Peptides targeting P300 degradation, drugs for the prevention and treatment of prostate cancer, preparation methods and applications

Abstract

This invention provides a peptide targeting the degradation of P300, a drug for the prevention and treatment of prostate cancer, a preparation method, and its applications, belonging to the field of protein peptide drug technology. The amino acid sequence of the peptide targeting the degradation of P300 is shown in SEQ ID NO: 1, and its binding constant to P300 protein is 78.3 nM, indicating a strong binding ability to P300 protein. Delivery of the peptide targeting the degradation of P300 to prostate cancer cells can significantly inhibit the proliferation of prostate cancer cells. Simultaneously, the peptide also has the ability to degrade P300. Compared with existing targeted drugs, the peptide drug conjugated with selenium nanoparticles of this invention provides a treatment strategy for drug-resistant and advanced castration-resistant prostate cancer, especially addressing the practical problem of a lack of therapeutic drugs for neuroendocrine prostate cancer.

Description

Technical Field

[0001] This invention belongs to the field of protein and polypeptide drug technology, specifically relating to a polypeptide that targets and degrades P300, a drug for the prevention and treatment of prostate cancer, its preparation method, and its application. Background Technology

[0002] Prostate cancer, one of the most common malignant tumors of the urinary system in men worldwide, is experiencing a rising incidence rate, with the vast majority of patients diagnosed at an advanced stage. Currently, treatments for prostate cancer primarily involve surgery and castration therapy (ADT) targeting the androgen receptor (AR) signaling pathway. However, while these therapies have shown some effectiveness, the vast majority of patients rapidly progress to castration-resistant prostate cancer (CRPC) due to drug resistance and other issues, leading to malignant tumor progression and recurrence. Neuroendocrine prostate cancer (NEPC) is a subtype of CRPC with an extremely poor prognosis, exhibiting resistance to ADT and poor response to radiotherapy and chemotherapy, with an average survival of less than one year, accounting for approximately 25% of deaths among CRPC patients. Therefore, developing novel treatment strategies for prostate cancer is urgently needed.

[0003] P300 is a known AR coactivator, playing a crucial role in activating the androgen receptor (AR) signaling pathway. Furthermore, P300 has been identified as an important target for treating various diseases, including malignant tumors. Therefore, developing drugs that inhibit P300 has become a hot research topic in the treatment of malignant tumors. However, currently developed P300 inhibitors suffer from numerous problems, including insufficient inhibitory efficacy, poor selectivity, weak affinity, high toxicity, and poor drug-likeness. Summary of the Invention

[0004] Therefore, the present invention aims to provide a polypeptide that targets and degrades P300, a drug for the prevention and treatment of prostate cancer, a preparation method thereof, and its application. The polypeptide has a strong binding ability to P300 protein, effectively degrades P300, and can effectively inhibit the proliferation of AR-positive, AR-negative, and NEPC prostate cancer cells. It can be used to prepare a drug for the prevention and treatment of prostate cancer.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a polypeptide for targeted degradation of P300, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Preferably, the binding constant of the polypeptide to P300 is 78.3 nM.

[0008] The present invention also provides a drug for the prevention and treatment of prostate cancer, the drug comprising the above-mentioned polypeptide and drug carrier.

[0009] Preferably, the drug carrier includes one or more of gold nanoparticles, liposomes, and selenium nanoparticles.

[0010] This invention also provides a method for preparing a peptide drug coupled with selenium nanoparticles, comprising the following steps:

[0011] The above-mentioned peptides that target the degradation of P300 were mixed with water, and then chitosan, sodium selenite and vitamin C were added and heated to react. After the reaction was completed, the residual reagents were removed by dialysis to obtain the peptide drug coupled with nano-selenium.

[0012] Preferably, the mass-to-volume ratio of the peptide targeting P300 degradation to water is 1 mg: 0.5–2 mL; the concentration of chitosan is 4%–6%; the molar concentration of sodium selenite is 45–55 mM; the molar concentration of vitamin C is 45–55 mM; and the volume ratio of chitosan, sodium selenite, vitamin C, to water is 0.5–0.7: 0.1–0.3: 1.5–2: 0.5–1.5.

[0013] Preferably, the temperature of the heating reaction is 45–55°C; and the heating reaction time is 15–25 min.

[0014] This invention also provides an application of the above-mentioned targeted peptide, drug, or preparation method for degrading P300, wherein the application is at least one of the following:

[0015] (1) The application of the peptide, drug or preparation method for targeting P300 degradation in the preparation of P300 protein degradation products;

[0016] (2) The application of the peptide, drug or preparation method that targets and degrades P300 in the preparation of drugs for the prevention and / or treatment of prostate cancer.

[0017] Preferably, the prostate cancer cells include one or more of AR-positive prostate cancer cells, AR-negative prostate cancer cells, and NEPC cell lines.

[0018] Preferably, the prostate cancer cells include at least one of the following prostate cells: C4-2, 22Rv1, PC3, DU145, and NE1.3.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention provides a peptide targeting the degradation of P300, a drug for the prevention and treatment of prostate cancer, a preparation method, and its applications. The peptide with the amino acid sequence shown in SEQ ID NO: 1 has a binding constant of 78.3 nM to the P300 protein, indicating that it has a strong binding affinity to the P300 protein. Delivery of the peptide to AR-positive, AR-negative, and NEPC prostate cancer cells significantly inhibits the proliferation of prostate cancer cells. The peptide also possesses the ability to degrade P300. Therefore, the peptide can be used in the preparation of drugs for the prevention and / or treatment of prostate cancer or in the preparation of reagents for degrading the P300 protein.

[0021] The present invention provides a peptide drug coupled with selenium nanoparticles. Compared with existing targeted drugs, the Se-P300pPROTAC drug can provide a new treatment strategy for the treatment of drug-resistant and advanced castration-resistant prostate cancer, especially to solve the practical problem of the lack of drugs for the treatment of neuroendocrine prostate cancer. Attached Figure Description

[0022] Figure 1 The identification results of the PROTAC peptide targeting P300 provided by the present invention are shown in the following figures: A is the spatial structure of the PROTAC peptide; B is the detection result of the binding ability of the PROTAC peptide to P300; and C is the detection result of the drug stability of the PROTAC peptide.

[0023] Figure 2 Characterization of Se-P300 pPROTAC drug: A shows the particle size analysis results of Se-P300 pPROTAC drug; B shows the transmission electron microscope image of Se-P300 pPROTAC drug; C shows the zeta potential detection results of Se-P300 pPROTAC drug.

[0024] Figure 3The results of the in vitro proliferative inhibitory activity of Se-P300 pPROTAC on prostate cells are shown below. A represents the effect of Se-P300 pPROTAC on the proliferation of 22RV1 cells; B represents the effect of Se-P300 pPROTAC on the proliferation of C4-2 cells; C represents the effect of Se-P300 pPROTAC on the proliferation of PC3 cells; D represents the effect of Se-P300 pPROTAC on the proliferation of DU145 cells; E-H represent the results of Western blotting detection of P300 degradation by Se-P300 pPROTAC in 22RV1, C4-2, PC3, and DU145 cells; I represents the results of immunocellular imaging detection of P300 protein degradation by Se-P300 pPROTAC in C4-2 cells; J and K represent the results of Western blotting detection of P300 protein degradation by Se-P300 pPROTAC in PC3 and C4-2 cells, respectively.

[0025] Figure 4 The results show the effects of Se-P300 pPROTAC on NE1.3 cells. A represents the effect of Se-P300 pPROTAC on the proliferation of NE1.3 cells; B represents the results of Western blotting on the degradation of P300 in NE1.3 cells by Se-P300 pPROTAC; and C represents the results of flow cytometry on the apoptosis-inducing effect of Se-P300 pPROTAC on NE1.3 cells.

[0026] Figure 5 The efficacy of Se-P300 pPROTAC was tested in animal experiments. A shows the statistical results of the effects of different drugs on tumor volume; B shows the results of the effects of different drugs on tumor growth; C shows the statistical results of the effects of different drugs on tumor weight; D shows the results of HE staining and Ki67 staining of tumor tissues treated with different drugs; E shows the scoring of Ki67 staining results after different drug treatments; F shows the detection results of ALT, AST, GGT, ALB, TBA, and ALP in the blood after different drug treatments; G shows the detection results of BUN and CREA in the blood after different drug treatments; and H shows HE staining images of heart, liver, spleen, and kidney tissues after different drug treatments. Detailed Implementation

[0027] This invention provides a polypeptide for targeted degradation of P300, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0028] In this invention, the amino acid sequence of the polypeptide is CPWIWDGDNKDDNSTDGGGSGGGTSFEQFWAWLWP (SEQ ID NO: 1), and the binding constant of the polypeptide to P300 is 78.3 nM. The polypeptide is synthesized using a solid-phase polypeptide synthesis method. This invention does not impose any particular limitation on the solid-phase polypeptide synthesis method; any polypeptide synthesis method well-known in the art can be used, such as Fmoc polypeptide synthesis. The protected amino acids for Fmoc were purchased from Jier Biochemical, HBTU, and the HOBT condensing agent was from Suzhou Haofan Biotechnology. The preparation method of the polypeptide of this invention includes:

[0029] (1) Deprotection: Fmoc-protected columns and monomers must have the amino protecting groups removed using an alkaline solvent (piperidine).

[0030] (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, forming a peptide bond. A large amount of ultra-concentrated reagent is used to drive the reaction in this step. Cycling: These two steps are repeated until the synthesis is complete.

[0031] (3) Elution and deprotection: The polypeptide is eluted from the column, and its protecting group is eluted and deprotected by a deprotecting agent (TFA) to obtain the crude product.

[0032] The peptides of this invention that target and degrade P300 exhibit strong binding affinity to P300. This invention does not impose any particular limitation on the source of the peptides targeting and degrading P300; any peptide source well-known in the art can be used. In the embodiments of this invention, the peptides are synthesized using a solid-phase peptide synthesis method. This invention does not impose any particular limitation on the solid-phase peptide synthesis method; any peptide synthesis method well-known in the art can be used, such as Fmoc peptide synthesis. The protected amino acids of Fmoc were purchased from Jier Biochemical, and the benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT) condensing agent were obtained from Suzhou Haofan Biotechnology.

[0033] The present invention also provides a drug for the prevention and treatment of prostate cancer, the drug comprising the above-mentioned polypeptide and drug carrier.

[0034] In this invention, the drug carrier includes one or more of gold nanoparticles, liposomes, and selenium nanoparticles.

[0035] This invention also provides a method for preparing a peptide drug coupled with selenium nanoparticles, comprising the following steps:

[0036] The above-mentioned peptides that target the degradation of P300 were mixed with water, and then chitosan, sodium selenite and vitamin C were added and heated to react. After the reaction was completed, the residual reagents were removed by dialysis to obtain the peptide drug coupled with nano-selenium.

[0037] In this invention, the peptides for targeting and degrading P300 described above are mixed with water. The mass-to-volume ratio of the peptides for targeting and degrading P300 to water is 1 mg: 0.5–2 mL, more preferably 1 mg: 0.6–1.5 mL, and even more preferably 1 mg: 1 mL.

[0038] The peptides targeting P300 degradation are mixed with water, and then chitosan, sodium selenite, and vitamin C are added for a heated reaction. The concentration of chitosan is preferably 4%–6%, more preferably 4.5%–5.5%, and even more preferably 5%, where the chitosan concentration is a mass-volume percentage. For example, 5% chitosan is prepared by dissolving 0.5g of chitosan in 10mL of pure water. The molar concentration of sodium selenite is preferably 45–55mM, more preferably 46–54mM, and even more preferably 50mM; the molar concentration of vitamin C is preferably 45–55mM, more preferably 47–53mM, and even more preferably 50mM; the volume ratio of chitosan, sodium selenite, vitamin C, and water is preferably 0.5–0.7:0.1–0.3:1.5–2:0.5–1.5, and even more preferably 0.6:0.2:1.6:1. The preferred temperature for the heating reaction is 45–55°C, more preferably 46–54°C, and even more preferably 50°C; the preferred heating reaction time is 15–25 min, more preferably 16–23 min, and even more preferably 20 min.

[0039] After heating and reacting, the mixture is cooled to room temperature to obtain a polypeptide coupled with selenium nanoparticles.

[0040] The peptide conjugated with selenium nanoparticles prepared by the above preparation method is CPWIWDGDNKDDNSTDGGGSGGGTSFEQFWAWLWP (SEQ ID NO: 1), with a particle size of 176.4±48.4 nm and a zeta potential of +47.3±5.25 mV.

[0041] The present invention delivers a peptide targeting P300 degradation to prostate cells, and it has been found to degrade the P300 protein in the cells and significantly inhibit the proliferation of prostate cancer cells. The peptide drug targeting P300 degradation has an IC50 value for C4-2 prostate cancer cells. 50 The IC50 value for 22Rv1 prostate cancer cells was 1693 nM. 50 The IC50 value for PC3 prostate cancer cells was 3064 nM.50 The IC50 value for DU145 prostate cancer cells was 1411 nM. 50 The IC50 value was 807.5 nM, and the IC50 value for NE1.3 prostate cancer cells was [missing value]. 50 It is 716.9 nM.

[0042] In this invention, the Se-p300 pPROTAC drug has been experimentally proven to be non-toxic at both the cellular and animal levels, exhibiting high safety.

[0043] Based on this, the present invention also provides an application of the above-mentioned targeted peptide, drug, or preparation method for degrading P300, wherein the application is at least one of the following:

[0044] (1) The application of the peptide, drug or preparation method for targeting P300 degradation in the preparation of P300 protein degradation products;

[0045] (2) The application of the peptide, drug or preparation method that targets and degrades P300 in the preparation of drugs for the prevention and / or treatment of prostate cancer.

[0046] In this invention, the prostate cancer cells preferably include one or more of AR-positive prostate cancer cells, AR-negative prostate cancer cells, and NEPC cell lines. The prostate cancer cells preferably include at least one of the following prostate cell lines: C4-2, 22Rv1, PC3, DU145, and NE1.3. The AR-positive prostate cancer cells include C4-2 and 22Rv1, the AR-negative prostate cancer cells include PC-3 and DU145, and the NEPC cells include NE1.3 cells.

[0047] The following detailed description, in conjunction with embodiments, illustrates a peptide targeting P300 provided by the present invention, its application, and a drug for the prevention and treatment of prostate cancer. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0048] Description of experimental materials: The experimental materials are divided into the following sections: Fmoc amino acids were purchased from Shanghai Jier Biochemical; DIEA, HOBT, and HBTU were purchased from Merck Life Sciences through Sigma-Aldrich. Chitosan, sodium selenite, and vitamin C in the nano-selenium preparation section were purchased from Sigma Reagents.

[0049] Glossary:

[0050] Se represents a single-nano selenium delivery system;

[0051] Se-p300 pPROTAC represents a finished product that conjugates a peptide drug and a nano-selenium delivery system.

[0052] peptide control represents naked peptide drugs without a coupled nano-selenium delivery system.

[0053] P300 represents the target protein P300.

[0054] Vinculin, a representative plaque protein, is used as an internal control in Western blotting.

[0055] Chx stands for cyclohexylimide, a protein synthesis inhibitor;

[0056] PROTAC represents a peptide that targets P300.

[0057] Example 1

[0058] The amino acid sequence of the P300-targeting peptide (PROTAC) of the present invention is CPWIWDGDNKDDNSTDGGGSGGGTSFEQFWAWLWP (SEQ ID NO:1).

[0059] The spatial structure of the peptide targeting P300 of this invention is shown in [reference needed]. Figure 1 A in the middle.

[0060] The rhodamine-labeled peptide targeting P300 was co-incubated with P300 protein for 30 min, followed by fluorescence polarization detection. The specific steps were as follows: First, the P300-targeting peptide was labeled with rhodamine at a concentration of 10 nM per well in a 384-well plate. Next, the P300 protein, expressed and purified in *E. coli*, was diluted sequentially to 10 nM–10 μM and co-incubated with the rhodamine-labeled peptide. After 30 min, the fluorescence polarization value was detected using a Tecan M1000 microplate reader.

[0061] The results are as follows Figure 1 The binding constant of PROTAC to P300 protein B is 78.3 nM.

[0062] Determination of half-life: 40 μL of a 1 mg / mL selenium-conjugated peptide drug was co-incubated with 60 μL of 20% FBS at 37 °C. Subsequently, after co-incubation for 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, 40 μL of the co-incubation liquid was added to an equal volume of acetonitrile for LC-MS analysis to determine the drug's half-life.

[0063] Depend on Figure 1 The results of C showed that the half-life of PROTAC was 10.19 hours.

[0064] Example 2

[0065] The preparation method of the peptide drug coupled with selenium nanoparticles (Se-p300 pPROTAC) includes the following steps:

[0066] 1 mg of the P300-targeting peptide from Example 1 was dissolved in 1 mL of pure water, followed by the addition of 0.6 mL of 5% chitosan (prepared by dissolving 0.5 g of chitosan in 10 mL of pure water), 0.2 mL of 50 mM sodium selenite, and 1.6 mL of 50 mM vitamin C. The reaction was heated at 50 °C for 20 min, and then cooled to room temperature to obtain the selenium-conjugated peptide drug Se-p300pPROTAC.

[0067] The morphology, zeta potential, and particle size of the peptide drug coupled with selenium nanoparticles were determined by transmission electron microscopy (TEM), zeta potential analysis, and DLS dynamic light scattering.

[0068] Figure 2 The results showed that the peptide drug with conjugated selenium nanoparticles prepared in this invention was spherical with a particle size of 176.4±48.4 nm and a zeta potential of +47.3±5.25 mV.

[0069] Example 3

[0070] Cellular level experiments of the Se-p300pPROTAC drug prepared in Example 2

[0071] 1. Cell culture methods: The culture conditions for prostate cancer C4-2, 22Rv1, PC3, and DU145 cell lines were all 1640 medium, 10% fetal bovine serum, saturated humidity of 5% CO2, adherent culture at 37℃, with a maximum cell density of 90% confluence. The culture conditions for NE1.3 cell line remained unchanged except that the 10% fetal bovine serum was replaced with 10% androgen-free fetal bovine serum.

[0072] 2. Experiment on the ability of drugs to inhibit cancer cell proliferation

[0073] The Cell Counting Kit-8 assay was used to analyze the ability of drugs to inhibit cancer cell proliferation.

[0074] The Cell Counting Kit-8 (CCK-8) is a rapid, highly sensitive assay kit based on WST-8, widely used for detecting cell proliferation and cytotoxicity. WST-8 is an MTT-like compound that, in the presence of an electron coupling reagent, can be reduced by certain dehydrogenases in mitochondria to produce an orange-yellow compound. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same number of cells, the color intensity is linearly related to the cell number.

[0075] In the experiment to detect cell viability, C4-2, 22Rv1, PC3, DU145, and NE1.3 cells were seeded at a density of 4500 cells / well into TC (Tissue Culture treated) 96-well plates, and 100 μL of cell culture was added to each well. After 24 hours of adherent cell culture, different concentrations of Se-p300 pPROTAC prepared in Example 3 (156 nM, 312.5 nM, 625 nM, 1250 nM, 2500 nM, 5000 nM, 10000 nM) were added to the cells for treatment. Different concentrations of naked selenium nanoparticles (Nano Se) and naked peptide drugs without a selenium nanoparticle delivery system were added as drug control groups (the drug control groups were substituted into the formula according to the drug-treated group). A complete control group (untreated group) consisting only of cells and culture medium without any drug treatment, a blank group (culture medium only without cells), and a background group (containing only (156 nM–10000 nM) Se-p300 pPROTAC without cells) were also set up. After 48 hours of treatment, 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. After color development, the absorbance of each well was measured at 450 nm and 690 nm using a microplate reader and a spectrophotometer. The absorbance of each well was then calibrated according to Formula I. Finally, cell viability was calculated using Formula II.

[0076] A = OD 450 -OD 690 Formula I

[0077] Cell viability (%) = [A(drug-treated) - A(background)] / [A(complete control) - A(blank)] × 100 Formula II

[0078] After drug treatment, the inhibitory effect of Se-p300 pPROTAC on the proliferation of prostate cancer cells was detected and calculated.

[0079] See results Figure 3 A, B, C, D and Figure 4 The A,Se-p300 pPROTAC drug showed dose-dependent growth inhibition in C4-2, 22Rv1, PC3, DU145, and NE1.3 cells, while naked selenium nanoparticles (Nano Se) and naked peptide control (without a nano-selenium delivery system) showed only slight inhibitory effects at high concentrations. The half-maximal inhibitory concentration (IC500) of Se-p300 pPROTAC drug in C4-2, 22Rv1, PC3, DU145, and NE1.3 cells was [not specified].50 The values ​​were 1693 nM, 3064 nM, 1411 nM, 807.5 nM and 716.9 nM, respectively.

[0080] 3. To investigate the degradation ability of the Se-p300 pPROTAC drug on P300, Western blotting (WB) analysis was used. The specific experimental procedure is as follows:

[0081] (1) Mix C4-2, 22Rv1, PC3, DU145, and NE1.3 at approximately 7 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well into 6-well plates treated with TC (tissue culture), with 2 mL of cell culture added to each well. After 24 h of adherent culture, different concentrations (0 μM, 0.08 μM, 0.15 μM, 0.3 μM, 0.6 μM, 1.25 μM, 2.5 μM, 5 μM) of Se-p300pPROTAC were added to the cells for treatment. After 24 h of treatment, RIPA lysis buffer containing a suitable protease inhibitor was added to each well, and the lysis buffer was collected.

[0082] (2) The total protein content in each sample group was quantified using the BCA quantitative kit, and the protein concentration in each sample group was made consistent by adjusting the sample volume. After adjusting the protein content, loading buffer was added, and the sample was in a 100°C metal bath for 10 minutes to completely denature the protein.

[0083] (3) Samples from different groups were separated by SDS-PAGE. A 10% polyacrylamide separating gel containing SDS and a 4% polyacrylamide stacking gel were prepared. The prepared samples and an equal volume of pre-stained protein samples were then added to the wells for electrophoretic separation. The electrophoresis conditions were as follows: the voltage was set to 90V, and separation was carried out for about 30 minutes until the bromophenol blue reached the separating gel. Then the voltage was adjusted to 120V, and separation was carried out for about 60 minutes until the bromophenol blue reached about 1 cm from the end of the separating gel. Electrophoresis was then stopped.

[0084] (4) Transfer the protein samples to a membrane. In this invention, all Western blotting experiments used PVDF membranes, arranged in the following order on the transfer apparatus: three layers of filter paper, PVDF membrane, gel, and three layers of filter paper. The transfer voltage was set to 120V, and the transfer time was 3 hours.

[0085] (5) Blocking. The PVDF membrane after transfer was immersed in a blocking solution containing 5% BSA and incubated at room temperature for 1 hour to remove the effects of non-specific adsorption.

[0086] (6) Primary antibody incubation. Prepare different antibody dilution solutions as needed, and then incubate at 4°C overnight to achieve the purpose of antibody recognition of specific antigens.

[0087] (7) Secondary antibody incubation. Prepare species-specific HRP-labeled secondary antibodies (anti-mouse or anti-rabbit) according to the different species from which the primary antibodies are derived, and dilute them 1:2000. Then incubate at room temperature for 1 hour.

[0088] (8) Color development. Prepare a color development solution, soak the PVDF membrane that has been fully incubated with the secondary antibody, and perform color analysis using a chemiluminescence analyzer.

[0089] Figure 3 E~H and Figure 4 The results in B showed that the Se-p300 pPROTAC drug can effectively degrade P300 in C4-2, 22Rv1, PC-3, DU145 and NE1.3 in a dose-dependent manner, with DC50 of approximately 500 nM, 300 nM, 1000 nM, 1000 nM and 500 nM, respectively.

[0090] 4. The method for determining the degradation of P300 by Se-p300pPROTAC is as follows:

[0091] Approximately 80,000 C4-2 cells were seeded in a laser confocal microplate. After the cells adhered for 24 hours, 500 nM of Se-p300 pPROTAC was added into the cells. After treatment for 0 h, 2 h, 4 h, 8 h, and 12 h, the cells were subjected to immunofluorescence assay.

[0092] The cell immunofluorescence detection method is as follows:

[0093] A. Fixed

[0094] (1) Rinse the sample twice with cold PBS.

[0095] (2) Fix the sample with 4% paraformaldehyde for 15 minutes.

[0096] (2) Rinse the sample twice with cold PBS.

[0097] B. Transparent

[0098] (1) Treat the sample with 0.1% Triton X-100 for 10 minutes.

[0099] (2) Rinse the cells with PBS 3 times.

[0100] C. Sealing and Incubation

[0101] (1) Cells were incubated in PBST containing 1% BSA for 30 minutes to block the non-specific binding of antibodies.

[0102] (2) Cells and antibodies (diluted with PBST containing 1% BSA) were incubated together in a humidified chamber overnight at 4°C.

[0103] (3) Discard the liquid and rinse the cells with PBS for 3-5 minutes.

[0104] (4) Incubate cells with secondary antibody in 1% BSA at room temperature for 1 hour, protected from light.

[0105] (5) Discard the secondary antibody solution, rinse the cells with PBS for 3-5 minutes, and protect from light.

[0106] D. Restaining

[0107] (1) Incubate cells with DAPI (DNA staining agent) for 1 minute.

[0108] (2) Rinse with PBS.

[0109] E. Sealing

[0110] (1) Add a drop of sealing liquid to the coverslip to seal it.

[0111] (2) Seal with a coverslip to prevent drying and move to a laser confocal microscope for observation.

[0112] The results showed that immunofluorescence staining of P300 in C4-2 cells after treatment with Se-p300 pPROTAC also confirmed that Se-p300 pPROTAC induced P300 degradation (see...). Figure 3 (I in the middle).

[0113] 5. Use Western blotting to detect whether the drug can accelerate the degradation of P300 and reduce the half-life of P300 protein. The detection method is as described in the Western blotting (WB) analysis method above.

[0114] Cycloheximine (CHX) effectively inhibits intracellular protein synthesis and is therefore commonly used in experiments to detect protein degradation half-life. Here, cells were treated with cycloheximine (CHX) containing or without 500 nM Se-p300 pPROTAC at different time points, and changes in protein levels were monitored. Gray-scale analysis of Western blotting showed that in both AR-negative PC-3 prostate cancer cell lines and AR-positive C4-2 prostate cancer cell lines, Se-p300 pPROTAC induced p300 degradation in a time-dependent manner (see [link to article]). Figure 3 (J and K in the middle).

[0115] 6. Se-p300 pPROTAC drug induces apoptosis in NE1.3 cells in vitro.

[0116] The ability of Se-p300pPROTAC to induce apoptosis in prostate cancer cells was assessed by flow cytometry using the FITC-Annexin V / PI assay kit (Biolegend). NE1.3 cells were cultured at 3 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of [number] cells / mL into 6-well plates and treated with different concentrations (500 nM, 1000 nM) of Se-p300 pPROTAC. A negative control group (containing 1000 nM naked selenium nanoparticles) and a control group (containing only cell culture medium) were also included. After 24 hours of culture, cells were collected and analyzed by flow cytometry. Each group was tested in triplicate.

[0117] Figure 4 The results showed that Se-p300 pPROTAC significantly induced apoptosis in the neuroendocrine prostate cancer cell line NE1.3. 500 nM Se-p300 pPROTAC induced an apoptosis rate of 65.97% in NE1.3 cells, and 1000 nM Se-p300 pPROTAC induced an apoptosis rate of 68.56%.

[0118] Example 4

[0119] Validating the effects of the Se-p300 pPROTAC drug prepared in Example 2 at the animal level

[0120] To evaluate the therapeutic effect of Se-p300 pPROTAC in vivo, 25 mice were randomly divided into 5 groups to establish a PC3 subcutaneous transplantation prostate cancer mouse model. When the tumor volume grew to 50-100 mm, the tumor was transplanted into the target tumor. 3 Drug treatment began at a specific time. Specifically, mice were treated every 3 days with the same dose of PBS, naked selenium nanoparticles, Se-p300 pPROTAC, peptide control, or I-CBP112 (a P300 inhibitor, used as a positive control) for 18 consecutive days. Tumor volume was continuously observed and recorded. Tumor volume was calculated using Formula III. All drugs were administered intraperitoneally every 3 days, with an injection volume of 100 μL and a final concentration of 5 mg / kg. Tumor size changes were recorded during injection. After 18 days of drug treatment, mice were euthanized, and the tumor sites and other major organs were removed and processed for HE staining and Ki67 staining.

[0121] 1. Tumor volume (V) = length × width 2 / 2Formula III.

[0122] 2. HE (HEMATOXYLIN-EOSIN STAINING) staining method

[0123] (1) Organizational stability.

[0124] Freshly removed tissue is placed in a pre-prepared 10% formalin fixative to denature and coagulate the proteins in the tissue and cells, preventing autolysis or bacterial decomposition after cell death, thus preserving the original morphology and structure of the cells.

[0125] (2) Tissue dehydration.

[0126] After fixation, the tissue was trimmed to 25px × 25px × 5px and rinsed with pure water to remove the fixative. Then, the tissue was gradually replaced with alcohol, starting from a low concentration and gradually increasing the concentration. The tissue block was then placed in xylene, a clearing agent soluble in both alcohol and paraffin, to replace the alcohol in the tissue block with xylene before paraffin embedding.

[0127] (3) Tissue embedding.

[0128] The transparent tissue block is placed in melted paraffin wax and kept warm in a paraffin melting box. After the paraffin wax has completely penetrated the tissue block, it is allowed to cool and solidify into a block.

[0129] (4) Tissue sections.

[0130] The embedded wax block is fixed on a microtome and cut into thin slices, typically 5–8 μm thick.

[0131] (5) Staining the sections.

[0132] Before staining, the paraffin in the sections needs to be removed again with xylene, followed by immersion in high-concentration to low-concentration alcohol, and finally in pure water to remove the alcohol. Then, staining begins by placing the sections in a hematoxylin solution for 10 minutes. Next, the sections are separated by acid and ammonia solutions for a few seconds each. After rinsing with pure water, the sections are dehydrated in 70% and 90% alcohol for 10 minutes each, and then stained with alcohol-eosin staining solution for 2 minutes.

[0133] (6) Slice and dehydrate.

[0134] The stained sections were dehydrated again using the same tissue dehydration method described above.

[0135] (7) Cover the film.

[0136] Apply resin to the cleared section, cover with a coverslip, and then observe and photograph it under a microscope.

[0137] 3. Ki67 Immunohistochemical Staining Method

[0138] (1) Tissue fixation and sectioning.

[0139] The same HE staining, fixation, and sectioning methods were used as described above.

[0140] (2) Dewaxing and hydration.

[0141] Paraffin sections were placed in fresh xylene and soaked for 15 minutes twice. After removing excess liquid, they were placed in anhydrous ethanol and soaked for 3 minutes twice. After removing excess liquid, they were placed in 95% ethanol and soaked for 3 minutes. After removing excess liquid, they were placed in 85% ethanol and soaked for 3 minutes. They were then rinsed with tap water for 1 minute and rinsed with PBS solution for 3 minutes three times.

[0142] (3) High-pressure heating of citric acid tissue antigen repair solution.

[0143] Heat the citric acid tissue antigen retrieval solution to boiling on a high-power induction cooker with the lid off. Place the dewaxed and hydrated sections on a high-temperature staining rack and put them into the pressure cooker. Cover the cooker, close the pressure valve, and continue heating until steam is released. Start timing for 2 minutes, then remove the pressure cooker from the heat source and allow it to cool naturally for 5 minutes. Rinse with tap water to cool it down, remove the valve and open the lid. After the liquid in the cooker has cooled to room temperature, remove the sections. Rinse with distilled water for 3 minutes x 2 times. Rinse with PBS solution for 3 minutes x 3 times.

[0144] (4) Block endogenous peroxidase.

[0145] Remove the PBS solution, add 100 μL of endogenous peroxidase inhibitor to the area of ​​tissue to be tested circled by the oil pen, and incubate at room temperature for 10 minutes. Rinse with PBS solution for 3 minutes × 3 times.

[0146] (5) Add Ki-67 antibody or blank control reagent.

[0147] Remove the PBS solution, add 100 μL of Ki-67 antibody or blank control reagent, and incubate at room temperature for 60 minutes. Rinse with PBS solution for 3 minutes each time.

[0148] (6) Add enzyme-labeled polymer.

[0149] Remove the PBS solution, add 100 μL of enzyme-labeled goat anti-mouse / rabbit IgG polymer, and incubate at room temperature for 15 minutes. Rinse with PBS solution for 3 minutes × 3 times.

[0150] (7) Color development.

[0151] Remove the PBS solution, add 100-200 μL of freshly prepared DAB chromogenic solution, incubate for 3-5 minutes, and observe the staining results under a light microscope for no more than 10 minutes.

[0152] (8) Re-dyeing.

[0153] Rinse with tap water, then incubate with 100–200 μL of hematoxylin somatic staining solution for 10–30 seconds. Rinse with PBS solution or tap water to restore blue color.

[0154] (9) Dehydration, clearing, and sealing.

[0155] Soak in 85% ethanol for 3 minutes; soak in 95% ethanol for 3 minutes; soak in anhydrous ethanol for 3 minutes; clear with xylene; seal with neutral resin and coverslip. Then observe and photograph under a microscope.

[0156] like Figure 5 As shown in Figures A through E, naked selenium nanoparticles (Nano-Se) and naked peptide drugs (Peptide Control) without conjugated nano-selenium delivery systems did not inhibit tumor growth. Se-p300pPROTAC showed high efficacy in the PC3 xenograft model, while ICBP-112 inhibited tumor growth in the PC3 xenograft model, but its efficacy was not as good as that of Se-p300pPROTAC.

[0157] To evaluate the safety of Se-p300 pPROTAC, mice were treated with the same dose of PBS, naked selenium nanoparticles (Nano-Vehicle), Se-p300 PROTAC (p300 PROTAC), peptide control, or I-CBP112 (a P300 inhibitor, serving as a positive control) every 3 days for 18 consecutive days. All drugs were administered intraperitoneally every 3 days, with an injection volume of 100 μL and a final concentration of 15 mg / kg. After drug treatment, blood was collected for subsequent blood biochemical assays, and mice were euthanized. Tissue sections and HE staining were performed on various organs.

[0158] like Figure 5 As shown in F to H, in this model, Se-p300 pPROTAC did not show significant toxicity in the blood system, heart, liver, spleen, kidneys, and other organs.

[0159] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide that targets and degrades P300, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The binding constant between the polypeptide and P300 is 78.3 nM.

3. A drug for the prevention and treatment of prostate cancer, characterized in that, The drug comprises the polypeptide and drug carrier as described in claim 1 or 2.

4. The drug according to claim 3, characterized in that, The drug carrier includes one or more of gold nanoparticles, liposomes, and selenium nanoparticles.

5. A method for preparing a polypeptide drug coupled with selenium nanoparticles, characterized in that, Includes the following steps: The peptide that targets and degrades P300 as described in claim 1 or 2 is mixed with water, and then chitosan, sodium selenite and vitamin C are added and heated to react. After the reaction is completed, the residual reagents are removed by dialysis to obtain the peptide drug coupled with nano-selenium.

6. The preparation method according to claim 5, characterized in that, The mass-to-volume ratio of the peptide targeting P300 degradation to water is 1 mg: 0.5~2 mL; the mass-to-volume percentage concentration of chitosan is 4%~6%; the molar concentration of sodium selenite is 45~55 mM; the molar concentration of vitamin C is 45~55 mM; and the volume ratio of chitosan, sodium selenite, vitamin C to water is 0.5~0.7:0.1~0.3:1.5~2:0.5~1.

5.

7. The preparation method according to claim 5, characterized in that, The heating reaction temperature is 45~55℃; the heating reaction time is 15~25min.

8. The use of the peptide targeting P300 degradation according to claim 1 or 2, the drug according to claim 3 or 4, or the peptide drug conjugated with selenium nanoparticles prepared by any one of claims 5 to 7 in the preparation of drugs for the prevention and / or treatment of prostate cancer.

9. The application according to claim 8, characterized in that, The cells mentioned in the prostate cancer include one or more of AR-positive prostate cancer cells, AR-negative prostate cancer cells, and NEPC cell lines.

10. The application according to claim 8, characterized in that, The prostate cancer cells include at least one of the following prostate cells: C4-2, 22Rv1, PC3, DU145, and NE1.3.

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