BRCA2 protein targeting polypeptide, application thereof and medicine for treating prostatic cancer

Abstract

The invention belongs to the technical field of protein polypeptides, and particularly relates to a polypeptide targeting BRCA2 protein, application of the polypeptide and a medicine for treating prostatic cancer. The amino acid sequence of the polypeptide is shown as SEQ ID NO.1, the polypeptide has high binding capacity with BRCA2 protein, cell proliferation and tumor growth can be effectively inhibited when the polypeptide is delivered to prostatic cancer, and meanwhile the polypeptide has the capacity of degrading BRCA2 in a targeted mode. The combined application of the polypeptide and the PARP inhibitor can enhance the sensitivity of prostate cancer cells to the PARP inhibitor. Therefore, the polypeptide can be applied to preparation of a medicine for treating metastatic castration-resistant prostate cancer or preparation of a reagent for degrading the BRCA2 protein. The BRCA2 targeting PROTAC medicine can provide a new treatment strategy for prostatic cancer treatment, and the PARP targeting inhibitor is combined to provide a PARP inhibitor synthesis lethal treatment effect for metastatic castration resistant prostatic cancer patients.

Description

Technical Field

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

[0002] Metastatic castration-resistant prostate cancer is an aggressive malignancy with a poor prognosis. Clinical trials have shown 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 have shown significant efficacy in patients carrying BRCA2 mutations. Unfortunately, only 19.3% of patients carry HR gene mutations, of which only 13% are BRCA2 gene mutations.

[0003] Targeting peptides are short peptides or proteins with specific binding ability, which can selectively bind to specific targets. However, general targeting peptides can only bind to targets and have limited effects on the degradation of targets. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a polypeptide targeting BRCA2 protein and its application and a drug for treating prostate cancer. The polypeptide has a high binding ability with 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 a first aspect of the present invention, a polypeptide targeting BRCA2 is provided, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0006] BRCA2 plays a vital role in maintaining the integrity of the genome. Figure 6 As shown, it participates in the repair of DNA double-strand breaks through the HR pathway. BRCA2 interacts with RAD51 and promotes RAD51 to load single-stranded DNA at DNA damage sites, 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 a 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. The present invention detects the ability of the polypeptide to bind to BRCA2 protein and DDB1 protein respectively through isothermal calorimetric titration experiments. The binding constant of the polypeptide to BRCA2 protein is 116nM, and the binding constant to DDB1 protein is 318nM±110nM. The polypeptide achieves BRCA2 degradation by binding to BRCA2 protein on the one hand, and enhances BRCA2 degradation in prostate cancer by inducing DDB1 protein to ubiquitinate BRCA2 on the other hand.

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

[0009] As a preferred embodiment of the drug of the present invention, the delivery system is selected from one of nano-gold, nano-selenium and liposome.

[0010] Furthermore, the drug also includes a PARP inhibitor.

[0011] Furthermore, the PARP inhibitor includes Olaparib.

[0012] The third aspect of the present invention provides use of the polypeptide in preparing an agent for degrading BRCA2 protein.

[0013] The fourth aspect of the present invention provides use of the polypeptide and the drug in preparing a drug for treating solid tumors.

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

[0015] Furthermore, 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 to prostate cancer cells, which can effectively inhibit cell proliferation. It can be seen that the polypeptide can be used in the preparation of drugs for treating prostate cancer or in the preparation of reagents for degrading BRCA2 protein.

[0018] The present invention also provides a cancer treatment drug, including the polypeptide and a delivery system. The drug can provide a new treatment strategy for drug-resistant and advanced prostate cancer tumors, especially can solve the problem that PARP inhibitors are not effective in patients who do not carry HR gene mutations, and fill the gap in drugs for polypeptide targeted degradation targeting BRCA2 worldwide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The design result of the polypeptide targeting BRCA2 provided by the present invention, wherein:

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

[0021] B is the computer prediction and sorting result of the amino acids binding to the BPD drug peptide. Protein-assisted design results.

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

[0023] D is the affinity test result of BPD drug peptide and BRCA2 protein.

[0024] E is the affinity test result between BPD drug peptide and DDB1 protein.

[0025] F is the protein stability test result of BPD drug relative to common peptides.

[0026] Figure 2 The results of the in vitro degradation of BRCA2 protein by BPD drugs, including:

[0027] A is the result of western blotting to detect drug degradation of BRCA2 in different cancer cells.

[0028] B is the result of western blotting to detect the degradation of BRCA2 in cancer cells by drugs at different treatment times.

[0029] C is a quantitative plot of B.

[0030] D is the result of western blotting to detect the degradation of BRCA2 in cancer cells by BPD drug peptide combined with MG132.

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

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

[0033] G is the γ-H2AX fluorescent focus in each C4-2 cell (the level of γ-H2AX fluorescent focus formation in the cell indicates the DNA damage of the cell).

[0034] Figure 3 The results of the research on the mechanism of action of BPD drugs include:

[0035] A shows the results of western blot detection of the binding of exogenously overexpressed DDB1 to BRCA2 in 293T cells in the presence of BPD drug.

[0036] B is the result of western blotting to detect the binding of exogenously overexpressed BRCA2 and DDB1 in 293T cells in the presence of BPD drug.

[0037] C is the result of western blotting to detect the binding of endogenously expressed DDB1 and BRCA2 in C4-2 cells in the presence of BPD drug.

[0038] D is the result of western blotting to detect the binding of endogenously expressed BRCA2 and DDB1 in C4-2 cells in the presence of BPD drug.

[0039] E is the result of western blotting detection of BPD drug combined with ubiquitination degradation of BRCA2 overexpressed by DDB1.

[0040] F is the result of western blotting detection of BRCA2 degradation in C4-2 cells by BPD drug combined with shDDB1.

[0041] G is the result of western blotting to detect the degradation of BRCA2 in C4-2 cells after treatment with BPD drug combined with shDDB1 for different time periods.

[0042] H is a quantitative plot of G.

[0043] I shows the results of BRCA2 degradation in C4-2 cells detected by cell immunofluorescence imaging using BPD drug combined with shDDB1 treatment.

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

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

[0046] L is the result of BRCA2 degradation in PC-3 cells detected by cell immunofluorescence imaging method using BPD drug combined with shDDB1 treatment.

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

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

[0049] Figure 4 The cell immunofluorescence experiment was used to detect the inhibitory effect of BPD drugs on homologous recombination (HR) of DNA damage in prostate cancer cells, where:

[0050] A is a cell immunofluorescence imaging method used to detect the recruitment of the control group and BPD drugs into the cell nucleus and degradation of BRCA2 under radiotherapy (IR) conditions.

[0051] B is the quantitative graph of A.

[0052] C is the level of RAD51 fluorescence focus formed in C4-2 cells under radiotherapy (IR) conditions detected by cell immunofluorescence imaging method in the control group and after BPD drug treatment.

[0053] D is a quantitative plot of C.

[0054] E is the level of RAD51 fluorescence focus formed in PC-3 cells under radiotherapy (IR) conditions detected by cell immunofluorescence imaging method in the control group and after BPD drug treatment.

[0055] F is a quantitative plot of E.

[0056] G is the protein level of BRCA1 and BRCA2 detected by western blotting in PC-3 cells after transfection with si Control (negative control group), si BRCA1, siBRCA2 and BPD drug treatment.

[0057] H is the DNA damage homologous recombination repair fluorescence detection system to detect the efficiency of homologous recombination repair function of PC-3 cells after transfection with si Control (negative control group), si BRCA1, si BRCA2 and BPD drug treatment. Among them, the treatment of si BRCA1, si BRCA2 and BPD significantly impaired the homologous recombination efficiency of cells.

[0058] Figure 5 To test the efficacy of BPD drugs combined with PARP inhibitors in cell and animal experiments, including:

[0059] A is a protein immunoblotting method to detect the degradation of BRCA2 in prostate cancer cells by BPD combined with PARP inhibitor.

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

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

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

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

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

[0065] G is a quantitative plot of J.

[0066] H is the growth inhibitory effect of the control group, PARP inhibitor and BPD drug on patient-derived xenograft tumors, respectively.

[0067] I is a quantitative graph of L.

[0068] J is the result of immunostaining of γ-H2AX (the expression level of γ-H2AX indicates the level of DNA damage in the tissue) for the tumor after drug treatment.

[0069] K is the immunostaining score result of the tumor after drug treatment.

[0070] Figure 6 Schematic diagram of the action of BRCA2 targeting PROTAC. DETAILED DESCRIPTION

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

[0072] The present invention provides a polypeptide targeting BRCA2, 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 restrictions on the source of the polypeptide, and any polypeptide source known in the art can be used. In the embodiment of the present invention, the polypeptide is synthesized by a polypeptide solid phase synthesis method. The present invention has no special restrictions on the polypeptide solid phase synthesis method, and any polypeptide synthesis method known in the art can be used, such as Fmoc polypeptide synthesis. Fmoc protected amino acids were purchased from Jier Biochemical, and HBTU and HIBT condensing agents were from Suzhou Haofan Biotechnology.

[0075] In the present invention, an isothermal calorimetric titration experiment is used to detect the ability of the polypeptide to bind to the BRCA2 protein, and the binding constant of the polypeptide to the BRCA2 protein is preferably 116 nM. The polypeptide binds to the BRCA2 protein and the DDB1 protein, induces the DDB1 protein to ubiquitinate BRCA2, and achieves the result of mediating BRCA2 degradation in prostate cancer.

[0076] In the present invention, the polypeptide is delivered to prostate cancer cells using nano-selenium and found to inhibit cell proliferation. IC of polypeptide drug for prostate cancer cell C4-2 50The IC value for prostate cancer cell PC-3 is 181nM. 50 The combined use of PARP inhibitors can significantly enhance the sensitivity of prostate cancer cells and transplanted tumor models to PARP inhibitors.

[0077] In view of the function of the polypeptide targeting 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 cells: C4-2, PC-3, LNCap, 22RV1 and DU145.

[0079] In view of the function of the polypeptide to degrade intracellular BRCA2 protein, the present invention provides the use of the polypeptide targeting BRCA2 protein in preparing an agent 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 any special restrictions on the type of the delivery system, and a delivery system well known in the art can be used, such as nanogold, liposomes, nanoselenium or other nano delivery systems well known in the art. In the embodiments of the present invention, nanoselenium is used as a delivery system as an example to illustrate the preparation method and efficacy of the drug. The present invention does not impose any special restrictions on the preparation method of the nanoselenium-polypeptide drug, and a method of nanoselenium coupling protein well known in the art can be used. The drug has been experimentally confirmed to be non-toxic at the cellular level and animal level, and has a high safety.

[0082] The following is a detailed description of a polypeptide targeting BRCA2 protein provided by the present invention, its application, and a drug for preventing and treating prostate cancer in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0083] Description of experimental materials:

[0084] The experimental materials are divided into the synthesis part: Fmoc amino acid was purchased from Shanghai Jier Biochemical, DIEA, HOBT, and HBTU were purchased from Sigma-Aldrich (a subsidiary of Merck Life Sciences). The nano-selenium preparation part is: branched PEI, and tetrachloroauric acid was purchased from Aladdin Reagent.

[0085] Glossary:

[0086] HR stands for DNA homologous recombination repair;

[0087] Se stands for single nanoselenium delivery system;

[0088] BPD represents the finished product coupled with peptide drug and nano-selenium delivery system;

[0089] Control represents the finished product coupled with the peptide negative control and the nano-selenium delivery system;

[0090] BRCA2 stands for target protein BRCA2;

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

[0092] β-actin represents actin and was used as a loading control in western blotting;

[0093] CHX stands for Cycloheximide, a protein production inhibitor.

[0094] Example 1

[0095] Protein-assisted design was used to obtain PROTAC peptides targeting the BRCA2 domain.

[0096] The peptide drug was synthesized according to the amino acid sequence using the peptide solid phase synthesis method (synthesized in my own laboratory). The synthesis method was the general Fmoc peptide synthesis. Fmoc protected amino acids were purchased from Jier Biochemical, and HBTU and HIBT condensation agents were from Suzhou Haofan Biotechnology. The synthesis method is as follows:

[0097] 1) Deprotection: Fmoc-protected columns and monomers must be treated with an alkaline solvent (piperidine) to remove the amino protecting 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. In this step, a large amount of superconcentrated reagents are used to drive the reaction to completion. Cycle: These two steps are repeated until the synthesis is complete.

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

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

[0101] Figure 1 A is the complex structure of the peptide targeting BRCA2 and BRCA2. Figure 1B and C are the results of protein-assisted design of BPD drug peptides. Figure 1 From D and E, we can see that the binding constant of BPD drug peptide to BRCA2 is 116nM±10.4nM, and the binding constant to DDB1 is 318nM±110nM. Figure 1 It can be seen from F that BPD drugs have stronger protein stability than ordinary peptides.

[0102] Example 2

[0103] Preparation of Se-BRCA2 PROTAC drug

[0104] The coupling method is as follows: 2 mg of the polypeptide is dissolved in 1 ml of pure water, 0.2 ml of 50 mM sodium selenite, 0.6 ml of 5% chitosan and 1.6 ml of 50 mM ascorbic acid are added, heated to 50 degrees, reacted for 20 minutes and cooled to room temperature to obtain the polypeptide drug BPD coupled with nano-selenium.

[0105] Example 3

[0106] Cell-level experiments on BPD drugs

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

[0108] 2. Experiment on the ability of drugs to inhibit cancer cell proliferation

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

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

[0111] In the experiment of detecting cell activity, C4-2 and PC-3 cells were cultured at 3×10 4The cells were seeded at a density of cells / mL in a 96-well plate treated with TC (Tissue Culture treated), and 100 μl of cell solution was added to each well. After the cells were cultured for 24 hours, different concentrations of drugs (10-2000 nM) were added to the cells for treatment, and the negative control was set as the finished product coupled with the peptide negative control and the nano-selenium delivery system. At the same time, a blank group, i.e., an untreated group, and a background group, i.e., a group in which only cell culture medium was added but no cells were added, were set. After 48 hours of treatment, the medium was replaced in each well, 200 μL of culture medium containing MTT was added, and the cells were incubated in a 37°C incubator for 4 hours. After the color development was completed, the absorbance of each well at a wavelength of 450 nm and 690 nm was measured using a spectrophotometer using an enzyme marker. After the measurement, the absorbance 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 detection and calculation showed that the finished product representing the conjugated peptide drug and nano-selenium delivery system had an inhibitory effect on the proliferation of prostate cancer cells.

[0115] Results Figure 5 B, C, D and E. BPD drug showed dose-dependent growth inhibition in both C4-2 and PC-3 cells, while the peptide control had no effect on prostate cancer cells. The half-maximal inhibitory concentration (IC50) values ​​of BPD drug on C4-2 and PC-3 cells were 181nM and 227nM, respectively. After BPD combined with PARP inhibitors to treat cells, the growth inhibition effect of PARP inhibitors on cells was significantly enhanced.

[0116] 3. In order to study the degradation ability of BPD drugs on BRCA2, immunoblotting (IB) was used to analyze BRCA2. The specific experimental process is as follows:

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

[0118] 2) The total protein content in each group of samples was quantified using a BCA quantification kit, and the protein concentration in each group of samples was made consistent by adjusting the sample volume. After the protein amount was adjusted, Loading Buffer was added and incubated at 100°C for 10 minutes to completely denature the protein.

[0119] 3) Perform SDS-PAGE separation on samples from different groups. Prepare 8% polyacrylamide separation gel containing SDS and 5% polyacrylamide concentration gel. Then add the prepared samples and the same volume of pre-stained protein samples into the loading wells for electrophoresis separation experiments. The electrophoresis conditions are: the voltage is set to 70V, and the separation is about 15min until the bromophenol blue reaches the separation gel. Then adjust the voltage to 120V, separate for about 60min until the bromophenol blue reaches about 1cm from the end of the separation gel, and stop the electrophoresis.

[0120] 4) Transfer the protein sample to a membrane. All Western Blot experiments in this project use PVDF membranes, which are placed in the following order on the membrane transfer instrument: three layers of filter paper, PVDF membrane, gel, and three layers of filter paper. Set the transfer current to 300mA and the transfer time to 2h.

[0121] 5) Blocking: The transferred PVDF membrane was immersed in a blocking solution containing 5% BSA and incubated at room temperature for 1 hour to remove the influence of non-specific adsorption.

[0122] 6) Primary antibody incubation. Prepare different antibody dilutions as required, and then incubate at 4°C overnight to achieve the purpose of antibody recognition of specific antigens.

[0123] 7) Secondary antibody incubation. Prepare species-specific HRP-labeled secondary antibodies (anti-mouse or anti-rabbit) according to the species of the primary antibody, dilute 1:2000, and incubate at room temperature for 1 hour.

[0124] 8) Color development: Prepare color development solution, soak the PVDF membrane that has been fully incubated with the secondary antibody, and use a chemiluminescence analyzer for color development analysis.

[0125] Results Figure 2 In A to D, BPD induced the degradation of BRCA2 in C4-2, PC-3, 22Rv1, LNCap and DU145 cells in a dose-dependent and time-dependent manner, while the peptide controls had no effect on cancer cells. Figure 3 As shown in Figures F to H, the ability of BPD drugs to degrade BRCA2 was inhibited after knocking down DDB1. Figure 4 As shown in Figure G, si BRCA1 can inhibit the protein expression level of BRCA1, while si BRCA2 and BPD can effectively reduce the protein level of BRCA2. Figure 5As shown in A, PARP inhibitors enhance the degradation effect of BPD drugs on BRCA2 in prostate cancer cells.

[0126] Example 4

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

[0128] The IP test method is as follows:

[0129] (1) C4-2, PC-3 cells treated with control peptide drugs or BPD drugs or cells transfected with BRCA2, DDB1 and Ub plasmids were harvested, and an appropriate amount of IP lysis buffer (containing proteasome inhibitors) was added. The cells were lysed on ice or at 4°C for 30 minutes, and the supernatant was collected by centrifugation at 12,000 g.

[0130] (2) Take a small amount of 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 3000 g for 5 minutes at 4°C to centrifuge the protein A / G beads to the bottom of the tube. Carefully aspirate the supernatant and wash the protein A / G beads 3-4 times with 1 ml of lysis buffer. Finally, add loading buffer and incubate at 100°C for 10 minutes.

[0132] (4) Perform Western Blot analysis using the same experimental method as above.

[0133] The results are as follows Figure 3 As shown in A to E, in the presence of BPD, BRCA2 is tightly bound to DDB1. The enhancement of ubiquitination is only in the presence of BPD, indicating that the enhancement of ubiquitination is through enhancing the binding of E3 ligase DDB1 to BRCA2.

[0134] Example 5

[0135] In order to study the effect of BPD drugs on BRCA2 degradation and the effect on the homologous recombination function of cell DNA damage, BRCA2, γ-H2AX and RAD51 were analyzed by cell immunofluorescence assay. The specific experimental process is as follows:

[0136] (1) Cell culture. C4-2 and PC-3 cells were cultured at 3×10 4 The cells were inoculated at a density of 1.5 cells / mL into a culture dish with a pre-treated coverslip placed thereon. After treatment with peptide control or BPD drug, the coverslip was removed and washed twice with PBS.

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

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

[0139] (4) Blocking: The cells were blocked with 0.5% BSA for 30 min.

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

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

[0142] (7) Sealing and testing: Add sealing agent, seal the slides, and examine under a fluorescence microscope.

[0143] After treatment with BPD drugs, 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] like Figure 2 As shown in Figure E, after treatment with BPD, the expression level of fluorescently labeled BRCA2 protein in prostate cells decreased significantly, indicating that BPD can effectively degrade BRCA2 protein. Figure 2 As shown in F and G, BPD triggered an increase in the level of γ-H2AX focus formation in cells. Figure 3 As shown in Figures I to N, after knocking down DDB1 in C4-2 and PC-3 cell lines, BPD drugs no longer triggered a decrease in the level of fluorescently labeled BRCA2 protein, indicating that the degradation of BRCA2 by BPD drugs is dependent on DDB1 protein. Figure 4 As shown in A to H, exogenous DNA damage induced by radiotherapy promotes the entry of BPD drugs into the cell nucleus, and BPD inhibits the recruitment of BRCA2 downstream protein RAD51 to form foci under DNA damage conditions.

[0145] Example 6

[0146] Verification of the inhibitory effect of BPD drugs on tumor growth at the animal level

[0147] 1. Mouse Prostate Cancer Xenograft Model Method

[0148] In order to evaluate the therapeutic effect of BPD drugs in vivo, a subcutaneous transplanted prostate cancer mouse model and a patient-derived xenografted subcutaneous tumor mouse model were established. The mice were randomly divided into 4 groups and treated with the same dose of peptide control, BPD drug (5 mg / kg), and Olaparib (PARP inhibitor, 50 mg / kg) every 3 days for 3 consecutive weeks. Continuous observation and recording of tumor volume were performed. When the tumor volume grew to 50-100 mm 3 Drug injection was performed at 1:10 pm. Tumor volume calculation was completed according to Formula III. All drugs were injected every other day, intraperitoneally, with an injection volume of 100 μl, and changes in tumor size were recorded at the same time. After 21 days of drug treatment, mice were dissected, and the tumor sites and other major organs were removed for HE staining and γ-H2AX immunohistochemical staining experiments.

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

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

[0151] 1) Tissue fixation.

[0152] The removed fresh tissue is placed in a pre-prepared 10% formalin fixative to denature and coagulate the proteins in the tissue and cells to prevent autolysis of the cells after death or decomposition by bacteria, thereby maintaining the original morphological structure of the cells.

[0153] 2) Tissue dehydration.

[0154] After fixation, the tissue is trimmed into 25px×25px×5px, and the fixative is removed by washing with pure water. Then the water in the tissue is gradually replaced with alcohol from low to high concentrations. The tissue block is then placed in xylene, a transparent agent that is soluble in both alcohol and paraffin, and the alcohol in the tissue block is replaced with xylene before wax embedding.

[0155] 3) Tissue embedding.

[0156] Place the transparent tissue block in the melted paraffin and put it in a wax melting box to keep warm. After the paraffin is completely immersed in the tissue block, let it cool and solidify into a block.

[0157] 4) Tissue sections.

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

[0159] 5) Staining of sections.

[0160] Before tissue staining, the paraffin in the slices needs to be removed with xylene, and then the alcohol is removed by immersing in pure water through high-concentration to low-concentration alcohol. Then start staining and place the slices in a hematoxylin aqueous solution for 10 minutes. Then separate the slices in acid water and ammonia water for a few seconds each. After rinsing with pure water, dehydrate in 70% and 90% alcohol for 10 minutes each, and then stain the slices in alcohol eosin staining solution for 2 minutes.

[0161] 6) Dehydrate the slices again.

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

[0163] 7) Seal the slides.

[0164] The transparent slices were dripped with gum and covered with coverslips. Then, the slices were observed under a microscope and photographed.

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

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

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

[0168]

[0169] 2) Immunohistochemistry antigen retrieval solution formula and preparation method

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

[0171] Trisodium citrate·2H2O 3g

[0172] Citric acid·H2O 0.4g

[0173] ddH2O 1000mL

[0174] 3) Gradient alcohol preparation (100mL)

[0175]

[0176] 4) Tissue fixation, slicing and baking

[0177] Tissue fixation and sectioning were performed in the same manner as described above for HE staining, and then the tissue sections were placed in an oven and baked at 60°C for 2 h.

[0178] 5) Dewaxing

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

[0180] Xylene Ⅰ 8min

[0181] Xylene Ⅱ 8min

[0182] Xylene Ⅲ 8min

[0183] 3) Alcohol gradient hydration

[0184] Remove the slices from xylene III and immediately place them in graded alcohol for hydration:

[0185]

[0186]

[0187] 6) PBS rinse

[0188] The slices were removed from the 50% alcohol and immediately placed in PBS for alcohol washing for 3 times, 3 minutes each time.

[0189] 7) Antigen retrieval

[0190] Take the slices out of PBS and place them in a histochemical slide box filled with 0.01M, pH=6.0 citrate buffer, then place the histochemical slide box in a digitally controlled hot antigen repair instrument for antigen repair at 121°C for 5 minutes. After antigen repair, naturally reduce the pressure, then take out the histochemical slide box and cool it naturally to room temperature.

[0191] 8) PBS rinse

[0192] The slices were removed from the citrate buffer and immediately placed in PBS for rinsing with citrate buffer for 3 times, 3 min each time.

[0193] 9) PBS rinse

[0194] The sections were placed in PBS for endogenous peroxidase washing, and the washing was performed 3 times, each time for 3 minutes.

[0195] 10) Endogenous peroxidase blocking

[0196] Remove the slices from PBS, wipe the PBS buffer around the tissue slices with filter paper, and then use a brush to outline the outside of the tissue chip. Use a pipette to absorb 1 mL of nonspecific blocking agent to cover the entire tissue on the tissue chip, and let it stand at room temperature for 30 minutes.

[0197] 11) PBS rinse

[0198] The sections were placed in PBS for nonspecific blocking agent washing, and washed 3 times, each time for 3 minutes.

[0199] 12) Primary antibody incubation

[0200] According to the antibody manual, dilute the primary antibody to an appropriate concentration with the primary antibody diluent. Remove the slice from the PBS, wipe the PBS buffer around the slice with filter paper, and then use a group brush to outline the outside of the slice. Use a pipette to draw 1mL of the prepared primary antibody solution to cover the entire tissue on the tissue chip, and set the primary antibody diluent as a negative control (i.e., no primary antibody is added to the primary antibody diluent). Place the slice with the primary antibody in a tissue humidification box and place it in a 4°C medical refrigerator to stand overnight.

[0201] 13) Rewarming

[0202] The tissue humidification box was taken out from the 4°C medical refrigerator and placed at room temperature for 1 hour.

[0203] 14) PBS rinse

[0204] The sections were placed in PBS and rinsed with the primary antibody solution for 3 times, each time for 3 minutes.

[0205] 15) Secondary antibody incubation

[0206] Remove the slice from PBS, wipe the PBS buffer around the tissue chip with filter paper, and then use a tissue brush to outline the outside of the tissue chip. Use a pipette to absorb the secondary antibody solution to cover the entire tissue on the slice, and place the slice with the secondary antibody in a tissue humidification box and place it at room temperature for 1 hour.

[0207] 16) PBS rinse

[0208] The sections were placed in PBS and rinsed with secondary antibody solution for 3 times, each time for 3 minutes.

[0209] 17) DAB color development

[0210] Remove the slice from PBS, wipe the PBS buffer around the slice with filter paper, and then use a brush to outline the outside of the slice. Use a pipette to absorb 1mL DAB dye solution (DAB: substrate buffer = 26μL: 1000μL) to cover the entire slice. Different primary antibodies have different color development times. Determine the termination time under a microscope and put it into deionized water to terminate the staining.

[0211] 18) PBS rinse

[0212] The sections were placed in PBS and rinsed with DAB staining solution for 3 times, each time for 3 minutes.

[0213] 19) Re-staining

[0214] Remove the slices from PBS, wipe the PBS buffer around the slices with filter paper, and then use a brush to outline the outside of the slices. Use a pipette to absorb 1 mL of hematoxylin stain, let it stand at room temperature for 5 minutes, and put it into deionized water to stop staining.

[0215] 20) PBS rinse

[0216] The prostate cancer clear cell carcinoma tissue chip was placed in PBS for hematoxylin staining and rinsed 3 times, each time for 3 minutes.

[0217] 21) Hydrochloric acid differentiation

[0218] Remove the slices from PBS, wipe the PBS buffer around the slices with filter paper, and then use a brush to outline the outside of the slices. Use a pipette to draw 1 mL of hydrochloric acid solution, let it stand at room temperature for 5 seconds, and then put it into deionized water to stop staining.

[0219] 22) Ammonia turns blue

[0220] After the slices were differentiated, they were immediately placed in 1% ammonia solution, left to stand at room temperature for 10 seconds, and then placed in deionized water to terminate the reaction.

[0221] 23) Dehydration with gradient alcohol and clearing with xylene

[0222] The prostate cancer clear cell carcinoma tissue chip was placed in gradient alcohol and xylene solutions for dehydration:

[0223]

[0224]

[0225] 24) Seal the film

[0226] The slices were taken out from xylene III, and after the xylene evaporated, neutral gum was added to the tissue area, and the tissue was covered with an 8 cm × 8 cm coverslip and placed in a fume hood to air dry, and then observed under a microscope and photographed.

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

[0228] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall 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 equivalents, 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 shown in SEQ ID NO.

1.

2. A cancer treatment drug, characterized in that: The invention comprises the polypeptide according to claim 1 and a delivery system.

3. The cancer treatment drug according to claim 2, characterized in that: The delivery system is selected from one of nano-gold, nano-selenium and liposome.

4. The cancer treatment drug according to claim 2, characterized in that: The drugs also include PARP inhibitors.

5. The cancer treatment drug according to claim 4, characterized in that: The PARP inhibitors include Olaparib.

6. Use of the polypeptide according to claim 1 in preparing an agent for degrading BRCA2 protein.

7. Use of the polypeptide according to claim 1 or the drug according to any one of claims 2 to 5 in the preparation of a drug for treating solid tumors.

8. The use according to claim 7, characterized in that: The solid tumors include prostate cancer.

9. The use according to claim 8, characterized in that: The prostate cancer is metastatic castration-resistant prostate cancer.

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