KR100758218B1 - METHOD FOR SCREENING PHARMACEUTIC AGENTS AGAINST METABOLIC DISORDER AND SKIN DISORDER BY USING PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR (PPAR delta;/beta;) AND PPAR CO-ACTIVATORS - Google Patents

Abstract

A method for screening pharmaceutical agents for treating metabolic disorder and skin disorder by using a peroxisome proliferator activated receptor(PPAR delta/beta) and PPAR co-activators is provided to screen a large quantity of candidate compounds simultaneously, and reduce the screening costs by detecting binding between PPAR delta/beta to PPAR co-activators. A method for screening pharmaceutical agents for treating metabolic disorder and skin disorder by using the peroxisome proliferator activated receptor (PPAR delta/beta) and PPAR co-activators comprises the steps of: (1) coating the PPAR co-activators on a plate; (2) adding a candidate compound together with PPAR delta/beta protein into the plate; (3) binding an antibody against PPAR delta/beta; (4) reacting a secondary antibody labeled with an enzyme capable of binding to the antibody against PPAR delta/beta; and (5) detecting the labeled secondary antibody, wherein the PPAR co-activator is selected from steroid receptor co-activator SRC-1, transcription mediator TIF-2 or p300 protein and the metabolic disorder is obesity, diabetes, hypertension, cardiovascular disease or hyperlipidemia.

Description

Method for screening pharmaceutic agents against metabolic disorder and skin disorder by using peroxisome proliferator using peroxysome proliferation factor activator receptor (PCR δ / β) and PAP's active cofactors activated receptor (PPAR δ / β) and PPAR co-activators

Figure 1 schematically shows the gene of the active cofactor of PPAR δ / β of the present invention.

Figure 2 shows the process of cloning the PPAR δ / β gene and its activity cofactor genes of the present invention and construct recombinant expression vectors.

Figure 3 shows the results confirmed by electrophoresis of the recombinant expression vectors containing the PPAR δ / β and its activity cofactor genes of the present invention, respectively.

Figure 4 shows the results confirmed by electrophoresis of the recombinant PPAR δ / β protein and its recombinant activator protein, respectively, produced using the recombinant expression vectors of the present invention.

Figure 5 schematically shows the principle of the search method using the PPAR δ / β, its active cofactor proteins and specific antibodies against PPAR δ / β of the present invention.

Figure 6 shows the result of measuring the degree of mutual binding and the addition of recombinant PPARδ / β protein diluted on a plate coated with the PPAR activator protein of the present invention.

Figure 7 shows the results of measuring the binding of the recombinant PPARδ / β protein and the addition of various candidate compounds on a plate coated with the PPARδ / β activity cofactor protein of the present invention.

The present invention provides a method for screening a drug candidate for improving or treating metabolic diseases and skin diseases using a peroxysome growth factor activating receptor (PPAR δ / β), an active cofactor of PPAR and a specific antibody to PPAR δ / β, and Search kits, and their use.

The peroxisom proliferator activated receptor (hereinafter abbreviated as "PPAR") is known as a kind of nuclear receptor protein. First, peroxisome is an intracellular microorganism involved in oxidative action, abundant in liver and kidney, oxidizing intracellular fatty acids and producing hydrogen peroxide to neutralize toxic substances. It also has the function of decomposing excessively generated hydrogen peroxide into water and oxygen using catalase, an oxidase. Second, peroxisome proliferator refers to compounds that can increase the number of peroxysomes numerically. These include fats, fatty acids, and hyperlipidemia, fibrate, and prostaglandin. Nuclear transcription factor receptors having such peroxysome growth factor as ligands are collectively called peroxysome growth factor activating receptors (PPARs).

Peroxysome growth factor activating receptors are largely divided into PPARα, PPARδ / β, and PPARγ. PPARα is mainly expressed in the liver and is involved in fatty acid oxidation or neutralization of toxic substances and is associated with an inflammatory response. PPARγ is known to be involved in the differentiation and fat accumulation of several cells, including adipocytes. PPARδ / β is not yet well known in its physiological function, but has been reported to be evenly distributed in vivo and involved in embryo implantation. The unique roles of each of these PPARs appear to be represented by binding to different ligands.

First, PPARα is mainly induced by eicosanoids, carbaprostacyclin, fibrates and nonsteroidal anti-inflammatory drugs (NSAIDs). PPARγ also binds specifically to thiazolidinediones (TZDs), an antidiabetic agent, and is a 15-deoxy-Δ ^12.14 -prostaglandin J ₂ (PGJ ₂ ), unsaturated fatty acid that is a metabolite of prostaglandin. The activity is induced by (polyunsaturated fatty acid) and the NSAID. PPARδ / β is also induced by several unsaturated fatty acids and eicosanoids.

This ligand-activated PPARα increased the expression of several catabolic enzymes involved in β-oxidation in mitochondria and peroxysomes and ω-oxidation in mitochondria. And thus promote the oxidation of fatty acids in the liver or muscles. Recent studies have reported that PPARα regulates lipid metabolism, inflammation and atherosclerosis. In addition, PPARγ increased the expression of genes important in lipogenesis and increased lipid droplets in adipocytes while also increasing the expression of genes involved in adipocyte differentiation. In addition, PPARγ is known to increase glucose uptake in adipocytes and muscle cells, inhibit the production and inflammatory action of inflammatory cytokines, and inhibit arteriosclerosis. In addition, PPARδ / β is reported to increase the expression of genes associated with lipid uptake and genes associated with lipid metabolism, and is responsible for intracellular lipid regulation. In particular, many studies on PPARδ / β have recently been conducted as a kind of target protein for improving and treating inflammation, arteriosclerosis, obesity and cancer.

Metabolic disease (metabolic disease or metabolic syndrome) is rapidly spreading, characterized by obesity (type 2 diabetes), hypertension (hypertension), cardiovascular disease and hyperlipidemia. These metabolic diseases are deeply linked to abnormalities in lipid constants due to excessive lipid intake and lack of exercise. Recent studies have reported that the activation of PPARδ / β activity in adipocytes and muscle cells increases the expression of genes involved in fatty acid oxidation and energy consumption. In particular, it has been reported that treatment of ligands or agonists against PPARδ / β in experimental obese mice reduced lipid accumulation (Wang YX et al., Cell , 113: 159, 2003). In addition, GW501516, the best known ligand of PPARδ / β, decreases triglyceride (TG) and insulin levels in obese monkeys, while increasing levels of high density lipoprotein (HDL). Known (Oliver WR et al., Proc. Natl. Acad. Sci. USA, 98: 5306, 2001). In addition, the ligand of PPARδ / β has been reported to stimulate fatty acid degradation in muscle cells of rats fed high fat to improve insulin resistance (Tanaka T. et al., Proc. Natl. Acad. Sci. USA, 100 : 15924, 2003). In addition, GW501516, a specific ligand for PPARδ / β, transports reverse cholesterol to move intracellular cholesterol out of cells when treated with human macrophage THP-1 cell line, skin fibroblasts 1BR3N cell line, and enterocyte FHS74 cell line. increase reverse cholesterol transport In addition, the ligand has an effect of normalizing blood HDL when treated in rhesus monkeys with symptoms of obesity and diabetes (Lee CH et al., Science , 302: 453, 2003). These studies report that PPARδ / β is widely involved in lipolysis and is an important target protein for metabolic diseases caused by obesity. Furthermore, ligands of PPARδ / β may have a high potential as a therapeutic agent for the metabolic disease.

PPARδ / β also has anti-inflammatory properties. Recent studies report that endothelial cells treated with PPARδ / β ligands decreased expression of VCAM-1 and MCP-1 genes, and macrophages treated with PPARδ / β ligands affected LPS. Expression of the COX-2 and iNOS genes induced was inhibited (Lival Y. et al., Eur. J. Pharmacol. , 435: 143, 2002; Welch JS et al., Proc. Natl. Acad. Sci. USA , 100: 6712, 2003).

In addition, there was an abnormality in the regulation of proliferation of keratinocytes in experimental rats in which PPARδ / β was lost (Peters JM et al., Mol. Cell. Biol ., 20: 5119, 2000). Interestingly, the expression of PPARδ / β is increased at the wound site of the damaged skin, and TNF-α, a primary inflammatory cytokine, regulates the expression level of PPARδ / β via the stress kinase signaling pathway and regulates PPARδ / β expression. Induces ligand production of / β (Tan NS et al., Genes Dev. 153: 263, 2001). These studies focus attention on the mechanism of PPARδ / β in the process of improving and treating skin diseases such as psoriasis and on the ligand of PPARδ / β as a new target for therapeutic purposes.

In general, immunological methods using the reaction of an antigen-antibody include various markers and devices that can recognize and measure the conjugate by making a specific antibody against the substance to be measured and binding it to the antigen. The bioactive substance is qualitatively or quantitatively analyzed by measuring the binding of the antigen-antibody and the degree of binding using the above.

The immunological measurement method of a bioactive substance which has been used so far can be classified according to the type of label used therein. Specifically, radioimmunoassay (RIA) using a radioisotope as a label, enzyme immunoassay (ELISA) and fluorescent immunoassay (FEIA) using a non-radioactive isotopic enzyme or fluorescent substance as a label, and the like are included. It is largely divided into non-radioactive immunoassays. The most widely used immunological method is radioimmunoassay, which is accurate, sensitive, easy to measure, easy to perform and fast. In addition, the devices used in this measurement process, such as gamma and beta meter has the advantage that the price is not very expensive. However, the immunoassay using radioisotopes has the biggest disadvantage of generating a lot of radioactive wastes that are harmful to the human body, and suffers from the inconvenience that the amount and type of radioisotopes are limited by regulations.

In order to overcome this problem, enzyme immunoassay (ELISA) is used as an alternative. The enzyme immunoassay is largely divided into direct label and indirect label according to the label format. Direct labeling is a method of directly labeling an antibody or a fragment of an antibody by a crosslinking agent, and indirect labeling is a method of binding a hapten to an antibody and then labeling a label component that recognizes the antibody. At this time, as a crosslinking agent used for direct labeling, N, N'- orthophenylenedimaleimide, 4- (N-maleimidomethyl) cyclohexane, N-succinimide ester, 6-maleimidohexane, N-succinate Imide esters, 4,4'-dithiopyridine and the like are known. In addition, heptins used for indirect labeling are known as biotin, dinitrophenyl pyridoxal or fluoresamine, and especially biotin is known to use avidin or streptavidin as a recognition ligand. In addition, horseradish peroxidase is mainly used as the enzyme used for the labeling because it can react with a large amount of substrate and can be easily bound to the antibody by the peroxanoic acid method.

In the process of identifying the labeled enzyme, horseradish peroxidase uses hydrogen peroxide (H ₂ O ₂ ) as the substrate solution, and 2,2'-azino-di- [3-ethylbenzothiane as a colorant. Sleepysulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, orthophenylenediamine, 4-aminoantipyrine, 3,3 ', 5,5'-tetramethylbenzidine and the like. In the case of using alkaline phosphatase, orthonitrophenyl phosphate, paranitrophenyl phosphate, etc. are used as a substrate, and when β-D-galactosidase is used, fluorescein-di- ( -D-galactopyranoside), 4-methyl unberphenylphenyl-β-D-galactopyranoside, and the like. In particular, enzyme immunoassay may be classified as sandwich enzyme immunoassay when the substance to be immobilized on the plate is an antibody.

Peroxysome growth factor activating receptor δ / β (PPAR δ / β) binds to partner RXRα in a cell to form a heterodimer and attaches to a specific gene binding site, PPRE (PPAR response elements). The PPAR δ / β complex acts as a transcriptional regulator that complexes with transcriptional regulators or co-activators to promote transcription of the gene of interest. The mechanism is that their transcriptional activity is regulated depending on the presence or absence of hydrophobic low molecules or angonists that specifically bind to PPARδ / β. Indeed, when the PPARδ / β protein binds to known ligands such as unsaturated fatty acids such as linoleic acid or GW501516, the steroid receptor SRC-, its active cofactor, is due to the morphological transformation of the PPARδ / β protein itself. 1 (steroid receptor co-activator-1), transcriptional mediator TIF-2 (transcriptional intermediary factor-2), and p300 proteins become stronger. These ligands not only increase the intracellular half life of the PPARδ / β protein, but also promote the binding of these cofactors to further raise the transcription of the gene of interest. As such, substances capable of improving the binding force between PPARδ / β protein and auxiliary proteins of transcriptional activity are referred to as agonists or ligands of PPARδ / β. It is attracting attention because it can be used as an agent for improving metabolic diseases, anti-inflammatory agents, anticancer agents and skin diseases.

Currently, the most widely used methods of screening modulators or ligands of PPARs include the yeast two hybrid system and luciferase assay, but these screening methods are specific cell lines. Another disadvantage is that yeast must be used and it is impossible to search for many candidates at the same time.

Also, by not using a specific cell line in vitro binding assay (in vitro binding assay) for may of the application of, in the conventional method is to attach a radioactive isotope, ligand measure the degree of competitive binding of the candidate substance Or a radioisotope attached to a PPARs protein or a radioisotope bound to an activity regulator to measure the binding force between the two proteins to the candidate substance. However, these methods also have the problem of using radioisotopes, and it is impossible to search many candidates simultaneously.

In addition, although there are methods for measuring the binding force between ligand-activated PPARs and activity regulators using precise instruments, these methods also have the disadvantage of being complex, expensive and incapable of rapidly searching many candidates at the same time. .

Accordingly, the present inventors have continued to develop a search system for new drug candidates for improving or treating metabolic diseases and skin diseases, and thus, (1) coating an active cofactor of PPAR δ / β; (2) adding candidate compounds with PPAR δ / β protein; (3) binding specific antibodies to the PPAR δ / β protein; As a process, it is possible to easily find candidate compounds for improving or treating various metabolic diseases, inflammation, cancer and skin diseases, etc., and (1) activity cofactors for PPARδ / β; (2) PPARδ / β protein; (3) specific antibodies against PPARδ / β; And (4) secondary antibodies that bind to specific antibodies to the PPARδ / β; The present invention has been successfully completed by developing a new drug candidate search kit for improving or treating metabolic diseases and skin diseases.

The present invention provides a drug candidate that can improve or treat metabolic diseases and skin diseases using peroxysome growth factor activating receptor δ / β (PPAR δ / β), PPAR activator, and specific antibodies against PPARδ / β. It is an object of the present invention to provide a method for searching for a substance, a kit for searching and a use thereof.

In order to achieve the above object, the present invention provides a new drug candidate search method that can improve or treat metabolic diseases and skin diseases using the peroxysome growth factor activator receptor δ / β (PPAR δ / β).

Specifically, the present invention

(1) coating an active cofactor for PPARδ / β on the plate;

(2) adding a drug candidate compound with PPARδ / β protein; And

(3) binding specific antibodies against PPARδ / β; The present invention provides a method for screening a new drug candidate for improving or treating a metabolic disease.

In addition, the present invention, in addition to the above process,

(4) reacting a secondary antibody labeled with an enzyme that binds to the specific antibody against PPARδ / β; And

(5) detecting the labeled secondary antibody; The present invention provides a method for screening a new drug candidate for improving or treating metabolic diseases in which the degree of binding between the PPARδ / β protein and active cofactors for the PPARδ / β is measured.

In addition to the metabolic disease, the present invention provides a method for searching for drug candidates that can improve or treat inflammation, cancer and skin diseases using the peroxysome growth factor activating receptor δ / β (PPAR δ / β). .

In addition, the present invention (1) activity cofactor for PPARδ / β; (2) PPARδ / β protein; (3) specific antibodies against PPARδ / β; And (4) secondary antibodies that bind to specific antibodies to the PPARδ / β; It provides a new drug candidate search kit configured to include.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for searching for a drug candidate using a peroxysome growth factor activating receptor δ / β (PPAR δ / β).

Specifically, the present invention

(1) coating an active cofactor for PPARδ / β on the plate;

(2) adding a drug candidate compound with PPARδ / β protein; And

(3) binding specific antibodies against PPARδ / β; The present invention provides a method for screening a new drug candidate for improving or treating a metabolic disease.

In addition, the present invention, in addition to the above process,

(4) reacting a secondary antibody labeled with an enzyme that binds to the specific antibody against PPARδ / β; And

(5) detecting the labeled secondary antibody; The process provides a search method such as candidate compounds for measuring the degree of binding between the PPARδ / β protein and the active cofactors to the PPARδ / β.

The PPARδ / β protein used in the present invention is strongly bound to cofactor proteins that assist in transcriptional activity when specific ligands bind. In particular, the PPARδ / β protein directly binds to the LXXLL site, which is a specific active site present in the activity cofactor. Accordingly, the present invention provides a method for determining the degree of binding between the peroxysome growth factor activator receptor δ / β (PPAR δ / β) and the transcriptional activator of PPAR δ / β to determine the candidate compounds of the drug. To explore the efficacy.

In the search method of the present invention, the activity cofactor for the PPARδ / β in step (1) is preferably at least one selected from steroid receptor activity cofactor SRC-1, transcription mediator TIF-2 or p300 protein, human More preferably it is a recombinant protein which is isolated from and comprises a specific active site of an activity cofactor for said PPARδ / β. In addition, in the above (2) process, the PPARδ / β protein is preferably a recombinant protein containing a histidine residue isolated from human. In addition, the pharmaceutical candidate compound in the process (3) includes plant extracts, functional foods and pharmaceutical compositions containing them as an active ingredient.

The search method of the present invention is preferably used to develop new drug candidates and the like that can improve or treat metabolic diseases related to glucose metabolism and lipid metabolism, including obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia.

In addition, the present invention provides a method for searching for a drug candidate compound that can improve or treat various inflammation, cancer and skin diseases by using the peroxysome growth factor activating receptor δ / β (PPAR δ / β) in addition to the metabolic disease. to provide.

In addition, the present invention (1) activity cofactor for PPARδ / β; (2) PPARδ / β protein; (3) specific antibodies against PPARδ / β; And (4) secondary antibodies that bind to specific antibodies to the PPARδ / β; It provides a new drug candidate search kit configured to include.

At this time, the (1) the active cofactor for PPARδ / β is preferably at least one selected from the steroid receptor activity cofactor SRC-1, transcription mediator TIF-2 or p300 protein, and is isolated from human and the PPARδ / β More preferred is a recombinant protein comprising a specific active site for PPARδ / β in an activity cofactor for the same. In addition, the (2) PPARδ / β protein is preferably a recombinant protein containing a histidine residue isolated from human.

The exploration kit of the present invention is used to develop a medicine for improving or treating metabolic diseases, inflammation, cancer, and skin diseases, including metabolic and lipid metabolism, including obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia, etc. It is desirable to.

Unlike the conventional method, the search method according to the present invention is a search system that can search for more candidate compound samples at the same time without using a radioisotope and determine a result more quickly. In other words, the search system of the present invention uses a recombinant antibody instead of a radioisotope, adopts a 96-well plate to simultaneously process a large number of candidate compound samples, and applies an enzyme coloration method. You can quantify it conveniently. Therefore, it is possible to determine excellent and quick results compared to the conventional method.

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Recombinant PPARδ / β Protein

The present invention isolated PPARδ / β genes derived from humans to prepare recombinant proteins to be used as antigens for PPARδ / β. First, a polymerase chain reaction (PCR) was performed using the spleen cDNA library, amplified the PPARδ / β gene, and then digested with restriction enzymes Bam HI and Xho I. This cleaved gene was mixed with E. coli expression vector pET28a (manufactured by Novagen) previously cleaved with the same restriction enzyme, and ligated at 16 ° C. for 3 hours by adding a binding enzyme thereto (see FIG. 2). As a result, a recombinant expression vector pET28a (PPARδ / β) was generated that can express a recombinant PPARδ / β protein to which six histidines residues are attached, including the PPARδ / β gene of the present invention.

Figure 2 shows the process of producing a recombinant expression vector by cloning the PPAR δ / β gene of the present invention.

The recombinant expression vector pET28a (PPARδ / β) of the present invention was transformed into E. coli BL21 (DE3) strain, and transformed E. coli was transformed using LB medium containing kanamycin antibiotic at a concentration of 100 μg / ml. Shake culture was carried out at ℃. When the E. coli culture reached an absorbance at 600 nm in the range of 0.4 to 0.6, 1 mM IPTG (isopropylthio-β-D-galactoside) was added again and incubated overnight at 20 ° C. to induce the expression of the PPARδ / β gene.

Then, the cell culture in which the expression of PPARδ / β protein was induced was centrifuged at 6,000 rpm for 20 minutes at 4 ° C., and the cells were harvested, and then the cell lysis solution (20 mM Tris (pH 7.4), 300 mM NaCl, 10 mM imidazole, 5 μg / ml aprotinin, 100 μM PMSF) was added and cells were completely disrupted using an ultrasonic crusher. The crushed cells were once again centrifuged at 12,000 rpm for 30 minutes at 4 ° C. and cell supernatants were recovered therefrom.

The supernatant recovered above binds to the histidine residue-attached PPARδ / β protein (His-PPARδ / β) using NTA agarose (pepttron, bead) beads having affinity for histidine residues. And then washed three times with washing solution (20 mM Tris pH 7.4, 300 mM NaCl, 20 mM imidazole) to reduce nonspecific binding. The recombinant protein bound to the agarose beads was recovered by separation in an elution solution (20 mM Tris pH 7.4, 300 mM NaCl, 250 mM imidazole). The recovered eluate was then electrophoresed on a 12% SDS-polyacrylamide gel to identify a recombinant His-PPARδ / β protein of the invention having a molecular weight of 60 kDa (see FIGS. 3 and 4).

Figure 3 shows the results confirmed by the electrophoresis of the recombinant expression vector containing PPAR δ / β of the present invention, Figure 4 is electrophoresis of the recombinant PPAR δ / β protein produced using the recombinant expression vector of the present invention It shows the result confirmed.

Example 2 Preparation of Recombinant Activity Cofactor Protein

The present invention provides a recombinant protein comprising an LXXLL site in a transcriptional activator factor to which the PPARδ / β protein directly reacts in order to obtain transcriptional activator proteins in which binding becomes stronger when a specific ligand binds to PPARδ / β. It was prepared as follows. The present invention selects steroid receptor co-activator-1 (SRC-1), transcriptional mediator TIF-2 (transcriptional intermediary factor-2), and p300 proteins, which are transcriptional activation cofactors, and includes a corresponding gene comprising an LXXLL site therefrom. Were isolated and cloned into E. coli expression vectors, respectively, and expressed and purified (see FIGS. 1, 2, 3 and 4).

Specifically, the LXXLL site of the steroid receptor SRC-1 was amplified by performing a polymerase chain reaction using a cDNA library of the spleen, separated and cleaved with restriction enzymes Bam HI and Xho I. This cleaved SRC-1 gene was cloned into E. coli expression vector pGEX4T-1 (manufactured by Amersham Pharmacia Biotech) cut with the same restriction enzyme.

In addition, the LXXLL region of the human transcription mediator TIF-2 was isolated by amplifying the whole mRNA using a human kidney cell line, followed by a polymerase chain reaction, and separated and digested with restriction enzymes Eco RI and Xho I. This cleaved TIF-2 gene was cloned into E. coli expression vector pGEX4T-1 cleaved with the same restriction enzyme.

In addition, the LXXLL region of the human p300 protein was amplified by performing a polymerase chain reaction using a spleen cDNA library and separated and digested with restriction enzymes Eco RI and Xho I. This cleaved p300 gene was cloned into the E. coli expression vector pGEX4T-1 digested with the same restriction enzyme.

Through the above procedure, each of the genes cloned into the E. coli expression vector was transformed into E. coli DH5α strain. E. coli DH5α (pGEX4T-1 / SRC-1), E. coli DH5α (pGEX4T-1 / TIF-2) and E. coli DH5α (pGEX4T-1 / p300) strains that produce these transcriptional activators in the form of GST-fused recombinant proteins Cell cultures were harvested from each of the cells, followed by ultrasonic disruption and centrifugation to separate cell supernatants. The cell supernatant thus obtained was combined with the recombinant GST-SRC-1 protein, GST-TIF-2 protein and GST-p300 proteins using glutathione sepharose 4B (manufactured by Amersham Pharmacia Biotech) beads, respectively. Wash three times with wash buffer (PBS + 0.5% Triton X100) to first reduce nonspecific binding. Recombinant proteins bound to the beads were isolated and purified using elution buffer (50 mM Tris pH 8.0, 20 mM GSH). The purified recombinant proteins were each electrophoresed in 12% SDS polyacrylamide gel to finally confirm that they were recombinant active cofactor proteins as shown in FIG. 4. At this time, the expression conditions and methods of all recombinant proteins were carried out in the same process as described in Example 1.

Example 3. Recombinant PPARδ / β Drug discovery using binding reaction between protein and recombinant activator protein

The present invention is to develop a method of screening a drug using a binding reaction between recombinant PPARδ / β protein and recombinant activator protein, PPARδ / using an ELISA plate that is more convenient and can accommodate a large number of samples We wanted to search for ligands for β.

As shown schematically in FIG. 5, the activity cofactor proteins were coated on an ELISA plate, and the E. coli lysate containing the PPARδ / β protein was treated with a candidate sample, and then the PPARδ / β An antibody was used to confirm the binding force between the transcriptional activator protein and the PPARδ / β protein. When the candidate sample contains a substance capable of becoming a ligand for PPARδ / β, the binding force between the transcriptional activator protein and the PPARδ / β protein is further increased, and the result is an ELISA reader. It was confirmed that the higher value than the control when measured with).

Example 4. Recombinant PPARδ / β Investigation of the Effect of PPARδ / β Concentration on the Binding Response Between Protein and Recombinant Activator Cofactor Proteins

The present invention is to investigate the effect of the PPARδ / β protein concentration on the binding between the cofactor protein and PPARδ / β protein, as shown in Figure 6 SRC-1, TIF-2 and p300 at a concentration of 0.8 ㎍ / ㎖ Proteins were coated in 96-well plates and E. coli lysates containing His-PPARδ / β protein were added in dilutions of 512, 256, 128, 64, 32, 16 and 8 times, respectively. In addition, the same experiment was repeated three times or more. As a result, as shown in Figure 6, when using the ELISA method it was found that the binding between the cofactor protein and PPARδ / β protein depending on the concentration of PPARδ / β. In addition, it was confirmed that the SRC-1 transcriptional activity coenzyme reacts more sensitively to the concentration of PPARδ / β protein among various cofactor proteins.

Example 5 Recombinant PPARδ / β Protein and Activator Cofactor Protein In vitro Establishment of Ligand Search System for PPARδ / β Using Binding Ability

The present invention is to examine the binding ability between the transcriptional activity cofactor protein and PPARδ / β protein present in the soluble fraction by ELISA method, recombinant activity cofactor protein isolated from E. coli, that is, SRC-1, TIF-2 and p300 Proteins were diluted using 0.1 M sodium carbonate buffer (pH 9.6) at approximately 800 ng / well concentrations, respectively. Protein solutions of each concentration were added to the plates for ELISA in 100 μl / well amounts and coated overnight at 4 ° C. The coated plates were washed three times using PBST buffer solution (PBS + 0.05% Tween20) and then treated with PBST buffer solution containing 3% skim milk overnight at 4 ° C. or for 2 hours at room temperature. A solution of 256-fold dilution of the soluble fraction of the recombinant PPARδ / β protein fused with His residue was added thereto at a concentration of 50 μl / well. Next, linoleic acid and GW501516 as an activity enhancing substance for PPARδ / β and ibuprofen as a substance that does not affect the PPARδ / β activity were 0, 4, 8, 16, 32, 64, 128 After addition to μM concentration, the reaction was slowly shaken for 1 hour at room temperature, and washed three times with PBST buffer again.

Then, in order to confirm the binding of the activator protein and PPARδ / β protein, the primary antibody is a PBST buffer containing 5% skim milk at a ratio of 1: 4,000 for a specific antibody that specifically binds to the PPARδ / β protein. Diluted solution was used, and the secondary antibody was used by diluting the anti-mouse IgG-HRP antibody with PBST buffer containing 5% skim milk at 1: 2,000. As a result, as shown in Figure 7, the binding force between the PPARδ / β protein and the activator cofactor protein was found to be dependent on the concentration of GW501516 and linolenic acid ligands for PPARδ / β. In addition, ibuprofen, which did not affect the activity enhancement of PPARδ / β as a control, had no effect on the binding strength between these recombinant proteins even at high concentrations. In particular, among the various transcriptional activity cofactors, SRC-1 reacted more sensitively to ligands for PPARδ / β, and among the ligands, GW501516 had the greatest effect on the binding capacity of the transcriptional activity cofactor and PPARδ / β. These results show that the specific binding of PPARδ / β and the transcriptional activator protein coated on the well plate is enhanced by specific morphological conversion by ligand to PPARδ / β. An ELISA system was established to more easily screen drug candidate compounds that specifically react to PPARδ / β.

As described above, the present invention provides a method for preparing an active cofactor of (1) PPAR δ / β; (2) adding a drug candidate compound with PPAR δ / β protein; And (3) binding specific antibodies to the PPAR δ / β protein; The present invention relates to a method for searching for new drug candidates, a search kit using the same, and a search method and a search kit for measuring the concentration of PPAR δ / β protein.

The methods and kits of the present invention provide a convenient search for a ligand for PPARδ / β in various functional foods and plant extracts, as well as metabolic diseases including obesity and diabetes, inflammation inhibition, cancer suppression and skin disease improvement. It is expected that the exploration of a number of substances related to the environment can be done quickly and environmentally friendly.

In addition, as a method and a search kit for searching for ligands using the only PPARδ / β protein to date, PPARδ / β protein as well as having a high competitiveness in the development and treatment of inflammatory, skin disease and metabolic diseases in the future It is expected to be widely used in related research.

Claims (12)

delete (1) coating an active cofactor for PPARδ / β on the plate; (2) adding a drug candidate compound with PPARδ / β protein; (3) bind specific antibodies against PPARδ / β; (4) reacting a secondary antibody labeled with an enzyme that binds to the specific antibody against PPARδ / β; And (5) detecting the labeled secondary antibody; Process for the detection of new drug candidates to improve or treat metabolic diseases, characterized in that for measuring the degree of binding between the PPARδ / β protein and active cofactors to the PPARδ / β. The method of claim 2, Wherein the activity cofactor for PPARδ / β is one or more selected from the steroid receptor activity cofactor SRC-1, transcription mediator TIF-2 or p300 protein new drug candidate screening method for improving or treating metabolic diseases. The method of claim 3, wherein Wherein the activity cofactor for PPARδ / β is a recombinant protein isolated from a human and comprising a specific active site for PPARδ / β new drug candidate for improving or treating metabolic diseases. The method of claim 2, The PPARδ / β protein is a new drug candidate for improving or treating metabolic diseases, characterized in that the recombinant protein isolated from human. The method of claim 2, The new drug candidate compound is a plant extract, a functional food, and a new drug candidate for improving or treating metabolic diseases, characterized in that it comprises a pharmaceutical composition comprising them as an active ingredient. The method according to any one of claims 2 to 6, The metabolic disease includes a metabolic disease associated with glucose metabolism and lipid metabolism, such as obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia. The method according to any one of claims 2 to 6, In addition to the metabolic disease, a new drug candidate searching method for improving or treating metabolic disease, characterized in that it comprises inflammation, cancer and skin diseases. (1) activity cofactors for PPARδ / β; (2) PPARδ / β protein; (3) specific antibodies against PPARδ / β; And (4) a secondary antibody that binds to a specific antibody to the PPARδ / β. The method of claim 9, The activity cofactor for the PPARδ / β is at least one selected from steroid receptor activity cofactor SRC-1, transcription mediator TIF-2 or p300 protein, characterized in that the recombinant protein comprising a specific active site isolated from human New drug candidate search kit. The method of claim 9, The PPARδ / β protein is a new drug candidate search kit, characterized in that the recombinant protein isolated from human. The method according to any one of claims 9 to 11, The new drug candidate material is a kit for searching for a new drug candidate, which is used to improve or treat metabolic diseases, including inflammation, cancer, and skin diseases, including obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia.

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