KR101090342B1 - The Composition for inhibiting cell death comprising Aldorase or Glyceraldehyde-3-phosphate as an active ingredient - Google Patents

Abstract

The present invention relates to a composition comprising an aldolase (Aldolase) or a metabolite thereof, a screening method using the material, specifically, the glyceraldehyde-3-phosphate (Glyceraldehyde-3) that is an aldorase or a metabolite thereof It relates to a composition for inhibiting apoptosis containing -phosphate (G-3-P) as an active ingredient, and a method for screening a substance for controlling apoptosis using aldorase or G-3-P. In the present invention, aldorase inhibits cell death, and in particular, G-3-P, which is a metabolite or substrate of aldorase and GAPDH, binds to caspase-3 and directly reduces cell activity. Since it has been confirmed to interfere with apoptosis, the compositions and methods of the present invention can be usefully used for the treatment of apoptosis-related diseases and the development of therapeutic agents.

Description

The composition for inhibiting cell death comprising Aldorase or Glyceraldehyde-3-phosphate as an active ingredient}

The present invention relates to a composition for inhibiting apoptosis and a method for screening apoptosis regulators.

The enzyme whose glycolysis converts glucose into pyruvic acid is the oldest in the network of molecular metabolism. Early research focused on the function of the enzyme in its action. Recent studies provide evidence that enzymes of a given glycolysis are more complex multifunctional proteins than simple components of the pathway of glycolysis (Chuang DM). et al ., 2005, Annu . Rev. Pharmacol . Toxicol . 45, 269-290; Sen N et al., 2008, Nat . Cell Biol . 10, 866-873). Several subtypes of hexokinase and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were involved in apoptosis (Chuang et al. al . , 2005; Robey RB and Hay N, 2006, Oncogene 25, 4683-4696). Based on studies on glucose metabolism closely bound to cell death and survival, it was speculated that apoptosis and its action were linked. However, the detailed mechanisms for the fundamental regulation of cell energetics during apoptosis are not yet known (Majors BS et al ., 2007, Metab . Eng . 9, 317-326).

Cell energy metabolism and apoptosis are two important determinants of survival. Many growth and survival factors stimulate energy metabolism, including glycolysis, and inhibit apoptosis (Garland JM et al ., 1997, J. Biol. Chem 272, 4680-4688; Plas DR and Thompson CB, 2002, Trends Endocrinol . Metab . 13, 75-78; Vander Heiden MG et al ., 2001, Mol . Cell . Biol . 21, 5899-5912). Removal of growth factors results in a decline in metabolism, which is marked by reduced glycolysis action, low oxygen consumption, reduced ATP levels and reduced protein synthesis. Unless reversed, these metabolic changes eventually lead to apoptosis. Recent studies show that ATP-dependent steps are required for apoptotic signal transduction, including apoptosome formation, chromatin aggregation and phosphorylation of pro-apoptotic proteins. The ATP level is regulated by the influx of glycolysis or the mitochondrial system. Other groups have previously suggested that ATP concentration in these cells acts as a switch between apoptosis and survival (Jiang X and Wang X, 2000, J. Biol . Chem . 275, 31199-31203; Kass GE et al ., 1996, Biochem . J. 318, 749-752; Leist m et al ., 1997, J. Exp . Med . 185, 1481-1486; Priault M et al ., 1999, Eur. J. Biochem . 260, 684-691). However, the mechanism has been found to be a mechanism involved in glucose metabolism, a major supplier of ATP in the apoptosis pathway.

Aldolase is a housekeeping enzyme that regulates the metabolic pathway of glycolysis that is ubiquitous and structurally expressed in mammalian cells. Although aldorase is overexpressed in some cancers, including lung, early colon and cervical carcinoma (Altenberg B et al ., 2004, Genomics 84, 1014-1020; Lu H et al ., 2002, J. Biol . Chem . 277, 23111-23115; Kilic m et al ., 2007, Oncogene 26, 2027-2038), its role in the apoptosis pathway is unknown.

Previous studies have reported that GAPDH is highly expressed in the early stages of apoptosis (Sen et. al . , 2008). Based on the findings associated with previous reports, Glyceraldehyde-3-phosphate (G-3), also known as triose phosphate or 3-phosphoglyceraldehyde -P) is a major intermediate in some important metabolic pathways including glycolysis and glucogenesis. G-3-P is isolated from fructose 1,6-bisphosphate (FBP) from aldorase and dihydroxyacetone phosphate from triose phosphate isomerase DHAP), isomerization and reversible reaction catalyzed by GAPDH. Aldorases are ubiquitous enzymes of high interest because of their ability to catalyze their carbon-carbon bond formation. Aldorase A (ALDOA) is found in skeletal muscle and red blood cells and is responsible for promoting reversible cleavage of FBP into trisaccharide phosphate, G-3-P and DHAP in its action. ALDOA is upregulated in various conditions of cancer, including human lung squamous epithelium and renal cell carcinoma (Li C et al ., 2006, Proteomics 6, 547-558; Unwin RD et al ., 2003, Proteomics 3, 1620-1632). Another important enzyme of glycolysis, GAPDH, is involved in the process of apoptosis and is involved in neuronal cell death in some neurodegenerative diseases. GAPDH is overexpressed in the nucleus during apoptosis in various cell types (Chuang et al . , 2005; Ishitani R et al ., 1996, J. Neurochem . 66, 928-935).

The present inventors assume that G-3-P concentration will be continuously changed according to the course of various conditions of cells including hypoxia, apoptosis and cancer, and the effect of G-3-P and aldorase on cell death As a result of studying the effects, aldorase inhibits cell death, especially G-3-P, a metabolite or substrate of aldorase and GAPDH, binds to caspase-3 and directly decreases apoptosis. The present invention has been completed by confirming the obstruction. Accordingly, the compositions and methods of the present invention can be usefully used for the treatment of cell death-related diseases and the development of therapeutic agents.

It is an object of the present invention to provide a composition for inhibiting apoptosis, which contains glyceraldehyde-3-phosphate (Glyceraldehyde-3-phosphate; G-3-P) or aldolase (Aldolase) as an active ingredient.

Another object of the present invention is to provide a method for screening apoptosis inducing agents or modulators using the mechanism of action of G-3-P or aldorase.

In order to achieve the above object, the present invention provides a composition for inhibiting apoptosis containing glyceraldehyde-3-phosphate (Glyceraldehyde-3-phosphate; G-3-P) or aldorase (Aldolase) as an active ingredient do.

In addition,

1) preparing an expression vector operably linked to a polynucleotide encoding an aldorase;

2) transducing and expressing the expression vector of step 1) in a host cell;

3) treating the test compound to the cells of step 2);

4) measuring the aldorase activity in the cells of step 3); And,

5) It provides a method for screening apoptosis inducer comprising the step of selecting a test compound having a reduced aldorase activity compared to the control.

In addition,

1) preparing an expression vector operably linked to a polynucleotide encoding an aldorase;

2) transducing and expressing the expression vector of step 1) in a host cell;

3) treating the test compound to the cells of step 2);

4) measuring the aldorase activity in the cells of step 3); And,

5) Provides a method for screening a therapeutic agent for apoptosis-related diseases comprising the step of selecting a test compound having reduced aldorase activity compared to the control.

In addition,

1) treating the test compound to the cells;

2) measuring the intracellular G-3-P concentration in the cells of step 1); And,

3) provides a screening method of apoptosis modulator comprising the step of selecting a test compound having a change in the concentration of G-3-P in the cell compared to the control.

In addition,

1) contacting G-3-P and Caspase-3 protein in vitro;

2) treating the test compound in the test tube of step 1);

3) measuring the degree of binding of G-3-P and caspase-3; And,

4) It provides a method for screening apoptosis inducer comprising the step of selecting a test compound that reduced the binding of G-3-P and caspase-3 compared to the control.

In addition,

1) contacting G-3-P and caspase-3 protein in vitro;

2) treating the test compound in the test tube of step 1);

3) measuring the degree of binding of G-3-P and caspase-3; And,

4) A method for screening a therapeutic agent for apoptosis-related diseases comprising the step of selecting a test compound having reduced binding of G-3-P and caspase-3 as compared to the control group.

In addition,

1) contacting G-3-P and caspase-3 protein in vitro;

2) treating the test compound in the test tube of step 1);

3) measuring the degree of activity of caspase-3; And,

4) It provides a method for screening apoptosis inducing agent comprising the step of selecting a test compound that reduced the activity of caspase-3 compared to the control.

In the present invention, aldolase (Aldolase) inhibits cell death, the metabolite of the glycerol-3-phosphate (Glyceraldehyde-3-phosphate; G-3-P) is a non-competitive method Since it was confirmed to inhibit apoptosis by directly binding to Caspase-3 and inhibiting the activity of caspase-3, the compositions and methods of the present invention can be used for the treatment and treatment of apoptosis-related diseases. It can be usefully used.

1 is a diagram showing the results of confirming whether or not aldolase (Aldolase) inhibits cell death.
Figure 2 shows the results confirming that G-3-P inhibits apoptosis in a concentration-dependent manner.
Figure 3 is a diagram showing the result confirmed by Western blot that the level of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in the nucleus is reduced by G-3-P.
Figure 4 is a diagram showing the results confirmed by the confocal fluorescence microscope that the level of GAPDH in the nucleus is reduced by G-3-P.
FIG. 5 is a diagram showing the results of confirming whether G-3-P affects PARP (Poly ADP ribose polymerase) cleavage and caspase-3 activation in apoptosis.
6 is a diagram showing the results of confirming the inhibition of caspase-3 activity by G-3-P.
FIG. 7 is a kinetic analysis result of the caspase-3 inhibition of G-3-P by a Michaelis-Menten plot.
FIG. 8 is a lineweaver-Burk plot of kinetic analysis of caspase-3 inhibition of G-3-P. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for inhibiting apoptosis containing glyceraldehyde-3-phosphate (Glyceraldehyde-3-phosphate; G-3-P) or aldolase (Aldolase) as an active ingredient.

The composition of the present invention can be used for the prevention or treatment of any one of apoptosis-related diseases selected from the group consisting of cancer, degenerative cerebral nervous system disease, stroke, immune system disease and inflammation, but is not limited thereto.

In a specific embodiment of the present invention, we have found that aldorase activity reduces cell death (see FIG. 1), which is because G-3-P, a metabolite of aldorase, inhibited apoptosis It was confirmed (see FIGS. 2 to 4). In addition, the inhibition of apoptosis is the result of G-3-P inhibiting the activity of Caspase-3 (see FIG. 5), and G-3-P is the active site of Caspase-3. Binding to other sites was confirmed to be directly inhibited by non-competitive methods (see Figures 6 to 8).

This is the first discovery that caspase activity was directly inhibited by intermediates of metabolism, and we found that the major molecule of several major metabolic pathways, G-3-P, was reduced during apoptosis, leading to a pro-apoptotic process. Has been shown to play a decisive role as a cell fate determinant.

The composition of the present invention is preferably a pharmaceutical composition, wherein the pharmaceutical composition may be administered orally or parenterally, and during parenteral administration, external or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection It is desirable to choose an injection or intrathoracic injection infusion.

The pharmaceutical composition according to the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents and the like. The disintegrants include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, chitosan, guar gum, low-substituted hydroxypropyl cellulose, magnesium aluminum silicate, and polyacryline Potassium and the like. In addition, the pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, Calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, Aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight based on the pharmaceutical composition.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and aerosols.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include powders, granules, tablets, capsules, sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc. It may be used in the form of a formulation, and preferably, an external skin pharmaceutical composition of cream, gel, patch, spray, ointment, warning agent, lotion agent, linen agent, pasta agent or cataplasma agent may be prepared and used. It is not limited to this. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but may be appropriately selected by those skilled in the art. have. For the desired effect, however, oral dosages generally give an adult an amount of 0.0001 to 100 mg / kg of the pharmaceutical composition of the present invention per kg of body weight per day, preferably 0.001 to 100 mg / kg per day. Good to do. Administration may be administered once a day or may be divided several times. The dose is not intended to limit the scope of the invention in any way. Since the pharmaceutical composition of the present invention has little toxicity and side effects, it is a composition that can be used safely even when taken for a long time.

In addition,

1) preparing an expression vector operably linked to a polynucleotide encoding an aldorase;

2) transducing and expressing the expression vector of step 1) in a host cell;

3) treating the test compound to the cells of step 2);

4) measuring the aldorase activity in the cells of step 3); And,

5) It provides a method for screening apoptosis inducer comprising the step of selecting a test compound having a reduced aldorase activity compared to the control.

In addition,

1) preparing an expression vector operably linked to a polynucleotide encoding an aldorase;

2) transducing and expressing the expression vector of step 1) in a host cell;

3) treating the test compound to the cells of step 2);

4) measuring the aldorase activity in the cells of step 3); And,

5) Provides a method for screening a therapeutic agent for apoptosis-related diseases comprising the step of selecting a test compound having reduced aldorase activity compared to the control.

The activity of aldorase-3 in step 3) may be measured by any one or more of the following methods 1) to 4), but is not limited thereto:

1) measuring the concentration of fructose 1,6-bisphosphatate (FBP) in cells;

2) measuring the concentration of G-3-P in cells;

3) measuring the concentration of triose phosphate in cells; And,

4) A method for measuring the concentration of dihydroxyacetone phosphate (DHAP) in the cell.

The host cell is a cell derived from a mammal, preferably a human, but is not limited thereto.

In addition, the apoptosis-related disease is preferably any one selected from the group consisting of cancer, degenerative neurological disease, stroke, immune system disease and inflammation, but is not limited thereto.

In addition, the test compound may be any one selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, bacteria or fungi metabolites and bioactive molecules, Now, but not limited to, any material that is unknown in the art for reducing the activity of aldorase can be.

In addition,

1) treating the test compound to the cells;

2) measuring the intracellular G-3-P concentration in the cells of step 1); And,

3) provides a screening method of apoptosis modulator comprising the step of selecting a test compound having a change in the concentration of G-3-P in the cell compared to the control.

The cell is a cell derived from a mammal, preferably a human, but is not limited thereto.

In addition, the concentration of the G-3-P is estimated by measuring GAPDH activity (Shimizu T et al ., 1998, Blood 71, 1130-1134) or HPLC, but is not limited thereto, and those skilled in the art will readily be able to infer a method for determining the concentration of the compound.

In addition, the test compound may be any one selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, bacteria or fungi metabolites and bioactive molecules, Now, but not limited to, any material whose activity to increase or decrease apoptosis is known in the art is possible.

In addition,

1) contacting G-3-P and Caspase-3 protein in vitro;

2) treating the test compound in the test tube of step 1);

3) measuring the degree of binding of G-3-P and caspase-3; And,

4) It provides a method for screening apoptosis inducer comprising the step of selecting a test compound that reduced the binding of G-3-P and caspase-3 compared to the control.

In addition,

1) contacting G-3-P and caspase-3 protein in vitro;

2) treating the test compound in the test tube of step 1);

3) measuring the degree of binding of G-3-P and caspase-3; And,

4) A method for screening a therapeutic agent for apoptosis-related diseases comprising the step of selecting a test compound having reduced binding of G-3-P and caspase-3 as compared to the control group.

The binding of G-3-P and caspase-3 in step 3) is performed by SPR, protein chip, gel shift assay, immunoprecipitation, co-immunoassay, fluorescence immunoassay and radioimmunoassay. It may be measured by any one method selected from the group consisting of, but is not limited to the above, the apoptosis-related disease may be any one selected from the group consisting of cancer, degenerative brain nervous system disease, stroke, immune system disease and inflammation. However, it is not limited thereto.

In addition,

1) contacting G-3-P and caspase-3 protein in vitro;

2) treating the test compound in the test tube of step 1);

3) measuring the degree of activity of caspase-3; And,

4) It provides a method for screening apoptosis inducing agent comprising the step of selecting a test compound that reduced the activity of caspase-3 compared to the control.

The activity of caspase-3 in step 3) can be measured by measuring the resolution of caspase-3 on Ac-DEVD-pNA, but is not limited thereto.

In addition, the test compound may be any one selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, bacteria or fungi metabolites and bioactive molecules, It is not limited now.

In a specific embodiment of the present invention, it was confirmed that aldorase inhibits cell death and G-3-P directly binds to caspase-3 to inhibit activity in a concentration-dependent manner. It can be usefully used for the screening of apoptosis inducing agents, apoptosis regulators or therapeutic agents for apoptosis-related diseases.

[Example]

Hereinafter, the present invention will be described in detail by the following Examples and Preparation Examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Preparation Examples.

Example  One. Aldorase  Preparation of Expression Vectors

The present inventors performed the following cloning to determine the effect of aldolase on apoptosis. First, cDNA (SEQ ID NO: 1) encoding aldorase A (ALDOA) was obtained from the Mammalian Gene Collection (NIH, USA), followed by PCR using Pfu polymerase (Stratagene, USA). Amplified and subcloned into the pFlag-CMV vector (Sigma, USA). Forward primer (5'-atg ccc tac caa tat cca-3 ') and reverse primer (5'-aat tat ccg cac caa tct ctg-3') as described in SEQ ID NO: 2 for PCR amplification Was used. PCR reaction conditions are as follows. Using a cDNA of the aldorase as a template, 94 ℃, was treated for 4 minutes, 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃, 4 minutes was repeated 25 times, and then extended to 72 ℃, 10 minutes. Amplified PCR products and vectors were digested and purified with Eco RI and Sal I, respectively. About 100 ng of the vector and the fragment to be inserted were used, and 1 unit of ligase (Roche) from Roche was added and reacted at 16 ° C. for 16 hours. After the ligation reaction, E. coli DH5α (Invitrogen, USA) was transformed, selected from ampicillin-containing plates, cut with appropriate restriction enzymes to obtain plasmids containing the desired DNA fragments, and DNA sequencing. Finally confirmed. The expression vector of the wild-type aldorase prepared above was named 'pFlag-ALDOA'.

In addition, the pFlag-ALDOA, SEQ ID NO: 4 (5'-CG GAA TCC A atg ccc tac caa tat cca-3 ') and SEQ ID NO: 5 (5'-GC GTC GAC aat tat ccg cac caa tct ctg-3') PFlag-ALDOA ^D33A , which expresses the D33A mutant aldorase (SEQ ID NO: 6) in which the 33rd aspartic acid of ALDOA is mutated to alanine using a primer pair of Prepared.

Example  2. Aldorase  Active Apoptosis  Check impact

<2-1> Cell Line Culture

Human leukemia cell line HL-60 and human embryonic kidney cell line BOSC 23 were purchased from the American Type Culture Collection (ATCC). The cell lines were incubated in RPMI 1640 and DMEM medium containing 10% FBS, respectively. At this time, it was incubated under the conditions of 37 ℃ and 5% CO ₂ .

<2-2> Aldorase  According to activity Cell death  Measure

Empty vector (negative control) using lipofectamine (Invitrogen) on BOSC 23 cells of Example 2-1, 'pcDNA3.1-ALDOA' and 'pcDNA3.1-ALDOA ^D33A ' prepared in Example 1 The vectors were each transduced according to the manufacturer's manual. Each transgenic cell line was treated with a known method of 100 μM of etoposide (Sigma) for 24 hours to induce apoptosis (Jang M et al ., 2008, Mol . Cells 26, 152-157; Jang M et al ., 2009, Oncogene 28, 1529-1536), CellTiter 96R Aqueous Non-radioactive Cell Proliferation analysis kit (Promega, USA) was used to measure cell viability according to the manufacturer's manual.

As a result, as shown in FIG. 1, approximately 42.5% of the wild-type aldorase-expressing cells were killed when compared to the cell killing rate (84.5%) of the negative control, while 76.4% of the cells expressing the mutant al-dorase. Was observed, and it was found that aldose activity inhibited cell death.

Example  3. G-3-P Apoptosis  Check impact

The inventors of the present invention, in order to confirm whether the inhibition of cell death by the aldorase confirmed in Example 2-2 is caused by the metabolite of aldorase, the glyceraldehyde-3-phosphate which is a metabolite of aldorase (Glyceraldehyde- Cell viability was measured in the same manner as in Example 2-2 above on BOSC 23 cells not treated with 3-phosphate; G-3-P) or treated with 0.5, 1 or 2 mM for 24 hours.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which affects cellular G-3-P levels, is known to overexpress and translocate into the nucleus during apoptosis. The present inventors performed BOB 23 cells without treatment of G-3-P or treated with 0.5 or 1 mM for 24 hours after nuclear division, and then subjected to western blot. Specifically, the cells treated with G-3-P were recovered and treated with NP-40 lysis buffer [137 mM NaCl, 20 mM Tris-HCl, pH 7.5), 1 mM EDTA, 10% glycerol, and 1% NP-, respectively. 40]. Approximately 10-40 μg of cell extracts were separated on SDS gels and then transferred to PVDF membranes. The membrane was blocked with TBST buffer (20 mM Tris-HCl, pH 7.6, 0.1369 M NaCl, and 0.1% Triton X-100) for 5 hours at room temperature, followed by anti-GAPDH (Sigma) antibody. Was treated. At this time, the nuclear expression level of the nuclear specific marker LaminB (LaminB) as a quantitative control was measured together using an anti-lamin B antibody (Calbiochem). The membrane was then treated with horseradish peroxidase-fusion secondary antibody (Sigma) for 1 hour, washed with TBST, and then photosensitized using an improved chemiluminescence (ECL) system (Pierce, USA). The amount of GAPDH was observed. In addition, BOSC 23 cells, which were not treated with G-3-P or treated with 1 mM for 24 hours, were immobilized on a slide with a fixed solution containing 10% glycerol, respectively, followed by anti-GAPDH antibody and GFP-fusion secondary antibody. Was treated to label intracellular GAPDH. At this time, the cells were stained at the same time using a DAPI staining reagent. The stained cells were observed using a laser confocal fluorescence microscope (Carl Zeiss, Germany). Each experiment was repeated three times and at least 300 cells were analyzed for each condition.

As a result, as shown in FIG. 2, cell death decreased in inverse proportion to G-3-P concentration. In addition, as shown in FIG. 3, the concentration of GAPDH in the nucleus was decreased in inverse proportion to the concentration of G-3-P. In addition, GAPDH was mainly present in the nucleus when G-3-P was not treated as shown in FIG. 4, but GAPDH was mainly observed in the cytoplasm when G-3-P was treated. The results showed that cell death was inhibited by G-3-P, a metabolite of aldorase, and the cell death was apoptosis.

Example  4. G-3-P Caspase -3 activation and PARP  Determine impact on cutting

Next, the present inventors performed the following experiment to determine whether G-3-P affects the caspase-3 activation and PARP (Poly ADP ribose polymerase) cleavage that induces apoptosis. Specifically, 2-5 × 10 ⁸ HL-60 cells cultured as in Example 2-1 were washed twice with PBS, and cold cell extract buffer (CEB; 20 mM HEPES-KOH, pH 7.5, 10 mM). KCl, 1.5 mM MgCl ₂ , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100 μM PMSF, 10 μg / ml lupetin, and 2 μg / ml aprotinin), followed by homogenizing the cells After transferring to a phase, two times the volume of the cell precipitate, cold CEB was added. The cells were reacted on ice for 15 minutes under low osmotic conditions, and then pulverized with a homogenizer. The cell extract was centrifuged at 15,000 g for 14 minutes at 4 ° C., and the supernatant was aliquoted and stored at −70 ° C. before being used for experiments. The cell extract was treated with a group of cytochrome c / dATP (2 mM dATP / MgCl ₂ and 50 μg / ml right heart cytochrome c) (positive control group) and none group, 0.25, 0.5, 1 or 2 mM. Each group was divided into a group treated with G-3-P and a group treated with z-DEVD-fmk (a caspase inhibitor, 100 μM, 24 h) (negative control). Thereafter, cell extracts of each group (10-15 mg / ml) were reacted at 37 ° C. to activate caspase-3, and then anti-PARP antibody (Sigma) and anti-caspase-3 antibody to each group. Western blot was performed in the same manner as in Example 3 using (Calbiochem, USA). At this time, the whole cell precipitate was observed by staining as a quantitative control, and Ac-DEVD-fmk was obtained from the enzyme system product.

As a result, as shown in FIG. 5, PARP cleavage and caspase-3 activation were inhibited in the positive control group, but PARP cleavage was inhibited in the group treated with 2 mM G-3-P. Activation was slightly inhibited. In addition, neither cleavage of PARP and activation of caspase were observed in the negative control. From this, it was found that G-3-P influences the pro-apoptotic pathway by preventing cleavage of the substrate by caspase-3.

Example  5. G-3-P Caspase -3 inhibits substrate degradation

The present inventors measured caspase-3 activity by a known method using Ac-DEVD-pNA, a chromogenic caspase-3 substrate, to confirm whether G-3-P inhibits the degradation of caspase-3. (Fang B et al ., 2006, J. Mol . Biol . 360, 654-666) was performed. Specifically, after adding 100 μM of Ac-DEVD-pNA to the HL-60 cell extract obtained in Example 4, the group treated with nothing, the group treated with 4 or 8 mM G-3-P, Each group was treated with 20 mM DHAP and 80 mM FBP, and treated for 24 hours. Subsequently, absorbance was measured at 410 nm using a DU 800 spectrophotometer (Beckman Coulter) to observe the caspase-3 activity of each group.

As a result, as shown in FIG. 6, caspase activity was strongly inhibited in inverse proportion to the concentration of G-3-P, and caspase activity was also slightly suppressed by FBP and DHAP, which are substrates of aldorase.

Example  6. Direct of G-3-P Caspase -3 suppression confirmation

We conducted the following experiments to determine whether G-3-P directly inhibits caspase-3. Specifically, human caspase-3 obtained by a known method is activated by incubating for 15 minutes in assay buffer (50 mM HEPES pH 7.4, 50 mM KCl, 2 mM MgCl ₂ , 1 mM EDTA, 10 mM DTT) and room temperature conditions. Then either nothing or 2, 4, 6 or 8 mM G-3-P. Each G-3-P concentration treatment group was treated with 0.7, 1.4, 2.8 or 5.6 mM Ac-DEVD-pNA for 20 minutes. Thereafter, caspase activity was measured in the same manner as in Example 5. In the above results, inhibition of caspase-3 was analyzed by Michaelis-Menten and Lineweaver-Burk plots, using the KaleidaGraph 4.0TM of Synergy Software, respectively, FIGS. 7 and 8. Shown graphically. The inhibition constant (K _i ) of G-3-P was determined using Equation 1. Data of the present invention are shown in the mean ± SD after three replicates in three sets, and statistical significance Student's t - determined the lowest level of significance at p <0.05 by analysis with a black (Student's t -test) .

In this case, Vmaxi represents the concentration of Vmax (Segal, 1975) and [I] represents the concentration of G-3-P in the presence of an inhibitor.

As a result, as shown in FIGS. 7 and 8, the maximum rate (Vmax) of caspase-3 activity was decreased by G-3-P (FIG. 7), but the KM value for the substrate (X axis, FIG. 8). Was not affected. The results support the possibility of binding G-3-P at a different position than the active site of the enzyme, suggesting that G-3-P is a noncompetitive inhibitor of caspase-3. The inhibition constant (K _i ) was determined to be 1.1 ± 0.12 mM by nonlinear recursion. As such, we have suggested that G-3-P is closely related to the regulation of apoptosis by directly inhibiting caspase activity.

Manufacturing example  1: Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

2 g of G-3-P or aldorase of the invention

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of G-3-P or aldorase of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of G-3-P or aldorase of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

1 g of G-3-P or aldorase of the invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of granules

150 mg of G-3-P or aldorase of the present invention

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

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Claims (13)

A composition for inhibiting apoptosis, comprising glyceraldehyde-3-phosphate (Glyceraldehyde-3-phosphate; G-3-P) as an active ingredient.
The composition according to claim 1, which is used for the prevention or treatment of any apoptosis-related diseases selected from the group consisting of cancer, degenerative neurological disease, stroke, immune system disease and inflammation.
The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
1) preparing an expression vector operably linked to a polynucleotide encoding an aldorase;
2) transducing and expressing the expression vector of step 1) in a host cell;
3) treating the test compound to the cells of step 2);
4) measuring the aldorase activity in the cells of step 3); And,
5) A method for screening an apoptosis inducer comprising the step of selecting a test compound having reduced aldorase activity compared to the control.
The method of claim 4, wherein the activity of aldorase-3 in step 3) is measured by any one or more of the following methods 1) to 4):
1) measuring the concentration of fructose 1,6-bisphosphatate (FBP) in cells;
2) measuring the concentration of G-3-P in cells;
3) measuring the concentration of triose phosphate in cells; And,
4) A method for measuring the concentration of dihydroxyacetone phosphate (DHAP) in the cell.
The method of claim 4, wherein the host cell is a cell derived from a mammal.
1) treating the test compound to the cells;
2) measuring the intracellular G-3-P concentration in the cells of step 1); And,
3) A method for screening apoptosis regulator comprising the step of selecting a test compound having a change in the concentration of G-3-P in the cell compared to the control.
8. The method of claim 7, wherein said cell is a cell derived from a mammal.
1) contacting G-3-P and caspase-3 protein in vitro;
2) treating the test compound in the test tube of step 1);
3) measuring the degree of binding of G-3-P and caspase-3; And,
4) A method for screening apoptosis inducing agent comprising the step of selecting a test compound that reduced the binding of G-3-P and caspase-3 compared to the control.
10. The method of claim 9, wherein the binding of G-3-P and caspase-3 in step 3) comprises SPR, protein chip, gel shift assay, immunoprecipitation, co-immunoassay, fluorescence immunoassay. It is measured by any one method selected from the group consisting of analytical methods and radioimmunoassay.
1) contacting G-3-P and caspase-3 protein in vitro;
2) treating the test compound in the test tube of step 1);
3) measuring the degree of activity of caspase-3; And,
4) A method for screening an apoptosis inducer comprising the step of selecting a test compound having reduced activity of caspase-3 compared to the control.
12. The method of claim 11, wherein the activity of caspase-3 in step 3) is determined by measuring the resolution of caspase-3 against Ac-DEVD-pNA.
The method of claim 4, 7, 9 or 11, wherein the test compound is a natural compound, synthetic compound, RNA, DNA, polypeptide, enzymes, proteins, ligands, antibodies, antigens, bacteria or A method characterized in that it is any one selected from the group consisting of metabolites of fungi and bioactive molecules.

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