Anti-tumor polypeptide and application thereof in tumor resistance

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-tumor polypeptide and application thereof in tumor resistance. The anti-tumor polypeptide provided by the invention comprises a first polypeptide and / or a second polypeptide, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 1; the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 2. The anti-tumor polypeptide is stable in chemical property, can selectively act on tumor cells through electrostatic interaction, then is inserted into a cell membrane phospholipid skeleton by utilizing a hydrophobic fragment in the structure of the anti-tumor polypeptide to destroy the integrity of a cell membrane, change membrane permeability, cause change of osmotic pressure of the tumor cells and finally cause cracking of the tumor cells, so that the anti-tumor effect is achieved. Good broad-spectrum anti-tumor activity is realized; the compound has small influence on normal cells, has good selectivity, and is suitable for being used as an active component of a clinical antitumor drug.

Description

Technical Field

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

[0002] Cancer has become the second leading cause of death worldwide, after cardiovascular disease. Chemotherapy is a systemic treatment for cancer. With the increasing variety of anti-tumor drugs and the continuous development of treatment strategies, chemotherapy has made considerable progress, especially in the treatment of leukemia and malignant lymphoma, significantly extending patient survival time. However, it has not achieved satisfactory results in treating solid tumors, which account for more than 90% of malignant tumors and pose the greatest threat to human life and health. Furthermore, anti-tumor drugs generally suffer from poor selectivity, significant toxic side effects, and easy drug resistance. Therefore, developing anti-tumor drugs with high selectivity for tumor cells and low toxicity has become a research hotspot in the field of cancer treatment. Summary of the Invention

[0003] The purpose of this invention is to provide an antitumor polypeptide that is chemically stable, capable of disrupting tumor cell membranes and inhibiting tumor cell growth, exhibiting good broad-spectrum antitumor activity; it also has low toxicity to normal cells and low hemolytic toxicity to erythrocytes, and good selectivity and better bioavailability.

[0004] This invention provides an antitumor polypeptide or a salt thereof, wherein the antitumor polypeptide comprises a first polypeptide and / or a second polypeptide; The amino acid sequence of the first polypeptide is shown in SEQ ID NO:1; The amino acid sequence of the second polypeptide is shown in SEQ ID NO:2.

[0005] Preferably, the first polypeptide is C-terminally amidated; the second polypeptide is C-terminally amidated.

[0006] The present invention also provides the application of the antitumor polypeptide or its salt described in the above technical solution in the preparation of antitumor products.

[0007] Preferably, the tumor includes one or more of cervical cancer, leukemia, and liver cancer.

[0008] The present invention also provides the application of the antitumor polypeptide or its salt described in the above technical solution in the preparation of products that disrupt tumor cell membranes and / or inhibit tumor cell growth.

[0009] Preferably, the tumor cells include one or more of cervical cancer cells, leukemia cells, and liver cancer cells.

[0010] Preferably, the cervical cancer cells include HeLa cervical cancer cells; The leukemia cells include leukemia cells MV-4-11; The liver cancer cells include HepG-2 liver cancer cells.

[0011] Preferably, the product includes a drug.

[0012] The present invention also provides an antitumor drug, wherein the active ingredient of the antitumor drug includes the antitumor polypeptide or its salt described in the above technical solution.

[0013] Preferably, the antitumor drug further includes pharmaceutically acceptable excipients.

[0014] Beneficial effects: The antitumor peptide provided by this invention is a short peptide, including a first peptide and / or a second peptide; the amino acid sequence of the first peptide is shown in SEQ ID NO:1; the amino acid sequence of the second peptide is shown in SEQ ID NO:2. The antitumor peptide or its salt provided by this invention has the following effects: (1) It is chemically stable and selectively acts on the negatively charged tumor cell membrane through electrostatic interaction, thus exhibiting good tumor selectivity. (2) It disrupts the integrity of the tumor cell membrane through hydrophobic fragments in its structure, causing changes in the osmotic pressure of the tumor cells, ultimately leading to tumor cell lysis and death. Since there is no specific target, the risk of drug resistance can be effectively reduced during use. (3) Compared with long peptides, it is easier to penetrate the vascular barrier and cell membrane of tumor tissue, resulting in higher bioavailability; at the same time, the risk of triggering allergies or immune reactions is lower, resulting in lower immunogenicity and easier safety control. (4) It can be prepared by microwave-promoted solid-phase synthesis, which has the advantages of high coupling efficiency and short synthesis cycle compared with traditional liquid-phase preparation, which is conducive to industrial synthesis. The antitumor polypeptide or its salt provided by this invention has antitumor activity, can destroy tumor cell membranes, and inhibit tumor cell growth, making it suitable as an active ingredient in clinical antitumor drugs. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 Hemolysis rate curves for different concentrations of polypeptides; Figure 2 LDH release rate of peptides at different concentrations; Figure 3 Tumor growth curves in vivo for HepG-2 in different treatment groups; where ** indicates P ≤0.01; Figure 4The tumor weight in vivo for HepG-2 in different treatment groups; where ** represents P ≤0.01. Detailed Implementation

[0017] The present invention provides an antitumor polypeptide or a salt thereof, the antitumor polypeptide comprising a first polypeptide and / or a second polypeptide; the amino acid sequence of the first polypeptide is shown in SEQ ID NO:1; the amino acid sequence of the second polypeptide is shown in SEQ ID NO:2.

[0018] As one embodiment, the salt described in this invention is a pharmaceutically acceptable salt.

[0019] In one embodiment, the first polypeptide of the present invention is subjected to C-terminal amidation. In another embodiment, the second polypeptide of the present invention is subjected to C-terminal amidation.

[0020] The antitumor polypeptide of this invention is chemically stable and can selectively act on tumor cells through electrostatic interactions. Then, by inserting hydrophobic fragments into the cell membrane phospholipid backbone, it disrupts the cell membrane integrity, alters membrane permeability, and causes changes in the osmotic pressure of tumor cells, ultimately leading to tumor cell lysis. It exhibits good broad-spectrum antitumor activity while having minimal impact on normal cells and good selectivity. This invention does not impose strict requirements on the preparation method of the antitumor polypeptide; conventional methods in the art can be used, such as microwave-promoted Fmoc / tBu orthogonal protected solid-phase synthesis.

[0021] The present invention also provides the application of the antitumor polypeptide or its salt described in the above technical solution in the preparation of antitumor products.

[0022] In one embodiment, the tumor described in this invention includes one or more of cervical cancer, leukemia, and liver cancer.

[0023] The present invention also provides the application of the antitumor polypeptide or its salt described in the above technical solution in the preparation of products that disrupt tumor cell membranes and / or inhibit tumor cell growth.

[0024] In one embodiment, the tumor cells of the present invention include one or more of cervical cancer cells, leukemia cells, and liver cancer cells. In one embodiment, the cervical cancer cells of the present invention include HeLa cervical cancer cells. In one embodiment, the leukemia cells of the present invention include MV-4-11 leukemia cells. In one embodiment, the liver cancer cells of the present invention include HepG-2 liver cancer cells.

[0025] As one implementation method, the product of this invention includes a drug.

[0026] The present invention also provides an antitumor drug, wherein the active ingredient of the antitumor drug includes the antitumor polypeptide or its salt described in the above technical solution.

[0027] As one implementation method, the antitumor drug of the present invention further includes pharmaceutically acceptable excipients. The present invention does not impose strict requirements on the specific composition of the pharmaceutically acceptable excipients; they can be selected conventionally based on the drug's dosage form.

[0028] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an antitumor polypeptide provided by the present invention and its application in antitumor treatment, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0029] The following abbreviations are used throughout this invention: DCM: Dichloromethane; NMP: N-methylpyrrolidone; DIPEA: N,N-diisopropylethylamine; TFA: Trifluoroacetic acid; EDT: Ethylene dithiol; Fmoc: N-9-fluorenylmethoxycarbonyl; HBTU: Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; HOBT: 1-hydroxy-benzotriazole; ESI-MS: Electrospray ionizer; Leu: Leucine; Lys: Lysine; Phe: Phenylalanine; Ile: Isoleucine; Tyr: Tyrosine; Arg: Arginine.

[0030] Example 1 The polypeptide shown in SEQ ID NO:1 (LysLysLeuPheArgArgIleLeuLysTyrLeu, KKLFRRILKYL) was synthesized, and its C-terminus was amidated to change from -COOH to -CONH2. The specific steps are as follows: (1) Swelling of resin Weigh 50 mg of Fmoc-Rink amide-MBHA Resin (substitution amount 0.4 mmol / g), swell with 7 mL of DCM for 30 min, filter to remove DCM, swell with 10 mL of NMP for 30 min, and finally rinse with 7 mL of NMP, DCM and NMP respectively.

[0031] (2) Microwave-promoted removal of Fmoc protecting groups The swollen resin was placed in a reactor, and 7 mL of a 25% piperidine / NMP (v / v) solution containing 0.1 M HOBT was added. The reaction was carried out in a microwave reactor for 1 min at a microwave power of 15 W, with the reaction temperature controlled below 50°C. Compressed air was used for cooling. After the reaction, the solution was filtered off. Another 7 mL of the 25% piperidine / NMP (v / v) solution containing 0.1 M HOBT was added, and the reaction was carried out in the microwave reactor for another 4 min at a microwave power of 25 W, with the reaction temperature controlled at 50°C. Compressed air was used for cooling. After the reaction, the solution was filtered off, and the resin was washed thoroughly with NMP. The resin with the initially attached Fmoc protecting group removed was obtained.

[0032] (3) Microwave-promoted synthesis of Fmoc-Leu-Rink amide-MBHA Resin Fmoc-Leu-OH (0.04 mmol), HBTU (0.04 mmol), HOBT (0.04 mmol), and DIPEA (0.08 mmol) were dissolved in 10 mL of NMP. This solution was then added to the resin and reacted in a microwave reactor for 7 min at a microwave power of 25 W. The reaction temperature was controlled at 50 °C, and compressed air was used for cooling. After the reaction was complete, the reaction solution was filtered off, and the resin was washed three times with 7 mL each of DCM and NMP.

[0033] (4) Detection of coupling efficiency Take a small amount of resin particles, wash them with ethanol, and place them in a transparent vial. Add 5% ninhydrin ethanol and 2×10 5 Add 2 drops each of mol / L KCN pyridine solution and 80% phenol ethanol solution, and heat at 80℃ for 5 min. If the resin turns blue, it is positive, indicating the presence of free amino groups, and the coupling time needs to be extended. If the resin turns white or light yellow, it is negative, the coupling is complete, and the next amino acid coupling cycle can begin.

[0034] (5) Elongation of peptide chains Following the order of the peptide chains, the above deprotection and coupling steps were repeated from the C-terminus to the N-terminus to connect the corresponding amino acids. The coupling reaction was promoted by microwave for 5 to 20 minutes to obtain the peptide-resin complex.

[0035] (6) Cleavage of peptides on resin The fatty acid chain-peptide-resin complex obtained above was placed in a reaction flask, and 10 mL of cleavage agent Reagent K (TFA / anisole / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added to each. The mixture was first shaken at 0 °C for 30 min, and then reacted at room temperature for 3 h. After the reaction was complete, the mixture was filtered, washed three times with a small amount of TFA and DCM, and the filtrates were combined. The filtrate was added to a large amount of ice-cold diethyl ether to precipitate a white flocculent precipitate, which was then centrifuged at freeze to obtain the crude target peptide. Finally, 45.3 mg of the crude target compound was obtained, with a yield of 81.3%.

[0036] (7) Purification of polypeptides The crude peptide was dissolved in 50% acetonitrile / water and purified by preparative liquid chromatography (HPLC). The chromatographic conditions were: C18 reversed-phase column (320 mm × 28 mm, 5 µm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40%–90%, 60 min; flow rate 5 mL / min; detection wavelength 214 nm. The collected solution was lyophilized to obtain 28.7 mg of pure product. The theoretical relative molecular mass was 1476.93. Electrospray ionization mass spectrometry (ESI-MS) m / z: calculate [M+2H] 2+ 739.47, [M+3H] 3+ 493.31; found [M+4H] 4+ 739.49, [M+5H] 5+ 493.28.

[0037] Example 2 Following the general method of Example 1, the polypeptide shown in SEQ ID NO:2 (LeuLysLysLeuPheLysLysIleLeuLysTyrLeuArgArg, LKKLFKKILKYLRR) was synthesized according to the corresponding sequence, and its C-terminus was amidated to change from -COOH to -CONH2. The molecular weight was confirmed by ESI-MS: the theoretical relative molecular mass is 1846.44. Electrospray ionization mass spectrometry (ESI-MS) m / z: calculate [M+2H] 2+ 924.22, [M+3H] 3+ 616.48; found [M+4H] 4+ 924.17, [M+5H] 5+ 616.51.

[0038] Test Example 1 Tumor cell selectivity assay The tumor cell selectivity of the peptides obtained in Examples 1 and 2 was determined using an in vitro cytotoxicity assay, and the specific steps are as follows: Cervical cancer cell line HeLa, leukemia cell line MV-4-11, liver cancer cell line HepG-2, human gastric mucosal cell line GES-1, and human umbilical vein endothelial cells HUVEC, all in good logarithmic growth phase, were selected and treated with 1×10⁻⁶ cells per cell line. 5 After seeding at a density of 1 / ml in 96-well plates and culturing for 12 h, peptides of different concentration gradients (0.625 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L, and 160 μmol / L) were added and incubated for 48 h. After incubation, 20 μl of CCK-8 solution was added to each well, and incubation was continued for 2 h. The OD value at 450 nm was then measured using a microplate reader. Finally, the IC50 of the test compound was calculated using GraphPad Prism 7.0. 50 The values ​​are shown in Table 1.

[0039] Table 1. In vitro cytotoxicity of different peptides (μmol / L)

[0040] As shown in Table 1, compared with normal cells GES-1 and HUVEC, the peptides obtained in Examples 1 and 2 all exhibited significant cytotoxicity against tumor cells HeLa, MV-4-11, and HepG-2. This indicates that the peptides obtained in Examples 1 and 2 possess good broad-spectrum antitumor activity, low cytotoxicity to normal cells, and good selectivity.

[0041] Test Example 2 Red blood cell hemolytic toxicity assay The hemolytic toxicity of the peptides obtained in Examples 1 and 2 to erythrocytes was determined using a hemolytic assay, and the specific steps are as follows: Take 20 ml of fresh rabbit blood into a 50 ml centrifuge tube containing anticoagulant, add 40 ml of PBS buffer, centrifuge at 1500 rpm for 15 min at 4°C, and discard the supernatant. Wash three times with ice-cold PBS buffer, then add 50 ml of PBS buffer and gently pipette to prepare a red blood cell suspension with a concentration of approximately 4%. Then add 200 μl of red blood cell suspension and 100 μl of peptides at different concentrations (40, 80, 120, 160, 200, and 240 μmol / L) to each well of a 48-well plate. PBS buffer is used as a negative control, and 1% Triton X-100 is used as a positive control. After incubation at 37℃ for 1 h, the 48-well plate was removed, centrifuged at 1500 rpm and 4℃ for 15 min, and 200 μl of supernatant was transferred to another 48-well plate. The OD value at 540 nm was measured using a microplate reader. The hemolysis rate (%) was calculated using the formula: (OD of drug-treated wells - OD of negative wells) / (OD of positive wells - OD of negative wells) × 100%. Finally, a concentration-hemolysis rate curve was plotted using GraphPad Prism 7.0. The results showed that when the concentration of the peptides obtained in Examples 1 and 2 was below 160 μmol / L, the hemolysis rate of erythrocytes was below 10%, indicating that the peptides obtained in Examples 1 and 2 had low hemolytic toxicity to erythrocytes. Figure 1 ).

[0042] Test Example 3 In vitro antitumor activity assay (membrane disruption effect) The in vitro antitumor activity of the peptides obtained in Examples 1 and 2 was determined using a lactate dehydrogenase (LDH) release assay, and the specific steps are as follows: MV-4-11 cells in the logarithmic growth phase were collected by centrifugation and prepared into a cell suspension. After cell counting, the concentration of the suspension was adjusted to 1×10⁻⁶. 5Cells were added at a concentration of 100 μl / ml to each well of a 96-well plate, and the plate was incubated in a 5% CO2, 37°C incubator for 24 h. Then, peptides of different concentration gradients (2, 4, 8, and 16 μmol / L) were added, and the plates were incubated for another 1 h. After centrifugation at 3000 rpm, 4°C for 5 min, the supernatant was collected. Each sample was tested using a lactate dehydrogenase assay kit. PBS buffer was used as a negative control, and 1% Triton X-100 was used as a positive control. The LDH release rate (%) was calculated using the formula: LDH release rate (%) = (OD-doped wells - OD-negative wells) / (OD-positive wells - OD-negative wells) × 100%. The results showed that as the concentration of the peptides obtained in Examples 1 and 2 increased, the LDH content in the cell supernatant gradually increased, indicating that the disruptive effect of the peptides obtained in Examples 1 and 2 on the tumor cell membrane gradually increased. Figure 2 ).

[0043] Test Example 4 In vivo antitumor activity assay The in vivo antitumor activity of the peptides obtained in Examples 1 and 2 was determined using a nude mouse tumor-bearing experiment. The specific steps are as follows: HepG-2 cells were subcutaneously in the right axilla of balb / c nude mice. Tumors were allowed to grow to 100 mm. 3 The animals were then randomly divided into a blank control group (Saline) and a treatment group. The drugs were administered intratumorally every two days. The treatment group received an injection of the corresponding peptide at a concentration of 20 mg / kg per injection, while the blank control group received an equal volume of physiological saline. The tumor volume V (mm²) was measured in mice the day after administration. 3 The antitumor effect of the compounds was dynamically observed, and tumor growth curves were plotted. Mice were sacrificed after seven administrations, and tumor masses were surgically removed and weighed. The results showed that the peptides obtained in Examples 1 and 2 significantly inhibited the growth of HepG-2 tumors in nude mice, exhibiting good in vivo antitumor activity. Figure 3 and Figure 4 ).

[0044] As can be seen from the above, the antitumor polypeptide provided by this invention has stable chemical properties, can destroy tumor cell membranes, and has good broad-spectrum antitumor activity, making it suitable as an active ingredient in clinical antitumor drugs.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antitumor polypeptide or a salt thereof, characterized in that, The antitumor polypeptide includes a first polypeptide and / or a second polypeptide; The amino acid sequence of the first polypeptide is shown in SEQ ID NO:1; The amino acid sequence of the second polypeptide is shown in SEQ ID NO:

2.

2. The antitumor polypeptide or its salt according to claim 1, characterized in that, The first polypeptide is subjected to C-terminal amidation; the second polypeptide is subjected to C-terminal amidation.

3. The use of the antitumor polypeptide or its salt as described in claim 1 or 2 in the preparation of antitumor products.

4. The application according to claim 3, characterized in that, The tumor includes one or more of cervical cancer, leukemia, and liver cancer.

5. The use of the antitumor polypeptide or its salt as described in claim 1 or 2 in the preparation of products that disrupt tumor cell membranes and / or inhibit tumor cell growth.

6. The application according to claim 5, characterized in that, The tumor cells include one or more of cervical cancer cells, leukemia cells, and liver cancer cells.

7. The application according to claim 6, characterized in that, The cervical cancer cells include HeLa cervical cancer cells; The leukemia cells include leukemia cells MV-4-11; The liver cancer cells include HepG-2 liver cancer cells.

8. The application according to any one of claims 3 to 7, characterized in that, The products include pharmaceuticals.

9. An antitumor drug, characterized in that, The active ingredient of the antitumor drug includes the antitumor polypeptide or its salt as described in claim 1 or 2.

10. The antitumor drug according to claim 9, characterized in that, The antitumor drugs also include pharmaceutically acceptable excipients.

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