A tumor-targeting peptide and application thereof

Abstract

The present application relates to a kind of polypeptides, and specifically relates to a kind of tumor targeting peptide and its application;Tumor targeting peptide p46-2 is the carboxyl end of the 361-382 fragment of human cell tumor antigen p53 by connecting peptide and tumor targeting fragment arginine-glycine-aspartic acid-arginine connection;Chemotherapy drugs include paclitaxel, epirubicin, 5 fluorouracil;Selected tumor types include breast cancer, colorectal cancer.Tumor targeting polypeptide and classic chemotherapy drug combination therapy tumor, can obviously achieve the effect of drug synergism.

Description

TECHNICAL FIELD

[0001] The present application relates to a polypeptide, in particular to a tumor targeting peptide and its application. BACKGROUND

[0002] TP53, also known as p53 gene, as a key tumor suppressor gene, has been the main focus of oncology research. The function of p53 protein encoded by TP53 gene is very rich, which regulates cell cycle, apoptosis and genome stability by activating the expression of multiple target genes. More and more evidences show that p53 helps tumor suppression in regulating cell metabolism, ferroptosis, tumor microenvironment, autophagy and so on.

[0003] At present, TP53 gene dysfunction exists in most human malignant tumors. There are mainly two reasons for this dysfunction: p53 gene mutation and MDM2 / MDM4-mediated down-regulation of wild-type p53. There are generally two strategies for these two cases: one is to reactivate mutant p53 back to wild-type structure, and the other is to inhibit the interaction between wild-type p53 and MDM2 / MDM4.

[0004] Studies have shown that using p53 C-terminal amino acid residues (361-382) as a drug can partially restore the functional activity of mutant p53, and the p53 C-terminal amino acid residues (361-382) are named p46. The R / KXXR / K motif is called CendR motif, which interacts with the b1 domain of neuropilin-1 (NRP-1). NRP-1 is a cell surface receptor that plays an important role in angiogenesis and vascular permeability regulation. NRP-1 is highly expressed in tumor cells, so CendR motif has a certain selectivity for tumor cells and can achieve penetration of extravascular tumor tissue. Angiogenesis is the process of generating new blood vessels on the basis of existing blood vessels, which is the basis of tumor growth and metastasis. Integrin is a dimer composed of two subunits, alpha and beta, which is a kind of transmembrane glycoprotein on the cell membrane that can mediate the adhesion between cells and the adhesion between cells and matrix. Integrin αvβ3 is highly expressed in the endothelial cell membrane of new blood vessels in various tumor tissues, but not expressed or lowly expressed in most normal tissues or mature blood vessel endothelial cells. RGDR peptide is a series of polypeptide fragments containing arginine-glycine-aspartic acid-arginine (Arg-Gly-Asp-Arg), which is the recognition site of integrin interacting with its ligand protein, mediating cell adhesion with extracellular matrix and intercellular adhesion. Based on the targeting and penetration of RGDR to tumors, connecting RGDR with drug molecules can help target drug delivery and promote drug accumulation in tumor tissues, which is one of the effective measures to enhance anti-tumor effect.

[0005] Based on the above theory, we further increase the targeting ability of p46 by adding the targeting sequence RGDR, and name the new polypeptide as p46-2.

[0006] Currently, the chemotherapeutic drugs used in clinical cancer treatment, such as paclitaxel, epirubicin, 5-fluorouracil, etc., many studies have shown that GSDME protein is activated by Caspase-3 cleavage under the stimulation of chemotherapy, thereby causing pyroptosis. Caspase-3, as a classic apoptosis effector protein, can be activated in multiple pathways of p53, so p46-2 can activate Caspase-3 by reactivating p53 protein, and then cleave GSDME. In addition, p53 protein itself can play a tumor suppressor role by affecting cell cycle, regulating cell metabolism, promoting ferroptosis, and changing tumor microenvironment.

[0007] Therefore, the application of p46-2 combined with chemotherapy can produce a significant inhibitory effect at a lower concentration of chemotherapeutic drugs, and thus the application of p46-2 combined with different chemotherapeutic drugs in various tumors is compared to reveal its effectiveness and superiority. SUMMARY

[0008] OBJECTIVE

[0009] The application aims to compare the inhibitory effects of different chemotherapeutic drug combinations on breast cancer and colorectal cancer by using p46-2 combined with common clinical chemotherapeutic drugs, and to screen the optimal drug combination scheme.

[0010] TECHNICAL SCHEME

[0011] A tumor-targeting peptide p46-2, characterized by having an amino acid sequence as shown in SEQ ID NO: 3. A pharmaceutical composition, taking p46-2 and a chemotherapeutic drug as active ingredients.

[0012] The chemotherapeutic drugs are paclitaxel, epirubicin, and 5-fluorouracil. The p46-2 includes a p46-2 compound or a pharmaceutically acceptable salt thereof.

[0013] The pharmaceutical composition is used for preparing a tumor treatment drug, and the tumor is breast cancer and colorectal cancer. The combination of p46-2 and a chemotherapeutic drug is used for preparing a breast cancer treatment drug.

[0014] The chemotherapeutic drugs are paclitaxel, epirubicin, and 5-fluorouracil. The combination of p46-2 and a chemotherapeutic drug is used for preparing a colorectal cancer treatment drug. The chemotherapeutic drugs are paclitaxel, epirubicin, and 5-fluorouracil.

[0015] ADVANTAGEOUS EFFECTS

[0016] The present application unexpectedly has synergistic effect when p46-2 is combined with several chemotherapy drugs (paclitaxel, epirubicin, 5-fluorouracil) at a specific dose ratio, for treating breast cancer, colorectal cancer, but has antagonistic effect out of the ratio range. Specifically, when the chemotherapy drugs are used at a concentration / dose that has a slight effect on tumors, the combination of p46-2 and the chemotherapy drugs can have synergistic effect. The pharmaceutical composition with p46-2 and the chemotherapy drugs as active ingredients shows good application prospect.

[0017] Specifically:

[0018] Different anti-tumor drugs combined with p46-2 have different effects on different tumor cells at different doses, that is, different doses have synergistic effect on some tumor cells and have antagonistic effect on others. When the tumor drugs are lower than the clinical dose, the synergistic effect of some drugs is obvious, which has important practical significance for reducing the side effects of anti-tumor drugs.

[0019] Specifically as follows:

[0020] For breast cancer: 0.0172 μM epirubicin combined with 31.25-500 μM p46-2 has synergistic effect;

[0021] For colorectal cancer: 11.7 μM paclitaxel combined with 500 μM p46-2 has synergistic effect;

[0022] 0.0172 μM epirubicin combined with 500 μM p46-2 has synergistic effect;

[0023] 0.0769 μM epirubicin combined with 31.25-500 μM p46-2 has synergistic effect; DETAILED DESCRIPTION

[0024] The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the experimental materials are commercially available from conventional biochemical reagent stores unless otherwise specified.

[0025] Combination index (CI) analysis: the median-effect principle is used, and the calculation is performed by Compusyn software. The average CI value range and interaction evaluation are shown in Table 1. Table 1. Average CI value range and interaction evaluation of compound combination

[0026]

[0027]

[0028] Example 1 Construction of tumor targeting peptide gene and prokaryotic expression vector:

[0029] Synthetic gene sequence and its complementary sequence (SEQ ID NO: 1):

[0030] ATGAGCGATAAAATTATTCATCTGACCGATGATAGCTTTGATACCGATGTGCTGAAAGCGGATGGCGCG

[0031] ATTCTGGTGGATTTTTGGGCGGAATGGTGCGGCCCGTGCAAAATGATTGCGCCGATTCTGGATGAAATT

[0032] GCGGATGAATATCAGGGCAAACTGACCGTGGCGAAACTGAACATTGATCAGAACCCGGGCACCGCGCCG

[0033] AAATATGGCATTCGCGGCATTCCGACCCTGCTGCTGTTTAAAAACGGCGAAGTGGCGGCGACCAAAGTG

[0034] GGCGCGCTGAGCAAAGGCCAGCTGAAAGAATTTCTGGATGCGAACCTGGCGGGCAGCGGCAGCGGCCAT

[0035] ATGCATCATCATCATCATCATAGCAGCGGCCTGGTGCCGCGCGGCAGCGGCATGAAAGAAACCGCGGCG

[0036] GCGAAATTTGAACGCCAGCATATGGATAGCCCGGATCTGGGCACCGATGATGATGATAAAGGCAGCCGC

[0037] GCGCATAGCAGCCATCTGAAAAGCAAAAAAGGCCAGAGCACCAGCCGCCATAAAAAAGGCGGCGGCGGC

[0038] CGCGGCGATCGC

[0039] Kpn I and Hind III enzyme digestion, cloning into pET32a(+) vector, transforming into E. coli, PCR screening positive transformants, nucleotide sequence determination analysis.

[0040] 2. Induction expression of recombinant bacteria:

[0041] The recombinant bacteria were inoculated and shaken overnight, inoculated into LB medium at 1% inoculation amount, and then 2 mmol / L lactose was added when OD600 reached 0.6 for induction for 6 hours. The culture medium was removed by centrifugation at 4°C, the bacteria were collected, resuspended, and the bacterial bodies were broken by ultrasonic treatment in an ice bath. The cell lysate supernatant was collected by centrifugation at 4°C again for further purification.

[0042] 3. Isolation and purification of recombinant protein:

[0043] The recombinant protein was purified using a Ni-NTA column, the crude protein was loaded onto the nickel column, and elution was performed using 0, 30, 50, 80, 100, 200, 250, and 300 mmol / L imidazole. The eluate was collected for subsequent SDS-PAGE analysis. According to the SDS-PAGE results, the high-purity eluate was retained, further desalted by Sephadex G-25 column chromatography, and the desalted product was further purified by nickel column after being cut by enterokinase. Finally, it was freeze-dried and stored in a -80°C refrigerator. The amino acid sequence of the fusion peptide p46-2 is (SEQ ID NO: 2)

[0044] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIA0PILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETA AAKFERQHMDSPDLGTDDDDKGSRAHSSHLKSKKGQSTSRHKKGGGGRGDR

[0045] The amino acid sequence of the tumor-targeting peptide p46-2 is (SEQ ID NO: 3)

[0046] GSRAHSSHLKSKKGQSTSRHKKGGGGRGDR. The tumor-targeting peptide p46-2 can also be synthesized by Shanghai Generay.

[0047] Example 2 Synergistic effect of p46-2 combined with chemotherapy drugs in the treatment of breast cancer

[0048] The breast cancer representative cell 4T1 cell was used as the research object, and the efficacy of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil was compared with that of single drug. The therapeutic effect of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil in inhibiting breast cancer was studied. As shown in Tables 2-6, the inhibition rate of breast cancer cells and the combination index after p46-2 and paclitaxel, epirubicin, and 5-fluorouracil were used alone or in combination.

[0049] Table 2: Inhibition rate of p46-2 single drug on 4T1 cells

[0050]

[0051] Table 3: Inhibition rate of paclitaxel, epirubicin, 5-fluorouracil single drug on 4T1 cells

[0052]

[0053]

[0054] Table 4: Combination index (CI) analysis of p46-2 combined with paclitaxel on breast cancer cells 4T1

[0055]

[0056] Table 5: Combination index (CI) analysis of p46-2 combined with epirubicin on breast cancer cells 4T1

[0057]

[0058] Table 6: Combination index (CI) analysis of p46-2 combined with 5-fluorouracil on breast cancer cells 4T1

[0059]

[0060] As shown in Tables 2-6, in 4T1 breast cancer cells, the combination of p46-2 and epirubicin has more obvious synergistic effect than paclitaxel and 5-fluorouracil, and epirubicin 0.0172 μM, p46-2 has synergistic effect at 31.25-500 μM, which can significantly improve the inhibition of tumor cells at a lower concentration of epirubicin.

[0061] In summary, the results confirm that p46-2 combined with epirubicin on breast cancer cells 4T1 can have strong synergistic effect or strong synergistic effect, which proves the advantage of p46-2 combined with epirubicin in preparing drug composition for treating breast cancer.

[0062] Example 3 Synergistic effect of p46-2 combined with chemotherapy drugs in treating mouse colorectal cancer

[0063] Taking mouse colorectal cancer CT26 as a representative, the efficacy of p46-2 combined with paclitaxel, epirubicin, 5-fluorouracil and single drug was compared, and the therapeutic effect of p46-2 combined with paclitaxel, epirubicin, 5-fluorouracil in inhibiting mouse colorectal cancer was studied. As shown in Tables 7-11, the inhibition rate of p46-2 and paclitaxel, epirubicin, 5-fluorouracil single or combined drug on mouse colorectal cancer cells.

[0064] Table 7: Inhibition rate of p46-2 single drug on CT26 cells

[0065]

[0066] Table 8: Inhibition rate of paclitaxel, epirubicin, 5-fluorouracil single drug on CT26 cells

[0067]

[0068] Table 9: Combination index (CI) analysis of p46-2 combined with paclitaxel on colorectal cancer cells CT26

[0069]

[0070] Table 10: Combination index (CI) analysis of p46-2 combined with epirubicin on colorectal cancer cells CT26

[0071]

[0072]

[0073] Table 11: Combination index (CI) analysis of p46-2 combined with 5-fluorouracil on colorectal cancer cells CT26

[0074]

[0075] As shown in Tables 7-11, in CT26 colorectal cancer cells, the combination of p46-2 and 5-fluorouracil has more obvious synergistic effect than paclitaxel and epirubicin, and 5-fluorouracil 0.0769 μM, p46-2 has synergistic effect at 31.25-500 μM, which can significantly improve the inhibition of tumor cells at a lower concentration of 5-fluorouracil.

[0076] In summary, the results confirm that p46-2 combined with 5-fluorouracil has strong synergistic effect or strong synergistic effect on colorectal cancer cells CT26, which proves the advantage of p46-2 combined with 5-fluorouracil in preparing drug composition for treating colorectal cancer.

[0077] Example 4 Synergistic effect of p46-2 combined with chemotherapy drugs on colorectal cancer in mice

[0078] Taking mouse colorectal cancer MC38 as a representative, the efficacy of p46-2 and paclitaxel, epirubicin, 5-fluorouracil combined drug and single drug was compared, and the therapeutic effect of p46-2 and paclitaxel, epirubicin, 5-fluorouracil combined drug on inhibiting mouse colorectal cancer was studied. As shown in Tables 12-16, the inhibition rate of p46-2 and paclitaxel, epirubicin, 5-fluorouracil single or combined drug on mouse colorectal cancer cells.

[0079] Table 12: Inhibition rate of p46-2 single drug on MC38 cells

[0080]

[0081]

[0082] Table 13: Inhibition rate of paclitaxel, epirubicin, 5-fluorouracil single drug on MC38 cells

[0083]

[0084] Table 14: Combination index (CI) analysis of p46-2 combined with paclitaxel on colorectal cancer cells MC38

[0085]

[0086] Table 15: Combination index (CI) analysis of p46-2 combined with epirubicin on colorectal cancer cells MC38

[0087]

[0088] Table 16: Combination index (CI) analysis of p46-2 combined with 5-fluorouracil on colorectal cancer cells MC38

[0089]

[0090]

[0091] As shown in Tables 12-16, in MC38 colorectal cancer cells, the combination of p46-2 and 5-fluorouracil has a more obvious synergistic effect than paclitaxel and epirubicin, and 5-fluorouracil 0.0769 μM, p46-2 has synergistic effect at 250-500 μM, which can significantly improve the inhibition of tumor cells at a lower concentration of 5-fluorouracil.

[0092] In summary, the results confirm that p46-2 combined with 5-fluorouracil can have a strong synergistic effect or a relatively strong synergistic effect on colorectal cancer cells CT26, proving the advantage of p46-2 combined with 5-fluorouracil in preparing a drug composition for treating colorectal cancer.

Claims

1. The application of a tumor-targeting peptide in combination with epirubicin in the preparation of a drug for treating breast cancer, characterized in that: The amino acid sequence of the tumor-targeting peptide is shown as SEQ ID NO:

3.

2. The use of a tumor targeting peptide in combination with 5-fluorouracil in the preparation of a medicament for the treatment of colorectal cancer, characterized in that: The amino acid sequence of the tumor-targeting peptide is shown as SEQ ID NO: 3.

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