A polypeptide and its application in preparing a drug for preventing and / or treating cancer

Abstract

The present invention discloses a polypeptide and its use in the preparation of a drug for preventing and / or treating cancer, wherein the amino acid sequence of the polypeptide is shown in SEQ ID No.1 or SEQ ID No.2. The cancer is at least one of breast cancer, colorectal cancer, leukemia, esophageal cancer, pancreatic cancer, glioma, renal cancer, skin cancer, osteosarcoma, ovarian cancer and prostate cancer. The present invention designs and synthesizes a polypeptide targeting the Staphylococcus nuclease domain 1 (SND1) and heteromucin (MTDH) tumor protein complex SND1-MTDH, and for the first time finds that the polypeptide has excellent inhibitory activity against a variety of different types of human cancer cells, providing an alternative for a broad-spectrum anticancer drug. The polypeptide synthesis process used in the present invention is simple and has low production cost.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, and in particular to a polypeptide and its application in preparing drugs for preventing and / or treating cancer. Background Art

[0002] Cancer is one of the major diseases that threaten human health, and its treatment remains a major challenge facing humanity. Chemotherapy is one of the main methods for treating cancer, but chemotherapy drugs have poor specificity, damage normal cells, and are prone to toxic side effects; long-term use of drugs is prone to multidrug resistance, which reduces the efficacy of drugs. Peptide drugs have the following advantages: strong targeting, able to target specific cancer proteins or cancer cells; good biocompatibility, composed of natural amino acids, low immunogenicity, and few adverse reactions; easy to synthesize and modify, can be easily synthesized and modified by methods such as solid phase synthesis, and are easy to mass produce.

[0003] The SND1-MTDH complex, formed by the binding of Staphylococcal nuclease domain 1 (SND1) and heteromucin (MTDH), is a tumor protein complex that plays a key role in the occurrence and development of various cancers. Studies have shown that disrupting the interaction between SND1 and MTDH can effectively inhibit the proliferation and migration of cancer cells, thereby preventing tumor progression. This complex promotes cancer development by enhancing tumor cell survival, promoting cell proliferation and migration, and inhibiting anti-tumor immune responses. Although targeting the SND1-MTDH complex is considered a promising therapeutic strategy, there is still a lack of targeted drugs that can specifically inhibit the binding of SND1 to MTDH. Summary of the invention

[0004] The present invention designs a polypeptide capable of inhibiting the interaction between SND1 and MTDH and degrading SND1 and MTDH, and the polypeptide has a good inhibitory effect on various cancer cells.

[0005] To solve the above technical problems, the technical solution of the present invention is as follows: a polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID No.1.

[0006] Preferably, the first amino acid Phe of the polypeptide is connected to a cell-penetrating peptide, and the polypeptide sequence after connection is as shown in SEQ ID No.2.

[0007] Preferably, the last amino acid Trp in the polypeptide sequence is protected by an amine group.

[0008] The present invention also provides the use of the polypeptide in preparing drugs for preventing and / or treating cancer.

[0009] Preferably, the cancer is at least one of breast cancer, colorectal cancer, leukemia, esophageal cancer, pancreatic cancer, glioma, kidney cancer, skin cancer, osteosarcoma, ovarian cancer and prostate cancer.

[0010] That is, the polypeptide can inhibit the proliferation of multiple cancer cells; the cancer cells are at least one of breast cancer, colorectal cancer, leukemia, esophageal cancer, pancreatic cancer, glioma, kidney cancer, skin cancer, osteosarcoma, ovarian cancer and prostate cancer cells.

[0011] Preferably, the polypeptide is used in combination with a pharmaceutically acceptable carrier. When the polypeptide is used as a drug, it can be used directly or in the form of a pharmaceutical composition. When the polypeptide is used, in order to ensure the efficacy, it is generally administered to a patient in need of treatment in the form of an injection.

[0012] Preferably, the polypeptide or a pharmaceutically acceptable salt thereof is used as a pharmaceutically active ingredient.

[0013] Preferably, the mass fraction of the pharmaceutically active component is 0.1% to 99%; further preferably, the mass fraction of the pharmaceutically active component is 0.1% to 20%.

[0014] Preferably, the polypeptide is used in combination with other drugs.

[0015] The characteristics of the present invention are as follows: The present invention designs and synthesizes multiple polypeptides targeting the Staphylococcus nuclease domain 1 (SND1) and heteromucin (MTDH) tumor protein complex SND1-MTDH. It is found through experiments that the polypeptide shown in SEQ ID No.1 has a good anti-cancer effect. Considering that small molecule polypeptides are difficult to enter cells to exert their effects, a membrane-penetrating peptide is introduced before the first amino acid of the SEQ ID No.1 polypeptide, and the connected polypeptide is shown in SEQ ID No.2. In order to increase the structural stability of the polypeptide, the carboxyl group of the last amino acid of the SEQ ID No.2 polypeptide reacts with an amino compound. Finally, the polypeptide obtained by the present invention has excellent inhibitory activity against various types of human cancer cells, providing an alternative for broad-spectrum polypeptide anticancer drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention designs a novel sequence-specific polypeptide, and for the first time finds that the polypeptide provided by the present invention has excellent inhibitory activity against various types of human cancer cells, providing an alternative for a broad-spectrum polypeptide anticancer drug. The polypeptide synthesis process used in the present invention is simple, the constituent amino acids are all natural amino acids, the production cost is low, and the safety is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the amino acid sequence and structural formula of the polypeptide;

[0018] Figure 2 The peptide inhibits the interaction of tumor protein complex SND1-MTDH, degrades SND1 and MTDH, and inhibits the proliferation of cancer cells; A is the immunoprecipitation detection of the peptide inhibiting the interaction between SND1 and MTDH in human breast cancer cells MDA-MB-231, and the protein immunoblotting detection of the peptide on the degradation of SND1 and MTDH in human breast cancer cells MDA-MB-231; B is the immunoprecipitation detection of the peptide inhibiting the interaction between SND1 and MTDH in human breast cancer cells HCC1937, and the protein immunoblotting detection of the peptide on the degradation of SND1 and MTDH in HCC1937; C is the human breast cancer cells MDA-MB-231 and HCC1937 cell lines were treated with different concentrations of peptides for 48 h, and then the total cell protein was extracted, and the protein content of SND1 and MTDH in the cells was detected by protein immunoblotting;

[0019] Figure 3 The results are the inhibitory effects of the peptide on various human cancer cell lines;

[0020] Figure 4 A is a comparison of the inhibitory activities of the two peptides on breast cancer cells; A is a comparison of the inhibitory activities of the two peptides at different concentrations on MDA-MB-231 cells; B is a comparison of the inhibitory activities of the two peptides at different concentrations on HCC1937 cells. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0022] The human breast cancer cells described in the following examples are human breast cancer cells MDA-MB-231, HCC1937, MCF7, HCC1806, BT549, SUM149PT, BT474, MDA-MB-468, SKBR3 and HCC1500, the human colorectal cancer cells are human colorectal cancer cells HCT-8 and RKO, the human leukemia cells are human acute T lymphocytic leukemia cells JurKat, the human esophageal cancer cells are human esophageal squamous cell carcinoma cells KYSE-180, the human pancreatic cancer cells are human pancreatic cancer cells SW1990, the human glioma cells are human glioblastoma cells U87, the human renal cancer cells are human renal clear cell carcinoma cells 786-O, the human skin cancer cells are human malignant melanoma cells A375, the human osteosarcoma cells are human osteosarcoma cells MG63, the human ovarian cancer cells are human ovarian cancer cells Hey, and the human prostate cancer cells are human prostate cancer cells PC3. The conventional cancer cells used above were all from Kunming Medical University.

[0023] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels. RPMI1640 culture medium, DMEM culture medium and fetal bovine serum were purchased from Biological Industries; phosphate buffered saline (PBS), 0.25% trypsin (containing EDTA) and CCK8 reagent were purchased from Bio-Tech Biotechnology Co., Ltd.

[0024] The cell lines were cultured in DMEM or RPMI1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2 and 90% humidity.

[0025] Example 1 Peptide Synthesis

[0026] The core amino acid sequence of the polypeptide provided by the present invention is shown in SEQ ID No. 1. The first amino acid Phe of the polypeptide is connected to a cell-penetrating peptide, and the polypeptide sequence after connection is shown in SEQ ID No. 2. The last amino acid Trp of the polypeptide sequence is protected by an amine group. The amino acid sequence and structural formula of the polypeptide are shown in Figure 1 shown.

[0027] The synthesis process of the polypeptide is as follows: the polypeptide is synthesized from the carboxyl end (C-terminus) of the polypeptide to the amino end (N-terminus) by using the FMOC solid phase method. First, the carboxyl group of the tyrosine (Tyr) at the C-terminus is connected to the polymer resin by covalent bond, and then the amino group of the tyrosine is deprotected and condensed with the carboxyl group of the next glycine (Gly) in the presence of a condensing agent to form an amide bond. After condensation → washing → deprotection → neutralization and washing → next round of condensation, arginine (Arg), lysine (Lys), lysine (Lys), arginine (Arg), arginine (Arg), glutamine (Gln), arginine (Arg), arginine (Arg), arginine (Arg), phenylalanine (Phe), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), proline (Pro), serine (Ser), glutamic acid (Glu), glutamic acid (Glu), tryptophan (Trp), phenylalanine (Phe) and tryptophan (Trp); tryptophan (Trp) is connected to the N-terminus of the polypeptide one by one.

[0028] Finally, piperidine was used to remove the FMOC protecting group at the N-terminus, and a primary amine (-NH2) group was introduced, and trifluoroacetic acid was used to cleave the polypeptide chain from the solid phase support.

[0029] The purity of the peptide was tested by high performance liquid chromatography (HPLC). Prepare buffer solutions of 0.1% trifluoroacetic acid in 100% acetonitrile (liquid A) and 0.1% trifluoroacetic acid in 100% water (liquid B). Install the C18 column on the HPLC system, adjust the column, mix 5% solution A + 95% solution B, wash the column at a flow rate of 40 mL / min for 15 min, then balance the column with 50% mixed solution (containing 0.1% trifluoroacetic acid) for 15 min, and then load the synthesized peptide into the column. The peptide was eluted with a linear gradient of 5% to 40% solution A and 95% to 60% solution B for 25 min, with a flow rate of 1.0 mL / min and a UV detection wavelength of 220 nm; the purity of the obtained peptide was 97.43% after testing.

[0030] Example 2 Test of polypeptide inhibiting the interaction between SND1 and MTDH and promoting the degradation of SDN1 and MTDH

[0031] 20 μM peptide was added to the lysate of human breast cancer cell lines MDA-MB-231 and HCC1937, and MTDH protein in the lysate was precipitated by MTDH antibody through co-immunoprecipitation technique. After elution, the content of SND1 protein co-precipitated with MTDH was detected by protein immunoblotting. Figure 2 A and Figure 2 As shown in B, compared with the DMSO control group, the amount of SND1 protein bound to MTDH in the peptide treatment group was significantly reduced, indicating that the peptide can inhibit the interaction between SND1 and MTDH.

[0032] Different concentrations of peptides were used to treat human breast cancer cell lines MDA-MB-231 and HCC1937 for 48 h, and then total cell protein was extracted. The protein content of SND1 and MTDH in the cells was detected by western blotting. Figure 2 As shown in C, with the increase of polypeptide concentration, the content of intracellular SND1 and MTDH proteins decreased in a dose-dependent manner, indicating that the polypeptide can promote the degradation of intracellular SND1 and MTDH proteins.

[0033] Example 3 Inhibition of Breast Cancer Cells by Peptides

[0034] The CCK-8 (Cell Counting Kit-8) method was used to detect the inhibitory effect of peptides on cancer cell growth. The CCK-8 kit contains a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt, which can be reduced by dehydrogenases in living cells to form an orange-yellow formazan dye, which produces an absorption peak at a wavelength of 450 nm. The absorbance value is linearly positively correlated with the number of cells, and can be used for quantitative detection of cell numbers.

[0035] The polypeptide prepared in Example 1 was dissolved in 50% PBS (phosphate buffer) + 50% DMSO (dimethyl sulfoxide) solution, and then the polypeptide was prepared into concentrates with concentrations of 2500 μM, 1500 μM, 1000 μM, 800 μM, 400 μM, 200 μM and 100 μM, respectively. Breast cancer cells MDA-MB-231, HCC1937, MCF7, HCC1806, BT549, SUM149PT, BT474, MDA-MB-468, SKBR3 and HCC1500 were seeded in 96-well plates, with 1×10 4 cells, cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours, and then different concentrations of polypeptides were added to the cell culture in each well. The final concentrations of polypeptide-treated cells were 25μM, 15μM, 10μM, 8μM, 4μM, 2μM and 1μM, respectively, and the final concentration of DMSO was 0.5%; the control group only added an equal volume of DMSO to the cell culture and incubated in an incubator for 72 hours. Then 10μL CCK8 solution was added to each well, and the cells were cultured in the incubator for another hour. The absorbance at 450nm was measured by an enzyme reader. The percentage of the absorbance value of the experimental group relative to the absorbance value of the control group represents the cell survival rate or the cell proliferation level, that is, the control group defaults to 100%, and the half inhibitory concentration (IC50) of the polypeptide on cell proliferation is calculated. 50 ), the results are shown in Figure 3 and Table 1.

[0036] Table 1 IC50 of peptides against human breast cancer cell lines 50 (μM)

[0037]

[0038] according to Figure 3 As shown in Table 1, the peptides have strong cytotoxic activity against human breast cancer cell lines MDA-MB-231, HCC1937, MCF7, HCC1806, BT549, SUM149PT, BT474, MDA-MB-468, SKBR3 and HCC1500, IC 50 are 2.839 μM, 0.889 μM, 19.06 μM, 15.87 μM, 9.378 μM, 15.04 μM, 21.87 μM, 9.904 μM, 11.48 μM and 6.534 μM respectively.

[0039] Example 4 Inhibition of colorectal cancer cells by polypeptide

[0040] The human breast cancer cells in Example 3 were replaced with human colorectal cancer cells HCT-8 and RKO, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 2.

[0041] Table 2 IC50 of peptides against human colorectal cancer cell lines 50 (μM)

[0042]

[0043] according to Figure 3 As shown in Table 2, the peptide has strong cancer cell toxicity against human colorectal cancer cell lines HCT-8 and RKO, IC 50 8.468 μM and 12.14 μM respectively.

[0044] Example 5 Inhibition of Leukemia Cells by Peptides

[0045] The human breast cancer cells in Example 3 were replaced with human leukemia cells Jurkat. Other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 3.

[0046] Table 3 IC50 of peptides against human leukemia Jurkat cell line 50 (μM)

[0047]

[0048] according to Figure 3 As shown in Table 3, the peptide has strong cytotoxic activity against human leukemia Jurkat cell line, IC 50 It is 15.03 μM.

[0049] Example 6 Inhibition of esophageal cancer cells by polypeptide

[0050] The human breast cancer cells in Example 3 were replaced with human esophageal cancer cells KYSE-180, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 4.

[0051] Table 4 IC50 of peptides against human esophageal cancer cell line KYSE-180 50 (μM)

[0052]

[0053] according to Figure 3As shown in Table 4, the peptide has strong cancer cell cytotoxicity against human esophageal cancer cell line KYSE-180, IC 50 is 8.185 μM.

[0054] Example 7 Inhibition of pancreatic cancer cells by polypeptide

[0055] The human breast cancer cells in Example 3 were replaced with human pancreatic cancer cells SW1990, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 5.

[0056] Table 5 IC50 of peptides against human pancreatic cancer cell line SW1990 50 (μM)

[0057]

[0058] according to Figure 3 As shown in Table 5, the peptide has strong cancer cell cytotoxicity against human pancreatic cancer cell line SW1990, IC 50 is 16.90 μM.

[0059] Example 8 Inhibition of Glioma by Peptides

[0060] The human breast cancer cells in Example 3 were replaced with human brain glioma cells U87, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 6.

[0061] Table 6 IC50 of peptides against human glioma cell line U87 50 (μM)

[0062]

[0063] according to Figure 3 As shown in Table 6, the peptide has strong cancer cell cytotoxicity against human glioblastoma U87 cell line, IC 50 It is 12.33 μM.

[0064] Example 9 Inhibition of renal cancer cells by polypeptide

[0065] The human breast cancer cells in Example 3 were replaced with human renal cancer cells 786-O, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 7.

[0066] Table 7 IC50 of peptides against human renal cancer cell line 786-O50 (μM)

[0067]

[0068] according to Figure 3 As shown in Table 7, the peptide has strong cancer cell toxicity against human renal cancer cell line 786-O, IC 50 It is 9.837 μM.

[0069] Example 10 Inhibition of skin cancer cells by polypeptides

[0070] The human breast cancer cells in Example 3 were replaced with human skin cancer cells A-375, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 8.

[0071] Table 8 IC50 of peptides against human skin cancer cell line A-375 50 (μM)

[0072]

[0073] according to Figure 3 As shown in Table 8, the peptide has strong cancer cell toxicity against human skin cancer cell line A-375, IC 50 is 4.62 μM.

[0074] Example 11 Inhibition of Osteosarcoma Cells by Peptides

[0075] The human breast cancer cells in Example 3 were replaced with human osteosarcoma cells MG63, and the other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 9.

[0076] Table 9 IC50 of the peptides against the growth inhibition of human osteosarcoma cell line MG63 50 (μM)

[0077]

[0078] according to Figure 3 As shown in Table 9, the peptide has strong cancer cell cytotoxicity against human osteosarcoma cell line MG63, IC 50 It is 4.981 μM.

[0079] Example 12 Inhibition of ovarian cancer cells by polypeptide

[0080] The human breast cancer cells in Example 3 were replaced with human ovarian cancer cells Hey, and the other operations were the same as in Example 3. Inhibitory Effect and IC50 The results of the values ​​are shown in Figure 3 and Table 10.

[0081] Table 10 IC50 of peptides against human ovarian cancer cell line Hey 50 (μM)

[0082]

[0083] according to Figure 3 As shown in Table 10, the peptide has strong cytotoxic activity against human ovarian cancer cell Hey cell line, IC 50 It is 18.91 μM.

[0084] Example 13 Inhibition of prostate cancer cells by polypeptide

[0085] The human breast cancer cells in Example 3 were replaced with human prostate cancer cells PC3, and other operations were the same as in Example 3. Inhibitory Effect and IC 50 The results of the values ​​are shown in Figure 3 and Table 11.

[0086] Table 11 IC50 of peptides against human prostate cancer cell line PC3 50 (μM)

[0087]

[0088] according to Figure 3 As shown in Table 11, the peptide has strong cytotoxic activity against human prostate cancer cell line PC3, IC 50 is 6.775 μM.

[0089] Example 14 Comparison of the inhibitory effects of the polypeptide provided by the present invention and the MTDH polypeptide on breast cancer cells

[0090] In order to evaluate the difference in the inhibitory activity of the polypeptide provided by the present invention (referred to as polypeptide I) and the polypeptide of MTDH binding to SND1 on breast cancer cell proliferation, the polypeptide of MTDH binding to SND1 was fused with a transmembrane peptide to obtain an MTDH polypeptide, the amino acid sequence of which is shown in SEQ ID No. 3, the last amino acid is protected by an amine group, and the structure is as follows: YGRKKRRQRRRDWNAPAEEWGNW-NH2, and the purity detected by HPLC is 95.07%. The test method is the same as that in Example 3. The concentrations of MTDH polypeptide for treating breast cancer cells MDA-MB-231 and HCC1937 are 25 μM, 15 μM, 10 μM, 8 μM, 4 μM, 2 μM and 1 μM, respectively, and the difference in the inhibitory activity of polypeptide I and MTDH polypeptide on breast cancer cells at the same concentration is tested.

[0091] The results are as follows Figure 4 As shown, the inhibition rate of peptide I with a concentration greater than 4 μM on MDA-MB-231 cells was greater than 50%, while the inhibition rate of MTDH peptide on MDA-MB-231 cells was less than 50%. At the same concentration, the inhibitory activity of MTDH peptide on MDA-MB-231 cells was significantly lower than that of peptide ( Figure 4 A). The inhibitory activity of 1-25 μM peptide I on HCC1937 cells was greater than 50%, while the inhibitory activity of MTDH peptide on HCC1937 cells was less than 50%. At the same concentration, the inhibitory activity of MTDH peptide on HCC1937 cells was significantly lower than that of peptide I ( Figure 4 B).

[0092] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, but rather, the present invention extends to all other methods and applications with the same functions.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. A polypeptide, characterized in that The first amino acid Phe of the polypeptide is connected to a cell-penetrating peptide, and the amino acid sequence of the polypeptide after connection is shown in SEQ ID No.

2.

3. A polypeptide, characterized in that The last amino acid Trp of the polypeptide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 is protected by an amine group.

4. Use of the polypeptide according to claim 3 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The cancer is at least one of breast cancer, colorectal cancer, leukemia, esophageal cancer, pancreatic cancer, glioma, kidney cancer, skin cancer, osteosarcoma, ovarian cancer and prostate cancer.

5. Use of the polypeptide according to claim 4 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The cancer is breast cancer.

6. Use of the polypeptide according to claim 4 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The polypeptide is used in combination with a pharmaceutically acceptable carrier.

7. Use of the polypeptide according to claim 4 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The polypeptide or a pharmaceutically acceptable salt thereof is used as a pharmaceutically active ingredient.

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