HER2 protein targeting covalent binding polypeptide molecule and application thereof

Abstract

The invention relates to the technical field of biological drugs, in particular to an HER2 protein targeting covalent binding polypeptide molecule and application thereof. The HER2 protein targeting covalent binding enzymatic self-assembly polypeptide molecule provided by the invention can be used as an HER2 protein covalent inhibitor to prepare antitumor drugs, provides a new choice for anti-HER2 targeting therapy of tumors, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological drugs, in particular to a HER2 protein targeting covalent binding polypeptide molecule and application thereof. BACKGROUND

[0002] Human epidermal growth factor receptor 2 (HER2) is a typical transmembrane receptor tyrosine kinase encoded by the ERBB2 gene and belongs to the EGF receptor family. HER2 protein is a key protein in regulating normal cell activities such as proliferation, differentiation and apoptosis in vivo, and is also a key factor in the occurrence, development and metastasis of various types of cancer. HER2 exerts its effects through homodimerization or heterodimerization with other members of the family, such as EGFR (HER1 / erbB1), HER3 / erbB3 and HER4 / erbB4. Specifically, when HER2 forms a dimer, it triggers autophosphorylation of tyrosine residues in the cytoplasmic domain and activates downstream signaling pathways, thereby regulating cell mitosis, proliferation, differentiation and apoptosis, and affecting life activities.

[0003] Gastric cancer is one of the most deadly malignant tumors in the world. In clinical practice, about 15%-30% of patients with advanced gastric cancer or gastroesophageal junction have HER2 amplification or overexpression. Overexpression of HER2 protein is not only closely related to the growth, invasion and metastasis of gastric cancer, but also significantly related to lymph node metastasis, tumor invasion depth and tumor stage and other clinicopathological features. In addition, studies have shown that HER2 overexpression can change the sensitivity of gastric cancer cells to chemotherapy, leading to more easily drug-resistant and metastatic and recurrent tumor cells and a poorer prognosis for patients. The TOGA study confirmed that the trastuzumab combined chemotherapy regimen can significantly improve the overall survival of HER2-positive gastric cancer patients, indicating that HER2 protein is an effective target for HER2-positive gastric cancer treatment. Thus, anti-HER2 targeted therapy has become a first-line treatment for HER2-positive gastric cancer patients. At present, FDA has approved a variety of anti-HER2-based targeted drugs, including monoclonal antibodies and antibody-drug conjugates. However, due to the strong heterogeneity of gastric cancer, patients are prone to develop drug resistance and off-target effects after long-term use of anti-HER-2 targeted drugs, and the incidence of adverse reactions such as cardiotoxicity is also increased, which all limit the effectiveness of anti-HER-2 therapy. Therefore, developing a new HER2-targeted therapy for gastric cancer is of great significance.

[0004] Covalent binding is an effective means to ensure the occurrence of residue-specific, irreversible interaction between binding agents and targets, which has a wide range of applications in drug development. The covalent inhibitors of EGFR used in clinical practice, such as afatinib, dacomitinib and neratinib, form covalent bonds with target protein residues, leading to complete inactivation of the protein, effectively overcoming the drug resistance problem of non-covalent inhibitors. However, the main problem of small molecule covalent inhibitors is the uncontrollable off-target. With the progress of covalent labeling technology and gene codon expansion technology, the application research of covalent binding has made breakthrough progress. For example, the team of Wang Lei from UCSF developed a protein covalent drug (PD-1-FSY) based on non-natural amino acid FSY. After the non-covalent binding of PD-1 with a nucleophilic group FSY to the surface of PD-L1 of tumor cells, FSY covalently reacts with amino acid residues near the binding site, effectively avoiding the dissociation of the two proteins, and showing strong anti-tumor activity. The team of Gao Yanfeng from Sun Yat-sen University also developed a lysosome-targeted protein degradation platform based on covalent polypeptides, effectively preventing the degradation and recycling of target proteins. Covalent binding can directly generate irreversible proteins or protein molecular complexes in living cells and even model animals, thereby selectively inhibiting or activating specific signaling pathways due to its zero dissociation rate. Covalent binding agents are usually divided into two parts: 1) electrophilic groups, such as the widely used FSY, pyridyl disulfide, etc., which can react with nucleophilic side chains mainly containing cysteine, lysine and histidine residues; 2) non-covalent binding ligands, which are usually protein antibodies with larger action areas. Small molecule ligands have higher specificity for targets, avoiding off-target. After high-specificity non-covalent interaction with a certain domain of the target protein, the electrophilic group will covalently bind to the amino acid residues adjacent to the receptor, forming a covalent complex. Therefore, it is of great significance to construct and develop suitable ligands for the high-selectivity covalent binding of covalent binding agents and target proteins.

[0005] Supramolecular self-assembled polypeptides have shown great application prospects in drug delivery, cancer treatment, and protein interaction regulation. Previous studies have shown that: 1) supramolecular self-assembly can regulate and maintain the secondary structure of polypeptide fragments, achieving specific binding of polypeptide assemblies to target proteins; 2) by changing the phosphorylation sites of the same polypeptide to regulate its pre-assembly state, the polypeptide assemblies obtained by enzymatic self-assembly have different spatiotemporal selectivity; 3) in situ self-assembly of polypeptides in response to tumor microenvironment can avoid the problems of insufficient permeability of similar protein antibody drugs in tumor tissues and insufficient residence time of small molecule agentia.

[0006] Therefore, how to provide a HER2 protein targeting covalent binding enzymatic self-assembly polypeptide molecule has become a technical problem to be solved in the art. SUMMARY

[0007] To solve the above technical problems, the present application first provides a phosphorylated polypeptide pS3, whose structural formula is as follows: The present application finds through screening of phosphorylation sites that the pre-assembly ability of the above-mentioned phosphorylated polypeptide is optimal, which presents long nanofibers arranged in order, and the assembly precursor and assembly body thereof are mainly in the form of beta-turn, and the binding effect with the extracellular domain of HER2 protein is optimal.

[0008] Further, the present application provides a polypeptide molecule, which is prepared by modifying a chemical group on the side chain of the phosphorylated polypeptide pS3; the purpose of the modified chemical group is to form a warhead for realizing covalent crosslinking with HER2 protein.

[0009] By modifying a chemical group on the side chain of the phosphorylated polypeptide pS3, covalent crosslinking of the polypeptide pS3 with HER2 protein can be realized by using the covalently bound warhead structure, thereby obtaining a HER2 protein targeting covalent binding polypeptide molecule.

[0010] In some embodiments, the chemical group is one of 4-mercaptopyridine, OPSS, 4-mercaptopyrimidine, 2-mercaptopyridine, mercaptoimidazole, and mercaptopurine.

[0011] Preferably, the chemical group is 4-mercaptopyridine.

[0012] The PDS formed by modifying 4-mercaptopyridine as a warhead can undergo a proximal covalent binding reaction with HER2 protein, and the stability of covalent binding is better.

[0013] Further, the present application provides a polypeptide molecule pS3-TP (supramolecular self-assembly covalent polypeptide), whose structural formula is as follows: The pre-assembly body of the above-mentioned polypeptide molecule pS3-TP is in situ self-assembled into a polypeptide nanofiber under the catalysis of alkaline phosphatase overexpressed on the membrane of a cancer cell; the secondary structure of the polypeptide nanofiber is mainly in the form of beta-turn, and the binding effect with the extracellular domain of HER2 protein is optimal.

[0014] After the polypeptide nanofiber specifically interacts with the HER2 protein, pyridine disulfide will undergo a thio exchange reaction with the adjacent cysteine residues on the HER2 protein to form a stable polypeptide-protein covalent complex. This not only improves the enrichment and residence level of the polypeptide nanofiber on the cell membrane surface of tumor cells and HER2 protein high expression cells, but also inhibits the proliferation, migration and invasion of tumor cells by interfering with the downstream PI3K / AKT signal pathway mediated by HER-2 protein. In addition, the polypeptide molecule pS3-TP of the present application also exhibits good tumor growth inhibition ability in vivo.

[0015] Further, the present application provides the use of the phosphorylated polypeptide pS3, or the polypeptide molecule, or the polypeptide molecule pS3-TP in the preparation of a drug.

[0016] Preferably, the drug is a HER2 protein covalent inhibitor.

[0017] Preferably, the drug is used for treating tumors; more preferably, the tumors are cancers; more preferably, the cancers are gastric cancers.

[0018] Further, the present application provides a drug containing the polypeptide molecule, or the polypeptide molecule pS3-TP.

[0019] In some embodiments, the drug further comprises other pharmaceutically acceptable excipients.

[0020] In some embodiments, the excipients include fillers, excipients, lubricants, wetting agents, diluents, etc.

[0021] In some embodiments, the drug further comprises other drugs for treating tumors.

[0022] In the specific implementation process, the HER2 protein covalent inhibitor of the present application can be combined with other anti-tumor drugs to obtain better therapeutic effect.

[0023] In some embodiments, the drug is used for treating tumors; preferably, the tumors are cancers; more preferably, the cancers are gastric cancers.

[0024] Compared with the prior art, the present application has the following advantages: The present application provides a HER2 protein targeting covalent binding enzyme self-assembly polypeptide molecule, which can be used as a HER2 protein covalent inhibitor to prepare an anti-tumor drug, and provides a new choice for anti-HER2 targeted therapy of tumors, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Molecular design and biochemical properties of different polypeptides; (A) Chemical structure of three polypeptides pS1, pS2 and pS3 and their chemical structure transformation after responding to ALP dephosphorylation; (B) Critical micelle concentration (CAC) of three polypeptides; (C) TEM images and Tyndall phenomenon of three polypeptides before and after dephosphorylation, white arrow represents nanofiber, red arrow represents nanoparticle; (D) Dephosphorylation kinetics and conversion level of three polypeptides (ALP 10 U / ml); (E) WST detection of HER2 protein affinity of three polypeptides before and after dephosphorylation.

[0026] Figure 2 Selectivity and targeting of enzymatic self-assembly polypeptides on cell membrane; (A) CLSM images of pS1, pS2 and pS3 co-incubated with N87 cells for 4h; (B) Colocalization of polypeptide pS3 on the surface of N87 cell membrane; (C) Biological scanning electron microscope images of N87 cells co-incubated with pS3 polypeptide for 4h (untreated group as control); (D) Time dependence of polypeptide pS3 targeting self-assembly behavior on the surface of N87 cell membrane; (E) Polypeptide pS3 targeting self-assembly behavior on the surface of N87 cell membrane is regulated by both ALP dephosphorylation process and HER2 targeting performance; (F) Flow cytometry detection of the binding amount of polypeptide pS3 on the cell membrane after co-culturing with different cells for 4h; (G) CCK8 detection of cell viability of N87 cells treated with different concentrations of polypeptide pS3.

[0027] Figure 3 Construction of supramolecular self-assembly covalent polypeptide and covalent binding of polypeptide-HER2 protein; (A) Chemical structure of supramolecular self-assembly covalent polypeptide pS3-TP; (B) Transmission electron microscope images of pS3-TP before and after dephosphorylation; (C) Liquid chromatogram of covalent binding of pS3-TP-Fmoc-Cys; (D) FITR spectrum of pS3-TP assembly before and after dephosphorylation and corresponding secondary structure analysis statistics; (E) Comparison of HER2 protein affinity of pS3-TP assembly and pS3 assembly, BSA as control; (F) Detection of pS3-TP-HER2 covalent complex by SDS-PAGE and fluorescence imaging after co-incubation of pS3-TP with HER2 protein in solution for 12h; (G) Detection of pS3-TP and N87 cell membrane HER2 protein covalent complex formation by SDS-PAGE and fluorescence imaging.

[0028] Figure 4Targeted self-assembly selectivity of supramolecular self-assembly covalent polypeptide on cell membrane and its inhibition of tumor cell growth; (A) CLSM images of covalent polypeptide pS3-TP after 4h co-incubation with N87 cells and GSE-1 cells; (B) Cell membrane localization of covalent polypeptide pS3-TP after 4h co-incubation with N87 cells; (C) Time dependence of covalent targeting of polypeptide pS3-TP to the surface of N87 cell membrane; (D) Comparison of biological scanning electron microscopy images of N87 cells after 4h co-incubation with pS3 polypeptide and pS3-TP covalent polypeptide (blue and red arrows represent nanofibers formed by pS3 and pS3-TP on the N87 cell membrane, respectively); (E) IC 50 value of pS3-TP polypeptide on NCI-N87 cells; (F) Comparison of cell viability of N87 cells treated with different concentrations of pS3 polypeptide and pS3-TP.

[0029] Figure 5 pS3-TP affects the malignant biological behavior of tumor cells by interfering with the HER-2 downstream PI3K / AKT signaling pathway; (A) Clonogenic assay to detect the proliferation ability of N87 cells in different treatment groups of PBS, pS3 and pS3-TP; (B) Transwell experiment to detect the migration and invasion ability of N87 cells in different treatment groups of PBS, pS3 and pS3-TP; (C) EDU experiment to detect the DNA synthesis ability of N87 cells in different treatment groups; (D) WB to detect the expression of key proteins related to EMT; (E) Clonogenic assay analysis; (F) and (G) Migration and invasion statistical analysis; (H) EDU experiment statistical analysis; (I) Flow cytometry apoptosis experiment statistical analysis; (J) Flow cytometry detection of the apoptosis rate of different treatment groups; (K) WB detection of key proteins downstream of PI3K / AKT.

[0030] Figure 6 Anti-tumor effect of pS3-TP in N87 cell xenograft tumor mice; (A) Schematic diagram of animal experiment, including inoculation time and drug administration period; (B) Subcutaneous transplanted tumor in different treatment groups; (C) Change of tumor volume in each group during treatment period; (D) Tumor weight of each treatment group after treatment; (E) Body weight change of mice in each treatment group; (F) HE staining of tumor tissue, Ki67 and TUNEL immunohistochemical detection of each treatment group; (G) HE staining of important organs (heart, liver, spleen, lung and kidney) of mice in each treatment group.

[0031] Figure 7 Blood routine and blood biochemical test results of PBS, pS3 and pS3-TP mice in different treatment groups. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application. In the embodiments provided in the present specification, the specific techniques or conditions not noted are implemented according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not noted the manufacturers are the conventional products that can be purchased through the regular channels.

[0033] All the chemical reagents and Fmoc amino acids in the following examples were purchased from Aladdin Reagent and Genview Biochemical (Shanghai). 2-Chlorotrityl chloride resin (1.1 mmol / g) was purchased from Nankai University Resin Co., Ltd. NBD-Cl (purity 99.52%) was purchased from MCE (China). Alkaline phosphatase (30 U / μL) was purchased from Shanghai Yisen Biological. HER2 antibody, BCA kit, Western blot antibody, etc. were purchased from CST, Biolegend, MCE, etc. The instruments included NanoTemper MST instrument, LC-MS, FTIR, TEM, CLSM, flow cytometer, etc.

[0034] Gastric cancer cells: NCI-N87, MKN-1, HGC-27, AGS; breast cancer cells: MDA-MB-468; normal gastric epithelial cells: GES-1. The above cells were respectively cultured in RPMI-1640 or DMEM medium containing 10% FBS and 1% double-antibiotic. BALB / c nude mice were purchased from Beijing SPF Biotechnology Co., Ltd. The experiment was approved by the Animal Ethics Committee of General Hospital of People's Liberation Army.

[0035] Example 1 Preparation of different phosphorylated serine polypeptides and characterization of self-assembly properties thereof The same self-assembly polypeptide can regulate its pre-assembly state by changing the phosphorylation site, which can affect the spatiotemporal selectivity of the polypeptide assembly process and the biological function of the assembly. Based on this, in the present embodiment, by changing the phosphorylation site of serine in the self-assembly polypeptide NBD-FFSCGKGGMSRTMSG, three phosphorylated polypeptides pS1, pS2 and pS3 were designed Figure 1A). In this set of self-assembling polypeptide sequences, 1) the fluorescent molecule NBD (nitrobenzoxadiazole) was used as a capping group to facilitate the observation of the spatial distribution of the assemblies at the cellular level, 2) FF as the core motif of self-assembly, 3) CG12 fragment (CGKGGMSRTMSG) is the HER2 protein targeting sequence. Three self-assembling polypeptides were synthesized by standard solid-phase synthesis method (SPPS), purified by high-performance liquid chromatography and characterized using high-resolution mass spectrometry.

[0036] The method of solid-phase synthesis of phosphorylated polypeptides pS1, pS2 and pS3 is as follows: The reaction used 2-chlorotrityl chloride resin as a solid-phase carrier, and N-Fmoc-protected amino acid coupling was performed. The Fmoc protecting group was removed using 20% piperidine / anhydrous N,N'-dimethylformamide (DMF). Subsequently, the Fmoc-protected amino acid (4 equivalents) was coupled with the free amino group under the condition of HBTU (4 equivalents) as a condensing agent.

[0037] The polypeptide was cleaved from the resin using a cleavage solution (95% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% water) at room temperature for 3 hours. After cleavage, the TFA was removed using a rotary evaporator, and the polypeptide was precipitated with cold ether. The crude peptide was further purified by reverse-phase high-performance liquid chromatography (HPLC), and the final product was characterized using high-resolution mass spectrometry (HR-MS).

[0038] Chemical structure of pS1 Chemical structure of pS2 Chemical structure of pS3 Next, we explored the pre-assembly state of the three polypeptides and the biophysical properties of the assemblies after catalysis by alkaline phosphatase. First, we detected the critical aggregation concentration (CAC) of the polypeptides by the pyrene fluorescence probe method, Figure 1 B As can be seen, compared with the CAC values of pS1 (188.38 μM) and pS2 (56.2 μM), pS3 has the smallest CAC value (26.51 uM). When the three polypeptides were dissolved in PBS solution (250 uM, pH = 7.4), they all presented transparent and clear yellow solutions without the addition of ALP, and the Tyndall effects were observed in the pS2 and pS3 polypeptide solutions, and the light path intensity of pS3 was stronger than that of pS2, but the Tyndall phenomenon was almost not observed in pS1, which was consistent with the CAC value results. The TEM images of the three polypeptides before the addition of ALP showedFigure 1 C), the pre-organization of pS1 was in the form of amorphous nanostructure, the pre-organization of pS2 was mainly in the form of short and randomly arranged nanofibers, and the pre-organization of pS3 was in the form of long and orderly arranged nanofibers. These results indicated that the pre-organization ability of the three polypeptides was in the order of pS3 > pS2 > pS1.

[0039] After adding ALP (10 U / mL) to the above polypeptide solution system and incubating at 37 °C for 4 h, pS1 changed from a clear solution to a brownish yellow liquid with visible precipitate, and obvious Tyndall effect was observed; while pS2 and pS3 polypeptides remained clear yellow liquid state, but the Tyndall effect of both was significantly enhanced compared with that before adding ALP. Figure 1 C). From the TEM images of dephosphorylated pS1, nanoparticles of different diameters (red arrows) and sparse nanofibers (white arrows) with a diameter of 8-15 nm were observed; in contrast, pS2 and pS3 formed dense and interlaced nanofibers Figure 1 C), in which the nanofibers formed by pS3 were more dense.

[0040] Further, we analyzed the dephosphorylation rate and level of the three polypeptides after adding ALP by LC-MS (liquid chromatography-mass spectrometry). First, we prepared pS1, pS2 and pS3 peptide PBS solutions (pH = 7.4) with a concentration of 250 μM at 37 °C for 1 h. Then, we added alkaline phosphatase (ALP, 10 U mL⁻¹) to each peptide solution, and at the set time point, we took out 50 μL of sample, diluted it with 300 μL of methanol, and then performed LC-MS detection and drew the dephosphorylation curve. From Figure 1 D, we found that the dephosphorylation conversion rate of pS3 reached about 90%, which was significantly higher than that of pS1 and pS2 (58.1% and 78.8%). The above results suggest that the pre-organization level of pS3, pS2 and pS1 is positively correlated with the dephosphorylation rate and degree Figure 1D). We speculate that the hydrophilic phosphate group in the chemical structure of pS1 is closer to the assembly factor FF, and this strong hydrophilic group may interfere with its π-π stacking process, thereby affecting the pre-assembly level of pS1. In addition, we believe that the amorphous pre-assembly precursor formed by it may be due to the relatively disordered arrangement of molecules, which may be the reason for the coexistence of nanoparticles, nanofibers, and even precipitates in various forms after dephosphorylation of pS1. The secondary structure of a polypeptide can determine its efficiency of docking and binding with a protein. A suitable secondary structure of a polypeptide can enter the "pocket" of the protein domain, thereby achieving effective binding. Therefore, we used Fourier transform infrared spectroscopy (FTIR) to detect the changes in the secondary structure of the polypeptide before and after assembly. FTIR spectra showed that the secondary structures of polypeptides pS1 and pS2 were similar before and after dephosphorylation, while the secondary structure of pS3 was different from them, and its assembly precursor and assembly were mainly in the form of β-turn.

[0041] The affinity between peptides and proteins was determined by microthermal mobility shift (MST) technology, which is an efficient and sensitive method. Different peptides were weighed and dissolved in PBS buffer (pH = 7.4) to prepare a peptide solution with a final concentration of 1 mM. Take 475 μL of each peptide solution and place it in an EP tube, add ALP (10 U / mL) to each tube, and incubate at 37 °C overnight; then use LC-MS to detect whether dephosphorylation is complete. Take another 475 μL of each peptide solution for standby. Incubate the peptides with recombinant HER2 protein (molecular weight 95 kDa) and negative control BSA protein (molecular weight 66 kDa) at room temperature for 1-2 hours (not less than 1 hour), then perform MST determination. By fitting the relationship curve between fluorescence intensity and ligand concentration, the binding constant KD value is calculated. In all six groups of molecular interaction analysis (MST) tests (pS1, pS2, pS3, pS1 assembly, pS2 assembly, and pS3 assembly), pS3 assembly showed the lowest dissociation constant (Kd value of 43.7 μM), indicating that only when the polypeptide maintains a β-turn conformation can it bind well with the extracellular domain of HER2 protein. The change in the binding constant of pS3 before and after assembly may be due to the conversion of phosphorylated serine in the HER2 targeting sequence region to serine under the catalysis of alkaline phosphatase (ALP), thereby restoring the standard HER2 targeting sequence. Therefore, we speculate that the pS3 polypeptide with the best pre-assembly performance may have the best assembly effect at the cellular level. Figure 1 E), pS3 assembly showed the lowest dissociation constant (Kd value of 43.7 μM), which indicated that only when the polypeptide maintained a β-turn conformation could it bind well with the extracellular domain of HER2 protein. The change in the binding constant of pS3 before and after assembly may be due to the conversion of phosphorylated serine in the HER2 targeting sequence region to serine under the catalysis of alkaline phosphatase (ALP), thereby restoring the standard HER2 targeting sequence. Therefore, we speculate that the pS3 polypeptide with the best pre-assembly performance may have the best assembly effect at the cellular level.

[0042] Example 2 In situ enzymatic polypeptide self-assembly on cancer cell membranes We detected the ALP responsiveness and HER2 protein affinity of three self-assembling polypeptides in solution. Subsequently, we used NCI-N87 cells (human gastric cancer cells) with high extracellular ALP activity and high HER2 protein expression to detect the self-assembly behavior of pS1, pS2 and pS3 at the cellular level. pS1, pS2 and pS3 (250 uM, pH = 7.4) were respectively incubated with NCI-N87 cells in a 37°C cell incubator containing 5% CO2 for 4 hours, and the confocal results (Fig. 6) Figure 2 A) show that after 4 hours of pS3 incubation, the obvious fluorescence signal is mainly concentrated on the NCI-N87 cell membrane. However, for pS1, only a small amount of fluorescence signal is observed on the cell membrane, and for pS2 polypeptide, almost no fluorescence signal can be observed on the NCI-N87 cell membrane or inside the cell. After co-incubation of pS3 polypeptide and NCI-N87 cells, we labeled the cell membrane by Dil dye (a commonly used cell membrane marker dye) for colocalization analysis, Figure 2 B shows that the green fluorescence almost completely coincides with the red fluorescence, indicating that the pS3 polypeptide is mainly combined and located on the cell membrane. Bio-SEM results show that after 4 hours of polypeptide pS3 treatment (250 uM), nanofibers with a diameter of about 15 nm are wrapped around the NCI-N87 cell membrane, which is different from the surface morphology of NCI-N87 cells before polypeptide treatment (Fig. 6) Figure 2 C). In summary, these results show at the cellular level that the phosphorylation site (or phosphorylation pattern) determines the self-assembly results of pS1, pS2 and pS3, and pS3 polypeptide performs best in generating membrane-bound nanofibers. In addition, the present application also found that the self-assembly of pS3 polypeptide on the cell membrane exhibits obvious concentration dependence and time dependence (Fig. 6) Figure 2 D). After NCI-N87 cells were incubated with different concentrations of pS3 polypeptide for 4 hours, when the pS3 concentration was 62.5 uM, which was higher than the measured CMC value and Kd value, obvious fluorescence signal could be observed on the cell membrane, and the fluorescence signal intensity increased with the increase of concentration, and reached equilibrium when the polypeptide concentration was 250 uM. When the fixed polypeptide concentration was 250 uM, the confocal results showed that pS3 polypeptide could quickly form membrane-bound nanofibers (~ 10 min), and reached equilibrium at 8 hours, and the formed nanofibers could long-term reside on the membrane for ~ 24 hours.

[0043] Next, we further verified whether the pS3 polypeptide membrane assembly process is jointly affected by ALP and HER-2 by adding ALP inhibitor (Na3VO4) or anti-HER-2 antibody, Figure 2FResults showed that the ability of pS3 to target and self-assemble on cell membrane was inhibited by adding ALP inhibitor (Na3VO4) or anti-HER-2 antibody, while almost no fluorescence signal of pS3 was observed on cell membrane when both of the above were added at the same time Figure 2 E). We used pS3 to treat different cell lines to verify the HER2 selectivity and targeting of pS3 in situ self-assembly. Results showed that unlike NCI-N87 cells, even after pS3 was incubated with normal gastric epithelial cells GES-1 (low expression of HER2) and triple-negative breast cancer cells MDA-MB-468 cells (no expression of HER2) and gastric cancer cell line MKN-1 (high ALP activity, low expression of HER2) for 24 hours, only weak fluorescence signal was observed on the cell membrane. At the same time, flow cytometry detection was performed after different cells were co-incubated with pS3 for 4 hours, and the results showed that the MFI on NCI-N87 cells was the largest Figure 2 F), which indicated that pS3 self-assembly had good cell selectivity and could self-assemble in situ on the cell membrane of cells with high expression of ALP and HER2 protein and form membrane-bound nanofibers. In addition, the results of CCK-8 experiment showed that pS3 polypeptide could significantly inhibit the growth of NCI-N87 cells only when the concentration was greater than 500 μM Figure 2 G), while for normal cells GSE1, pS3 polypeptide could even promote their growth.

[0044] In summary, based on the results of solution experiments and cell experiments, we screened the best phosphorylation mode of HER2 targeting self-assembly polypeptide pS3, and the pS3 polypeptide with β-turn secondary structure and capable of rapidly enriching on the surface of NCI-N87 provided a strategy based on enzymatic polypeptide self-assembly (EISA) for the preparation of HER2 protein covalent inhibition ligand.

[0045] Example 3 Construction of supermolecular self-assembly covalent polypeptide and covalent binding of HER2 protein-polypeptide Cysteine targeting strategy is the main direction of the development of covalent drugs at present, and the extracellular domain II of HER2 protein is a cysteine-rich region. Based on the design concept of covalent binding agent, we modified 4-mercapto pyridine to the side chain thiol of pS3 polypeptide to prepare pS3-TP polypeptide molecule containing pyridine disulfide group Figure 3 A), and the PDS formed by the self-assembly of the enzyme as the warhead and the HER2 protein had a proximal covalent binding reaction.

[0046] The construction steps of pS3-TP and pS2-TP are as follows: pS3 peptide (18.65 mg, 0.01 mmol) was dissolved in 1 mL of a 1:1 mixture of water and acetonitrile, followed by the addition of 2,2′-dipyridine disulfide (2.2 mg, 0.01 mmol). The reaction mixture was stirred overnight at 25 °C, and the reaction progress was monitored by HPLC. After the reaction was complete, the crude product was purified by HPLC and freeze-dried to obtain a pale yellow solid pS3-TP (18.32 mg, yield 92.8%). The synthesis of pS2-TP was performed using the same procedure as pS3-TP.

[0047] Chemical structural formula of pS3-TP Chemical structural formula of pS2-TP The dephosphorylation experiment was conducted using the same procedure as above. Transmission electron microscopy images showed that pS3-TP preassembled into regularly arranged nanofibers, and the nanofiber density significantly increased after dephosphorylation. Figure 3 B), which is similar to the morphological changes before and after pS3 assembly. To verify that the pS3-TP assembly can bind to cysteine, we first incubated it with ALP and pS3-TP solution (250 μM) to dephosphorylate it, and then co-incubated it with Fmoc-cysteine ​​(Fmoc-Cys) (250 μM) for 12 h. High-performance liquid chromatography (HPLC) showed that peak 1 (tR = 13.0 min) disappeared and peak 2 (tR = 22.7 min) appeared. Mass spectrometry further confirmed peak 2, which is the pS3-TP-Fmoc-Cys covalent complex (…). Figure 3 C). FITR analysis of the secondary structure of the pS3-TP assembly before and after dephosphorylation showed that, similar to the secondary structure of pS3, the secondary structure of both the pS3-TP precursor and assembly was also predominantly β-turn. Figure 3 (D) indicates that the modification of 4-mercaptopyridine has almost no effect on the secondary structure of the polypeptide.

[0048] Next, the affinity of pS3-TP for HER2 protein before and after ALP treatment was detected using the MST assay method described above, with BSA protein serving as a control group. The results showed that the affinity of the pS3-TP assembly for HER2 protein was 7 times that of the pS3-TP pre-assembled assembly. The dissociation constant (Kd value) of the pS3-TP assembly was 22.022 μM, approximately half that of the pS3 peptide assembly (43.742 μM). In terms of thermophoretic range, the pS3-TP assembly was also significantly higher than the pS3 assembly. Figure 3E), which indicated that the PDS group significantly increased the affinity of the polypeptide assembly to the HER2 protein. pS3 and pS3-TP almost did not bind to the BSA protein, which indicated that the covalent polypeptide specifically interacted with HER2. To verify whether the pS3-TP assembly could form a covalent bond with the HER2 protein, we incubated the polypeptide assembly (pS3-TP+ALP) with the HER2 protein, Figure 3 FThe results showed that the fluorescent group of NBD appeared on the SDS-PAGE, and the incubation group without pS3-TP failed to appear fluorescence, which indicated that the self-assembled polypeptide pS3-TP could covalently react with HER2 and form a covalent complex. Further, a thiol-disulfide exchange reaction was used to verify the covalent binding reaction between pS3-TP and Fmoc-cysteine. To verify whether the supramolecular self-assembled covalent polypeptide could covalently bind to the HER2 protein on the cell membrane, we incubated pS3-TP with N87 cells, and then collected the cell proteins and detected the generation of the covalent complex by SDS-PAGE. Figure 3 GThe results showed that compared with the untreated cells, the fluorescent bands obviously appeared in the polypeptide-treated cell samples, which indicated that the polypeptide assembly had a thiol exchange reaction with the HER2 protein and formed a polypeptide-HER2 protein covalent complex. In addition, we speculated that the formation of the pS3-TP and HER2 protein covalent complex might be related to the secondary structure of the covalent polypeptide. Therefore, we also introduced 4 thiol pyridine into the side chain of pS2 to construct pS2-TP to further verify our speculation. In contrast, after treating N87 cells with pS2-TP, extensive fluorescence appeared on the N87 cell membrane and inside the cells (penetrated the membrane), and extensive green fluorescence also appeared inside and outside the cells after co-incubating pS2-TP with GES1 cells for 4 h. After collecting the N87 cells treated with pS2-TP, extracting the proteins, and performing SDS-PAGE electrophoresis, the WB results and fluorescence imaging results showed that pS2-TP could bind to most proteins to emit fluorescence, indicating that its binding to proteins was not selective and specific. The above results indicated that the difference in the secondary structure of pS3-TP and pS2-TP polypeptides might cause the difference in selectivity, and the β-turn structure could selectively bind to the HER2 protein.

[0049] Example 4 Targeting self-assembly selectivity of supramolecular self-assembled covalent polypeptide on the cell membrane and its inhibition of tumor cell growth After incubating pS3-TP (250 μM) with NCI-N87 cells for 4 h, it was observed by laser confocal microscopy that the fluorescence of pS3-TP polypeptide showed a larger area of distribution around the NCI-N87 cell membrane than that of pS3 (see Figure 4 A). The Dil staining results showed that the polypeptide assembly was significantly enriched on the cell membrane, which was consistent with the results of pS3 (see Figure 4B). Similarly, pS3-TP exhibited good time dependence, and it could obviously target to N87 cell membrane at about 10 min, target to almost all cell membrane surface at about 30 min, and reach equilibrium at about 4 hours. Figure 4 C). Bio-SEM images showed that pS3-TP formed a more dense nanonetwork on the cell membrane surface than pS3 Figure 4 D), indicating that covalent reaction significantly enhanced the interaction of polypeptide assembly with cell membrane.

[0050] To verify the inhibitory effect of covalent polypeptides on tumor cells, NCI-N87 cells were co-incubated with polypeptides for 24 hours, and then CCK8 experiment was performed. It was found that at a concentration of 125 uM, it could obviously inhibit the viability of tumor cells, and at a concentration of 250 uM, the cell inhibition rate was about 50% Figure 4 E), however, pS3 could hardly inhibit tumor cell growth at the same concentration Figure 4 F). More importantly, pS3-TP did not significantly affect the viability of normal gastric epithelial cells GES-1. This indicates that under the dual regulation of ALP and HER2 proteins, pS3-TP has good cell selectivity (cell compatibility).

[0051] In summary, pS3-TP exhibits better enzymatic self-assembly performance and membrane targeting binding capacity. The rapid reaction of nucleophilic groups with HER-2 proteins adjacent to the cell membrane surface may disrupt the protein conformation and affect the normal function and viability of tumor cells.

[0052] Example 5 pS3-TP affects the malignant biological behavior of tumor cells by interfering with the HER-2 downstream PI3K / AKT signaling pathway The most basic feature of malignant tumor is its sustained proliferation ability. The proliferation ability of pS3 and pS3-TP on NCI-N87 cells was tested by plate colony formation experiment. NCI-N87 cells in logarithmic growth phase were inoculated in 6-well plates at a density of 800 cells per well to make the cells evenly distributed. Each group had 3 replicate wells. The cells were incubated in a 37 °C, 5% CO2 incubator. After the cells were completely adherent, they were treated with 250 μM of pS3 and pS3-TP polypeptides for 12 hours, with PBS as the control. The drug solution was discarded, and after washing with PBS, fresh complete medium was added for continuous culture. When the number and size of the colonies reached the experimental requirements, they were fixed with 4% paraformaldehyde for 15 minutes, and then stained with 0.1% crystal violet for 30 minutes. After removing the excess dye, they were air-dried at room temperature and photographed, and finally data statistical analysis and plotting were performed. The results showed that compared with the control group and pS3 treatment group, the number of NCI-N87 cell colonies in the pS3-TP treatment group was significantly reducedFigure 5 A, Figure 5 E).

[0053] Transwell assay was used to evaluate the migration and invasion of NCI-N87 cells treated with pS3 and pS3-TP. NCI-N87 cells treated with pS3 and pS3-TP were collected and resuspended in serum-free medium. 5x10 4 cells were added to the upper chamber of Transwell and 200 μL of complete medium containing 20% FBS was added to the lower chamber. Each group had 3 replicates. If invasion assay was performed, the upper chamber of Transwell was coated with Matrigel beforehand. Then the cells were incubated in the incubator for 48-72 hours. The medium was carefully discarded and the cells were fixed with 4% paraformaldehyde for 15-30 minutes and stained with 0.1% crystal violet for 20-30 minutes. The excess dye was removed by gently wiping the surface of the upper chamber with a wet cotton swab and washing with PBS for 2-3 times. After air-drying at room temperature, the cells were counted under an inverted microscope. The results showed that the migration and invasion of NCI-N87 cells treated with pS3-TP were significantly inhibited Figure 5 B, Figure 5 F, Figure 5 G).

[0054] In addition, we detected the changes of DNA synthesis ability of tumor cells in different treatment groups by EDU labeling technology. NCI-N87 cells were inoculated in 96-well plates and cultured overnight. Cells were treated with 250 μM pS3 and pS3-TP for 12 h. Then, an appropriate amount of 50 μM EdU working solution was prepared with complete culture medium; the original culture medium was removed, and the EdU working solution was added and returned to the incubator for continuous incubation for 12 h. After incubation, the culture medium was removed, 50 μL of PBS containing 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 min; the fixing solution was discarded, 50 μL of PBS containing 3% BSA was added to each well, and the cells were washed 3 times for 5 min each time, and the supernatant was discarded. 50 μL of PBS containing 0.3% Triton X-100 was added to each well, and the cells were permeabilized at room temperature for 20 min; after discarding the solution, the cells were washed with 3% BSA / PBS for 3 times for 5 min each time. The Click reaction solution was prepared according to the kit instructions, 100 μL was added to each well, and the liquid was gently shaken to completely cover the cells, and the reaction was carried out at room temperature for 30 min; the reaction solution was discarded, and the cells were washed with 3% BSA / PBS for 3 times for 5 min each time. The DAPI working solution was prepared according to the number of samples, 100 μL was added to each well, and the cells were incubated at room temperature for 5-10 min; after discarding the solution, the cells were washed with 3% BSA / PBS for 3 times for 5 min each time. Finally, the cells were observed and photographed under a fluorescence microscope. From the results of the above experiments, we can know that the DNA synthesis ability of tumor cells in the pS3-TP treatment group decreased significantly, which was consistent with the above clonal formation results. Figure 5 C and Figure 5 From the above experiments, we can know that the DNA synthesis ability of tumor cells in the pS3-TP treatment group decreased significantly, which was consistent with the above clonal formation results.

[0055] The loss of adhesion ability of epithelial tumor cells and the acquisition of mesenchymal cell migration ability can promote the process of tumor metastasis and drug resistance. Therefore, we detected the expression of EMT key proteins E-cadherin, N-cadherin and Vimentin in each treatment group by Western blot. The results showed that compared with the control group, the expression of E-cadherin in N87 cells in the pS3-TP treatment group was up-regulated, and the expression of N-cadherin and Vimentin was down-regulated (Fig. 6). Figure 5D). Flow cytometry apoptosis assay showed that the percentage of apoptosis in N87 cells treated with pS3-TP was about 9.5 times higher than that in untreated cells, and 5.5 times higher than that in pS3-treated cells (Fig. I, Fig. J). Therefore, we speculate that after pS3-TP targets the HER-2 protein on the membrane of tumor cells, it may affect the downstream signaling pathway, leading to the impact on the malignant biological phenotype. Further, WB assay showed that the relative expression of p-PI3K and pAKT in pS3-TP-treated tumor cells was significantly lower than that in PBS- and pS3-treated cells (Fig. K). Figure 5 K). We can infer that after pS3-TP targets the HER-2 protein on the membrane of gastric cancer cells through enzymatic self-assembly to form nanofibers, it affects the conduction of the downstream PI3K / AKT signaling pathway, thereby affecting the proliferation, migration, and invasion of gastric cancer NCI-N87 cells, inhibiting the occurrence of EMT, and thus promoting apoptosis.

[0056] Example 6 Anti-tumor effect of pS3-TP in vivo To further study the anti-tumor effect of pS3-TP in vivo, we constructed a subcutaneous tumor model of NCI-N87 in male Balb / c nude mice (5 weeks old). The steps are as follows: 4-6-week-old male BALB / c nude mice weighing 18-22 g were selected, and the experiment was started after 1 week of adaptive feeding. The logarithmic growth period of gastric cancer NCI-N87 cells was taken, and after trypsin digestion, the cells were centrifuged at 1000 rpm for 5 min, washed with PBS twice, and the cell density was adjusted to 1×10 7 cells / mL with PBS. 100 μL of cell suspension (1×10 6 cells per mouse) was injected subcutaneously on the back of the nude mice, avoiding blood vessels. After inoculation, the mice were numbered and observed for tumor formation. When the subcutaneous tumor volume reached 100 mm 3 , it was recorded as Day 0, and the mice were randomly divided into groups of 5. On Day 0, 2, 4, and 6, the corresponding treatment was given. During the experiment, the tumor volume and body weight were measured and recorded every 2 days. After the last observation, the mice were sacrificed, and tumor and major organ specimens were collected for subsequent detection.

[0057] Tumor volume (TV) and tumor inhibition rate (TGI) calculation formula: TV (mm 3 ) = length (L) × width (W) 2 × 0.5 TGI (%) = (1 − treatment group tumor weight / control group tumor weight) × 100% As shown in Figure 6 A, we constructed a subcutaneous tumor model, and when the tumor volume reached about 100 mm 3Mice were then randomly divided into three groups, receiving PBS, pS3 (10 mg / kg), and pS3-TP (10 mg / kg) treatments, respectively. The effects of each treatment on tumors were observed. In the untreated group (PBS) and the pS3-treated group, tumor volume increased rapidly, and both groups exhibited relatively consistent tumor growth curves, indicating that the pS3 treatment group failed to effectively inhibit tumor growth in vivo. In contrast, the pS3-TP treatment group significantly inhibited tumor growth, with both tumor volume and weight significantly reduced. Figure 6 BD). The tumor growth inhibition indices (TGIs) in the pS3 treatment group and the pS3-TP treatment group were 11.3% and 63.3%, respectively. Furthermore, there was no statistically significant difference in body weight change among the three groups during treatment. Figure 6 E).

[0058] Mice were euthanized after treatment, and tumor tissues and vital organs (heart, liver, spleen, lung, and kidney) from each treatment group were collected for HE staining and immunohistochemical staining. HE staining of tumor tissues revealed that, compared to PBS, the tumor tissues in the pS3 treatment group were richly vascularized and showed focal tumor necrosis. In contrast, the tumor tissues in the pS3-TP treatment group showed extensive tumor necrosis with a small number of remnants of degenerated cells. Ki-67 expression was significantly decreased in the tumor tissues of the pS3-TP treatment group, while its expression was significantly increased by TUNEL staining. These results indicate that the proliferation capacity of tumor cells in the pS3-TP treatment group was significantly reduced, and apoptosis was significantly increased. Figure 6 F). HE staining of vital organs (heart, liver, spleen, lung, and kidney) tissues from mice in each treatment group showed no significant abnormalities. Figure 6 G). Blood samples were collected from the tail vein of mice for complete blood count and blood biochemistry tests. No significant differences were observed among the three groups. Figure 7 This indicates that the pS3-TP treatment group initially demonstrated good biocompatibility in in vivo tumor treatment. In summary, pS3-TP can inhibit tumor proliferation and promote apoptosis in vivo, significantly inhibiting tumor growth and demonstrating good therapeutic effects.

[0059] In summary, the present application develops a novel supramolecular self-assembly covalent polypeptide, which provides a new idea for anti-HER2 targeted therapy of tumor. By using the advantage of in-situ assembly of enzyme-catalyzed self-assembly polypeptide, the present application constructs self-assembly polypeptide responding to ALP and targeting HER2, and confirms the influence of different phosphorylation sites on the assembly ability of polypeptide and HER2 targeting. The pS3 polypeptide with the best responding to ALP and HER2 targeting affinity is screened. By using the feature of covalent combination of protein through mercapto pyridine nucleophilic group to avoid off-target, the supramolecular self-assembly covalent polypeptide pS3-TP is synthesized. The in-vivo and in-vitro experiments confirm that pS3-TP targets HER2 protein on the membrane surface of gastric cancer NCI-N87 cells with high selectivity, affects tumor cell proliferation, migration and invasion by down-regulating PI3K / AKT signaling pathway, inhibits EMT, and promotes tumor cell apoptosis. Therefore, the present application provides a novel treatment option for HER2 targeted therapy of tumor.

[0060] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A phosphorylated polypeptide pS3, characterized in that, Its structural formula is as follows: 。 2. A polypeptide molecule, characterized in that, It is prepared by modifying the side chain of the phosphorylated polypeptide pS3 of claim 1 with chemical groups; the purpose of the modified chemical groups is to form a warhead for covalent cross-linking with HER2 protein.

3. The polypeptide molecule according to claim 2, characterized in that, The chemical group is one of 4-mercaptopyridine, OPSS, 4-mercaptopyrimidine, 2-mercaptopyridine, mercaptoimidazole, and mercaptopurine.

4. The polypeptide molecule pS3-TP, characterized in that, Its structural formula is as follows: 。 5. The use of the phosphorylated polypeptide pS3 of claim 1, or the polypeptide molecule of claim 2 or 3, or the polypeptide molecule pS3-TP of claim 4 in the preparation of a drug.

6. The application according to claim 5, characterized in that, The drug is a covalent inhibitor of the HER2 protein.

7. The application according to claim 5, characterized in that, The drug is used to treat tumors; preferably, the tumor is cancer; more preferably, the cancer is gastric cancer.

8. A drug, characterized in that, It contains the polypeptide molecule as described in claim 2 or 3, or the polypeptide molecule pS3-TP as described in claim 4.

9. The medicament according to claim 8, characterized in that, The drug also includes other pharmaceutically acceptable excipients; And / or, the drug may also include other drugs for treating tumors.

10. The medicament according to claim 8 or 9, characterized in that, The drug is used to treat tumors; preferably, the tumor is cancer; more preferably, the cancer is gastric cancer.

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