JP2018194299A - Screening method of glycolytic metabolic control substance and glycolytic metabolic regulator - Google Patents

Abstract

To provide a novel screening method capable of finding selective control substances that can specifically control glycolytic metabolism in cancer and selective control substances that can specifically control the glycolytic metabolism of cells and tissues whose aging is desired to be suppressed, or glycolytic metabolic control substances, and also to provide glycolytic metabolic regulators found by the method thereof.SOLUTION: The present invention provides a screening method of glycolytic metabolic substances which are test substances being evaluated to be controlling glycolytic metabolism when inhibiting or promoting the binding between PGAM and Chk1. Specifically, in the screening method of the present invention, the amount of binding between PGAM and Chk1 is measured in the presence and absence of a test substance, and the presence or absence of glycolytic metabolic control activity of the test substance is determined from the ratio of the binding amount in each case.SELECTED DRAWING: None

Description

  The present invention relates to a method for screening a glycolytic metabolism controlling substance and a glycolytic metabolism controlling agent.

  Glycolytic metabolism is a general term for metabolic processes that synthesize high-energy molecules called ATP by degrading glucose taken up by cells. There are two pathways in glycolytic metabolism: the process of synthesizing lactic acid from its intermediate product, pyruvate, and the process of oxidative phosphorylation in mitochondria. The former is called anaerobic glycolytic metabolism because oxygen respiration is not required, and the latter is called aerobic glycolytic metabolism because ATP is synthesized by mitochondrial oxygen respiration. This glycolytic metabolism plays an important role in energy metabolism in most normal cells and is indispensable for an individual to live. Therefore, abnormalities in glycolytic metabolism are closely related to human diseases.

  A decrease in glycolytic metabolism causes neurodegenerative diseases, decreased cardiac function, diabetes, muscle atrophy, anemia, and the like, while several pathological conditions in which glycolytic metabolism is enhanced are known. Examples of the pathological condition in which glycolytic metabolism is enhanced include cancer, ischemia (reduction in blood flow), local inflammation, and the like, but particularly in many cancer cells, glycolytic metabolism is enhanced (Warburg effect; Non-Patent Document 1), which is known as one of the cell biological characteristics of cancer. In fact, in current clinical settings, using this property, FDG-PET is useful for evaluating the size of tumors in the patient's body and the presence or absence of metastasis, and is widely spread.

  On the other hand, in cell aging research, it became clear that even young cells were aged by various stresses such as oxidative stress and DNA damage from around 1997, and such aging has come to be known as “stress aging”. . The stress that induces stress aging may be collectively referred to as “stress aging signal”. Cancer cells have been re-recognized as cells that are refractory to “stress aging signals” and are less susceptible to stress aging. The present inventors have been engaged in stress aging research since around 2001, and reported that when glycolytic metabolism is enhanced, stress aging becomes difficult (see Non-Patent Document 2). That is, it has been found that increased glycolytic metabolism can be one of the causes of cell canceration by reducing the responsiveness to stress aging signals. The present inventors have also found that one of the main reasons is attenuation of mitochondrial oxidative stress (see Non-Patent Document 3), and conversely, when glycolytic metabolism decreases, cell senescence is caused. (Refer nonpatent literature 2). Therefore, it was concluded that an important factor for converting cells from “aging” to “canceration” or from “canceration” to “aging” is the increase or decrease in glycolytic metabolism.

  The present inventors have found phosphoglycerate mutase (PGAM) as a glycolytic metabolic enzyme responsible for contact between cell aging and canceration as described above (see Non-Patent Document 2). The glycolytic metabolic pathway consists of a sequential reaction of 10 glycolytic enzymes, and PGAM catalyzes the conversion reaction from 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) as its eighth enzyme. The present inventors have found that by strongly expressing PGAM in a cell, an effect of enhancing glycolysis and reducing oxidative stress is induced, and the cell becomes difficult to age, whereas inactivation of PGAM induces stress aging. (Refer nonpatent literature 2).

  In the conventional interpretation, PFK and GAPDH that are subject to allosteric control are known as rate-limiting enzymes in glycolytic metabolic pathways, and PGAM was considered not to be a rate-limiting enzyme. Therefore, the importance of PGAM in glycolytic metabolism remained unknown. Furthermore, HIF-1 has been reported as a transcription factor that enhances glycolytic metabolism under hypoxia, that is, in an environment where mitochondrial oxygen respiration is not possible. In observation with HIF-1 knockout cells, HIF-1 It has been clarified that PGAM is the only exception glycolytic enzyme that is not controlled by However, the upstream factor of PGAM remained a mystery. Recently, the present inventors have worked on elucidating the upstream regulatory mechanism of PGAM and found that activation of stress aging signal promotes ubiquitination-dependent proteolysis of PGAM. It has also been found that the ubiquitin ligase enzyme responsible for ubiquitination of PGAM is the oncogene Mdm2 (see Non-Patent Document 4). Thus, it has been reached that the reason why PGAM is not controlled by the transcription factor HIF-1 is that post-transcriptional control (in this case ubiquitination) is more important. At the same time, PGAM is an unstable protein that is susceptible to ubiquitination due to stress, and it was suggested that the increase or decrease in the amount of PGAM protein may affect the overall glycolytic metabolism. In fact, it has also been clarified that when a mutant in which PGAM is difficult to undergo ubiquitination is produced, glycolytic metabolism is easily increased and carcinogenesis is likely to occur (see Non-Patent Document 4). As described above, the present inventors have found the importance of the glycolytic enzyme PGAM, which has not been conventionally known, in aging and canceration.

Warburg O. , Science, 1956, 124: p.269-270. H. Kondoh et al. , Cancer Research, 2005, 65 (1), p. 177-185 h. Kondoh et al. Antioxidid Redox Signal, 2007, 9 (3), p. 293-299 T.A. Mikawa et al. J. et al. Cell Biol. , 2014, 204 (5), p. 729-745

  As described above, glycolytic metabolism has been known to be enhanced in cancer for a long time, and the possibility of a target for anticancer agents has been pointed out. In fact, glycolytic inhibitors can suppress the growth of many cancer cells, but practical therapeutic applications are still halfway. The reason is that glycolytic metabolism has essential physiological functions even in normal cells, and mere metabolic inhibition has the risk of causing serious side effects, and it is difficult to establish the effectiveness as a cancer treatment. In cancer treatment targeting glycolysis, it is essential to overcome this problem. For example, it is possible to specifically inhibit glycolysis metabolism in cancer while preserving glycolysis metabolism in normal cells as much as possible. Discovery of such selective inhibitors is desired.

  Therefore, the present invention provides a selective control substance that can specifically control glycolytic metabolism in cancer, and a selective control that can specifically control glycolytic metabolism of cells and tissues in which aging is to be suppressed. It is an object to provide a novel screening method capable of finding a substance, that is, a glycolytic metabolic control substance. In addition, this screening method enables new solutions such as substances that specifically inhibit glycolytic metabolism in cancer and substances that exhibit anti-aging effects (anti-aging effects) in cells and tissues where cell aging is to be suppressed. The purpose is to find a sugar metabolism control substance.

As a result of diligent research to solve the above problems, the present inventors have found that PGAM and Chk1 bind in cells, and that these physical bonds play an important role in glycolytic metabolism, It has been found that the glycolysis can be controlled by controlling these physical bonds, and the present invention has been completed. That is, the present invention is as follows.
[1] Screening for a glycolytic metabolism control substance that evaluates that the test substance is a substance that controls glycolytic metabolism when the test substance inhibits or promotes the binding between PGAM and Chk1 Method.
[2] Measure the binding amount of PGAM and Chk1 in the presence and absence of the test substance, and determine the presence or absence of glycolytic metabolic control activity of the test substance from the ratio of the binding amount in each case The screening method according to [1], wherein:
[3] The screening method according to [1] or [2], comprising the following steps;
1) a step of measuring the amount of binding between PGAM and Chk1 when a test substance is added to a sample containing PGAM and Chk1;
2) a step of measuring the amount of binding between PGAM and Chk1 when no test substance is added, and 3) a step of comparing the measurement value of step 1) with the measurement value of step 2).
[4] The screening method according to any one of [1] to [3], wherein PGAM and Chk1 are labeled with a tag capable of binding to each other.
[5] The screening method according to any one of [1] to [4], wherein the glycolytic metabolism regulator is used as an anticancer agent.
[6] The screening method according to any one of [1] to [4], wherein the glycolytic regulator is used in an anti-aging composition.
[7] A glycolytic metabolism regulator comprising a substance that inhibits or promotes the binding between PGAM and Chk1.
[8] A substance that inhibits or promotes the binding between PGAM and Chk1,
The glycolysis according to [7], which is at least one selected from the group consisting of i) an antisense nucleic acid or siRNA against PGAM gene or Chk1 gene, ii) an antibody specific for PGAM or Chk1, and iii) natrin System metabolism regulator.
[9] The glycolytic metabolism control agent according to [7] or [8], which is used as an anticancer agent.
[10] The glycolytic metabolism control agent according to [7] or [8], which is used in an anti-aging composition.

  The screening method for glycolytic metabolism control substances of the present invention is based on the fact that the present inventors have first shown that PGAM and Chk1 bind in cells and that these bonds play an important role in glycolytic metabolism. It became possible by having found. The cooperative glycolytic system control mechanism by PGAM and Chk1 does not involve the enzyme activity of PGAM, and physical binding is important. In the present invention, by targeting the function of PGAM different from such glycolytic enzyme activity, the original PGAM activity can be preserved, and the glycolytic system can be specifically identified by the effect of only PGAM-Chk1 binding control. It is possible to screen for substances that can be inhibited. Further, according to this method, a substance effective for symptoms and diseases that can be prevented, treated, and treated by controlling glycolytic metabolism can be found. For example, the glycolytic metabolism regulator found by the screening method of the present invention is effectively used for a novel anticancer agent and anti-aging composition.

FIG. 1 is a schematic diagram of gene design of a PGAM knockout mouse. FIG. 2 is a diagram showing the results of an IPGTT test for PGAM-TG mice. FIG. 3 is a diagram showing the results of an IPGTT test for PGAM hetero KO mice. FIG. 4 is a diagram showing a decrease in glycolytic mRNA expression in the skin tissue of PGAM hetero KO mice. FIG. 5 is a diagram showing the influence of PGAM siRNA treatment on glycolytic mRNA expression in cancer cell lines. FIG. 6 is a diagram showing the results of an inflammation induction test on the skin of PGAM-TG mice. FIG. 7 is a diagram showing the results of a malignant tumor induction test on the skin of PGAM-TG mice. FIG. 8 is a diagram showing the expression of glycolytic mRNA in malignant tumors formed on the skin of PGAM-TG mice. FIG. 9 shows phosphorylated p53 in PGAM-TG mice. FIG. 10 is a diagram showing that PGAM and Chk1 are physically coupled. FIG. 11 shows the effect of Chk1 inactivation or activation on glycolytic mRNA expression. FIG. 12 is a diagram showing the results of analyzing the protein phosphatase activity of PGAM. FIG. 13 is a diagram showing the glycolytic enhancement effect by Chk1 in PGAM-R90W mutant TG mice. FIG. 14 is a diagram schematically showing the Chk1 binding site and the Mdm2 binding site in the PGAM protein. FIG. 15 is a diagram showing the inhibitory effect of natrin on the binding of PGAM and Chk1. FIG. 16 is a diagram showing the effect of suppressing the expression of glycolytic mRNA by the treatment with natriline of cancer cell line H1299.

  Hereinafter, the method for screening a glycolytic metabolism controlling substance and the glycolytic metabolism controlling agent of the present invention will be described in detail.

<Screening method for glycolytic metabolism control substances>
In the method for screening a glycolytic metabolism controlling substance of the present invention, when the test substance inhibits or promotes the binding between PGAM and Chk1, the test substance is a substance that controls glycolytic metabolism. It is characterized by evaluating. The present invention has been made possible by the first discovery by the present inventors that PGAM and Chk1 bind in cells and that these bonds play an important role in glycolytic metabolism. It is.

  In the present invention, PGAM is phosphoglycerate mutase, an enzyme responsible for the eighth reaction among the sequential reactions of ten glycolytic enzymes in the glycolytic metabolic pathway. From 3-phosphoglycerate (3PG) It refers to an enzyme that catalyzes the conversion reaction to 2-phosphoglycerate (2PG). PGAM is a contact point between cell aging and canceration. There are two isoforms of PGAM, PGAM1 and PGAM2. Although PGAM1 and PGAM2 have a difference in tissue expression pattern, they have high homology in amino acid sequence and are considered to have the same functions and enzyme activities. The amino acid sequences of human PGAM1 and PGAM2 are shown in the sequence listing as SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the cDNA sequences are shown as SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

  In the present invention, Chk1 is a serine threonine protein kinase, which is a checkpoint kinase that plays a role in activation of DNA damage checkpoints in the G2 phase of the cell cycle. Chkl was found by the present inventors to play an important role in glycolytic metabolism. The amino acid sequence of human Chk1 is shown as SEQ ID NO: 5, and the cDNA sequence is shown as SEQ ID NO: 6 in the sequence listing.

  The glycolytic metabolism in the present invention is a general term for metabolic processes in which ATP is synthesized by degrading glucose taken up by cells. In glycolytic metabolism, there are two pathways: a pathway that breaks down glucose into its intermediate product, pyruvate, and synthesizes lactic acid, and a pathway that leads to oxidative phosphorylation in mitochondria. The former is called anaerobic glycolytic metabolism because oxygen respiration is not required, and the latter is called aerobic glycolytic metabolism because ATP is synthesized by mitochondrial oxygen respiration. This glycolytic metabolism plays an important role in energy metabolism in most normal cells and is indispensable for an individual to live.

  The glycolytic metabolism controlling substance in the present invention refers to a substance that suppresses or enhances glycolytic metabolism. It has been found by the present inventors that the binding of PGAM and Chk1 plays an important role in the process of glycolytic metabolism. A substance that affects the binding between PGAM and Chk1. The glycolytic metabolism controlling substance in the present invention may be one that suppresses or inhibits the binding between PGAM and Chk1, or may enhance it. The glycolytic metabolism control substance in the present invention may be a substance that binds to PGAM and / or Chk1 itself and affects the binding between PGAM and Chk1, or affects the expression of PGAM and / or Chk1. The substance which controls the coupling | bonding of PGAM and Chk1 by this may be sufficient.

  Specifically, the screening method of the present invention measures the amount of binding between PGAM and Chk1 in the presence and absence of a test substance, and determines the glycolytic metabolism of the test substance from the ratio of both binding quantities. It is characterized by determining the presence or absence of control activity. Such a screening method of the present invention preferably includes the following steps 1) to 3).

1) Step of measuring the binding amount of PGAM and Chk1 when a test substance is added to a sample containing PGAM and Chk1 2) Measuring the binding amount of PGAM and Chk1 when the test substance is not added Step 3) A step of comparing the measured value of the above step 1) with the measured value of the above step 2)

  In step 1) of the screening method of the present invention, first, a test substance is added to a sample containing PGAM and Chk1, thereby bringing the test substance into contact with PGAM and / or Chk1.

  PGAM and Chk1 used in the screening method of the present invention are as described above. The PGAM and Chk1 used in the screening method of the present invention include proteins functionally equivalent to the above-mentioned known PGAM and Chk1. Examples of such proteins include mutants of PGAM and Chk1, alleles, variants, homologs, partial peptides of PGAM and Chk1, fusion proteins with other proteins, those labeled with tags, etc. It is not limited to.

  The mutants of PGAM and Chk1 in the present invention are naturally-occurring proteins comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described above, Mention may be made of proteins functionally equivalent to proteins consisting of amino acid sequences. A protein that is encoded by a naturally-derived DNA that hybridizes under stringent conditions with a DNA comprising the above-described base sequence, and that is functionally equivalent to a protein comprising the amino acid sequence described above, is also PGAM. And Chk1 mutants.

  In the present invention, the number of amino acids to be mutated is not particularly limited, but is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids). The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), a sulfur atom-containing side Amino acids having chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by another amino acid sequence of one amino acid sequence to a certain amino acid sequence maintains its biological activity. ing.

  In the present invention, functionally equivalent means that the target protein has a biological function or biochemical function equivalent to PGAM or Chk1. The biological function or biochemical function of PGAM is a function as a glycolytic enzyme, specifically, a function that catalyzes a conversion reaction from 3-phosphoglycerate (3PG) to 2-phosphophosphorate (2PG). Can be mentioned. In addition, examples of the biological function and biochemical function of Chk1 include a function as a serine threonine protein kinase.

  In order to prepare DNA encoding a protein functionally equivalent to the target protein, methods well known to those skilled in the art include methods using hybridization technology and polymerase chain reaction (PCR) technology. That is, for those skilled in the art, DNA having a high homology with PGAM or Chk1 is simply obtained using the base sequence of PGAM or Chk1 or a part thereof as a probe and using an oligonucleotide that specifically hybridizes to PGAM or Chk1 as a primer. Separation is something that can usually be done. Thus, DNA encoding a protein having a function equivalent to PGAM or Chk1 that can be isolated by a hybridization technique or a PCR technique is also included in the DNA of the present invention.

  The tag-labeled PGAM and Chk1 used in the screening method of the present invention are preferably those in which an antibody against them or a resin capable of specifically binding exists, for example, FLAG tag, HA tag, Myc Tags, His tags, GST tags and the like.

  The biological species from which PGAM and Chk1 used in the screening method of the present invention are derived is not limited to a specific biological species. For example, human, monkey, mouse, rat, guinea pig, pig, cow, yeast, insect and the like can be mentioned.

  The state of PGAM and Chk1 used in the screening method of the present invention is not particularly limited, and may be, for example, a purified state, a state expressed in a cell, a state expressed in a cell extract, or the like.

  The test substance in the present invention is not particularly limited. For example, a single substance such as a natural compound, an organic compound, an inorganic compound, a nucleic acid, a protein (including an antibody), a peptide; a compound library, a nucleic acid library, and a peptide Library, gene library expression product; cell extract, cell culture supernatant, fermented microbial product, marine organism extract, plant extract, prokaryotic cell extract, eukaryotic single cell extract or animal cell extract And the like. Further, the test substance in the present invention may be a mixture thereof. In addition, these test substances can be labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label.

  In the present invention, the contact is performed according to the states of PGAM and Chk1. For example, if PGAM and Chk1 are in a purified state, it can be performed by adding a test substance to the purified sample. In addition, if it is expressed in a cell or in a cell extract, it is added by adding a test substance to the cell culture solution or cell extract, or by direct administration to a laboratory animal, respectively. It can be carried out. When the test substance is a protein, for example, a vector containing DNA encoding the protein is introduced into a cell expressing PGAM and Chk1, or the vector is introduced into a cell extract expressing PGAM and Chk1. It is also possible to carry out by adding. Further, for example, a two-hybrid method using yeast or animal cells can be used.

  In the present invention, PGAM and / or Chk1 may be brought into contact with the test substance by adding the test substance to a sample containing PGAM and Chk1. Moreover, after adding a test substance to the sample containing either PGAM or Chk1, you may make the other and the test substance contact each other which was not included.

  In the present invention, next, the amount of binding between PGAM and Chk1 is measured. In step 2) of the screening method of the present invention, the amount of binding between PGAM and Chk1 when no test substance is added to the sample containing PGAM and Chk1 is measured. Specifically, the amount of binding between PGAM and Chk1 when the test substance is added (when contacted) and when the test substance is not added (when not contacted) is measured, and the screening method of the present invention In step 3), these values are compared. When the test substance is added, if the amount of binding between PGAM and Chk1 is increased or decreased as compared with the case where the test substance is not added, this test substance controls the binding between PGAM and Chk1. It can be judged that there is. Then, it can be determined that a test substance having an effect of increasing the amount of binding between PGAM and Chk1 has an effect of enhancing glycolytic metabolism. On the other hand, a test substance having an effect of reducing the amount of binding between PGAM and Chk1 can be determined to have an effect of suppressing or inhibiting glycolytic metabolism.

  Specific examples of the method for measuring the amount of binding between PGAM and Chk1 include the following methods. PGAM2-FLAG protein is immunoprecipitated from a protein extract of cells co-expressed with PGAM2-FLAG and Chk1-myc-His expression vectors using a FLAG tag antibody conjugated to protein G agarose. At this time, Chk1-myc-His protein that binds to PGAM2-FLAG protein and co-precipitates is detected and quantified by Western blotting. Alternatively, Chk1-myc-His protein is precipitated from the same protein extract using Ni-NTA agarose beads. At this time, the PGAM2-FLAG protein that binds to the Chk1-myc-His protein and coprecipitates is detected and quantified by Western blotting.

  As a method for measuring the amount of binding between PGAM and Chk1, for example, the following method using a Nanobit system can be used. In Nanobit, a large subunit and a small subunit of a photoprotein produced based on luciferase are attached to PGAM and Chk1, respectively, as tags. These tags have a structure that can bind to each other. Each subunit does not emit light by itself, but emits a luminescent signal when the large subunit and the small subunit associate with each other by the binding of PGAM and Chk1 to form a complete photoprotein. By measuring the intensity of the luminescence signal at this time, the amount of binding can be measured with high sensitivity and high quantitativeness.

<Glycolytic metabolism regulator>
The glycolytic metabolism controlling agent in the present invention contains a glycolytic metabolism controlling substance found by the above-described screening method of the present invention as an active ingredient.

Examples of the glycolytic metabolism regulator found by the screening method of the present invention include:
i) antisense nucleic acid or siRNA against PGAM gene or Chk1 gene, ii) an antibody specific for PGAM or Chk1, and iii) a compound such as natrin.

(Antisense nucleic acid or siRNA against PGAM gene or Chk1 gene)
The antisense nucleic acid in the present invention is an antisense nucleic acid complementary to a transcription product of DNA encoding PGAM or Chk1. It is preferred that the antisense nucleic acid is an antisense nucleic acid that inhibits the transcription, splicing or translation process and suppresses the expression of the target gene.

  As an aspect of the antisense sequence used in the present invention, if an antisense sequence complementary to the untranslated region near the 5 ′ end of PGAM or Chk1 mRNA is designed, it is effective in inhibiting gene translation. Conceivable. However, sequences complementary to the coding region or the 3 'untranslated region can also be used. Thus, not only a translation region of a gene but also a sequence complementary to an untranslated region can be used. Thus, not only the translation region of a gene but also the nucleic acid containing the antisense sequence of the sequence of an untranslated region is included in the antisense nucleic acid used in the present invention. The antisense nucleic acid preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. The length of the antisense RNA that effectively inhibits the expression of the target gene using the antisense sequence is not particularly limited.

  The siRNA in the present invention means a double-stranded RNA consisting of short strands in a range not toxic to cells, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 21 base pairs. It can be 30 base pairs. ShRNA is RNA in which single-stranded RNA constitutes a double strand through a hairpin structure.

  The siRNA need not be completely identical to the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more sequence homology.

  The part of double-stranded RNA in which RNAs in siRNA paired with each other is not limited to a perfect pair, but includes mismatches (corresponding bases are not complementary), bulges (no bases corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be contained in the double-stranded RNA region where the RNAs in the dsRNA pair.

(An antibody specific for PGAM or Chk1)
The antibody specific for PGAM or Chk1 in the present invention can be obtained as a polyclonal antibody or a monoclonal antibody using known means. The origin of the antibody used in the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a human-derived antibody. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody inhibits the binding between PGAM and Chk1 by binding to PGAM or Chk1.

  Antibody-producing hybridomas can be basically produced using known techniques. That is, using PGAM or Chk1 as a sensitizing antigen, immunizing it according to a normal immunization method, fusing the resulting immune cells with a known parent cell by a normal cell fusion method, It can be produced by screening monoclonal antibody-producing cells.

  As a method for obtaining a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a normal method and obtained as a culture supernatant, or the hybridoma is administered to a mammal compatible therewith to proliferate, A method of obtaining ascites is employed. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies. Further, as a monoclonal antibody, a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing it into a host, and producing it using a gene recombination technique can be used.

  In the present invention, a recombinant antibody that has been artificially modified for the purpose of reducing xenoantigenicity to humans, such as a chimeric antibody, a humanized antibody, a human antibody, etc. Can be used. These modified antibodies can be produced using methods known to those skilled in the art.

(Natrine and other compounds)
When tested on a compound library using the screening method of the present invention, it was found that natrin significantly inhibited the binding of PGAM to Chk1. When this natrin is added to H1299 cells to confirm whether it affects glycolytic metabolism, glycolytic enzyme expression is markedly reduced and functions in a suppressive manner against glycolytic metabolism. It became clear. That is, natrin is a glycolytic metabolism controlling substance, and a composition containing it can be used effectively as a glycolytic metabolism controlling agent.

  Similarly, single substances such as natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins, peptides; compound libraries, nucleic acid libraries, peptide libraries, gene library expression products; cell extracts, cell cultures PGAM and Chk1 can be obtained by subjecting Qing, fermenting microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extract extracts to the screening method of the present invention. It is possible to find a compound that affects the binding of glycan, that is, a compound that affects glycolytic metabolism.

  In addition to the above active ingredients, the glycolytic metabolism control agent of the present invention includes one or two or more carriers, excipients, bindings, which are pharmacologically or physiologically acceptable in terms of formulation, as other optional ingredients. An agent, a disintegrant, a lubricant, a dispersant, a surfactant, a plasticizer, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, a bacteria inhibitor, and the like may be contained. In addition, other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, saccharides such as amino acids, polysaccharides and monosaccharides, carbohydrates, and sugar alcohols may be included. Furthermore, you may contain the other active ingredient as needed.

  The glycolytic metabolism control agent of the present invention can be produced by any conventionally known method in the fields of pharmaceuticals, quasi drugs, foods and the like. In the production process, the active ingredient and other optional ingredients may be added and mixed by any known method.

  In the case of an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and appropriate dissolution You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc. If necessary, it may further contain a diluent, a solubilizer, a pH adjuster, a soothing agent, a sulfur-containing reducing agent, an antioxidant and the like.

  Moreover, it is encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or used as a colloid drug delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.). You can also. Furthermore, a method of using a drug as a sustained-release drug is also known and can be applied to the present invention.

  When the glycolytic metabolism control agent of the present invention is used as a medicine for humans or other animals, in addition to administering these substances themselves to patients, they are formulated and administered by a known pharmaceutical method. It is also possible. When formulating, the above-described pharmaceutically acceptable ingredients may be added.

  All drugs in the present invention can be administered in the form of pharmaceuticals, and can be administered systemically or locally orally or parenterally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc. can be selected, and the administration method should be selected appropriately depending on the age and symptoms of the patient Can do. The effective dose is selected in the range of 0.001 mg to 100 mg per kg of body weight per time. Alternatively, a dose of 0.1 to 1000 mg, preferably 0.1 to 50 mg per patient can be selected. As specific examples, 0.1 mg to 40 mg, preferably 1 mg to 20 mg per 1 kg body weight per month (4 weeks) is divided into 1 to several times, for example, 2 times / week, 1 time / week, It is administered by a method such as intravenous injection such as infusion, subcutaneous injection, oral administration, etc., on a dosing schedule such as once / two weeks or once / 4 weeks. The administration schedule is 2 times / week or once / week to once / 2 weeks, once / 3 weeks, once / 4 weeks, etc. while observing the state after administration and observing the trend of blood test values It is also possible to adjust such as extending the administration interval.

  The present invention will be specifically described in the following examples, but the present invention is not limited to the examples.

1. Analysis of PGAM model mice (peritoneal glucose tolerance test)
In order to verify the in vivo effect of PGAM, PGAM model mice (systemic expression type transgenic mice (TG) and knockout mice (cKO) mice) were analyzed. FIG. 1 shows a schematic diagram of gene design of a PGAM knockout mouse. Global Pgam1 heterozygous KO mice were produced by crossing CAG-Cre transgenic mice with Pgam1 flox / + mice, and after crossing of Pgam1 heterozygous KO mice, homozygous and heterozygous KO mice survived. Investigate possible offspring. Among the 155 offspring obtained, the ratio of wild type: heterozygote KO: isozygous KO was 52: 103: 0 (Table 1). Therefore, Pgam1 homozygous KO mice are embryonic lethal and are not observed at embryonic day 13.5 (E13.5). The initial lethality of Pgam1 − / − mice is similar to that observed in homozygous KO mice with other glycolytic enzymes. Since PGAM homo KO mice were embryonic lethal (Table 1), the following analysis was performed with hetero KO mice.

  Considering that PGAM is a glycolytic enzyme, in order to investigate the presence or absence of diabetes or abnormal sugar metabolism, PGAM-TG mice, PGAM hetero KO mice and wild type mice were used to perform an intraperitoneal glucose tolerance test (IPGTT). ) That is, after fasting for 16 hours for each mouse, glucose was intraperitoneally administered at 1.5 g / kg body weight, blood was collected after 0, 15, 30, 60, and 120 minutes, and the glucose level in whole blood was analyzed. As a result, in vivo glucose metabolism parameters were almost normal in both PGAM-TG mice (FIG. 2) and PGAM hetero KO mice (FIG. 3).

2. Analysis of PGAM model mice; tissue-specific analysis of mRNA (glycolytic mRNA) of substances related to glycolytic metabolism Glycolytic mRNA profile by organ using PGAM model mice (systemic expression type TG and cKO mice) Was analyzed. In PGAM-TG mice, the examined tissues / organs (skin, liver, kidney, muscle, white fat, lung, heart) were almost normal. On the other hand, in PGAM hetero knockout mice, glycolysis mRNA profile was normal in many organs (liver, kidney, muscle, white fat, lung, brain, heart), but in skin, Hk2, Gpi, Pfkm, A significant decrease in Tpi, Aldoa, Gapdh, Pgk1, and Pkm1, and a moderate decrease in Eno3 (p = 0.08) were found, indicating that the overall glycolytic mRNA was greatly reduced (Figure). 4).

3. Analysis of PGAM in cancer cultured cells; reduction of glycolytic mRNA by PGAM siRNA PGAM siRNA was used to inactivate PGAM in HeLa, H1299, and HSC1 (cancer cultured cells). That is, when each of HeLa, H1299, and HSC1 was transfected with PGAM siRNA, the expression of PGAM mRNA was suppressed by 95% or more. In these cells, a decrease in the mRNA profile of many glycolytic enzymes was observed (FIG. 5).

4). Inflammation induction test in skin of PGAM-TG mice As described above, glycolysis mRNA expression under normal conditions was normal in the skin of PGAM-TG mice. However, it was found that the skin of PGAM-TG mice is more susceptible to inflammation due to treatment with TPA drugs than wild type mice. That is, the ear of a PGAM-TG mouse was treated with 10 ng of TPA (12-O-tetradecanoylphorbol-13-acetate), and then the state of the ear was observed for 8 hours. A tinged change was shown (Figure 6). Further, when the degree of ear thickening was measured with a constant pressure thickness measuring instrument, it was found that the degree of ear thickening was more prominent in PGAM-TG mice than in wild-type mice, as shown in FIG.

5). Malignant tumor induction test in the skin of PGAM-TG mice The following chemical carcinogenesis experiments were performed on the skins of PGAM-TG mice and wild-type mice. That is, 100 μg of DMBA (7,12-dimethylbenz (a) anthracene) was applied to the skin of each mouse, and then 12.5 μg of TPA was applied twice a week for a total of 20 weeks. As a result, the total number of tumors that appeared by this treatment was slightly higher in PGAM-TG mice than in wild-type mice, but the tumor size was significantly larger in PGAM-TG mice (FIG. 7). In addition, all the skin tumors formed in wild type mice were benign papillomas, but large tumors (6 mm) appeared in PGAM-TG mice, and about 10% of all received pathologically malignant diagnosis. (Table 2). Furthermore, when glycolytic mRNA was compared in the formed tumor, a more significant enhancement was observed in TG mice. In conclusion, PGAM-TG mice are prone to skin cancer, and glycolytic mRNA enhancement (Warburg effect) was observed at that time (FIG. 8).

6). Phosphorylation of tumor suppressor gene p53 in PGAM-TG mice In order to verify the carcinogenic mechanism in PGAM-TG mice, attention was paid to the tumor suppressor gene p53. p53 is the most deleted (about 50%) tumor suppressor gene in human cancer, and is regulated by post-transcriptional modification (phosphorylation or ubiquitination). In PGAM-TG MEF cells, we found that phosphorylation of serine23, one of p53-activated phosphorylation, was reduced in the presence of carcinogenic stress due to the expression of constitutively active mutation Ras (Ras-G12V). (FIG. 9). Next, since phosphorylation of serine23 is carried by Chk1 kinase, the profile of Chk1 kinase was verified. As a result, in PGAM-TG MEF cells, the amount of Chk1 kinase protein and activated phosphorylation were not changed, but PGAM and Chk1 were found to be bound intracellularly (FIG. 10).

7). Binding of PGAM and Chk1 The effects of intracellular binding of PGAM and Chk1 on glycolytic metabolism were examined. It was confirmed that glycolysis mRNA was decreased by Chk1 inactivation (siRNA or inhibitor UCN01 treatment) as in PGAM inactivation (FIG. 11A). On the other hand, due to Chk1 activation, glycolytic mRNA was normal in wild-type cells, but enhanced glycolytic mRNA was observed in PGAM-TG cells (FIG. 11B). From the above, it was found that PGAM and Chk1 cooperatively control the glycolysis.

8). Contribution of enzyme activity to cooperative glycolytic regulation by PGAM and Chk1 An experiment was conducted to elucidate the molecular mechanism of cooperative glycolytic regulation by PGAM and Chk1. First, when the possibility that PGAM functions as protein phosphatase was verified, a negative result was obtained in the in vitro reaction (FIG. 12). FIG. 12 shows phosphorylated peptides used in the assay. ΛPPase was used as a positive control for protein phosphatase. Specifically, the phosphorylated peptide was reacted with PGAM recombinant protein or λPPase, and phosphoric acid dissociated from the phosphorylated peptide was measured. As shown in FIG. 12, phosphatase activity was not observed in the PGAM recombinant protein.

  Next, PGAM-R90W mutant TG mice were created by suspecting the involvement of PGAM glycolytic metabolic enzyme activity. The PGAM-R90W mutation is a mutation that has already been reported to inactivate PGAM activity in human patient families (Tsujino et al. Muscle Nerple Suppl 1995). Contrary to expectations, even in PGAM-R90W mutant MEF cells, enhanced glycolysis was observed when Chk1 was activated by introducing a Chk1 expression vector (FIG. 13).

9. Contribution of physical binding to cooperative glycolytic regulation by PGAM and Chk1 The possibility that the physical binding of PGAM and Chk1 is important for promoting glycolytic metabolism was examined. We noticed that there is a natrin target sequence (Nicholson et al. Cell Signal 2014) on the N-terminal side of PGAM that binds to Chk1 (FIG. 14). Natrin is a kind of anticancer agent developed as a chemical substance that inhibits the binding between p53 and Mdm2. MEF cells expressing PGAM-FLAG and Chk1-myc-his are treated with natrin, and Chk1-myc-his is recovered from the protein extract using Ni-NTA beads, and the amount of bound PGAM-FLAG is examined. As a result, it was observed that the binding of PGAM and Chk1 was inhibited by natrin treatment (FIG. 15). At the same time, it was confirmed that glycolytic mRNA expression was significantly reduced in the cancer cell H1299 by the treatment with natrin (FIG. 16). From the above, it was concluded that PGAM phosphatase activity and glycolytic metabolic enzyme activity are not involved in cooperative enhancement of glycolysis by PGAM and Chk1, and physical binding is important. Further, by using the screening method of the present invention, it was possible to find natrin as a glycolytic metabolism controlling substance capable of specifically controlling glycolytic metabolism.

10. Screening of glycolytic metabolic control substances using the binding of PGAM and Chk1 as an indicator Screening using the binding of PGAM and Chk1 as an indicator is performed by the following method using the Nanobit system. In Nanobit, a large subunit and a small subunit of a photoprotein produced based on luciferase are attached to PGAM and Chk1, respectively, as tags. Each subunit does not emit light by itself, but emits a luminescent signal when the large subunit and the small subunit associate with each other by the binding of PGAM and Chk1 to form a complete photoprotein. At this time, the luminescence signal decreases in the presence of a substance that inhibits the binding between PGAM and Chk1, such as natrin. Therefore, it is possible to screen for compounds that inhibit the binding of PGAM and Chk1 from the drug library using the decrease in the luminescence signal as an index. It is also possible to screen for compounds that enhance this luminescence signal, that is, compounds that promote the binding between PGAM and Chk1.

  In addition, screening using PGAM and Chk1 binding as an index was performed by collecting and binding Chk1-myc-his using Ni-NTA beads from a protein extract of cultured cells expressing PGAM-FLAG and Chk1-myc-his. The amount of PGAM-FLAG is evaluated by Western blotting. By treating a candidate compound with cells in culture, it is possible to screen for a binding control compound using as an index the decrease or increase in the amount of PGAM-FLAG binding to Chk1-myc-his.

  The screening method for glycolytic metabolism control substances of the present invention is based on the fact that the present inventors have first shown that PGAM and Chk1 bind in cells and that these bonds play an important role in glycolytic metabolism. It became possible by having found. According to this method, a substance effective for symptoms and diseases that can be prevented, treated, or treated by controlling glycolytic metabolism can be found. For example, the glycolytic metabolism regulator found by the screening method of the present invention is effectively used for a novel anticancer agent and anti-aging composition.

Claims (10)

  A screening method for a glycolytic metabolism controlling substance, wherein when the test substance inhibits or promotes the binding between PGAM and Chk1, the test substance is evaluated as a substance that controls glycolytic metabolism.   Measuring the binding amount of PGAM and Chk1 in the presence and absence of the test substance, and determining the presence or absence of glycolytic metabolism control activity of the test substance from the ratio of the binding amount in each case The screening method according to claim 1, wherein the screening method is characterized. The screening method according to claim 1 or 2, comprising the following steps;
1) a step of measuring the amount of binding between PGAM and Chk1 when a test substance is added to a sample containing PGAM and Chk1;
2) a step of measuring the amount of binding between PGAM and Chk1 when no test substance is added, and 3) a step of comparing the measurement value of step 1) with the measurement value of step 2).   The screening method according to any one of claims 1 to 3, wherein PGAM and Chk1 are labeled with a tag capable of binding to each other.   The screening method according to any one of claims 1 to 4, wherein the glycolytic metabolism regulator is used as an anticancer agent.   The screening method according to any one of claims 1 to 4, wherein the glycolytic regulator is used in an anti-aging composition.   A glycolytic metabolism regulator comprising a substance that inhibits or promotes the binding between PGAM and Chk1. A substance that inhibits or promotes the binding between PGAM and Chk1,
The glycolysis according to claim 7, which is at least one selected from the group consisting of i) an antisense nucleic acid or siRNA against PGAM gene or Chk1 gene, ii) an antibody specific for PGAM or Chk1, and iii) natrin. System metabolism regulator.   The glycolytic metabolism control agent according to claim 7 or 8, which is used as an anticancer agent.   The glycolytic metabolism regulator according to claim 7 or 8, which is used in an anti-aging composition.

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