KR101796891B1 - Method for Determination of metabolites changed by Translocator protein 18 kDa in hypothalamus cells - Google Patents

Abstract

The present invention relates to a method for analyzing metabolic changes by TSPO (translocator protein) in a cell, and more particularly, to a method for analyzing a metabolic change by TSPO (translocator protein) -TOFMS, and provide the final phenotype for the function of TSPO.
The method of analyzing the metabolites by TSPO of the present invention can identify the metabolic structure and secretion change pattern by TSPO according to treatment with TSPO ligand (PK11195), so that identification of specific metabolites changed after TSPO ligand treatment is performed, Can be used as a marker for the metabolic regulation of TSPO by presenting the final phenotype for the function of TSPO which has not yet been clarified.

Description

METHODS FOR DETERMINING METABOLIC CHANGE BY TSPO IN BRAIN SIXTH SEMICONDUCTORS BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a method for analyzing metabolism by TSPO in brain hypothalamus cells. More particularly, the present invention relates to a method for analyzing metabolic changes in brain hypothalamic cells by LC / Q-TOFMS And provide the final phenotype for the function of TSPO.

Endogenous metabolites present in the human body are altered by factors such as diet, environment, genetics, disease, medicines, etc., and metabolism (metabolism), which is a field of studying human metabolism, It is possible to predict the biochemical metabolite changes involved. Genomics, transcriptomics, and proteomics, which are areas of Omics, require a lot of time and effort to analyze and estimate the number of genes or proteins to be about 40,000 and more than 150,000 There are many difficulties in interpreting later, but in the case of metabolomics, the number of analytes to be analyzed is less than about 2,500, which is easy to interpret. In general, metabolism is related to diverse physiological, pathological, and genetic conditions of the metabolic pathway, including the composition and changes of low molecular endogenous metabolites in vivo using various advanced analytical instruments and statistical algorithms.

Global metabolomics profiling method, which is one of metabolism analysis methods, is a method of analyzing metabolites as a whole without specifying a specific group, and it is known that mass spectrometry (MS) is mainly used. Mass spectrometry is a high-sensitivity method that allows a large amount of information from analytes to be obtained from a small amount of sample and allows analysis of metabolites with a wide range of molecular weights. Among them, time-of-flight mass spectrometry (TOFMS) is based on the assumption that all ions receive the same energy in the acceleration field and accelerate to the detector, (Liquid chromatography / mass spectrometry), it is possible to combine various separation techniques and detectors by selecting a column or combining a moving phase.

On the other hand, in the basic research stage, it is considered that it is important not only to study the candidate substance (ligand) of a new drug but also to find a target protein binding to the ligand and to understand its function. The translocator protein (TSPO) is known to form complexes with other proteins such as disulfide-dependent anion channel (VDAC) and adenine nucleotide translocation enzyme (ANT) in membrane proteins expressed in mitochondrial outer membrane. TSPO is different from benzodiazepine receptor of central nervous system (GABA), which is functionally and structurally, and is distributed in peripheral organs, especially liver, adrenal glands and hematopoietic organs, but in normal central nervous system Only a small amount exists.

Recently, ligands of TSPO have attracted attention as new drug candidate compounds such as neurodegenerative disorders, inflammation or anxiety neuropathy. Among them, PK11195 [1- (2-chloro-phenyl) -N-methyl-N- (1-methylpropyl) -isoquinoline carboxamide] is known to selectively bind to TSPO. Although the expression of TSPO has been reported to increase in leptin-deficient mice (Giannaccini et al., BMC Neuroscience , 12:18, 2011), the role of TSPO has not yet been elucidated, and TSPO ligands PK11195) has not been reported in the literature.

Therefore, the present inventors have made intensive efforts to confirm the constitutive and secretory changes of metabolites for the role of TSPO in brain hypothalamus cells. As a result, the brain hypothalamus cells were treated with TSPO ligand PK11195, followed by LC / Q-TOFMS As a result, significant changes of metabolites were observed according to the role of TSPO, and the above-mentioned analysis method was used to identify specific metabolites which were changed after TSPO ligand treatment, so that it was useful for diseases related pathology, pathology of diseases, And can be used as a marker for the regulation of metabolic processes of TSPO by presenting the final phenotype for the function of TSPO which has not yet been clearly clarified. Thus, the present invention has been completed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for analyzing TSPO metabolite composition and secretion change pattern by TSPO ligand treatment.

A second object of the present invention is to provide a chemical control index for the role of TSPO including the TSPO metabolite analyzed by the above-described metabolite analysis method.

Disclosure of the Invention In order to solve the first problem of the present invention, the present invention provides a method for producing a TSPO metabolite, comprising: treating a cell with a translocator protein (TSPO) ligand and then isolating a cell extract containing a TSPO metabolite; And analyzing the TSPO metabolite by comparing the metabolite concentration contained in the cell extract with a control group not treated with TSPO ligand; Lt; RTI ID = 0.0 > TSPO < / RTI >

According to a preferred embodiment of the present invention, the cell may be a brain hypothalamic cell.

According to another preferred embodiment of the present invention, the translocator protein (TSPO) ligand is selected from the group consisting of PK11195 [1- (2-chloro-phenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinoline carboxamide ].

According to another preferred embodiment of the present invention, the metabolite concentration analysis is performed using LC / Q-TOFMS (time-of-flight mass spectrometry) using time of flight (TOF) Can be used.

According to another preferred embodiment of the present invention, the method for analyzing a metabolite by TSPO comprises the steps of: (a) treating a cell with a translocator protein (TSPO) ligand for 3 to 6 hours, ≪ / RTI > (b) adding reserpine as an internal standard substance to the cell extract, followed by centrifugation by treating with an organic solvent; (c) separating the aqueous solution layer and the organic solvent layer from the aqueous phase (upper layer), protein layer (intermediate layer) and organic solvent layer (lower layer) Analyzing the protein concentration contained in the cell extract with Q-TOFMS; And (d) comparing the metabolites contained in the cell extracts not treated with the TSPO ligand and the metabolites contained in the cell extract treated with the TSPO ligand to select the metabolites with increased or decreased expression by the TSPO ligand The method comprising the steps of:

According to another preferred embodiment of the present invention, the cell extract in step (a) may be extracted using a mixed solvent of methanol and water.

According to another preferred embodiment of the present invention, the organic solvent of step (b) may be chloroform.

According to another preferred embodiment of the present invention, the TSPO metabolite is L-oleandrosyl-oleandolide, C8-Dihydroceramide, (BD-Glucopyranuronic acid), N2-Succinyl-L-arginine, Uracil, Phytyl diphosphate, S- (hydrosiphenylacetoxyhydroximoyl (S-Hydroxyphenylacetothiohydroximoyl) -L-cysteine, Urocortisone, Tetracosahexaenoyl-CoA, cortisone acetate, 16 (S) -HETE, Phytosphingosine, 6- (2-chloro-5 (trifluoromethyl) phenyl) -1-phenyl-2-thiovaiurea 1-phenyl-2-thiobiurea, triphenylphosphine oxide, nicotinuric acid, CMP-N-glycoylenylaninate (CMP-N-glycoloylneuraminate), 1-hexadecanol, homoisocitrate, N-palmitoyl tryptophan, N-stearoyl glutamic acid acid, N-acetyllactosamine, L-urobilin, 7a-hydroxy-3-oxocolest-4-ene- oxocholest-4-en-27-oic acid, phosphatidylethanolamine, and triglyceride.

In order to solve the second problem, the present invention may include a TSPO metabolic regulation marker including the TSPO metabolism analyzed by the TSPO metabolism analysis method, and the TSPO metabolite analyzed by the TSPO metabolism analysis method And measuring the expression level of the TSPO activity.

According to a preferred embodiment of the present invention, the TSPO metabolite is selected from the group consisting of L-oleandrosyl-oleandolide, C8-Dihydroceramide, bD-glucopyranuronic acid (bD-Glucopyranuronic acid), N2-Succinyl-L-arginine, Uracil, Phytyl diphosphate, S- (hydrosiphenylacetoxyhydroximoyl) Cysteine, S-Hydroxyphenylacetothiohydroximoyl-L-cysteine, Urocortisone, Tetracosahexaenoyl-CoA, cortisone acetate, 16 (S) -HETE, Phytosphingosine, 6- (2-chloro-5 (trifluoromethyl) phenyl) -1-phenyl-2-thiovaiurea phenyl-2-thiobiurea, triphenylphosphine oxide, nicotinuric acid, CMP-N-glycoylinuraminate (CMP-Ng lycloylneuraminate, 1-hexadecanol, homoisocitrate, N-palmitoyl tryptophan, N-stearoyl glutamic acid, N- N-acetyllactosamine, L-urobilin, 7a-hydroxy-3-oxocholest-4-ene -27-oic acid, phosphatidylethanolamine, and triglyceride.

The method of analyzing a metabolite by TSPO of the present invention can confirm the metabolite composition and secretion change pattern by TSPO according to the TSPO ligand treatment, so that identification of a specific metabolite changed after TSPO ligand treatment is performed, , Disease pathology, and physiology, and can be used as a marker for the regulation of TSPO metabolism by presenting a final phenotype for the function of TSPO that has not yet been clarified.

Figure 1 is data showing the chromatographic pattern and intensity of the internal standard material (reserpine).
FIG. 2 is a graph showing changes in principal component analysis patterns in the water-soluble layer of brain nerve cell extract (0h) and PK11195-treated brain nerve cell extract (4h), which were not treated with PK11195, depending on whether or not the quality control sample (QC) .
FIG. 3 is a graph showing changes in principal component analysis pattern of each group in the organic layer of the brain nerve cell extract (0h) not treated with PK11195 and the brain nerve cell extract (4h) treated with PK11195 according to the presence or absence of the quality control sample (QC).
FIG. 4 is a data showing a loading plot in a water-soluble layer of a brain nerve cell extract (control group) not treated with PK11195 and a brain cell extract treated with PK11195.
FIG. 5 is a data showing loading plots in an organic layer of a brain neuron cell extract (control group) not treated with PK11195 and a brain cell extract treated with PK11195.
6 is data showing a heat map for confirming the clustering between the metabolites of the PK11195-untreated control group and the PK11195 treated group (the fold change cut-off between the two groups of 2.0 or higher Selective analysis).
7 shows the R software code used in the heat map analysis.
FIG. 8 is a schematic diagram of a hypothetical metabolite network that activates the action mechanism according to the TSPO role. FIG.
FIG. 9 shows MS / MS pattern data of 6- (2-chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea identified in a standard product and a brain nerve cell sample.
10 is data showing MS / MS patterns of Triphenylphosphine oxide identified in the standard product and in the brain nerve cell sample.
FIG. 11 shows MS / MS pattern data of cortisone acetate identified in the standard and brain nerve cell samples.

Hereinafter, the present invention will be described in more detail.

As described above, recently, ligands of TSPO have been attracting attention as new drug candidate compounds such as neurodegenerative disorders, inflammation or anxiety neuropathy, and compounds capable of selectively detecting TSPO have been studied, but the roles of TSPO , And no study has been reported on the metabolic change pattern for TSPO role identification.

The present invention sought to solve the above-mentioned problems by providing a method for analyzing TSPO metabolites by treatment with a translocator protein (TSPO) ligand. In this way, identification of specific metabolites that are changed after TSPO ligand treatment can be performed, and the final phenotype of the function of TSPO can be suggested by grasping the metabolic change pattern for TSPO role identification. The TSPO Metabolites can be used as markers for the regulation of TSPO metabolism and for measuring TSPO activity.

Accordingly, the present invention includes a method for analyzing a TSPO metabolite by treatment with a translocator protein (TSPO) ligand. More specifically, the present invention relates to a method for producing a TSPO metabolite by treating a cell with a translocator protein (TSPO) Isolating the cell extracts comprising the cells; Analyzing the metabolite concentration contained in the cell extract, and then analyzing metabolites by TSPO through comparison with a control group not treated with TSPO ligand; Lt; RTI ID = 0.0 > TSPO < / RTI >

The 'translocator protein' (TSPO) is a transgene protein (18 kDa) known as a peripheral benzodiazepine receptor (PBR), which contains an adenine nucleotide carrier (ANC) (30 kDa) The mitochondrial permeability transition pore (MPTP) can be formed by forming a triple complex with a voltage-dependent anion channel (VDAC). TPSO is distinguished from a central benzodiazepine receptor (CBR) by its unique structure, physiological function, and intracellular location on the mitochondrial outer membrane. TSPO has been associated with numerous biological processes, but some aspects of its physiological role have not yet been clearly elucidated.

TSPO is closely distributed in most peripheral organs, such as the lungs, heart and kidneys, but only in the brain parenchyma. After nerve injury or infection, TSPO expression in the parenchyma is dramatically increased. By in vitro autoradiography and immunohistochemistry, it was found that elevated TSPO binding in this region is directly related to the appearance of activated microglia. Recently, by in vivo positron emission tomography (PET) imaging of patients suffering from Alzheimer's disease (AD) and multiple sclerosis (MS), TSPO binding in the brain parenchyma is activated It was confirmed that it is restricted to the glial cells.

The term " metabolome " refers to the total amount of metabolism present in a cell, and refers to a substance produced as a result of a substance change in a living body. The 'TSPO metabolite' or 'metabolite by TSPO' of the present invention means a metabolite which is changed after the TSPO ligand treatment, and the TSPO ligand selectively binds to TSPO and reduces the activity of TSPO in the cells. That is, as the TSPO metabolite analysis method of the present invention is reduced to TSPO activity by TSPO ligand treatment, it is possible to select metabolites and to metabolize the metabolites according to TSPO activity, The final phenotype for the function can be presented.

In the present invention, the cells may be brain hypothalamic cells, but not limited thereto, TSPO metabolites in various cells can be analyzed using the TSPO metabolite analysis method of the present invention.

In the present invention, the translocator protein (TSPO) ligand may be PK11195 [1- (2-chloro-phenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinoline carboxamide] The antagonist capable of specifically binding to TSPO can be used without limitation.

In the present invention, the metabolism analysis can use LC / Q-TOFMS (liquid chromatography / quadrupole time-of-flight mass spectrometry) using a time of flight (TOF) of a metabolite material.

The TSPO metabolite analysis method will be described in more detail.

(a) treating cells with a translocator protein (TSPO) ligand for 3 to 6 hours, and then obtaining a cell extract containing the TSPO metabolite;

(b) adding reserpine as an internal standard substance to the cell extract, followed by centrifugation by treating with an organic solvent;

(c) separating the aqueous solution layer and the organic solvent layer from the aqueous phase (upper layer), protein layer (intermediate layer) and organic solvent layer (lower layer) Analyzing the protein concentration contained in the cell extract with Q-TOFMS; And

(d) comparing the metabolites contained in the TSPO ligand-treated cell extract with the metabolites contained in the TSPO ligand-treated cell extract to select metabolites with increased or decreased expression by the TSPO ligand The method comprising the steps of:

In step (a), the cell is treated with a translocator protein (TSPO) ligand for 3 to 6 hours to obtain a cell extract containing a TSPO metabolite. In the present invention, The hypothalamic A2 / 29 cells were treated with TSPO ligand PK11195 (10 [mu] M) and reacted for about 4 hours to obtain a cell extract.

In the present invention, the cells are brain hypothalamic cells. The cell extract can be extracted using a general protein extraction method known in the art. In the present invention, a mixture of methanol and water is preferably used.

Next, in step (b), reserpine is added to the cell extract as an internal standard, followed by centrifugation by treating with an organic solvent.

The internal standard (IS) is a compound known to be added to a background sample, a reference material for a calibration curve, a sample or a sample extract immediately before analyzing the sample. (IS) should be used to check retention time, relative response, and the amount of analyte present in each sample.

In the present invention, a quality control sample is used to quickly evaluate how well the analysis of the metabolism profiling method used in the present invention is accomplished visually. The metabolism profiling analysis method requires many sample preprocessing steps, In addition to graphical techniques, the mass spectrometer's response affects the degree of performance degradation due to column contamination over time. Quality control samples are used to quickly assess how well these analyzes are visually performed, and these quality control samples are usually scattered randomly at the beginning, middle, and end of the sample set, and in the middle of the analysis. Quality control samples provide information on both the suitability of the system and the stability of the system, including problems caused by contamination of the system or equipment errors such as reduced sensitivity, changes in retention times, or changes in mass accuracy.

In one embodiment of the present invention, the first five of the quality control samples were used to perform system suitability tests at the beginning of the analysis. The reproducibility of the analyzed quality control samples was confirmed by satisfying the same chromatographic pattern and the coefficient of variation of the intensity of the internal reference material. Reserpine was used as an internal reference material, and the coefficient of variation (CV,%) of the internal standard substance in the quality control sample was 5.76%. The chromatographic pattern and strength of the internal reference material in the quality control sample are as shown in Fig.

As the organic solvent of step (b), any solvent capable of extracting a protein corresponding to a metabolite from cells may be used without limitation, and chloroform may be preferably used.

In one aspect of the present invention, a method of isolating a protein or a lipid-containing metabolite contained in a cell extract is carried out using a Bligh-Dyer extraction method (Bligh EG & Dyer WJ, Can. J. Biochem. Physiol. 37: 911, 1959).

Next, in step (c), an aqueous solution layer and an organic solvent in an aqueous phase (upper layer), a protein layer (intermediate layer) and an organic solvent layer (lower layer) Layer is separated and the concentration of protein contained in the cell extract is analyzed by LC / Q-TOFMS.

When the modified Bligh-Dyer extraction method is used, the cell extract of step (b) is treated with a solvent such that chloroform: methanol: water is finally mixed at a ratio of 1: 2: 1.8, It is divided into an aqueous phase (upper layer), a protein layer (intermediate layer) and an organic phase (lower layer). Half of the separated organic solvent layers were sequentially subjected to an acid extraction step and a base extraction step and the previously extracted aqueous phase (upper layer) and organic solvent phase (lower layer) were selected, Metabolic analysis was performed.

In the present invention, a global metabolomics profiling method for analyzing metabolites in vivo using LC / Q-TOFMS was used for the analysis of metabolites, and UPLC (Agilent Technologies 1290 Infinity) and Q-TOFMS (liquid chromatography / quadrupole time- -flight mass spectrometry), the main components contained in the aqueous solution layer and the organic solvent layer were confirmed, and the metabolite change patterns between the control and experimental groups were analyzed.

First, the changes of the principal component analysis patterns of the experimental group treated with the TSPO ligand PK11195 and the control group treated with the PK11195 were analyzed in the aqueous solution layer and the organic solvent layer, respectively. As a result, as shown in Figs. 2 and 3, separation of the principle component corresponding to TSPO ligand treatment (0 hour) and TSPO ligand treatment (4 hours) was significantly inhibited in the group treated with TSPO ligand (QC) were gathered. Therefore, the stability was analyzed without any change of sensitivity, change of retention time, and change of mass accuracy during analysis, and reliability was secured.

As a result of analyzing a loading plot for each group of the experimental group treated with TSPO ligand PK11195 and the control group not treated with PK11195 in each of the aqueous solution layer and the organic solvent layer, as shown in FIGS. 4 and 5, The points show that after treatment with PK11195, there is a significant change in the metabolite depending on the role of TSPO in both aqueous and organic solvent layers.

Finally, in step (d), the expression of the TSPO ligand is increased or decreased by the TSPO ligand through comparison of the metabolites contained in the cell extracts not treated with the TSPO ligand and the metabolites contained in the cell extract treated with the TSPO ligand Which is a step for selecting metabolites.

In one embodiment of the present invention, the significant difference between the control group treated with PK11195 and the treated group treated with PK11195 in the aqueous solution layer or the organic solvent layer of A2 / 29 cell of brain hypothalamic cell was performed by unpaired t-test Clustering analysis using Heat map analysis technique was performed.

FIG. 6 is a heat map for confirming a significant difference between two groups and clustering between metabolites. When the degree of metabolism was compared before TSPO ligand treatment (0 hour) and after 4 hours of TSPO ligand treatment, The area marked in red means that the amount of metabolism is decreased, and the area marked in green means that the amount of metabolism is increased. Among the total metabolites, 723 groups were found in the aqueous solution layer and 2233 in the organic solvent layer when the fold change cut-off between the two groups was 2.0 or more. Among them, candidates having the highest correlation with the role of TSPO 35 substances in the aqueous solution layer and 50 in the organic solvent layer were selected, and a total of 85 substances were selected as heat maps (Tables 1 to 3 and 6).

The heat map for confirming the significant difference between the two groups and the clustering between the metabolites was performed using the R software code shown in FIG.

Identification of the exact m / z of the metabolite from the mass spectrometer data is important for the correlation between the amount of metabolite and the role of TSPO, which is called metabolite identification. In the present invention, the entire pathway enrichment analysis was performed using a human Metabolome Database (HMDB) based on a metabolite having a high correlation with the role of TSPO, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) database using metabolic pathway information and interaction information that are related to cellular mechanisms of increased metabolism (enrich) metabolism Metabolites were selected as surrogate markers by evaluation of integrated correlation with the mechanism of TSPO in the candidate endogenous metabolites.

Metabolic networks can be said to be the task of completing biochemical and physical characterization of metabolism and physical processes in cells. This network is a chemical regulator of metabolism. Metabolic networks are not only a tool for modeling and studying metabolic processes, but also play a role in determining the relationship between the role of TSPO and the phenotype in the brain. Each node represents a specific metabolite, and the degree of correlation with other metabolites determines its location.

FIG. 8 is a schematic diagram of a hypothetical metabolic network that activates the action mechanism according to the role of TSPO. In FIG. 8, a node attribute is input in a hypothetical network model constructed using cytospec, We identified virtual subnetworks and identified the main modules that activate the mechanism of action according to the role of TSPO.

L-cysteine, Urocortisone, Tetracosahexaenoyl-L-arginine, N-Succinyl-L-arginine, Uracil, Phytyl diphosphate, S-Hydroxyphenylacetothiohydroximoyl- (2-chloro-5-trifluoromethyl) phenyl-1-phenyl-2-thiobiurea and triphenylphosphine oxide were increased by the addition of nicotinuric acid acid, CMP-N-glycoloylneuraminate, 1-hexadecanol, Homoisocitrate, N-palmitoyl tryptophan, N-stearoyl glutamic acid, N-acetyllactosamine, L-urobilin, 7a-hydroxy-3-oxocholest- The expression of metabolites such as phosphatidylethanolamine and triglyceride decreased

In addition, in one embodiment of the present invention, in order to clarify the relationship between the role of TSPO and the phenotype in the brain neuron, a standard product is prepared by using the characteristics of precursor ion and cleavage ion (product ion) Metabolites in vivo were identified. The identified metabolites are 6- (2-chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea, triphenylphosphine oxide and cortisone acetate. These metabolites showed that these metabolites increased in the ligand - treated group when TSPO ligand (PK11195) was treated with brain hypothalamic cell (A2 / 29 cell) for 4 hours.

9 to 11 show the MS / MS patterns of 6- (2-chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea, triphenylphosphine oxide and cortisone acetate identified in the standard and brain nerve cell samples , The retention time of 6- (2-chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea was 12 minutes and 327.0128 of m / , And collision energy of 40 eV, it can be confirmed that it is split into 174.9806 m / z and 98.9845 m / z.

As shown in FIGS. 10 and 11, the retention time of triphenylphosphine oxide was 11 minutes, and when the collision energy of m / z 279.0916, which is the parent ion, was 10, 20, and 40 eV, it was 201.0455 m / z The retention time of cortisone acetate was 17 minutes. It was confirmed that the collision energy of 403.1977 m / z of the parent ion was 10, 20, and 40 eV, and it was cleaved at 283.2603 m / z have.

Thus, when the TSPO ligand (PK11195) is treated with the TSPO ligand (PK11195) for 4 hours, metabolites associated with the role of TSPO can be presented. In the present invention, metabolites associated with the role of TSPO are L- L-oleandrosyl-oleandolide, C8-Dihydroceramide, bD-Glucopyranuronic acid, N2-succinyl-L-arginine -Arginine, Uracil, Phytyl diphosphate, S- (Hydroxyphenylacetothiohydroximoyl) -L-cysteine, Urocortisone, , Tetracosahexaenoyl-CoA, cortisone acetate, 16 (S) -HETE, Phytosphingosine, 6- (2-chloro-5 (trifluoromethyl) Phenyl-1-phenyl-2-thiobiurea, 6- (2-chloro-5- (CMP-N-glycoloylneuraminate), 1-hexadecanol, homoisocatecholate (1-hexadecanol), triphenylphosphine oxide, Homoisocitrate, N-palmitoyl tryptophan, N-stearoyl glutamic acid, N-acetyllactosamine, L-urobilin, 7a-hydroxy-3-oxocholest-4-ene-27-oic acid, phosphatidylethanolamine, and triglyceride. , Preferably 6- (2-chloro-5 (trifluoromethyl) phenyl) -1-phenyl-2-thiovaiurea 2-thiobiurea, triphenylphosphine oxide, or cortisone acetate.

The present invention may also include a TSPO metabolic control marker including the TSPO metabolite analyzed by the TSPO metabolism analysis method, and may further include a method of analyzing the expression level of the TSPO metabolite analyzed by the TSPO metabolism analysis method And measuring the TSPO activity.

According to a preferred embodiment of the present invention, the TSPO metabolite is selected from the group consisting of L-oleandrosyl-oleandolide, C8-Dihydroceramide, bD-glucopyranuronic acid (bD-Glucopyranuronic acid), N2-Succinyl-L-arginine, Uracil, Phytyl diphosphate, S- (hydrosiphenylacetoxyhydroximoyl) Cysteine, S-Hydroxyphenylacetothiohydroximoyl-L-cysteine, Urocortisone, Tetracosahexaenoyl-CoA, cortisone acetate, 16 (S) -HETE, Phytosphingosine, 6- (2-chloro-5 (trifluoromethyl) phenyl) -1-phenyl-2-thiovaiurea phenyl-2-thiobiurea, triphenylphosphine oxide, nicotinuric acid, CMP-N-glycoylinuraminate (CMP-Ng lycloylneuraminate, 1-hexadecanol, homoisocitrate, N-palmitoyl tryptophan, N-stearoyl glutamic acid, N- N-acetyllactosamine, L-urobilin, 7a-hydroxy-3-oxocholest-4-ene -27-oic acid, phosphatidylethanolamine, and triglyceride.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Before transfer protein (translocator protein, TSPO) ligand treatment and cell extracts can benefit

1-1: TSPO ligand treatment

In the present invention, the TSPO ligand was treated in the brain cells of the brain, the upper and lower brain cells, in order to identify the role of TSPO according to the metabolic change pattern after TSPO ligand treatment.

First, upper and lower brain cells are used in A2 / 29 cells (CELLutions Biosystems Inc., Adults Mouse Hypothalamus Cell line mHypoA-2/29). 0.5 × 10 ⁶ cells were inoculated on a 100 × 20 mm plate (cell culture container; BD Falcon, Cat # 353003, USA) and incubated for 48 hours at 37 ° C., 5% CO ₂ , DMEM (Sigma, Cat # D5796, USA) (Cat. No. C0424, Sigma, USA), which was then cultured and adhered in a medium containing 10% FBS (HyClone, Cat # SH30071.03, USA) + 1% Peniciliin (HyClone, Cat # SV30010, USA) ) Was treated at a concentration of 10 μM and cultured at 37 ° C and 5% CO ₂ for 4 hours. In addition, a group in which A2 / 29 cells were cultured under the same conditions as above without treating the TSPO ligand was used as a control.

1-2: Obtain cell extract

To obtain cell extracts containing metabolites in the control group of Example 1-1 and the PK11195 treatment group, the Bligh-Dyer extraction method (Bligh EG & Dyer WJ, Can. J. Biochem . Physiol. 37: 911, 1959).

First, in order to obtain about 2 mg of protein per cell sample, cells were cultured so as to be at least 0.5 × 10 ⁶ cells. In order to slow the metabolic rate in the cell extraction process, the cell culture container was placed on a wet ice , The medium was removed and the cells were washed twice with cold DPBS (Ca 2 -free, Mg 2 -free Phosphate Buffered Saline; Gibco, Cat. No. 14190-144, USA). Then, the cells were frozen with the cell culture container placed on dry ice, and then treated with 525 μl of a methanol: water (2: 0.8) mixed solvent (JT Baker, Deventer, Holland). Then, the cell extract was recovered using a scraper (Cat. No. 90040, SPL, Korea), and the cells were immediately stored at -80 ° C.

In order to isolate the TSPO metabolite-containing protein from the cell extract, the cell extract stored at -80 ° C was subjected to sonication, and 25 μl of reserpine (50-55- (CHCl ₃ ; 67-66-3, Fisher Sci., UK) was added to a 1: 2 mixture of chloroform: methanol: water : 0.8. Then, 1 part of chloroform was further added to make a ratio of chloroform: methanol: water at a ratio of 2: 2: 1.8, followed by centrifugation at 4 DEG C and 5000 rpm for 30 minutes. When centrifugation is carried out, it is separated into an aqueous phase (upper layer), a protein layer (protein layer), and an organic solvent phase (lower layer). The organic solvent layer in the lower layer is divided into halves And the other half was subjected to acid extraction.

Dry down process was performed for acid extraction and then redissolved in a mixed solvent of methanol and 1% formic acid (pH 2.0) at a ratio of 2: 0.8. Thereafter, 1 part of chloroform was added, and the ratio of chloroform: methanol: water was adjusted to 1: 2: 0.8. Then, 1 part of water was added, and the ratio of chloroform: methanol: water was 1: 2: 1.8. One part of chloroform was further added to make a final ratio of chloroform: methanol: water of 2: 2: 1.8, followed by centrifugation at 5000 rpm for 30 minutes at 4 ° C. After centrifugation, the organic solvent layer of the lower layer was divided into halves in a solvent separated into an aqueous solution layer, a protein layer and an organic solvent layer, and the half was stored and the other half was subjected to base extraction.

For the base extraction, a dry down process was carried out in the same manner as the above-mentioned acid extraction, and then methanol and 0.1% ammonium hydroxide (pH 9.0) were mixed using a mixed solvent at a ratio of 2: 0.8 And reused. Thereafter, 1 part of chloroform was added, and the ratio of chloroform: methanol: water was adjusted to 1: 2: 0.8. Then, 1 part of water was added, and the ratio of chloroform: methanol: water was 1: 2: 1.8. One part of chloroform was further added to make a final ratio of chloroform: methanol: water of 2: 2: 1.8, followed by centrifugation at 5000 rpm for 30 minutes at 4 ° C.

After centrifugation, saponified lipid was present in the precipitate of the bottom layer (organic solvent layer), which was finally extracted in a solvent separated into an aqueous solution layer, a protein layer and an organic solvent layer, so that it was not used for analysis and was not used And the previously extracted aqueous solution layer and the organic solvent layer were redissolved in a mixed solvent of methanol and mobile phase A (mixed solvent of water and 0.1% formic acid) at a ratio of 1: 1, and then analyzed

Metabolism profiling using UPLC / Q-TOFMS

The UPLC / MS analytical instrument was an Agilent Technologies 1290 Infinity UPLC (Agilent, USA) and an Agilent Technologies 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer Lt; / RTI >

RP columns composed of C18-bonded silica, which is a typical silica-bound, separable, medium-polar and non-polar metabolite for cellular extracts, And the present invention is also used for the analysis. The solvent and mobile phase used in the analysis are HPLC grade reagents. In gradient conditions, pH modifiers such as formic acid may be added as a method of reducing the pH of the mobile phase to inhibit ionization of weak organic acids and improve retention have. The mobile phase A was prepared by adding 0.1% formic acid to water in the UPLC condition and the mobile phase B was prepared by adding 0.1% formic acid to acetonitrile.

The linear gradient conditions were used at 90:10 (v / v) to 50:50 (v / v) for the initial 5 minutes to 10 minutes and 10:90 (v / v) , The composition was changed to 90:10 (v / v) for 30 minutes, and the same composition was maintained for the remaining 5 minutes. In the analysis, the column was maintained at 4 ° C using an Agilent poroshell 120 EC-C18 (2.7 μ, 2.1 × 100 mm) column, and the injection volume was adjusted to 10 μl at 0.3 ml / min.

When LC-MS-based metabolomic analysis is performed carefully, it can provide delicate, accurate, and reproducible results over a wide range of metabolites present in vivo. Due to the diversity of metabolites present in vivo, it is not always possible to obtain a comprehensive, systematic, high-quality analysis. The structural information obtained by the MS can be used to identify potential biomarkers for further study and hypothesis generation.

For the analysis of the metabolism, electrospray ionization (ESI) method was used for mass spectrometer ionization method under MS conditions. Jet stream method was used to gather atomized ionization and transfer it quickly. In the positive ionization mode, the gas temperature was set at 350 ° C, the skimmer voltage was set at 65V, and the fragmentation voltage was set at 175V.

Data were standardized from LC / MS analytical instruments and processed using Masshunter Mass profiler software (CA, USA) for identification of candidate substances.

Establish condition of quality control sample

In the present invention, TSPO ligand (PK11195) was treated with A2 / 29 cell of brain hypothalamus and then the global profile analysis of metabolites using UPLC / Q-TOFMS with excellent sensitivity, reproducibility, mass accuracy and data rate .

For this, optimal quality control conditions were established in brain hypothalamic cells. The quality control sample is made up of a subdivision of all samples and the integrated quality control sample from each sample represents the sample mean, including all analyzes.

The first five of the quality control samples were used to conduct conformance testing of the system at the beginning of the analysis and the remaining three were used at the middle and end of the sample batch to evaluate the performance of the analysis from beginning to end of the sample batch. The reproducibility of the analyzed quality control samples was confirmed by satisfying the same chromatographic pattern and the coefficient of variation of the internal reference material intensity. The internal reference material is reserpine.

As a result, the internal standard substance intensity coefficient value (CV,%) in the quality control sample was 5.76%, which satisfied the analytical criteria, and the chromatographic pattern and intensity ) Are as shown in Fig.

Principal component analysis pattern change, loading plot of the TSPO ligand treatment (loading plot) analysis

In the present invention, the aqueous solution layer and the organic solvent layer obtained in Example 1 were analyzed by the UPLC / Q-TOFMS analysis method of Example 2 in order to confirm the change in the principal component analysis pattern of each group according to the TSPO ligand treatment.

First, 5 samples per group were divided into control group and experimental group (group treated with TSPO ligand in cranial nerve cells). The sample obtained through sample pretreatment was classified into an aqueous phase (aqueous phase) using UPLC / Q- ) And an organic solvent phase. The raw data was analyzed by using Masshunter Mass profiler software (USA) for statistical analysis such as peak recognition, alignment and identification of candidate substances. Statistical analysis in metabolomics aims to simplify the patterns represented by complex mixtures of metabolites to identify groups of observational populations, or to identify the markers of specific populations (groups that have treated TSPO ligands).

4-1: Principal component analysis pattern change

First, the endogenous metabolite changes according to the metabolic change pattern and the role of TSPO in the control and experimental groups were observed in the brain neurons.

As a result, the separation of the principle component corresponding to the time before the TSPO ligand treatment (0 hour) and the time after the TSPO ligand treatment (4 hours), as shown in FIG. 2 (aqueous solution layer) In the group treated with TSPO ligand compared with the control group, a significant improvement was observed in the TSPO ligand group. As a result, quality control samples (QC) were gathered and it was analyzed stably without decrease in sensitivity, change in retention time, Respectively.

4-2: Analysis of loading plots

As shown in FIG. 4 (aqueous solution layer) and FIG. 5 (organic solvent layer), the loading plot of each group of the experimental group treated with TSPO ligand PK11195 and the control group not treated with PK11195 in the aqueous solution layer and the organic solvent layer, ), Square points show that there is a significant change in the metabolite depending on the role of TSPO in both aqueous and organic solvent layers after treatment with PK11195.

Clustering analysis using heat map analysis

The unpaired t-test was used to analyze the significant differences between the control group treated with PK11195 and the treated group treated with PK11195 in the aqueous solution layer or the organic solvent layer of the brain hypothalamic cell, A2 / 29 cell, Clustering analysis was performed using the analysis method. The p-values were calculated using the asymptotic analysis, benjamini Hochberg FDR method, and statistically significant at P-value <0.05.

[Table 1]

Changes in Metabolite Expression by TSPO Ligand Treatment

[Table 2]

Changes in Metabolite Expression by TSPO Ligand Treatment

[Table 3]

Changes in Metabolite Expression by TSPO Ligand Treatment

Among the total metabolites, 723 groups were found in the aqueous solution layer and 2233 in the organic solvent layer when the fold change cut-off between the two groups was 2.0 or more. Among them, candidates having the highest correlation with the role of TSPO 35 substances in the aqueous solution layer and 50 in the organic solvent layer were selected, and a total of 85 substances were selected as heat maps (Tables 1 to 3, FIG. 6).

The heat map for confirming the significant difference between the two groups and the clustering between the metabolites was performed using the R software code shown in FIG.

Analysis of hypothetical metabolite network activating mechanism of action according to TSPO role

In the present invention, the entire pathway enrichment analysis was performed using a human Metabolome Database (HMDB) based on a metabolite having a high correlation with the role of TSPO.

Metabolic networks can be said to be the task of completing biochemical and physical characterization of metabolism and physical processes in cells. This network is a chemical regulator of metabolism. Metabolic networks are not only a tool for modeling and studying metabolic processes, but also play a role in determining the relationship between the role of TSPO and the phenotype in the brain. Each node represents a specific metabolite, and the degree of correlation with other metabolites determines its location.

Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database operated by the Kyoto University of Japan, we analyzed pathway information and interaction information that corresponded to the enrichment of metabolism, and the role of TSPO And metabolic substances were selected as surrogate markers by evaluating the integrated correlation with the mechanism of TSPO in the candidate endogenous metabolites.

In the hypothetical network model constructed using Cytoscape, the node attribute is input to identify the subnetwork corresponding to each cell mechanism and the main module that activates the action mechanism according to the role of TSPO (Fig. 8).

As a result, as shown in FIG. 8, L-oleandrosyl-oleandolide, C8-Dihydroceramide, bD-Glucopyranuronic acid, N2-Succinyl-L- arginine, Uracil, Phytyl diphosphate, S- (Hydroxyphenylacetothiohydroximoyl) Metabolism of L-cysteine, Urocortisone, Tetracosahexaenoyl-CoA, cortisone acetate, 16 (S) -HETE, Phytosphingosine, 6- (2-chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea and Triphenylphosphine oxide N-stearoyl glutamic acid, N-acetyllactosamine, L-urobilin, 7a-hydroxy-3-oxocholest-4-ene, en-27-oic acid, phosphatidylethanolamine, and triglyceride, and the expression of the metabolites was decreased.

Metabolic verification and tandem mass spectrometry (MS / MS)

To investigate the relationship between the role of TSPO and the phenotype in brain neurons, in vivo metabolites were analyzed by MS / MS using the characteristics of precursor ion and cleavage ion (product ion) Respectively. The metabolites were obtained from brain hypothalamic cells (A2 / 29 cells) using 6- (2-chloro-5- trifluoromethyl) phenyl-1-phenyl-2-thiobiurea, triphenylphosphine oxide, and cortisone acetate. Of the TSPO ligand (PK11195) for 4 hours, these metabolites increase in the group treated with the ligand.

The MS / MS analysis showed a mass to charge ratio (m / z) in the range of 50 to 2000 with a full scan spectrum in a positive mode and a full scan with varying collision energy The pattern in which ions are sculpted was analyzed.

The impact energy is an important parameter for the production of the product ion in MS / MS, and the metabolites and standard products analyzed in the present invention gave three collision energies of 10 eV, 20 eV and 40 eV.

9, the retention time of 6- (2-chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea was 12 minutes and 327.0128 of m / When collision energy is given at 40 eV, it can be confirmed that it is split into 174.9806 m / z and 98.9845 m / z.

10 and 11, the retention time of triphenylphosphine oxide was 11 minutes. When the collision energy of m / z 279.0916, which is the parent ion, was given at 10, 20 and 40 eV, it was split at 201.0455 m / z . The retention time of cortisone acetate was 17 minutes. The collision energy of m / z 403.1977 at 10, 20, and 40 eV was found to be 283.2603 m / z.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (1)

(a) treating brain hypothalamic cells with a TSPO ligand;
(b) separating cell extracts containing TSPO metabolites from the brain hypothalamus cells prepared in step (a);
(c) analyzing TSPO metabolism of the cell extract prepared in step (b) by LC / Q-TOFMS; And
(d) one or more TSPO metabolites selected from the group consisting of (i) below are increased in comparison with the TSPO ligand-treated experimental group, and one or more TSPO metabolites selected from the group consisting of A method for screening a TSPO ligand comprising the step of determining a ligand binding to TSPO when shedding is decreased,
(i) L-oleandrosyl-oleandolide, C8-Dihydroceramide, bD-Glucopyranuronic acid, N2-succinyl- L-arginine, Uracil, Phytyl diphosphate, S- (Hydroxyphenylacetothiohydroximoyl) -L (S- (Hydroxyphenylacetothiohydroximoyl) -L -cysteine, Urocortisone, Tetracosahexaenoyl-CoA, cortisone acetate, 16 (S) -HETE, Phytosphingosine, 6- (2- Chloro-5- (trifluoromethyl) phenyl) -1-phenyl-2-thiobiurea), triphenylphosphine Triphenylphosphine oxide,
(ii) at least one compound selected from the group consisting of nicotinuric acid, CMP-N-glycoloylneuraminate, 1-hexadecanol, homoisocitrate, N-palmitoyl tryptophan, N-stearoyl glutamic acid, N-acetyllactosamine, L-urobilin, 7a-hydroxy-3 4-en-27-oic acid, phosphatidylethanolamine and triglyceride.

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