Methods for modulating kinase activity and uses thereof

Abstract

Use of an active ingredient that targets and regulates the expression level of a metabolite or blocks the binding site between LKB1 protein and a metabolite in the prevention and / or treatment of an AMPK kinase-mediated metabolic-related disease, wherein the metabolic-related disease is selected from obesity, diabetes, tumors, inflammatory diseases, or a combination thereof.

Description

[Technical Field]

[0001] The present invention relates to the field of biochemistry, and in particular, the present invention relates to methods and uses for modulating kinase activity. [Background technology]

[0002] Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPPK) is an AMP-dependent protein kinase and an important signaling molecule that regulates energy metabolism in the body. It exerts its physiological function mainly by phosphorylating downstream protein substrates through the action of protein kinase activity. Therefore, regulating AMPK kinase activity is an important method for intervening in AMPK kinase and treating metabolic diseases (such as obesity, diabetes, tumors, and inflammatory diseases). AMPK exists in the body as a trimeric complex and exerts kinase function, comprising one α-catalytic subunit, one β-regulatory subunit, and one γ-regulatory subunit. Different species may encode multiple highly homologous α, β, and γ subunits. For example, the human genome encodes two highly homologous AMPK α-catalytic subunits (designated α1 and α2, respectively). Activation of AMPK kinase is mediated by the α-catalytic subunit protein FLR. T Dependent on phosphorylation of threonine residues (underlined) within the SC motif (e.g., T183 site in human AMPK α1 protein and T172 site in AMPK α2 protein), LKB1 and CaMKKβ are known as two important upstream kinases for phosphorylating this site.

[0003] LKB1 kinase (Liver kinase B1), also known as STK11 (Serine-Threonine Kinase 11) or PJS protein (Peutz-Jeghers syndrome, PJS), forms complexes with other proteins (e.g., MO25 and STRAD1) and catalyzes the phosphorylation of multiple kinases, including the AMPK kinase α-catalytic subunit, thereby regulating physiological activities such as cellular metabolism, proliferation, and autophagy. Therefore, LKB1 is an important target point for disease regulation. It has been reported that LKB1 may sense physiological signals, such as cellular metabolic status, but the specific mechanism remains unknown. Previous studies have demonstrated that metabolic intermediates in the glycolytic pathway, such as 3-phosphoglycerate (C3H7O7P, CAS#820-11-1), 2-phosphoglycerate (C3H7O7P, CAS#2553-59-5), and pyruvate (also known as 2-oxopropionic acid, acetoformic acid, pyruvic acid, or pyruvate; chemical formula C3H4O3, CAS#127-17-3), and their corresponding salt forms, can directly bind to LKB1 kinase or the LKB1 kinase complex and inhibit the phosphorylation of the AMPK kinase α-catalytic subunit. Therefore, LKB1 kinase or the LKB1 kinase complex can directly sense intermediates in glucose metabolism and influence the activating effect of LKB1 kinase or the LKB1 kinase complex on AMPK kinase. Therefore, there is an urgent need in the art to develop new active ingredients that target and regulate the expression levels of metabolites, which can be used to prepare pharmaceutical compositions, and the pharmaceutical compositions are used to prevent and / or treat AMPK kinase-mediated metabolic-related diseases, such as obesity, diabetes, tumors, inflammatory diseases, etc. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide a new use of an active ingredient capable of targeting and regulating the expression level of a metabolite, which can be used to prepare a pharmaceutical composition for preventing and / or treating AMPK kinase-mediated metabolic-related diseases. [Means for solving the problem]

[0005] A first aspect of the present invention provides the use of an active ingredient for targeting and regulating the expression level of a metabolite or blocking the binding site between LKB1 protein and a metabolite, which is used in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition is used for the prevention and / or treatment of an AMPK kinase-mediated metabolic-related disease, and the metabolite is selected from the group consisting of 3-phosphoglycerate, 2-phosphoglycerate, pyruvate, or a combination thereof. In another preferred embodiment, the active ingredient is used to down-regulate the metabolite. In another preferred embodiment, the AMPK kinase-mediated metabolic-related disease is selected from the group consisting of obesity, diabetes, tumors, inflammatory diseases, or a combination thereof.

[0006] In another preferred embodiment, the active ingredient is used to downregulate the production of the metabolite or to block the binding of the metabolite to LKB1 kinase or the LKB1 kinase complex. In another preferred embodiment, the blocking is competitive blocking or non-competitive blocking. In another preferred embodiment, the active ingredient is used to activate the LKB1-AMPK pathway. In another preferred embodiment, the active ingredient is used to inhibit the activity of a metabolic enzyme that produces the metabolite. In another preferred embodiment, the metabolic enzyme is phosphoglycerate kinase PGK1, PGK2, Phosphoglycerate mutase PGAM1, PGAM2, PGAM3, PGAM4, PGAM5, Pyruvate kinase PKM1, PKM2, PKRL, Pyruvate dehydrogenase PDHK1, PDHK2, PDHK3, PDHK4, or a combination thereof.

[0007] In another preferred embodiment, the active ingredient is used to activate metabolic pathways that consume 3-phosphoglycerate, 2-phosphoglycerate and / or pyruvate or salts thereof.

[0008] In another preferred embodiment, the active ingredient is also used to inhibit the transcriptional expression of metabolic enzymes, which are used to catalyze the production of 3-phosphoglycerate, 2-phosphoglycerate and / or pyruvate or salts thereof.

[0009] In another preferred embodiment, the active ingredient is selected from the group consisting of a small molecule drug, an antibody, a molecular adhesive, Protac, an mRNA-containing delivery vector, an antisense nucleic acid sequence RNA-containing delivery vector, or a combination thereof. In another preferred embodiment, the active ingredient is compound S33. [ka]

[0010] A second aspect of the present invention provides a method for non-therapeutic modulation of the LKB1-AMPK pathway in vitro, comprising culturing cells or tissues in the presence of an active ingredient that targets and modulates the expression level of a metabolite, The metabolite is selected from the group consisting of 3-phosphoglycerate, 2-phosphoglycerate, pyruvate, or a combination thereof. [Effects of the Invention]

[0011] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0012] [Figure 1] Panel A shows that SUN739 liver cancer cell line and HeLa cell line were transfected with PGK1 siRNA to knock down PGK1, and the activation status of the AMPK signaling pathway was detected by Western blot.

[0013] (B) siRNAs against PGK1, PGMA1, and PKM2 were transfected into HEK293T cells to knock down the three endogenous metabolic enzymes, and the cells were harvested and the activation status of the AMPK signaling pathway was detected by Western blotting.

[0014] (C) Cultured HEK293T cells were glucose-starved, then treated with various concentrations of 3-phosphoglycerate (3-PG) as shown, harvested, and analyzed by Western blotting to detect AMPK activation.

[0015] (D) Cultured HEK293T cells were subjected to glucose starvation, and then 200 μM each of 3-phosphoglycerate (3PG), 2-phosphoglycerate (2PG), and pyruvate (PYR) was added to the cells. The cells were then harvested and the activation status of AMPK was detected by Western blotting. E represents the structural formula of compound S33. F shows the enzyme activity change curve of PGK1 under the treatment of compound S33 and the calculated IC 50 Indicates the value.

[0016] G shows the changes in specific proteins and phosphorylation sites of the AMPK α-catalytic subunit in HeLa and HEK293T cell lines under the treatment with compound S33, as well as the phosphorylation status of ACC1, a typical downstream substrate of the AMPK pathway.

[0017] (H) The LKB1 complex-related plasmid (GST-LKB1 / FLAG-STRAD1 / Myc-MO25) was overexpressed in HEK293T cells and purified by immunoprecipitation to obtain the LKB1 complex. The LKB1 complex was then co-incubated with recombinantly expressed His-AMPK protein in E. coli for in vitro kinase experiments. Simultaneously, 100 μM each of the metabolites 3-phosphoglycerate (3PG) or 3-phosphoglyceraldehyde (GAP) or the corresponding solvent control (veh) was added, and the effect on LKB1 kinase complex-mediated AMPK phosphorylation was detected by Western blot analysis.

[0018] Panel I shows that an in vitro kinase experiment similar to that shown in Figure 1E was performed using the purified LKB1 complex described above, and three concentrations of 3-phosphoglycerate (3PG) were added as shown in the figure, and the effect on LKB1 kinase-mediated AMPK phosphorylation was detected by Western blot.

[0019] J shows that GST-LKB1 was overexpressed in HEK293T cells, purified by immunoprecipitation to obtain LKB1 kinase, and then co-incubated with His-AMPK protein recombinantly expressed in E. coli to perform in vitro kinase experiments. At the same time, 100 μM each of 3-phosphoglycerate (3PG), 2-phosphoglycerate (2PG), and pyruvate (PYR) was added, and the effects on LKB1 kinase-mediated AMPK phosphorylation were detected by Western blot.

[0020] K~M show that large-scale purification of LKB1 kinase was performed using HEK293F cells, and co-incubated with various concentrations of 3-phosphoglycerate (3PG), 2-phosphoglycerate (2PG), and pyruvate (PYR). The dissociation constants (Kd values) between LKB1 protein and metabolites were measured and calculated using a microthermophoresis device. DETAILED DESCRIPTION OF THE INVENTION

[0021] Through extensive and thorough research and extensive screening, the present inventors have for the first time developed a novel active ingredient that can target and regulate the expression levels of metabolites, which can be used to prepare pharmaceutical compositions for the prevention and / or treatment of AMPK kinase-mediated metabolic diseases, and have completed the present invention based on this.

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions generally follow conventional conditions or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated by weight. Experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0023] General Experimental Methods 1. siRNA Transfection We used the siRNA dry powder synthesized by Takuzen Co., Ltd. and dissolved it in DEPC water. We performed transfection using X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's instructions. The cell density required for siRNA transfection is approximately 30%. In this lab, we used Roche X-tremeGENE siRNA transfection reagent. Prepare two 1.5 mL RNase-free centrifuge tubes, add 250 μL of Opti-MEM to each tube, and add 5 μL of X-tremeGENE siRNA transfection reagent and 6.25 μL of 20 μM siRNA. Gently pipette to mix thoroughly and leave at room temperature for 5 minutes. Mix the Opti-MEM containing X-tremeGENE siRNA transfection reagent and the Opti-MEM containing siRNA, gently pipette to mix thoroughly, and leave for 15 minutes. The 293T cells were removed, the culture medium was aspirated, and the cells were washed once with PBS buffer, followed by the addition of 2 mL of Opti-MEM. The Opti-MEM mixture of plasmid and transfection reagent was slowly added dropwise to the cells, and gently shaken to ensure uniform distribution of the siRNA. After 5 hours, 2 mL of DMEM complete medium containing serum and antibiotics was added to the transfected cells. Normal DMEM cell culture medium was replaced with that of the transfected cells. After 36 hours, the cells were harvested and total cellular protein was extracted. Changes in specific proteins and changes in specific site phosphorylation were detected by Western blot using commercially available antibodies.

[0024] 2. Metabolite-treated cells Cells were glucose-starved for 2 hours using glucose-free medium. The corresponding metabolites were added to the medium as illustrated (e.g., Figures 1C and 1D) until their concentrations reached the labeled level. In the absence of labeling, the metabolite concentration in the medium was 200 μM. After treatment, cells were harvested and lysates were prepared. Western blot analysis was performed to detect changes in specific proteins and phosphorylation at specific sites. 3. In vitro enzyme activity of PGK1 and IC of compounds 50 Inhibition curve plot

[0025] Prepare a substrate buffer mixture with final concentrations of 10 mM KH2PO4, pH 7.0, 2 mM GAP, 0.6 mM β-NAD, 0.4 mM ADP, 10 mM MgSO4, 200 mM glycine, and 0.01 μg / μL GAPDH. Dilute purified hPGK1 recombinant protein to 0.2 ng / μL. Add inhibitor to the protein to set up a concentration gradient. Add 100 μl of substrate and 100 μl of the hPGK1 protein and inhibitor mixture to each reaction. Incubate at 37°C for 25 minutes. Centrifuge at 10,000 rpm at 4°C for 10 minutes and collect 100 μl of the supernatant. For the ATP detection kit (Beyotime S0026), dilute the ATP detection solution with the ATP detection diluent at a ratio of 1:10. Add 100 μl of diluted detection solution to each well of a 96-well white plate, leave at room temperature for 5 minutes, add 100 μl of enzyme activity reaction supernatant, let react for 5 seconds, and read the fluorescence value on a microplate reader. Calculate the relative enzyme activity based on the fluorescence value and calculate the IC using GraphPad nonlinear regression fitting. 50 Calculate the value.

[0026] 4. In Vitro Kinase Experiments The final reaction system for kinase experiments was 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, and 100 μM ATP. HEK293T cells were co-transfected with equal amounts of GST-LKB1, FLAG-STRAD, and MYC-MO25 plasmids, or FLAG-LKB1 plasmid alone. Cells were harvested and co-immunoprecipitation was performed using GST or FLAG beads. The cells were washed three times with cell lysate containing NP-40 and twice with kinase reaction buffer. His-AMPK protein was induced and purified using the sensitive E. coli BL21 strain. Before the reaction, the kinase was co-incubated with 100 μM metabolites at room temperature for 15 minutes. The reaction buffer and His-AMPK protein were then added and incubated at 30°C for 30 minutes. The phosphorylation status of AMPK protein was then detected by Western blot analysis.

[0027] 5. Microscale Thermophoresis Experiments LKB1 protein was purified and fluorescently labeled with NHS dye. A 1 mM metabolite solution was prepared and diluted stepwise in a 2-fold gradient, for a total of 16 gradients. The labeled protein was diluted to 25 nM and mixed with the metabolite solution in a 1:1 ratio. The solution was then aspirated into a glass capillary tube and detected using the NT.115 microthermophoresis apparatus. The data was then directly processed using the data acquisition software MO.Control to obtain the dissociation constant.

[0028] 6.Cell culture The HEK293T cell line, SUN739 liver cancer cell line, and HeLa cell line were all cultured in DMEM medium in a cell incubator at 37°C / 5% CO2. They were passaged every 2-3 days and required revival after approximately 30 generations.

[0029] Example 1. Study on the regulatory mechanism of PGK1 knockdown in the LKB1-AMPK pathway siRNA dry powder synthesized by Takuzen Co., Ltd. was dissolved in DEPC water. PGK1 knockdown experiments were performed using X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's instructions. For each siRNA transfection, prepare two 1.5 mL RNase-free centrifuge tubes, add 250 μL of Opti-MEM to each tube, and add 5 μL of X-tremeGENE siRNA transfection reagent and 6.25 μL of 20 μM siRNA. Mix thoroughly by gently pipetting and leave at room temperature for 5 minutes. Mix Opti-MEM with X-tremeGENE siRNA transfection reagent and Opti-MEM with siRNA, gently pipette until uniform, and leave for 15 minutes. The liver cancer cell line SNU739 and cervical cancer cell line HeLa cells to be transfected were removed, the culture medium was aspirated, the cells were washed once with PBS buffer, and 2 mL of Opti-MEM was added. The Opti-MEM mixture of plasmid and transfection reagent was slowly added dropwise to the 293T cells and gently shaken to ensure uniform distribution of the siRNA. After 5 hours, 2 mL of DMEM complete medium containing serum and antibiotics was added to the transfected cells. The transfected cells were replaced with normal DMEM cell culture medium. After 36 hours, the cells were harvested and total cellular protein was extracted. Changes in specific proteins and changes in specific site phosphorylation were detected by Western blot using commercially available antibodies.

[0030] Small RNA interference (SiRNA) experiments were performed in the liver cancer cell line SNU739 and the cervical cancer cell line HeLa targeting phosphoglycerate kinase 1 (PGK1), a key metabolic enzyme that catalyzes the production of 3-phosphoglycerate during glycolysis. Western blot results showed that, upon PGK1 knockdown via siRNA transfection, the band signal characterizing the phosphorylation level of the AMPK α-catalytic subunit (p-AMPK signal represents the phosphorylation level at T183 in AMPK α1 and T172 in AMPK α2) was upregulated, and the phosphorylation level of ACC1 downstream of AMPK was increased, indicating activation of the AMPK pathway (Figure 1A). Knocking down the metabolic enzymes PGAM1 and PKM2 downstream of PGK1 also activated the AMPK pathway (Figure 1B).

[0031] Example 2. Study on the regulatory mechanism of 3-phosphoglycerate in the AMPK pathway PGK1 catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate (3PG), PGAM1 catalyzes the conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate, and PKM2 catalyzes the conversion of phosphoenol pyruvate to pyruvate (PYR). When the expression and function of these metabolic enzymes are inhibited, their corresponding products decrease. Therefore, we speculate that a mechanism exists whereby AMPK is regulated by metabolites in the glycolytic pathway, and that AMPK activation is related to the amount of metabolites. To test this hypothesis, we added various doses of exogenous 3-phosphoglycerate (3-PG) to glucose-starved 293T cells (which activate the AMPK signaling pathway). 293T cells were glucose-starved for 2 hours under normal culture conditions (37°C / 5% CO2) using glucose-free DMEM medium. Add 3-PG to the medium as described in the illustration (e.g., Figure 1C) until its concentration in the medium reaches the labeling level (100-800 µM) (Figure 1C) or reaches 200 µM (Figure 1D). After treatment, collect the cells and prepare lysates. Western blot analysis is then performed to detect changes in specific proteins and the AMPK α-catalytic subunit phosphorylation site.

[0032] The results show that 3-phosphoglycerate significantly inhibited the phosphorylation signaling of the intracellular AMPK α-catalytic subunit in a dose-dependent manner (Fig. 1C), and exogenously added 2-phosphoglycerate (2-PG) and pyruvate (PYR) also had similar inhibitory effects on the phosphorylation signaling of the AMPK α-catalytic subunit (Fig. 1D).

[0033] Example 3. Studies on the regulation of PGK1 inhibitors in the AMPK pathway The compound reported in the above patent (S33, patent number: ZL201610826187.X) is a PGK1 inhibitor (Figure 1E), and the inhibitory activity of compound S33 against PGK1 was measured. Recombinant human PGK1 protein was purified using E. coli, and S33 compound was added to set up a concentration gradient. 100 μl of substrate and 100 μl of a mixture of hPGK1 protein and inhibitor were added to each reaction, where the hPGK1 recombinant protein was diluted to 0.2 ng / ml, and seven gradient S33 concentrations were set (0, 1, 3, 5, 7, 10, and 30 μM, respectively). A substrate buffer mixture was then prepared: 10 mM KH2PO4, pH 7.0, 2 mM GAP, 0.6 mM βNAD, 0.4 mM ADP, 10 mM MgSO4, 200 mM glycine, and 0.01 μg / μL GAPDH. Incubate at 37°C for 25 minutes. Centrifuge at 10,000 rpm for 10 minutes at 4°C and remove 100 μl of supernatant. For the ATP detection kit (Beyotime S0026), dilute the ATP detection solution with the ATP detection diluent at a ratio of 1:10. Add 100 μl of the diluted detection solution to each well of a 96-well white plate and leave at room temperature for 5 minutes. Add 100 μl of the enzyme activity reaction supernatant, incubate for 5 seconds, and read the fluorescence value using a microplate reader. Calculate the relative enzyme activity based on the fluorescence value and obtain the IC using GraphPad nonlinear regression fitting. 50 The values ​​are calculated (FIG. 1F). The above data indicate that compound S33 is an effective inhibitor of PGK1.

[0034] To study the regulatory effect of PGK1 inhibition on the AMPK pathway, various concentrations of S33 (2, 5, and 10 μM, respectively) were added to the culture supernatants of HeLa and 293T cells for 2 hours. After treatment, the cells were harvested and lysed, and Western blot analysis was performed to detect changes in specific proteins and the phosphorylation sites of the AMPK α-catalytic subunit, as well as the phosphorylation status of ACC1, a typical downstream substrate of the AMPK pathway.

[0035] The results show that the PGK1 inhibitor S33 can dose-dependently upregulate the phosphorylation of the AMPK α-catalytic subunit and ACC1, suggesting that inhibition of PGK1 enzyme activity by the compound can activate the AMPK pathway (Figure 1G).

[0036] Example 4. Study on the regulatory mechanism of 3-phosphoglycerate in the LKB1-AMPK pathway Since LKB1 is known to be an important upstream kinase that phosphorylates and activates the AMPK α subunit, we speculated that the above metabolites may affect the LKB1 kinase or the LKB1 kinase complex on the AMPK α subunit phosphorylation kinase. To test this hypothesis, we transfected GST-LKB1, FLAG-STRAD1, and MYC-MO25 into 293T cells (2 μg of each expression plasmid was used in approximately 7 × 10 6 LKB1-STRAD1-MO25 kinase complexes were co-transfected into cells. After 48 hours, cells were harvested and lysed. The LKB1-STRAD1-MO25 kinase complex was affinity-purified using glutathione gel particles and co-incubated with recombinantly expressed His-AMPK α1 subunit in E. coli for in vitro kinase experiments. The final reaction mixture for kinase experiments was 50 mM Tris-HCl, 10 mM MgCl, 1 mM DTT, and 100 μM ATP. Simultaneously, the metabolites 3-phosphoglyceraldehyde (GAP) or 3-phosphoglycerate (3-PG) were added to 100 μM as shown (e.g., Figure 1H) or to concentrations of 50–400 μM as shown (e.g., Figure 1I). Before the reaction, the kinase complex was pre-incubated with the metabolites at room temperature for 15 minutes, and then the reaction buffer and His-AMPK protein were added and reacted at 37°C for 30 minutes, and the phosphorylation status of the AMPK protein was detected by Western blot.

[0037] The results show that 3-phosphoglycerate strongly inhibits AMPKα1 phosphorylation induced by LKB1 kinase complex inhibition, whereas 3-phosphoglyceraldehyde has no effect (Figure 1H). Furthermore, the inhibitory effect of 3-phosphoglycerate on AMPKα1 phosphorylation by the LKB1 kinase complex is dose-dependent (Figure 1I).

[0038] Example 5. Metabolites that inhibit phosphorylation of AMPKα1 kinase by LKB1 kinase in vitro To further investigate whether the three metabolites, 3-phosphoglycerate (3-PG), 2-phosphoglycerate (2-PG), and pyruvate (PYR), act directly on LKB1 kinase and affect the downstream phosphorylation and activation of AMPK kinase, we transfected 293T cells, prepared LKB1 kinase (free from STRAD1 and MO25) by immunoprecipitation, and performed in vitro kinase experiments by co-incubating it with His-AMPK α1 subunit recombinantly expressed in E. coli. The final reaction system for kinase experiments was 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, and 100 μM ATP. Simultaneously, the same concentrations (100 μM) of the following metabolites were added, as shown in the diagram (e.g., Figure 1J): fructose 1,6-bisphosphate (FBP), 3-phosphoglyceraldehyde (GAP), 3-phosphoglycerate (3-PG), 2-phosphoglycerate (2-PG), and pyruvate (PYR). The kinase complex was preincubated with the metabolites for 15 minutes at room temperature, followed by the addition of reaction buffer and His-AMPK protein. The reaction was incubated at 37°C for 30 minutes, and the phosphorylation status of AMPK was then detected by Western blot analysis.

[0039] The results show that 100 μM 3-phosphoglycerate, 2-phosphoglycerate (2-PG), and pyruvate (PYR) can all significantly inhibit the phosphorylation of AMPKα1 by LKB1 kinase, whereas fructose 1,6-bisphosphate (FBP) and 3-phosphoglyceraldehyde (GAP) have no effect (Figure 1J).

[0040] Example 6. Study on the regulatory mechanism of three metabolites in the LKB1-AMPK pathway To further explore whether LKB1 directly interacts with the three metabolites 3-phosphoglycerate, 2-phosphoglycerate (2-PG), and pyruvate (PYR), we prepared LKB1 kinase protein by large-scale transfection and affinity purification in 293T cells and molecular screening. Microscale thermophoresis experiments were then performed with varying concentrations of the three metabolites to calculate the binding profiles between LKB1 protein and the metabolites. First, a 1 mM metabolite solution was prepared and serially diluted 2-fold to create a total of 16 concentration gradients. The purified LKB1 protein was then fluorescently labeled with NHS dye. The labeled protein was diluted to 25 nM and mixed 1:1 with the metabolite solution. The fluorescently labeled protein was then transferred to a glass capillary tube and detected using the NT.115 microthermophoresis system. The data were then directly processed using the MO.Control data acquisition software to obtain the dissociation constants.

[0041] The results show that LKB1 protein has strong interactions with three metabolites: 3-phosphoglycerate (3-PG), 2-phosphoglycerate (2-PG), and pyruvate (PYR), and their equilibrium dissociation constants (Kd) were calculated (Figure 1K-1M).

[0042] To summarize the above results, three metabolites, 3-phosphoglycerate, 2-phosphoglycerate, and pyruvate, directly bind to LKB1, thereby inhibiting the activation effect of LKB1 kinase on the AMPK α subunit. Therefore, altering the content of these metabolites by targeting and regulating the metabolic enzymes that produce them, or blocking the binding sites between LKB1 and these metabolites, can be used to regulate AMPK kinase activity and be used to regulate physiological functions and treat diseases.

[0043] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. 1. Use of an active ingredient for targeting and modulating the expression level of a metabolite or for blocking the binding site between the LKB1 protein and a metabolite, comprising: the use is for the preparation of a pharmaceutical composition, the pharmaceutical composition is used for the prevention and / or treatment of an AMPK kinase-mediated metabolic-related disease, and the metabolite is selected from the group consisting of 3-phosphoglycerate, 2-phosphoglycerate, pyruvate, or a combination thereof; Use of an active ingredient to target and regulate the expression level of said metabolite or to block the binding site between the LKB1 protein and the metabolite.

2. The AMPK kinase-mediated metabolic disease is selected from the group consisting of obesity, diabetes, tumors, inflammatory diseases, or a combination thereof.

2. The use according to claim 1.

3. The active ingredient is used to downregulate the production of the metabolite or to block the binding of the metabolite to LKB1 kinase or the LKB1 kinase complex.

2. The use according to claim 1.

4. The blocking is characterized in that it is competitive blocking or non-competitive blocking.

4. The use according to claim 3.

5. The active ingredient is characterized in that it is used to activate the LKB1-AMPK pathway.

2. The use according to claim 1.

6. The active ingredient is characterized in that it is used to inhibit the activity of metabolic enzymes that produce the metabolites.

2. The use according to claim 1.

7. The metabolic enzymes include phosphoglycerate kinases PGK1 and PGK2, Phosphoglycerate mutase PGAM1, PGAM2, PGAM3, PGAM4, PGAM5, Pyruvate kinase PKM1, PKM2, PKRL, Pyruvate dehydrogenase PDHK1, PDHK2, PDHK3, PDHK4, or a combination thereof.

7. The use according to claim 6.

8. The active ingredient is characterized in that it is used for activating metabolic pathways that consume 3-phosphoglyceric acid, 2-phosphoglyceric acid and / or pyruvic acid or salts thereof.

2. The use according to claim 1.

9. The active ingredient is also used to inhibit the transcriptional expression of metabolic enzymes, and the metabolic enzymes are used to catalyze the production of 3-phosphoglyceric acid, 2-phosphoglyceric acid and / or pyruvic acid or salts thereof.

2. The use according to claim 1.

10. The active ingredient is selected from the group consisting of a small molecule drug, an antibody, a molecular adhesive, Protac, an mRNA-containing delivery vector, an antisense nucleic acid sequence RNA-containing delivery vector, or a combination thereof.

2. The use according to claim 1.

11. A method of non-therapeutic modulation of the LKB1-AMPK pathway in vitro comprising: The method comprises culturing cells or tissues in an environment in which an active ingredient that targets and regulates the expression level of a metabolite is present, A method of non-therapeutic modulation of the LKB1-AMPK pathway in vitro, wherein the metabolite is selected from the group consisting of 3-phosphoglycerate, 2-phosphoglycerate, pyruvate, or combinations thereof.

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