Maternal structure of PKM2 agonist and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a parent structure of a PKM2 agonist and application of the PKM2 agonist, the PKM2 agonist is hypaconitine screened from a monkshood alkaloid part, the chemical formula of the PKM2 agonist is C33H45NO10, and the structural formula of the PKM2 agonist is shown in the description. The hypaconitine has the functions of inhibiting phosphorylation of tumor cell PKM2 protein, inhibiting conversion from tetramer to dimer, enhancing PKM2 activity and inhibiting PKM2 nuclear translocation; the parent structure of the hypaconitine is composed of diterpenoid skeleton atoms, and the parent nucleus of the hypaconitine is C19 diterpenoid alkaloid; according to the invention, a PKM2 activating component in monkshood alkaloid is screened by using computer-aided drug design, and it is reported for the first time that hypaconitine has a PKM2 activating effect; a certain foundation is laid for the research on the anti-tumor mechanism of the radix aconiti carmichaeli, a basis is provided for the clinical application of the anti-tumor of the radix aconiti carmichaeli, and a new parent structure is provided for the research and development of a PKM2 agonist.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a parent structure of a PKM2 agonist and an application thereof. Background Art

[0002] PKM2 can maintain aerobic glycolysis in tumor cells to adapt to cell growth and proliferation. Targeting PKM2 aims to reduce the generation and accumulation of glycolytic intermediates and disrupt aerobic glycolysis. Given that PKM2 in tumor cells mostly exists as a dimer with low activity, and oncogene proteins, tyrosine phosphorylation and oxidative stress reactions can promote the formation of low-activity PKM2 dimers. Inhibiting dimer formation, improving tetramer stability and reversing glycolysis may correct tumor cell metabolism to the level of normal cell metabolism. my country has abundant resources of traditional Chinese medicine with rich and diverse chemical structures, which is an important source for the discovery of target compounds.

[0003] Aconite is the subroot of Aconitum carmichaeli Debx., a plant of the genus Aconitum in the family Ranunculaceae. It was first recorded in the Shennong's Herbal Classic and can "break up hard masses and blood masses". In traditional Chinese medicine theory, tumors belong to the categories of masses, accumulations, and hiccups. Many doctors believe that yang deficiency and cold stagnation are the main causes of masses and accumulations, and warming yang is the preferred treatment method. Aconite is one of the yang-warming drugs commonly used in the treatment of tumors in clinical practice. The pathogenesis of advanced tumors is a mixture of deficiency and excess. Aconite is the main drug, which takes the effect of warming yang and dispersing masses, and has a better effect on patients with advanced tumors.

[0004] Aconite contains alkaloids, polysaccharides, and other ingredients. Modern pharmacological studies have shown that the alkaloids in Aconite have cardiotonic, analgesic, anti-inflammatory, anti-tumor, and anti-rheumatic effects. Alkaloids are divided into diterpenes, amides, quaternary ammonium salts, aporphines, etc. according to their structural mother nucleus, mainly diterpenoid alkaloids. Diterpenoid alkaloids can be divided into C 18 , C 19 , C 20 Type diterpene alkaloids. 19 Type 2 diterpenoid alkaloids are the most studied alkaloids in Aconitum, including aconitine, metaaconitine, hypaconitine, benzoylmeaconitine, benzoylhypaconitine, benzoylaconitine, and neorin.

[0005] PKM2 is a key rate-limiting enzyme that regulates aerobic glycolysis and tricarboxylic acid cycle, and may be involved in the regulation of aconite alkaloids on tumor energy metabolism. Its mechanism may be that aconite alkaloid components inhibit PKM2 protein phosphorylation by binding to PKM2, regulate the conversion of PKM2 dimers / tetramers, and inhibit PKM2 nuclear translocation, thereby correcting tumor cell glycolysis. Therefore, based on the activation and regulatory effects of aconite alkaloids on PKM2, the present invention further studies the anti-tumor mechanism of aconite alkaloid sites, and screens monomer components in aconite alkaloid sites that can activate PKM2. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a parent structure of a PKM2 agonist and its application.

[0007] In order to achieve the purpose of the present invention, the present invention will be implemented by adopting the technical solution described below.

[0008] A parent structure of a PKM2 agonist, wherein the PKM2 agonist is aconitine screened from the alkaloid part of aconite, and its chemical formula is C 33 H 45 NO 10 The hypoaconitine has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers to dimers, enhancing the activity of PKM2 and inhibiting the nuclear translocation of PKM2; the parent structure of the hypoaconitine is composed of diterpene skeleton atoms, and its parent nucleus is C 19 Diterpene alkaloids; the chemical structural formula of the aconitine is:

[0009]

[0010] As a preferred embodiment of the present invention, the aconite alkaloid fraction has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers to dimers, enhancing PKM2 activity and inhibiting PKM2 nuclear translocation.

[0011] As a preferred solution of the present invention, the method for determining the function comprises the following steps:

[0012] S31, extracting the aconite alkaloid fraction from the aconite root, purifying it using D101 macroporous resin, and determining the content of the aconite alkaloid fraction using HPLC method;

[0013] S32, MTT test was used to detect the effect of the alkaloid fraction of Aconite on the proliferation ability of tumor cells;

[0014] S33, Western blot technology was used to evaluate the effects of the alkaloid fractions of Aconite on the phosphorylation, nuclear translocation, and oligomerization of PKM2 in tumor cells;

[0015] S34. CETSA and DARTS techniques were used to analyze the effects of the alkaloid fractions of Aconite on the thermal stability and hydrolytic stability of PKM2 in tumor cells.

[0016] S35. ShRNA transfection technology was used to verify that the alkaloids of Aconitum carmichaelii inhibited the growth of tumor cells by stimulating PKM2.

[0017] As a preferred embodiment of the present invention, the screening method of hypaconitine comprises the following steps:

[0018] S41, using Autodock Vina to screen for aconite alkaloid components with stronger binding to PKM2 than the activator TEPP-46 from the aconite alkaloid site;

[0019] S42, using an MST test to detect the ability of the aconite alkaloid components screened out in step S41 to bind to PKM2, and obtaining aconite alkaloid components with strong binding ability;

[0020] S43. Using a pyruvate kinase activity test, the activation effect of the aconite alkaloid components with strong binding ability detected in step S42 on PKM2 was evaluated, and hypaconitine was screened out.

[0021] As a preferred solution of the present invention, the function verification method comprises the following steps:

[0022] S51, MTT test was used to detect the effect of hypaconitine on tumor cell proliferation;

[0023] S52, Western blot was used to evaluate the effects of hypoaconitine on the phosphorylation, nuclear translocation and oligomerization of PKM2 in tumor cells;

[0024] S53. Molecular dynamics simulation was used to analyze the binding mode of aconitine to PKM2.

[0025] The invention discloses an application of a parent structure of a PKM2 agonist in anti-tumor treatment, wherein the hypoaconitine has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers to dimers, enhancing the activity of PKM2 and inhibiting the nuclear translocation of PKM2; the parent structure of the hypoaconitine is composed of diterpene skeleton atoms, and its parent nucleus is C 19 Diterpene alkaloids; the chemical structural formula of the aconitine is:

[0026]

[0027] As a preferred embodiment of the present invention, the parent structure of the PKM2 agonist is used to correct glycolysis in tumor cells.

[0028] As a preferred embodiment of the present invention, the parent structure of the PKM2 agonist is used to inhibit the growth of lung cancer cells.

[0029] Beneficial Effects

[0030] Hypaconitine screened out by the present invention can increase the activity of pyruvate kinase, inhibit PKM2 phosphorylation, hinder its nuclear translocation, increase tetramer and reduce dimer form;

[0031] The present invention uses computer-aided drug design to screen PKM2 activating components in aconite alkaloids, and reports for the first time that hypaconitine has a PKM2 activating effect; it lays a certain foundation for the study of the anti-tumor mechanism of aconite, provides a basis for the clinical application of aconite anti-tumor, and provides a new parent structure for the research and development of PKM2 agonists. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effect of the alkaloid fraction of Radix Aconiti Lateralis Preparata on the viability of lung cancer cells;

[0033] Figure 2 The effect of the alkaloid fraction of Radix Aconiti Lateralis Preparata in the present invention on the nuclear and cytoplasmic localization of PKM2 in lung cancer cells;

[0034] Figure 3 The effect of the alkaloid part of Radix Aconiti Lateralis Preparata in the present invention on the oligomeric state of PKM2 in lung cancer cells;

[0035] Figure 4 The effect of the alkaloid fraction of Radix Aconiti Lateralis Preparata on the expression of PKM2 protein in lung cancer cells;

[0036] Figure 5 The effect of the aconite alkaloid part on the nuclear translocation of PKM2 in the present invention;

[0037] Figure 6 The effect of the alkaloid fraction of Radix Aconiti Lateralis Preparata on the thermal stability of PKM2 protein in lung cancer cells;

[0038] Figure 7 The effect of the alkaloid fraction of Radix Aconiti Lateralis Preparata on the stability of PKM2 protein in lung cancer cells;

[0039] Figure 8 The effect of knocking down PKM2 expression in lung cancer cells in the present invention on the inhibition of lung cancer cell viability by the aconite alkaloid site;

[0040] Fig. 9The left side is the molecular docking diagram of Crassicauline A and 4G1N in the present invention, wherein: A1 is the binding mode diagram, and A2 is the two-dimensional action mode diagram; the right side is the molecular docking diagram of TEPP-46 and 4G1N in the present invention, wherein: B1 is the binding mode diagram, and B2 is the two-dimensional action mode diagram;

[0041] Fig.10 This is the binding curve between the alkaloid component of Radix Aconiti Lateralis Preparata and the PKM2 protein in the present invention;

[0042] Fig.11 The effects of the various components of the aconite alkaloids in the present invention on the activity of PKM2 kinase;

[0043] Fig.12 The effect of the hypaconitine in the present invention on the PKM2 kinase activity in lung cancer cells;

[0044] Fig.13 The effect of the hypaconitine in the present invention on the viability of lung cancer cells;

[0045] Fig.14 The effect of the hypaconitine in the present invention on the expression of PKM2 protein in lung cancer cells;

[0046] Fig.15 The effect of the hypaconitine in the present invention on the nuclear and cytoplasmic localization of PKM2 in lung cancer cells;

[0047] Fig.16 The effect of the hypoaconitine in the present invention on the oligomeric state of PKM2 in lung cancer cells;

[0048] Fig.17 The effect of the hypaconitine in the present invention on the nuclear translocation of PKM2;

[0049] Fig.18 It is a molecular dynamics simulation diagram of the interaction between hypaconitine and PKM2 protein in the present invention, wherein: A is the root mean square variance curve of the hypaconitine-PKM2 complex; B is the root mean square fluctuation curve of the hypaconitine-PKM2 complex; C is the protein gyration radius curve of the hypaconitine-PKM2 complex; D is the solvent accessible surface area curve of the hypaconitine-PKM2 complex; E is the hydrogen bond analysis diagram of the hypaconitine-PKM2 complex.

[0050] Fig.19 Schematic diagram of the experimental process and molecular mechanism for screening the PKM2 agonist aconitine from the alkaloid part of aconite in the present invention. DETAILED DESCRIPTION

[0051] The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] As an embodiment of the present invention, a parent structure of a PKM2 agonist, wherein the PKM2 agonist is aconitine screened from the alkaloid part of aconite, and its chemical formula is C 33 H 45 NO 10 The hypoaconitine has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers to dimers, enhancing the activity of PKM2 and inhibiting the nuclear translocation of PKM2; the parent structure of the hypoaconitine is composed of diterpene skeleton atoms, and its parent nucleus is C 19 Diterpene alkaloids; the chemical structural formula of the aconitine is:

[0053]

[0054] As an embodiment of the present invention, the hypaconitine is screened out from aconite alkaloids by computer-aided drug design.

[0055] As an embodiment of the present invention, a method for screening the parent structure of a PKM2 agonist comprises the following steps:

[0056] Effects of the parts and components of Aconite alkaloids on PKM2 in tumor cells

[0057] 1. Methods

[0058] 1.1. Preparation and component analysis of Aconite alkaloids

[0059] Take about 200g of Heishun tablets powder, add 4 times the amount of 85% ethanol solution and soak for 24h, add 4 times the amount of 85% ethanol solution to the residue, heat and reflux to extract 4 times, 2h each time, recover ethanol, add distilled water to make a 10g / mL mother liquor. D101 resin is used to purify total alkaloids, the sample volume flow rate is 1.5mL / min, 5BV distilled water is used to wash away impurities, and 8BV ethanol is eluted at a volume flow rate of 1mL / min. The HPLC method is used to determine the content of the alkaloid part of Fuzi.

[0060] 1.2. MTT method to study the effect of aconite alkaloids on tumor cell proliferation

[0061] Lung cancer cells were inoculated in 96-well plates. After overnight culture, the alkaloid parts and components of Aconite were added and incubated for 24, 48 and 72 hours respectively. After the culture was completed, the medium was discarded and MTT solution was added to each well. The culture was continued for 4 hours. After discarding, DMSO was added and shaken for 10 minutes. The absorbance was measured and the inhibition rate of the drug on cell viability was calculated.

[0062] 1.3. Western blot technique to detect the effect of aconite alkaloids on PKM2 protein nuclear translocation

[0063] Lung cancer cells were inoculated in 6-well plates and cultured overnight. Different concentrations of aconite alkaloid parts and components were added and incubated for 24 hours. The cytoplasmic protein and nuclear protein in the cells were extracted using a nuclear and cytoplasmic protein extraction kit, and the protein concentration was determined using a BCA kit. Western blot technology was used to analyze the effects of aconite alkaloid parts and components on the nuclear translocation of PKM2 protein.

[0064] 1.4. Glutaraldehyde cross-linking method to detect the effect of aconite alkaloids on the oligomeric state of PKM2 protein

[0065] Lung cancer cells were inoculated in 6-well plates, cultured overnight, and then different concentrations of aconite alkaloid parts and components were added and incubated for 24 hours. The protein concentration of the extracted protein lysate was determined using a BCA kit; 0.025% glutaraldehyde was added to each 4 mg / mL protein; incubated in a 37°C water bath for 3 minutes; 1M Tris-HCl (pH 8.0) was added to terminate the reaction to a final concentration of 50mM; the protein loading amount was 50μg, and an appropriate amount of loading buffer was added, and the mixture was denatured in a boiling water bath for 10 minutes, and then Western blot technology was used to analyze the effects of aconite alkaloid parts and components on the oligomeric state of PKM2 protein.

[0066] 1.5 Immunofluorescence detection of the effect of aconite alkaloids on PKM2 protein nuclear translocation

[0067] Lung cancer cells were inoculated in 12-well plates and treated with different concentrations of aconite alkaloid parts and components for 24 hours. Wash with PBS and add pre-cooled 4% paraformaldehyde, fix at 4°C for 30 minutes, add pre-cooled PBS to wash 3 times, add appropriate amount of 0.2% TritonX-100, shake for 10 minutes; discard TritonX-100, add 1% BSA solution and shake at room temperature for 30 minutes; wash with pre-cooled PBS 3 times, 10 minutes each time. Incubate cells with primary antibody at 4°C overnight, and then incubate with fluorescent secondary antibody in the dark at room temperature for 2 hours. Seal the slices directly with anti-fluorescence quenching sealing agent containing DAPI, and then take pictures with a fluorescence microscope.

[0068] 1.6. CETSA evaluation of the effect of aconite alkaloids on the thermal stability of PKM2

[0069] Lung cancer cells were taken, washed twice with pre-cooled PBS, and lysed on ice for 30 minutes with RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. The supernatant was centrifuged and divided into two equal parts. 100 μg / mL of aconite alkaloid fraction or DMSO was incubated with the cell lysate supernatant at room temperature for 1 hour. The respective lysates were divided into 10 equal parts, each with 50 μL, and heated at different temperatures of 42, 45, 48, 51, 54, 57, 60, 63, 66, and 69°C for 3 minutes, and cooled at room temperature for 3 minutes. Centrifuged, 30 μL of supernatant was added to 5× loading buffer, and boiled for 10 minutes for Western blot analysis.

[0070] 1.7. DARTS evaluation of the effect of aconite alkaloids on the proteolytic stability of PKM2

[0071] Take lung cancer cells, wash the cells twice with ice-cold PBS, add 200μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, and lyse on ice for 30min. Centrifuge and take the supernatant and incubate with different concentrations of aconite alkaloid fractions at room temperature for 1h. After incubation, add 10mg / mL pronase E to treat the sample (enzyme / protein = 1:800), mix well and hydrolyze at 25℃ for 20min. Add various protease inhibitor mixtures to stop the reaction. Take 30μL of sample and add 5× loading buffer, boil for 10min for SDS-PAGE and Western blot analysis.

[0072] 1.8. Effect of knocking down PKM2 expression in lung cancer cells by cell transfection technology on the inhibitory effect of aconite alkaloids on lung cancer cells

[0073] Lung cancer cells were cultured in 12-well plates, and polybrene was added when they grew to 50%. Lentivirus particles were thawed on ice, gently mixed and set aside. shPKM2 and shControl lentivirus particles were added to the culture medium to infect the cells and incubated overnight. The culture medium was discarded and replaced with complete culture medium without polybrene, and the cells were cultured overnight. Lung cancer cells that stably expressed shPKM2 were selected using 0.1 μg / mL puromycin hydrochloride, and fresh culture medium containing puromycin was replaced every 3 days until a resistant cell population could be identified. Several cells were selected, expanded and subjected to Western blot analysis to obtain lung cancer cells that stably expressed shPKM2. The role of PKM2 in the inhibition of aerobic glycolysis in lung cancer cells was further verified.

[0074] 1.9. Molecular docking technology predicts the binding ability of aconite alkaloid components to PKM2

[0075] According to the resolution of protein crystals, co-crystallization ligands and mutations, a suitable protein structure is selected. The reference of the present invention selects 4G1N protein based on the investigation results of wild-type PKM2 protein crystals in the PDB database. Pymol software is used to remove water molecules and ligand NZT in protein crystals, and Auto Dock Tools 1.5.6 software is used to add non-polar hydrogen and calculate Gasteiger charge. The 3D structure of the ligand small molecule is searched from Pubchem, and the small molecule that cannot directly obtain the 3D structure uses ChemDraw to convert its 2D structure into a 3D structure. The molecular docking uses AutodockVina software, selects 4G1N protein as a receptor, and uses 45 kinds of aconite alkaloid components screened in the preliminary study of this topic as ligands. The present invention sets the docking box Gridbox coordinates according to the binding site of the original ligand NZT of the 4G1N protein in the PDB database, that is, center_x=1.359, center_y=-12.452, center_z=46.87, and other parameters are default. The Lamarckian algorithm was used for molecular docking, and PyMOL was used for visualization analysis.

[0076] 1.10. MST technology to determine the binding ability of alkaloid components of Aconitum carmichaelii to PKM2

[0077] Each compound of aconite alkaloid prepared in DMSO was diluted to 2 mM with PBST solution, and 16 PCR tubes were taken and numbered 1 to 16 respectively; 20 μL of 2 mM compound solution was added to tube 1, and 10 μL of PBST was added to PCR tubes 2 to 16; 10 μL of the solution in tube 1 was pipetted into tube 2, and mixed by pipetting, and gradient dilution was performed at a volume ratio of 1:1 until tube 16, and the gradient dilution was completed; then 10 μL of protein-dye mixture was added to each tube, and gently mixed, centrifuged to remove bubbles, and the sample was aspirated with a capillary and then tested on the machine.

[0078] 1.11 Effects of Aconite alkaloids on the pyruvate kinase activity of PKM2 recombinant protein

[0079] Pyruvate kinase catalyzes phosphoenolpyruvate (PEP) and ADP to generate ATP and pyruvate, and lactate dehydrogenase (LDH) further catalyzes NADH and pyruvate to generate lactate and NAD + The kinase activity can be reflected by measuring the decreasing rate of NADH at 340 nm. Based on this principle, the pyruvate kinase activity detection kit (Beijing Solebow Technology Co., Ltd.) was selected for detection in this experiment.

[0080] 1.12 Effects of Aconite Alkaloids on Intracellular Pyruvate Kinase Activity

[0081] Lung cancer cells in the logarithmic growth phase were inoculated in a 6-well plate. After overnight culture, the culture medium was discarded, and different concentrations of aconitine (50, 100, 200 μM) were added and cultured for 24 hours. The culture medium was discarded, and the cells were washed with pre-cooled PBS and then added with the extract. After ultrasonic disruption of the cells, the supernatant was centrifuged to obtain the cell lysate. The effect of aconitine on the activity of PKM2 in the cells was then detected according to the instructions of the pyruvate kinase activity detection kit.

[0082] 1.13 Molecular dynamics simulation analysis of the binding ability of aconite alkaloids to PKM2

[0083] Gromacs 2022.3 software was used to analyze the binding ability of alkaloid components of Aconite to PKM2. AmberTools22 was used to add GAFF force field to small molecules, and Gaussian 16W was used to calculate the RESP potential of small molecules. Proteins and small molecules were simulated using gmx grompp and gmx mdrun commands. The simulation conditions were set to a static temperature of 300K and normal pressure (1Bar), using the Amber99sb-ildn force field and water molecules (Tip3p water model) as the model. By adding an appropriate amount of Na + ions to neutralize the total charge of the simulation system. The simulation system was energy minimized by the steepest descent method, followed by isothermal isochoric ensemble (NVT) equilibrium and isothermal isobaric ensemble (NPT) equilibrium, with a coupling constant of 0.1ps and a duration of 100ps. Finally, a free molecular dynamics simulation was run, with a total of 5,000,000 steps, a step size of 2fs, and a total duration of 100ns.

[0084] 2. Experimental Results

[0085] 1. Effects of Aconite alkaloid fractions on tumor cells and PKM2

[0086] 1.1. Content of mono- and diester alkaloids in the alkaloid fraction of Aconite

[0087] Six batches of alkaloid fractions from Radix Aconiti Lateralis Preparata were prepared repeatedly, and the contents of monoester and diester alkaloids were determined. The results are as follows: the content of monoester alkaloids was 851.37±30.23μg / g, the content of diester alkaloids was 67.19±5.27μg / g, and the total content of these five components was 918.56±35.69μg / g. As shown in Table 1.

[0088] Table 1 Contents of mono- and diester-type alkaloids in alkaloid parts of different batches of Aconite (μg / g)

[0089]

[0090] 1.2 Effects of Aconite alkaloid fractions on tumor cell proliferation

[0091] like Figure 1 As shown, when NCI-H460 cells were incubated with different concentrations of aconite alkaloid fractions for 24, 48 and 72 hours, the inhibition rate increased significantly, such as 10.94μg / mL to 700μg / mL aconite alkaloid fractions were treated for 24, 48 and 72 hours, the inhibition rate increased from 12.43% to 69.55%, from 26.78% to 91.60% and from 41.88% to 99.16%, respectively. When NCI-H1299 cells were incubated with different concentrations of aconite alkaloid fractions for 24, 48 and 72 hours, the inhibition rate increased significantly, such as 10.94μg / mL to 700μg / mL aconite alkaloid fractions were treated for 24, 48 and 72 hours, the inhibition rate increased from 28.47% to 84.98%, from 17.79% to 92.75% and from 26.29% to 98.06%, respectively. When A549 cells were incubated with different concentrations of aconite alkaloid fractions for 24, 48 and 72 h, the inhibition rate change trend was consistent with that of NCI-H460 and NCI-H1299. For example, when treated with 10.94 μg / mL to 700 μg / mL aconite alkaloid fractions for 24, 48 and 72 h, the inhibition rate increased from -1.08% to 11.14%, from 7.48% to 73.51%, and from 5.08% to 88.59%, respectively.

[0092] 1.3 Effects of Aconite alkaloid fractions on the nuclear and cytoplasmic localization of PKM2 in lung cancer cells

[0093] PKM2 usually exists in the cytoplasm as a tetramer and acts as a metabolic kinase with high catalytic activity; in the nucleus, the dimeric PKM2 can phosphorylate the Y705 site of STAT3, enhance the transcriptional activity of STAT3, promote aerobic glycolysis, and regulate the growth, survival and metastasis of tumor cells as a protein kinase that stimulates transcription. The effect of the alkaloid part of Aconite on the cytoplasm and nuclear PKM2 of A549 was analyzed by Western blot technology. Figure 2 As shown in the figure, after treatment with the alkaloid part of Aconitum carmichaelii, the PKM2 in the cytoplasm of A549 was significantly increased, and the PKM2 in the nucleus was significantly decreased, and the downstream protein p-STAT3 was also significantly decreased, indicating that the alkaloid part of Aconitum carmichaelii affects the localization of PKM2 in the cytoplasm and nucleus, affects its dimer / tetramer existence form, and inhibits transcriptional activity.

[0094] 1.4 Effects of Aconite alkaloid fractions on PKM2 oligomerization in lung cancer cells

[0095] The balance between the dimer and tetramer conformations of PKM2 is crucial for the occurrence and development of tumors. The results of the glutaraldehyde cross-linking experiment showed that the alkaloid site of Aconitum inhibited the dimerization of PKM2 in lung cancer cells and promoted the formation of tetramers. Activating PKM2 and converting it from a dimer state to a highly active tetramer form can allow more glucose metabolic intermediates to enter the energy-generating pathway instead of turning to anabolism, reduce lactic acid production, reverse the Warburg effect, and thus prompt cells to resume normal metabolic activities. At the same time, the PKM2 activator TEPP-46 was used as a positive control. After treatment with TEPP-46, the PKM2 dimer decreased and the tetramer increased. Figure 3 As shown, the results of cell viability assay indicated that TEPP-46 treatment also aggravated the inhibitory effect of the alkaloid fraction of Aconite on the proliferation of lung cancer cells.

[0096] 1.5 Effects of Aconite alkaloid fractions on PKM2 protein expression in tumor cells

[0097] like Figure 4 As shown in the figure, after treatment with the concentration of the alkaloid fraction of Aconite, the expression level of p-PKM2 protein in lung cancer cells decreased significantly, indicating that the alkaloid fraction of Aconite can significantly inhibit the expression of p-PKM2 protein. The study found that PKM2 tyrosine 105 (Y105) phosphorylation reduces the activity of PKM2 pyruvate kinase by destroying the formation of PKM2 tetramers; eliminating Y105 phosphorylation can increase the activity of PKM2 pyruvate kinase, reduce the production of lactic acid, increase oxygen consumption, and inhibit the formation of xenograft tumors. The experiment showed that the alkaloid fraction of Aconite can significantly inhibit the phosphorylation of PKM2 (Y105), indicating that the alkaloid fraction of Aconite increases the activity of PKM2 pyruvate kinase, thereby correcting tumor glycolysis.

[0098] 1.6 Effects of Aconite alkaloid fractions on PKM2 nuclear translocation in lung cancer cells

[0099] We then used immunofluorescence assays to further explore whether the alkaloid fraction of Aconite could affect the nuclear translocation of PKM2. Figure 5 As shown, compared with the blank control group, with the increase of the concentration of the aconite alkaloid fraction, the PKM2 in the cell nucleus was significantly reduced, and PKM2 was mainly present in the cytoplasm, indicating that the aconite alkaloid fraction inhibited the nuclear translocation of PKM2 in lung cancer cells, which was consistent with the above Wenternblot results.

[0100] 1.7. CETSA evaluates the effect of the alkaloid fraction of aconite on the thermal stability of PKM2 protein in lung cancer cells. CETSA is often used to determine the target. In order to further verify that the alkaloid fraction of aconite directly binds to PKM2 to stabilize its structure, this part of the experiment used a method combined with CETSA and Western blot technology. The results of CETSA showed that compared with the DMSO control group, the alkaloid fraction of aconite at 100 μg / mL was Figure 6 As shown, there are obvious differences in the PKM2 protein bands within the temperature range of 42-69°C, the corresponding melting curve shifts significantly to the right, and the Tm also increases from 49.76°C to 57.99°C, indicating that co-incubation of cell lysate with the aconite alkaloid fraction can prevent the thermal denaturation of PKM2 at various temperatures.

[0101] 1.8. DARTS evaluation of the effects of aconite alkaloid fractions on the proteolytic stability of PKM2 in lung cancer cells

[0102] DARTS test results, such as Figure 7 As shown in the figure, compared with the control group without pronase E, pronase E had a significant hydrolysis effect on the protein (p < 0.05). After treatment with different concentrations of aconite alkaloid fractions and cell lysate for 1 hour, the PKM2 protein band was higher than that in the DMSO group, indicating that the aconite alkaloid fraction can inhibit the hydrolysis of PKM2 protein in a concentration-dependent manner, suggesting that the aconite alkaloid fraction can bind to the PKM2 protein to improve the stability of the protein during the hydrolysis process.

[0103] 1.9. shRNA transfection technology verifies that the alkaloids of Aconite inhibit tumor cell growth by stimulating PKM2

[0104] To further verify the role of PKM2 in the inhibition of aerobic glycolysis in lung cancer cells by aconite alkaloids, we used cell transfection technology to knock down the expression of PKM2 in lung cancer cells. Figure 8 As shown in the figure, the expression of PKM2 was reduced by PKM2-shRNA, indicating that the expression of PKM2 was successfully knocked down in lung cancer cells (p < 0.01). The shPKM2 and shControl cells were treated with the aconite alkaloid fraction, and the cell viability was detected. The results showed that in lung cancer cells with PKM2 knockdown, the cell proliferation inhibition after treatment with the aconite alkaloid fraction was significantly reversed (p < 0.01). The above results indicate that PKM2 is involved in the inhibition of lung cancer cell proliferation by the aconite alkaloid fraction.

[0105] 1.10 Summary and Discussion

[0106] After testing, the content of monoester alkaloids was 851.37±30.23μg / g, the content of diester alkaloids was 67.19±5.17μg / g, and the total content of these five components was 918.56±34.83μg / g. In addition, the RSD of the content of each component in different batches of aconite alkaloids was less than 10%, indicating that the content of each component in different batches of alkaloids was slightly different, and the preparation process was stable and feasible. The results of the MTT test showed that the aconite alkaloid site inhibited the proliferation of five lung cancer cells, including NCI-H1299, A549, NCI-H1975, NCI-H1650, and NCI-H260, in a concentration-dependent manner, and inhibited the proliferation of NCI-H1299, A549, and NCI-H460 cells in a time-dependent manner. Western blot analysis and immunofluorescence test results showed that the alkaloids of Aconite inhibited the phosphorylation of PKM2 protein in lung cancer cells, inhibited the conversion of tetramers to dimers, increased tetramers, enhanced PKM2 activity, inhibited PKM2 nuclear translocation, thereby correcting tumor cell glycolysis and ultimately exerting an anti-lung cancer effect. The target stability test of cell thermal shift and drug affinity reaction showed that there was an obvious interaction between the alkaloids of Aconite and PKM2 protein. Cell transfection technology verified that the alkaloids of Aconite exerted anti-lung cancer effects by targeting PKM2. In summary, the alkaloids of Aconite can effectively inhibit the growth of lung cancer. The mechanism may be that the alkaloids of Aconite bind to PKM2, reduce PKM2 protein phosphorylation, reduce the conversion of tetramers to dimers, and inhibit PKM2 nuclear translocation, thereby correcting tumor cell glycolysis and ultimately exerting an anti-lung cancer effect. This article takes the regulation of glycolysis by the key kinase PKM2 as the starting point to explore the new anti-lung cancer mechanism of the alkaloid part of Aconitum carmichaelii, provide a scientific basis for its clinical application, provide a foundation for future preparation development, and provide new ideas for the anti-tumor research of warming and yang-tonifying drugs.

[0107] 2. Effects of Aconite alkaloid components on PKM2 in tumor cells

[0108] 2.1 Molecular docking technology predicts the binding ability of aconite alkaloid components to PKM2

[0109] Binding free energy (ΔGbind) was selected as the analysis index of docking results. The smaller the ΔGbind, the more stable the binding of drug small molecules to protein macromolecule receptors. Using AutoDock Vina molecular docking software, 45 kinds of aconite alkaloid components were molecularly docked with PKM2 protein. The docking results are shown in Table 2. It is generally believed that when the docking binding energy is less than -7.0 kcal / mol, the small molecule is considered to have good binding with the target protein. As shown in Table 2, all 45 kinds of aconite alkaloid components can achieve good binding with PKM2 protein; at the same time, the known PKM2 agonist TEPP-46 was docked with 4G1N protein, and its binding force was -8.7 kcal / mol. Based on this standard, the binding ability of aconite alkaloids with 4G1N protein was screened. A total of 34 compounds had better binding ability with 4G1N protein than TEPP-46. Among them, aconitine A, inertin, delphinidin, neoaconitine, and jungaraconitamine had better binding ability.

[0110] like Fig. 9 As shown in Figures A1 and A2 in Figure 1, aconitine A forms hydrogen bond interactions with amino acid residues such as Lys311B, Leu353A, and Asn350B, forms hydrophobic interactions such as π-sigma, alkyl or π-alkyl with Ile389A, Met30A, Leu394B, Phe26B, and Phe26A, and forms electrostatic interactions with Asp354B. Fig. 9 As shown in Figures B1 and B2 in , TEPP-46 forms hydrogen bond interactions with Asn350B, Asp354B, Asp354A, and Leu27B amino acid residues, forms π-cation electrostatic interactions with Lys311B, forms π-π or π-alkyl hydrophobic interactions with Phe26B and Met394A, and forms weak van der Waals forces with Leu353B, Lys311A, Asn350A, Leu353A, Ile389A, Phe26A, Tyr390A, and Gln393A.

[0111] Table 2 Binding free energy of alkaloid components of Aconite and PKM2

[0112]

[0113] 2.2 MST determination of the binding ability of alkaloid components of Aconitum carmichaelii to PKM2

[0114] In order to verify whether there is interaction between 14 alkaloid components and PKM2 recombinant protein and the strength of the interaction, the present invention conducts MST test on 14 alkaloid compounds and PKM2 protein. The binding ability of each alkaloid component with PKM2 is at the micromolar level, indicating that the binding ability with PKM2 protein is good. Among them, the binding ability of aconitine and mesaconitine reaches the nanomolar level, which is 0.05±0.03μM and 0.09±0.01μM. The binding ability of aconitine, benzoyl aconitine, songguoling, and aconitine with PKM2 is at the same level, which are 1.36±0.69μM, 1.72±0.46μM, 5.05±5.13μM, and 9.42±2.78μM, respectively. The binding forces of benzoylhypaconitine, benzoylmeaconitine, hypaconitine, and neorin to PKM2 were 18.70±4.38μM, 19.77±6.44μM, 27.23±7.90μM, and 52.53±24.95μM, respectively. The binding forces of talasamine, aconitine, demethylsaccharine, and aconitine to PKM2 were 320.00±131.06μM, 331.33±161.00μM, 352.00±120.22μM, and 483.33±37.19μM, respectively. Fig.10 As shown, the binding curves of neoaconitine, hypoaconitine and aconite with PKM2 protein, the binding curves of the three components fit well with PKM2.

[0115] 2.3. Study on the activation effect of each component of aconite alkaloids on PKM2

[0116] like Fig.11 As shown in the figure, after the 14 alkaloid components were incubated with PKM2 recombinant protein at a concentration of 10 μM for 1 hour, aconitine, benzoylaconitine and mesaconitine activated the PKM2 recombinant protein, and mesaconitine had the strongest activation effect on PKM2. Table 3 shows the dissociation constants, activation effects and lung cancer cell inhibition rates of each alkaloid component with PKM2. The pyruvate kinase activity of normal lung cancer cells is 907.85±50.25U / mgprot. As shown in Table 3, aconitine and benzoylaconitine have PKM2 activation effects, but have little effect on lung cancer cell growth; demethylsaccharine has a strong inhibitory effect on lung cancer, but has no PKM2 activation effect; and mesaconitine has the strongest ability to activate PKM2 and has certain anti-tumor activity, which may be the effective component of aconite alkaloids that exerts anti-tumor effects through PKM2.

[0117] Table 3 Dissociation constants, activation abilities and cell inhibition rates of each component of Aconite alkaloids with PKM2 (100 μM)

[0118]

[0119]

[0120] 2.4 Activation of PKM2 by Hypoaconitine in Lung Cancer Cells

[0121] Next, we continued to investigate whether hypaconitine has an activating effect on PKM2 in lung cancer cells. After treating lung cancer cells with different concentrations of hypaconitine for 24 hours, the activity of pyruvate kinase in the cells was detected. Fig.12 As shown, compared with the blank group, the pyruvate kinase activity of lung cancer cells was significantly enhanced after being treated with hypaconitine, and was concentration-dependent, indicating that hypaconitine can enhance the pyruvate kinase activity in lung cancer cells.

[0122] 2.5. Inhibitory effect of hypoaconitine on lung cancer cell proliferation

[0123] In order to study the effect of hypaconitine on the proliferation of lung cancer cells, A549 and NCI-H1299 cells were treated with different concentrations of hypaconitine, and the inhibition rate was detected and calculated using the MTT method. As shown in Table 4, Fig.13 As shown in the results, the inhibition rate of 12.5μM-200μM hypoaconitine on A549 and NCI-H1299 cells was between 20% and 30%, and the 48h IC50 of hypoaconitine on A549 and NCI-H1299 cells was 302.60μM and 545.10μM, respectively. Therefore, the above results show that hypoaconitine has a certain inhibitory effect on the proliferation of lung cancer cells

[0124] Table 4 Effect of aconitine on the growth inhibition rate of A549 and H1299 cells

[0125]

[0126] 2.6 Effect of Hypoaconitine on Intracellular PKM2 Protein Expression

[0127] After treating lung cancer cells with different concentrations of hypaconitine for 24 h, the changes in PKM2 protein expression were detected by Western blot. Fig.14 As shown in the results, hypoaconitine had no significant effect on the expression of total PKM2 protein, but inhibited the phosphorylation of PKM2 protein at the amino acid site Tyr105. It has been reported that the activity of PKM2 protein phosphorylated at the amino acid site Tyr105 is lower than that of PKM2 in its physiological state.

[0128] 2.7 Effect of Hypoaconitine on Nuclear and Cytoplasmic Localization of PKM2 in Lung Cancer Cells

[0129] PKM2 usually exists in the cytoplasm as a tetramer and has a high catalytic activity as a metabolic kinase. PKM2 exists in the nucleus as a dimer and acts as a protein kinase that stimulates transcription to regulate the growth, survival and metastasis of tumor cells. The effect of hypaconitine on PKM2 in the cytoplasm and nucleus of lung cancer was analyzed by Western blot. Fig.15 As shown, after treatment with hypoaconitine, the PKM2 in the nucleus of lung cancer cells was significantly reduced, while there was no obvious change in PKM2 in the cytoplasm, indicating that hypoaconitine affects the expression of PKM2 in the cell nucleus.

[0130] 2.8 Effect of Hypoaconitine on the Oligomeric State of PKM2 in Lung Cancer Cells

[0131] PKM2 can exist in tetrameric, monomeric or dimer forms. The tetrameric form is believed to have pyruvate kinase activity, while the dimer has protein kinase activity. Fig.16 As shown, hypoaconitine can increase the tetramer form of PKM2 and reduce its dimer form in lung cancer cells, indicating that hypoaconitine can reduce the conversion of PKM2 tetramer to dimer.

[0132] 2.9 Effect of Hypoaconitine on Nuclear Translocation of PKM2 Protein in Lung Cancer Cells

[0133] The results are as follows Fig.17 As shown in the figure, in the blank control group, the boundary between the nucleus and the cytoplasm was unclear, and the green fluorescence around the nucleus was stronger, while after treatment with hypoaconitine, the boundary between the nucleus and the cytoplasm was clear, and the green fluorescence was evenly distributed in the cytoplasm. This indicates that hypoaconitine can inhibit PKM2 nuclear translocation, which is consistent with the above Western blot results.

[0134] 2.10 Molecular dynamics simulation to explore the binding mode of hypoaconitine and PKM2

[0135] RMSD is an indicator for evaluating protein structural changes, indicating the distance between the same atoms in different structures. The trend of RMSD changes is an important indicator for determining whether the simulation has reached stability. Fig.18 As shown in Figure A, RMSD gradually stabilizes within 76-100ns and stabilizes between 0.37-0.48nm, indicating that after 100ns of simulation, the aconitine-PKM2 system has reached equilibrium and the RMSD is less than 1nm. RMSF is an indicator for evaluating protein dynamics, which calculates the change in each atom relative to its average position. Amino acids far away from the active site will show higher RMSF values. At the same time, RMSF is also related to protein flexibility. The more flexible the region, the larger the RMSF value. Fig.18As shown in Figure B, the amino acid residues 20-30 and 300-350 of the PKM2-hypoaconitine complex fluctuate slightly, and the fluctuation is small at the binding site, while there is a large fluctuation at residues 100-200, indicating that the amino acid residues 100-200 have greater flexibility. It can be seen that the binding of small molecules has a certain impact on the stability of the protein. Fig.18 As shown in Figure C, the Rg of the complex gradually stabilizes, indicating that the overall structure of the complex gradually becomes tighter and more robust. SASA is used to evaluate the surface area of ​​protein molecules exposed to the solution. It calculates the solvent accessible surface area of ​​each atom in the protein and evaluates the interactions between them and the stability of the protein structure. Fig.18 As shown in Figure D, after 60-100 ns, the SASA of the PKM2 and aconitine binding system gradually stabilizes. Hydrogen bonding is a strong non-covalent interaction, and HBond is used to evaluate the number of hydrogen bonds within a protein. Fig.18 As shown in Figure E, PKM2 and the aconitine complex form stable hydrogen bonds within 0-50ns, and up to 4 hydrogen bonds can be formed, indicating that a strong interaction can be formed between PKM2 and aconitine.

[0136] 2.11 Summary and Discussion

[0137] Studies have shown that PKM2 exists mainly in the form of low-activity dimers in tumor cells. Although this low-activity state reduces the energy produced by glycolysis, it is more likely to accumulate intermediates in the glycolysis process, meeting the needs of rapid tumor proliferation. The dimer form of PKM2 translocates to the cell nucleus and regulates the growth of tumor cells as a protein kinase that stimulates transcription. The tetramer form of PKM2 has a high affinity for PEP, has pyruvate kinase activity, participates in enzyme catalysis reactions and produces energy. Therefore, increasing PKM2 tetramers, improving pyruvate kinase activity, and reducing dimers to inhibit protein kinase action have become potential ways to inhibit tumor cell growth. Fig.19 As shown, the present invention extracts and purifies the alkaloid fraction of Aconitum carmichaelii, further screens the PKM2 agonist components in Aconitum carmichaelii alkaloids, and finds that hypaconitine can inhibit PKM2 phosphorylation, hinder the conversion of tetramer to dimer, and inhibit PKM2 nuclear translocation.

[0138] Computer-aided drug design can not only analyze the physicochemical interactions between molecules and targets, but also accelerate the discovery of new bioactive compounds. Hypoaconitine is a type of diester alkaloid in Aconite, and is often used as a quality control component in Aconite. PKM2 in tumor cells is often in a low-activity state, which causes the accumulation of glycolysis intermediates, resulting in these products entering lipid and protein synthesis, and producing more biomacromolecules to meet the needs of rapid tumor proliferation. Therefore, increasing the activity of PKM2 pyruvate kinase and allowing more glucose metabolism intermediates to enter energy generation rather than anabolism has become a potential way to inhibit tumor cell growth. The present invention molecularly docks the known PKM2 ligand TEPP-46 with the 4G1N protein, and a total of 34 Aconite alkaloid components have better binding to PKM2 than TEPP-46. And the binding curves of the alkaloid components and the PKM2 protein are well fitted, indicating that the 14 Aconite alkaloid components have a direct binding effect with the PKM2 recombinant protein. Hypoaconitine can increase the activity of PKM2 in cells, and it is dose-dependent. Pyruvate kinase activity detection found that hypoaconitine had the strongest PKM2 activation effect. Western blot experiments showed that hypoaconitine could increase the tetramer form of PKM2 in cells, reduce the dimer form, and hinder its nuclear translocation. Molecular dynamics simulation results showed that hypoaconitine and PKM2 could form a strong interaction, and the complex structure was stable. In summary, hypoaconitine could inhibit PKM2 phosphorylation, hinder the conversion of tetramer to dimer, and inhibit PKM2 nuclear translocation. Computer-aided drug design was used to screen PKM2 activating components in aconite alkaloids, and it was reported for the first time that hypoaconitine had PKM2 activation effect. This laid a certain foundation for the study of the anti-tumor mechanism of aconite, provided a basis for the clinical application of aconite anti-tumor, and provided a new parent structure for the research and development of PKM2 agonists.

Claims

1. A parent structure of a PKM2 agonist, wherein the PKM2 agonist is aconitine screened from the alkaloid part of aconite, and its chemical formula is C 33 H 45 NO 10 , characterized in that, The hypoaconitine has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers to dimers, enhancing the activity of PKM 2 and inhibiting the nuclear translocation of PKM2; the parent structure of the hypoaconitine is composed of diterpene skeleton atoms, and its parent nucleus is C 19 Diterpene alkaloids; the chemical structural formula of the aconitine is:

2. The parent structure of a PKM2 agonist according to claim 1, characterized in that: The aconite alkaloid part has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers into dimers, enhancing the activity of PKM2 and inhibiting the nuclear translocation of PKM2.

3. The parent structure of a PKM2 agonist according to claim 2, characterized in that: The method for determining the function comprises the following steps: S31, extracting the aconite alkaloid fraction from the aconite root, purifying it using D101 macroporous resin, and determining the content of the aconite alkaloid fraction using HPLC method; S32, MTT test was used to detect the effect of the alkaloid fraction of Aconite on the proliferation ability of tumor cells; S33, Western blot technology was used to evaluate the effects of the alkaloid fractions of Aconite on the phosphorylation, nuclear translocation, and oligomerization of PKM2 in tumor cells; S34. CETSA and DARTS techniques were used to analyze the effects of the alkaloid fractions of Aconite on the thermal stability and hydrolytic stability of PKM2 in tumor cells. S35. ShRNA transfection technology was used to verify that the alkaloids of Aconitum carmichaelii inhibited the growth of tumor cells by stimulating PKM2.

4. The parent structure of a PKM2 agonist according to claim 1, characterized in that: The screening method of hypaconitine comprises the following steps: S41, using Autodock Vina to screen for aconite alkaloid components with stronger binding to PKM2 than the activator TEPP-46 from the aconite alkaloid site; S42, using an MST test to detect the ability of the aconite alkaloid components screened out in step S41 to bind to PKM2, and obtaining aconite alkaloid components with strong binding ability; S43. Using a pyruvate kinase activity test, the activation effect of the aconite alkaloid components with strong binding ability detected in step S42 on PKM2 was evaluated, and hypaconitine was screened out.

5. The parent structure of a PKM2 agonist according to claim 1, characterized in that: The function verification method comprises the following steps: S51, MTT test was used to detect the effect of hypaconitine on tumor cell proliferation; S52, Western blot was used to evaluate the effects of hypoaconitine on the phosphorylation, nuclear translocation, and oligomerization of PKM2 in tumor cells; S53. Molecular dynamics simulation was used to analyze the binding mode of aconitine to PKM2.

6. Use of a parent structure of a PKM2 agonist in anti-tumor treatment, characterized in that: The hypoaconitine has the functions of inhibiting the phosphorylation of PKM2 protein in tumor cells, inhibiting the transformation of tetramers to dimers, enhancing PKM2 activity and inhibiting PKM2 nuclear translocation; the parent structure of the hypoaconitine is composed of diterpene skeleton atoms, and its parent nucleus is C 19 Diterpene alkaloids; the chemical structural formula of the aconitine is:

7. The use of the parent structure of a PKM2 agonist in anti-tumor according to claim 6, characterized in that: The parent structure of the PKM2 agonist is used to correct glycolysis in tumor cells.

8. The use of the parent structure of a PKM2 agonist in anti-tumor according to claim 7, characterized in that: The application of the parent structure of the PKM2 agonist in inhibiting the growth of lung cancer cells.

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