Anti-tumor polypeptide and application thereof

Abstract

The invention belongs to the technical field of polypeptide drugs, and particularly relates to an anti-tumor polypeptide and application thereof. According to the preparation method, Rink amide MBHA amino resin is taken as a solid phase carrier, modification and transformation are carried out according to an amino acid sequence of a template polypeptide H-0 (hymenochirin-1Pa): Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASAMA-NH2, and on the basis that key amino acid residues are reserved, original amino acids are replaced by S5 at the positions of i and i + 4 amino acids, so that the target stapling peptide is obtained. Pharmacological experiments show that the synthesized stapled peptide can significantly inhibit growth and proliferation of tumor cells of liver cancer, lung cancer, glioma and colon cancer, and has an application prospect of being developed into a novel anti-cancer drug.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug technology, specifically relating to an anti-tumor polypeptide and its applications. Background Technology

[0002] Malignant tumors have become one of the leading causes of death worldwide, posing a continuous and severe challenge to human health. Although traditional chemotherapy, targeted therapy, and immunotherapy have made some progress in clinical practice, the emergence of multidrug-resistant tumors, the non-selective toxicity of therapeutic drugs, and the complexity of the tumor microenvironment still severely limit clinical treatment outcomes. Common malignant tumors such as non-small cell lung cancer, breast cancer, and colorectal cancer often fail to respond to chemotherapy drugs, and traditional drugs lack the ability to distinguish between tumor cells and normal cells, easily causing serious side effects such as bone marrow suppression and gastrointestinal reactions, significantly reducing patients' quality of life. Therefore, developing anti-tumor drugs with high tumor selectivity and strong metabolic stability has become a core problem urgently needing to be solved in the field of tumor pharmacology.

[0003] Host-defense peptides (HDPs) are key components of the innate immune system and are widely found in organisms such as amphibians and mammals, with frog skin secretions being a major natural source. These peptides are characterized by their small molecular weight, diverse structures, and unique mechanisms of action. Their anti-tumor activity is mainly achieved by disrupting the integrity of tumor cell membranes, regulating tumor cell apoptosis pathways, or activating the body's anti-tumor immune response. Compared to traditional chemotherapy drugs, peptides, with their non-specific "membrane disruption" mechanism, are less likely to induce drug resistance in tumor cells, and some peptides retain activity against multidrug-resistant tumor cells. Therefore, they are considered highly promising templates for the development of novel anti-tumor drugs and have unique development prospects in targeted cancer therapy.

[0004] Hymenochirin-1Pa (sequence LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2), an antimicrobial peptide (HDP) isolated from the skin secretions of the Merlin clawed frog, exhibits significant broad-spectrum antibacterial activity (effective against methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and other multidrug-resistant bacteria) and antitumor activity. Structurally, hymenochirin-1Pa forms a characteristic amphiphilic α-helix structure in a membrane-mimicking environment, containing two helical domains (K6-G17 and G21-M28), and forming a 93° bend structure through the GXXXG motif between G17 and G21. This structure is believed to be closely related to peptide insertion into the cell membrane and the exertion of biological activity. Although natural HDPs such as hymenochirin-1Pa have shown good antitumor potential, their inherent defects severely limit clinical translation. First, natural HDPs generally suffer from insufficient tumor cell selectivity. Second, the α-helix structure of natural peptides is easily degraded by proteases in the physiological environment, resulting in insufficient stability and affecting their in vivo half-life and efficacy. Therefore, the antitumor activity and enzyme stability of hymenochirin-1Pa still need further optimization to meet the dosage requirements of clinical treatment. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an antitumor polypeptide. This antitumor polypeptide is a staple peptide. Compared to the template polypeptide, this staple peptide exhibits enhanced antitumor activity and improved enzyme stability.

[0006] Another object of the present invention is to provide pharmaceutical use of the said antitumor polypeptide.

[0007] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows: An antitumor polypeptide, wherein the antitumor polypeptide is a staple peptide, and the staple peptide is: H-1: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 5P and 9D are replaced by S5. H-2: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 10T and 14V are replaced by S5. H-3: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 14V and 18A are replaced by S5. H-4: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 17G and 21G are replaced by S5. H-5: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 18A and 22A are replaced by S5. H-6: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 19I and 23I are replaced by S5. H-7: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 21G and 25I are replaced by S5. H-8: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 22A and 26A are replaced by S5. H-9: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 23I and 27S are replaced by S5. H-10: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 24A and 28M are replaced by S5. H-11: The peptide template is Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2, in which amino acid residues 25I and 29A are replaced by S5.

[0008] Preferably, in the above-mentioned antitumor polypeptide, the staple peptide is: H-4: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 17G and 21G are replaced by S5. H-5: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 18A and 22A are replaced by S5. H-6: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 19I and 23I are replaced by S5. H-7: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 21G and 25I are replaced by S5. H-10: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 24A and 28M are replaced by S5. H-11: The peptide template is Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2, in which amino acid residues 25I and 29A are replaced by S5.

[0009] More preferably, in the above-mentioned antitumor polypeptide, the stapled peptide is: H-5: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 18A and 22A are replaced by S5. H-7: The peptide template is Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2, in which amino acid residues 21G and 25I are replaced by S5.

[0010] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows: The above-mentioned staple peptides are used in the preparation of anticancer drugs.

[0011] Preferably, the tumor is liver cancer.

[0012] Preferably, the tumor is lung cancer.

[0013] Preferably, the tumor is a glioma.

[0014] Preferably, the tumor cells are colon cancer.

[0015] In this invention, the abbreviations are explained as follows: Fmoc: fluorenemethyloxycarbonyl; DCE: 1,2-dichloroethane; Oxyme: ethyl 2-oxime cyanoacetate; DCM: dichloromethane; DMF: N,N-dimethylformamide; DIC: N,N-diisopropylcarbodiimide; S5: 2-amino-2-methyl-9-heptenic acid; TFA: trifluoroacetic acid; EDT: 1,2-ethylenedithiol; GrubbsⅠ: phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride; CCK-8: CellCounting Kit-8; PBS: phosphate buffer; MS: mass spectrometry; HR-Q-TOF-MS: high-resolution matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; Huh7: human hepatocellular carcinoma cells; A549 cells: human alveolar basal epithelial cells of lung cancer; U87: glioma cells; T84: colon cancer cells.

[0016] The advantages of this invention are: 1. This invention uses Rink amide MBHA amino resin as a solid-phase support and modifies the template polypeptide H-0 (hymenochirin-1Pa) according to its amino acid sequence: Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2. While retaining key amino acid residues, S5 is used to replace the original amino acids at positions i and i+4 to obtain the target staple peptide. Pharmacological experiments show that the synthesized staple peptide can significantly inhibit the growth and proliferation of liver cancer, lung cancer, glioma, and colon cancer cells, and has the potential to be developed into a novel anticancer drug.

[0017] 2. Except for H-8 and H-9, the other 9 peptides obtained in this invention all showed significantly higher inhibitory activity than H-0 in at least one tumor cell line (P<0.05), indicating that the anti-tumor activity was significantly improved through structural modification. Among them, compared with the template peptide H-0, H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against Huh7 (human liver cancer cells) proliferation (P<0.001); H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against A549 cells (human alveolar basal epithelial cells of lung cancer) proliferation (P<0.001); H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against U87 (glioma cells) proliferation (P<0.001); and H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against T84 (colon cancer cells) proliferation (P<0.001). H-7's IC50 against Huh7 (human liver cancer cells), A549 cells (non-small cell lung cancer cells), and U87 (glioma cells) 50 A concentration below 1 μM indicates significant H-7 activity.

[0018] 3. The results of the enzymatic stability test show that the staple peptide H-7 of the present invention has significantly improved stability against chymotrypsin hydrolysis and trypsin hydrolysis compared with the template peptide H-0. Attached Figure Description

[0019] Figure 1 The diagram shows the amino acid sequence of H-0 and its characterization spectrum, where A is the amino acid sequence of H-0, B is the HPLC chromatogram of H-0, and C is the mass spectrum of H-0.

[0020] Figure 2 The diagram shows the amino acid sequence of H-1 and its characterization spectrum, where A is the amino acid sequence of H-1, B is the HPLC chromatogram of H-1, and C is the mass spectrum of H-1.

[0021] Figure 3 The diagram shows the amino acid sequence of H-2 and its characterization spectrum, where A is the amino acid sequence of H-2, B is the HPLC chromatogram of H-2, and C is the mass spectrum of H-2.

[0022] Figure 4 The diagram shows the amino acid sequence of H-3 and its characterization spectrum, where A is the amino acid sequence of H-3, B is the HPLC chromatogram of H-3, and C is the mass spectrum of H-3.

[0023] Figure 5 The diagram shows the amino acid sequence of H-4 and its characterization spectrum, where A is the amino acid sequence of H-4, B is the HPLC chromatogram of H-4, and C is the mass spectrum of H-4.

[0024] Figure 6 The diagram shows the amino acid sequence of H-5 and its characterization spectrum, where A is the amino acid sequence of H-5, B is the HPLC chromatogram of H-5, and C is the mass spectrum of H-5.

[0025] Figure 7 The diagram shows the amino acid sequence of H-6 and its characterization spectrum, where A is the amino acid sequence of H-6, B is the HPLC chromatogram of H-6, and C is the mass spectrum of H-6.

[0026] Figure 8 The diagram shows the amino acid sequence of H-7 and its characterization spectrum, where A is the amino acid sequence of H-7, B is the HPLC chromatogram of H-7, and C is the mass spectrum of H-7.

[0027] Figure 9 The diagram shows the amino acid sequence of H-8 and its characterization spectrum, where A is the amino acid sequence of H-8, B is the HPLC chromatogram of H-8, and C is the mass spectrum of H-8.

[0028] Figure 10 The diagram shows the amino acid sequence of H-9 and its characterization spectrum, where A is the amino acid sequence of H-9, B is the HPLC chromatogram of H-9, and C is the mass spectrum of H-9.

[0029] Figure 11 The diagram shows the amino acid sequence of H-10 and its characterization spectrum, where A is the amino acid sequence of H-10, B is the HPLC chromatogram of H-10, and C is the mass spectrum of H-10.

[0030] Figure 12 The diagram shows the amino acid sequence of H-11 and its characterization spectrum, where A is the amino acid sequence of H-11, B is the HPLC chromatogram of H-11, and C is the mass spectrum of H-11.

[0031] Figure 13 The graph shows the degradation kinetics of H-0 and H-7 trypsins.

[0032] Figure 14 The degradation kinetic curves of H-0 and H-7 chymotrypsin are shown. Detailed Implementation

[0033] The present application will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present application. However, these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. That is, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0034] This invention designs and synthesizes 11 staple peptides based on the amino acid sequence of template peptide H-0 (hymenochirin-1Pa): Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2. The amino acid sequence, HPLC chromatogram, and mass spectrum of template peptide H-0 are shown below. Figure 1 .

[0035] The experimental materials involved in the embodiments of this invention were sourced as follows: Fmoc-amino acids and RinkamideMBHA amino resin were purchased from Nankai Synthetic Co., Ltd.; Fmoc-amino acids, N,N-dimethylformamide, N,N-diisopropylcarbodiimide, and ethyl 2-oxime cyanoacetate were purchased from Jier Biochemical (Shanghai) Co., Ltd.; trifluoroacetic acid, acetonitrile (chromatographic grade), benzyl sulfide, 1,2-ethanedithiol, anhydrous diethyl ether, dichloromethane, 1,2-dichloroethane, piperidine, and phenol were all analytical grade and purchased from Shanghai Titan Technology Co., Ltd.

[0036] Example 1: Preparation of H-O-based staple peptide 1. General Synthesis Process All stapled peptides (H-1 to H-11) were prepared using a solid-phase peptide synthesis method based on the Fmoc (fluorenemethyloxycarbonyl) protection strategy. This method used Rink amide MBHA resin as the solid-phase support at a loading capacity of 0.35 mmol / g. Synthesis was carried out in solid-phase synthesis reaction tubes, and the entire process included resin swelling, Fmoc protecting group removal, amino acid condensation, N-terminal acetylation, olefin metathesis cyclization, and final peptide cleavage.

[0037] (1) Resin pretreatment and activation: Take 400 mg of amino resin and soak it in dichloromethane solvent for 30 minutes to allow it to swell fully. Then, treat it twice (5 minutes each time) with 7 mL of 20% piperidine N,N-dimethylformamide solution at 35°C to remove the Fmoc protecting groups on the resin surface. Finally, wash the resin three times each with N,N-dimethylformamide, dichloromethane and N,N-dimethylformamide in sequence.

[0038] (2) Amino acid condensation: Following the sequence of the template polypeptide H-0 (Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2), amino acids were sequentially linked from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus). For common amino acids, 1 mmol of Fmoc-protected amino acid, 142 mg of ethyl 2-oxime cyanoacetate, and 200 μL of N,N-diisopropylcarbodiimide were dissolved in 7 mL of N,N-dimethylformamide, activated at 37°C for 15 minutes, and then added to a reaction tube for coupling with resin. The reaction was carried out at 60°C for 20 minutes. For non-natural amino acid S5, the amount used was 0.2 mmol, and the condensing agent was 43 mg of ethyl 2-oxime cyanoacetate and 60 μL of N,N-diisopropylcarbodiimide. After activation in the same solvent, the reaction was carried out at 60°C for 3 hours or at 37°C overnight to ensure complete coupling. After each amino acid is attached, the Fmoc protecting group must be removed with a 20% piperidine N,N-dimethylformamide solution, followed by washing.

[0039] (3) N-terminal acetylation: After sequence synthesis, the Fmoc protecting group of the terminal amino acid was removed using a 20% piperidine N,N-dimethylformamide solution. Then, 10 mL of acetylation reagent (diisopropylethylamine:acetic anhydride:N,N-dimethylformamide = 1:1:8, V / V / V) was added, and the reaction was carried out at 37°C for 5 minutes to acetylate the N-terminus of the peptide. After the reaction, the resin was dried and washed.

[0040] (4) Olefin metathesis reaction (cyclization): The resin was rinsed three times with 1,2-dichloroethane. A solution of 56 mg of phenylmethylene bis(tricyclohexylphosphine) ruthenium dichloride dissolved in 6 mL of 1,2-dichloroethane was added, and the reaction was carried out at room temperature for 8 hours. This caused a ring-closing metathesis reaction between the introduced non-natural amino acid side chain olefins, forming a full-carbon scaffold, thereby stabilizing the α-helical conformation of the peptide. The resin was thoroughly washed after the reaction was completed.

[0041] (5) Peptide cleavage and purification: The resin was placed in a 50 mL centrifuge tube, and 20 mL of cleavage reagent K (trifluoroacetic acid: water: 1,2-ethylenedithiol: benzyl sulfide: phenol = 82.5:5:2.5:5:5, V / V / V / V / V) was added. The mixture was shaken at 37°C for 3 hours. After the reaction was complete, the cleavage solution was collected, dried under nitrogen, and concentrated. The crude peptide was then precipitated with pre-cooled ice-cold ether. After centrifugation (3500 r / min, 3 min), the supernatant was discarded, and the precipitate was air-dried to obtain the crude target peptide. The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and its structure was identified by mass spectrometry.

[0042] 2. Specific synthesis of each binding peptide The general synthetic procedure described above applies to all staple peptides from H-1 to H-11. The difference between each staple peptide lies in the substitution of a non-natural amino acid S5 at a specific position in its template sequence, as shown in Table 1 below. These substitutions are the basis for the olefin metathesis reaction to proceed and form the specific "staple" structure.

[0043] Table 1. Specific amino acid substitution sites for each binding peptide Note: S5 is a specific non-natural amino acid, which is introduced for subsequent olefin metathesis reactions.

[0044] 2. Purification of staple peptide samples The crude peptide was dissolved in a mixed solvent of acetonitrile and water, and purified by reversed-phase preparative RP-HPLC to obtain the purified staple peptide product. The separation conditions were as follows: Instrument: Shimadzu LC-20A reversed-phase high-performance liquid chromatograph; Column: UltimateXB-C18, 21.2 × 250 mm, 5 μm; Mobile phase: Mobile phase A is an acetonitrile solution of 0.1% trifluoroacetic acid by volume, and mobile phase B is an aqueous solution of 0.1% trifluoroacetic acid by volume; Procedure and parameters: Elute with 90% B for 3 min, then elute with 90% B to 25% B for 55 min; flow rate is 8 mL / min, injection volume is 3 mL, and detection wavelengths are 214 nm and 254 nm.

[0045] Each peptide is purified individually.

[0046] During gradient elution, when the volume fraction of mobile phase A rises to approximately 56%, compound H-0 is eluted to obtain H-0, with a separation rate of 32.5%.

[0047] During gradient elution, when the volume fraction of mobile phase A rises to approximately 60%, compound H-1 is eluted to obtain H-1, with a separation rate of 23.8%.

[0048] During gradient elution, when the volume fraction of mobile phase A increased to approximately 64%, compound H-2 was eluted to obtain H-2, with a separation rate of 14.4%.

[0049] During gradient elution, when the volume fraction of mobile phase A increased to approximately 69%, compound H-3 was eluted to obtain H-3, with a separation rate of 12.9%.

[0050] During gradient elution, when the volume fraction of mobile phase A increased to approximately 73%, compound H-4 was eluted to obtain H-4, with a separation rate of 24.8%.

[0051] During gradient elution, when the volume fraction of mobile phase A increased to approximately 73%, compound H-5 was eluted to obtain H-5, with a separation rate of 19.4%.

[0052] During gradient elution, when the volume fraction of mobile phase A increased to approximately 60%, compound H-6 was eluted to obtain H-6, with a separation rate of 27.4%.

[0053] During gradient elution, when the volume fraction of mobile phase A increased to approximately 65%, compound H-7 was eluted to obtain H-7, with a separation rate of 17.3%.

[0054] During gradient elution, when the volume fraction of mobile phase A increased to approximately 69%, compound H-8 was eluted to obtain H-8, with a separation rate of 19.4%.

[0055] During the gradient elution process, when the volume fraction of mobile phase A increased to approximately 58%, compound H-9 was eluted to obtain H-9, with a separation rate of 8.9%.

[0056] During gradient elution, when the volume fraction of mobile phase A increased to approximately 59%, compound H-10 was eluted to obtain H-10, with a separation rate of 21.4%.

[0057] During gradient elution, when the volume fraction of mobile phase A increased to approximately 60%, compound H-11 was eluted to obtain H-11, with a separation rate of 15.4%.

[0058] Identification and structural analysis of the product in Example 2 The product obtained in step 2 of Example 1 was identified by reversed-phase HPLC. Analytical column: Welch C18; mobile phase A was an acetonitrile solution of 0.1% trifluoroacetic acid (v / v), and mobile phase B was an aqueous solution of 0.1% trifluoroacetic acid (v / v). Gradient elution was used (0–2 min, mobile phase B: 90%; 3–25 min, mobile phase B: 90%–10%); flow rate: 1.0 mL / min. -1 The detection wavelengths were 214 nm and 254 nm, and the injection volume was 24 μL. The peak elution time was consistent with that of the crude product, and the purity of the staple peptide prepared by this method was >95%. The HPLC chromatogram of the H-1-H-11 staple peptide is shown below. Figures 2-12 .

[0059] Structural analysis was performed using HR-Q-TOF-MS, and the mass spectrometry analysis results of the obtained H-1-H-11 binding peptide are shown below. Figures 2-12 The structure of the staple peptide obtained after analysis is shown in Table 2.

[0060] Table 2. Sequences of the template peptide and the modified binding peptide used in this invention. The amino acid sequences of the template peptides and the obtained stapler peptides involved in Tables 1 and 2 of this invention are shown in SEQ ID NO: 1-12.

[0061] Example 3: Experiment on the inhibition of Huh7, A549, U87, and T84 cell proliferation by the staple peptide of the present invention. The cell proliferation activity of four cell lines (Huh7, A549, U87, and T84) treated with peptides was detected using the Cell Counting Kit-8 (CCK-8) assay. Huh7, A549, U87, and T84 cells were cultured at 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 100 cells / well into 96-well plates and cultured overnight to allow for full cell adhesion. The culture medium was then discarded, and 200 μL of complete culture medium containing different concentrations of the peptide drug (peptide drug concentrations: 0, 0.625, 1.25, 2.5, 5, 10, 20 μM) was added to each well. The plates were incubated for 24 h. The culture medium was then discarded, and the cells were washed 2-3 times with PBS. 100 μL of 1640 medium (containing 10 μL of CCK-8 solution) was then added to each well. The 96-well plates were incubated at 37 °C for approximately 1-3 h. The absorbance at 450 nm was then measured using a microplate reader. Finally, the IC50 values ​​of the drugs were statistically analyzed using PRISM software. In preliminary screening at the cellular level, compounds with IC50 values ​​below 10 μM are generally considered to have research value, while those below 1 μM indicate significant activity. The results are shown in Table 3.

[0062] Data analysis methods: One-way ANOVA was used, followed by Dunnett's multiple comparison test. The template peptide H-0 group was used as the control group. The significance of the inhibitory activity of each binding peptide (H-1 to H-11) in four tumor cell lines (Huh7, A549, U87, T84) was examined. P < 0.05 was used to indicate a statistically significant difference.

[0063] Table 3. Results of experiments on the inhibition of Huh7, A549, U87, and T84 cell proliferation by the peptide of this invention. Note: Compared with group H-0 P<0.001, P<0.0001.

[0064] The results in Table 3 show that, except for H-8 and H-9, the other nine peptides obtained in this invention exhibited significantly higher inhibitory activity than H-0 in at least one tumor cell line (P<0.05), indicating that the antitumor activity was significantly enhanced through structural modification. Among them, compared with the template peptide H-0, H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against Huh7 (human liver cancer cells) proliferation (P<0.001); H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against A549 cells (human alveolar basal epithelial cells of lung cancer) proliferation (P<0.001); H-1, H-2, H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against U87 (glioma cells) proliferation (P<0.001); and H-3, H-4, H-5, H-6, H-7, H-10, and H-11 significantly enhanced the inhibitory activity against T84 (colon cancer cells) proliferation (P<0.001). H-7's IC50 against Huh7 (human liver cancer cells), A549 cells (non-small cell lung cancer cells), and U87 (glioma cells) 50 A concentration below 1 μM indicates significant H-7 activity.

[0065] The above embodiments demonstrate that the present invention successfully prepared a modified staple peptide based on H-O. Through experiments on inhibiting tumor cell proliferation, it was proven that the synthesized staple peptide can significantly inhibit the growth and proliferation of liver cancer, lung cancer, glioma, and colon cancer tumor cells, and has the potential to be developed into a novel anticancer drug.

[0066] Example 4: Stability Experiment of the Bound Peptide Enzymatic Hydrolysis of the Present Invention 1. Methods and results for determining the stability of trypsin digestion (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH=7.4) until it is fully dissolved, so that the final concentration is 2 mM.

[0067] (2) Preparation of trypsin solution: Weigh a small amount of trypsin and dissolve it in the prepared buffer solution to make the final solubility 0.01 ng / μL.

[0068] (3) Preparation of peptide solution: Weigh 1 mg of template peptide H-0 and H-7, the optimal activity of the staple peptide derivative modified by the staple peptide strategy, and add them to the buffer solution to make the final solubility 1 mM.

[0069] (4) Reaction and Sampling: 1950 μL of trypsin solution was placed in a 5 mL centrifuge tube, and 50 μL of the prepared 1 mM polypeptide solution was added to the centrifuge tube containing the trypsin solution to carry out the trypsin degradation experiment. At time points of 0 min, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min during the experiment, 60 μL of the reaction mixture was taken and 20 μL of concentrated hydrochloric acid (1M) was added to quench and terminate the enzymatic hydrolysis reaction.

[0070] (5) Results analysis: Take 50 μL for HPLC analysis, determine the residual amount of peptide by integrating the peak area of ​​peptide, calculate the percentage of peak area at each time point to peak area at time 0 and draw a curve to obtain the analysis chart of peptide antitrypsin hydrolysis stability.

[0071] The principle of trypsin degradation of peptides is that trypsin's main cleavage site is the peptide bond at the carboxyl terminus of arginine or lysine, which can specifically cleave the peptide bond formed at the carboxyl terminus of arginine or lysine in the peptide chain.

[0072] The results are shown in Tables 4 and 5. Figure 13 .

[0073] Table 4. Stability data of H-0 trypsin Table 5. Stability data of H-7 trypsin The results in Tables 4 and 5 show that, among the staple peptides of the present invention, staple peptide H-7, which exhibits the best inhibitory activity against tumor cell proliferation, still retains 1.77% of its staple peptide intact after 3 hours of trypsin digestion. The trypsin hydrolysis stability test demonstrates that staple peptide H-7 has significantly improved stability against trypsin hydrolysis compared to the template peptide H-0.

[0074] 2. Methods and results for determining the stability of chymotrypsin hydrolysis (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH=7.4) until it is fully dissolved, so that the final concentration is 2 mM.

[0075] (2) Preparation of chymotrypsin solution: Weigh a small amount of chymotrypsin and dissolve it in the prepared buffer solution to make the final solubility 0.01 ng / μL.

[0076] (3) Preparation of peptide solution: Weigh 1 mg of template peptide H-0 and H-7, the optimal activity of the staple peptide derivative modified by the staple peptide strategy, and add them to the buffer solution to make the final solubility 1 mM.

[0077] (4) Reaction and Sampling: 1950 μL of chymotrypsin solution was placed in a 5 mL centrifuge tube, and 50 μL of the prepared 1 mM polypeptide solution was added to the centrifuge tube containing the chymotrypsin solution to carry out the chymotrypsin degradation experiment. At time points of 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min during the experiment, 60 μL of the reaction mixture was taken and 20 μL of concentrated hydrochloric acid (1M) was added to quench and terminate the enzymatic hydrolysis reaction.

[0078] (5) Results analysis: Take 50 μL for HPLC analysis, determine the residual amount of peptide by integrating the peak area of ​​peptide, calculate the percentage of peak area at each time point to peak area at time 0 and draw a curve to obtain the analysis chart of peptide antichymotrypsin hydrolysis stability.

[0079] The principle behind chymotrypsin's degradation of peptides is that chymotrypsin's main cleavage sites are tryptophan, tyrosine, phenylalanine, and threonine, which can specifically cleave the peptide bonds formed at the carboxyl ends of tryptophan, tyrosine, phenylalanine, and threonine in the peptide chain.

[0080] The results are shown in Tables 6 and 7. Figure 14 .

[0081] Table 6. Hydrolysis data of H-0 chymotrypsin Table 7. Hydrolysis data of H-7 chymotrypsin The results in Tables 6 and 7 show that the staple peptide H-7, which exhibits the best anti-tumor cell proliferation activity among the staple peptides of this invention, was not completely degraded after 3 hours of exposure to chymotrypsin, retaining 14% of its original content. The chymotrypsin hydrolysis stability test indicates that, compared to the template peptide H-0, staple peptide H-7 lacks chymotrypsin cleavage sites in its polypeptide sequence, thus significantly enhancing its stability against chymotrypsin hydrolysis.

Claims

1. An antitumor polypeptide, characterized in that, The antitumor polypeptide is a stapler peptide, and the stapler peptide is: H-1: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 5P and 9D are replaced by S5. H-2: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 10T and 14V are replaced by S5. H-3: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 14V and 18A are replaced by S5. H-4: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 17G and 21G are replaced by S5. H-5: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 18A and 22A are replaced by S5. H-6: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 19I and 23I are replaced by S5. H-7: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 21G and 25I are replaced by S5. H-10: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 24A and 28M are replaced by S5. H-11: The peptide template is Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2, in which amino acid residues 25I and 29A are replaced by S5.

2. The antitumor polypeptide according to claim 1, characterized in that, The stapler peptide is: H-4: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 17G and 21G are replaced by S5. H-5: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 18A and 22A are replaced by S5. H-6: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 19I and 23I are replaced by S5. H-7: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 21G and 25I are replaced by S5. H-10: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 24A and 28M are replaced by S5. H-11: The peptide template is Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2, in which amino acid residues 25I and 29A are replaced by S5.

3. The antitumor polypeptide according to claim 2, characterized in that, The stapler peptide is: H-5: Using Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2 as a peptide template, amino acid residues 18A and 22A are replaced by S5. H-7: The peptide template is Ac-LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2, in which amino acid residues 21G and 25I are replaced by S5.

4. The use of the antitumor polypeptide according to claim 1, 2 or 3 in the preparation of antitumor drugs.

5. The application according to claim 4, characterized in that, The tumor is liver cancer.

6. The application according to claim 4, characterized in that, The tumor is lung cancer.

7. The application according to claim 4, characterized in that, The tumor is a glioma.

8. The application according to claim 4, characterized in that, The tumor is colon cancer.

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