Buforin llb or derivative peptide thereof as ARF6 inhibitor and application

Abstract

The invention provides Buforin llb or a derivative peptide thereof as an ARF6 inhibitor and application. One purpose of the present application is to provide an ARF6 inhibitor comprising: (1) a peptide capable of binding to ARF6; (2) a nucleic acid molecule encoding the peptide; or (3) a biological material containing the peptide or nucleic acid molecule; the peptide is buforin IIb or a derivative peptide of the buforin IIb, and the buforin IIb or the derivative peptide of the buforin IIb can be combined with ARF6. The amino acid sequence of the buforin IIb is as shown in SEQ ID NO.1, the derivative peptides comprise derivative peptides B1 to B3 and derivative peptides C1 to C4, and the amino acid sequences of the derivative peptides B1 to B3 and the derivative peptides C1 to C4 are as shown in SEQ ID NO.2 to SEQ ID NO.8. The Buforin IIb or the derivative peptide thereof can be used as an ARF6 polypeptide inhibitor, and the polypeptide inhibitor provided by the invention has higher targeting property and is not easy to accumulate in vivo.

Description

Technical Field

[0001] The present application relates to the technical field of polypeptide drugs, and in particular to Buforin llb or its derivative peptides as ARF6 inhibitors and their applications. Background Art

[0002] ARF6, the full name of ADP-ribosylation factor 6, is a member of the guanosine triphosphatase (ARF) family. It is widely expressed in mammalian cells and exists in the plasma membrane and endosomes. It has the function of regulating membrane transport (such as endocytosis, exocytosis and endosomal recycling) and actin remodeling. ARF6 is highly expressed in patients with various clinical cancers. The activation and high expression of ARF6 protein are significantly correlated with tumor invasion and metastasis. Existing ARF6 inhibitors are all small molecule compounds, such as NAV-2729, brefeldin A, etc., which have severe in vivo toxicity, limiting the clinical application of ARF6 targeted drugs.

[0003] At present, the treatments for malignant tumors include surgery, chemotherapy, radiotherapy, targeted therapy and immunotherapy. Among them, immunotherapy has changed the previous concept of tumor treatment. It no longer relies on external forces to kill tumor cells, but mobilizes the body's own immune system to inhibit or eliminate tumor cells. Immunogenic cell death (ICD) is a defense pathway that activates the body's immune system to fight tumors. Drug-induced ICD in tumor cells will release three key signaling molecules: calreticulin (CRT), adenosine triphosphate (ATP) and high mobility group protein B1 (HMGB1). These three molecules can act as "immune adjuvants" to significantly enhance the presentation of tumor antigens by dendritic cells and the attack activity of cytotoxic T cells, promoting immune killing of tumors.

[0004] Peptide compounds also have the ability to induce ICD. Compared with small molecule compounds, peptide compounds have high selectivity, high affinity, biocompatibility and less toxic side effects. The development of peptide drugs targeting ARF6 will contribute to clinical cancer treatment, and it has a stronger potential for drug development. At the same time, ARF6 is activated in a variety of inflammatory diseases, such as nephritis, pulmonary fibrosis, pulmonary hypertension, neuroinflammation, sepsis, atherosclerosis, respiratory inflammation, and neutrophil- and macrophage-mediated cardiovascular diseases. Therefore, the development of ARF6 inhibitory peptides often has application prospects in many inflammatory diseases. Therefore, the development of new peptide drugs targeting ARF6 has practical clinical needs and great social significance. Summary of the invention

[0005] One of the purposes of the present application is to provide an ARF6 inhibitor, comprising ① a peptide capable of binding to ARF6; ② a nucleic acid molecule encoding the peptide; or ③ a biological material containing the aforementioned peptide or nucleic acid molecule;

[0006] The peptide is buforin IIb or a peptide derived therefrom, and the buforin IIb or a peptide derived therefrom can bind to ARF6.

[0007] Preferably, the amino acid sequence of buforin IIb is as shown in SEQ ID NO.1, which is RAGLQFPVGRLLRRLLRRLLR,

[0008] The derivative peptides include B1 to B3 and derivative peptides C1 to C4, and their amino acid sequences are shown in SEQ ID NO.2 to SEQ ID NO.8;

[0009] Derivative peptide B1: RLLRQWPIQRLLRRLLR (SEQ ID NO. 2),

[0010] Derivative peptide B2: RAQLQFPVQRLLRRLLR (SEQ ID NO. 3),

[0011] Derivative peptide B3: RAQLQFPVQRLLRRLLRRLLR (SEQ ID NO. 4),

[0012] Derivative peptide C1: RGGLQFPVGRLLRRLLRRLLR (SEQ ID NO. 5),

[0013] Derivative peptide C2: R(Ail)GLQFPVGRLLRRLLRRLLR (SEQ ID NO.6), Ail is 2-aminoisobutyric acid,

[0014] Derivative peptide C3: RPQLQFPVQRLLRRLLRRLLR (SEQ ID NO. 7),

[0015] Derivative peptide C4: R(Ail)QLQFPVQRLLRRLLRRLLR (SEQ ID NO. 8).

[0016] Preferably, the biological material includes antibodies (such as monoclonal antibodies, etc.), cells (such as mammalian cells, insect cells, etc.), bacteria (such as engineered bacteria, probiotics, etc.), microcapsules or microparticles containing the nucleic acid molecule or expressing the peptide.

[0017] Preferably, the biological material comprises a gene expression element or expression cassette containing the nucleic acid molecule. The expression cassette is a recombinant nucleic acid molecule obtained by connecting regulatory elements for transcription and translation upstream and downstream of the nucleic acid molecule.

[0018] Preferably, the biomaterial comprises a conjugate formed by connecting the buforin IIb or its derivative peptide to a carrier. The carrier is a polypeptide (including various tumor high affinity polypeptides, such as endogenous hormones), a compound (such as folic acid), a monoclonal antibody, an immunoglobulin G4 heavy chain fragment (Fc), human albumin, and nanoparticles such as liposomes, microcapsules, and microparticles that carry and are capable of targeted therapy. The biomaterial comprises a modified product obtained by labeling or cyclization of the buforin IIb or its derivative peptide, specifically, by D-amino acids, β-amino acids, sulfamides or side chain mimics, N-methylation and N-terminal acetylation, peptide design, sulfide bonds or side chain mimics, N-methylation and N-terminal acetylation, peptone design, disulfide bond mimics, PEGylation, lipidation and stapling to stabilize α-helices and form cyclic peptides, etc., to improve drugability, and improve the in vivo stability, bioavailability and therapeutic effect of peptide drugs.

[0019] Preferably, the conjugate is a conjugated peptide formed by the Buforin IIb or a peptide derived therefrom and GE11.

[0020] The second purpose of the present application is to provide the use of any of the above-mentioned ARF6 inhibitors in the preparation of tumor vaccines, anti-cancer drugs or inflammatory disease drugs, which can be used alone or in combination.

[0021] Preferably, the tumor vaccine is a whole tumor lysate antigen vaccine, and the anticancer drug is an agent or drug that promotes the release of anti-tumor immune adjuvants.

[0022] Tumor therapeutic vaccines are an important strategy for immunotherapy. Whole tumor cell lysates contain all tumor cell antigen information and can be used to prepare targeted personalized tumor vaccines by obtaining clinical tumor tissues. This advantage has attracted much attention. In this process, tumor cells undergo immunogenic cell death, surface exposure of calreticulin, extracellular release of ATP, protein high-mobility group protein B1, heat shock proteins and other molecules. These immunogenicities promote the activation of dendritic cells DC, the absorption and maturation of antigens, and ultimately promote T cell activation.

[0023] Preparation methods for whole tumor cell lysates include physical, chemical and drug treatments. Physical methods include ultraviolet irradiation, repeated freeze-thaw cycles, high temperature and high hydrostatic pressure (HPP). Chemical methods include hypochlorous acid oxidation. Drug methods can use doxorubicin (DOX) pretreatment.

[0024] The third object of the present application is to provide the use of any of the above-mentioned ARF6 inhibitors in reducing anti-tumor drug-induced cardiotoxicity and myocarditis.

[0025] A fourth object of the present application is to provide a drug comprising any of the above-mentioned ARF6 inhibitors.

[0026] The sequence of buforin IIb is RAGLQFPVGRLLRRLLRRLLR (SEQ ID NO.1). At present, the research on buforin IIb focuses on its broad-spectrum antibacterial and anticancer effects. The present application shows that buforin IIb or its derivative peptides can bind to ARF6, inhibit the transfer and expression of EGFR from the Golgi apparatus to the cell membrane, thereby inhibiting the growth, movement and invasion / metastasis of tumor cells or cancer cells, and is a new type of ARF6 targeted peptide drug. The buforin IIb derivative peptides are obtained by replacing the amino acids of buforin IIb, including replacement of G (glycine) to Q (glutamine), replacement of F (phenylalanine) to W (tryptophan), replacement of V (valine) to I (isoleucine), and replacement of A (alanine) to Ail (aminoisobutyric acid) or P (proline).

[0027] The beneficial effects of this application are at least:

[0028] First, buforin IIb or its derivative peptides can be used as ARF6 polypeptide inhibitors. Compared with existing ARF6 inhibitors, the polypeptide inhibitors of the present application have higher targeting, are not easy to accumulate in the body, have stronger biological activity, require smaller dosages, have lower toxic side effects and have significant therapeutic effects.

[0029] Second, buforin IIb or its derivative peptides and other substances can be used as ARF6 inhibitors to prepare tumor vaccines, anticancer drugs or inflammatory disease drugs. For example, it has the effect of enhancing tumor immunogenicity and is used for whole tumor vaccines; for example, it can efficiently induce tumor ICD and is used to promote the release of anti-tumor immune adjuvants. Among them, buforin IIb derivative peptides B1 and B2 have lower hemolysis and higher therapeutic index than buforin IIb, and buforin IIb derivative peptide B3 has better anti-tumor cell activity than buforin IIb. Buforin IIb derivative peptide C2 has better target arf6 affinity than buforin IIb, and derivative peptides C3 and C4 have better target arf6 affinity than derivative peptide B3. Buforin IIb or its derivative peptides are connected to the carrier to form a conjugate or a modified product obtained by labeling modification or cyclization modification, which improves the drugability of the drug and improves the in vivo stability, bioavailability and therapeutic effect of peptide drugs.

[0030] Third, the ARF6 inhibitor of the present application can reduce the cardiotoxicity and myocarditis induced by traditional anti-tumor drugs and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the docking score diagram of the anti-tumor peptide and ARF6 protein (PDB ID: 2A5D).

[0032] Figure 2 The binding capacity of buforin IIb and ARF6 was determined by ultrafiltration mass spectrometry.

[0033] Figure 3 The changes in tumor volume after administration of buforin IIb and DOX.

[0034] Figure 4 The tumor occurrence after administration of buforin IIb and DOX (n=6-8).

[0035] Figure 5 This is the CRT exposure ratio after administration of buforin IIb and DOX in Example 3.

[0036] Figure 6 This is the extracellular ATP content after administration of buforin IIb and DOX in Example 3.

[0037] Figure 7 This is the HMGB1 content after administration of buforin IIb and DOX in Example 3.

[0038] Figure 8 This is a graph showing the detection result that buforin IIb inhibits the transfer of EGFR from the Golgi apparatus to the cell membrane.

[0039] Fig. 9 This is the synthetic route of buforin IIb-fatty acid FA.

[0040] Fig.10 This is the infrared spectrum of buforin IIb-fatty acid FA.

[0041] Fig.11 The synthetic route for deriving peptide B2-fatty acid FA.

[0042] Fig.12 The mass spectrum of the derived peptide B2-fatty acid FA.

[0043] Fig.13 NH2-SS-PEG 600 -Synthesis route of SS-NH2.

[0044] Fig.14 It is a hydrogen nuclear magnetic resonance spectrum.

[0045] Fig.15 A synthetic route for self-assembling materials.

[0046] Fig.16 Schematic diagram of the structure of buforin IIb and three derived peptides.

[0047] Fig.17 is the inhibition rate of buforin IIb (BFP2) and its derivative peptides B1-B3 on HepG2 cells.

[0048] Fig.18 This is an investigation on the hemolytic activity and therapeutic index of the three derivative peptides. DETAILED DESCRIPTION

[0049] The following examples are used to illustrate the present application, but are not intended to limit the scope of the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0050] Example 1 Buforin IIb can bind to ARF6

[0051] 1. Molecular docking screening

[0052] The docking receptor is ARF6 protein (PDB ID: 2A5D), and the docking ligands are 406 antitumor peptides;

[0053] The docking program was HPEPDOCK 2.0. Peptides with docking scores less than -200 were selected for cell penetration prediction. 65 peptides with docking scores less than -200 were used for cell penetration prediction. The docking program was ChemRF-CPPred. CPP represents the possibility of cell penetrating peptides, and non-CPP represents the possibility of non-cell penetrating peptides, such as Figure 1 As shown, buforin IIb was found to have good cell permeability and a high Arf6 affinity score.

[0054] 2. Buforin IIb and ARF6 binding experiment

[0055] Control group: 10μg / mL buforin IIb standard solution. BSA control group: 10μg / mL buforin IIb standard solution and 10μg BSA protein incubation. Experimental group: 10μg / mL buforin IIb standard solution and 10μg recombinant ARF6 protein incubation. The samples of each group were incubated at 4℃ and room temperature for 30min, respectively. After incubation, the samples were transferred to the upper layer of the ultrafiltration tube and centrifuged at 8,000rpm, 4℃ for 30min. Buffer 450μL was added to the upper layer of the ultrafiltration tube, centrifuged at 8,000rpm, 4℃ for 30min, and the remaining solution in the upper layer was transferred to a new ultrafiltration tube. Buffer 450μL was added to the upper layer of the ultrafiltration tube again, centrifuged at 8,000rpm, 4℃ for 30min, and the upper layer solution was collected, concentrated by centrifugation, evaporated, and re-dissolved. DTT was added to the sample to 5mM and incubated at 37℃ for 30min. Add IAA to 15 mM and incubate at 37°C in the dark for 45 min. Add trypsin to the sample and incubate at 37°C overnight. Add formic acid to stop digestion. Desalt the sample, concentrate and evaporate, add 0.1% formic acid water to reconstitute, and then test.

[0056] LC MS / MS detection conditions

[0057] Liquid phase conditions: EASY-nLC 1200 nanoliter liquid chromatograph (Thermo Scientific), chromatographic column: homemade capillary column (75 μm × 400 mm, 1.9 μm C18, ), mobile phase A: water (0.1% formic acid), B: acetonitrile (0.1% formic acid), flow rate 300nL / min, injection volume 2μL. Gradient elution conditions: 0min, 5% B; 0-90min, 5%-20% B; 90-108min, 20%-30% B; 108-115min, 30%-90% B; 115-120min, 90% B.

[0058] Mass spectrometry conditions: Q Exactive PlusTM high-resolution mass spectrometer was used for detection in positive ion scanning mode, with a primary mass spectrometry scanning range of m / z 300-1700, a resolution of 70,000, an automatic gain control target (AGC target) of 3e6, and a maximum dwell time (Maximum IT) of 50ms. The secondary mass spectrometry scanning range was m / z 200-2,000, the mass spectrometry resolution was 17,500, the automatic gain control target was 1e5, the maximum dwell time was 100ms, the parent ion isolation width (Isolation window) was 2m / z, the dynamic exclusion time was 30s, and the high energy collision (HCD) mode was used.

[0059] The binding of buforin IIb to ARF6 was determined by ultrafiltration mass spectrometry. The binding strength was evaluated based on the buforin IIb remaining in the upper layer of the ultrafiltration tube. Figure 2 As shown, compared with the control group, there was no significant change in the buforin IIb content in the upper layer of ultrafiltration after interaction with the negative control BSA, while the content of buforin IIb in the upper layer was significantly increased after ultrafiltration after interaction with ARF6, indicating that buforin IIb can target and bind to ARF6.

[0060] Example 2 Buforin IIb as an ARF6 inhibitor for the preparation of tumor therapeutic vaccines

[0061] Drug-treated tumor lysates have a better ability to induce immune killing than other methods. In this example study, we evaluated the use of buforin IIb as a drug treatment method, mixed with tumor cells in vitro, and used for the preparation of whole tumor cell lysate vaccines. The results showed that tumor cell lysates treated with buforin IIb can efficiently induce tumor immunogenic death ICD and have better safety than DOX.

[0062] 1. This experiment used different doses of buforin IIb to incubate mouse liver cancer cells Hepa1-6 (purchased from Wuhan Pronocell Life Science Technology Co., Ltd.) and Hepa1-6 as a control. Whole-cell lysates were prepared by ultrasonic disruption as a vaccine, which was transplanted into the left side of the mouse to observe the effect on the growth of tumors on the right side of the mouse and to evaluate the potential of buforin IIb to induce the preparation of whole-tumor vaccines.

[0063] Animal grouping and modeling: Before the experiment, the mice were depilated of their armpit hair and randomly divided into DOX control group, model group, buforin IIb low, medium and high dose groups (4, 8, 16 μg / mL) according to their body weight, with 6-8 mice in each group. Hepa1-6 mouse liver cancer cells were transplanted in the left armpit of the mice, and 4.0 μg / mL DOX was injected subcutaneously in the DOX control group; 5×10 Hepa1-6 cells were injected subcutaneously in the model group. 5 The low-dose group was subcutaneously injected with 5×10 Hepa1-6 cells treated with 4.0 μg / mL buforin IIb. 5 The medium-dose group was subcutaneously injected with 5×10 Hepa1-6 cells treated with 8.0 μg / mL buforin IIb. 5 The high-dose group was subcutaneously injected with 5×10 Hepa1-6 cells treated with 16.0 μg / mL buforin IIb 5 After five days, 1×10 tumor cell suspension was inoculated subcutaneously in the right axilla. 7After inoculation, the tumor size of the mice was observed and measured every day. On the 13th day, the mice were euthanized, the subcutaneous tumors were removed, the tumor weight was weighed, and the tumor inhibition rate was calculated (inhibition rate = (tumor weight of the model group - tumor weight of the drug administration group) / tumor weight of the model group. After the tumor grew, the maximum long diameter (A) and the maximum transverse diameter (B) of the tumor in the perpendicular direction of the tumor-bearing mice were measured every day, and the similar tumor volume (V) was calculated according to the formula V (cm 3 )=(A×B 2 ) / 2.

[0064] like Figure 3 As shown, after the liver cancer cells Hepa1-6 were treated with DOX control and injected into the left side of the mouse, the occurrence of newly inoculated tumors on the right side was significantly inhibited after five days. Tumor cell lysates treated in vitro with Buforin IIb (4, 8, 16 μg / ml) were inoculated into the left side of the mouse, which could effectively induce tumor immunogenic death ICD and significantly inhibit the occurrence and growth of liver cancer on the right side. The inhibition rates of the low, medium and high dose groups of buforin IIb were 38.45%, 70.69% and 95.58% respectively (P<0.001).

[0065] like Figure 4 As shown, all mice in the model group developed tumors on the right side 5 days after inoculation with Hepa1-6 tumor cells, while only one mouse developed a tumor during the 13-day observation period after inoculation with tumor cell lysate treated with high-dose buforin IIb (16 μg / mL). The tumor lysate vaccine treated with high-dose buforin IIb has a good inhibitory effect on the occurrence of liver cancer.

[0066] Clinical intravenous administration of DOX has significant vascular irritation, and most patients will develop phlebitis during medication, with pain, swelling and induration at the injection site. In addition, doxorubicin has strong tissue and organ toxicity. The above results show that buforin IIb peptide has the effect of enhancing tumor immunogenicity and can be used for the preparation of whole tumor cell lysate antigen vaccine. Compared with doxorubicin, peptide drugs have no organ toxicity and are safer.

[0067] Example 3 Buforin IIb as an ARF6 inhibitor induces liver cancer cells to release immune adjuvant molecules

[0068] 1. Detection methods of immune adjuvant molecules

[0069] CRT detection on cell membrane surface:

[0070] Normally grown HepG2 liver cancer cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were seeded in a 24-well plate, and 1×10 5 / well. After the cells adhere, add drugs and incubate for 4 hours. Discard the supernatant and wash the cells 3 times with PBS. Add 100μL 4% paraformaldehyde and fix at 4℃ for 20 minutes. Discard the supernatant and wash the cells 3 times with PBS. Add 100μL 0.2% Triton X-100 and fix at room temperature for 5 minutes. Discard the supernatant and wash the cells 3 times with PBS. Add 100μL 1% BSA and block at room temperature for 1 hour. Discard the supernatant, add 100μL Anti-CRT and incubate at room temperature for 1 hour. Discard the supernatant and wash the cells 3 times with PBS. Add 100μL rabbit anti-IgG and incubate at room temperature for 1 hour. Discard the supernatant and wash the cells 3 times with PBS and protect from light. Add 100μL DAPI solution to counterstain the nucleus and incubate at room temperature for 5 minutes in the dark. Discard the supernatant and wash the cells 3 times with PBS and protect from light. Add anti-fluorescence quenching sealing liquid on the slide, and place the slide upside down on the slide. Observe with a Zesis fluorescence microscope. Quantify with Image Pro, and use the ratio of the fluorescence area to the total area as the percentage of fluorescence data results.

[0071] Extracellular ATP detection:

[0072] Normally growing HepG2 liver cancer cells were seeded in a 96-well plate, and 5×10 3 / well. Add drugs after cells adhere to the wall and incubate for 12h. Collect cell fluid, centrifuge at 3,000rpm at room temperature for 20min, and collect supernatant. Pour Reconstitution Buffer into rL / L reagent bottle as detection reagent and let stand to dissolve. Press 1×10 -7 M, 2×10 -8 M, 4×10 -9 M, 8×10 -10 M, 1.6×10 -10 M, 3.2×10 -11 M, 6.4×10 -12 M.

[0073] 1.28×10 -12 A standard curve was prepared with a concentration of 100 M. 50 μL of ATP detection reagent was added to every 50 μL of sample and standard, and fluorescence detection was completed within 2-10 s.

[0074] Extracellular HMGB1 detection:

[0075] Normally growing HepG2 liver cancer cells were seeded in a 96-well plate, and 5×10 3 / well. After the cells adhere to the wall, add drugs and incubate for 24 hours. Collect the cell fluid, centrifuge at room temperature at 3,000rpm for 20 minutes, and collect the supernatant. Prepare standard curves at concentrations of 2.5μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, and 30μg / mL. After the sample is diluted 5 times and measured, add the sample and standard to the bottom of the ELISA plate well. After sealing the plate with a sealing film, incubate at 37℃ for 30 minutes. Remove the sealing film, discard the liquid, add washing solution to each well, let it stand for 30s, and then discard it. Repeat 5 times. Add 50μL of enzyme labeling reagent to each well. After sealing the plate with a sealing film, incubate at 37℃ for 30 minutes. Remove the sealing film, discard the liquid, add washing solution to each well, let it stand for 30s, and then discard it. Repeat 5 times. Add 50μL of color developer A and 50μL of color developer B to each well, and color at 37℃ in the dark for 15 minutes. Add 50 μL of stop solution to terminate the reaction. Measure the absorbance at 450 nm using an ELISA reader within 15 min.

[0076] 2. Experiments and Results

[0077] Immunofluorescence assay revealed that buforin IIb significantly induced the exposure of CRT on the cell surface of HepG2 cells. Figure 5 As shown. Bioluminescence assay showed that buforin IIb significantly induced the level of ATP released by HepG2 cells, as shown Figure 6 As shown in Figure 2, ELISA enzyme-linked immunosorbent assay revealed that buforin IIb significantly promoted the level of HMGB1 released from extracellular space of HepG2 liver cancer cells. Figure 7 shown.

[0078] Overexpression of epidermal growth factor (EGFR) receptor is an important driving force for tumor proliferation and weakens anti-tumor immune response. Buforin IIb, as an ARF6 inhibitor, can inhibit the transfer of ARF6-EGFR complex from the Golgi apparatus to the cell membrane by binding to ARF6. Figure 8 Buforin IIb inhibits EGFR through a novel mechanism, thereby promoting anti-tumor immune response.

[0079] Example 4 Preparation of Buforin IIb-GE11 coupled nanoparticles

[0080] 1. Synthesis and characterization of Buforin IIb (Buff IIb)-hexadecanedioic acid (FA)

[0081] Weigh FA (10 mg, 0.035 mmol) and dissolve it in DMF. Add EDCI (13.4 mg, 0.07 mmol) and HOBT (9.5 mg, 0.07 mmol) and stir for 24 h. Then add Buff IIb (6 mg, 0.0024 mmol) and continue stirring for 24 h. After the reaction is completed, transfer the reaction solution to a dialysis bag for dialysis to purify the sample. After dialysis, use a concentrator to dry to obtain a brown-yellow powder. The synthetic route of Buff IIb-FA is as follows: Fig. 9 shown.

[0082] Deuterated DMSO was used as solvent to characterize the compound. The vibration peak of some dimethyl hydrogen in hexadecanedioic acid appeared at 1.24 ppm by 1HNMR spectrum analysis. Fig.10 As shown, infrared spectrum analysis showed that Buff IIb-FA had two asymmetric double peaks at 2850-2960 cm-1, which were the characteristic absorption peaks of the methylene group in FA, indicating that FA was coupled to the Buff IIb peptide.

[0083] 2. Design of B2-FA

[0084] The synthesis was carried out by referring to the synthesis steps of Buff IIb-FA. FA (20 mg, 0.070 mmol) was dissolved in DMF, EDCI (19 mg, 0.099 mmol) and NHS (27 mg, 0.200 mmol) were added, and stirred overnight. Then 6 mg of B2 derivative peptide was added and stirring was continued for 24 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag for separation and finally dried using a freeze concentrator. The product was named B2-FA. The synthesis route is as follows: Fig.11 As shown. Fig.12 As shown, mass spectrometry analysis showed that the compound existed in a multi-charged form, and the molecular weight was determined to be 1216.13, which was consistent with the molecular weight of the compound in the case of two charges. The above results indicate that B2-FA has been successfully synthesized.

[0085] 3. Disulfide bond design, synthesis, and characterization

[0086] In order to achieve targeted release of drugs at the tumor site, disulfide bonds were selected as responsive materials for coupling. Disulfide bonds can remain stable in the normal environment in the body, and when they reach the tumor site, they can respond to the high concentration of reactive oxygen in the tumor microenvironment and break, thereby releasing the target drug. At the same time, PEGylation was performed to increase the stability of the drug in the body.

[0087] In order to prevent unnecessary side reactions of cystamine dihydrochloride during the reaction, one of the amino groups of cystamine dihydrochloride (CYS) is protected. The specific steps are as follows: first, weigh 1g of CYS and dissolve it in 40mL of methanol, add about 2mL of TEA, and stir for 30min under ice bath conditions to convert it into a non-hydrochloride form. Then 1g of (BOC)2O is dissolved in 10mL of methanol and added dropwise to the above CYS methanol solution using a constant pressure dropping funnel. The reaction solution is stirred overnight at room temperature. Concentrate under reduced pressure and dissolve in 1M / L NaOH solution, then extract three times with DCM, combine the organic phases, dry with anhydrous sodium sulfate, and finally purify by column chromatography (elution gradient: 30% Me / DCM—20% Me / DCM). The product is named BOC-SS-NH2. Then, 500mg of HOOC-PEG 600 -COOH was dissolved in methanol, and 160 mg of EDCI and 113 mg of HOBT were added respectively. The mixture was reacted at room temperature for 24 h. Then 420 mg of the prepared BOC-SS-NH2 was added to the reaction system and the reaction was continued for 48 h. After the reaction was completed, the mixture was transferred to a dialysis bag and separated by methanol solution for 24 h to obtain the product, which was named BOC-SS-PEG. 600 -SS-BOC. Then 2M / L HCl was added and stirred overnight to remove the protecting group to obtain the product, which was named NH2-SS-PEG 600 -SS-NH2, the synthetic route is as follows Fig.13 Then, the target peptide is coupled with the compound to prepare a redox responsive material.

[0088] Deuterated DMSO was used as solvent for the reaction of BOC-SS-PEG 600 -SS-BOC is characterized. Fig.14 As shown, the vibration peak of -CH3 hydrogen in the protecting group appeared at 1.36-1.42ppm; the vibration peak of hydrogen in the amide bond appeared at 5.74-5.81ppm; the vibration peak of -CH2- hydrogen in -SS-BOC appeared at 2.71-2.78ppm and 3.15-3.23ppm; the vibration peak of PEG appeared at 4.13-4.17ppm 600 The vibration peak of -CH2- hydrogen.

[0089] 4. Design and synthesis of coupled EGFR targeting peptides

[0090] GE11 peptide is considered as a candidate for targeting EGFR to treat tumors. By modifying Buff IIb peptide with GE11 peptide, it can target EGFR and enhance its targeting to tumors.

[0091] First, 1 eq of GE11 peptide was weighed and dissolved in DMF, and 1.5 eq of EDCI and NHS were added and stirred at room temperature for 24 h. Then, 1.5 eq of the prepared NH2-SS-PEG 600 -SS-NH2 was added to the reaction solution and stirred for 48 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag and separated for 24 hours using a mixed solution of DMF and distilled water as the dialysis medium. The dialysis medium was then replaced with distilled water and the separation was continued for another 24 hours. The product was then dried using a freeze concentrator and named GE11-SS-PEG. 600 -SS-NH2. Then, 1 eq of Buff IIb was weighed and dissolved in DMF, and 1.5 eq of EDCI and NHS were added for activation for 24 h. 600 -SS-NH2 was reacted for 24 hours. Then 1eq of (BOC)2O was added and stirred overnight. After the reaction was completed, the mixture was separated using a dialysis bag. Subsequently, 1eq of hexadecanedioic acid was weighed and dissolved in DMF, 3eq of EDCI and HOBT were added, and the mixture was activated for 24 hours. Then 1eq of the prepared GE11-SS-PEG was added. 600 -SS-NH2 was reacted for 24 hours and then separated using a dialysis bag to obtain the product BOC-GE11-SS-PEG 600 -SS-Buff IIb-FA. The synthetic route of self-assembly materials is as follows Fig.15 shown.

[0092] 5. Buforin IIB-GE11 Nanoparticles Encapsulating OSI

[0093] The prepared amphiphilic material BOC-GE11-SS-PEG 600 -SS-Buff IIb-FA was dissolved in DMSO, and then osimertinib (OSI) dissolved in DMSO was added dropwise to the solution. After the addition was completed, it was stirred at room temperature for 24 hours. The reaction solution was then transferred to a dialysis bag and the solvent DMSO was removed using water as the dialysis medium. Finally, it was filtered through a 0.45 μm filter membrane to obtain Buforin IIB-GE11 coupled nanoparticles (NPs) containing OSI.

[0094] Example 5 Effect of Buforin IIb combined with osimertinib (OSI) on tumor cell proliferation

[0095] Normally grown Lewis lung cancer cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were seeded in a 96-well plate, and 5×10 3 / well. Incubate overnight at 37℃, 5% CO2. Discard the culture medium, add different concentrations of drugs, and divide them into buforin IIb (20μg / ml) group, OSI (100ng / ml) group, buforin IIb (20μg / ml) + OSI (100ng / ml) mixture, NP nanoparticle group (containing 20μg / ml buforin IIb and 100ng / ml OSI), and culture at constant temperature for 72h. Add 20μL 5.0mg / mL MTT solution to each well in the dark and culture for 4h. Discard the supernatant, add 150μL DMSO to each well, and shake for 5min. Measure the absorbance at 490nm with an enzyme marker. Calculate the cell survival rate (%) = absorbance of the drug well / absorbance of the blank well.

[0096] Table 1 Toxicity of each drug to Lewis cells of lung cancer (n=4)

[0097]

[0098] **P<0.01, significantly different from the OSI group. ## P<0.01, there was a very significant difference between the NP group and the Buforin IIb+OSI group.

[0099] The results are shown in Table 1. The anti-tumor cell proliferation activity of the Buforin IIb+OSI group was significantly higher than that of Buforin IIb and OSI alone. This indicates that Buforin IIb targets ARF6 upstream to inhibit the transport of EGFR to the cell membrane, while osimertinib inhibits the activity of EGFR tyrosine kinase downstream, and the two act at different links to produce synergy. The anti-tumor cell activity of the NP nanoparticles described in detail in this example was experimentally evaluated, and it was found that the nanoparticles NP can show a better therapeutic effect than the free drug Buforin IIb peptide, free drug OSI and physical mixture Buforin IIb+OSI.

[0100] Example 6 Effect of Buforin IIb on Myocarditis Induced by Antitumor Drug OSI

[0101] Normally grown H9C2 cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were seeded in 96-well plates, and 2×10 4 / well. Incubate overnight at 37°C, 5% CO2. Discard the culture medium, add different concentrations of drugs, and divide into Buforin IIb (10μg / ml) group, OSI (10μg / ml) group, OSI (10μg / ml) + Buforin IIb (10μg / ml) group, OSI (10μg / ml) + Buforin IIb (100μg / ml) group, and culture at constant temperature for 72h. Aspirate the cell supernatant and use LDH kit to determine the enzyme activity. Then add 20μL MTT solution to each well and incubate for 4h. Measure the absorbance at 490nm with an enzyme marker. Calculate the cell survival rate (%) = absorbance of the drug well / absorbance of the blank well.

[0102] Table 2 Effect of Buforin IIb on OSI-induced injury of rat cardiomyocytes H9C2 (n=4)

[0103]

[0104] **P<0.01, significantly different from the OSI group.

[0105] The results are shown in Table 2. In the rat cardiomyocyte H9C2 model cultured in vitro, 10 μg / ml OSI can induce a decrease in cardiomyocyte viability and myocardial injury, which can lead to the release of lactate dehydrogenase LDH. Buforin IIb can significantly inhibit myocardial cell injury and the release of inflammatory mediator LDH at doses of 10 and 100 μg / ml.

[0106] Example 7 Evaluation of the therapeutic properties of Buforin IIb-derived peptides

[0107] Experimental methods:

[0108] 1. Buforin IIb structural modification design

[0109] Buforin IIb was structurally modified by adjusting the number of α-helices and replacing similar amino acids, aiming to improve the drugability of Buforin IIb. The adjustment of the number of α-helices includes the increase or decrease of the simulated helical sequence of the tail and the replacement of the helical sequence of the irregular coil part of the head; similar amino acid replacement mainly replaces the amino acids that will destroy the stability and safety of the α-helix in Buforin IIb, including the replacement of G (glycine) to Q (glutamine), F6 (phenylalanine) to W6 (tryptophan), and V8 (valine) to I8 (isoleucine). The derivative peptides were screened by predictive calculation of peptide property parameters and molecular docking technology. The property prediction includes charge number, helicity, solubility properties, instability index, aliphatic index and total hydrophilicity average coefficient; molecular docking includes the binding of the modified peptide to ARF6. The second round of derivative peptide design was carried out according to the peptide sequences hit by molecular docking screening.

[0110] 2. Detection of the inhibitory effect of derived peptides on HepG2 cell proliferation

[0111] Normally growing HepG2 liver cancer cells were seeded in a 96-well plate, and 2×10 4 After the cells adhered to the wall, the drug was added and incubated for 48 hours. Then, 20 μL of 5 mg / mL MTT working solution was added to each well and incubated for 4 hours. The supernatant was discarded, 150 μL DMSO was added to each well, and after reacting for 10 minutes, the absorbance at 490 nm was measured using an ELISA reader. Inhibition rate = 1-(absorbance of the drug well / absorbance of the blank well)*100%. Graphpad Prism 8 was used to calculate the IC of each peptide 50 .

[0112] 3. Hemolytic activity detection

[0113] Fresh mouse red blood cells were used for hemolysis experiments. Fresh mouse blood was drawn, centrifuged at 3000rpm for 5min, and the upper plasma and middle white blood cells were discarded. Use about ten times the volume of normal saline for washing, invert and mix, centrifuge at 3000r for 5min, discard the supernatant, and repeat the washing until the supernatant is clear. Take the red blood cells and prepare a 2% fresh red blood cell suspension with normal saline. Take 190μL of red blood cell suspension, add 10μL of buforin IIb and derivative peptides at different concentrations to make the concentrations 200, 100, 50, and 25μg / mL respectively. 2% Triton X-100 was used as a positive hemolysis control, red blood cells containing only normal saline were used as negative controls, and 10μL of deionized water was added as a blank group. Incubate at 37℃ for 1h. Centrifuge at 3000rpm for 5min, draw 150μL of supernatant and measure the absorbance at 540nm with an ELISA reader. Hemolytic activity = (OD sample - OD blank) / (OD positive - OD blank) * 100%. Graphpad Prism 8 was used to fit and calculate the dose of each peptide at which the hemolytic activity was 50%.

[0114] 4. Calculation of therapeutic index

[0115] Therapeutic index (TI) = toxic dose / effective dose. This study will use the measured hemolytic activity value and IC 50 The therapeutic index of the derived peptide was calculated as follows: The therapeutic index = the dose with 50% hemolytic activity / IC for tumor cells HepG2 50 .

[0116] Experimental results:

[0117] 1. Docking screening of Buforin IIb-derived peptides binding to ARF6

[0118] The buforin IIb derived peptide sequences are shown in Table 3.

[0119] Table 3

[0120]

[0121]

[0122] HPEPDOCK uses collective docking to consider the flexibility of peptides, and rigidly docks 1000 peptide structures generated by the peptide structure rapid generation program MODPEP to the binding site. As shown in Table 4, the results show that the original peptide docking score is -203.918. The lower the molecular docking score of the software, the higher the molecular affinity, indicating that the modified R1, R3, R4, R5, and R6 have higher affinity for interacting proteins than the original peptide, and the order is R5>R3>R6>R4>R1.

[0123] MVD is weaker than HEPDOCK in predicting peptide-protein docking, and its data serves as a supplement. The results obtained from the supplementary MVD docking show that the docking score of the original peptide is -263.184. The lower the molecular docking score of the software, the higher the affinity. The derivative peptides with the lowest MolDock Score are R3, R4, and R5. The Rerank Score is lower than that of the original peptide except for R2. The comprehensive results show that R3, R4, and R5 have a higher affinity with the interacting protein arf6.

[0124] Table 4 Docking screening of Buforin IIb derived peptides R1-R7 and arf6 protein

[0125]

[0126] 2. Results of efficacy and safety evaluation of derived peptides

[0127] According to the prediction calculation of polypeptide property parameters and molecular docking technology, three derivative peptides (R3, R4, R5) with better parameter prediction than the original peptide and higher affinity with the docking molecule than the original peptide were selected, corresponding to the derivative peptide B1: RLLRQWPIQRLLRRLLR (SEQ ID NO.2), derivative peptide B2: RAQLQFPVQRLLRRLLR (SEQ ID NO.3), derivative peptide B3: RAQLQFPVQRLLRRLLRRLLR (SEQ ID NO.4) mentioned above, and their structures are shown in Fig.16 shown.

[0128] The MTT method was used to detect the in vitro anti-HepG2 cell proliferation ability of the derived peptide and the original peptide. Within the dosage concentration range, the original peptide and the derived peptide inhibited the growth of liver cancer cells in a dose-dependent manner. Fig.17 The IC50 values ​​of the original peptide, B1 peptide, B2 peptide and B3 peptide against HepG2 were 12.13 μM, 82.58 μM, 41.59 μM and 9.04 μM, respectively, and the anti-HepG2 proliferation ability was ranked as B3>Buforin IIb>B2>B1.

[0129] The plasma safety of the original peptide and its derivatives was investigated by measuring the hemolytic activity. Fig.18 As shown in A, within the concentration range of 25-200 μg / mL, peptides B1 and B2 showed weak hemolytic activity, peptide B3 was hemolytic, but still significantly lower than the original peptide, and the original peptide showed high hemolytic activity at four doses. The doses of 50% hemolytic activity of each peptide were calculated and fitted, and the original peptide, B1 peptide, B2 peptide, and B3 peptide were 33.36 μg / mL, 11229 μg / mL, 20366 μg / mL, and 64.35 μg / mL, respectively. The hemolytic safety ranking is B2>B1>B3>Buforin IIb.

[0130] The hemolytic activity of B2-FA was also tested in the experiment. B2-FA had no hemolytic toxicity at concentrations of 100, 200 and 400 μg / mL, indicating that fatty acid modification can further improve the safety of B2 peptide.

[0131] The therapeutic index is used to measure the safety of original peptides and derived peptides, such as Fig.18 As shown in B, the original peptide, B1 peptide, B2 peptide, and B3 peptide are 1.07, 59.46, 226.33, and 2.63, respectively, and the therapeutic index is ranked as B2>B1>B3>Buforin IIb. The results show that the derivative peptides B1 and B2 have lower hemolytic properties and higher therapeutic indexes than buforin IIb.

[0132] Example 8 Effect of alanine substitution on the binding of BFP2 and its derivative peptides to the target protein Arf6

[0133] In the aforementioned molecular docking study, it was found that the alanine site in the buforin IIb sequence plays an important role in stabilizing the peptide in the target pocket through hydrophobic interaction with the Arf6 group. In view of this, we considered further changing the hydrophilicity of this site, specifically replacing alanine (A) with more hydrophobic aminoisobutyric acid (Ail) or proline (P), and replacing alanine (A) with less hydrophobic glycine (G). The affinity of the above-mentioned peptide to Arf6 was determined by ultrafiltration-mass spectrometry (see the experimental part of Example 1 for details of the method).

[0134] The experiment was to mix and incubate 10μg / mL buforin IIb standard solution, 10μg / mL derivative peptide B3 standard solution and derivative peptide C1, derivative peptide C2, derivative peptide C3, derivative peptide C4 with 10μg recombinant ARF6 protein, and then ultrafiltration centrifugation. The peptides that do not bind to the target protein are present in the lower layer of the ultrafiltration tube, while the peptides that bind to the target protein will remain in the upper layer of the ultrafiltration tube, and their affinity is evaluated by mass spectrometry analysis.

[0135] The results are shown in Table 5 below. Alanine substitution has a significant effect on target affinity. Affinity order: derived peptide C2>Buforin IIb>derived peptide C1; derived peptide C3>derived peptide C4>derived peptide B3. For the original buforin IIb peptide, replacing alanine (A) with aminoisobutyric acid (Ail) is better than replacing it with glycine (G). For the derived peptide B3, replacing alanine (A) with proline (P) is better than aminoisobutyric acid (Ail). In general, replacing alanine (A) with aminoisobutyric acid (Ail) or proline (P) can increase the affinity of this type of peptide with the target arf6.

[0136] Table 5 Effect of alanine substitution on the binding of buforin IIb and its derivative peptides to the target protein Arf6

[0137]

[0138] **P<0.01, compared with buforin IIb group, ##P<0.01, compared with derivative peptide B3 group, there are extremely significant differences.

[0139] For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description, and it is not necessary and impossible to list all implementation methods here. Therefore, modifications or improvements made without departing from the spirit of this application are within the scope of protection required by this application.

Claims

1. An ARF6 inhibitor, It is characterized in that The invention comprises ① a peptide capable of binding to ARF6; ② a nucleic acid molecule encoding the peptide; or ③ a biological material containing the aforementioned peptide or nucleic acid molecule; The peptide is buforin IIb or a peptide derived therefrom, and the buforin IIb or a peptide derived therefrom can bind to ARF6.

2. An ARF6 inhibitor according to claim 1, It is characterized in that The amino acid sequence of buforin IIb is shown in SEQ ID NO.1, which is RAGLQFPVGRLLRRLLRRLLR. The derivative peptides include B1 to B3 and derivative peptides C1 to C4, and their amino acid sequences are shown in SEQ ID NO.2 to SEQ ID NO.8; Derivative peptide B1: RLLRQWPIQRLLRRLLR (SEQ ID NO. 2), Derivative peptide B2: RAQLQFPVQRLLRRLLR (SEQ ID NO. 3), Derivative peptide B3: RAQLQFPVQRLLRRLLRRLLR (SEQ ID NO. 4), Derivative peptide C1: RGGLQFPVGRLLRRLLRRLLR (SEQ ID NO. 5), Derivative peptide C2: R(Ail)GLQFPVGRLLRRLLRRLLR (SEQ ID NO.6), Derivative peptide C3: RPQLQFPVQRLLRRLLRRLLR (SEQ ID NO. 7), Derivative peptide C4: R(Ail)QLQFPVQRLLRRLLRRLLR (SEQ ID NO. 8).

3. An ARF6 inhibitor according to claim 1, It is characterized in that The biological material includes antibodies, cells, bacteria, microcapsules or microparticles containing the nucleic acid molecule or expressing the peptide.

4. An ARF6 inhibitor according to claim 1, It is characterized in that The biological material includes a gene expression element or expression cassette containing the nucleic acid molecule.

5. An ARF6 inhibitor according to claim 1, It is characterized in that The biomaterial includes a conjugate formed by connecting the buforin IIb or its derivative peptide with a carrier, and a modified product obtained by labeling or cyclization modification of the buforin IIb or its derivative peptide.

6. An ARF6 inhibitor according to claim 5, It is characterized in that The conjugate is a conjugated peptide formed by the BuforinIIb or its derivative peptide and GE11.

7. Use of an ARF6 inhibitor according to any one of claims 1 to 6 in the preparation of tumor vaccines, anticancer drugs or inflammatory disease drugs.

8. The use according to claim 7, It is characterized in that The tumor vaccine is a whole tumor lysate antigen vaccine, and the anticancer drug is a reagent or drug that promotes the release of antitumor immune adjuvants.

9. Use of an ARF6 inhibitor according to any one of claims 1 to 6 in reducing anti-tumor drug-induced cardiotoxicity and myocarditis.

10. A drug, It is characterized in that The invention comprises the ARF6 inhibitor according to any one of claims 1 to 6.

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