Nano glycopeptide activator based on PKM2 as well as preparation method and application of nano glycopeptide activator

Abstract

The invention relates to a PKM2-based nano glycopeptide activator and application thereof.The nano glycopeptide activator comprises three functional units, namely a PKM2 targeted activation unit R3, a self-assembly unit R2 and a response unit R1, and the chemical structure of the nano glycopeptide activator is shown in the formula (I). After administration, the nano glycopeptide activator can be enriched to a tumor site through targeting of sugar receptor transporter protein or an EPR effect; after entering tumor cells, the polypeptide is subjected to in-situ fibrosis deformation under the hydrolysis action of glycohydrolase, so that specific targeting and retention are realized; the nano-fiber with the PKM2 targeting activation unit can promote conversion of dimer PKM2 into tetramer PKM2, inhibit nucleation of PKM2, and prevent the DNA damage repair process of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a PKM2-based nanoglycopeptide activator, its preparation method, and its application. Background Technology

[0002] It is well known that cancer cells promote tumor growth through aerobic glycolysis, with pyruvate kinase 2 (PKM2) being a key intervention protein. PKM2 is a protein encoded by the human PKM2 gene and is an isoenzyme of glycolytic pyruvate kinase. PKM2 is expressed in various tissues and organs, such as the lungs, adipose tissue, retina, and pancreatic islets, as well as in all cells that synthesize large amounts of nucleic acids, such as normally proliferating cells, embryonic stem cells, and especially tumor cells. Dimerized PKM2 is mainly found in the nucleus of tumor cells and plays a crucial role in tumor cell proliferation, invasion, and metastasis. PKM2 can be activated by serine, inducing tetramerization, reducing glycolytic intermediates that can serve as biosynthetic precursors, preventing dimerized PKM2 from entering the nucleus, and inhibiting the STAT3 pathway. Therefore, activating PKM2 is considered an effective strategy for cancer treatment.

[0003] CN104817490A discloses a novel aminodithiocarbamate compound, its preparation method, and its application. This aminodithiocarbamate compound can target PKM2 and is an agonist of PKM2. It has been found that this type of compound produces an anti-tumor effect by preventing PKM2 from entering the cell nucleus, and has a good anti-tumor effect and good selectivity for tumor cells.

[0004] CN116249531A discloses compositions and methods for activating pyruvate kinase, and provides compositions and methods for using pyruvate kinase activators to treat diseases or conditions (e.g., eye diseases, blood disorders, or cancer).

[0005] However, there are currently few existing strategies for utilizing PKM2 activation. In recent years, the rapid development of nanomaterials has led to their widespread attention and deepening application areas. Therefore, it is very meaningful to develop a novel strategy for anti-tumor activity by activating PKM2. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a PKM2-based nanoglycopeptide activator, its preparation method, and its applications. This nanoglycopeptide activator can be selectively hydrolyzed by glycolytic enzymes, specifically targeting and accumulating at tumor sites. It effectively promotes the conversion of PKM2 dimers in tumor cells into tetramers, thereby altering the metabolic pathways of tumor cells and achieving anti-tumor and chemotherapy sensitization effects.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a PKM2-based nano-glycopeptide activator, the nano-glycopeptide activator comprising three functional units: a PKM2-targeting activation unit R3, a self-assembly unit R2, and a response unit R1, the chemical structure of which is shown in formula (I):

[0009]

[0010] Among them, R3 comes from an amino acid sequence that activates PKM2 tetramerization; R2 comes from a fibropeptide with multiple hydrogen bonds in the molecule; R1 comes from glucose, acetylglucosamine, mannose, fucose, galactose or folic acid; and X comes from serine or threonine.

[0011] This invention, through precise structural design, enables the nano-glycopeptide activator to accumulate at the tumor site after administration via glycoreceptor transport protein targeting or EPR effect. Upon entering tumor cells, it undergoes in-situ fibrosis and deformation through hydrolysis by glycolytic enzymes, achieving specific targeting and retention. The nanofibers containing PKM2 targeting activation units promote the conversion of dimer PKM2 into tetramer PKM2, inhibiting PKM2 nuclear translocation and preventing DNA damage repair processes in tumor cells. This enables the inhibition of glycolytic metabolic pathways in chemotherapy-resistant and highly metastatic solid tumors, achieving anti-tumor effects and enhancing sensitization to chemotherapy drugs.

[0012] This nano-glycopeptide activator can be stably dispersed in aqueous solution in the form of nanomicelles. After being hydrolyzed by glycolytic enzymes, it undergoes a fiber structure transformation. The nanomicelles have a particle size of 20-50 nm, the fibers have a diameter of 5-15 nm, and a length of 5-50 μm. They can be flexible or rigid, and can be solid or hollow.

[0013] Preferably, the amino acid sequence having the function of activating PKM2 tetramerization is a polypeptide with 6-10 amino acids (e.g., 6, 7, 8, 9, 10); its C-terminus and N-terminus are independently selected from H (histidine), K (lysine), or Dab; ​​the intermediate amino acid is independently selected from A (alanine), V (valine), L (leucine), I (isoleucine), F (phenylalanine), W (tryptophan), M (methionine), and S (serine); wherein Dab represents 2,4-diaminobutyric acid;

[0014] Preferably, the amino acid sequence having the function of activating PKM2 tetramerization has a binding constant Kd > 10 with PKM2. -5 Polypeptides.

[0015] More preferably, the amino acid sequence having the function of activating PKM2 tetramerization is selected from any one of the following:

[0016] (1) SEQ ID NO.1: HHHFFTTK; (2) SEQ ID NO.2: HFFFVK; (3) SEQ ID NO.3: KFFFVH; (4) SEQ ID NO.4: HFFTTH; (5) SEQ ID NO.5: KFTWWK; (6) SEQ ID NO.6: HLLTWH; (7) SEQ ID NO.7: KFVVVWK; (8) SEQ ID NO.8: HFFFFK; (9) SEQ ID NO.9: KFVWWK; (10) SEQ ID NO.10: HFSTWH; (11) SEQ ID NO.11: HTSAWFH; (12) SEQ ID NO.12: HFSWWK; (13) SEQ ID NO.13: HFFWWVKKH; (14) SEQ ID NO.14: HFFTTIHH; (15) SEQ ID NO.15: HFFLTH; (16) SEQ ID NO.16: HFFWWK; (17) SEQ ID NO.17: HFFLSH; (18) SEQ ID NO.18: HFFTVH; (19) SEQ ID NO.19: KFTWWK; (20) The product of the connection between SEQ ID NO.20 and DabK, wherein SEQ ID NO.20: KTWWW, and W is connected to Dab; ​​(21) SEQ ID NO.21: HFSWWK; (22) SEQ ID NO.22: KFSWWH; (23) SEQ ID NO.23: HFWSWK; (24) SEQ ID NO.24: HFFWWK; (25) SEQ ID NO.25: KFFWWH; (26) SEQ ID NO.26: HFTFTK; (27) SEQ ID NO.27: HVFWVH; (28) SEQ ID NO.28: KFFVFH; (29) SEQ ID NO.29: HFTFTSKKH; (30) SEQ SEQ ID NO. 30: KWFTWK; (31) SEQ ID NO. 31: HLLWTH; (32) KDabDab connected to SEQ ID NO. 32, wherein SEQ ID NO. 32: FVWVK, and Dab is connected to F; (33) SEQ ID NO. 33: KFFFFH; (34) SEQ ID NO. 34: KFWIIVWK; (35) SEQ ID NO. 35: HFSWTH; (36) SEQ ID NO. 36: HTWFSH; (37) HKDab connected to SEQ ID NO. 37, wherein SEQ ID NO. 37: WSWWK, and Dab is connected to WS; (38) SEQ ID NO.38: HFWFWH; (39) The product of the connection between SEQ ID NO.39 and DabH, wherein SEQ ID NO.39: HFFTL, and L is connected to Dab; ​​(40) SEQ ID NO.40: HFWFWK; (41) SEQ ID NO.41: HKFFSLH; (42) SEQ ID NO.42: HFFVTKKH; (43) SEQ ID NO.43: KFWTWK; (44) SEQ ID NO.44: KWTWWK; (45) SEQ ID NO.45: HFWSWK; (46) SEQ ID NO.46: KFWSWH; (47) SEQ ID NO.47: HFLFLH; (48) SEQ ID NO.48: HFWFWK; (49) SEQ ID NO.49: KFWFWSSH; (50) The product of the connection between HDab and SEQ ID NO.50, wherein SEQ ID NO.50: LFWSWKK, where L is connected to Dab.

[0017] Preferably, the fibropeptide with multiple hydrogen bonds within the molecule is a polypeptide with 4-8 amino acids (e.g., 4, 5, 6, 7, 8); its amino acids are independently selected from A (alanine), V (valine), L (leucine), I (isoleucine), F (phenylalanine), D (aspartic acid), N (asparagine), G (glycine), P (proline), E (glutamic acid), and T (threonine).

[0018] This invention optimizes the assembly sequence to achieve in-situ fiber structure regulation, exposing activation structures at multiple sites and achieving more efficient PKM2 activation.

[0019] More preferably, the fibropeptide with multiple hydrogen bonds within the molecule is selected from any one of the following:

[0020] (1) FFA; (2) FTI; (3) YFT; (4) FAG; (5) FFV; (6) SEQ ID NO.51: FFED; (7) SEQ ID NO.52: FFAF; (8) SEQ ID NO.53: TDNL; (9) SEQ ID NO.54: FTITD; (10) SEQ ID NO.55: ITFVV; (11) SEQ ID NO.56: YFTEF; (12) SEQ ID NO.57: LVFFA; (13) SEQ ID NO.58: ITDNL; (14) SEQ ID NO.59: LDFPI; (15) SEQ ID NO.60: FAGFT; (16) SEQ ID NO.61: FGFDP; (17) SEQ ID NO.62: FFVDF; (18) SEQ ID NO.63: FFGFF; (19) SEQ ID NO.64: LVFF.

[0021] More preferably, R1 is derived from glucose, glucosamine, or folic acid.

[0022] In a second aspect, the present invention provides a method for preparing a PKM2-based nano-glycopeptide activator according to the first aspect, the preparation method comprising:

[0023] The PKM2-based nano-glycopeptide activator was synthesized using amino acids with both terminal and side chain amino groups protected, as well as response unit materials, via a solid-phase synthesis method.

[0024] Preferably, the preparation method includes: on a polymer resin, following the amino acid sequence of the polypeptide molecule, starting from the carboxyl terminus, sequentially linking amino acids into a specific sequence, repeating the operation (condensation → washing → deprotection → neutralization and washing → next round of condensation) until the desired peptide chain length is reached, then coupling the Fmoc sugar amino acid to the N-terminus using the same condensation method, then removing the Lys side chain amino protecting group adjacent to the sugar amino acid, and coupling the targeted polypeptide sequence using the same condensation method, finally cleaving the peptide chain from the resin, removing the acetyl protection of the sugar molecule, and after purification and other treatments, the desired glycopeptide is obtained.

[0025] Thirdly, the present invention provides the use of the PKM2-based nanoglycopeptide activator according to the first aspect in the preparation of antitumor drugs.

[0026] Preferably, the tumor is selected from prostate cancer, kidney cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, glioma, and melanoma.

[0027] Preferably, the tumor is a chemotherapy-resistant and highly metastatic tumor.

[0028] More preferably, the tumor is selected from highly metastatic prostate cancer, intracranial glioma, triple-negative epithelial breast cancer, and highly metastatic clear adenocarcinoma of the kidney.

[0029] Preferably, the tumor is a solid tumor whose main metabolic pathway is glycolysis, and the tumor overexpresses glycolytic enzymes and PKM2.

[0030] Preferably, the administration method of the drug includes intravenous administration, subcutaneous administration, and intraperitoneal administration.

[0031] Preferably, the dosage form of the drug is any pharmaceutically acceptable dosage form.

[0032] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0033] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0034] Fourthly, the present invention provides the use of the PKM2-based nanoglycopeptide activator according to the first aspect in the preparation of tumor chemotherapy drug enhancers.

[0035] Fifthly, the present invention provides an antitumor combination drug composition comprising the PKM2-based nanoglycopeptide activator and a chemotherapeutic drug as described in the first aspect.

[0036] Preferably, the chemotherapeutic agent includes paclitaxel or a pharmaceutically acceptable salt thereof, doxorubicin or a pharmaceutically acceptable salt thereof, or temozolomide or a pharmaceutically acceptable salt thereof.

[0037] Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0038] Preferably, the combined pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

[0039] Preferably, the formulation is any pharmaceutically acceptable dosage form.

[0040] Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0041] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention, through precise structural design, enables the nano-glycopeptide activator to accumulate at the tumor site after administration via sugar receptor transporter targeting or the EPR effect. Upon entering tumor cells, it undergoes in-situ fibrosis and deformation through hydrolysis by glycolytic enzymes, achieving specific targeting and retention, resulting in a stronger anti-tumor effect. The nanofibers containing PKM2 targeting activation units promote the conversion of dimer PKM2 into tetramer PKM2, inhibiting PKM2 nuclear translocation and preventing the DNA damage repair process in tumor cells. This nano-glycopeptide activator can achieve universal growth inhibition of highly metastatic and chemotherapy-resistant solid tumors by inhibiting the main aerobic glycolysis pathway of tumor growth, and also provides universal sensitization to various first-line chemotherapy drugs. Furthermore, this nano-glycopeptide activator can cross the blood-brain barrier (BBB) ​​via sugar transporter receptors, effectively entering brain tumors with high drug utilization and long duration of action. Attached Figure Description

[0044] Figure 1 This is the mass spectrum of the nano-glycopeptide activator prepared in Example 1;

[0045] Figure 2 This is the mass spectrum of the nano-glycopeptide activator prepared in Example 2;

[0046] Figure 3 This is the mass spectrum of the nano-glycopeptide activator prepared in Example 3;

[0047] Figure 4 This is the mass spectrum of the nano-glycopeptide activator prepared in Example 4;

[0048] Figure 5 This is the mass spectrum of the nano-glycopeptide activator prepared in Example 5;

[0049] Figure 6 This is the mass spectrum of the nano-glycopeptide activator prepared in Example 6;

[0050] Figure 7 These are transmission electron microscopy (TEM) images of the nano-glycopeptide activators prepared in Examples 1 and 2 before and after morphological transformation.

[0051] Figure 8 This is a diagram showing the verification results of the nano-glycopeptide activators prepared in Examples 1-3 promoting PKM2 tetramerization in cells;

[0052] Figure 9 The graph shows the results of the assay on the inhibition of U87 cell viability by the nano-glycopeptide activators prepared in Examples 1-3.

[0053] Figure 10 The graph shows the results of the assay on the inhibition of U118 cell viability by the nano-glycopeptide activators prepared in Examples 4-6.

[0054] Figure 11 These are anatomical images of the tumors in each group of mice;

[0055] Figure 12 This is a graph showing the statistical results of tumor volume in each group of mice;

[0056] Figure 13 This is a graph showing the cytotoxicity test results of 50 amino acid sequences that have the function of activating PKM2 tetramerization;

[0057] Figure 14 This is a graph showing the binding constant detection results of 50 amino acid sequences that have the function of activating PKM2 tetramerization. Detailed Implementation

[0058] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0059] Example 1

[0060] This embodiment provides a nano-glycopeptide activator, the chemical structure of which is as follows:

[0061]

[0062] In this sequence, R1 is folic acid, R2 is the polypeptide sequence YFTEF (where the Y end is the linker), R3 is the polypeptide sequence KTWWWDabK (where the KT end is the linker), and X is threonine.

[0063] Its preparation method is as follows:

[0064] Weigh the resin and add it to the peptide solid-phase synthesis tube (hereinafter referred to as the reactor). Add 10 mL of DMF to swell for 120 minutes. Remove the DMF and perform the Fmoc deprotection reaction with the deprotection solution. Place the tube on a shaker for 10 minutes, remove the deprotection solution, and wash three times with DMF and DCM. Take a small amount of resin (5 mg) from the reactor into a test tube, wash twice with ethanol, and test positive (deep blue) by the ninhydrin method. Then prepare to add the next amino acid (according to the target sequence) and enter the amino acid condensation reaction.

[0065] Following the polypeptide sequence, the corresponding amino acids and HBTU (amino acid:HBTU = 1:1) were dissolved in the reaction solution and added to the reactor. The mixture was stirred and reacted. After 1 hour, a small amount of resin was taken from the reactor and placed in a test tube. The resin was washed twice with ethanol and tested using the ninhydrin method. A negative result (no color change) confirmed the successful condensation reaction. The liquid in the reactor was removed, and the resin was washed twice each with DMF and DCM to obtain the peptide resin after the condensation of the first amino acid.

[0066] The above "Fmoc deprotection-amino acid condensation" reaction steps were repeated on the obtained peptide resin until the last amino acid was reacted to obtain the polypeptide moiety. Appropriate amounts of folic acid, threonine, and HBTU were weighed, added to the reaction solution, and poured into the above reactor to synthesize the glycopeptide moiety. After the reaction was completed, the Dde protecting group of K was removed. The deprotecting agent used was 10 mL of (hydrazine hydrate / DMF = 2 / 98), which was poured into the reactor and reacted for 15 min. Then, the resin was washed three times each with DMF and DCM. The result of the ninhydrin method was negative. After that, the side chain-coupled polypeptide sequence, HBTU, and reaction solution were poured into the reactor. After the reaction was completed, the resin was washed three times each with DMF and DCM, and washed twice with methanol. The mixture was dried for another 15-20 min, and the synthesized peptide resin was taken out from the reactor.

[0067] The peptide was lysed for two hours at 25°C in a lysis buffer (which was first placed in an ice bath for 20 min). After filtration through the resin, the peptide was evaporated to dryness using a rotary evaporator and washed three times with anhydrous diethyl ether (in an ice bath). The crude peptide was purified using preparative reversed-phase HPLC, and the purity was >90% as determined by HPLC. The obtained pure peptide was identified by mass spectrometry (MS, electrospray).

[0068] The prepared nano-glycopeptide activator was characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 1 As shown, by Figure 1 As can be seen from the main peak of the mass spectrum, the molecular weight of the glycopeptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the nano-glycopeptide activator with the above structure has been successfully synthesized.

[0069] Example 2

[0070] This embodiment provides a nano-glycopeptide activator, the chemical structure of which is as follows:

[0071]

[0072] Wherein, R1 is glucose, R2 is the polypeptide sequence ITFVV (where the I end is the linker), R3 is the polypeptide sequence HFFFVK (where the H end is the linker), and X is serine.

[0073] The preparation method is the same as in Example 1.

[0074] The prepared nano-glycopeptide activator was characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 2 As shown, by Figure 2 As can be seen from the main peak of the mass spectrum, the molecular weight of the glycopeptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the nano-glycopeptide activator with the above structure has been successfully synthesized.

[0075] Example 3

[0076] This embodiment provides a nano-glycopeptide activator, the chemical structure of which is as follows:

[0077]

[0078] Wherein, R1 is acetylglucosamine, R2 is the polypeptide sequence FFVDF (where the FF end is the linker), R3 is the polypeptide sequence HFSWWK (where the H end is the linker), and X is threonine.

[0079] The preparation method is the same as in Example 1.

[0080] The prepared nano-glycopeptide activator was characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 3 As shown, by Figure 3 As can be seen from the main peak of the mass spectrum, the molecular weight of the glycopeptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the nano-glycopeptide activator with the above structure has been successfully synthesized.

[0081] Example 4

[0082] This embodiment provides a nano-glycopeptide activator, the chemical structure of which is as follows:

[0083]

[0084] In this sequence, R1 is acetylglucosamine, R2 is the polypeptide sequence LVFF (where the L end is the linker), R3 is the polypeptide sequence KWWSFH (where the K end is the linker), and X is serine.

[0085] The preparation method is the same as in Example 1.

[0086] The prepared nano-glycopeptide activator was characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 4 As shown, by Figure 4 As can be seen from the main peak of the mass spectrum, the molecular weight of the glycopeptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the nano-glycopeptide activator with the above structure has been successfully synthesized.

[0087] Example 5

[0088] This embodiment provides a nano-glycopeptide activator, the chemical structure of which is as follows:

[0089]

[0090] In this sequence, R1 represents folic acid, R2 represents the polypeptide sequence FFA (with the F-terminus being the linker), R3 represents the polypeptide sequence KWWSFH (with the K-terminus being the linker), and X represents serine.

[0091] The preparation method is the same as in Example 1.

[0092] The prepared nano-glycopeptide activator was characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 5 As shown, by Figure 5 As can be seen from the main peak of the mass spectrum, the molecular weight of the glycopeptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the nano-glycopeptide activator with the above structure has been successfully synthesized.

[0093] Example 6

[0094] This embodiment provides a nano-glycopeptide activator, the chemical structure of which is as follows:

[0095]

[0096] Wherein, R1 is glucose, R2 is the polypeptide sequence LDFPI (where the L end is the linker), R3 is the polypeptide sequence HDabLFWSWKK (where the H end is the linker), and X is threonine.

[0097] The preparation method is the same as in Example 1.

[0098] The prepared nano-glycopeptide activator was characterized by mass spectrometry. The mass spectrometry characterization results are as follows: Figure 6 As shown, by Figure 6 As can be seen from the main peak of the mass spectrum, the molecular weight of the glycopeptide material is basically consistent with that of the design. This indicates that the target molecule has been synthesized, demonstrating that the nano-glycopeptide activator with the above structure has been successfully synthesized.

[0099] Test Example 1

[0100] TEM characterization:

[0101] The nano-glycopeptide activators prepared in Examples 1 and 2 were dissolved in DMSO solvent (the concentration of the self-assembled material was 5 × 10⁻⁶). -3M), take 10 μL of the above solution and place it in a centrifuge tube, then slowly add 990 μL of deionized water to the centrifuge tube to obtain the sample solution. First, drop the sample solution onto a copper grid. Next, remove the upper liquid layer, let it stand for 10 minutes, and then stain the copper grid with 2% uranium acetic acid for 1 minute. Finally, wash the surface of the copper grid with distilled water. Let the copper grid stand overnight, and then observe it with TEM. The morphology of the nano-glycopeptide activators prepared in Example 1 and Example 2 was characterized by transmission electron microscopy using the two sample solutions. The results are as follows. Figure 7 As shown, by Figure 7 It can be seen that the nano-glycopeptide activators prepared in Examples 1 and 2 formed self-assembled particles in aqueous solution, with particle sizes of 36.3±16.5nm and 33.5±12.3nm, respectively.

[0102] Then, O-GlcNAc glycosidase was added to the mixed solution, and the morphological transformation of the obtained nano-glycopeptide activators was characterized by transmission electron microscopy. The results are as follows: Figure 7 As shown, by Figure 7 It can be seen that the morphology of the nano-glycopeptide activators prepared in Examples 1 and 2 has changed, forming fibrous structures.

[0103] Test Example 2

[0104] Verification of how nano-glycopeptide activators promote PKM2 tetramerization in cells:

[0105] Validation Methods: U87 cell lines treated with the nanoglycopeptide activators prepared in Examples 1, 2, and 3 were lysed using sodium dodecyl sulfate (SDS) cell lysis buffer (P0013G; Beyotime) containing a protease inhibitor mixture (87785; Thermo Scientific) to obtain proteins. Protein concentrations were determined using a Pierce BCA protein assay kit (PC0020; Solarbio). Protein bands were visualized following conventional Western blotting procedures. The presence and abundance of target proteins were determined using secondary antibody detection combined with the primary antibody.

[0106] The results are as follows Figure 8 As shown in the figure, the nano-glycopeptide activators prepared in Examples 1, 2 and 3 can tetramerize PKM2 in U87 cells.

[0107] Test Example 3

[0108] Validation of the antitumor activity of nano-glycopeptide activators:

[0109] (1) U87 cell inhibition assay: Cytotoxicity in U87 cells was assessed using the CCK-8 assay. First, cells were loaded at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C and 5% CO2 for 24 h. Subsequently, the nanoglycopeptide activators prepared in Examples 1-3 were diluted to a series of different concentrations and incubated with the cells for 24 h. Finally, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance of blank wells (Ab), sample wells (As), and control wells (Ac) was measured using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm. Cell viability (%) was calculated using the formula: (As-Ab) / (Ac-Ab) × 100%. All experiments were repeated three times, and the mean and standard deviation are reported.

[0110] The results are as follows Figure 9 As shown in the figure, the IC50 of glycopeptide 1 (Example 1) is... 50 The IC50 of glycopeptide 2 (Example 2) is 86.36 μm. 50 The IC50 of glycopeptide 3 (Example 3) is 106.3 μm. 50 It is 85.43 μm.

[0111] (2) U118 cell inhibition assay:

[0112] Cytotoxicity was assessed in U118 cells using the CCK-8 assay. First, cells were loaded at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C and 5% CO2 for 24 h. Subsequently, the nanoglycopeptide activators prepared in Examples 4-6 were diluted to a series of different concentrations and incubated with the cells for 24 h. Finally, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance of blank wells (Ab), sample wells (As), and control wells (Ac) was measured using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm. Cell viability (%) was calculated using the formula: (As-Ab) / (Ac-Ab) × 100%. All experiments were repeated three times, and the mean and standard deviation are reported.

[0113] The results are as follows Figure 10 As shown in the figure, the IC50 of glycopeptide 4 (Example 4) is... 50 IC50 of glycopeptide 5 (Example 5) with a particle size greater than 100 μm 50 The IC50 of glycopeptide 6 (Example 6) is 66.09 μm. 50 It is 56.04 μm.

[0114] Test Example 4

[0115] Validation of the antitumor activity of nano-glycopeptide activators in combination with chemotherapy drugs:

[0116] (1) Animal grouping: (i) Control group; (ii) Glycopeptide 6 + DOX; (iii) Glycopeptide 3 + TMZ; (iv) Glycopeptide 2 + Paclitaxel; (v) Glycopeptide 4 + DOX; (vi) Glycopeptide 1 + TMZ.

[0117] (2) Model Construction Method: Animal experiments were conducted according to national and international guidelines. All animal studies were conducted at Beijing Tiantan Hospital and approved by the Beijing Tiantan Committee. U87 cells (4 × 10⁻⁶) were used. 6 The solution was suspended in 100 μl of PBS and injected subcutaneously into the back of each female nude mouse (model Balb / c nude).

[0118] (3) Administration method: Intraperitoneal administration daily: (i) Control group: normal saline daily; (ii) Glycopeptide 6 + DOX group: 6 mg / kg glycopeptide 6 and 5 mg / kg DOX per nude mouse daily; (iii) Glycopeptide 3 + TMZ group: 6 mg / kg glycopeptide 3 and 5 mg / kg TMZ per nude mouse daily; (iv) Glycopeptide 2 + Paclitaxel group: 6 mg / kg glycopeptide 2 and 5 mg / kg paclitaxel per nude mouse daily; (v) Glycopeptide 4 + DOX group: 6 mg / kg glycopeptide 4 and 5 mg / kg DOX per nude mouse daily; (vi) Glycopeptide 1 + TMZ group: 6 mg / kg glycopeptide 1 and 5 mg / kg TMZ per nude mouse daily. Administration continued for 5 days.

[0119] The anatomical appearance of tumors in each group of mice is as follows: Figure 11 As shown in the figure, the tumor volume statistics of each group of mice are as follows: Figure 12 As shown in the figure, compared with the control group, each drug group has a better inhibitory effect on glioma, and the glycopeptide 1+TMZ group has the best effect in treating glioma.

[0120] Test Example 5

[0121] Investigation into the cytotoxicity and binding constant of PKM2 targeting the activating unit R3:

[0122] The cytotoxicity and binding constant of the 50 amino acid sequences listed in the aforementioned specification that have the function of activating PKM2 tetramerization were detected, as follows:

[0123] (1) Cytotoxicity test:

[0124] Cytotoxicity was assessed in U87 cells using the CCK-8 assay. First, cells were loaded at 5 × 10⁶ cells per well. 3Cells were seeded at a density of 100 μm / well in 96-well plates and incubated at 37°C and 5% CO2 for 24 h. Subsequently, 100 μm of 50 amino acid sequences capable of activating PKM2 tetramerization were added to each well and incubated with the cells for another 24 h. Finally, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance of blank wells (Ab), sample wells (As), and control wells (Ac) was measured using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm. Cell viability (%) was calculated using the formula: (As-Ab) / (Ac-Ab) × 100%. All experiments were repeated three times, and the mean and standard deviation are reported.

[0125] The results are as follows Figure 13 As shown in the figure (numbered sequentially according to the amino acid sequence), a total of 10 short peptides showed better tumor cell killing effects than the positive control molecule DASA-58, namely 9, 21, 22, 32, 34, 37, 44, 45, 46, and 47.

[0126] (2) Binding Constant Detection Assay: SPR measurements were performed on a Biacore 8K instrument (GE Healthcare, Piscatavir, NJ, USA). Briefly, purified PKM2 protein (200 mg / mL, pH 8.0) was immobilized on an S-series sensor chip (w10000RU) according to a standard amine coupling procedure. PBS (AR1155, pH 7.2e7.4, Boster) containing 3% DMSO was used as the immobilization run buffer. After immobilization, the stock solution was serially diluted with electrophoresis buffer to prepare solutions of 50 short peptides at different concentrations. Seven concentrations of GPS were simultaneously injected at a flow rate of 65 mL / min, and a binding period of 60 s was performed at 25°C. The final graph was obtained by subtracting the blank sensor plot. Experimental data were collected and analyzed using Biacore 8K management software (GE Healthcare, Piscataway, New Jersey, USA) to fit an appropriate binding model to obtain the equilibrium dissociation constant (KD).

[0127] The results are as follows Figure 14 As shown in the figure (the amino acid sequences are listed in order and numbered accordingly), the KD values ​​of all 50 peptides are in the μm range. Peptides 37 and 38 have the lowest KD values, at 0.03 × 10⁻⁶. -6 and 0.08×10 -6 .

[0128] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0129] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0130] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A PKM2-based nano-glycopeptide activator, characterized in that, The nano-glycopeptide activator comprises three functional units: a PKM2-targeting activation unit R3, a self-assembly unit R2, and a response unit R1, the chemical structure of which is shown in formula (I): Among them, R3 comes from an amino acid sequence that activates PKM2 tetramerization; R2 comes from a fibropeptide with multiple hydrogen bonds in the molecule; R1 comes from glucose, acetylglucosamine, mannose, fucose, galactose or folic acid; and X comes from serine or threonine.

2. The PKM2-based nano-glycopeptide activator according to claim 1, characterized in that, The amino acid sequence having the function of activating PKM2 tetramerization is a polypeptide with 6-10 amino acids; its C-terminus and N-terminus are independently selected from H, K or Dab; ​​the intermediate amino acid is independently selected from A, V, L, I, F, W, M, S; wherein Dab represents 2,4-diaminobutyric acid; Preferably, the amino acid sequence having the function of activating PKM2 tetramerization has a binding constant Kd > 10 with PKM2. -5 Polypeptides.

3. The PKM2-based nano-glycopeptide activator according to claim 2, characterized in that, The amino acid sequence having the function of activating PKM2 tetramerization is selected from any one of the following: HHHFFTTK, HFFFVK, KFFFVH, HFFTTH, KFTWWK, HLLTWH, KFVVVWK, HFFFFK, KFVWWK, HFSTWH, HTSAWFH, HFSWWK, HFFWWVKKH ,HFFTTIHH,HFFLTH,HFFWWK,HFFLSH,HFFTVH,KFTWWK,KTWWWDabK,HFSWWK,KFSWWH,HFWSWK,HFFWWK,KFFWWH,HFTFTK,H VFWVH, KFFVFH, HFTFTSKKH, KWFTWK, HLLWTH, KDabDabFVWVK, KFFFFH, KFWIIVWK, HFSWTH, HTWFSH, HKDabWSWWK, HFWFWH ,HFFTLDabH,HFWFWK,HKFFSLH,HFFVTKKH,KFWTWK,KWTWWK,HFWSWK,KFWSWH,HFLFLH,HFWFWK,KFWFWSSH,HDabLFWSWKK.

4. The PKM2-based nano-glycopeptide activator according to claim 1, characterized in that, The fibrous peptide with multiple hydrogen bonds within the molecule is a polypeptide with 4-8 amino acids; its amino acids are independently selected from A, V, L, I, F, D, N, G, P, E, and T.

5. The PKM2-based nano-glycopeptide activator according to claim 4, characterized in that, The fibropeptide with multiple hydrogen bonds within the molecule is selected from any one of the following: FFA, FTI, YFT, FAG, FFV, FFED, FFAF, TDNL, FTITD, ITFVV, YFTEF, LVFFA, ITDNL, LDFPI, FAGFT, FGFDP, FFVDF, FFGFF, LVFF.

6. The PKM2-based nano-glycopeptide activator according to claim 1, characterized in that, R1 comes from glucose, acetylglucosamine, or folic acid.

7. The method for preparing the PKM2-based nano-glycopeptide activator according to any one of claims 1-6, characterized in that, The preparation method includes: The PKM2-based nano-glycopeptide activator was synthesized using amino acids with both terminal and side chain amino groups protected, as well as response unit materials, via a solid-phase synthesis method.

8. The use of the PKM2-based nanoglycopeptide activator according to any one of claims 1-6 in the preparation of antitumor drugs; Preferably, the tumor is selected from prostate cancer, kidney cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, glioma, and melanoma; Preferably, the tumor is a chemotherapy-resistant and highly metastatic tumor; Preferably, the tumor is a solid tumor whose main metabolic pathway is glycolysis, and the tumor overexpresses glycolytic enzymes and PKM2.

9. The use of the PKM2-based nanoglycopeptide activator according to any one of claims 1-6 in the preparation of tumor chemotherapy drug potentiators.

10. A combination pharmaceutical composition for antitumor use, characterized in that, The antitumor combination drug composition comprises the PKM2-based nanoglycopeptide activator and the chemotherapeutic drug as described in any one of claims 1-6; Preferably, the chemotherapeutic agent includes paclitaxel or a pharmaceutically acceptable salt thereof, doxorubicin or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof; Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations; Preferably, the combined pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially. Preferably, the formulation is any pharmaceutically acceptable dosage form; Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

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