A polypeptide targeting BRCA2 protein, its application and a drug for treating prostate cancer

Abstract

The present invention belongs to the technical field of protein polypeptides, and particularly relates to a polypeptide targeting the BRCA2 protein, its application, and a drug for treating prostate cancer. The amino acid sequence of the polypeptide is shown as SEQ ID NO.1, which has a strong binding ability with the BRCA2 protein. When delivered to prostate cancer, it can effectively inhibit cell proliferation and tumor growth, and at the same time has the ability to target and degrade BRCA2. The combined application of the polypeptide and a PARP inhibitor can enhance the sensitivity of prostate cancer cells to the PARP inhibitor. It can be seen that the polypeptide can be applied to the preparation of a drug for treating metastatic castration-resistant prostate cancer or a reagent for degrading the BRCA2 protein. The BRCA2-targeted PROTAC drug can provide a new treatment strategy for prostate cancer treatment. In combination with a PARP-targeted inhibitor, it can provide a synthetic lethal treatment effect of PARP inhibitor for patients with metastatic castration-resistant prostate cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein polypeptides, and particularly relates to a polypeptide targeting BRCA2 protein, its application, and a drug for treating prostate cancer. Background Art

[0002] Metastatic castration-resistant prostate cancer is an aggressive malignant tumor with a poor prognosis. Clinical trials have demonstrated that PARP inhibitors can significantly improve the overall survival, objective response rate, and progression-free survival of patients with homologous recombination repair (HR) gene defects. Compared with mutations in other HR genes, PARP inhibitors exhibit significant efficacy in patients carrying BRCA2 mutations. Unfortunately, only 19.3% of patients carry HR gene mutations, and only 13% of them are BRCA2 gene mutations.

[0003] A targeting peptide is a short peptide or protein with specific binding ability that can selectively bind to a specific target. However, general targeting peptides can only target and bind to the target, and have limited degradation effect on the target. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a polypeptide targeting BRCA2 protein, its application, and a drug for treating prostate cancer. The polypeptide has a high binding ability to BRCA2 protein, can efficiently degrade BRCA2, and effectively inhibit the proliferation of prostate cancer cells, and can be used to prepare a drug for treating prostate cancer.

[0005] In the first aspect of the present invention, a polypeptide targeting BRCA2 is provided, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1.

[0006] BRCA2 plays a crucial role in maintaining genomic integrity. As Figure 6 shown, it participates in the repair of DNA double-strand breaks through the HR pathway. BRCA2 interacts with RAD51, promotes the loading of single-stranded DNA by RAD51 at the DNA damage site, thereby completing DNA strand exchange and high-fidelity DNA repair. BRCA2 gene mutations can lead to abnormal DNA double-strand repair, enhance the sensitivity of prostate cancer and other tumors to PARP inhibitors, and achieve the pharmacological mechanism of synthetic lethality.

[0007] The polypeptide targeting BRCA2 provided by the present invention has an amino acid sequence as shown in SEQ ID NO.1. Through isothermal titration calorimetry experiments, the present invention respectively detected the binding abilities of the polypeptide to BRCA2 protein and DDB1 protein. The binding constant of the polypeptide to BRCA2 protein is 116 nM, and the binding constant to DDB1 protein is 318 nM ± 110 nM. On the one hand, the polypeptide binds to BRCA2 protein to achieve the degradation of BRCA2. On the other hand, by inducing the ubiquitination of BRCA2 by DDB1 protein, the enhancement of BRCA2 degradation in prostate cancer is achieved.

[0008] In the second aspect of the present invention, a cancer treatment drug is provided, comprising the polypeptide and a delivery system.

[0009] As a preference of the drug of the present invention, the delivery system is selected from one of gold nanoparticles, selenium nanoparticles and liposomes.

[0010] Furthermore, the drug further comprises a PARP inhibitor.

[0011] Even further, the PARP inhibitor comprises Olaparib.

[0012] In the third aspect of the present invention, an application of the polypeptide in preparing a reagent for degrading BRCA2 protein is provided.

[0013] In the fourth aspect of the present invention, an application of the polypeptide and the drug in preparing a drug for treating solid tumors is provided.

[0014] Furthermore, the solid tumor includes prostate cancer.

[0015] Even further, the prostate cancer is metastatic castration-resistant prostate cancer.

[0016] The present invention has the following beneficial effects:

[0017] The present invention delivers the polypeptide targeting BRCA2 into prostate cancer cells, which can effectively inhibit cell proliferation. It can be seen that the polypeptide can be applied in preparing a drug for treating prostate cancer or a reagent for degrading BRCA2 protein.

[0018] The present invention also provides a cancer treatment drug, comprising the polypeptide and a delivery system. This drug can provide a new treatment strategy for the treatment of drug-resistant and advanced prostate cancer tumors. In particular, it can solve the problem that the treatment effect of PARP inhibitors is not significant in patients without HR gene mutations, filling the gap of drugs targeting the targeted degradation of BRCA2 polypeptide globally. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Design results of the BRCA2-targeting polypeptide provided by the present invention, wherein:

[0020] A is the structure of the complex of the BRCA2-targeting polypeptide and BRCA2.

[0021] B is the computer-predicted sorting result of the amino acids bound by the BPD drug polypeptide. Protein-assisted design result.

[0022] C is the design result of the predicted sequence mutation of the BPD drug polypeptide.

[0023] D is the result of the affinity detection of the BPD drug polypeptide and the BRCA2 protein.

[0024] E is the result of the affinity detection of the BPD drug polypeptide and the DDB1 protein.

[0025] F is the result of the protein stability detection of the BPD drug relative to the ordinary polypeptide.

[0026] Figure 2 Is the result of the detection of the degradation of the BRCA2 protein by the BPD drug in vitro, wherein:

[0027] A is the result of detecting the degradation of BRCA2 in different cancer cells by the drug using Western blot.

[0028] B is the result of detecting the degradation of BRCA2 in cancer cells by the drug at different treatment times using Western blot.

[0029] C is the quantitative graph of B.

[0030] D is the result of detecting the degradation of BRCA2 in cancer cells by the combination of the BPD drug polypeptide and MG132 using Western blot.

[0031] E is the result of detecting the degradation effect of the BPD drug on the BRCA2 protein in C4-2 cells using the cell immunofluorescence imaging method.

[0032] F is the average fluorescence intensity of the BRCA2 protein in C4-2 cells.

[0033] G is the γ-H2AX fluorescence focus in each C4-2 cell (the level of γ-H2AX fluorescence focus formation in the cell will indicate the DNA damage situation of the cell).

[0034] Figure 3 Is the result of the study on the mechanism of action of the BPD drug, wherein:

[0035] A is the result of detecting the binding of overexpressed exogenous DDB1 and BRCA2 in 293T cells using Western blot in the presence of the BPD drug.

[0036] Panel B shows the results of detecting the binding of exogenous overexpressed BRCA2 to DDB1 in 293T cells by Western blotting in the presence of the BPD drug.

[0037] Panel C shows the results of detecting the binding of endogenous DDB1 to BRCA2 in C4-2 cells by Western blotting in the presence of the BPD drug.

[0038] Panel D shows the results of detecting the binding of endogenous BRCA2 to DDB1 in C4-2 cells by Western blotting in the presence of the BPD drug.

[0039] Panel E shows the results of detecting the ubiquitination and degradation of exogenous overexpressed BRCA2 by DDB1 by Western blotting in the presence of the BPD drug.

[0040] Panel F shows the results of detecting the degradation of BRCA2 in C4-2 cells by Western blotting in the presence of the BPD drug combined with shDDB1.

[0041] Panel G shows the results of detecting the degradation of BRCA2 in C4-2 cells by Western blotting at different time points after treatment with the BPD drug combined with shDDB1.

[0042] Panel H is the quantification graph of Panel G.

[0043] Panel I shows the results of detecting the degradation of BRCA2 in C4-2 cells by cellular immunofluorescence imaging after treatment with the BPD drug combined with shDDB1.

[0044] Panel J shows the average fluorescence intensity of DDB1 in C4-2 cells after treatment with the BPD drug combined with shDDB1.

[0045] Panel K shows the average fluorescence intensity of BRCA2 in PC-3 cells after treatment with the BPD drug combined with shDDB1.

[0046] Panel L shows the results of detecting the degradation of BRCA2 in PC-3 cells by cellular immunofluorescence imaging after treatment with the BPD drug combined with shDDB1.

[0047] Panel M shows the average fluorescence intensity of DDB1 in PC-3 cells after treatment with the BPD drug combined with shDDB1.

[0048] Panel N shows the average fluorescence intensity of BRCA2 in PC-3 cells after treatment with the BPD drug combined with shDDB1.

[0049] Figure 4 For detecting the inhibitory effect of the BPD drug on DNA damage homologous recombination (HR) in prostate cancer cells by cellular immunofluorescence experiments, where:

[0050] A shows the detection by cell immunofluorescence imaging method of the recruitment of the control group and BPD drug to the cell nucleus for degrading BRCA2 under radiotherapy (IR) conditions.

[0051] B is the quantitative graph of A.

[0052] C shows the detection by cell immunofluorescence imaging method of the level of RAD51 forming fluorescent foci in C4-2 cells under radiotherapy (IR) conditions after treatment with the control group and BPD drug.

[0053] D is the quantitative graph of C.

[0054] E shows the detection by cell immunofluorescence imaging method of the level of RAD51 forming fluorescent foci in PC-3 cells under radiotherapy (IR) conditions after treatment with the control group and BPD drug.

[0055] F is the quantitative graph of E.

[0056] G shows the detection by Western blot of the protein levels of BRCA1 and BRCA2 in PC-3 cells after transfection with si Control (negative control group), si BRCA1, siBRCA2 and treatment with BPD drug.

[0057] H shows the detection by DNA damage homologous recombination repair fluorescence detection system of the efficiency of homologous recombination repair function in PC-3 cells after transfection with si Control (negative control group), si BRCA1, si BRCA2 and treatment with BPD drug, and the treatment with si BRCA1, si BRCA2 and BPD significantly impairs the homologous recombination efficiency of the cells.

[0058] Figure 5 Cell and animal experiments were conducted to detect the efficacy of BPD drug combined with PARP inhibitor treatment, where:

[0059] A shows the detection by Western blot of the degradation of BRCA2 in prostate cancer cells by BPD combined with PARP inhibitor.

[0060] B shows the inhibition of the growth of C4-2 cells by the control group and BPD drug.

[0061] C shows the inhibition of the growth of PC-3 cells by the control group and BPD drug.

[0062] D shows the inhibition of the growth of C4-2 cells by PARP inhibitor and BPD drug combined with PARP inhibitor treatment.

[0063] E shows the inhibition of the growth of PC-3 cells by PARP inhibitor and BPD drug combined with PARP inhibitor treatment.

[0064] F shows the inhibitory effects of the control group, PARP inhibitor, and BPD drug on the growth of subcutaneous xenografts of prostate cancer cells, respectively.

[0065] G is the quantitative graph of J.

[0066] H shows the inhibitory effects of the control group, PARP inhibitor, and BPD drug on the growth of patient-derived xenografts, respectively.

[0067] I is the quantitative graph of L.

[0068] J shows the results of γ-H2AX (the expression level of γ-H2AX indicates the level of DNA damage in tissues) immunostaining of the tumor bodies after drug treatment.

[0069] K shows the results of immunostaining score of the tumor bodies after drug treatment.

[0070] Figure 6 is the schematic diagram of the action of BRCA2-targeted PROTAC. Detailed implementation manners

[0071] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention.

[0072] The present invention provides a polypeptide targeting BRCA2, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1:

[0073] SEQ ID NO.1: LLDEEDDSEEGGSGGTSSLLFSLWE.

[0074] The present invention has no special limitation on the source of the polypeptide, and the well-known polypeptide sources in the art can be used. In the embodiments of the present invention, the polypeptide is synthesized by the solid-phase synthesis method of polypeptides. The present invention has no special limitation on the solid-phase synthesis method of polypeptides, and the well-known polypeptide synthesis methods in the art can be used, such as Fmoc polypeptide synthesis. Fmoc-protected amino acids are purchased from Gil Biochemical, and HBTU and HIBT condensing agents are from Suzhou Haofan Biotechnology.

[0075] In the present invention, the binding ability of the polypeptide to BRCA2 protein is detected by isothermal titration calorimetry experiment, and the binding constant of the polypeptide to BRCA2 protein is preferably 116 nM. The polypeptide binds to BRCA2 protein and DDB1 protein, induces the ubiquitination of BRCA2 by DDB1 protein, and realizes the result of mediating the degradation of BRCA2 in prostate cancer.

[0076] In the present invention, the polypeptide is delivered into prostate cancer cells by nano-selenium, and it is found that it can inhibit cell proliferation. The IC of the polypeptide drug for prostate cancer cell C4-2 50was 181 nM, and the IC for prostate cancer cell line PC-3 50 was 227 nM. When used in combination with PARP inhibitors, it can significantly enhance the sensitivity of prostate cancer cells and xenograft models to PARP inhibitors.

[0077] In view of the function of the polypeptide targeting and binding to BRCA2, inhibiting the proliferation of prostate cancer cells, and enhancing the sensitivity of prostate cells to PARP inhibitors, the present invention provides the use of the polypeptide targeting BRCA2 protein in the preparation of a drug for treating metastatic castration-resistant prostate cancer.

[0078] In the present invention, the prostate cancer preferably includes one or more of the following prostate cancer cell lines: C4-2, PC-3, LNCap, 22RV1, and DU145.

[0079] In view of the function of the polypeptide degrading intracellular BRCA2 protein, the present invention provides the use of the polypeptide targeting BRCA2 protein in the preparation of a reagent for degrading BRCA2 protein.

[0080] The present invention provides a drug for treating metastatic castration-resistant prostate cancer, comprising the polypeptide and a delivery system.

[0081] The present invention does not impose special restrictions on the type of the delivery system, and any well-known delivery system in the art can be used, such as gold nanoparticles, liposomes, selenium nanoparticles, or other well-known nanodelivery systems in the art. In the embodiments of the present invention, taking selenium nanoparticles as the delivery system as an example, the preparation method and drug efficacy of the drug are illustrated. The present invention does not impose special restrictions on the preparation method of the selenium nanoparticle-polypeptide drug, and any well-known method for coupling selenium nanoparticles with proteins in the art can be used. The drug has been experimentally proven to be non-toxic at the cellular level and animal level and has high safety.

[0082] The following examples are used to illustrate in detail a polypeptide targeting BRCA2 protein, its application, and a drug for preventing and treating prostate cancer provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0083] Description of experimental materials:

[0084] The test materials are divided into two parts: Fmoc amino acids were purchased from Shanghai Gil Biochemical Co., Ltd., and DIEA, HOBT, and HBTU were purchased from Sigma-Aldrich (a company under Merck Life Sciences). The materials for preparing selenium nanoparticles were branched PEI and chloroauric acid, which were purchased from Aladdin Reagents.

[0085] Explanation of terms:

[0086] HR represents DNA homologous recombination repair;

[0087] Se represents a single-nanometer selenium delivery system;

[0088] BPD represents the finished product conjugated with a polypeptide drug and a selenium nanometer delivery system;

[0089] Control represents the finished product conjugated with a polypeptide negative control and a selenium nanometer delivery system;

[0090] BRCA2 represents the target protein BRCA2;

[0091] PROTAC is the abbreviation of Proteolysis-Targeting Chimeras;

[0092] β-actin represents actin and serves as an internal reference in Western blot;

[0093] CHX represents Cycloheximide, which is a protein synthesis inhibitor.

[0094] Example 1

[0095] Use protein-assisted design to obtain a PROTAC polypeptide targeting the BRCA2 domain.

[0096] Use the solid-phase peptide synthesis method to synthesize a polypeptide drug according to the amino acid sequence (synthesized in our own laboratory). The synthesis method is general Fmoc peptide synthesis. Fmoc-protected amino acids are purchased from GL Biochem, and HBTU and HIBT condensing agents are from Suzhou Haofan Biotech. The synthesis method is as follows:

[0097] 1) Deprotection: The Fmoc-protected column and monomer must be treated with a basic solvent (piperidine) to remove the protecting group of the amino group.

[0098] 2) Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to cross-link and form a peptide bond. A large amount of super-concentrated reagent is used in this step to drive the reaction to completion. Cycle: These two reactions are repeated until the synthesis is completed.

[0099] 3) Elution and deprotection: The polypeptide is eluted from the column, and its protecting group is removed by a deprotecting agent (TFA) to obtain the crude product.

[0100] The amino acid sequence of the PROTAC polypeptide is LLDEEDDSEEGGSGGTSSLLFSLWE (SEQ ID NO.1).

[0101] Figure 1 A is the structure of the complex of the polypeptide targeting BRCA2 and BRCA2, Figure 1B and C are the results of protein-assisted design of BPD drug polypeptides. As can be seen from Figure 1 D and E, the binding constant of the BPD drug polypeptide with BRCA2 is 116 nM ± 10.4 nM, and the binding constant with DDB1 is 318 nM ± 110 nM. As can be seen from Figure 1 F, the BPD drug has stronger protein stability than ordinary polypeptides.

[0102] Example 2

[0103] Preparation of Se-BRCA2 PROTAC Drug

[0104] The coupling method is as follows: Dissolve 2 mg of polypeptide in 1 ml of pure water, add 0.2 ml of 50 mM sodium selenite, 0.6 ml of 5% chitosan, and 1.6 ml of 50 mM ascorbic acid. Heat to 50 °C, react for 20 minutes, and cool to room temperature to obtain the polypeptide drug BPD conjugated with nano-selenium.

[0105] Example 3

[0106] Cell-level Experiment of BPD Drug

[0107] 1. The cell culture method is as follows: The culture conditions for C4-2, PC-3, LNCap, and 22RV1 cell lines are all 1640 medium, 10% fetal bovine serum, 5% CO2 saturated humidity, and adherent culture at 37 °C. The maximum cell culture density is up to 90% confluence. The culture conditions for the DU145 cell line are DMEM medium, and the other conditions are the same as above.

[0108] 2. Experiment on the Ability of the Drug to Inhibit Cancer Cell Proliferation

[0109] The MTT assay was used to analyze the ability of the drug to inhibit cancer cell proliferation.

[0110] MTT is a rapid and highly sensitive detection kit widely used in cell proliferation and cytotoxicity assays. In the presence of an electron-coupling reagent, MTT can be reduced by some dehydrogenases in mitochondria to form blue-violet crystalline formazan. The more and faster the cell proliferation, the darker the color. For the same cells, the depth of color is linearly related to the number of cells.

[0111] When performing the experiment to detect cell viability, seed C4-2 and PC-3 cells at 3×10 4Cells were seeded into a TC (Tissue Culture treated) 96-well plate at a density of [[ID=]] cells / mL, and 100 μL of cell suspension was added to each well. After 24 h of adherent culture, different concentrations of drugs (10 - 2000 nM) were added to the cells for treatment. A negative control was set as the product conjugated with the polypeptide negative control and the nano-selenium delivery system. At the same time, a blank group (untreated group) and a background group (only cell culture medium without cells added) were set. After 48 h of treatment, the medium in each well was changed, 200 μL of medium containing MTT was added, and the plate was incubated in an incubator at 37 °C for 4 h. After color development, the absorbance values of each well at 450 nm and 690 nm wavelengths were measured using a microplate reader with a spectrophotometer. After measurement, the absorbance value of each well was calibrated according to Formula I. Finally, the cell viability was calculated according to Formula II.

[0112] A = OD 450 - OD 690 Formula I.

[0113] Cell viability (%) = (A (drug added) - A (background) / A (control) - A (blank)) × 100 Formula II.

[0114] After drug treatment, the inhibitory effect of the product conjugated with the polypeptide drug and the nano-selenium delivery system on the proliferation of prostate cancer cells was detected and calculated.

[0115] The results are shown in Figure 5 B, C, D, and E in. The BPD drug showed dose-dependent growth inhibitory effects in both C4-2 and PC-3 cells, while the polypeptide control had no effect on prostate cancer cells. The half-maximal inhibitory concentration (IC50) values of the BPD drug for C4-2 and PC-3 cells were 181 nM and 227 nM, respectively. After treating the cells with BPD combined with a PARP inhibitor, the growth inhibitory effect of the PARP inhibitor on the cells was significantly enhanced.

[0116] 3. To study the degradation ability of the BPD drug on BRCA2, immunoblotting (IB) was used to analyze BRCA2. The specific experimental procedure was as follows:

[0117] 1) C4-2 / PC-3 cells were seeded into a TC (Tissue Culture treated) 12-well plate at a density of 3 × 10 4 cells / mL, and 1 mL of medium was added to each well. After 24 h of adherent culture, different drugs were added to the cells for treatment. After 24 h of treatment, 50 μL of RIPA lysis buffer containing protease inhibitor was added to each well, and the lysate was collected.

[0118] 2) Quantify the total protein content in each group of samples using a BCA quantification kit, and adjust the protein concentration in each group of samples to be consistent by adjusting the sample volume. After adjusting the protein amount, add Loading Buffer and incubate at 100 °C for 10 minutes to completely denature the protein.

[0119] 3) Separate the samples from different groups by SDS-PAGE. Prepare an 8% polyacrylamide separating gel containing SDS and a 5% polyacrylamide stacking gel. Then add the prepared samples and the same volume of prestained protein samples into the sample wells for electrophoresis separation experiments. The electrophoresis conditions are as follows: set the voltage to 70 v and separate for about 15 min until the bromophenol blue reaches the separating gel. Then adjust the voltage to 120 v and separate for about 60 min until the bromophenol blue reaches about 1 cm from the end of the separating gel, and stop electrophoresis.

[0120] 4) Transfer the protein samples. In all Western Blot experiments in this project, PVDF membranes are used. Arrange them in order on the transfer apparatus: three layers of filter paper, PVDF membrane, gel, three layers of filter paper. Set the transfer current to 300 mA and the transfer time to 2 h.

[0121] 5) Blocking. Immerse the PVDF membrane after transfer in a blocking solution containing 5% BSA and incubate at room temperature for 1 h to remove the influence of non-specific adsorption.

[0122] 6) Incubate with primary antibody. Prepare dilution solutions of different antibodies according to requirements, and then incubate overnight at 4 °C to achieve the purpose of antibody recognition of specific antigens.

[0123] 7) Incubate with secondary antibody. Prepare a species-specific HRP-labeled secondary antibody (anti-mouse or anti-rabbit) according to the source species of different primary antibodies, and dilute it 1:2000. Then incubate at room temperature for 1 h.

[0124] 8) Develop color. Prepare a color developing solution, soak the PVDF membrane after complete incubation with the secondary antibody, and perform color development analysis using a chemiluminescence instrument.

[0125] The results are shown in Figure 2 A - D in. The BPD drug induces the degradation of BRCA2 in C4-2, PC-3, 22Rv1, LNCap, and DU145 cells in a dose-dependent and time-dependent manner, while the polypeptide control has no effect on cancer cells. As Figure 3 shown in F - H of, the degradation ability of the BPD drug on BRCA2 is inhibited after knocking down DDB1. As Figure 4 shown in G of, si BRCA1 can inhibit the protein expression level of BRCA1, while si BRCA2 and BPD can both effectively reduce the protein level of BRCA2. As Figure 5As shown in A, the PARP inhibitor enhanced the degradation of BRCA2 by the BPD drug in prostate cancer cells.

[0126] Example 4

[0127] To study the mechanism of action of the BPD drug, the binding ability of BRCA2 and DDB1 in C4-2, PC-3, and 293T cell lines after treatment with the BPD drug was analyzed.

[0128] The IP test method is as follows:

[0129] (1) Harvest C4-2, PC-3 cells treated with the control polypeptide drug or the BPD drug, or cells transfected with BRCA2, DDB1, and Ub plasmids. Add an appropriate amount of IP lysis buffer (containing a proteasome inhibitor), lyse on ice or at 4°C for 30 minutes, and centrifuge at 12,000 g to take the supernatant.

[0130] (2) Take a small amount of the lysate for Western Blot analysis. Add 1 μg of antibody and 10 - 50 μl of protein A / G beads to the remaining lysate, and incubate slowly overnight.

[0131] (3) After the immunoprecipitation reaction, centrifuge at 3,000 g for 5 minutes at 4°C to centrifuge the protein A / G beads to the bottom of the tube. Carefully aspirate the supernatant. Wash the protein A / G beads 3 - 4 times with 1 ml of lysis buffer, and finally add the loading buffer, 100°C, 10 minutes.

[0132] (4) Perform Western Blot analysis, and the experimental method is as above.

[0133] The results are as Figure 3 shown in A - E. In the presence of the BPD drug, BRCA2 and DDB1 bind tightly. The enhancement of ubiquitination occurs only in the presence of the BPD drug, indicating that the enhancement of ubiquitination is through enhancing the binding of the E3 ligase DDB1 to BRCA2.

[0134] Example 5

[0135] To study the degradation effect of the BPD drug on BRCA2 and its effect on the homologous recombination function of cellular DNA damage, immunofluorescence experiments were used to analyze BRCA2, γ-H2AX, and RAD51. The specific experimental procedure is as follows:

[0136] (1) Cell spreading on slides. Seed C4-2 and PC-3 cells at a density of 3×10 4 cells / mL into a culture dish pre-placed with treated cover slips. After treatment with the polypeptide control or the BPD drug, take out the cover slips and wash them twice with PBS.

[0137] (2) Fixation. Fix the cells on the coverslip with 4% paraformaldehyde. Wash with PBS three times.

[0138] (3) Permeabilization. Treat the cells with 0.2% Triton-X100 for 15 min and wash with PBS three times.

[0139] (4) Blocking. Block the cells with 0.5% BSA for 30 min.

[0140] (5) Primary antibody binding. Incubate at room temperature for 1 h or overnight at 4°C. Rinse three times with PBST.

[0141] (6) Secondary antibody binding. Incubate the FITC- or Cy3-fluorescently labeled HRP-conjugated secondary antibody in the dark at room temperature for 1 h. Rinse three times with PBST.

[0142] (7) Mounting and detection. Drop the mounting medium, mount the coverslip, and examine under a fluorescence microscope.

[0143] After treatment with the BPD drug, the DNA homologous recombination repair function mediated by BRCA2 in prostate cancer cells was inhibited, and the level of DNA damage in the cells increased.

[0144] As Figure 2 shown in E, after treatment with the BPD drug, the expression level of the fluorescently labeled BRCA2 protein in prostate cells decreased significantly, indicating that BPD can effectively degrade the BRCA2 protein. At the same time, as Figure 2 shown in F and G, the BPD drug induced an increase in the formation level of γ-H2AX foci in the cells. As Figure 3 shown in I-N, after knocking down DDB1 in the C4-2 and PC-3 cell lines, the BPD drug could no longer induce a decrease in the level of the fluorescently labeled BRCA2 protein, indicating that the degradation of BRCA2 by the BPD drug is dependent on the DDB1 protein. As Figure 4 shown in A-H, the exogenous DNA damage induced by radiotherapy promoted the entry of the BPD drug into the nucleus, and BPD inhibited the recruitment and formation of foci of the BRCA2 downstream protein RAD51 under DNA damage conditions.

[0145] Example 6

[0146] Verify the effect of the BPD drug on inhibiting tumor growth at the animal level

[0147] 1. Method for mouse prostate cancer xenograft model

[0148] To evaluate the therapeutic effect of BPD drugs in vivo, a subcutaneous xenograft prostate cancer mouse model and a patient-derived xenograft subcutaneous tumor mouse model were established. The mice were randomly divided into 4 groups, and the same dose of polypeptide control, BPD drug (5 mg / kg), and Olaparib (PARP inhibitor, 50 mg / kg) was administered every 3 days for 3 consecutive weeks. Continuous observation was carried out, and the tumor volume was recorded. When the tumor volume increased to 50-100 mm 3 at that time, drug injection was performed. The tumor volume was calculated according to formula III. All drugs were injected every other day via intraperitoneal injection with a volume of 100 μl, and the changes in tumor size were recorded simultaneously. 21 days after drug treatment, the mice were dissected, and the tumor sites and other major organs were removed and subjected to HE staining and γ-H2AX immunohistochemical staining experiments.

[0149] Tumor volume (V) = length × width 2 / 2 Formula III.

[0150] 2. HE (HEMATOXYLIN-EOSIN STAINING) staining method

[0151] 1) Tissue fixation.

[0152] The freshly obtained tissue was placed in a pre-prepared fixing solution of 10% formalin to denature and coagulate the proteins of the tissue and cells, preventing autolysis after cell death or decomposition by bacteria, thereby maintaining the original morphological structure of the cells.

[0153] 2) Tissue dehydration.

[0154] The fixed tissue was trimmed to 25px × 25px × 5px and rinsed with pure water to remove the fixing solution in the tissue. Then, the tissue was gradually replaced with alcohol from low concentration to high concentration to replace the water in the tissue with alcohol. The tissue block was then placed in xylene, a clearing agent that is soluble in both alcohol and paraffin, to replace the alcohol in the tissue block with xylene before being infiltrated with wax and embedded.

[0155] 3) Tissue embedding.

[0156] The cleared tissue block was placed in the melted paraffin and kept warm in a wax melting box. After the paraffin had completely infiltrated the tissue block, it was allowed to cool and solidify into a block.

[0157] 4) Tissue sectioning.

[0158] The embedded wax block was fixed on a microtome and cut into thin slices, generally 5-8 μm thick.

[0159] 5) Section staining.

[0160] Before tissue staining, the paraffin in the sections needs to be removed again with xylene, followed by alcohol from high concentration to low concentration, and finally immersed in pure water to remove alcohol. Then start staining. Place the sections in hematoxylin aqueous solution and stain for 10 minutes. Then differentiate the sections in acid water and ammonia water for several seconds each. After rinsing with pure water, dehydrate the sections in 70% and 90% alcohol for 10 minutes each, and then stain the sections with alcoholic eosin staining solution for 2 minutes.

[0161] 6) Dehydrate the sections again.

[0162] Dehydrate the stained sections again according to the above tissue dehydration method.

[0163] 7) Mount the slides.

[0164] Drop gum on the cleared sections, cover with a cover slip to mount the slides. Then observe and take pictures under the microscope.

[0165] 3. Immunohistochemical γ-H2AX staining experimental method

[0166] 1) Immunohistochemical PBS reagent formula and preparation method

[0167] The following formula takes the reagents required to prepare 1000 mL of 0.01 M potassium-free PBS buffer for immunohistochemical staining, pH = 7.4 as an example:

[0168]

[0169] 2) Immunohistochemical antigen repair solution formula and preparation method

[0170] The following formula takes the reagents required to prepare 1000 mL of 0.01 M citrate buffer for immunohistochemical staining antigen repair solution, pH = 6.0 as an example:

[0171] Trisodium citrate·2H2O 3 g

[0172] Citric acid·H2O 0.4 g

[0173] ddH2O 1000 mL

[0174] 3) Preparation of gradient alcohol (100 mL)

[0175]

[0176] 4) Tissue fixation, sectioning and baking

[0177] Tissue fixation and sectioning are the same as the above HE staining fixation and sectioning methods. Then place the tissue sections in an oven and bake at 60 °C for 2 h.

[0178] 5) Deparaffinization

[0179] Take out the sections from the oven and immediately place them successively in xylene for dewaxing:

[0180] Xylene Ⅰ 8 min

[0181] Xylene Ⅱ 8 min

[0182] Xylene Ⅲ 8 min

[0183] 3) Hydration with gradient alcohol

[0184] Take out the sections from xylene Ⅲ and immediately place them successively in gradient alcohol for hydration:

[0185]

[0186]

[0187] 6) Rinse with PBS

[0188] Take out the sections from 50% alcohol and immediately place them in PBS for alcohol rinsing, rinsing 3 times, 3 min each time.

[0189] 7) Antigen retrieval

[0190] Take out the sections from PBS and place them in a histochemistry slide box containing 0.01 M citric acid buffer with pH = 6.0. Then place the histochemistry slide box in a numerical control thermal antigen retrieval instrument for antigen retrieval at 121 °C for 5 min. After antigen retrieval, let the pressure drop naturally, and then take out the histochemistry slide box and let it cool to room temperature naturally.

[0191] 8) Rinse with PBS

[0192] Take out the sections from citric acid buffer and immediately place them in PBS for citric acid buffer rinsing, rinsing 3 times, 3 min each time.

[0193] 9) Rinse with PBS

[0194] Place the sections in PBS for endogenous peroxidase rinsing, rinsing 3 times, 3 min each time.

[0195] 10) Block endogenous peroxidase

[0196] Take out the sections from PBS, dry the PBS buffer around the tissue sections with filter paper, and then outline the outside of the tissue microarray with a histology brush. Use a pipette to aspirate 1 mL of non-specific blocker to cover the tissue on the entire tissue microarray and let it stand at room temperature for 30 min.

[0197] 11) Rinse with PBS

[0198] Place the sections in PBS and rinse them with a non-specific blocker three times for 3 minutes each.

[0199] 12) Primary antibody incubation

[0200] Dilute the primary antibody to an appropriate concentration with the primary antibody diluent according to the antibody instruction manual. Take the sections out of PBS, dry the PBS buffer around the sections with filter paper, and then outline the outside of the sections with a tissue brush. Pipette 1 mL of the prepared primary antibody solution to cover the tissues on the entire tissue microarray, and set the primary antibody diluent as the negative control (i.e., no primary antibody is added to the primary antibody diluent). Place the sections with the added primary antibody in a humidified chamber and put it in a 4°C medical refrigerator overnight.

[0201] 13) Rewarming

[0202] Take the humidified chamber out of the 4°C medical refrigerator and place it at room temperature for 1 hour.

[0203] 14) PBS rinsing

[0204] Place the sections in PBS and rinse them with the primary antibody solution three times for 3 minutes each.

[0205] 15) Secondary antibody incubation

[0206] Take the sections out of PBS, dry the PBS buffer around the tissue microarray with filter paper, and then outline the outside of the tissue microarray with a tissue brush. Pipette the secondary antibody solution to cover the tissues on the entire section. Place the sections with the added secondary antibody in a humidified chamber and incubate at room temperature for 1 hour.

[0207] 16) PBS rinsing

[0208] Place the sections in PBS and rinse them with the secondary antibody solution three times for 3 minutes each.

[0209] 17) DAB color development

[0210] Take the sections out of PBS, dry the PBS buffer around the sections with filter paper, and then outline the outside of the sections with a tissue brush. Pipette 1 mL of DAB staining solution (DAB:substrate buffer = 26 μL:1000 μL) to cover the entire section. The color development time for different primary antibodies is different. Judge the termination time under the microscope and stop the staining by placing it in deionized water.

[0211] 18) PBS rinsing

[0212] Place the sections in PBS and rinse them with the DAB staining solution three times for 3 minutes each.

[0213] 19) Counterstaining

[0214] Take the sections out of PBS, dry the PBS buffer around the sections with filter paper, and then outline the outside of the sections with a group paintbrush. Pipette 1 mL of hematoxylin staining solution, let it stand at room temperature for 5 min, and then place it in deionized water to terminate the staining.

[0215] 20) Rinse with PBS

[0216] Place the prostate cancer clear cell carcinoma tissue microarray in PBS for hematoxylin staining rinsing, rinse 3 times, 3 min each time.

[0217] 21) Differentiate with hydrochloric acid

[0218] Take the sections out of PBS, dry the PBS buffer around the sections with filter paper, and then outline the outside of the sections with a group paintbrush. Pipette 1 mL of hydrochloric acid solution, let it stand at room temperature for 5 s, and then place it in deionized water to terminate the staining.

[0219] 22) Blue with ammonia water

[0220] After the differentiation of the sections is completed, immediately place them in 1% ammonia water solution, let it stand at room temperature for 10 s, and then place it in deionized water to terminate the reaction.

[0221] 23) Dehydrate with gradient alcohol and clear with xylene

[0222] Place the prostate cancer clear cell carcinoma tissue microarray in gradient alcohol and xylene solutions in sequence for dehydration treatment:

[0223]

[0224]

[0225] 24) Mount the sections

[0226] Take the sections out of xylene III, wait for the xylene to volatilize, then drop neutral balsam onto the area where the tissue is located, cover the tissue with an 8 cm × 8 cm coverslip, place it in a fume hood to air dry, and then observe and take pictures under a microscope.

[0227] As Figure 5 shown in F-I, nano-selenium particles have no therapeutic effect on tumor growth. The BPD drug shows high efficacy in both the PC-3 xenograft model and the patient-derived xenograft subcutaneous tumor model. Olaparib (PARP inhibitor) also shows similar results of inhibiting tumor growth to the low-concentration BPD drug in both models. The combined use of the BPD drug and Olaparib shows a more efficient effect of inhibiting tumor growth in both models. Immunohistochemistry (IHC) analysis of γ-H2AX in each group in the patient-derived xenograft subcutaneous tumor model confirmed that the BPD drug can effectively induce DNA damage in tumor cells ( Figure 5 in J and K).

[0228] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0229] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A polypeptide targeting BRCA2, characterized in that, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

1.

2. A therapeutic drug for prostate cancer, characterized in that, It includes the polypeptide described in claim 1 and a nano-selenium delivery system.

3. The prostate cancer treatment drug according to claim 2, characterized in that, The drug further includes a PARP inhibitor.

4. The prostate cancer therapeutic drug according to claim 3, characterized in that, The PARP inhibitor includes Olaparib.

5. Use of the prostate cancer therapeutic drug described in claim 2 in the preparation of a reagent for degrading BRCA2 protein.

6. Use of the prostate cancer therapeutic drug according to any one of claims 2 to 4 in the preparation of a drug for treating prostate cancer.

7. The application according to claim 6, wherein The prostate cancer is metastatic castration-resistant prostate cancer.

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