present invention provides a method for treating female menopausal disease, which comprises administering to a female in need thereof an effective amount of at least one compound selected from the group consisting of osteoporosis, hyperthermia, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, Joint pain, muscular pain, poor circulation and weight gain.

In order to accomplish still another object of the present invention, there is provided a composition for promoting osteoblast differentiation comprising a PDK2 inhibitor as an active ingredient.

In order to achieve another object of the present invention, there is provided a method for screening a test substance, comprising the steps of (a) contacting a test substance to a cell, (b) selecting a test substance that decreases the expression or activity of PDK2 protein as compared to a control substance not contacted with the test substance, (c) contacting the test substance selected in the step (b) with osteoblasts to select a test substance that promotes differentiation, thereby screening a substance for promoting osteoblast differentiation promotion.

In order to accomplish still another object of the present invention, there is provided a composition for preventing or treating female menopausal disease comprising PDK2 inhibitor as an active ingredient.

According to another aspect of the present invention, there is provided a composition, wherein the PDK2 inhibitor is siRNA or antisense RNA for PDK2.

In order to achieve another object of the present invention, the present invention provides a composition, wherein the PDK2 has an amino acid sequence represented by SEQ ID NO: 1.

In order to achieve the other object of the present invention, the present invention provides a method for treating female menopausal disease, which comprises administering to a female in need thereof an effective amount of at least one compound selected from the group consisting of osteoporosis, hyperthermia, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, Arthralgia, muscular pain, poor circulation, weight gain, and the like.

Hereinafter, the present invention will be described in detail.

In the present invention, the relationship between PDK2 and female menopausal disease was examined by observing the degree of osteoblast differentiation by infecting the osteoblast cell line MC3T3E1 with retrovirus PDK2. As a result, it was confirmed that PDK2-overexpressed osteoblast cells had decreased cell differentiation (see Example 1).

In addition, in the present invention, the bone marrow stromal cells of PDK2-deficient mice were primary cultured, and their degree of differentiation was observed. As a result, in the mice lacking PDK2, the primary culture of bone marrow stem cells was different from that of primary cultured bone marrow stem cells in mice in which PDK2 was normally expressed, and the progenitor of osteoblasts ) Were also increased (see Examples 2 and 3).

In addition, in the present invention, ovariectomy was performed on mice lacking PDK2 and mice in which PDK2 was normally expressed, and after establishing a menopausal animal model, weight gain and bone density were observed. As a result, it was confirmed that the menopausal animal model in which PDK2 was deficient was suppressed in weight gain and had less bone loss than the menopausal animal model in which PDK2 was normally expressed (see Examples 4 to 7).

Based on the results of the above-mentioned examples, it was found that PDK2 is closely related to menopausal women's disease, and thus it was concluded that a substance inhibiting PDK2 would be effective for the prevention or treatment of menopausal women's disease .

Accordingly, the present invention provides a method for the treatment of female menopausal symptoms, comprising the steps of: (a) contacting a test substance to a cell; and (b) selecting a test substance that reduces the expression or activity of PDK2 protein as compared to a control A method for screening a disease preventing or treating agent.

The cell is a cell expressing PDK2 or a cell having an increased expression of PDK2, which is originally a cell containing a gene encoding PDK2 or a cell intentionally introduced with a gene encoding PDK2. Methods for introducing a gene encoding PDK2 into cells can be used without limitation as those known in the art. For example, the cells may be transfected with an expression vector comprising a polynucleotide encoding the PDK2 protein, or PDK2 may be infected with a retrovirus.

According to one preferred embodiment, the cell may be an osteoblast cell, preferably a mouse osteoblast cell line MC3T3E1 cell.

The cells include all cells of an animal origin such as human or cow, goat, pig, mouse, rabbit, hamster, guinea pig and the like, and are primary cultures from primary cells, secondary cells, immortalized cells, And the like.

The test substance means an unknown substance used for screening in order to check whether it decreases the expression or activity of PDK2 protein. The test substance may be any one selected from the group consisting of a chemical substance, an antisense oligonucleotide complementary to the mRNA of the PDK2 gene, an siRNA (short interfering RNA), an shRNA (short hairpin RNA), an RNAi and a natural product extract, A compound that binds complementarily to a protein, a peptide, a peptide mimetic, and an antibody, but the present invention is not limited thereto.

When it is determined that the expression level of the PDK2 protein or the activity of the PDK2 protein is decreased in the cell to which the test substance has been contacted as compared with the control substance to which the test substance is not contacted after the test substance is contacted with the cell, Can be judged as a preventive or therapeutic agent.

As a method for confirming the expression or activity of PDK2, general techniques used for confirmation of protein expression in the art can be used. For example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, sandwich assay, Western blot on polyacrylamide gel, immunoprecipitation analysis, complement fixation assay, flow cytometry using antibody against PDK2, (FACS), protein chips, and combinations thereof may be used, but are not limited thereto.

Alternatively, the step of (a) or (b) may further comprise the step of administering the selected test substance to the individual to thereby select a test substance which is inhibited from a decrease in bone density. In this case, the subject may be a rat or a mouse, and may be an animal model in which ovariectomy is performed according to a method known in the art to induce female menopausal disease. The method of administering the test substance to an individual may be, but not limited to, oral administration, transdermal administration, intravenous administration or intraperitoneal administration.

The present invention also provides a method characterized in that the cell of step (a) is a PDK2-expressing cell or an PDK2-expressing cell.

It may be a cell originally containing a gene encoding PDK2 or a cell intentionally introduced with a gene encoding PDK2. Methods for introducing a gene encoding PDK2 into cells can be used without limitation as those known in the art. For example, the cells may be transfected with an expression vector comprising a polynucleotide encoding the PDK2 protein, or the PDK2 may be infected with a retrovirus.

In the present invention, the female menopausal disease includes, but is not limited to, osteoporosis, hyperthermia, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, memory loss, headache, nervousness, dizziness, palpitations, tachycardia, nausea, , Poor circulation and weight gain, but is preferably osteoporosis.

The term "female menopausal disease" means a disease caused by or caused by the following: o) a symptom caused by a change in vascularity; ii) a symptom caused by an ovarian dysfunction; Symptoms due to musculoskeletal changes, iii) symptoms due to genitourinary changes, iv) symptoms due to changes in the cranial nervous system, and v) symptoms due to general changes.

The symptoms due to the vascular change include facial flushing, hyperthermia, tachycardia, hyperhidrosis, poor circulation or headache, and the symptoms due to the musculoskeletal changes include muscle pain, arthralgia or back pain, Or urinary incontinence. The symptoms due to the cranial nervous system change include memory loss, depression, loss of concentration, insomnia, nervous irritation, nausea and dizziness. The symptoms caused by the general change include visual loss, weight gain, , But the present invention is not limited thereto.

The osteoporosis may include, but is not limited to, menopausal osteoporosis triggered by postmenopausal female hormone reduction, primary osteoporosis resulting from aging, or osteoporosis following oophorectomy.

The present invention also provides a composition for stimulating osteoblast differentiation comprising a PDK2 inhibitor as an active ingredient.

The PDK2 inhibitor may be any one selected from the group consisting of a chemical compound, an antisense oligonucleotide complementary to mRNA of the PDK2 gene, an siRNA (short interfering RNA), an shRNA (short hairpin RNA), an RNAi and a natural product extract, A compound that binds complementarily to a protein, a peptide, a peptide mimetic, and an antibody, but the present invention is not limited thereto.

The composition may be a health functional food composition or a pharmaceutical composition for promoting osteoblast differentiation, but is not limited thereto.

When the composition is a health functional food composition, it includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. For example, as a health food, the PDK2 itself of the present invention can be produced in the form of tea, juice, and drink and then consumed, granulated, encapsulated, and powdered. In addition, PDK2 of the present invention may be mixed with a known substance or active ingredient known to have osteoblast differentiation promoting effect to form a composition.

The composition of the present invention is effective for prevention and improvement of diseases due to bone loss, and the disease caused by bone loss is preferably one selected from the group consisting of osteoporosis, rheumatoid arthritis, periodontitis, no. In addition, the composition of the present invention may be effective for bone loss due to other diseases such as hyperparathyroidism, Cushing's disease, chronic kidney disease, myeloma or antiepileptic drug, and diseases caused by bone loss known to those skilled in the art are all included in the present invention do.

(B) selecting a test substance that reduces the expression or activity of PDK2 protein as compared to a control substance not contacted with the test substance; and (c) contacting the test substance selected in step b) with osteoblasts to select a test substance that promotes differentiation, and a method for screening a substance for promoting osteoblast differentiation.

The cell of step (a) may be a cell expressing PDK2 or a cell having an increased expression of PDK2, which is originally a cell containing a gene encoding PDK2 or a cell intentionally introduced with a gene encoding PDK2. Methods for introducing a gene encoding PDK2 into cells can be used without limitation as those known in the art. For example, the cells may be transfected with an expression vector comprising a polynucleotide encoding the PDK2 protein, or PDK2 may be infected with a retrovirus.

According to one preferred embodiment, the cell may be an osteoblast cell, preferably a mouse osteoblast cell line MC3T3E1 cell.

The cells include all cells of an animal origin such as human or cow, goat, pig, mouse, rabbit, hamster, guinea pig and the like, and are primary cultures from primary cells, secondary cells, immortalized cells, And the like.

The test substance refers to an unknown substance used for screening in order to test whether or not it promotes osteoblast differentiation. The test substance may be any one selected from the group consisting of a chemical substance, an antisense oligonucleotide complementary to the mRNA of the PDK2 gene, an siRNA (short interfering RNA), an shRNA (short hairpin RNA), an RNAi and a natural product extract, A compound that binds complementarily to a protein, a peptide, a peptide mimetic, and an antibody, but the present invention is not limited thereto.

As a method for confirming the expression or activity of PDK2 in step (b), general techniques used for confirmation of protein expression in the art may be used. For example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, sandwich assay, Western blot on polyacrylamide gel, immunoprecipitation analysis, complement fixation assay, flow cytometry using antibody against PDK2, (FACS), protein chips, and combinations thereof may be used, but are not limited thereto.

Thereafter, when it is confirmed that osteoblast differentiation is promoted in the cells to which the test substance is contacted as compared with the control group in which the test substance is not contacted after the test substance is contacted with osteoblast, the test substance is used as a substance for promoting osteoblast differentiation Can be determined.

As a method for confirming the differentiation of osteoblast cells, general techniques used for confirming osteoblast differentiation in the art can be used. Examples include, but are not limited to, ALP staining, vonKossa staining, or measurement of osteogenic gene expression.

The present invention also provides a composition for the prevention or treatment of female menopausal disease comprising a PDK2 inhibitor as an active ingredient.

The PDK2 inhibitor may be any one selected from the group consisting of a chemical compound, an antisense oligonucleotide complementary to mRNA of the PDK2 gene, an siRNA (short interfering RNA), an shRNA (short hairpin RNA), an RNAi and a natural product extract, A compound that binds complementarily to a protein, a peptide, a peptide mimetic, and an antibody, but the present invention is not limited thereto.

The pharmaceutical composition according to the present invention may contain the composition of the present invention, or a pharmaceutically acceptable salt thereof, alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition for preventing or treating female menopausal disease of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI > Preferably, the pharmaceutical composition of the present invention can be transdermally administered. In the above, transdermal administration refers to administration of the pharmaceutical composition of the present invention to cells or skin to allow the active ingredient contained in the composition of the present invention to be delivered into the skin. For example, the pharmaceutical composition of the present invention can be administered in a scanning type formulation, pricking the skin lightly with a 30 gauge needle, or directly applying it to the skin.

The pharmaceutical composition of the present invention may be formulated into oral or parenteral dosage forms according to the route of administration as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent. In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of a pharmaceutical composition comprising a PDK2 inhibitor of the present invention can be administered to a patient in a single dose and can be administered by a fractionated treatment protocol administered at multiple doses over a prolonged period of time ≪ / RTI > In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.01 μg to 1,000 mg, and most preferably from 0.1 μg to 100 mg, per kg body weight of the patient per day. However, the dosage of the pharmaceutical composition of the present invention is not limited to the administration route and the number of treatments of the pharmaceutical composition, and may be varied depending on various factors such as the patient's age, body weight, health condition, sex, severity of disease, It will be understood by those skilled in the art that the pharmaceutical composition of the present invention can be used to determine an appropriate effective dose depending on the specific use as a preventive or therapeutic agent for female menopausal disease will be. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The present invention also provides a composition for the prevention or treatment of female menopausal disease, wherein the PDK2 inhibitor is siRNA or antisense RNA for PDK2.

The antisense RNA means an RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein. The antisense sequence of the present invention refers to an RNA sequence that is complementary to PDK2 mRNA and capable of binding to PDK2 mRNA and is essential for translation of PDK2 mRNA, translocation into the cytoplasm, maturation, or any other overall biological function The activity can be inhibited.

The length of the antisense RNA may be 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases. The antisense RNA may be synthesized in vitro by conventional methods and administered in vivo or may be used to synthesize antisense RNA in vivo. One example of the synthesis of antisense RNA in vitro is RNA polymerase I. One example of the synthesis of antisense RNA in vivo is the use of a vector in which the origin of the multiple cloning site (MCS) is in the opposite direction so that the antisense RNA is transcribed. The antisense RNA is preferably such that translation stop codon is present in the sequence so that it is not translated into the peptide sequence. The design of the antisense RNA which can be used in the present invention can be easily produced by a method known in the art with reference to the nucleotide sequence of the PDK2 gene.

Said siRNA means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see International Publication Nos. 00/44895, 01/36646, 99/32619, 01/29058, 99/07409 and 00/44914). siRNA is provided as an efficient gene knock-down method or gene therapy method since it can inhibit the expression of a target gene. Such siRNAs for PDK2 can be easily prepared from known gene sequences of known databases by methods known to those skilled in the art.

The present invention also relates to a method for the treatment and prophylaxis of osteoporosis, osteoporosis, hyperhidrosis, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, memory loss, headache, neuropsychiatric disorder, palpitations, tachycardia, nausea, arthralgia, Or a pharmaceutically acceptable salt thereof. The present invention also provides a composition for the prevention or treatment of female menopausal symptoms.

The present invention also provides a composition characterized in that the osteoporosis is osteoporosis triggered by ovariectomy.

INDUSTRIAL APPLICABILITY As described above, the present invention provides a method for screening for a prophylactic or therapeutic agent for female menopausal disease, a composition for promoting osteoblast differentiation promotion comprising a PDK2 inhibitor as an active ingredient, a screening method for promoting osteoblast differentiation, and a PDK2 inhibitor as an active ingredient Or a pharmaceutically acceptable salt thereof. The prophylactic or therapeutic agent for female menopausal disease according to the present invention may be effective for preventing or treating female menopausal diseases such as osteoporosis by decreasing the expression or activity of PDK2.

FIG. 1 shows the results of experiments in which PDK2 overexpression was inhibited in osteoblast differentiation (A: PDK2 expression was confirmed by western blotting, B: ALP staining, C: vonKossa staining, D: Gene expression).
FIG. 2 shows the results of experiments in which primary bone marrow stem cells were cultured to determine the degree of cell differentiation according to expression of PDK2 (WT: bone marrow stem cells of wild-type mouse in which PDK2 is normally expressed, PDK2 KO: PDK2 deficient A: ALP staining, B: vonKossa staining, C: osteogenic gene expression).
FIG. 3 shows the results of ALP staining of cells expressing PDK2 in mouse bone marrow stem cells and its precursor fibroblast (WT: cells of wild-type mouse in which PDK2 is expressed normally, PDK2 KO: PDK2 deficient CFU-F: colony forming unit fibroblast, CFU-O: colony forming unit osteoblast, CFU-F: colony forming unit fibroblast.
FIG. 4 shows the results of a comparison of body weight change and bone loss according to the expression of PDK2 in ovariectomized mice (WT: wild-type mice in which PDK2 is expressed normally, 2KO: mice lacking PDK2, A: Weight change, B: Comparison of bone loss in the lumbar spine, C: Comparison of bone loss in the femur): OVX: Ovariectomized mouse.
FIG. 5 shows the results of a comparison of loss of trabecular according to the expression of PDK2 in ovariectomized mice using micro CT (WT: wild-type mouse in which PDK2 is expressed normally, 2KO: PDK2 deficient Mouse, sham: ovariectomized mouse, OVX: ovariectomized mouse, BMD: bone mineral density).
FIG. 6 shows the results of osteogenesis and bone loss according to the expression of PDK2 in ovariectomized mice (OC assay: osteocalcin assay, CTX assay: c-terminal telopeptide assay).
FIG. 7 shows the results of an experiment in which the degree of osteoclast activity induced by ovariectomy was compared according to the expression of PDK2 (WT: wild-type mouse with normal expression of PDK2, mouse with 2KO: PDK2 deficient, sham: ovary Mice not undergoing resection, OVX: mice undergoing ovariectomy).

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Assessment of osteoblast differentiation according to PDK2 expression

<1-1> Culture and differentiation of MC3T3E1 cells

MC3T3E1 cells (MC3T3E1 Subclone 4, ATCC, CRL-2593TM) were cultured in α-MEM supplemented with 10% FBS and antibiotics (Gibco, A1049001). For the osteoblast differentiation, 3 × 10 ⁵ cells were placed in a 35 mm cell culture multiplate and cultured in α-MEM medium supplemented with 10 mM b-glycerol phosphate and 50 μg / ml ascorbic acid for 7 to 35 days Respectively. The bone morphogenetic genes were measured on days 7, 14, 21, and 28, ALP stainin between 7 and 10 days, and von Kossa staining between 30 and 35 days. The medium was changed every 3 days. Retrovirus was used to infect MC3T3E1 cells with PDK2. ALP staining and von Kossa staining were performed according to methods known in the art.

<1-2> RNA extraction and RT-PCR (Real-time PCR)

Total RNA was extracted from the cells using the RNeasy® Lipid Tissue Mini Kit (QIAGEN, 74804) according to the manufacturer's instructions. CDNA was synthesized using 5 ug of total RNA using QuantiTect Reverse Transcription Kits (QIAGEN, Cat.205310) according to the manufacturer's instructions, and 25 to 50 ng of cDNA was synthesized using SYBR® Green (Applied Biosystem, Rf.4367659) Real-time-PCR was performed with cDNA. More specifically, 25-50 ng of cDNA and 2 pmol of forward and reverse primers were mixed with 5 μl SYBR® Green Master mix to give a total of 10 μl of Rx. vol. In the ViiA ™ 7 Real-Time PCR System (Life Technologies) system, initial denaturation at 95 ° C for 2 minutes, denaturation at 95 ° C for 15 seconds, and anealing at 60 ° C for 1 minute were repeated 40 cycles. The relative amounts of mRAN were compared using the DNA amplification curves plotted in the system during the cycling cycle. The primers used in the experiments are shown in Table 1 below.

Table 1. RT-PCR, primers used for real time PCR

After the PDK2 was infected with MC3T3E1 cells using retrovirus, expression of PDK2 was confirmed by Western blotting (Fig. 1, A). In order to compare the degree of osteoblast differentiation according to the expression of PDK2, ALP staining was performed on the 8th day after induction of osteoblast differentiation, and von Kossa staining was performed on the 35th day. As a result, PDK2- (Fig. 1, B, C). As shown in Fig. 1, the osteoblast differentiation was significantly suppressed in cells overexpressing PDK2. RT-PCR was performed on days 7, 14, 21 and 28 after induction of osteoblast differentiation to confirm the association of PDK2 with genes involved in bone formation. As a result, BMP2 (bone morphogenetic protein 2 ) And OC (osteocalcin) expression was inhibited in cells overexpressing PDK2 (Fig. 1, D). Based on the above results, it can be confirmed that PDK2 has a function of inhibiting osteoblast differentiation.

&Lt; Example 2 &gt;

Evaluation of differentiation of primary bone marrow stem cells according to PDK2 expression

After removing red blood cells from wild-type C57BL6 / J female mice in which PDK2 was normally expressed and C57BL6 / J female mice deficient in PDK2, the cells were filtered with a 70 μm nylon mash (BD Falcon, 352350) -MEM &lt; / RTI &gt; medium for 3 days. Half of the medium was changed 3 times for 7 days. Bone marrow stem cells were cultured in α-MEM medium containing 10% FBS, 8 mM b-gp and 50 μg / ml ascorbic acid, and the medium was changed every 3 days. On day 3, the cells were fixed with 4% paraformaldehyde and ALP staining was performed using ALP solution (SIGMA, No. 86). On day 14, cells were fixed with 4% paraformaldehyde and von Kossa staining was performed with 5% silver nitrate solution. The degree of osteogenesis-related gene expression was confirmed by RT-PCR.

ALP staining and von Kossa staining for confirming the differentiation of bone marrow stem cells revealed that the cell differentiation was significantly increased in bone marrow stem cells of PDK2-deficient mice compared with bone marrow stromal cells of mice in which PDK2 was normally expressed (Figs. 2A and 2B). RT-PCR revealed the expression level of the gene related to osteogenesis. As a result, it was found that the expression of osteogenic genes in the PDK2-deficient mice was significantly increased as the culture time of the bone marrow stem cells was longer than that of the wild type 2, C). Based on the above results, it can be seen that when PDK2 is deficient, the differentiation of bone marrow stem cells of an animal is increased, and symptoms of osteoporosis, which is one of women's menopausal diseases, can be alleviated.

&Lt; Example 3 &gt;

Comparison of the degree of differentiation of osteoblast precursors according to the expression of PDK2

After removing red blood cells from wild-type C57BL6 / J female mice in which PDK2 was normally expressed and C57BL6 / J female mice deficient in PDK2, the cells were filtered with a 70 μm nylon mash (BD Falcon, 352350) -MEM &lt; / RTI &gt; medium for 3 days. Half of the medium was changed 3 times for 7 days. Bone marrow stem cells were cultured in α-MEM medium containing 10% FBS, 8 mM b-gp and 50 μg / ml ascorbic acid, and the medium was changed every 3 days. To observe the formation of colony formation units (CFUs), cells were fixed and ALP staining was performed on the 3rd and 7th day of cell culture to detect CFU-osteoblast (CFU-OB) cells. Only colony size greater than 1mm was counted. The same plate was stained with a 0.2% crystal violet solution in 2% ethanol for 1 hour to detect CFU-fibroblast (CFU-F). The plate was washed and then dried naturally. Only the colonies having a diameter of 1 mm or more were counted.

As a result of evaluating the degree of differentiation of fibroblasts, which are precursors of osteoblasts, it was found that the differentiation of fibroblasts primary cultured in PDK2-deficient mice was significantly increased compared with that of wild-type mice in which PDK2 was normally expressed (Fig. 3).

<Example 4>

Changes in body weight and bone resorption according to PDK2 expression in ovariectomized mice

Wild type C57BL6 / J female mice in which PDK2 was normally expressed and C57BL6 / J female mice in which PDK2 was deficient were cultured and ovariectomized in a conventional manner in the art to induce a female climacteric animal model. The animals were anesthetized with avertin once a week, and the animals were anesthetized once weekly with a Piximus instrument and software version 1.4 (GE Lunar, Madison, WI) bone mineral density, BMD) were measured.

(i) In wild-type mice in which PDK2 was normally expressed, the body weight was increased in the ovariectomized group compared to the normal group, and the weight increase phenomenon, which is one of female ovariectomized female ovarian disease, was observed. On the other hand, in the case of mice lacking PDK2, it was confirmed that the body weight of the mice deficient in PDK2 remained the same even after ovariectomy (FIG. 4, A). (ii) In wild-type mice in which PDK2 is normally expressed, the BMD of the lumbar and femur tend to decrease in the ovariectomized group compared to the normal group, and osteoporosis, which is one of the female ovariectomized menopausal diseases, Respectively. On the other hand, in the case of mice lacking PDK2, BMD of the lumbar spine and femur were maintained at the same level compared to the normal group after ovariectomy (Fig. 4, B, C). Based on the above results, it can be seen that the expression of PDK2 is inhibited, and it is possible to prevent or treat women's menopausal diseases such as weight gain and osteoporosis.

&Lt; Example 5 &gt;

Evaluation of trabecular loss according to PDK2 expression in ovariectomized mice

The left femur was dissected and fixed with 4% formalin for 24 hours. MicroCT analysis was performed by exchanging the fixative with 80% ethanol. Histomorphometric analysis was performed using a Micro-CT system (eXplore Locus SP scanner, GE Healthcare) to analyze the three-dimensional structure of animal bone. All morphometric parameters were measured using eXplore MicroView, version 2.2 (GE Healthcare).

In the case of wild-type mice in which PDK2 was normally expressed, trabeculae were significantly reduced in the ovariectomized mouse group compared to the normal group, whereas ovariectomy was performed in the mice deficient in PDK2 It was confirmed that a mouse group and a normal group maintained the same trabecular level (Fig. 5). Based on these results, it was found that PDK2 inhibition can prevent or treat osteoporosis, which is one of the female menopausal diseases.

&Lt; Example 6 &gt;

Bone formation and bone resorption according to PDK2 expression in ovariectomized mice Evaluation of Indicators

The osteocalcin (OC), an index of bone formation, and the C-terminal telopeptide (CTX), an index of bone resorption, were measured to evaluate bone formation and bone resorption according to PDK2 expression.

As a result of measuring the blood level of OC, which is an index of osteogenesis in ovariectomized mice, the blood level of OC was increased more in mouse group in which PDK2 was deficient than in wild-type mouse group in which PDK2 was normally expressed (FIG. 6, A). Further, the blood content of CTX, which is an index of bone resorption, was measured. As a result, the blood content of CTX in the mouse group in which PDK2 was deficient compared to the wild type mouse group in which PDK2 was normally expressed (Fig. 6, B). The results show that inhibition of PDK2 expression increases osteogenesis and decreases bone resorption. Inhibiting PDK2 inhibits osteoporosis, one of female osteoporosis diseases Or treatment may be possible.

&Lt; Example 7 &gt;

Activity of osteoclasts according to PDK2 expression in ovariectomized mice evaluation

The degree of osteoclast activity was evaluated by TUNEL staining. TUNEL staining was performed according to the manufacturer's instructions with a commercially available kit (Roche 11 684 795 910). Fluorescence microscopy was used to determine the presence of a green sphere as a positive reaction.

In wild-type mice in which PDK2 was normally expressed, the osteoclast-treated mouse group showed an increase in osteoclast activity as compared with the normal group. In the case of mice lacking PDK2, ovariectomized mice The osteoclast activity was maintained at an equivalent low level compared to the normal group (Fig. 7).

Comprehensive evaluation of the above results indicates that inhibiting the expression or activity of PDK2 alleviates the female menopausal disease.

As described above, PDK2 is closely related to women's menopausal disease, and when the expression or activity of PDK2 is inhibited, women's menopausal disease can be alleviated, thereby screening substances capable of inhibiting the expression or activity of PDK2, The present invention is highly likely to be industrially applicable in that a composition containing PDK2 inhibitor as an active ingredient can be used as a preventive or therapeutic agent for female menopausal diseases.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Screening method for pharmaceutical compound for preventing or          treating menopause using PDK2 and pharmaceutical composition for          preventing or treating menopause comprising pharmaceutical          compound using the same. <130> OP14-0113PCT <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 407 <212> PRT Pyruvate dehydrogenase kinase, isozyme 2 isoform 1 precursor [Homo sapiens] <400> 1 Met Arg Trp Val Trp Ala Leu Leu Lys Asn Ala Ser Leu Ala Gly Ala   1 5 10 15 Pro Lys Tyr Ile Glu His Phe Ser Lys Phe Ser Pro Ser Pro Leu Ser              20 25 30 Met Lys Gln Phe Leu Asp Phe Gly Ser Ser Asn Ala Cys Glu Lys Thr          35 40 45 Ser Phe Thr Phe Leu Arg Gln Glu Leu Pro Val Arg Leu Ala Asn Ile      50 55 60 Met Lys Glu Ile Asn Leu Leu Pro Asp Arg Val Leu Ser Thr Pro Ser  65 70 75 80 Val Gln Leu Val Gln Ser Trp Tyr Val Gln Ser Leu Leu Asp Ile Met                  85 90 95 Glu Phe Leu Asp Lys Asp Pro Glu Asp His Arg Thr Leu Ser Gln Phe             100 105 110 Thr Asp Ala Leu Val Thr Ile Arg Asn Arg His Asn Asp Val Val Pro         115 120 125 Thr Met Ala Gln Gly Val Leu Glu Tyr Lys Asp Thr Tyr Gly Asp Asp     130 135 140 Pro Val Ser Asn Gln Asn Ile Gln Tyr Phe Leu Asp Arg Phe Tyr Leu 145 150 155 160 Ser Arg Ile Ser Ile Arg Met Leu Ile Asn Gln His Thr Leu Ile Phe                 165 170 175 Asp Gly Ser Thr Asn Pro Ala His Pro Lys His Ile Gly Ser Ile Asp             180 185 190 Pro Asn Cys Asn Val Ser Glu Val Val Lys Asp Ala Tyr Asp Met Ala         195 200 205 Lys Leu Leu Cys Asp Lys Tyr Tyr Met Ala Ser Pro Asp Leu Glu Ile     210 215 220 Gln Glu Ile Asn Ala Ala Asn Ser Lys Gln Pro Ile His Met Val Tyr 225 230 235 240 Val Pro Ser His Leu Tyr His Met Leu Phe Glu Leu Phe Lys Asn Ala                 245 250 255 Met Arg Ala Thr Val Glu Ser His Glu Ser Ser Leu Ile Leu Pro Pro             260 265 270 Ile Lys Val Met Val Ala Leu Gly Glu Glu Asp Leu Ser Ile Lys Met         275 280 285 Ser Asp Arg Gly Gly Gly Val Pro Leu Arg Lys Ile Glu Arg Leu Phe     290 295 300 Ser Tyr Met Tyr Ser Thr Ala Pro Thr Pro Gln Pro Gly Thr Gly Gly 305 310 315 320 Thr Pro Leu Ala Gly Phe Gly Tyr Gly Leu Pro Ile Ser Arg Leu Tyr                 325 330 335 Ala Lys Tyr Phe Gln Gly Asp Leu Gln Leu Phe Ser Met Glu Gly Phe             340 345 350 Gly Thr Asp Ala Val Ile Tyr Leu Lys Ala Leu Ser Thr Asp Ser Val         355 360 365 Glu Arg Leu Pro Val Tyr Asn Lys Ser Ala Trp Arg His Tyr Gln Thr     370 375 380 Ile Gln Glu Ala Gly Asp Trp Cys Val Pro Ser Thr Glu Pro Lys Asn 385 390 395 400 Thr Ser Thr Tyr Arg Val Ser                 405

Claims (10)

A method for screening for a preventive or therapeutic agent for female menopausal disease comprising the steps of:
(a) contacting a test substance with a cell;
(b) selecting a test substance that reduces the expression or activity of the PDK2 protein as compared to a control substance not contacted with the test substance The method according to claim 1, wherein the cell of step (a) is a PDK2-expressing cell or an PDK2-expressing cell. The method of claim 1 or 2, wherein the female menopausal disease is selected from the group consisting of osteoporosis, hyperthermia, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, memory loss, headache, neuropathy, dizziness, palpitations, tachycardia, Muscular pain, poor circulation, and weight gain. A composition for promoting the differentiation of osteoblasts comprising PDK2 inhibitor as an active ingredient. A method for screening a substance for promoting osteoblast differentiation comprising the steps of:
(a) contacting a test substance with a cell;
(b) selecting a test substance that reduces the expression or activity of PDK2 protein as compared to a control substance not contacted with the test substance; And
(c) contacting the test substance selected in the step (b) with osteoblasts to select a test substance which is promoted to differentiate. A composition for preventing or treating female menopausal symptoms comprising PDK2 inhibitor as an active ingredient. 7. The composition of claim 6, wherein said PDK2 inhibitor is siRNA or antisense RNA for PDK2. 7. The composition of claim 6, wherein the PDK2 has the amino acid sequence of SEQ ID NO: 1. 9. The method according to any one of claims 6 to 8, wherein the female menopausal disease is selected from the group consisting of osteoporosis, hyperthermia, hyperhidrosis, depression, insomnia, urinary incontinence, edema, facial flushing, memory loss, headache, Nausea, arthralgia, muscular pain, poor circulation and weight gain. 10. The composition of claim 9, wherein the osteoporosis is osteoporosis induced by ovariectomy.

Images