Polypeptide, preparation method and application thereof, and medicine for treating head and neck cancer

Abstract

The invention relates to a polypeptide, a preparation method and application thereof, and a drug for treating head and neck cancer, the polypeptide is an iRGD-TRP-PK1 fusion peptide, and comprises an iRGD targeting peptide sequence and a TRP-PK1 sequence; the iRGD-TRP-PK1 fusion peptide can specifically target an alpha v beta 3 integrin highly expressed on the surface of a tumor cell, and induces death of the tumor cell through a potassium leakage channel formed by TRP-PK1. The polypeptide provided by the invention can be combined with head and neck cancer cells with high expression of integrin alphaVbeta3, so that the operation complexity of a targeted delivery system is effectively reduced, the stability is improved, and a new treatment strategy and application prospect are provided for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and provides a polypeptide, a preparation method and application thereof, and a medicine for treating head and neck cancer. Background Art

[0002] Head and neck cancer is a complex disease that often requires multiple treatments, including surgery, radiation therapy, and chemotherapy. Chemotherapy is a common treatment modality, but it not only kills tumor cells but also damages healthy cells, leading to side effects. To reduce the side effects of chemotherapy, researchers have been looking for ways to achieve more precise targeted therapy to minimize damage to healthy cells. Summary of the Invention

[0003] The applicant has discovered that iRGD targeting peptide is a biomolecule for drug delivery with specific tumor targeting. TRP-PK1 is the fourth transmembrane segment of the transient receptor potential vanilloid 4 (TRPV4) protein, which can autonomously insert into biological membranes and self-assemble into potassium (K + )-like ion channels, thereby forming potassium leak channels, mediating K + From high concentration to low concentration. Therefore, TRP-PK1 is linked to the iRGD targeting peptide, leveraging the potassium leakage channel properties of TRP-PK1 to achieve targeted killing of tumor cells, thus providing a potential method for the clinical treatment of head and neck cancer. The technical problem to be solved by the present invention is to provide a polypeptide that can specifically target tumor sites. In addition to being able to target tumor cells, the present invention can also inhibit tumor cell growth.

[0004] The present invention is achieved through the following technical solutions:

[0005] The first object of the present invention is to provide a polypeptide with targeting and anti-tumor capabilities, wherein the polypeptide is an iRGD-TRP-PK1 fusion peptide, comprising an iRGD targeting peptide sequence and a TRP-PK1 sequence; the iRGD-TRP-PK1 fusion peptide can specifically target the αvβ3 integrin highly expressed on the surface of tumor cells and induce tumor cell death through the potassium leak channel formed by TRP-PK1.

[0006] As an embodiment of the present invention, the iRGD targeting peptide and TRP-PK1 sequences are:

[0007] TRP-PK1:AYLAVMVFALVLGWMNALYFTRGL

[0008] iRGD targeting peptide: CRGDKGPDC.

[0009] The second object of the present invention is to provide a preparation method, wherein the iRGD-TRP-PK1 fusion peptide is prepared by chemically linking the iRGD peptide and the TRP-PK1 peptide by gene fusion to form a fusion polypeptide with tumor targeting and cell killing activity.

[0010] The third invention objective of the present invention is to provide the use of the above polypeptide in the preparation of drugs for treating head and neck cancer.

[0011] The fourth invention objective of the present invention is to provide a drug for treating head and neck cancer, wherein the drug contains the above-mentioned polypeptide.

[0012] The beneficial effects of the present invention are: based on the characteristic of high expression of αvβ3 in tumor tissue, combined with its classical ligand iRGD, targeted therapy is achieved for mice with various tumor types, including head and neck cancer, providing new treatment strategies and application prospects for tumor treatment.

[0013] TRP-PK1 is an α-helix in the fourth transmembrane region of the calcium channel TRPV4, composed of 24 amino acids, and has the ability to automatically insert into the lipid bilayer. Linking TRP-PK1 to iRGD to form iRGD-TRP-PK1 can bind to head and neck cancer cells that overexpress integrin αVβ3, effectively reducing the operational complexity of the targeted delivery system and increasing its stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Representative images of integrin αvβ3 expression in NP69, CNE1, HN4, CNE2, HONE1, and 5-8F cells.

[0015] Figure 2 To detect the killing effect of iRGD-TRP-PK1 on head and neck cancer cells. Figure 2 A is the detection of HN4 cell activity after iRGD-TRP-PK1 treatment, Figure 2 B is the detection of tumor tissue size after iRGD-TRP-PK1 was injected into the CDX model mice established by HN4 through the tail vein. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0017] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0018] Example 1

[0019] Provided is a polypeptide with targeting and anti-tumor capabilities, namely an iRGD-TRP-PK1 fusion peptide, which comprises an iRGD targeting peptide sequence and a TRP-PK1 sequence; the iRGD-TRP-PK1 fusion peptide can specifically target the αvβ3 integrin highly expressed on the surface of tumor cells and induce tumor cell death through the potassium leak channel formed by TRP-PK1.

[0020] Among them, iRGD targeting peptide and TRP-PK1 are used to prepare fusion peptides, and their sequences are:

[0021] TRP-PK1 sequence:AYLAVMVFALLVLGWMNALYFTRGL

[0022] iRGD targeting peptide sequence: CRGDKGPDC.

[0023] The iRGD-TRP-PK1 fusion peptide is formed by connecting the iRGD peptide and the TRP-PK1 peptide through chemical connection or gene fusion to form a fusion peptide with tumor targeting and cell killing activity.

[0024] This application provides a specific implementation method, including the following contents:

[0025] Starting from the C-terminus, amino acids were added one by one according to the iRGD (CRGDKGPDC) sequence. Each cycle included the following steps: the Fmoc-protected amino acids were mixed with HBTU / HOBt and DIPEA, activated in an ice bath for 5 minutes, the activated amino acid solution was added to the resin, and stirred at room temperature for 1 hour until the coupling was complete. The resin was washed with 20% piperidine / DMF to remove the Fmoc protecting group; and the TRP-PK1 sequence (AYLAVMVFALVLGWMNALYFTRGL) was gradually added along the C-terminus of iRGD.

[0026] Example 2

[0027] The experiment is as follows:

[0028] 1. Verification of the targeting ability of iRGD-TRP-PK1 on head and neck cancer cells

[0029] Specific experimental steps:

[0030] 1. Western Blot

[0031] A. To determine the expression of the αvβ3 receptor of the iRGD targeting peptide in various head and neck cancer cells, we collected whole-cell lysates from NP69, CNE1, HN4, CNE2, HONE1, and 5-8F cells. The whole-cell lysates were mixed with 5× loading buffer at a ratio of 4:1 and boiled in a metal bath at 100°C for 10 minutes.

[0032] B. Sample loading: 100 μg of protein was loaded;

[0033] C. Electrophoresis: Electrophoresis was performed at a constant current of 35 mA for 30 minutes, then switched to a constant current of 60 mA and continued for 2 hours.

[0034] D. Transfer: Soak a 0.45 μm PVDF membrane in methanol and place it on a black plate, mesh pad, two layers of filter paper, gel, PVDF membrane, two layers of filter paper, mesh pad, and white plate. Transfer the membrane using a constant current of 200 mA for 120 minutes.

[0035] E. Blocking: Take out the membrane and place it in blocking solution, and block on a shaker for 60 minutes;

[0036] F. Primary antibody incubation: Wash three times with PBST, 5 minutes each time, and incubate with primary antibodies against αvβ3 (1:1000) and GAPDH (1:5000) at 4°C overnight.

[0037] G. Secondary antibody incubation: Wash three times with PBST, 5 minutes each time, and incubate with secondary antibody (dilution ratio of 1:10,000) on a shaker for 2 hours;

[0038] H. Image acquisition: Wash three times with PBST, 5 minutes each time, and use ECL developer to expose and acquire images.

[0039] Result analysis: Since small peptides cannot be observed using fluorescence, the expression of iRGD target αvβ3 in cells was detected. Figure 1 The results showed that αvβ3 expression was significantly increased in head and neck cancer cells HN4. The high expression of αvβ3 indicates that iRGD can effectively target these cells.

[0040] 2. Verification of the killing ability of iRGD-TRP-PK1 on head and neck cancer cells

[0041] iRGD-TRP-PK1 was treated with head and neck cancer cells for 24 hours or injected into the tail vein of mice with tumor models established with head and neck cancer cells at 2μM or 2mg / kg, once every three days for a total of one month. After the treatment, the tumor tissues of the mice were collected and the size of the tumor tissues was detected.

[0042] Result analysis: According to the attached Figure 2It can be determined that iRGD-TRP-PK1 can inhibit the activity of HN4 cells and the growth of tumor tissue in the established CDX model mice.

[0043] Specific experimental steps:

[0044] 1. Cell activity detection: The experiment used HN4 cells to study the activity of HN4 cells after iRGD-TRP-PK1 peptide treatment. 5 HN4 cells were seeded in a 96-well plate and treated with PBS (control group) or iRGD-TRP-PK1 (2 μM) for 24 h. 10 μl of CCK-8 dye (MedChemExpress, USA) was added to each well, and the absorbance at 450 nm was measured with a microplate reader. The absorbance value is proportional to the cell activity.

[0045] 2. Tumor mouse model and treatment of head and neck cancer cells: BALB / c nude mice (about 7 weeks old) were used to study the anti-tumor efficacy of iRGD-TRP-PK1 peptide in vivo. HN4 (1×10 6 ) cells were mixed with matrix gel (1:1) (MedChemExpress, USA) and injected subcutaneously into mice. The mice were randomly divided into two groups. Subsequently, the following treatments were injected intravenously every three days: PBS (control group) and iRGD-TRP-PK1 (2 mg / kg). Tumor volume was calculated as follows: Tumor volume (mm 3 ) = 1 / 2 × length × width 2 .

[0046] Result analysis: According to the attached Figure 2 It can be determined that iRGD-TRP-PK1 can effectively kill tumor cells, thereby inhibiting the growth of head and neck cancer cells.

[0047] Example 3

[0048] Provided is a use of the polypeptide described in Example 1 in the preparation of a drug for treating head and neck cancer.

[0049] Also provided is a drug for treating head and neck cancer, which is the polypeptide described in Example 1, or a drug prepared by adding an adjuvant. The drug can be in any dosage form applicable in the field of using polypeptides to treat tumors, and the method for preparing the drug can be prepared using existing public technology.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A polypeptide with targeting and anti-tumor capabilities, characterized in that: The polypeptide is an iRGD-TRP-PK1 fusion peptide, which comprises an iRGD targeting peptide sequence and a TRP-PK1 sequence; the iRGD-TRP-PK1 fusion peptide can specifically target the αvβ3 integrin highly expressed on the surface of tumor cells and induce tumor cell death through the potassium leakage channel formed by TRP-PK1.

2. The polypeptide having targeting and anti-tumor capabilities according to claim 1, characterized in that: The iRGD targeting peptide and TRP-PK1 sequences are: TRP-PK1:AYLAVMVFALVLGWMNALYFTRGL iRGD targeting peptide: CRGDKGPDC.

3. A method for preparing a polypeptide with targeting and anti-tumor capabilities, characterized in that: The method comprises the following steps: connecting the iRGD peptide and the TRP-PK1 peptide by chemical connection or gene fusion to form a fusion polypeptide with tumor targeting and cell killing activity.

4. Use of the polypeptide according to claim 1 or 2 in the preparation of a drug for treating head and neck cancer.

5. A drug for treating head and neck cancer, characterized in that: The medicine contains the polypeptide according to claim 1 or 2.

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