WO2014171206A1 - Nouvelle méthode de criblage de médicament contre le diabète - Google Patents

Nouvelle méthode de criblage de médicament contre le diabète Download PDF

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WO2014171206A1
WO2014171206A1 PCT/JP2014/055528 JP2014055528W WO2014171206A1 WO 2014171206 A1 WO2014171206 A1 WO 2014171206A1 JP 2014055528 W JP2014055528 W JP 2014055528W WO 2014171206 A1 WO2014171206 A1 WO 2014171206A1
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sir
concentration
insulin receptor
glucose
screening
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Japanese (ja)
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智之 湯浅
洋介 蛯名
誠一 橋田
宗英 松久
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Yuasa Tomoyuki
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

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  • the present invention relates to a screening method for a therapeutic drug for diabetes having a novel mechanism. More specifically, the present invention relates to a screening method for an inhibitor of a process in which cleavage of an insulin receptor present on a cell membrane is promoted by hyperglycemia.
  • Type 2 Diabetes Mellitus is a type of diabetes that is common in adults. In Japan, 95% of diabetic patients are said to have type 2 diabetes. In patients with type 2 diabetes, the response to insulin in the living body is reduced (referred to as insulin resistance), and in severe cases, it is necessary to administer insulin to control blood glucose levels. That is, it can be said that type 2 diabetes is a state in which carbohydrate metabolism is abnormal due to insulin action failure.
  • the type 2 diabetes mellitus improving drug is a drug for returning a problematic carbohydrate metabolism (glucose metabolism) to normal.
  • glucose uptake in muscle and adipose tissue is carried out via a glucose transporter (glucose transporter: GLUT) on the cell membrane, and the amount of glucose uptake depends on the amount of glucose transporter. It is prescribed.
  • the main glucose transporter of these tissues is called glucose transporter 4 (GLUT4), but it is usually present inside the cell and moves (translocates) onto the cell membrane by insulin stimulation. It is taken up into the cell through the carrier.
  • GLUT4 glucose transporter 4
  • the liver is responsible for glucose metabolism through regulating the expression of genes related to glucose metabolism.
  • the insulin receptor plays a role in transmitting the insulin stimulation to GLUT4. That is, the insulin receptor is one of tyrosine kinase-coupled receptors, and an ⁇ subunit that binds to insulin and a ⁇ subunit that penetrates the cell membrane and contains tyrosine kinase in the cytoplasm are linked by a disulfide (SS) bond. Protein. This insulin receptor binds to insulin to initiate glucose uptake into cells and signal transduction that initiates glycogen synthesis.
  • SS disulfide
  • Insulin tyrosine kinase is activated by binding to the receptor and is autophosphorylated.
  • Insulin receptor substrate 1 IRS1 binds to the phosphorylated tyrosine, and this IRS1 is phosphorylated.
  • Phosphorylated IRS1 binds to phosphatidylinositol (PI) -3-kinase (PI-3kinase) and causes activation.
  • PI-3 kinase further attracts protein kinase B (PKB) to the cell membrane and activates it.
  • PKB protein kinase B
  • the activated PKB moves GLUT4 to the cell membrane in muscle and adipose tissue and takes glucose into the cell.
  • Non-patent Document 2 This phenomenon is considered to be one of the causes of worsening type 2 diabetes because it causes inactivation of serum insulin and worsens carbohydrate metabolism. Therefore, it was thought that if a drug that inhibits the release of the insulin receptor extracellular domain is screened, a novel mechanism for improving diabetes can be created. In this way, a screening system has been examined and reported (Patent Document 1), but the reproducibility is poor and it has not been an appropriate evaluation system. Therefore, it was a situation where establishment of a new evaluation system was required.
  • An object of the present invention is to provide a novel screening method for a therapeutic drug for diabetes. That is, an object of the present invention is to provide a screening method for a therapeutic drug for diabetes based on a novel mechanism based on the inhibitory action on the release of the extracellular domain of insulin receptor.
  • Non-patent Document 2 This cleaved molecule (soluble insulin receptor; sIR) was thought to contain an insulin binding site (insulin receptor ⁇ subunit) and bind insulin and inactivate insulin. The release of sIR into the blood along with the disappearance of the insulin receptor is considered to be a cause of insulin resistance. Therefore, as shown in FIG. 1, the present inventors proposed on the cell membrane surface. Elucidation of the mechanism of insulin receptor cleavage is an important issue. Thus, the present inventors constructed an in vitro system for a model for releasing insulin receptor cleavage.
  • sIR soluble insulin receptor
  • the ultrasensitive ELISA method (ICT-EIA method) of Patent Document 2 that can be quantified even at an extremely low concentration was used.
  • the suitability of four types of human-derived cultured cell lines (HeLa, HEK293, A549, HepG2) and human primary cultured hepatocytes was verified.
  • the above five types of human-derived cultured cells there are those in which sIR is released or not in the culture solution even though insulin receptor is expressed as shown in FIG. It was issued. That is, sIR could be detected in the culture medium only in the case of HepG2 cells and human primary cultured hepatocytes.
  • a human-derived cultured cell line that can be a model for cleaving release of insulin receptor is HepG2 cells derived from liver.
  • the physical properties of sIR released into the culture medium were identified by gel filtration, and as shown in FIG. 2, it was confirmed that it was almost the same as sIR in human serum. It was similar to the insulin receptor extracellular domain molecule. That is, sIR derived from HepG2 cells is considered to be the same molecule as sIR present in human serum.
  • the following items were checked in order to evaluate the identity of the insulin receptor cleavage release mechanism. a) The amount of sIR present in human serum increases, reflecting the hyperglycemic state.
  • Patent Document 1 uses cultured cells in which insulin receptor is overexpressed in CHO cells.
  • HepG2 human-derived cultured cell line
  • an endogenously expressed insulin receptor is used.
  • the present inventors have further studied and found the following. c) When high glucose treatment was performed for a long time using HepG2 cells, the insulin receptor was cleaved depending on the glucose concentration, and the amount of sIR in the culture medium increased. d) As shown in FIG.
  • the present inventors have used a cultured cell system of HepG2 close to that of diabetic patients as a drug evaluation system, and constructed a drug screening method that suppresses the release of sIR.
  • a screening method for inhibitors of calcium-dependent protease responsible for cleaving the insulin receptor as follows: 2) A screening method for inhibitors of the O-type glycosylation reaction that regulates insulin receptor cleavage has become feasible. This makes it possible to screen a completely new concept of an antidiabetic drug that improves insulin resistance by inhibiting insulin receptor cleavage.
  • the gist of the present invention is as follows.
  • a screening method for an insulin receptor cleavage inhibitor comprising the following steps: a) culturing human-derived cultured cell HepG2 under high glucose concentration conditions; b) culturing under high glucose conditions in which the calcium ion concentration is 1 to 10 mM or the PUGNAC concentration is 20 to 200 ⁇ M in the presence of the evaluation compound; c) a step of measuring the soluble insulin receptor (sIR) contained in the culture supernatant by ultrasensitive ELISA method (ICT-EIA method) after culturing under high glucose concentration conditions; and d) measuring by c) Comparing the concentration of the obtained sIR with a control to evaluate the cleavage inhibitory activity of the test compound (2)
  • the screening method according to (1) wherein the high glucose condition is a glucose concentration of 6 to 27.5 mM.
  • the screening method of the present invention by culturing the human-derived cultured cell HepG2 under high glucose conditions, the mechanism of release and release of the insulin receptor performed on the human cell membrane could be reproduced in HepG2 cells. . Therefore, by using a drug screening system using HepG2 cells, it has become possible to select a compound that inhibits insulin receptor cleavage and release in a screening system close to a living body. Furthermore, by allowing calcium ions and PUGNac to coexist in the screening system, it has become possible to screen for inhibitors of calcium-dependent proteases or inhibitors of O-type glycosylation. Thus, the drug provided by the screening method of the present invention was considered to be a new therapeutic agent compatible with the actual pathogenesis of diabetes.
  • FIG. 1 is a conceptual diagram showing the function of an insulin receptor and how the extracellular domain is cleaved under the high blood glucose level of the insulin receptor.
  • Fig. 2 shows that the insulin receptor extracellular domain (HIR SspI) expressed in CHO cells by gene recombination technology and released into the culture medium and the sIR released from HepG2 cells have almost the same physical properties by gel filtration. It is the column elution figure which showed that there was.
  • FIG. 3 is a graph showing that sIR released from HepG2 cells increases in a time-dependent manner under high glucose concentration conditions.
  • FIG. 4 shows that the behavior of sIR released from HepG2 cells at a high glucose concentration is dependent on the glucose concentration.
  • FIG. 1 is a conceptual diagram showing the function of an insulin receptor and how the extracellular domain is cleaved under the high blood glucose level of the insulin receptor.
  • FIG. 2 shows that the insulin receptor extracellular domain (HIR SspI) expressed in CHO cells by gene
  • FIG. 5 shows that sIR released from HepG2 cells was not released based on osmotic pressure due to high glucose.
  • other sugars mannitol, sorbitol
  • electrolytes NaCl
  • FIG. 7 shows that sIR released from HepG2 cells is suppressed by metal ion chelating agents EDTA and EGTA.
  • FIG. 8 shows that sIR released from HepG2 cells in culture in a high glucose medium is higher than matrix metalloproteinase (MMP) and disintegrin & metalloproteinase (ADAM) compared to medium with control (D; DMSO). It is shown that the specific inhibitor to inhibit (G in the figure; GM6001) and the lead ion chelator TPEN (T in the figure) are not suppressed.
  • MMP matrix metalloproteinase
  • ADAM disintegrin & metalloproteinase
  • FIG. 9 shows that sIR released from HepG2 cells is suppressed by EGTA, which is a metal ion chelator, which is eliminated by supplementation with calcium chloride, and even when calcium chloride is added compared to controls, It promotes the release of sIR.
  • FIG. 10 shows that calcium ions are required instead of iron ions and magnesium ions contained in the medium in order to release sIR from HepG2 cells.
  • FIG. 11 shows that when sIR is released from HepG2 cells, the calcium ion concentration curve shows sigmoid.
  • FIG. 12 shows that BAPTA-AM, which is a chelating agent for intracellular calcium ions, and ionomycin that releases calcium ions into cells do not affect sIR release from HepG2 cells.
  • FIG. 13 shows that when sIR is released from HepG2 cells in culture in a high glucose medium, DON acts as a glutamine: fructose-6-phosphate amide transferase (GFAT) antagonist and suppresses the production of sIR.
  • FIG. 14 is a schematic diagram of an O-type glycosylation reaction from the hexosamine synthesis pathway.
  • FIG. 15 shows that when OGT or OGA is knocked down using an appropriate siRNA, the O-GlcNAc addition reaction decreases and increases. Show.
  • FIG. 16 shows that as a result of using an appropriate siRNA, the OGT knockout inhibited the enzymatic addition reaction of the O-GlcNAc moiety and decreased the amount of sIR.
  • the enzymatic removal reaction of the O-GlcNAc moiety was inhibited, and the sIR concentration was significantly increased.
  • FIG. 17 is a diagram showing that the behavior of sIR released from HepG2 cells at a high glucose concentration is reversible. In other words, the HepG2 cells cultured for 72 hours at a high glucose concentration (11 mM) remain sIR production increased in the initial 24 hours even when switching to the culture with normal glucose concentration (5.5 mM).
  • FIG. 18 shows that in HepG2 cells, the release of sIR promoted by PUGNAc that promotes enzymatic addition reaction of high glucose concentration (16.5 mM) or O-type sugar chain is suppressed by EDTA, which is a metal ion chelator. Is shown.
  • FIG. 19 shows that when HepG2 cells are cultured in normal glucose concentration medium (5.5 mM) with and without PUGNAc (100 ⁇ M) (used for control), enzymatic addition reaction of O-type sugar chain is promoted. This shows that the addition of PUGNAc promotes the release of sIR.
  • the “insulin receptor” of the invention is a receptor protein having a heterotetrameric structure composed of two subunits ⁇ and ⁇ .
  • the ⁇ subunit is extracellular and the ⁇ subunit penetrates the cell membrane.
  • the ⁇ subunit is linked to the extracellular domain of the ⁇ subunit by an SS bond via the SH group of the Cys residue on the C-terminal side.
  • tyrosine residues in the intracellular domain of the ⁇ subunit are autophosphorylated, and an insulin signal is transmitted to the cell.
  • the insulin receptor found in the cell membrane is taken up into the cell by endocytosis after binding to insulin (the receptor half-life is 7 hours). The number of insulin receptors decreases with increasing insulin concentration.
  • Insulin receptor cleavage in the present invention means that the N-terminal side of the transmembrane domain of the insulin receptor ⁇ subunit is cleaved immediately above the cell membrane.
  • the “soluble insulin receptor (sIR)” of the present invention refers to a heterotetramer released to the outside of the cell in a state where the entire insulin receptor ⁇ -subunit and the N-terminal of the ⁇ -subunit are bound by an SS bond.
  • the “HepG2” of the present invention is a human liver cancer cell line, and is widely used as a cell line retaining the characteristics of human hepatocytes.
  • the “high glucose condition” of the present invention is a glucose concentration condition corresponding to a high glucose value in a diabetic patient.
  • the normal value of glucose concentration in human plasma is considered to be 4 to 6 mM, and a patient having a glucose concentration higher than that is considered to be hyperglycemic. So in this screening system.
  • As the high glucose condition 6 to 27.5 mM was selected.
  • Preferred high glucose conditions include 16-22 mM.
  • the “ultra-sensitive ELISA method (ICT-EIA method)” of the present invention is a measurement method developed to reduce non-specific binding of labeled antibodies, which has become a background and hinders high sensitivity. By transferring only specific immune complexes to the second solid phase while leaving the non-specifically bound labeled antibody on the first solid phase, the background is greatly reduced, enabling highly sensitive measurement. This is a measurement method. As a result, ultrahigh sensitivity measurement was possible, and zmol (1 ⁇ 10 ⁇ 21 mol; 600 molecules) could be detected (see Japanese Patent No. 2607722, JP-A 2009-85685).
  • the “screening method” of the present invention refers to measuring the soluble insulin receptor (sIR) released into the culture supernatant after culturing HepG2 cells under high glucose concentration conditions and then contacting the evaluation compound with HepG2 cells. Thus, it is a method for evaluating and measuring the action of the evaluation compound as compared with the control example.
  • the method for measuring the soluble insulin receptor (sIR) is not particularly limited, but specifically, the ultrasensitive ELISA method (ICT-EIA method) specific to the human insulin receptor ⁇ subunit established by the inventors. ) Is preferable as a measuring method.
  • the concentration of soluble insulin receptor (sIR) observed in cells that did not receive the evaluation compound can be used as a control.
  • sIR soluble insulin receptor
  • Example 1 Identification of sIR released from HepG2 cells (gel filtration method)
  • gel filtration was used to compare with the insulin receptor extracellular domain (HIR SspI) expressed in CHO cells and released into the culture medium by genetic recombination techniques.
  • HIR SspI insulin receptor extracellular domain
  • Example 2 Effect of high glucose concentration on HepG2 cells According to the culture method of Example 1, HepG2 cells were cultured, but the glucose concentration conditions and pretreatment culture time were changed.
  • the glucose concentration was set in 8 steps of 3 mM, 4 mM, 5.5 mM, 8.25 mM, 11 mM, 16.5 mM, 22 mM and 27.5 mM.
  • the pretreatment culture was carried out at the above eight concentrations for 48 hours and further cultured for 24 hours, and the amount of sIR released into the culture solution was measured and evaluated by the ICT-EIA method. The result is shown in FIG.
  • the concentration of sLR released in the culture solution of HepG2 depends on the glucose concentration (3-27.5 mM), and the higher the glucose concentration in the culture solution, the more the release of sIR is promoted. It showed that.
  • Example 3 Influence of culture time of HepG2 cells under high glucose concentration conditions
  • the glucose concentration conditions were fixed at two conditions of 5.5 mM and 27.5 mM.
  • the pretreatment culture time was changed and set in six stages of 0 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours.
  • each culture was carried out according to Example 1 for 24 hours.
  • the amount of sIR released in each culture solution was measured and evaluated by the ICT-EIA method. The result is shown in FIG.
  • the concentration of sLR released in the culture solution of HepG2 shows time dependency (0-120 hr) with respect to the glucose treatment time.
  • Example 4 Effect of culture solution osmotic pressure on HepG2 cells According to the method of Example 1, instead of glucose, other carbohydrates (mannitol, sorbitol) or electrolyte (NaCl) are used, and HepG2 cells are used. Then, the presence or absence of the influence of the osmotic pressure of the culture solution was confirmed.
  • the amount of sIR released from the HepG2 cells was clearly different from that in the culture medium supplemented with mannitol, sorbitol or sodium chloride set to the same osmotic pressure as the 27.5 mM glucose concentration. This reveals that the release of sIR from the cell membrane did not occur based on the effect of osmotic pressure.
  • Free concentration of sIR in culture medium of human-derived cultured cell lines As human-derived cultured cell lines, HeLa (derived from cervix), HEK293 (derived from kidney), A549 (derived from lung), HepG2 (derived from liver) And, using human primary cultured hepatocytes, the cells were cultured in serum-free DMEM medium for 24 hours.
  • the amount of sIR released to the medium was measured and evaluated by the ICT-EIA method of Test Example 1. The result is shown in FIG. As shown in FIG. 6, among the above-mentioned four types of human-derived cultured cells, only the liver-derived HepG2 cells and human primary cultured hepatocytes have significant insulin receptor cleavage and release of sIR into the culture medium. It was. (Example 6) Calcium-dependent proteolytic enzyme responsible for the cleavage of insulin receptor Matrix metalloproteinase (MMP) and disintegrin & metalloproteinase (ADAM) degrade many membrane proteins. It is thought to be a proteolytic enzyme responsible for receptor cleavage.
  • MMP Matrix metalloproteinase
  • ADAM disintegrin & metalloproteinase
  • Example 7 Effect of O-type glycosylation reaction that regulates insulin receptor cleavage Since glucose is metabolized in HepG2 cells and cleaves insulin receptor, O-linked glycosylation occurs at the time of insulin receptor cleavage. It was evaluated whether or not the addition reaction of N-acetylglucosamine (O-GlcNAc) was mediated.
  • O-GlcNAc N-acetylglucosamine
  • DON acts as a glutamine: fructose-6-phosphate amidotransferase (GFAT) antagonist resulting in a decrease in O-GlcNAc. When this DON was added, as shown in FIG.
  • OGT knockout decreased insulin receptor cleavage due to high glycolol, but sIR concentration significantly increased in OGA knockdown. From the above, it has been clarified that when the O-GlcNAc addition reaction increases, the production of sIR increases, and when the O-GlcNAc addition reaction decreases, the production of sIR decreases. Screening for inhibitors of the O-type glycosylation reaction using the increase or decrease of this O-GlcNAc addition reaction as an index makes it possible to screen for a therapeutic agent for diabetes to protect the insulin receptor.
  • Example 8 Screening method for compounds that inhibit insulin receptor cleavage (1) Screening method 5.5 mM glucose-containing cell culture medium (DMEM) (bovine serum (FBS) concentration is 1%) Is used as a comparative control, and a cell culture medium containing high glucose up to 22 mM glucose is used. The increase in sIR concentration due to treatment with high glucose relative to 5.5 mM glucose as a control requires a minimum of 48 hours of pretreatment after medium change every 24 hours. Therefore, two samples are prepared, one in which the compound to be evaluated is added at 10 ⁇ M in the high glucose-containing cell culture medium (with bovine serum (FBS) concentration of 1%) and the one without addition (used for control).
  • DMEM glucose-containing cell culture medium
  • FBS bovine serum
  • HepG2 cells were cultured in 24-well plates. Since HepG2 cells tend to aggregate, fully isolated cells are used. Moreover, since the increase in the release of sIR by the cell culture medium containing high glucose depends on the cell density, 3 ⁇ 10 5 cells / well (24-well plate) were used. Under all conditions including a high glucose-containing medium containing a compound to be evaluated, the medium was changed every 24 hours, and a high glucose pretreatment was performed for a minimum of 48 hours. After pretreatment for 48 hours or more, the medium was replaced with 500 ⁇ l of fresh glucose-containing cell culture medium, and 400 ⁇ l of the culture solution after 24 hours of treatment was collected in a 1.5 ml plastic tube.
  • the concentration of sIR released to the medium is reduced by the addition of the compound, this indicates that the insulin receptor cleavage reaction could be suppressed. That is, such an evaluation compound may be useful as a drug that improves insulin resistance.
  • a compound that decreases the sIR concentration and suppresses the insulin receptor cleavage is selected in the above screening, the O-type glycosylation reaction responsible for the insulin receptor cleavage or the calcium dependence according to the following Example 9 or 10
  • the mechanism of action of the drug can be clarified by further screening for the mechanism of the action of the sex protease.
  • Example 9 Screening Method for Inhibitors of O-Type Glycosylation Reaction (1) Screening Method According to Example 8, with addition of 100 ⁇ M PUGNac to 5.5 mM glucose-containing cell culture medium (DMEM) Two were prepared (used for control), and the latter was added with 10 ⁇ M of the compound to be evaluated and the other was not added. Using these three cell culture media, HepG2 cells were cultured in 24-well plates. (2) Evaluation A 5.5 mM glucose-containing medium as a comparative control is compared with a 5.5 mM glucose-containing medium to which PUGNAC is added, and an increase in the concentration of sIR due to PUGNAC is confirmed.
  • DMEM glucose-containing cell culture medium
  • Example 10 Screening method for inhibitors of calcium-dependent protease (1) Screening method HepG2 cells are cultured according to Example 8, and the change in sIR concentration by addition of 5 mM calcium chloride is measured with ultrasensitive sIR. The method (ICT-EIA) was evaluated. (2) Evaluation The concentration of sIR released to the medium increases with the addition of calcium chloride. It was evaluated how the concentration of sIR released in the medium changes depending on whether or not the compound to be evaluated is added in the calcium chloride-containing medium.
  • Monoclonal anti-hIR ⁇ antibody (Ab-2; Clone 83-7) is also available from LAB VISION Corp. (CA, USA).
  • Rabbit anti-2,4-dinitrophenyl group (DNP) -bovine serum albumin (BSA) serum was purchased from Shibayagi (Gunma).
  • the specimen was evaluated using the immune complex transfer measurement method (ICT-EIA method) described in Patent Document 2.
  • ICT-EIA method The culture supernatant sample described in Example 1 was diluted twice with 0.01 M sodium phosphate buffer (pH 7.0) and used for measurement by the ICT-EIA method.
  • the present invention can provide a new screening method for insulin receptor cleavage inhibitors. That is, a screening method for inhibitors of calcium-dependent protease and a screening method for inhibitors of O-type glycosylation reaction.
  • the screening method of the present invention is a highly versatile screening method because it can be performed with simple operations and good reproducibility. As a result, the screening method of the present invention has made it possible to develop a new therapeutic agent for diabetes that can suppress insulin receptor cleavage and improve insulin resistance.

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Abstract

L'objet de la présente invention est de pourvoir à une méthode de criblage d'inhibiteurs en vue d'inhiber le clivage de récepteurs de l'insuline sur des membranes cellulaires. Il a été découvert que lorsque des cellules HepG2 sont sélectionnées et mises en culture dans un état de concentration élevée en glucose, de la sirtuine (sIR) est libérée dans le surnageant de culture de façon dépendante de la concentration en glucose et du temps de contact élevé avec le glycol. Par le biais de l'addition de davantage d'ions calcium ou de PUGNAc afin de mettre en œuvre une culture en présence du composé évalué et d'évaluer la variation de sIR sur la base de l'observation susmentionnée, il est possible de cribler des inhibiteurs de protéase dépendants du calcium et des inhibiteurs de réaction d'O-glycosylation.
PCT/JP2014/055528 2013-04-17 2014-02-26 Nouvelle méthode de criblage de médicament contre le diabète WO2014171206A1 (fr)

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