A polypeptide inhibitor for treating triple-negative breast cancer and its preparation method and use

Abstract

The present invention discloses a polypeptide inhibitor, whose sequence is: D‑W‑Cyclo(isoD‑AP‑Dap)‑E‑E‑W‑G‑N‑W‑V‑D‑E or Cyclo(isoD‑RR‑Dap)‑D‑W‑N‑A‑P‑A‑E‑E‑W‑G‑N‑W‑V‑D‑E. The present invention also provides a method for preparing the above-mentioned polypeptide inhibitor and its use in the preparation of drugs for interfering with the formation of MTDH / SND1 complex and treating triple-negative breast cancer. The polypeptide inhibitor of the present invention introduces a terminal aspartic acid nucleation-induced stable polypeptide ring closure method, so that the polypeptide can maintain a suitable binding conformation when binding to the target. Experiments have shown that the present invention has good cell permeability and serum stability, and exhibits good anti-tumor activity and extremely low nonspecific cytotoxicity in triple-negative breast cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of chemistry and pharmacy, and relates to a polypeptide, specifically a polypeptide inhibitor for treating triple-negative breast cancer, and a preparation method and use thereof. Background Art

[0002] Breast cancer is one of the most common cancers among women worldwide, seriously endangering women's health. Triple-negative breast cancer refers to breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2. This type of breast cancer is highly invasive, has a higher risk of distant metastasis, and has a worse prognosis than other types. Currently, there are no specific drugs for triple-negative breast cancer. Treatment methods mainly rely on conventional breast cancer treatments, which lack targeting and specificity, and their therapeutic effects are often limited. Therefore, it is urgent to develop specific drugs for the treatment of triple-negative breast cancer.

[0003] MTDH protein (Metadherin), also known as AEG-1 or LYRIC, is a single transmembrane protein with a molecular weight of 64kDa, which is composed of 582 amino acids. Its transmembrane region is located at 49-72. In addition, MTDH protein also contains 3 nuclear localization signal peptide sequences, which are located at 79-91; 432-451 and 561-580 amino acids. The expression level of MTDH in most normal tissues is very low. Studies have shown that MTDH is not an essential protein for maintaining normal cell growth, and its role in normal tissues is currently unclear. However, MTDH protein is overexpressed in many malignant tumors, such as breast cancer, liver cancer, melanoma, prostate cancer, malignant glioma, etc., and plays a very important role in promoting tumor proliferation, invasion and drug resistance. SND1 protein (Staphylococcal Nuclease Domain containing 1), also known as p100 protein or Tudor-SN (Tudor-Staphylococcal Nuclease) protein, is a conserved multifunctional protein. The SND1 protein consists of four consecutive SN (Staphylococcal Nuclease, SN) domains at the N-terminus and an extended Tudor domain at the C-terminus. SND1 was first discovered as a transcriptional co-activator that can promote the binding of transcription factors to the basal transcription complex, such as c-Myc, STAT6, RNA polymeraseⅡ (RNA polymeraseⅡ) STAT6 and CBP (CREB-binding protein, CEBP binding protein). Subsequent studies have also shown that the SN domain of the SND1 protein can bind to the 3'UTR region of mRNA, thereby enhancing the translation and stability of mRNA. In recent years, a large number of studies have been conducted on the function of SND1 in tumorigenesis. For example, in liver cancer, SND1, as a member of RISC, binds to MTDH to promote the degradation of tumor suppressor mRNAs.

[0004] MTDH can interact with SND1 to form the MTDH / SND1 complex. Professor Kang Yibin's team found that the complex plays a very important role in the occurrence and development of breast cancer and the prognosis and treatment of drug resistance, and screened a series of small molecule compounds for the complex. After further exploration, they screened compounds with satisfactory therapeutic effects and found that small molecule drugs can effectively interfere with the formation of the MTDH / SND1 complex, inhibit the proliferation and metastasis of triple-negative breast cancer, and increase the sensitivity of tumor cells to chemotherapy drugs, bringing hope to the development of triple-negative breast cancer drugs.

[0005] Compared with small molecules, the structure of peptides has a larger size and high enough flexibility, which can better bind to large and flat protein-protein interaction interfaces, reduce off-target effects and thus reduce toxicity. Secondly, the diversity of peptides can be achieved by changing the number and type of amino acids, and in vitro synthesis and modification are also easier to complete. The solid phase peptide synthesis method (SPPS) and peptide purification and separation technology proposed by Merrifield in 1963 made the separation and purification of peptides simpler and more economical, and the quality was easier to control. In addition, the rich post-translational modification (PTM) of amino acids further enriched the possibility of functional assumption. Artificial non-natural amino acids can also enrich the recognition ability of peptides. At the same time, through a large number of mutations at the interaction sites that bind to proteins, it is easier to establish a combinatorial chemical library to screen out peptides with high selectivity and affinity. The methods currently used include phage display, ribosome and mRNA display, yeast display and other evolutionary technologies. In addition, compared with biological macromolecules such as antibodies, its production cost is lower, its immunogenicity is weaker, and its smaller size makes it easier for peptides to enter cells to perform biological functions.

[0006] In view of the above characteristics, polypeptides have unique advantages in regulating protein-protein interactions and have always been a hot topic in drug research and development. In the application of polypeptides regulating protein-protein interactions, stabilizing polypeptides of specific sequences by appropriate stabilization methods has become one of the very important means. Based on the structural characteristics of the MTDH / SND1 complex, the present invention uses an N-terminal aspartic acid nucleation template (TD) to perform cyclization modification on the original sequence to maintain the binding conformation of the original sequence in a complex system, making it more conducive to exerting biological effects, and bringing new possibilities for the treatment of triple-negative breast cancer. Summary of the invention

[0007] In view of the above-mentioned technical problems in the prior art, the present invention provides a polypeptide inhibitor for treating triple-negative breast cancer, a preparation method and use thereof. The polypeptide inhibitor for treating triple-negative breast cancer, a preparation method and use thereof are intended to solve the technical problem that the prior art drugs are not effective in treating triple-negative breast cancer.

[0008] The present invention provides a polypeptide inhibitor (MS2D-cyc4 and MS2D-cyc6), whose amino acid sequence is: DW-Cyclo(isoD-AP-Dap)-EEWGNWVDE(MS2D-cyc4) or Cyclo(isoD-RR-Dap)-DWNAPAEEWGNWVDE(MS2D-cyc6).

[0009] Its structural formula is as follows:

[0010] or

[0011] R is Ac or H or FITC, that is, the N-terminal amino group of the polypeptide inhibitor is modified with FITC fluorescent dye or acetylation.

[0012] The present invention also provides a method for preparing the above polypeptide inhibitor, comprising the following steps:

[0013] 1. For MS2D-cyc4, the synthesis steps are as follows:

[0014] 1.1 Preparation of oligopeptide solid phase resin: The target oligopeptide is loaded on the MBHA resin by a standard solid phase synthesis method;

[0015] 1.2 Preparation of Compound I: This step is carried out on a resin, and the target oligopeptide is subjected to the action of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to remove the Alloc (allyloxycarbonyl) and Allyl (allyl) protecting groups to obtain Compound I;

[0016] 1.3 Preparation of compound II: Compound I is subjected to a ring-closing reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HoBt) and N-methylmorpholine to generate compound II;

[0017] 1.4 Preparation of compound III: The Fmoc protecting group of compound II was removed by 50% by volume morpholine and then directly connected to two amino acids D and W by a standard solid phase synthesis method;

[0018] 1.5 Preparation of MS2D-cyc4 modified peptide: The Fmoc protecting group of compound III is removed by 50% by volume concentration of morpholine, and then the compound can be directly acetylated or modified with FITC fluorescent dye.

[0019] The process of the above reaction is as follows:

[0020]

[0021] x:Fmoc-Dap(Alloc)-OH

[0022] z:Fmoc-isoAsp(OH)-OAllyl

[0023] 2. For MS2D-cyc6, the synthesis steps are as follows:

[0024] 1.1 Preparation of oligopeptide solid phase resin: The target oligopeptide is loaded on the MBHA resin by a standard solid phase synthesis method;

[0025] 1.2 Preparation of Compound I: This step is carried out on a resin, and the target oligopeptide is subjected to the removal of Alloc (allyloxycarbonyl) and Allyl (allyl) protecting groups in the presence of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to obtain Compound I;

[0026] 1.3 Preparation of compound II: Compound I is subjected to a ring-closing reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HoBt) and N-methylmorpholine to generate compound II;

[0027] 1.4 Preparation of MS2D-cyc6 modified polypeptide: The Fmoc protecting group of compound II is removed by 50% volume percent morpholine and then directly acetylated or modified with FITC fluorescent dye.

[0028] The process of the above reaction is as follows:

[0029]

[0030] x:Fmoc-Dap(Alloc)-OH

[0031] z:Fmoc-isoAsp(OH)-OAllyl

[0032] The present invention also provides the use of the above polypeptide inhibitor in preparing a drug for interfering with the formation of the MTDH / SND1 complex.

[0033] The present invention also provides the use of the above polypeptide inhibitor in the preparation of a drug for treating triple-negative breast cancer.

[0034] The present invention also provides a pharmaceutical composition for treating triple-negative breast cancer, comprising the above-mentioned polypeptide inhibitor and paclitaxel.

[0035] Specifically, the molar concentration ratio of the above-mentioned polypeptide inhibitor to paclitaxel is (5000-20000):1.

[0036] Starting from the MTDH / SND1 crystal structure, the present invention selects the shortest sequence (DWNAPAEEWGN) in the MTDH protein that binds to SND1 as the starting point, combines molecular docking technology with experimental methods such as mutation and cyclization, and designs and optimizes a series of stable polypeptide inhibitors based on terminal aspartic acid cyclization.

[0037] The present invention uses a computational simulation method to build a model based on the main binding of the polypeptide derived from the MTDH sequence and the SND1 protein, and attempts to explain the polypeptide-protein binding interface. In the original MTDH sequence, two tryptophans Trp394 and Trp403 can extend into the two hydrophobic pockets of the SND1 protein, playing a crucial role in binding to the SND1 protein. Therefore, in this system, we mainly focus on the binding interface between the two key amino acids Trp394 and Trp403 and the SND1 protein. On the basis of this effect, the binding of other amino acids to the SND1 protein is enhanced by sequence extension and other methods.

[0038] The present invention proves that the stable polypeptide can effectively bind to the SND1 protein through a series of experiments and molecular docking methods, and finds that the two tryptophans (Trp394 and Trp403) of the stable polypeptide also play a leading role in the binding interface. The two tryptophans are mutated to Ala, and the binding of the polypeptide to the SND1 protein is almost lost. In addition, the present invention also proves that adding negatively charged amino acids near the C-terminus can enhance the binding of the polypeptide to the SND1 protein, because the negatively charged amino acids can interact with the alkaline surface of SND1 through hydrogen bonds, electrostatic effects, etc. The stable polypeptide inhibitor of the present invention can effectively interfere with the interaction of MTDH / SND1 proteins, and can effectively penetrate cells and resist the degradation of proteases in serum, and has the potential to be applied to in vivo research. The stable polypeptide inhibitor of the present invention has extremely low cytotoxicity, can selectively inhibit the growth and migration of triple-negative breast cancer, and brings new possibilities for breast cancer treatment.

[0039] Compared with the existing technology, the present invention has significant technical progress. At present, small molecules and polypeptide drugs targeting the MTDH / SND1 complex are still very limited, and stable polypeptide inhibitors have never been reported. The present invention provides a polypeptide inhibitor targeting the MTDH / SND1 complex developed based on the terminal aspartic acid nucleation-induced stable polypeptide (TD) method, and discloses a polypeptide (MS2D-cyc4 and MS2D-cyc6) whose conformation is stabilized by specific chemical modification (TD strategy); through a series of biological experiments, it is proved that the above-mentioned polypeptide can effectively interfere with the formation of the MTDH / SND1 protein complex, thereby inhibiting the growth and migration of triple-negative breast cancer cells. This type of polypeptide has no cytotoxicity to normal cells, and can improve the therapeutic effect of paclitaxel when combined with a low dose of the anticancer small molecule paclitaxel, while also reducing the potential toxicity of high-dose paclitaxel, expanding the therapeutic window of paclitaxel; the TD strategy retains the free amino group at the N-terminus of the polypeptide, which can be further chemically modified and then applied to further structural optimization; this type of polypeptide may be used as a potential drug for the treatment of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 .MTDH / SND1 crystal structure analysis diagram (PDB: 4QMG).

[0041] Figure 2 .The action mode of MS1-cyc1 and MS1-cyc8 with the target SND1 protein.

[0042] Figure 3 .The action mode of MS2D-cyc4 and MS2D-cyc6 with the target SND1 protein.

[0043] Figure 4 .The cell membrane penetration ability of MS2D-cyc4 and MS2D-cyc6, their co-localization with SND1 in cells, and their interference with the MTDH / SND1 complex.

[0044] Figure 5 . Serum stability of stable peptide inhibitors.

[0045] Figure 6 .Effects of stable peptide inhibitors on cell proliferation and lactate dehydrogenase release.

[0046] Figure 7 .Analysis of hemolysis of stable peptide inhibitors.

[0047] Figure 8 .The therapeutic effect of stable peptide inhibitor combined with paclitaxel on triple-negative breast cancer. DETAILED DESCRIPTION

[0048] Example 1 Design and optimization of stable polypeptide inhibitors

[0049] The present invention starts from the MTDH / SND1 crystal structure (PDB: 4QMG) and selects the shortest sequence (DWNAPAEEWGN) in the MTDH protein that binds to SND1 as the starting point ( Figure 1 ), a series of optimizations such as the position of cyclization and mutation were made to this original sequence. In order to test the binding strength of the stable polypeptide inhibitor to the SND1 protein, the present invention synthesized a series of polypeptides labeled with FITC according to the polypeptide solid phase synthesis method and expressed and purified the SND1 protein (16-339). Subsequently, the fluorescence polarization experiment (fluorescence polarization, FP) was used to test the binding ability of the polypeptide to the SND1 protein (16-339). The experimental results showed that fixing the cyclization position at the N-terminus of this sequence and not inserting this sequence (Cyclo (isoD AA Dap) DWNAPAEEWGN, named MS1-cyc8) can better maintain the basic skeleton of the original sequence, and the two key tryptophans can extend into the hydrophobic pocket of the SND1 protein, thereby retaining the binding strength of the original sequence to the SND1 protein (~200nM), while directly closing the cyclization at its end (Cyclo (isoD WN Dap) PAEEWGN, named MS1-cyc1) greatly reduced the binding ability ( Figure 2 A). Further molecular docking technology revealed that directly closing the -WN- ring at the end would cause the overall structure of the peptide to be relatively distorted and unable to effectively interact with the SND1 surface. The most direct manifestation is that the two key Trp residues are exposed on the surface of the SND1 protein and cannot be anchored in the hydrophobic pocket of the SND1 protein, greatly reducing its binding ability. Therefore, it was decided not to change the original 11 amino acid peptide sequence (DWNAPAEEWGN), but to directly add the amide ring of the terminal aspartic acid at the N-terminus of the sequence to make its structure more stretched and able to bind to the target ( Figure 2 B).

[0050] On the basis of the above sequence (Cyclo(isoD AA Dap)DWNAPAEEWGN), in order to further improve the binding ability of the peptide, the amino acid sequence at the C-terminus was extended to enhance the interaction with the basic surface of SND1. In addition, peptides with closed rings at different positions were screened to compare the differences in binding between cyclization and targets at different positions. The results showed that the extended cyclic peptides DW-Cyclo(isoD-AP-Dap)-EEWGNWVDE(MS2D-cyc4) and Cyclo(isoD-RR-Dap)-DWNAPAEEWGNWVDE(MS2D-cyc6) bound to SND1 at ~74nM(FP) and ~82nM, respectively, and the binding ability was improved compared with the previous peptides. While enhancing the binding ability of the original linear peptide, the two stable peptides can also effectively interfere with the formation of the MTDH / SND1 protein complex ( Figure 4 C). Molecular docking showed that tryptophan Trp394 and Trp403 in the two polypeptides can both extend into the two hydrophobic pockets of the SND1 protein. At the same time, another tryptophan residue near the C-terminus may also participate in the hydrophobic interaction. In addition, the acidic amino acids at the C-terminus can also interact with the basic pocket of the SND1 protein, thereby enhancing hydrogen bonding and electrostatic interactions ( Figure 3 ).

[0051] Example 2

[0052] The present invention also provides a method for preparing the above polypeptide inhibitor, comprising the following steps:

[0053] 1. For MS2D-cyc4, the synthesis steps are as follows:

[0054] 1.1 Preparation of oligopeptide solid phase resin: The target oligopeptide is loaded on the MBHA resin by a standard solid phase synthesis method;

[0055] 1.2 Preparation of Compound I: This step is carried out on a resin, and the target oligopeptide is subjected to the action of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to remove the Alloc (allyloxycarbonyl) and Allyl (allyl) protecting groups to obtain Compound I;

[0056] 1.3 Preparation of compound II: Compound I is subjected to a ring-closing reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HoBt) and N-methylmorpholine to generate compound II;

[0057] 1.4 Preparation of compound III: The Fmoc protecting group of compound II was removed by 50% by volume morpholine and then directly connected to two amino acids D and W by a standard solid phase synthesis method;

[0058] 1.5 Preparation of MS2D-cyc4 modified peptide: The Fmoc protecting group of compound III is removed by 50% by volume concentration of morpholine, and then the compound can be directly acetylated or modified with FITC fluorescent dye.

[0059] The process of the above reaction is as follows:

[0060]

[0061] x:Fmoc-Dap(Alloc)-OH

[0062] z:Fmoc-isoAsp(OH)-OAllyl

[0063]

[0064] 2. For MS2D-cyc6, the synthesis steps are as follows:

[0065] 1.1 Preparation of oligopeptide solid phase resin: The target oligopeptide is loaded on the MBHA resin by a standard solid phase synthesis method;

[0066] 1.2 Preparation of Compound I: This step is carried out on a resin, and the target oligopeptide is subjected to the removal of Alloc (allyloxycarbonyl) and Allyl (allyl) protecting groups in the presence of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to obtain Compound I;

[0067] 1.3 Preparation of compound II: Compound I is subjected to a ring-closing reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HoBt) and N-methylmorpholine to generate compound II;

[0068] 1.4 Preparation of MS2D-cyc6 modified polypeptide: The Fmoc protecting group of compound II is removed by 50% volume percent morpholine and then directly acetylated or modified with FITC fluorescent dye.

[0069] The process of the above reaction is as follows:

[0070]

[0071] x:Fmoc-Dap(Alloc)-OH

[0072] z:Fmoc-isoAsp(OH)-OAllyl

[0073]

[0074] Example 3 Cell penetrating ability and serum stability of stable polypeptide inhibitors

[0075] The present invention uses flow cytometry to evaluate the cell penetration of cyclic peptides MS2D-cyc4 and MS2D-cyc6, and their cell penetration ability in triple-negative breast cancer cells is stronger than that of other cyclic peptides and corresponding linear peptides ( Figure 4 A). Furthermore, laser confocal scanning microscopy was used to observe the binding of the peptides to the target sites in the cells. The results showed that the two peptides could effectively bind to the SND1 protein in the cytoplasm ( Figure 4 B). In order to evaluate the ability of peptides to resist protease, the above cyclic peptides MS2D-cyc4 and MS2D-cyc6 were incubated with mouse serum for 72 hours. The results showed that compared with linear peptides, cyclic peptides were more stable and more resistant to protease degradation ( Figure 5 A), the duration of action was extended to 7 days. At 7 days, more than 50% of MS2D-cyc4 and MS2D-cyc6 remained stable ( Figure 5 B), indicating that cyclic peptides have the potential for application and in vivo research, and also provide conditions for the subsequent evaluation of the effects at the animal level.

[0076] Example 4 Application of stable polypeptide inhibitors in triple-negative breast cancer cells

[0077] The present invention uses three breast cancer cells (triple negative breast cancer cells MDA-MB-231, MDA-MB-468 and ERα-positive T47D cells) and a normal cell line (MCF10A) to verify the cellular effects of MS2D-cyc4 and MS2D-cyc6 ( Figure 6 A). The results of CCK8 showed that the peptides were more effective against triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 than ERα-positive T47D cells, which may be related to the expression of the MTDH / SND1 complex. The IC50 values ​​of the two peptides were close to 50μM, and the growth inhibition rate of MDA-MB-231 cells could reach about 50% at 100μM. Despite this, the peptides had almost no effect on the growth and proliferation of normal cells at 200μM ( Figure 6 A), which further confirmed the extremely low cytotoxicity of the polypeptide. The present invention then measured the release of cellular lactate dehydrogenase ( Figure 6 B) and hemolysis of mouse red blood cells ( Figure 6 C) It was further shown that the stable peptide inhibitor had extremely low cytotoxicity and hemolytic activity, making it a candidate peptide drug for inhibiting triple-negative breast cancer.

[0078] Triple-negative breast cancer is highly invasive. The present invention uses a scratch assay to evaluate the effect of a stable peptide inhibitor on the migration of triple-negative breast cancer cells ( Figure 7 ), and observed for 24 and 48 hours respectively. The experimental results showed that both MS2D-cyc4 and MS2D-cyc6 could effectively inhibit the migration of MDA-MB-231 cells, further proving that the polypeptide inhibitor proposed in the present invention has the potential to inhibit the metastasis and invasion of triple-negative breast cancer.

[0079] Example 5 Application of a stable polypeptide inhibitor combined with paclitaxel in triple-negative breast cancer cells

[0080] Paclitaxel is a widely used clinical anticancer drug, but its therapeutic window is relatively narrow due to its potential toxicity at high concentrations. In view of the extremely low cytotoxicity and hemolytic properties of the stable peptide inhibitor of the present invention, we considered combining the stable peptide inhibitor with the paclitaxel small molecule to reduce the cytotoxicity while reducing the dose of paclitaxel, while maintaining a good therapeutic effect. CCK8 experiments showed that the combination of peptide and paclitaxel had a more significant tumor cell growth inhibitory effect than a single dose of peptide or paclitaxel at the same concentration, but had little effect on the growth of normal cells ( Figure 8 ), which also confirmed that the combination of peptides and low-dose paclitaxel (the molar concentration ratio of peptides to paclitaxel is (10000:1) can effectively reduce the potential toxicity caused by high concentrations of paclitaxel while maintaining good therapeutic effects, broadening the application of stable peptide inhibitors and providing a new solution for the treatment of triple-negative breast cancer.

Claims

1. A polypeptide inhibitor, characterized in that Its structural formula is, or ； R is Ac or H or FITC.

2. A polypeptide inhibitor according to claim 1, characterized in that: The N-terminal amino group was modified with FITC fluorescent dye or acetylation.

3. The method for preparing a polypeptide inhibitor according to claim 1, characterized in that The steps include: 1) Preparation of oligopeptide solid phase resin: Load the target oligopeptide on MBHA resin by standard solid phase synthesis method; 2) Preparation of Compound I: This step is carried out on a resin, and the target oligopeptide is subjected to tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to remove the Alloc and Allyl protecting groups to obtain Compound I; 3) Preparation of compound II: Compound I is subjected to a ring-closing reaction in a DMF solution of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine to generate compound II; 4) Preparation of compound III: The Fmoc protecting group of compound II was removed by using 50% by volume morpholine and then directly connected to two amino acids D and W by a standard solid phase synthesis method; 5) Preparation of modified peptides: The Fmoc protecting group of compound III is removed by using 50% volume percent morpholine, and then the compound can be directly acetylated or modified with FITC fluorescent dye.

4. The method for preparing a polypeptide inhibitor according to claim 1, characterized in that The steps include: 1) Preparation of oligopeptide solid phase resin: Load the target oligopeptide on MBHA resin by standard solid phase synthesis method; 2) Preparation of Compound I: This step is carried out on a resin, and the target oligopeptide is subjected to tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to remove the Alloc and Allyl protecting groups to obtain Compound I; 3) Preparation of compound II: Compound I undergoes a ring-closing reaction in a solution of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine to generate compound II; 4) Preparation of modified peptides: The Fmoc protecting group of compound II is removed by using 50% volume percent morpholine, and then the compound can be directly acetylated or modified with FITC fluorescent dye.

5. Use of the polypeptide inhibitor according to claim 1 in the preparation of a drug for treating triple-negative breast cancer.

6. A pharmaceutical composition for treating triple-negative breast cancer, characterized in that: Contains the polypeptide inhibitor according to claim 1 and paclitaxel.

7. A pharmaceutical composition for treating triple-negative breast cancer according to claim 6, characterized in that: The molar concentration ratio of the polypeptide inhibitor to paclitaxel is (5000-20000):1.

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