Tumor targeting peptide and application thereof

Abstract

The invention relates to a polypeptide, in particular to a tumor targeting peptide and application thereof. The tumor targeting peptide p46-2 is formed by connecting carboxyl terminals of 361-382 fragments of a human cell tumor antigen p53 with a tumor targeting fragment arginine-glycine-aspartic acid-arginine through a connecting peptide, and the carboxyl terminals of the 361-382 fragments of the human cell tumor antigen p53 are connected with the tumor targeting fragment arginine-glycine-aspartic acid-arginine through a connecting peptide. The chemotherapeutic drugs comprise paclitaxel, epirubicin and 5 fluorouracil; the selected tumor types comprise breast cancer and colorectal cancer. When the tumor targeting polypeptide and the classical chemotherapeutic drug are combined to treat tumors, the synergistic effect of the drugs can be obviously achieved.

Description

Technical Field

[0001] The invention relates to a polypeptide, in particular to a tumor targeting peptide and application thereof. Background Art

[0002] TP53, also known as the p53 gene, is a key tumor suppressor gene and has always been the main focus of oncology research. The p53 protein encoded by the TP53 gene has a wide range of functions. It regulates the cell cycle, apoptosis and genome stability by activating the expression of multiple target genes. Currently, more and more evidence shows that p53 contributes to tumor suppression in regulating cell metabolism, ferroptosis, tumor microenvironment, autophagy and other aspects.

[0003] Currently, TP53 gene dysfunction exists in most human malignancies. There are two main reasons for this dysfunction: p53 gene mutation and MDM2 / MDM4-mediated downregulation of wild-type p53. There are generally two strategies for these two situations, one is to reactivate mutant p53 back to the wild-type structure, and the other is to inhibit the interaction between wild-type p53 and MDM2 / MDM4.

[0004] Studies have shown that the p53 C-terminal amino acid residues (361-382) can partially restore the functional activity of mutant p53 as a drug. Here, the p53 C-terminal amino acid residues (361-382) are named p46. The R / KXXR / K motif is called the CendR motif, which interacts with the neuropilin-1 (NRP-1) b1 domain. NRP-1 is a cell surface receptor that plays an important role in angiogenesis and vascular permeability regulation. Tumor cells generally highly express NRP-1, so the CendR motif has a certain selectivity for tumor cells and can achieve penetration into extravascular tumor tissues. 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, α and β, and is a type of transmembrane glycoprotein on the cell membrane that can mediate adhesion between cells and between cells and matrix. Integrin αvβ3 is highly expressed in the membrane of new blood vessel endothelial cells in various tumor tissues, but is not expressed or expressed at a low level in most normal tissues or mature vascular 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 for the interaction between integrin and its ligand protein, mediating the adhesion between cells and extracellular matrix and between cells. Based on the targeting and penetration of RGDR to tumors, connecting RGDR to drug molecules helps in the targeted delivery of drugs and promotes the accumulation of drugs in tumor tissues, which is one of the effective measures to enhance the anti-tumor effect.

[0005] Based on the above theory, we further increased the targeting ability of p46 by adding the targeting sequence RGDR, and named the new peptide p46-2.

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

[0007] In summary, the combination of p46-2 and chemotherapy can produce significant inhibitory effects at lower chemical drug concentrations. Therefore, under the above theoretical background, the present invention compares the application of p46-2 in combination with different chemotherapy drugs on various tumors to reveal its effectiveness and superiority. Summary of the invention

[0008] Purpose of the invention:

[0009] The purpose of the present invention is to compare the inhibitory effects of different chemotherapy drug compositions on breast cancer and colorectal cancer by combining p46-2 with common clinical chemotherapy drugs, and to screen out the optimal drug combination scheme.

[0010] Technical solution:

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

[0012] The chemotherapy drugs include paclitaxel, epirubicin, and 5-fluorouracil. In the pharmaceutical composition, the p46-2 includes a p46-2 compound or a pharmaceutically acceptable salt thereof.

[0013] The pharmaceutical composition is used in preparing drugs for treating tumors, wherein the tumors are breast cancer and colorectal cancer. The p46-2 is used in combination with a chemotherapy drug in preparing drugs for treating breast cancer.

[0014] The chemotherapy drugs are paclitaxel, epirubicin, and 5-fluorouracil. The application of p46-2 and chemotherapy drugs in the preparation of drugs for treating colorectal cancer. The chemotherapy drugs are paclitaxel, epirubicin, and 5-fluorouracil.

[0015] Beneficial effects:

[0016] The unexpected p46-2 of the present invention can produce a synergistic effect when used in combination with several chemotherapeutic drugs (paclitaxel, epirubicin, 5-fluorouracil) at a specific dosage ratio to treat breast cancer and colorectal cancer, but it produces an antagonistic effect when it is not within the ratio range. Specifically, at a concentration / dose where the chemotherapeutic drug has a slight effect on the tumor alone, the combined administration of p46-2 and the above chemotherapeutic drugs can produce a synergistic effect. The pharmaceutical composition with p46-2 and chemotherapeutic drugs as active ingredients shows good application prospects.

[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 effects on different tumor cells, while others have antagonistic effects. When the tumor drug is lower than the clinical dosage, the synergistic effect of some drugs is obvious, which has important practical significance for reducing the side effects of anti-tumor drugs.

[0019] The details are as follows:

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

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

[0022] The combination of 0.0172 μM epirubicin and 500 μM p46-2 had a synergistic effect;

[0023] The combination of 0.0769 μM epirubicin and 31.25-500 μM p46-2 had a synergistic effect; DETAILED DESCRIPTION

[0024] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

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

[0026]

[0027]

[0028] Example 1 Tumor targeting peptide gene cloning and prokaryotic expression vector construction:

[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] The gene was digested with KpnⅠ and HindⅢ, cloned into pET32a(+) vector, transformed into Escherichia coli, screened positive transformants by PCR, and analyzed by nucleotide sequence determination.

[0040] 2. Induced expression of recombinant bacteria:

[0041] Inoculate the recombinant bacteria, shake overnight, and inoculate into LB medium at a 1% inoculum. When OD600 reaches 0.6, add 2mmol / L lactose for induction for 6 hours. Centrifuge at 4℃ to remove the medium, collect the bacteria, resuspend, and disrupt the cells in an ice bath with ultrasound. Centrifuge again at 4℃, and collect the supernatant of the cell lysate 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 a nickel column and eluted with 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 and further desalted by Sephadex G-25 column chromatography. The desalted product was further purified by a nickel column after being cut with enterokinase and finally lyophilized. The lyophilized product was stored in a -80°C refrigerator. The amino acid sequence of the fusion peptide p46-2 is (SEQ ID NO: 2)

[0044] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIA0PILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETA 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 commissioned to Shanghai Bioengineering for synthesis.

[0047] Example 2 Study on the synergistic effect of p46-2 combined with chemical drugs in the treatment of breast cancer

[0048] The representative breast cancer 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 drugs, to study the therapeutic effect of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil in inhibiting breast cancer. As shown in Table 2-6, the inhibition rate and joint action index of p46-2 and paclitaxel, epirubicin, and 5-fluorouracil alone or in combination on breast cancer cells are shown.

[0049] Table 2: Inhibitory effect of p46-2 on 4T1 cells

[0050]

[0051] Table 3: Inhibitory effects of paclitaxel, epirubicin, and 5-fluorouracil on 4T1 cells

[0052]

[0053]

[0054] Table 4: Analysis of the combined effect index (CI) of p46-2 combined with paclitaxel on breast cancer cell line 4T1

[0055]

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

[0057]

[0058] Table 6: Analysis of the combined effect index (CI) of p46-2 combined with 5-fluorouracil on breast cancer cell line 4T1

[0059]

[0060] As shown in Table 2-6, in 4T1 breast cancer cells, compared with paclitaxel and 5-fluorouracil, the synergistic effect of the combination of p46-2 and epirubicin is more obvious. At epirubicin 0.0172 μM, p46-2 has a synergistic effect at 31.25-500 μM, which can significantly improve the inhibitory effect on tumor cells at lower epirubicin concentrations.

[0061] In summary, the results confirmed that p46-2 combined with epirubicin can have a strong or relatively strong synergistic effect on breast cancer cells 4T1, proving the advantage of p46-2 combined with epirubicin in preparing a pharmaceutical composition for treating breast cancer.

[0062] Example 3 Study on the synergistic effect of p46-2 combined with chemical drugs in the treatment of colorectal cancer in mice

[0063] Taking mouse colorectal cancer CT26 as an example, the efficacy of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil and the efficacy of p46-2 alone were compared to study the therapeutic effect of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil in inhibiting mouse colorectal cancer. As shown in Table 7-11, the inhibition rate of mouse colorectal cancer cells after p46-2 and paclitaxel, epirubicin, and 5-fluorouracil alone or in combination was shown.

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

[0065]

[0066] Table 8: Inhibitory effects of paclitaxel, epirubicin, and 5-fluorouracil on CT26 cells

[0067]

[0068] Table 9: Analysis of the combined effect index (CI) of p46-2 combined with paclitaxel on colorectal cancer cell CT26

[0069]

[0070] Table 10: Analysis of the combined effect index (CI) of p46-2 combined with epirubicin on colorectal cancer cell CT26

[0071]

[0072]

[0073] Table 11: Analysis of the combined effect index (CI) of p46-2 combined with 5-fluorouracil on colorectal cancer cell CT26

[0074]

[0075] As shown in Tables 7-11, in CT26 colorectal cancer cells, the synergistic effect of the combination of p46-2 and 5-fluorouracil was more obvious than that of paclitaxel and epirubicin. At 0.0769 μM of 5-fluorouracil, p46-2 had a synergistic effect at 31.25-500 μM, which could significantly improve the inhibitory effect on tumor cells at lower 5-fluorouracil concentrations.

[0076] In summary, the results confirm that p46-2 combined with 5-fluorouracil can have a strong or relatively strong synergistic effect on colorectal cancer cells CT26, proving the advantages of p46-2 combined with 5-fluorouracil in preparing a pharmaceutical composition for the treatment of colorectal cancer.

[0077] Example 4: Synergistic effect of p46-2 combined with chemical drugs in the treatment of colorectal cancer in mice

[0078] Taking mouse colorectal cancer MC38 as an example, the efficacy of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil and their use alone was compared to study the therapeutic effect of p46-2 combined with paclitaxel, epirubicin, and 5-fluorouracil in inhibiting mouse colorectal cancer. As shown in Tables 12-16, the inhibition rate of mouse colorectal cancer cells after p46-2 and paclitaxel, epirubicin, and 5-fluorouracil were used alone or in combination.

[0079] Table 12: Inhibitory effect of p46-2 alone on MC38 cells

[0080]

[0081]

[0082] Table 13: Inhibitory effects of paclitaxel, epirubicin, and 5-fluorouracil on MC38 cells

[0083]

[0084] Table 14: Analysis of the combined effect index (CI) of p46-2 combined with paclitaxel on colorectal cancer cell MC38

[0085]

[0086] Table 15: Analysis of the combined effect index (CI) of p46-2 combined with epirubicin on colorectal cancer cell MC38

[0087]

[0088] Table 16: Analysis of the combined effect index (CI) of p46-2 combined with 5-fluorouracil on colorectal cancer cell MC38

[0089]

[0090]

[0091] As shown in Tables 12-16, in MC38 colorectal cancer cells, the synergistic effect of the combination of p46-2 and 5-fluorouracil was more obvious than that of paclitaxel and epirubicin. At 0.0769 μM of 5-fluorouracil, p46-2 had a synergistic effect at 250-500 μM, which could significantly improve the inhibitory effect on 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 or relatively strong synergistic effect on colorectal cancer cells CT26, proving the advantages of p46-2 combined with 5-fluorouracil in preparing a pharmaceutical composition for the treatment of colorectal cancer.

Claims

1. A tumor targeting peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO:

3.

2. A pharmaceutical composition, characterized in that Contains the tumor targeting peptide according to claim 1 and pharmaceutically acceptable excipients.

3. Use of the tumor targeting peptide according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of drugs for treating tumors.

4. The use according to claim 3, characterized in that: The tumor is breast cancer or colorectal cancer.

5. Use of the tumor targeting peptide according to claim 1 or the pharmaceutical composition according to claim 3 in combination with a chemotherapeutic drug in the preparation of a drug for treating tumors.

6. The use according to claim 5, characterized in that: The tumor is breast cancer or colorectal cancer.

7. The use according to claim 5, characterized in that: The chemotherapy drug is paclitaxel, epirubicin or 5-fluorouracil.

8. The use according to claim 7, characterized in that: For breast cancer: 0.0172μM epirubicin combined with 31.25-500μM p46-2 has a synergistic effect; for colorectal cancer: 11.7μM paclitaxel combined with 500μM p46-2 has a synergistic effect; The combination of 0.0172 μM epirubicin and 500 μM p46-2 had a synergistic effect; The combination of 0.0769 μM epirubicin and 31.25-500 μM p46-2 has a synergistic effect.

Images