Polypeptide self-assembly PROTAC degradation agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a polypeptide self-assembly PROTAC degradation agent and a preparation method and application thereof. The polypeptide self-assembly PROTAC degradation agent comprises a self-assembly group, an E3 ligase recognition group, a cathepsin response group, an FOXM1 targeting binding group, a connecting peptide and an integrin alpha 6 targeting peptide, the amino acid sequence of the FOXM1 targeted binding group is as shown in SEQ ID NO: 1. According to the polypeptide-based nano delivery system, the drug delivery efficiency is remarkably improved, meanwhile, the risk of side effects is reduced to the minimum through controllable release of active fragments, and a basis is provided for preparation of tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide self-assembly PROTAC degradation agent and a preparation method and application thereof. Background Art

[0002] FOXM1 (ForkheadBox M1) is a transcription factor that plays a key role in cell cycle regulation, proliferation, DNA damage repair, and tumorigenesis and development. Moreover, FOXM1 is overexpressed in many cancers, including breast cancer, lung cancer, liver cancer, colon cancer, and prostate cancer. FOXM1 overexpression is closely related to the proliferation, metastasis, drug resistance, and poor prognosis of various tumors. FOXM1 has great potential as an anti-tumor target, and therapeutic strategies targeting FOXM1 are expected to provide new treatment options for cancer patients. The occurrence and development of most diseases are related to abnormal expression or aggregation of proteins. For the pathological mechanisms of such diseases, the traditional drug development idea is to develop small molecule inhibitors or protein inhibitors to occupy and block the active action sites of target proteins and inhibit the functional activity of proteins. Multiple preclinical studies have shown that inhibiting the activity of FOXM1 can inhibit tumor growth and metastasis and prolong survival time. For example, small molecules such as Thiostrepton and FDI-6 have been shown to effectively inhibit the activity of FOXM1 and inhibit the proliferation of tumor cells. Although some progress has been made in the research on FOXM1, the application of FOXM1 inhibitors in clinical pharmacology is still challenging due to the lack of clear drug binding sites and large interaction interfaces. On the one hand, small molecule compound drugs require a large dose and sufficient affinity to ensure sufficient binding rate, but larger doses may produce toxic side effects and off-target effects; on the other hand, small molecule inhibitors are prone to cause compensatory protein increase or gene mutation, leading to drug resistance. FOXM1 also plays an important role in normal cell cycle regulation. How to specifically inhibit FOXM1 in tumor cells without affecting normal cell function is a challenge.

[0003] Protein degradation targeting chimeras (PROTACs) are an emerging class of promising therapeutic approaches that induce ubiquitination and degradation of target proteins by linking them to E3 ubiquitin ligases. Compared with traditional small molecule inhibitors, PROTACs can degrade a variety of mutant proteins and reduce the occurrence of drug resistance. Since PROTACs can be recycled, they can theoretically achieve efficient degradation at lower doses. PROTAC technology can potentially be applied to target the degradation of any intracellular protein, including so-called undruggable targets such as transcription factors, scaffold proteins, or non-enzyme proteins in cells. Notably, several small molecule PROTACs have entered clinical trials for the treatment of various types of cancer. However, PROTAC molecules still face many potential problems and challenges: (1) Small molecule PROTACs have a hook effect, and this dose-independent protein degradation greatly limits the clinical translation of PROTAC molecules. (2) Potential off-target effects cause the distribution of PROTAC molecules in non-targeted normal tissues and organs, resulting in toxicity, which limits the clinical translation of PROTAC molecules.

[0004] Rationally designed drug delivery systems can improve PROTACs' physicochemical properties or serve as transport carriers, increase their tissue specificity and intracellular accumulation, improve degradation efficiency and reduce side effects, thereby improving target specificity and anti-tumor activity. In recent years, peptide PROTACs have the advantages of high specificity and low toxicity and have become a powerful protein targeted degradation technology. Among them, the PROTAC nanoplatform based on peptide self-assembly has been developed to meet the above challenges. The platform has the characteristics of easy screening and editable ligand ratio. The peptide-based self-assembly nanoplatform has potential flexibility for multi-module selection and can be programmed for specific purposes. It can achieve rapid target protein degradation and formulation optimization, which greatly promotes the development and application of PROTAC technology. Enzymes that are often overexpressed under pathological conditions, such as alkaline phosphatase, matrix metalloproteinases, cathepsins, tyrosinases, etc. In view of this, stimulus-activated PROTAC prodrugs have recently been used to limit the precise protein degradation of favorable targets, which may overcome the adverse effects of PROTAC due to uncontrolled protein degradation.

[0005] Therefore, it is of great significance to develop a dose-dependent and selective protein ubiquitination degrader. Summary of the invention

[0006] The object of the present invention is to provide a polypeptide self-assembly PROTAC degradation agent and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a polypeptide self-assembly PROTAC degrader, wherein the polypeptide self-assembly PROTAC degrader comprises a self-assembly group, an E3 ligase recognition group, a cathepsin response group, a FOXM1 targeting binding group, a connecting peptide and an integrin α6 targeting peptide;

[0009] The amino acid sequence of the FOXM1 targeting binding group is shown in SEQ ID NO:1.

[0010] Preferably, the amino acid sequence of the self-assembling group is as shown in SEQ ID NO:2.

[0011] Preferably, the amino acid sequence of the E3 ligase recognition group is shown in SEQ ID NO:3.

[0012] Preferably, the amino acid sequence of the tissue protease response group is shown in SEQ ID NO:4.

[0013] Preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 5;

[0014] The amino acid sequence of the integrin α6 targeting peptide is shown in SEQ ID NO:6.

[0015] Preferably, the structural formula of the polypeptide self-assembling PROTAC degrader is as shown in formula (I):

[0016]

[0017] The present invention also provides a method for preparing the polypeptide self-assembly PROTAC degrader, comprising the following steps:

[0018] (1) synthesizing a self-assembly group, an E3 ligase recognition group, a cathepsin response group, a FOXM1 targeting binding group, a connecting peptide and an integrin α6 targeting peptide by a peptide solid phase synthesis method to obtain a peptide NFTP;

[0019] (2) Mixing the peptide NFTP with hexafluoroisopropanol, reacting for 20 to 28 hours, and drying to obtain a peptide self-assembling PROTAC degrader.

[0020] Preferably, the mass volume ratio of the mixture of the polypeptide NFTP and hexafluoroisopropanol in step (2) is 1 mg: 18-22 μL; the reaction temperature in step (2) is 35-39° C., and the reaction speed is 20-50 rpm.

[0021] The present invention also provides the use of the polypeptide self-assembling PROTAC degrader or the polypeptide self-assembling PROTAC degrader prepared by the preparation method in preparing a preparation for targeted degradation of FOXM1 or preparing a drug with anti-tumor efficacy;

[0022] The tumor is a tumor caused by overexpression of FOXM1, including breast cancer, non-small cell lung cancer, kidney cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer or gastric cancer;

[0023] The drug contains 20 to 100 μM of the polypeptide self-assembly PROTAC degrader.

[0024] The present invention also provides a nanoparticle for targeted degradation of FOXM1, wherein the nanoparticle comprises the polypeptide self-assembly PROTAC degrader or the polypeptide self-assembly PROTAC degrader prepared by the preparation method;

[0025] The particle size of the nanoparticles is 100 to 150 nm;

[0026] The preparation method of the nanoparticles is: dissolving the polypeptide self-assembly PROTAC degradation agent in water, separating, and obtaining the nanoparticles.

[0027] The present invention provides a polypeptide self-assembly PROTAC degrader and a preparation method and application thereof. The polypeptide self-assembly PROTAC degrader NFTP is prepared by adding integrin α6 targeting peptide to enhance its tumor targeting ability. Due to the low expression of cathepsin response group (CatB) in normal tissues, PROTACs remain inactive when attached to nanoassemblies, and can only recover activity after CatB cleavage and release after reaching tumor tissues. NFTP can be brought to tumor cells by integrin α6 targeting peptide and be taken up efficiently and selectively, and can significantly reduce the concentration of FOXM1, thereby inhibiting tumors and reducing systemic toxicity caused by off-target effects.

[0028] The polypeptide self-assembly PROTAC degrader provided by the present invention provides a new idea for solving the structure-activity relationship limitations in the development process of small molecule PROTACs. Small molecule PROTACs generally face the problem that at high concentrations, binary complexes of PROTAC and target protein or E3 ligase will be preferentially formed, rather than ternary complexes, which affects the degradation efficiency of proteins; in the present invention, the structure of the self-assembly nano-peptide degrader breaks the non-concentration dependence of small molecule PROTACs protein degradation; at high concentrations, the topological structure of the polypeptide assembly is further extended, providing a larger surface area for binding to the target protein and the E3 ligase recognition group, so as to achieve the formation of an efficient and stable ternary complex.

[0029] The polypeptide self-assembly PROTAC degradation agent of the present invention can be specifically triggered in the tumor area, has specificity and selectivity, and significantly reduces off-target toxicity. This strategy can achieve efficient degradation of target proteins at the cellular level and animal level, thereby causing apoptosis of tumor cells, thereby inhibiting tumor growth. The polypeptide self-assembly PROTAC of the present invention significantly inhibits the growth of 4T1 breast cancer, and exerts a stronger inhibitory effect than the linear polypeptide PROTAC at the same dose. The polypeptide nanodegrader particles can be prepared by chemical synthesis, which is simple and easy, not easy to produce drug resistance, will not produce obvious side effects in the body, has good biocompatibility, and avoids the toxic and side effects of small molecule inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the structural formula of the peptide self-assembly PROTAC degrader NFTP;

[0031] Figure 2 Synthesis and characterization of nanoparticles of the peptide self-assembly PROTAC degrader NFTP (A represents the molecular formula of the peptide NFTP; B represents the morphology of the NFTP nanoparticles; C represents the particle size of the NFTP nanoparticles; D represents the potential of the NFTP nanoparticles; E represents the ultraviolet absorption spectrum test results of the NFTP nanoparticles; F represents the structural group of the self-assembly of the NFTP nanoparticles; G represents the Fourier infrared spectrum test results);

[0032] Figure 3 Analysis of tumor cell uptake of NFTP nanoparticles (A represents the fluorescence microscopy imaging results; B and C represent the uptake of NFTP nanoparticles by tumor cells);

[0033] Figure 4 The results of the analysis of the degradation of FOXM1 protein by NFTP nanoparticles in vitro (A represents the effect of LFTP on the expression of FOXM1 protein; B represents the effect of NFTP nanoparticles on the expression of FOXM1 protein; C represents the effect of different concentrations of drugs on the level of FOXM1 protein; D represents the effect of NFTP nanoparticles on the expression of FOXM1 protein; E represents the amount of FOXM1 protein at different times; F represents the content of FOXM1 protein in HepG2 cells after different treatments; G represents the results of qPCR; H represents the total ubiquitination level in HepG2 cells after NFTP treatment);

[0034] Figure 5NFTP nanoparticles induce apoptosis of tumor cells (A represents the effect of different drugs on cell viability; B represents the effect of different drugs on tumor cell apoptosis; C represents the effect of NFTP nanoparticles on cell cycle; D represents the effect of NFTP nanoparticles on abnormal cell cycle of tumor cells verified by detecting markers; E represents the effect of NFTP nanoparticles on cell viability of various tumor cells);

[0035] Figure 6 Evaluation of the anti-tumor effect of NFTP nanoparticles in vivo (A represents the in vivo detection flowchart of mice; B represents the effect of NFTP nanoparticles on tumor volume; C represents the effect of NFTP nanoparticles on tumor weight; D represents the picture of tumor size after treatment with different drugs; E represents HE sections and apoptosis staining of tumor tissue; F represents the number of Ki67-positive cells in tumor tissue; G represents immunohistochemical staining of FOXM1 in tumors; H represents WB detection of the degradation level of FOXM1 in tumors; I represents the effect of different drugs on mouse body weight). DETAILED DESCRIPTION

[0036] In the embodiment of the present invention, the amino acid sequence of the FOXM1 targeting binding group is shown in SEQ ID NO: 1, SEQ ID NO: 1: GLSSMHSAPPLR;

[0037] The amino acid sequence of the self-assembling group is shown in SEQ ID NO: 2, SEQ ID NO: 2: KLVFFGAK;

[0038] The E3 ligase recognition group is VHL-Ligand, and the amino acid sequence is shown in SEQ ID NO: 3, SEQ ID NO: 3: PIYPALA;

[0039] The amino acid sequence of the cathepsin response group (CatB) is shown in SEQ ID NO:4, SEQ ID NO:4: GGLFG;

[0040] The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 5, SEQ ID NO: 5: GSGSG;

[0041] The amino acid sequence of the integrin α6 targeting peptide is shown in SEQ ID NO: 6, SEQ ID NO: 6: CRWYDENAC.

[0042] Integrin α6 targeting peptide is expressed on the surface of various tumor cells, and integrin α6 targeting peptide can recognize and target tumor cells.

[0043] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0044] The experimental instruments and materials used in the embodiments of the present invention are:

[0045] N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, FOXM1 inhibitor FDI-6, tetramethyluronium hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), and ethanedithiol (EDT) were obtained from conventional channels;

[0046] HepG2, SKBR3, MDA-MB-231, A549 and 4T1 cell lines were obtained from our laboratory.

[0047] Example 1

[0048] Synthesis and characterization of peptide self-assembling PROTAC degrader NFTP

[0049] The polypeptide self-assembly PROTAC degrader NFTP is composed of a self-assembly group, an E3 ligase recognition group, a cathepsin response group, a FOXM1 targeting binding group, a connecting peptide and an integrin α6 targeting peptide; the amino acid sequence of the FOXM1 targeting binding group is shown in SEQ ID NO: 1; the amino acid sequence of the self-assembly group is shown in SEQ ID NO: 2; the amino acid sequence of the E3 ligase recognition group (VHL-Ligand) is shown in SEQ ID NO: 3; the amino acid sequence of the cathepsin response group (CatB) is shown in SEQ ID NO: 4; the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 5; the amino acid sequence of the integrin α6 targeting peptide is shown in SEQ ID NO: 6.

[0050] The amphiphilic peptide NFTP was synthesized by solid phase synthesis. The structural formula of the peptide NFTP is as follows: Figure 1 shown.

[0051] The peptide NFTP (1 mg) was dissolved in hexafluoroisopropanol (HFIP) (20 μL), and then slowly dripped into sterile enzyme-free water to finally obtain a reaction solution with a concentration of 1 mmol. The reaction solution was placed on a shaker at 37 ° C and slowly shaken (speed of 30 rpm) for 24 h for self-assembly. After the self-assembly was completed, the obtained clear solution was lyophilized to remove impurities, and the obtained white powder solid was the peptide self-assembly PROTAC degrader, which was stored at -80 ° C for standby use. The self-assembled peptide self-assembly PROTAC degrader NFTP powder was dissolved in sterile enzyme-free water to a liquid with a concentration of 1 mmol, diluted 1:100, and 20 μL was dripped into a 300 mesh copper mesh and allowed to stand for 10 min. The copper mesh was counterstained with 2% uranyl acetate solution for 35 s, and then dried at room temperature to obtain nanoparticles (NFTP nanoparticles). The morphology of the nanoparticles was analyzed by transmission electron microscopy (Tecnai G2, FEI, US). 1 mL of the 1:100 diluted nanoparticle solution was injected into the Elvin sample cell, and the size, polydispersity index (PDI) and Zeta potential of the nanoparticles were analyzed by a dynamic light scattering particle size analyzer (DLS, Zetasizer, Malvern Panalytical, UK). The prepared nanoparticle solution was diluted to a sample solution with a concentration of 0.01 wt%, and 10 μL of the sample solution was dropped on the infrared window (intrinsic Si wafer) and detected using the transmission mode of a Fourier transform micro-infrared spectrometer (iN10, Thermo Fisher Scientific, US), with a spectral detection range of 675 to 4000 cm -1 . Take 1mL of nanoparticle sample solution with a concentration of 0.01wt%, transfer it to a quartz cuvette (0.1mm thick), and use a circular dichroism spectrometer (J-1500, JASCO, Japan) for detection. The spectral detection range is 180nm to 300nm, the scanning speed is 200nm / min, the bandwidth is set to 2nm, and the entire detection process is carried out in a nitrogen protection environment. Use a UV-visible spectrophotometer (TU-1901, Puxi General, China) to scan the spectrum at 250-500nm.

[0052] Results: The peptide NFTP with self-assembly performance was synthesized by solid phase synthesis method, the sequence is KLVFFGAKCRWYDENACGGLFGPIYPALAGSGSGGLSSMHSAPPLR (SEQ ID NO: 7), the molecular formula is as follows Figure 2As shown in A. Through dynamic light scattering detection, in the HFIP / ddH2O system, the peptide NFTP formed a spherical structure with a size of 100nm (2B~C). In addition, through ultraviolet absorption spectrum detection of nanoparticles, the experiment found that NFTP nanoparticles showed a characteristic absorption peak at a wavelength of 270nm ( Figure 2 E), indicating that NFTP nanoparticles have chromophores with lone pair electrons. Circular dichroism detection found that NFTP nanoparticles have a maximum peak at about 190nm, suggesting the presence of β-sheets, indicating that NFTP nanoparticles are mainly assembled through β-sheets to form nanostructures ( Figure 2 F). In the Fourier infrared spectrum, the highest absorption peak was detected at 1633.84nm, which also proved the existence of β-sheet ( Figure 2 G). Thus, the present invention designs and obtains a nanoparticle with high delivery efficiency, precise targeting on tumor cells, and targeted degradation of FOXM1 through controllable release of active ingredients.

[0053] Example 2

[0054] Analysis of tumor cell uptake of the self-assembling degrader NFTP

[0055] In order to trace NFTP nanoparticles, diphenylcyclooctyne-cyanine dye (DBCO-Cy5) was conjugated to NFTP nanoparticles by copper-free click chemistry. NFTP nanoparticles and DBCO-Cy5 were mixed in ddH2O at a molar ratio of 1:1 and placed on a shaker at room temperature for 15 min to obtain Cy5-labeled NFTP (NFTP-Cy5). HepG2 cells in the logarithmic growth phase were cultured at 2×10 4 The cells were seeded in 8-well chambers at a density of 10 cells per well. After the cells were fully attached to the wall, they were incubated with NFTP-Cy5. The cell nuclei were labeled and stained with Hoechst 33342. The images were taken using a laser confocal fluorescence microscope (TCS SP8, Leica, Germany). The cell uptake of NFTP was analyzed using Image J software. HepG2 cells in the logarithmic growth phase were cultured at a density of 1×10 6 Cells were seeded at a density of 10 cells / well in a six-well plate. After the cells were fully attached, they were incubated with NFTP-Cy5 for 1, 2, and 4 hours. The cells were digested with 0.25% trypsin and then resuspended in PBS. Fluorescence quantitative analysis of the cells was performed by flow cytometry. FlowJo X software was used to analyze the uptake of NFTP by the cells.

[0056] Confocal fluorescence microscopy imaging ( Figure 3A) The analysis found that the average fluorescence intensity of Cy5-NFTP in cells reached 119.90±30.09 after 1h of incubation. This indicates that NFTP nanoparticles have a high cellular uptake rate, which is attributed to the modified integrin α6 targeting peptide. Compared with the average fluorescence intensity of 139.20±13.27 after 2h of incubation, Cy5-NFTP was further taken up by cells after 4h of incubation, with an average fluorescence intensity of 190.30±19.85. In addition, to further verify the cellular uptake of NFTP, flow cytometry showed ( Figure 3 BC), at 1 hour, 98.7% of the cells took up NFTP-Cy5. In the negative control cell line HEK-293T cells, even after incubation for 4 hours, the average fluorescence intensity of NFTP-Cy5 was only 89.70±5.33. These experiments show that NFTP nanoparticles can be efficiently taken up by tumor cells through cell surface receptor-mediated endocytosis.

[0057] Example 3

[0058] Evaluation of the effect of self-assembling PROTAC degrader NFTP on FOXM1 protein degradation in vitro

[0059] HepG2 cells were co-cultured with PBS, NFTP nanoparticles, and LFTP peptide (including the targeting binding group shown in SEQ ID NO:1, the E3 ligase recognition group described in SEQ ID NO:3, and the connecting peptide described in SEQ ID NO:5, synthesized according to the solid phase synthesis method, and the specific sequence is PIYPALAGSGSGGLSSMHSAPPLR (SEQ ID NO:8)) for 24 to 36 hours. The protein lysate RIPA was mixed with 100mM protease inhibitor (PMSF) at a ratio of 100:1 to prepare the cell lysate, and the total protein was extracted at 4°C. The FOXM1 protein concentration of the supernatant was determined using a BCA protein assay kit. The FOXM1 protein sample was separated by SDS-polyacrylamide gel electrophoresis. The protein sample was electrotransferred to a PVDF membrane and blocked with 5% skim milk powder at room temperature for 2 hours. FOXM1 primary antibody, Ubiquitin primary antibody, and internal reference β-Actin antibody were added to the PVDF membrane bound to the protein sample in sequence. For primary antibodies derived from different species, the corresponding secondary antibodies were incubated. Data quantification was performed using Image J software.

[0060] Different concentrations of LFTP and NFTP nanoparticles were added to HepG2 cells and incubated for 24 hours before Western Blotting analysis. The results are shown in Figure ( Figure 4AB) showed that both LFTP and NFTP nanoparticles can effectively degrade FOXM1 protein in a dose-dependent manner. The fitted drug dose-response curve showed ( Figure 4 C), the half degradation concentration value of LFTP (DC 50 ) was 65.02 μM, while the DC of NFTP nanoparticles 50 The degradation efficiency of NFTP nanoparticles was better due to the more efficient delivery efficiency of self-assembled nanoparticles and the more efficient drug uptake by cells assisted by cell-targeted peptides. The degradation of FOXM1 mediated by NFTP nanoparticles was also time-dependent ( Figure 4 D). At 36 h after administration, NFTP nanoparticles almost completely degraded FOXM1 ( Figure 4 E). MG-132 is a proteasome inhibitor that can effectively inhibit the degradation of ubiquitinated proteins by the proteasome. In the presence of MG-132, HepG2 cells treated with NFTP nanoparticles can restore the expression of FOXM1. This shows that NFTP nanoparticles achieve specific degradation of FOXM1 with the help of the ubiquitin proteasome system. CA-074 is an effective inhibitor of Cat B. Since the activity of Cat B is affected by CA-074, NFTP nanoparticles that enter the cell cannot release active LFTP peptides through enzymatic cleavage, and ultimately cannot degrade FOXM1 ( Figure 4 F). In addition, the results of qPCR showed that ( Figure 4 G), the effects of NFTP nanoparticles and LFTP on FOXM1 only remain at the protein level and do not cause significant changes in FOXM1 gene expression. The total ubiquitination level in HepG2 cells treated with NFTP nanoparticles was detected ( Figure 4 H). The results showed that the overall ubiquitination level in the NFTP nanoparticle-treated group increased. This is because the VHL ligand in the NFTP nanoparticles effectively binds to VHL and activates the subsequent cascade amplification reaction, which ultimately leads to increased polymerization of ubiquitin molecules. These results show that NFTP nanoparticles use the Cat B enzyme expressed in the cells to release active LFTP, and then use the ubiquitin-proteasome system to achieve targeted degradation of FOXM1.

[0061] Example 4

[0062] Self-assembling PROTAC degrader NFTP induces apoptosis of tumor cells

[0063] The effects of NFTP nanoparticles, LFTP, and commercial FOXM1 inhibitor (FDI-6) on cell viability were evaluated using a CCK-8 kit. HepG2 cells in the logarithmic growth phase were cultured at 1×104 The cells were seeded in a 96-well plate at a density of 10 cells per well. After the cells were fully attached, they were incubated with different concentrations (0.5, 1, 5, 10, 20, 40, 60, 80, 100 μM) of NFTP nanoparticles, LFTP and FDI-6 for 24 hours. The CCK-8 solution was diluted 10 times with complete culture medium. The culture medium in the well plate was discarded, and then 100 μL of the diluted CCK-8 solution was added to each well and incubated at 37°C for 1 to 4 hours. After the water-soluble orange-yellow formazan was generated, the absorbance of each well was detected at OD450 nm using an enzyme reader to calculate the cell activity.

[0064] HepG2 cells in logarithmic growth phase were cultured at 1×10 6 The cells were seeded at a density of 1:1 in a 6-well plate. After the cells adhered to the bottom, 50 μM NFTP nanoparticles, LFTP and FDI-6 were added and co-cultured for 24 hours. Then, the cells were treated according to the cell cycle assay kit and Annexin V-FITC apoptosis detection kit protocols and detected by flow cytometry.

[0065] The effect of NFTP on the activity of HepG2 cells was studied using CCK-8. Figure 5 As shown in A, NFTP nanoparticles can significantly inhibit the activity of HepG2 cells, and its inhibitory effect is similar to that of LFTP and FDI-6. Annexin V and PI double staining method found that compared with the 7.04% apoptosis rate of the PBS-treated group, LFTP can cause 30.23% of cells to undergo apoptosis, while NFTP nanoparticles can induce up to 79.3% of tumor cells to undergo varying degrees of apoptosis ( Figure 5 B). In order to explore the underlying reasons for the increase in cell apoptosis after NFTP nanoparticle treatment, flow cytometry was used to detect the effect of NFTP nanoparticles on the cell cycle. Compared with the control group, the proportion of cells arrested in the G2 / M phase in the NFTP group increased relatively ( Figure 5 C). The detection of mRNA of these cell cycle-related markers, Cyclin B, CENPE and PCNA, also confirmed that NFTP nanoparticles can improve the abnormal cell cycle of tumor cells ( Figure 5 D). These results indicate that NFTP can lead to cell cycle arrest by degrading FOXM1, and ultimately increase cell apoptosis. Not only for HepG2 cells, but also for a variety of tumor cells, NFTP nanoparticles can have a significant inhibitory effect on cell viability ( Figure 5 E), including human breast cancer cells SK-BR-3, human poorly differentiated colon cancer RKO, and human malignant melanoma cells A-375. These results indicate that NTFP nanoparticles have therapeutic potential in multiple cancer types.

[0066] Example 5

[0067] Evaluation of the anti-tumor effect of self-assembled PROTAC degraders in vivo

[0068] 4T1 cells (2×10 6 cells) were inoculated subcutaneously into the back of the right leg of BALB / C mice. 3 At the same time, the mice were randomly divided into a control group, an NFTP group, an LFTP group, and an FDI-6 group, with 5 mice in each group. Each group of mice was treated by tail vein injection of NFTP nanoparticles, LFTP, and FDI-6 (dosage of 30 mg / kg, 0.15 mL) once every two days for 14 days. The control group was intravenously injected with normal saline containing PBS. The body weight and tumor volume of the mice were measured every two days. 12 hours after the last treatment, the mice were killed and the tumors were weighed. Blood was collected through the orbital plexus vein for blood biochemistry and blood routine tests. Tumor tissues and major organs were removed for tissue sections, immunohistochemistry, immunofluorescence experiments, and western blotting experiments. The analysis process is as follows Figure 6 As shown in A.

[0069] from Figure 6 It can be seen that LFTP and FDI-6 have tumor inhibition rates of 18.13% and 23.1%, respectively, while the tumor inhibition rate of NFTP reached a significant 52.12%. These conclusions all indicate that self-assembled PROTAC degraders have more significant anti-tumor effects ( Figure 6 BD). H&E results showed that ( Figure 6 E) NFTP nanoparticles can induce tumor cell volume reduction and cell nucleus shrinkage and rupture after treatment. Ki-67 ( Figure 6 The reduction of red fluorescence in F) indicates that NFTP nanoparticles inhibited the proliferation of tumor cells to a certain extent. The IHC test results of FOXM1 support that NFTP nanoparticles can effectively induce the targeted degradation of FOXM1 in vivo. Further WB detection found that after treatment with NFTP nanoparticles, the FOXM1 level in the tumor was effectively degraded ( Figure 6 H). The biosafety of NFTP nanoparticles for in vivo treatment was evaluated by monitoring the weight changes of mice throughout the treatment process. There was no significant difference in the weight changes of mice in the NFTP treatment group compared with the control group ( Figure 6 I).

[0070] As can be seen from the above examples, the present invention provides a polypeptide self-assembly PROTAC degrader and its preparation method and application. The polypeptide-based nanodelivery system of the present invention significantly improves the drug delivery efficiency, while the controlled release of active fragments minimizes the risk of side effects. In addition, NFTP accurately targets and degrades FOXM1, thereby regulating the cell cycle, reducing the proliferation and invasion of tumor cells, and ultimately promoting tumor apoptosis. A good protein degradation effect was achieved at the animal tissue level, achieving a good tumor inhibition effect on mice and reducing side effects outside the tumor.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide self-assembly PROTAC degradation agent, characterized in that: The polypeptide self-assembly PROTAC degrader includes a self-assembly group, an E3 ligase recognition group, a cathepsin response group, a FOXM1 targeting binding group, a connecting peptide and an integrin α6 targeting peptide; The amino acid sequence of the FOXM1 targeting binding group is shown in SEQ ID NO:

1.

2. The polypeptide self-assembly PROTAC degradation agent according to claim 1, characterized in that The amino acid sequence of the self-assembling group is shown in SEQ ID NO:

2.

3. The polypeptide self-assembly PROTAC degradation agent according to claim 2, characterized in that The amino acid sequence of the E3 ligase recognition group is shown in SEQ ID NO:

3.

4. The polypeptide self-assembly PROTAC degradation agent according to claim 3, characterized in that The amino acid sequence of the cathepsin responsive group is shown in SEQ ID NO:

4.

5. The polypeptide self-assembly PROTAC degradation agent according to claim 4, characterized in that The amino acid sequence of the connecting peptide is shown in SEQ ID NO: 5; The amino acid sequence of the integrin α6 targeting peptide is shown in SEQ ID NO:

6.

6. The polypeptide self-assembling PROTAC degradation agent according to any one of claims 1 to 5, characterized in that The structural formula of the polypeptide self-assembly PROTAC degrader is shown in formula (I):

7. The method for preparing the polypeptide self-assembling PROTAC degradation agent according to any one of claims 1 to 6, characterized in that: The steps include: (1) synthesizing a self-assembly group, an E3 ligase recognition group, a cathepsin response group, a FOXM1 targeting binding group, a connecting peptide and an integrin α6 targeting peptide by a peptide solid phase synthesis method to obtain a peptide NFTP; (2) Mixing the peptide NFTP with hexafluoroisopropanol, reacting for 20 to 28 hours, and drying to obtain a peptide self-assembling PROTAC degrader.

8. The preparation method according to claim 7, characterized in that: The mass volume ratio of the polypeptide NFTP and hexafluoroisopropanol mixed in step (2) is 1 mg: 18-22 μL; the reaction temperature in step (2) is 35-39° C., and the reaction speed is 20-50 rpm.

9. Use of the polypeptide self-assembling PROTAC degrader according to any one of claims 1 to 6 or the polypeptide self-assembling PROTAC degrader prepared by the preparation method according to any one of claims 7 to 8 in the preparation of a preparation for targeted degradation of FOXM1 or in the preparation of a drug with anti-tumor efficacy; The tumor is a tumor caused by overexpression of FOXM1, including breast cancer, non-small cell lung cancer, kidney cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer or gastric cancer; The drug contains 20 to 100 μM of the polypeptide self-assembly PROTAC degrader.

10. A nanoparticle for targeted degradation of FOXM1, characterized in that: The nanoparticles include the polypeptide self-assembling PROTAC degrader according to any one of claims 1 to 6 or the polypeptide self-assembling PROTAC degrader prepared by the preparation method according to any one of claims 7 to 8; The particle size of the nanoparticles is 100 to 150 nm; The preparation method of the nanoparticles is: dissolving the polypeptide self-assembly PROTAC degradation agent in water, separating, and obtaining the nanoparticles.

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