PKM2-based nanoglycopeptide activator, preparation method therefor and use thereof

Abstract

Provided are a PKM2-based nanoglycopeptide activator and a use thereof. The nanoglycopeptide activator comprises three functional units, namely a PKM2-targeted activation unit R3, a self-assembly unit R2, and a response unit R1. The chemical structure of the nanoglycopeptide activator is as shown in formula (I). Upon administration, the nanoglycopeptide activator can be enriched at a tumor site by means of glucose receptor transporter-mediated targeting or the EPR effect; after entering tumor cells, the nanoglycopeptide activator undergoes in-situ fibrotic deformation under the hydrolysis action of glycosidase, thereby achieving specific targeting and retention; and nanofibers having the PKM2-targeted activation unit promote the conversion of dimeric PKM2 into tetrameric PKM2, inhibit the nuclear translocation of PKM2 , and block the DNA damage repair process in the tumor cells.

Description

PKM2-based nano glycopeptide activator and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a PKM2-based nano glycopeptide activator and a preparation method and application thereof. BACKGROUND

[0002] It is known that cancer cells promote tumor growth through aerobic glycolysis, and important intervention proteins include pyruvate kinase 2 (PKM2). PKM2 is a protein encoded by the human PKM2 gene and is a glycolytic pyruvate kinase isozyme. PKM2 is expressed in different tissue organs, such as lung, adipose tissue, retina and islet, and in all cells with a large amount of nucleic acid synthesis, such as normally proliferating cells, embryonic stem cells, and especially tumor cells. Dimeric PKM2 mainly exists in the nucleus of tumor cells and plays a key role in tumor cell proliferation, invasion and metastasis. Among them, PKM2 can be activated by serine to induce the tetramerization of PKM2, reduce the glycolysis intermediates that can be used as biosynthetic precursors, prevent dimeric PKM2 from entering the nucleus, and inhibit the STAT3 pathway. Therefore, activating PKM2 is considered to be an effective strategy for treating cancer.

[0003] CN104817490A discloses a novel aminodithiocarbamate compound, a preparation method and application thereof. The aminodithiocarbamate compound can target PKM2, is an agonist of PKM2, and is found to have good anti-tumor effect by preventing PKM2 from entering the nucleus, and has 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., ocular diseases, hematological conditions, or cancer).

[0005] At present, there are few strategies for activating PKM2 in the prior art, and in recent years, the rapid development of nanomaterials has attracted widespread attention, and the application field of nanomaterials is also deepening. Therefore, it is very meaningful to develop a new strategy for resisting tumors by activating PKM2.

[0006] SUMMARY

[0007] The application provides a PKM2-based nanoglycopeptide activator, a preparation method and application thereof. The nanoglycopeptide activator can be selectively hydrolyzed by a glycosylase, specifically targeted to accumulate at a tumor site, effectively promote the conversion of dimeric PKM2 of tumor cells into tetrameric PKM2, thereby changing the metabolic mode of tumor cells, and play an anti-tumor and chemotherapy sensitization effect.

[0008] In a first aspect, the application provides a PKM2-based nanoglycopeptide activator, which comprises three functional units, namely a PKM2-targeted activation unit R3, a self-assembly unit R2 and a response unit R1. The chemical structure of the nanoglycopeptide activator is shown in formula (I):

[0009] wherein R3 is derived from an amino acid sequence having a function of activating PKM2 tetramerization; R2 is derived from a fibrous peptide having multiple hydrogen bonds in the molecule; R1 is derived from glucose, acetylglucosamine, mannose, fucose, galactose or folic acid; and X is derived from serine or threonine.

[0010] The application, through precise structural design, enables the nanoglycopeptide activator to be targeted to a tumor site by a sugar receptor transporter protein or EPR effect (enhanced permeability and retention effect) after administration; after entering tumor cells, the nanoglycopeptide activator is deformed in situ by fibration through the hydrolysis of a glycosylase, thereby achieving specific targeting and retention; the nanofiber with the PKM2-targeted activation unit promotes the conversion of dimeric PKM2 into tetrameric PKM2, inhibits the nuclear entry of PKM2 and prevents the DNA damage repair process of tumor cells, thereby achieving glycolysis metabolic pathway inhibition for chemotherapy-resistant and high-metastasis solid tumors, anti-tumor effect and chemotherapy sensitization.

[0011] The nanoglycopeptide activator can be stably dispersed in the form of a nanomicelle in an aqueous solution, and after being hydrolyzed by a glycosylase, the fibrous structure is transformed. The nanomicelle has a particle size of 20-50 nm, the fiber has a diameter of 5-15 nm and a length of 5-50 μm, and can be flexible or rigid, and can be solid or hollow.

[0012] Preferably, the amino acid sequence having a function of activating PKM2 tetramerization is a polypeptide with an amino acid number of 6-10 (for example, 6, 7, 8, 9 or 10); the C-terminal and N-terminal of the polypeptide are independently selected from H (histidine), K (lysine) or Dab; the middle amino acid of the polypeptide is independently selected from A (alanine), V (valine), L (leucine), I (isoleucine), F (phenylalanine), W (tryptophan), M (methionine), S (serine); wherein Dab represents 2,4-diaminobutyric acid;

[0013] Preferably, the amino acid sequence having the function of activating the tetramerization of PKM2 has a binding constant Kd> 10 -5 to PKM2.

[0014] Further preferably, the amino acid sequence having the function of activating the tetramerization of PKM2 is selected from any one of the following:

[0015] (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: KWWSFH; (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 the W end; (38) SEQ ID NO. 38: HFWFWH; (39) the connection product of 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 connection product of HDab and SEQ ID NO. 50, wherein SEQ ID NO. 50: LFWSWKK, and L is connected to Dab.

[0016] Preferably, the fibrous peptide having multiple intramolecular hydrogen bonds is a polypeptide with 4-8 (e.g. 4, 5, 6, 7, 8) amino acids; the amino acids thereof 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), T (threonine).

[0017] The application can realize in-situ regulation of fibrous structure by optimizing the assembly sequence, expose multiple sites of activated structure, and realize more efficient PKM2 activation.

[0018] Further preferably, the fibrous peptide having multiple intramolecular hydrogen bonds is selected from any one of the following:

[0019] (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.

[0020] Further preferably, R1 is derived from glucose, acetylglucosamine or folate.

[0021] In a second aspect, the present application provides a method for preparing the PKM2-based nanoglycopeptide activator according to the first aspect, the method comprising:

[0022] The PKM2-based nanoglycopeptide activator is synthesized by solid-phase synthesis using amino acids with both terminal and side-chain amino groups protected and a response unit material as raw materials.

[0023] Preferably, the method comprises: on a polymer resin, amino acids are sequentially connected into a specific sequence from the carboxyl end according to the amino acid sequence of the polypeptide molecule, and the operation is repeated (condensation → washing → deprotection → neutralization and washing → next round of condensation) until the desired length of the peptide chain is reached, then Fmoc sugar amino acid is coupled to the N-terminus, the condensation method is the same as above, then the Lys side-chain amino protecting group adjacent to the sugar amino acid is removed, the same condensation method is used to couple the target polypeptide sequence, finally the peptide chain is cleaved from the resin, the acetyl protecting group of the sugar molecule is removed, and the desired glycopeptide is obtained after purification and other treatments.

[0024] In a third aspect, the present application provides use of the PKM2-based nanoglycopeptide activator according to the first aspect in the preparation of an anti-tumor drug.

[0025] Preferably, the tumor is selected from prostate cancer, renal cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, glioma, or melanoma.

[0026] Preferably, the tumor is a chemotherapy-resistant and highly metastatic tumor.

[0027] More preferably, the tumor is selected from high metastatic prostate cancer, intracranial glioma, triple negative epithelial breast cancer, or high metastatic renal clear cell adenocarcinoma.

[0028] Preferably, the tumor is a solid tumor with glycolysis as the main metabolic mode, and the tumor has overexpression of glycolytic enzymes and PKM2.

[0029] Preferably, the administration of the drug includes intravenous administration, subcutaneous administration, or intraperitoneal administration.

[0030] Preferably, the dosage form of the drug is any one of pharmaceutically acceptable dosage forms.

[0031] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0032] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, coloring agents, pH regulators, antioxidants, bacteriostatic agents, or buffers.

[0033] In a fourth aspect, the present application provides a use of the PKM2-based nano-glycopeptide activator according to the first aspect in the preparation of a tumor chemotherapy drug sensitizer.

[0034] In a fifth aspect, the present application provides an anti-tumor combination drug composition, which is composed of the PKM2-based nano-glycopeptide activator according to the first aspect and a chemotherapy drug.

[0035] Preferably, the chemotherapy drug includes paclitaxel or a pharmaceutically acceptable salt thereof, doxorubicin or a pharmaceutically acceptable salt thereof, or temozolomide or a pharmaceutically acceptable salt thereof.

[0036] Preferably, the combination drug composition is a single complex preparation or a combination of two separate preparations.

[0037] Preferably, the combination drug composition is a combination of two separate preparations, which are administered simultaneously or sequentially.

[0038] Preferably, the preparation is any one of pharmaceutically acceptable dosage forms.

[0039] Preferably, the combination drug composition further contains pharmaceutically acceptable excipients.

[0040] 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.

[0041] Compared with the prior art, this application has the following advantages:

[0042] This application utilizes precise structural design to enable the nano-glycopeptide activator to accumulate at the tumor site after administration via glycan 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 glycan transporter receptors, effectively entering brain tumors with high drug utilization and long duration of action. Attached Figure Description

[0043] Figure 1 is a mass spectrum of the nano-glycopeptide activator prepared in Example 1.

[0044] Figure 2 is a mass spectrum of the nano-glycopeptide activator prepared in Example 2.

[0045] Figure 3 is the mass spectrum of the nano-glycopeptide activator prepared in Example 3.

[0046] Figure 4 is the mass spectrum of the nano-glycopeptide activator prepared in Example 4.

[0047] Figure 5 is the mass spectrum of the nano-glycopeptide activator prepared in Example 5.

[0048] Figure 6 is the mass spectrum of the nano-glycopeptide activator prepared in Example 6.

[0049] Figure 7 shows the transmission electron microscopy (TEM) images of the nanoglycopeptide activators prepared in Examples 1 and 2 before and after morphological transformation.

[0050] Figure 8 shows the verification results of the nano-glycopeptide activators prepared in Examples 1-3 promoting PKM2 tetramerization in cells.

[0051] Figure 9 shows the results of the assay on the inhibition of U87 cell viability by the nano-glycopeptide activators prepared in Examples 1-3.

[0052] Figure 10 is a graph of the results of the U118 cell viability inhibition test of the nanoglycopeptide activators prepared in Examples 4-6.

[0053] Figure 11 is a graph of the tumor dissection appearance of each group of mice.

[0054] Figure 12 is a graph of the tumor volume statistics of each group of mice.

[0055] Figure 13 is a graph of the cytotoxicity detection results of 50 amino acid sequences with the function of activating PKM2 tetramerization.

[0056] Figure 14 is a graph of the binding constant detection results of 50 amino acid sequences with the function of activating PKM2 tetramerization. DETAILED DESCRIPTION

[0057] The technical solutions of the present application are further illustrated below by means of specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0058] Example 1

[0059] The present embodiment provides a nanoglycopeptide activator, which has the following chemical structure:

[0060] wherein R1 is folic acid, R2 is the polypeptide sequence YFTEF (SEQ ID NO. 56, wherein Y is a connecting end), R3 is the polypeptide sequence KTWWW (SEQ ID NO. 20)-DabK (wherein KT is a connecting end), and X is threonine.

[0061] The preparation method is as follows:

[0062] The resin is weighed and put into a polypeptide solid-phase synthesis tube (hereinafter referred to as a reactor), 10 mL of DMF is added for swelling for 120 minutes, the DMF is removed, a deprotection solution is used for Fmoc deprotection reaction, and the reactor is placed on a shaker for 10 minutes. The deprotection solution is removed, and the reactor is washed with DMF and DCM for 3 times. A small amount of resin (5 mg) is taken from the reactor into a test tube, washed with ethanol for 2 times, and tested by the ninhydrin method. If the result is positive (dark blue), the next amino acid (according to the target sequence) is prepared for coupling reaction.

[0063] According to the polypeptide sequence, the corresponding amino acid is taken, HBTU (amino acid:HBTU = 1:1), and the reaction solution is used for dissolution and put into the reactor for stirring reaction. After 1 hour, a small amount of resin is taken from the reactor into a test tube, washed with ethanol for 2 times, and tested by the ninhydrin method. If the result is negative (no color change), it proves that the coupling reaction is successful. The liquid in the reactor is removed, and the reactor is washed with DMF and DCM for 2 times to obtain the peptide resin after the coupling of the first amino acid.

[0064] The above "Fmoc deprotection-amino acid condensation" reaction steps are repeated on the obtained peptide resin until the last amino acid is reacted, to obtain a polypeptide moiety; an appropriate amount of folic acid, threonine and HBTU are weighed and added to the reaction solution, and poured into the above reactor to synthesize a glycopeptide moiety; after the reaction is completed, the Dde protecting group of K is removed, and the deprotection agent used is (hydrazine hydrate: DMF = 2:98) 10 mL, which is poured into the reactor, and reacted for 15 min, then the resin is washed with DMF and DCM each 3 times, and the result is negative by ninhydrin method, then the side chain coupled polypeptide sequence and HBTU and the reaction solution are poured into the reactor, after the reaction is completed, the resin is washed with DMF and DCM each 3 times, and methanol 2 times, and the synthesis of the peptide resin is completed.

[0065] The resin is filtered and dried on a rotary evaporator, washed with anhydrous ether (ice bath) 3 times, and the crude peptide is purified by preparative reverse phase HPLC, and the purity is detected by HPLC to be >90%, and the obtained pure peptide is identified by mass spectrometry (MS, electrospray).

[0066] The prepared nanoglycopeptide activator is characterized by mass spectrometry, and the mass spectrometry characterization result is shown in Figure 1. As can be seen from Figure 1, the main peak of the mass spectrum shows that the molecular weight of the glycopeptide material is basically consistent with the design, so it is concluded that the target molecule is synthesized, indicating that the nanoglycopeptide activator with the structure as shown above is successfully synthesized.

[0067] Example 2

[0068] This example provides a nanoglycopeptide activator, which has the following chemical structure:

[0069] Wherein, R1 is glucose, R2 is polypeptide sequence ITFVV (SEQ ID NO. 55, wherein I end is a connecting end), R3 is polypeptide sequence HFFFVK (SEQ ID NO. 2, wherein H end is a connecting end), and X is serine.

[0070] The preparation method thereof is referred to Example 1.

[0071] The prepared nanoglycopeptide activator is characterized by mass spectrometry, and the mass spectrometry characterization result is shown in Figure 2. As can be seen from Figure 2, the main peak of the mass spectrum shows that the molecular weight of the glycopeptide material is basically consistent with the design, so it is concluded that the target molecule is synthesized, indicating that the nanoglycopeptide activator with the structure as shown above is successfully synthesized.

[0072] Example 3

[0073] The embodiment provides a kind of nanoglycopeptide activator, its chemical structure is as follows:

[0074] Wherein, R1 is acetylglucosamine, R2 is polypeptide sequence FFVDF (SEQ ID NO.62, wherein FF end is connecting end), R3 is polypeptide sequence HFSWWK (SEQ ID NO.12, wherein H end is connecting end), X is threonine.

[0075] Its preparation method refers to embodiment 1.

[0076] The nanoglycopeptide activator prepared is characterized by mass spectrum, and mass spectrum characterization result is as shown in Figure 3, as can be seen from Figure 3: the main peak of mass spectrum can be seen and the molecular weight of glycopeptide material when designing is basically identical, so as to synthesize target molecule, indicate that the nanoglycopeptide activator of structure as above formula is successfully synthesized.

[0077] Embodiment 4

[0078] The embodiment provides a kind of nanoglycopeptide activator, its chemical structure is as follows:

[0079] Wherein, R1 is acetylglucosamine, R2 is polypeptide sequence LVFF (SEQ ID NO.64, wherein L end is connecting end), R3 is polypeptide sequence KWWSFH (SEQ ID NO.21, wherein K end is connecting end), X is serine.

[0080] Its preparation method refers to embodiment 1.

[0081] The nanoglycopeptide activator prepared is characterized by mass spectrum, and mass spectrum characterization result is as shown in Figure 4, as can be seen from Figure 4: the main peak of mass spectrum can be seen and the molecular weight of glycopeptide material when designing is basically identical, so as to synthesize target molecule, indicate that the nanoglycopeptide activator of structure as above formula is successfully synthesized.

[0082] Embodiment 5

[0083] The embodiment provides a kind of nanoglycopeptide activator, its chemical structure is as follows:

[0084] Wherein, R1 is folic acid, R2 is polypeptide sequence FFA (wherein F end is connecting end), R3 is polypeptide sequence KWWSFH (SEQ ID NO.21, wherein K end is connecting end), X is serine.

[0085] Its preparation method refers to embodiment 1.

[0086] The prepared nano glycopeptide activator was characterized by mass spectrometry, and the mass spectrometry characterization result is shown in Figure 5. As shown in Figure 5, the main peak of the mass spectrum shows that the molecular weight of the glycopeptide material is basically consistent with the design, and it is concluded that the target molecule is synthesized, indicating that the nano glycopeptide activator with the structure as shown above is successfully synthesized.

[0087] Example 6

[0088] This example provides a nano glycopeptide activator, which has the following chemical structure:

[0089] Wherein, R1 is glucose, R2 is polypeptide sequence LDFPI (SEQ ID NO. 59, wherein L end is a connecting end), R3 is polypeptide sequence HDab-LFWSWKK (SEQ ID NO. 50) (wherein H end is a connecting end), and X is threonine.

[0090] The preparation method thereof refers to Example 1.

[0091] The prepared nano glycopeptide activator was characterized by mass spectrometry, and the mass spectrometry characterization result is shown in Figure 6. As shown in Figure 6, the main peak of the mass spectrum shows that the molecular weight of the glycopeptide material is basically consistent with the design, and it is concluded that the target molecule is synthesized, indicating that the nano glycopeptide activator with the structure as shown above is successfully synthesized.

[0092] Test Example 1

[0093] TEM characterization:

[0094] The nano glycopeptide activators prepared in Example 1 and Example 2 were respectively dissolved in DMSO solvent (the concentration of the self-assembled material was 5×10 -3 M), 10 μL of the above solution was taken and placed in a centrifuge tube, and then 990 μL of deionized water was slowly added into the centrifuge tube to obtain a sample solution. First, the sample solution was dropped onto a copper mesh, then the upper liquid layer was removed, and after standing for 10 minutes, the copper mesh was stained with 2% uranyl acetate for 1 minute. Finally, the surface of the copper mesh was washed with distilled water. The copper mesh was placed overnight, and then observed by TEM. The morphology of the nano glycopeptide activators prepared in Example 1 and Example 2 was characterized by transmission electron microscopy, and the results are shown in Figure 7. As shown in Figure 7, the nano glycopeptide activators prepared in Example 1 and Example 2 formed self-assembled particles in aqueous solution, and the particle sizes were 36.3±16.5 nm and 33.5±12.3 nm, respectively.

[0095] Then O-GlcNAc glycosidase was added into the mixed solution, and the morphology transformation of the obtained nano glycopeptide activator was characterized by transmission electron microscopy, and the results are shown in Figure 7. As shown in Figure 7, the morphology of the nano glycopeptide activators prepared in Example 1 and Example 2 was changed, and fibrous structures were formed.

[0096] Test Example 2

[0097] Verification of the promotion of PKM2 tetramerization in cells by the nano-glycopeptide activator:

[0098] Verification method: U87 cell lines treated with the nano-glycopeptide activator prepared in Example 1, Example 2, and Example 3 were lysed with a sodium dodecyl sulfate (SDS) cell lysis buffer (P0013G; Beyotime) containing a protease inhibitor cocktail (87785; Thermo Scientific) to obtain proteins. The protein concentration was determined using a Pierce BCA Protein Assay Kit (PC0020; Solarbio). The protein bands were visualized following the traditional procedure of Western blotting. The presence and abundance of the target protein were determined by detecting the bound primary antibody using a secondary antibody.

[0099] The results are shown in Figure 8. As can be seen from the figure, the nano-glycopeptide activators prepared in Example 1, Example 2, and Example 3 can cause the PKM2 in U87 cells to tetramerize.

[0100] Test Example 3

[0101] Verification of the anti-tumor activity of the nano-glycopeptide activator:

[0102] (1) U87 cell inhibition test: CCK-8 was used to evaluate the cytotoxicity in U87 cells. First, cells were seeded into a 96-well plate at a density of 5 x 10 3 cells per well and incubated at 37°C in a 5% CO2 environment for 24 h. Subsequently, the nano-glycopeptide activators prepared in Examples 1-3 were diluted into a series of different concentrations and incubated with the cells for 24 hours. Finally, 10 μL of CCK-8 solution was added to each well, and incubation was continued for 2 h. The absorbance of the 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. The cell survival rate (%) was calculated using the formula: (As-Ab) / (Ac-Ab) x 100%. All experiments were repeated three times, and the average value and the respective standard deviation were reported.

[0103] The results are shown in Figure 9. As can be seen from the figure, the IC 50 of glycopeptide 1 (Example 1) was 86.36 μm, the IC 50 of glycopeptide 2 (Example 2) was 106.3 μm, and the IC 50 of glycopeptide 3 (Example 3) was 85.43 μm.

[0104] (2) U118 cell inhibition test:

[0105] Cytotoxicity was evaluated in U118 cells using CCK-8 assay. First, cells were seeded into 96-well plates at a density of 5 x 10 3 cells per well and incubated at 37 °C in a 5% CO2 environment for 24 h. Subsequently, the nanoglycopeptide activators prepared in Examples 4-6 were diluted into 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 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 survival rate (%) was calculated using the formula: (As-Ab) / (Ac-Ab) x 100%. All experiments were repeated three times and the average value and the respective standard deviation were reported.

[0106] The results are shown in Figure 10. As can be seen from the figure, the IC 50 of glycopeptide 4 (Example 4) was greater than 100 μm, the IC 50 of glycopeptide 5 (Example 5) was 66.09 μm, and the IC 50 of glycopeptide 6 (Example 6) was 56.04 μm.

[0107] Test Example 4

[0108] Verification of the anti-tumor activity of the combination of nanoglycopeptide activators and chemotherapeutic drugs:

[0109] (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.

[0110] (2) Model construction method: Animal experiments were conducted in accordance with national and international guidelines. All animal studies were conducted at Beijing Tiantan Hospital and were approved by the Beijing Tiantan Committee. U87 cells (4 x 10 6 ) were suspended in 100 μL of PBS and injected subcutaneously into the back of each female nude mouse (Balb / c nude model).

[0111] (3) Administration mode: intraperitoneal administration every day: (i) Control group: physiological saline was administered every day; (ii) Glycopeptide 6+DOX group: 6 mg / kg of glycopeptide 6 and 5 mg / kg of DOX were administered to each nude mouse every day; (iii) Glycopeptide 3+TMZ group: 6 mg / kg of glycopeptide 3 and 5 mg / kg of TMZ were administered to each nude mouse every day; (iv) Glycopeptide 2+paclitaxel group: 6 mg / kg of glycopeptide 2 and 5 mg / kg of paclitaxel were administered to each nude mouse every day; (v) Glycopeptide 4+DOX group: 6 mg / kg of glycopeptide 4 and 5 mg / kg of DOX were administered to each nude mouse every day; (vi) Glycopeptide 1+TMZ group: 6 mg / kg of glycopeptide 1 and 5 mg / kg of TMZ were administered to each nude mouse every day. The administration was continuously performed for 5 days.

[0112] The tumor dissection appearance of each group of mice is shown in FIG. 11, and the tumor volume statistical results of each group of mice are shown in FIG. 12. As can be seen from the figures, compared with the control group, each administration group has a better glioma inhibition effect, and the glycopeptide 1+TMZ group has the best effect on treating glioma.

[0113] Test Example 5

[0114] Cytotoxicity and binding constant exploration of PKM2-targeting activation unit R3:

[0115] The cytotoxicity and binding constant of the 50 amino acid sequences with the function of activating PKM2 tetramerization listed in the foregoing description were detected, and the details are as follows:

[0116] (1) Cytotoxicity test:

[0117] CCK-8 was used to evaluate the cytotoxicity in U87 cells. First, the cells were seeded into a 96-well plate at a density of 5×10 3 cells per well and incubated at 37°C in a 5% CO2 environment for 24 h. Subsequently, 50 amino acid sequences with the function of activating PKM2 tetramerization 100 μm were incubated with the cells for 24 hours. Finally, 10 μL of CCK-8 solution was added to each well, and 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. The cell survival rate (%) was calculated using the formula: (As-Ab) / (Ac-Ab)×100%. All experiments were repeated three times, and the average value and the respective standard deviation were reported.

[0118] The results are shown in FIG. 13 (in the order of the number corresponding to the sequence of amino acid sequences in the figure), and as can be seen from the figure, a total of 10 short peptides have a better killing effect on tumor cells than the positive control molecule DASA-58, which are 9, 21, 22, 32, 34, 37, 44, 45, 46 and 47, respectively.

[0119] (2) Binding constant assay test: SPR measurement was performed on a Biacore 8K instrument (GE Healthcare, Piscataway, NJ, USA). Briefly, purified PKM2 protein (200 mg / mL, pH 8.0) was immobilized on a Series S sensor chip (GE Healthcare, Piscataway, NJ, USA) (w10000 RU) according to the standard amine coupling procedure. PBS (AR1155, pH 7.2-7.4, Boster) containing 3% DMSO was used as the running buffer for immobilization. After immobilization, 50 different concentrations of solutions of short peptides were prepared by serial dilution of the stock solution with electrophoresis buffer. Seven concentrations of GPS were injected simultaneously at a flow rate of 65 mL / min for a 60 s association period at 25°C. The final graph was obtained by subtracting the blank sensorgram. Experimental data were collected and analyzed using Biacore 8K management software (GE Healthcare, Piscataway, NJ, USA) to fit the appropriate binding model to obtain the equilibrium dissociation constant (KD).

[0120] The results are described in Figure 14 (in the order of the amino acid sequence listed in the figure, corresponding to the figure with a digital number), and from the figure, it can be seen that the KD values of the 50 polypeptides are all in the μm level, and the KD values of No. 37 and No. 38 short peptides are the smallest, respectively 0.03 x 10 -6 and 0.08 x 10 -6 .

[0121] The applicant declares that the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0122] The above describes the preferred embodiments of the present application, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0123] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable means without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A PKM2-based nanoglycopeptide activator comprising three functional units, PKM2-targeting activation unit R3, self-assembly unit R2 and response unit R1, respectively, wherein, The chemical structure of the nano-glycopeptide activator is shown in formula (I): wherein R3 is from an amino acid sequence having a function of activating PKM2 tetramerization; R2 is from a fibrous peptide having multiple hydrogen bonds in a molecule; R1 is from glucose, acetylglucosamine, mannose, fucose, galactose or folic acid; and X is from serine or threonine.

2. The PKM2-based nanoglycopeptide activator of claim 1, wherein, The amino acid sequence having a function of activating PKM2 tetramerization is a polypeptide with 6-10 amino acids; the C-terminal and N-terminal of the polypeptide are independently selected from H, K or Dab; the middle amino acid of the polypeptide 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 the tetramerization of PKM2 has a binding constant Kd>10 -5 to PKM2.

3. The PKM2-based nanoglycopeptide activator of claim 2, wherein, The amino acid sequence having a function of activating PKM2 tetramerization is selected from any one of the following: (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 connection product of SEQ ID NO. 20 and DabK: KTWWW-DabK; (21) SEQ ID NO. 21: KWWSFH; (22) SEQ ID NO. 22: KFSWWH; (23) SEQ ID NO. 23: HFWSWK; (24) SEQ ID NO. 24: (1) SEQ ID NO. 1: KFFWVH; (2) SEQ ID NO. 2: KFFWVWK; (3) SEQ ID NO. 3: KFFWVWH; (4) SEQ ID NO. 4: KFFWVWK; (5) SEQ ID NO. 5: KFFWVWK; (6) SEQ ID NO. 6: KFFWVWK; (7) SEQ ID NO. 7: KFFWVWK; (8) SEQ ID NO. 8: KFFWVWK; (9) SEQ ID NO. 9: KFFWVWK; (10) SEQ ID NO. 10: KFFWVWK; (11) SEQ ID NO. 11: KFFWVWK; (12) SEQ ID NO. 12: KFFWVWK; (13) SEQ ID NO. 13: KFFWVWK; (14) SEQ ID NO. 14: KFFWVWK; (15) SEQ ID NO. 15: KFFWVWK; (16) SEQ ID NO. 16: KFFWVWK; (17) SEQ ID NO. 17: KFFWVWK; (18) SEQ ID NO. 18: KFFWVWK; (19) SEQ ID NO. 19: KFFWVWK; (20) SEQ ID NO. 20: KFFWVWK; (21) SEQ ID NO. 21: KFFWVWK; (22) SEQ ID NO. 22: KFFWVWK; (23) SEQ ID NO. 23: KFFWVWK; (24) SEQ ID NO. 24: KFFWVWK; (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 ID NO. 30: KWFTWK; (31) SEQ ID NO. 31: HLLWTH; (32) The ligation product of KDabDab and SEQ ID NO. 32: KDabDab-FVWVK; (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) The ligation product of HKDab and SEQ ID NO. 37: HKDab-WSWWK; (38) SEQ ID NO. 38: HFWFWH; (39) The ligation product of SEQ ID NO. 39 and DabH: HFFTL-DabH; (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, or (50) The ligation product of HDab and SEQ ID NO. 50: HDab-LFWSWKK.

4. The PKM2-based nanoglycopeptide activator of claim 1, wherein, The fibrous peptide with multiple hydrogen bonds in the molecule is a polypeptide with 4-8 amino acids; the amino acids are independently selected from A, V, L, I, F, D, N, G, P, E, T.

5. The PKM2-based nanoglycopeptide activator of claim 4, wherein, The fibrous peptide with multiple hydrogen bonds in the molecule is selected from any one of the following: (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.

6. The PKM2-based nanoglycopeptide activator of claim 1, wherein, R1is derived from glucose, acetylglucosamine or folic acid.

7. A method for preparing the PKM2-based nano-glycopeptide activator according to any one of claims 1-6, comprising: synthesizing the PKM2-based nano-glycopeptide activator by solid phase synthesis using amino acids with both terminal and side chain amino groups protected and response unit materials as raw materials.

8. Use of the PKM2-based nano-glycopeptide activator according to any one of claims 1-6 in the preparation of an anti-tumor drug. Preferably, the tumor is selected from prostate cancer, renal cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, glioma, melanoma; Preferably, the tumor is a chemotherapy-resistant and high-metastatic tumor. Preferably, the tumor is a solid tumor with glycolysis as the main metabolic mode, and the tumor overexpresses glycolytic enzymes and PKM2.

9. Use of the PKM2-based nano-glycopeptide activator according to any one of claims 1-6 in the preparation of a tumor chemotherapy drug potentiator.

10. An anti-tumor combination pharmaceutical composition consisting of the PKM2-based nano-glycopeptide activator according to any one of claims 1-6 and a chemotherapy drug. Preferably, the chemotherapy drug comprises paclitaxel or a pharmaceutically acceptable salt thereof, doxorubicin or a pharmaceutically acceptable salt thereof, temozolomide or a pharmaceutically acceptable salt thereof. Preferably, the combination pharmaceutical composition is a single complex preparation or a combination of two separate preparations. Preferably, the combination pharmaceutical composition is a combination of two separate preparations, which are administered simultaneously or sequentially. Preferably, the preparation is any one of pharmaceutically acceptable dosage forms. Preferably, the combination pharmaceutical composition further contains a pharmaceutically acceptable excipient. Preferably, the pharmaceutically acceptable excipient comprises any one or a combination of at least two of a carrier, a diluent, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure adjusting agent, a surfactant, a coating material, a colorant, a pH adjusting agent, an antioxidant, a bacteriostatic agent, or a buffer.

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