An anti-angiogenic peptide and its screening method and application

Abstract

The present invention belongs to the field of biomedicine and relates to an anti-angiogenic peptide, screening method, and application thereof. The anti-angiogenic peptide is polypeptide 1 or polypeptide 2; the amino acid sequence of polypeptide 1 is shown in SEQ ID NO. 1; polypeptide 2 is a polypeptide derived from polypeptide 1 by substitution, deletion, and / or addition of one or two amino acid residues and having the same glycosyltransferase function. The anti-angiogenic peptide provided by the present invention has good targeting and anti-angiogenic activity, and therefore has great practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to an anti-angiogenic peptide and a screening method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] The theoretical basis for anti-angiogenic tumor therapy is the "tumor starvation" theory, which states that in the early stages of tumor growth, tumors primarily rely on diffusion to obtain nutrients from their surroundings. During this period, tumor growth is slow and metastasis is less likely to occur. However, when a tumor grows to a certain size and the nutrients it receives are insufficient to meet its growth needs, the tumor and its surrounding stromal cells express various angiogenic factors, which promote the growth of endothelial cells toward the tumor tissue, ultimately generating new tumor microvessels and promoting tumor cell growth and invasion.

[0004] After decades of research, it has been discovered that vascular endothelial growth factor (VEGF) is a major factor promoting angiogenesis. In various tumor cells, VEGF is overexpressed upon significant stimulation, thereby promoting angiogenesis and tumor growth. Numerous studies have shown that VEGF binds to its receptor, VEGFR-2, and can induce tyrosine phosphorylation of VEGFR-2, thereby triggering a series of downstream signaling events, thereby promoting angiogenesis. Based on this, blocking the signaling pathway of vascular endothelial growth factor and its receptor (VEGF / VEGFR), thereby blocking angiogenesis, is an important direction for tumor treatment. Therefore, it is necessary to use this as a target in the search and development of more anti-angiogenic active substances. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the present invention aims to provide an anti-angiogenic peptide and a screening method and application thereof. The anti-angiogenic peptide provided by the present invention has good targeting and anti-angiogenic activity, and therefore has good practical application value.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In a first aspect, an anti-angiogenic peptide is polypeptide 1 or polypeptide 2;

[0008] The amino acid sequence of the polypeptide 1 is shown in SEQ ID NO.1;

[0009] The polypeptide 2 is a polypeptide derived from polypeptide 1 which has one or two amino acid residues substituted and / or deleted and / or added and has the same glycosyltransferase function.

[0010] In a second aspect, a method for screening the above-mentioned anti-angiogenic peptides comprises using a phage random twelve-peptide library to screen specific targeting polypeptides with VEGF as the target protein, performing affinity screening using phage in at least one adsorption-elution-amplification process to obtain monoclonal phage that can specifically bind to the specific targeting polypeptide, and obtaining the amino acid sequence of the specific targeting polypeptide through the monoclonal phage; and then further screening peptides with affinity activity using ELISA detection and surface plasmon resonance (SPR).

[0011] In a third aspect, a method for preparing the above-mentioned anti-angiogenic peptide is provided, comprising:

[0012] The anti-angiogenic peptide is prepared by a microbial fermentation method; or,

[0013] The anti-angiogenic peptide is prepared by genetic engineering; or

[0014] The anti-angiogenic peptide is synthesized by chemical methods.

[0015] In some embodiments, the chemical method is solid phase synthesis.

[0016] In a fourth aspect, a use of the above-mentioned anti-angiogenic peptide in the preparation of a drug, wherein the drug is an anti-angiogenic drug or an anti-tumor drug.

[0017] In some embodiments, the medicament is a composition.

[0018] Specifically, the composition comprises one or more anti-angiogenic peptides.

[0019] Specifically, the composition includes pharmaceutical excipients. More specifically, the pharmaceutical excipients can be pharmaceutical carriers, such as natural polymers (lipids, carbohydrates, proteins, etc.), synthetic polymers (polycyanoacrylates, polylactic acid, etc.), small molecule organic materials (such as stearic acid, etc.), etc.; or excipients, such as binders (syrup, gum arabic, sodium carboxymethyl cellulose, etc.), fillers (lactose, starch, etc.), disintegrants (sodium carboxymethyl starch, microcrystalline cellulose, cross-linked polyvinylpyrrolidone, etc.), solubilizers (polysorbate, poloxamer, etc.), preservatives (benzyl alcohol, benzoic acid, salicylic acid, etc.), pH adjusters, stabilizers, etc.

[0020] In some embodiments, the dosage form of the drug is tablets, granules, lyophilized powder injections, injections, etc.

[0021] In some embodiments, the drug is administered orally, subcutaneously, intravenously, or intramuscularly.

[0022] In some embodiments, the diseases treated by anti-angiogenic drugs include but are not limited to diabetic retinopathy, coronary heart disease, degenerative arthritis, etc.

[0023] In some embodiments, the tumor includes but is not limited to skin cancer, head and neck cancer, lung cancer, esophageal cancer, cervical cancer, uterine cancer, pancreatic cancer, breast cancer, kidney cancer, ureteral cancer, bladder cancer, squamous cell carcinoma, basal cell carcinoma, melanoma, tongue cancer, pharyngeal squamous cell carcinoma or malignant lymphoma, laryngeal squamous cell carcinoma, lung squamous cell or small cell carcinoma, esophageal squamous cell carcinoma, cervical cancer, digestive tract tumors, reproductive system tumors, lymphomas, bone tumors, head and neck tumors, etc.

[0024] The beneficial effects of the present invention are:

[0025] The anti-angiogenic peptide provided by the present invention has a good affinity for VEGF. Lumen formation experiments have demonstrated that the peptide has excellent anti-angiogenic activity, facilitating tumor treatment by blocking angiogenesis. Furthermore, cell proliferation experiments have shown that the peptide can directly inhibit the proliferation of mouse breast cancer cells, demonstrating excellent anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is a sensorgram analyzing the interaction between different concentrations of the V19 polypeptide and VEGF in Example 1 of the present invention;

[0028] Figure 2 This is a graph showing the affinity fitting results of the interaction between the V19 polypeptide and VEGF in Example 1 of the present invention;

[0029] Figure 3 This is a graph showing the experimental results of the V19 polypeptide screened in Example 2 of the present invention inhibiting endothelial cell lumen formation in vitro.

[0030] Figure 4 This is a graph showing the experimental results of the V19 polypeptide in Example 3 of the present invention inhibiting the proliferation of mouse breast cancer cells in vitro. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1:

[0033] 1. Four rounds of panning, DNA extraction and sequencing

[0034] (1) VEGF was prepared into a 10 μg / mL solution using 0.1 M sodium bicarbonate solution (pH 8.6). 100 μL of the solution was added to a 96-well ELISA plate and shaken at 75 rpm at 4°C overnight.

[0035] (2) Discard the coating solution, fill with BSA buffer blocking solution, and place at 4°C for 90 minutes.

[0036] (3) Discard the buffer solution and wash the plate 6 times with TBST 0.1% Tween 20, 1 minute each time, by tapping vigorously.

[0037] (4) 1×10 11 90 μL TBS and 10 μL phage peptide library stock solution were added to the coated wells, mixed, and shaken at room temperature for 60 minutes.

[0038] (5) Discard the phage solution and tap vigorously. To wash away unbound phage, wash six times with TBST 0.1% Tween 20 for 1 minute each time and tap dry.

[0039] (6) To elute the phages specifically bound to the monoclonal antibody, add 100 μL of glycine buffer and shake slowly and evenly at room temperature for 60 minutes.

[0040] (7) Transfer the eluate to a 1 mL sterile microcentrifuge tube and add 15 μL of Tris-HCl buffer to obtain the first round of phage eluate. Perform phage titer determination and amplification on the first round of eluate.

[0041] (8) Repeat the above process to perform rounds 2 to 4 of phage peptide library screening. In the last two rounds of screening, the Tween concentration in the washing step needs to be increased to 0.5% (v / v), and the eluent is changed to a free target molecule solution.

[0042] (9) The overnight culture of ER 2738 was diluted 1:100 and inoculated into LB medium. 10 mL was aliquoted into a sterile culture flask.

[0043] (10) Pick blue plaques from the fourth round of phage plate (the total number of plaques does not exceed 100) and transfer them to the above culture tube. Incubate on a shaker at 37°C for 4.5 h.

[0044] (11) Transfer the culture into a sterile centrifuge tube, centrifuge at 10,000 rpm for 30 seconds, remove the supernatant, and centrifuge again for 30 seconds.

[0045] (12) Transfer the supernatant above 80% into a new EP tube, which is the amplified phage storage solution.

[0046] (13) After amplifying the selected phage clone, centrifuge and collect 500 μL of the phage supernatant. Add 200 μL of PEG / NaCl, mix thoroughly by inversion, and let stand at room temperature for 10 minutes. Centrifuge at 10,000 rpm at 4°C for 10 minutes and discard the supernatant.

[0047] (14) Resuspend in 100 μL of iodide buffer, centrifuge briefly, add 250 μL of anhydrous ethanol, and let stand at room temperature for 10 minutes. Centrifuge at 10,000 rpm for 10 minutes at 4°C and discard the supernatant.

[0048] (15) Add 70% ethanol to wash the precipitate, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant.

[0049] (16) Add 30 μL of TE buffer and sequence the monoclonal phage.

[0050] (17) The results of monoclonal phage DNA sequencing were analyzed, and an insertion sequence was found between the GGTACC Kpn I restriction site and the CGGCCGEag I restriction site. The amino acid sequence of the corresponding inserted polypeptide was then deduced using the genetic code table.

[0051] 2. Molecular Docking

[0052] The target protein and the screened peptide were docked using the ZDOCK module in the Discovery Studio Visualizer client software, and the ZDock Score in the result was used as the scoring function. The larger the absolute value of the ZDock Score, the stronger the binding ability between the receptor and the ligand. The steps are as follows:

[0053] (1) The protein structure of VEGF was retrieved and downloaded from the Protein Data Bank (PDB) database as the receptor protein in the molecular docking experiment. The structure of the selected peptides was predicted from the PEP-FOLD website, and the prediction results were saved and used as the ligand protein in the molecular docking experiment.

[0054] (2) Open the Discovery Studio Visualizer client software, find the target protein and peptide files in the File menu, and open them. Select Prepare Protein in the expanded menu bar, and click the Clean Protein shortcut tool in the Manual Preparation panel group to pre-treat the target protein and screening peptide by removing ligand molecules and water molecules from the protein crystal structure, removing protein multiple conformations, adding incomplete amino acid residues, and hydrogenating the protein.

[0055] (3) Use ZDOCK to dock the screened peptide molecules with the processed target protein. Open the receptor and ligand proteins in the same 3D window, click Macromolecules in the Tool toolbar, expand the Dock and Analyze Protein Complexes tool, click Dock Proteins (ZDOCK), and the parameter setting dialog box will open. Click the Input Receptor Protein parameter and select 2ptn:2ptn; click the Input Ligand Protein parameter and select 2ptn:2sta; click the Angular Step Size parameter and select 15. Expand the Clustering parameter group, click the RMSD Cutoff parameter and set the value to 6; click the Interface Cutoff parameter and set the value to 9; click the Maximum Number of Clusters parameter and set the value to 60. Click Run and wait for the results to complete.

[0056] (4) When the calculation is finished, DS will display the Job Completed dialog box. In the Job Explorer, double-click the completed calculation task and DS will open a new Report.htm file. In the Output Files section, click the ZDockResults.dsv link. In the Data Table, click Protein Pose. Click Macromolecule in the Tools toolbar, select the Dock and Analyze Protein Complexes tool, click Browse in Browse Poses, and select Top Poses in Largest Clusters in the drop-down menu. This will display the top 100 highest-scoring conformations in the 10 largest clusters. Select Chart|3D Point Plot. This will open a Choose Plot Axes dialog box. Select ZDock Score for the X-axis, Cluster for the Y-axis, and Density for the Z-axis. Click OK. In the results, select the Pose with a higher ZDock Score. The corresponding conformation in the molecule window will be selected, and you can now see the ZDock Score corresponding to the selected Pose. This study selected ZDock Score as the scoring function. The larger the absolute value of ZDock Score, the stronger the binding ability between the receptor and the ligand.

[0057] The absolute values ​​of ZDock Score were sorted from large to small, and the top 30 screening peptides were selected for the next experiment.

[0058] 3. ELISA experiment

[0059] Perform ELISA test on the top 30 phage clones from the molecular docking results to identify the binding ability of the phage-displayed peptide to the target protein. The steps are as follows:

[0060] (1) When amplifying plaques for DNA sequencing, store the remaining plaque-containing supernatant at 4°C.

[0061] (2) Dilute the overnight culture of ER2738 into 20 mL of LB medium at a ratio of 1:100. Add 5 μL of phage supernatant to each tube of ER2738 culture medium and incubate at 37°C with aeration for 4.5 h.

[0062] (3) Transfer the culture to a centrifuge tube and centrifuge at 10,000 rpm for 10 minutes. Transfer the supernatant to a fresh centrifuge tube and centrifuge again.

[0063] (4) Take 80% of the supernatant and place it in a fresh centrifuge tube. Add 1 / 6 volume of PEG / NaCl and let it precipitate overnight at 4°C.

[0064] (5) Centrifuge at 10,000 rpm for 15 min at 4°C, discard the supernatant, centrifuge briefly again, and aspirate the remaining supernatant.

[0065] (6) The pellet was resuspended in 1 mL of TBS, the suspension was transferred to a microcentrifuge tube, and centrifuged at 4°C for 5 min to remove residual cells in the pellet.

[0066] (7) Transfer the supernatant to a fresh microcentrifuge tube and add 1 / 6 volume of PEG / NaCl to reprecipitate. Incubate on ice for 15-60 min. Centrifuge at 4°C for 10 min, discard the supernatant, centrifuge briefly again, and aspirate the remaining supernatant.

[0067] (8) Resuspend the pellet in 50 μL TBS and measure the phage titer. Store at 4°C.

[0068] (9) Coat each well of the ELISA plate with 100 μL of 1 μg / mL target molecule, with three wells coated for each clone to be identified. Coating was carried out overnight at 4°C in a sealed humidified chamber.

[0069] (10) Discard excess target molecule solution and invert the plate on a paper towel to remove any residual liquid. Fill each well with blocking solution and block at 4°C for 1 h. In addition, add blocking solution to each uncoated well of the clone to be identified to test the binding affinity of the selected sequence to the BSA-coated plastic plate.

[0070] (11) Discard the blocking solution and wash the plate six times with TBST 0.5% Tween 20, each time inverting the plate on a clean paper towel to remove the solution.

[0071] (12) 1×10 9 Add the phages to the wells coated with the target molecule and shake at room temperature for 1 hour.

[0072] (13) Wash the plate six times with TBST 0.5% Tween 20. Dilute HRP-labeled anti-M13 antibody at a ratio of 1:5000 in blocking buffer. Add 100 μL of diluted antibody to each well and shake at room temperature for 1 hour.

[0073] (14) Wash the plate 6 times with TBST 0.5% Tween 20. Add 200 μL of HRP substrate solution to each well and incubate at room temperature for 1 hour.

[0074] (15) Record the absorbance at 405-415 nm using a plate reader.

[0075] The results showed that the phages with OD values ​​more than 2 times higher than those of the negative control group, lower affinity to the blocking solution and higher affinity to the target protein were regarded as positive phages that specifically bound to the target protein.

[0076] IV. SPR Experiment

[0077] The peptides of the top three phage clones according to the ELISA test results were synthesized, and the peptide with the strongest affinity to VEGF was determined by surface plasmon resonance (SPR).

[0078] (1) System Check: Replace the buffer with 1.05× PBS and the chip with a new one. In the Tools module of the Biacore X100plus software, select the System Check and Pump Calibration option. Follow the prompts to perform a system check using BIAtest solution and ultrapure water. Proceed to the next step if all tests pass.

[0079] (2) Find the optimal pH for coupling protein to the chip: Use the FindImmobilization pH module in the Kinetics / Affinity module to determine the optimal pH for coupling the target protein. According to the system instructions, dilute the protein to 50 μg / mL with 10 mM sodium acetate buffer solutions at pH 4.0, 4.5, 5.0, and 5.5, respectively. Use 50 mM NaOH solution as a cleaning solution and inject the sample for 180 seconds. After the end, the result graph pops up. According to the response value and curve shape of the sample under each pH condition, select the appropriate pH as the pH condition for the final coupling protein.

[0080] (3) Coupling protein: Select the Immobilize option in the Kinetics / Affinity module, and immobilize the target protein solution at the optimal pH selected in the previous step on the CM5 chip by the amino coupling method. Flow Cell 2 is used as the sample channel, and the blank Flow Cell 1 is used as the reference channel. The maximum protein coupling amount Rmax is set to 100RU according to the formula, and the ligand coupling level RL is calculated according to the following formula, where SM is the stoichiometric ratio, which is usually set to 1. The actual coupling amount in the experiment is 1.5 times RL.

[0081]

[0082] (4) Affinity (KD value) determination: Since the peptide drug used in SPR is dissolved in less than 0.5% DMSO, and very low concentrations of DMSO have little effect on SPR results, a calibration curve is not required for this experiment. 100 μM of the peptide to be tested is prepared, and the affinity of the peptide to the target protein is measured in a series of concentration gradients (half-fold dilution method). The RU at the binding saturation period is obtained, and the binding affinity of the target protein and the peptide is obtained using a single-site interaction model.

[0083] Figure 1 The sensorgrams for analyzing the interaction between V19 peptide and VEGF at different concentrations are shown in FIG. Figure 2 This is the affinity fitting result for the interaction between the V19 peptide and VEGF. The vertical line represents the fitted KD value, and its presence within the sample concentration range indicates that the fitted data is reliable. Table 1 shows the affinity fitting data for the interaction between the V19 peptide and VEGF. The smaller the affinity fitting KD value, the stronger the affinity between the peptide and the protein. As can be seen from the results, the KD value of V19 is 9.803E-5, indicating that the V19 peptide has a good affinity for VEGF.

[0084] Table 1 Fitting results of the interaction between V19 polypeptide and VEGF

[0085] KD(M) Rmax(RU) <![CDATA[Chi 2 (RU2)]]> 9.803E-5 26.76 0.710

[0086] The amino acid sequence of the selected polypeptide V19 is RTSPSPRGKHGE, as shown in SEQ ID NO. 1. It was commissioned to Hangzhou Baige Pharmaceutical Technology Co., Ltd. for synthesis.

[0087] Example 2 Effect of V19 peptide on endothelial cell lumen formation

[0088] (1) Use a pre-cooled pipette tip to add the melted Matrigel into a pre-cooled 48-well plate (80 μL / well). Be careful to avoid creating bubbles.

[0089] (2) Collect EAhy 926 cells in the logarithmic growth phase and adjust the cell suspension concentration to 11×10 4 / well, and add them into the well plate covered with matrix gel.

[0090] (3) The wells containing no drug but only DMEM medium were used as blank control groups, the wells containing serum-free DEME medium with 10 ng / mL VEGF were used as negative control groups, the wells containing serum-free DEME medium with 10 ng / mL VEGF and ES2 (an anti-angiogenic peptide) were used as positive control groups, and the wells containing serum-free DEME medium with 10 ng / mL VEGF and V19 peptide were used as experimental groups. The drug concentrations were set at 500 μg / mL and 1 mg / mL, and three replicate wells were set for each group.

[0091] (4) After the addition of drugs, the well plate was cultured for another 6 h, and the formation of the cell vascular network was observed and photographed using an inverted fluorescence microscope. The photos were processed using Image J software.

[0092] The results are as follows Figure 3 As shown, compared with the negative control group, both V19 and ES2 can significantly inhibit the formation of endothelial cell tubes. The endothelial cells in the blank control group and the negative control group showed a clear lattice-like structure, while the endothelial cells in the V19 experimental group and the ES2 positive control group mostly showed a monolayer adherent state with only a small amount of tubular structures formed. This result shows that the V19 polypeptide has a strong anti-angiogenic ability in vitro.

[0093] Example 3 Effect of V19 peptide on proliferation of mouse breast cancer cells

[0094] 4T1 cells in logarithmic phase were collected, cell suspension was adjusted to an appropriate concentration, and 5×10 3Cells were seeded into 96-well plates and placed in a carbon dioxide incubator. The 96-well plates were cultured overnight at 37°C until the cells adhered. V19 and ES2 (an anti-angiogenic peptide that can inhibit endothelial cell proliferation but has no effect on tumor cells, used as a control group) were then added at drug concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, and 1 mg / mL, respectively. Eight replicates were set for each drug. Wells containing only DMEM medium were set as blank controls, and wells with cells but no drug-containing medium were used as negative controls. The 96-well plate was placed in a carbon dioxide incubator and incubated for 48 h. The culture medium was discarded under light-proof conditions, and CCK-8 solution was added to the 96-well plate, 10 μL per well. The 96-well plate was placed in an incubator and taken out after the culture medium color turned orange. The OD value of each well under the condition of 450 nm wavelength was then detected by a microplate reader, and the cell proliferation rate was calculated as follows: cell proliferation rate = [(experimental group - blank control group) / (negative control group - blank control group)] × 100%.

[0095] The results are as follows Figure 4 As shown, it can be seen that compared with the control group ES2 polypeptide, the cell proliferation rate of the V19 polypeptide group showed a significant downward trend with the increase of concentration, which indicates that the V19 polypeptide has a significant inhibitory effect on the proliferation of 4T1 cells and thus has good in vitro anti-tumor activity.

[0096] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An anti-angiogenic peptide, characterized in that It is polypeptide 1; The amino acid sequence of the polypeptide 1 is shown in SEQ ID NO.

1.

2. A method for preparing the anti-angiogenic peptide according to claim 1, characterized in that: The method is: The anti-angiogenic peptide is prepared by genetic engineering; or The anti-angiogenic peptide is synthesized by chemical methods.

3. The method according to claim 2, wherein: The chemical method is a solid phase synthesis method.

4. Use of the anti-angiogenic peptide according to claim 1 in the preparation of a drug, wherein the drug is an anti-breast cancer drug.

5. The use according to claim 4, characterized in that: The medicine is a composition.

6. The use according to claim 5, characterized in that: The composition includes pharmaceutical excipients.

7. The use according to claim 4, characterized in that: The dosage form of the medicine is tablet, granule or injection.

8. The use according to claim 7, characterized in that: The dosage form of the medicine is freeze-dried powder injection.

9. The use according to claim 4, characterized in that: The drug is administered orally, by subcutaneous injection, intravenous injection or intramuscular injection.

Images