A bicyclic peptide targeting degradation of foxa1 protein and preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, specifically disclosing a bicyclic peptide for targeted degradation of FOXA1 protein, its preparation method, and its applications. The amino acid sequence of the bicyclic peptide for targeted degradation of FOXA1 protein is shown in SEQ ID NO.1. The bicyclic peptide for targeted degradation of FOXA1 protein provided by this invention can effectively degrade FOXA1 protein and exhibits the ability to significantly inhibit the proliferation of prostate cancer cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a bicyclic peptide (FTBPA: Foxa1 Targeting Bicyclic Peptide Autotac drug) that targets and degrades FOXA1 protein, its preparation method, and its application. Background Technology

[0002] Prostate cancer is one of the most common cancers in men, ranking second in incidence among solid malignant tumors. With an aging population and changes in dietary habits, prostate cancer is becoming one of the malignant tumors seriously affecting men's quality of life and health. The androgen receptor (AR) is a core driving factor in the occurrence and development of prostate cancer. Currently, the main treatments for prostate cancer are surgery and castration therapy (ADT) targeting the androgen receptor (AR) signaling pathway. However, while these therapies have achieved some success, a significant proportion of prostate cancer patients develop resistance after a period of treatment, thus progressing to castration-resistant prostate cancer (CRPC), which has a poor prognosis. Therefore, developing novel prostate cancer treatment strategies is urgently needed. Summary of the Invention

[0003] To develop a novel treatment strategy for prostate cancer, this invention provides a bicyclic peptide (FTBPA) that targets and degrades FOXA1 protein, its preparation method, and its applications. The FTBPA provided by this invention can effectively degrade FOXA1 protein and exhibits the ability to significantly inhibit the proliferation of prostate cancer cells.

[0004] This invention provides a bicyclic peptide that targets and degrades FOXA1 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] The bicyclic peptide for targeting and degrading FOXA1 protein provided by this invention can effectively degrade FOXA1 protein and has shown the ability to significantly inhibit the proliferation of prostate cancer cells.

[0006] Furthermore, the preparation process of the bicyclic peptide targeting the degradation of FOXA1 protein is as follows: the peptide shown in SEQ ID NO.1 is synthesized using a peptide solid-phase synthesis method, and the peptide is induced to form a bicyclic peptide using bismuth bromide.

[0007] The present invention also provides a method for preparing the bicyclic peptide that targets and degrades FOXA1 protein, comprising the following steps:

[0008] The polypeptide shown in SEQ ID NO.1 was synthesized using a solid-phase polypeptide synthesis method.

[0009] Bismuth bromide-induced peptide to form bicyclic peptide: The peptide shown in SEQ ID NO.1 was mixed with PBS and bismuth bromide was added. The mixture was reacted at 20℃~25℃ for 2h~4h. After purification, a bicyclic peptide that targets and degrades FOXA1 protein was obtained.

[0010] Furthermore, the final concentration of bismuth bromide is 0.5 mg / ml to 2 mg / ml.

[0011] Furthermore, the final concentration of bismuth bromide is 0.95 mg / mL to 1 mg / mL.

[0012] The present invention also provides the use of the bicyclic peptide that targets and degrades FOXA1 protein in the preparation of drugs for the treatment and / or prevention of prostate cancer.

[0013] Furthermore, the prostate cancer cells are at least one of CWR22Rv1, C4-2, LNCaP, and VCaP cells.

[0014] Furthermore, the drug uses a bicyclic peptide that targets and degrades the FOXA1 protein as its sole active ingredient.

[0015] The present invention also provides the application of the bicyclic peptide that targets and degrades FOXA1 protein in the preparation of products that degrade FOXA1 protein.

[0016] Furthermore, the degraded FOXA1 protein product uses a polypeptide or bicyclic peptide with an amino acid sequence as shown in SEQ ID NO.1 as the sole active ingredient.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides a bicyclic peptide (FTBPA) conjugated with bismuth bromide, the amino acid sequence of which is shown in SEQ ID NO.1. The binding constant of this bicyclic peptide (FTBPA) to FOXA1 protein is 197 nM, indicating that the bicyclic peptide (FTBPA) has a strong binding affinity to FOXA1 protein. Delivery of the bicyclic peptide (FTBPA) to prostate cancer cells effectively degrades FOXA1 protein and demonstrates a significant inhibitory effect on prostate cancer cell proliferation. FTBPA exhibits dose-dependent growth inhibition in CWR22Rv1, C4-2, LNCaP, and VCaP cells. Treatment with 10 mg / kg FTBPA showed high efficacy in a prostate cancer PDX model and significantly reduced the levels of FOXA1 and AR in tumor tissue. The half-maximal inhibitory concentration (IC50) of FTBPA in the prostate cancer PDO model is [not specified in the original text]. 50 The value is 1.8 μM.

[0019] FTXA1-targeting bicyclic peptides (FTBPA) can be used in the preparation of drugs for the prevention and / or treatment of prostate cancer or in the preparation of products that degrade FTXA1 protein. They can provide a new treatment strategy for drug-resistant and advanced castration-resistant prostate cancer, and in particular, can solve the practical problem of the lack of FTXA1-targeting drugs. They have very broad application value and drug development prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Characterization and evaluation of the bicyclic peptide (FTBPA) for targeted degradation of FOXA1 protein provided by the present invention;

[0022] In the figure, A represents the spatial structure of FTBPA predicted by Alphafold3;

[0023] B represents the binding affinity test between FTBPA and FOXA1;

[0024] C represents the ability of FTBPA to be taken up by C4-2 and CWR22Rv1 cells using confocal microscopy.

[0025] D represents the ability of FTBPA to be taken up by C4-2 and CWR22Rv1 cells using flow cytometry.

[0026] Figure 2 The results are for the in vitro assay of FTBPA's inhibitory activity against proliferation.

[0027] In the figure, A shows the concentration-dependent results and statistical analysis of drug degradation of FOXA1 protein in prostate cancer cells CWR22Rv1 by Western blotting (treated with different concentrations of FTBPA for 24 hours).

[0028] B shows the concentration-dependent results and statistical analysis of FOXA1 protein in prostate cancer cells C4-2 detected by Western blotting (treated with different concentrations of FTBPA for 24 hours).

[0029] C represents the concentration-dependent results and statistical analysis of FOXA1 protein in LNCaP prostate cancer cells detected by Western blotting (treated with different concentrations of FTBPA for 24 hours).

[0030] D represents the concentration-dependent results and statistical analysis of FOXA1 protein in VCaP of prostate cancer cells detected by Western blotting (treated with different concentrations of FTBPA for 24 hours).

[0031] E represents the ability of drugs to inhibit the proliferation of prostate cancer cells (CWR22Rv1, C4-2, LNCaP, VCaP) as detected using the CCK-8 assay;

[0032] F represents the time-dependent results and statistical analysis of drug-induced degradation of FOXA1 protein in prostate cancer cells CWR22Rv1 by Western blotting (treatment with 1000 nM FTBPA for different times).

[0033] G represents the time-dependent results and statistical analysis of drug-induced degradation of FOXA1 protein in prostate cancer cells C4-2 by Western blotting (treatment with 1000 nM FTBPA for different times).

[0034] H represents the results and statistical analysis of drug degradation of FOXA1 protein in prostate cancer cells (CWR22Rv1, C4-2) using immune cell imaging methods.

[0035] Figure 3 To detect the in vivo efficacy of FTBPA in a nude mouse subcutaneous xenograft tumor model;

[0036] In the figure, A represents the statistical results of the effects of different drugs on tumor volume;

[0037] B represents the effects of different drugs on tumor growth;

[0038] C represents the statistical results of the impact of different drugs on tumor quality;

[0039] D represents the representative results of HE staining, FOXA1 staining, AR staining, and Ki67 staining of tumor tissues after different drug treatments;

[0040] E represents the scoring results of FOXA1 staining (left), AR staining (middle), and Ki67 staining (right) after different drug treatments.

[0041] Figure 4 Images of heart, liver, spleen, lung, and kidney tissues after treatment with different drugs, stained with hematoxylin and eosin (HE).

[0042] Figure 5 The results of in vivo efficacy assays of FTBPA in patient-derived tumor xenograft models (PDX) and patient-derived organoid models (PDO);

[0043] In the figure, A represents the effects of different drugs on tumor growth;

[0044] B represents the statistical results of the effects of different drugs on tumor volume;

[0045] C represents the statistical results of the impact of different drugs on tumor quality;

[0046] D represents the representative results of FOXA1 staining in tumor tissues treated with different drugs;

[0047] E represents the AR staining results of tumor tissues treated with different drugs;

[0048] F represents the representative results of Ki67 staining of tumor tissues after different drug treatments;

[0049] G represents the scoring of FOXA1 staining results after different drug treatments;

[0050] H represents the scoring of AR staining results after different drug treatments;

[0051] I represents the scoring of Ki67 staining results after different drug treatments;

[0052] J represents the results of prostate cancer organoid formation assays on day 0 and day 4 after treatment with different concentrations of FTBPA.

[0053] K represents the viability assay results of prostate cancer organoids treated with different concentrations of FTBPA. Detailed Implementation

[0054] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0055] This invention provides a bicyclic peptide (FTBPA) for targeted degradation of FOXA1 protein, its preparation method and application, wherein the amino acid sequence of the bicyclic peptide (FTBPA) for targeted degradation of FOXA1 protein is shown in SEQ ID NO.1.

[0056] SEQ ID NO. 1: CREEECQYYPPFSDDAC.

[0057] The binding constant of the bicyclic peptide to the FOXA1 protein is 197 nM. The bicyclic peptide is synthesized using a solid-phase peptide synthesis method. This invention does not impose any particular limitation on the solid-phase synthesis method of the bicyclic peptide; any peptide synthesis method well-known in the art can be used, such as Fmoc peptide synthesis.

[0058] The bicyclic peptide (FTBPA) for targeted degradation of FOXA1 protein prepared in this invention was delivered to prostate cells and found to degrade FOXA1 protein in the cells and significantly inhibit the proliferation of prostate cancer cells. FTBPA showed an IC50 inhibitory effect on CWR22Rv1 prostate cancer cells. 50 The IC50 value for C4-2 prostate cancer cells was 2.8 μM. 50 The IC50 value for LNCaP prostate cancer cells was 1.3 μM. 50 The IC50 value for VCaP prostate cancer cells was 1.7 μM. 50 The concentration is 3.6 μM. In this invention, the FTBPA has been experimentally verified to be non-toxic at the animal level and has high safety.

[0059] In this invention, the prostate cancer cells include at least one of the following prostate cancer cells: CWR22Rv1, C4-2, LNCaP, and VCaP. This invention also provides a drug for the prevention and treatment of prostate cancer, the drug comprising the above-mentioned bicyclic peptide conjugated with bismuth bromide (FTBPA).

[0060] Based on this, the present invention also provides an application of the above-mentioned bicyclic peptide (FTBPA) that targets and degrades FOXA1 protein, wherein the application is at least one of the following:

[0061] (1) Application of the bicyclic peptide (FTBPA) that targets and degrades FOXA1 protein in the preparation of FOXA1 protein degradation products.

[0062] (2) The use of the bicyclic peptide (FTBPA) that targets and degrades FOXA1 protein in the preparation of drugs for the prevention and / or treatment of prostate cancer.

[0063] The following examples illustrate the preparation method and application of the FOXA1 protein-targeting bicyclic peptide (FTBPA) provided by the present invention, but these should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1: A bicyclic peptide (FTBPA) for targeted degradation of FOXA1 protein and its preparation method.

[0065] I. Preparation method of bicyclic peptide for targeted degradation of FOXA1 protein

[0066] 1. Experimental materials

[0067] Synthesis Section: Fmoc protected amino acids were purchased from Shanghai Jier Biochemical; Rink Amide MBHA resin was purchased from Haipu Functional Materials Co., Ltd.; dichloromethane (DCM), N,N-dimethylformamide (DMF), N,N-diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), anhydrous diethyl ether, acetonitrile, and bismuth bromide were purchased from Sigma-Aldrich, a subsidiary of Merck Life Sciences. Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT) condensing agents were from Suzhou Haofan Biotechnology.

[0068] The bismuth bromide in the coupled bismuth bromide portion was purchased from Sigma Reagents.

[0069] Explanation of English names in this invention:

[0070] FTBPA stands for bicyclic peptide (FTBPA) that targets and degrades FOXA1 protein.

[0071] Linear Peptide represents a peptide that targets and degrades the FOXA1 protein;

[0072] FOXA1 represents the target protein FOXA1.

[0073] Vinculin stands for focal adhesion protein, which serves as an internal control in Western blotting.

[0074] Both FTBPA and Linear Peptide target and degrade FOXA1 protein.

[0075] 2. Synthesis Method

[0076] The polypeptide was synthesized using a solid-phase polypeptide synthesis method according to the amino acid sequence shown in SEQ ID NO.1. The specific synthesis method is as follows:

[0077] (1) Resin swelling and pretreatment

[0078] Based on the desired peptide sequence targeting the degradation of FOXA1 protein, Rink Amide MBHA resin with a substitution value of 0.30-0.40 mmol / g was selected as the solid-phase synthesis support. 1 g of Rink Amide MBHA resin was weighed and placed in a solid-phase synthesis column. 10 mL of dichloromethane (DCM) was added to completely submerge the resin. After swelling at room temperature (25°C) for 30 min, the liquid was drained. Then, 5 mL of deprotection solution (prepared by mixing piperidine and DMF at a volume ratio of 1:4) was added, and the reaction was carried out at room temperature for 8 min. The liquid was then drained, and this process was repeated twice to remove the Fmoc groups from the resin. The column was then thoroughly washed with DMF (6 times, 5 mL each time) until no piperidine odor was detected, and the column was drained to await the next reaction step.

[0079] (2) Ligation and deprotection of starting amino acids

[0080] The Fmoc-protected amino acid corresponding to the first amino acid at the C-terminus of SEQ ID NO.1 (2 mmol, equivalent to 5 molar equivalents of the resin loading), condensing agent HBTU (2 mmol, 5 molar equivalents), HOBT (2 mmol, 5 molar equivalents), and DIEA (2 mmol, 5 molar equivalents) were added to 5 mL of DCM and activated for 5 min. The activated solution was then added to the swollen and deprotected resin from step (1), and nitrogen gas was introduced while stirring. The reaction was carried out at room temperature for 40 min. After the reaction, the column was thoroughly washed with DMF (6 times, 5 mL each time) to remove unreacted raw materials and byproducts. Then, 5 mL of deprotection solution was added, and the reaction was carried out at room temperature for 8 min, followed by draining the liquid. This process was repeated twice to completely remove the Fmoc protecting group of the starting amino acid. The column was then thoroughly washed with DMF (6 times, 5 mL each time) until no piperidine odor was detected, and the column was drained to await the next reaction step.

[0081] (3) Stepwise coupling of amino acids

[0082] For each subsequent amino acid in SEQ ID NO.1, the steps in (2) are repeated using the corresponding Fmoc protected amino acid cycle until all designed amino acids are sequentially added to complete the stepwise assembly of the polypeptide chain and obtain the polypeptide-resin complex.

[0083] (4) Peptide cutting and crude product acquisition

[0084] The synthesized peptide-resin complex was transferred to a dry container. Then, 8 mL of cleavage mixture (TFA and water at a volume ratio of 19:1) was added, and the mixture was stirred at room temperature for 3 hours. After the reaction, the resin was washed with 2 mL of TFA, and the filtrates were combined. The filtrate was then slowly added dropwise to 100 mL of pre-cooled (4 °C) anhydrous diethyl ether, resulting in the formation of a white flocculent precipitate. The precipitate was centrifuged at 4 °C and 5000 rpm for 10 min, and the supernatant was carefully discarded. The precipitate was washed and centrifuged twice more with 20 mL of pre-cooled (4 °C) anhydrous diethyl ether (4 °C, 5000 rpm, 10 min) to thoroughly remove residual TFA and organic impurities. The crude peptide targeting the degradation of FOXA1 protein was obtained by freeze-drying at -60 °C for 48 h and stored at -20 °C.

[0085] (5) Polypeptide purification and identification

[0086] The crude peptide was dissolved in 10 mL of 20% acetonitrile (acetonitrile and water were mixed at a volume ratio of 1:4), and purified by high-performance liquid chromatography (HPLC). HPLC was performed using a C18 reversed-phase column, with the mobile phases being 0.1% TFA aqueous solution (TFA and water were mixed at a volume ratio of 1:999) and 0.1% TFA-acetonitrile solution (TFA and acetonitrile were mixed at a volume ratio of 1:999), respectively. After mass spectrometry identification, the purified peptide (Linear Peptide) targeting the degradation of FOXA1 protein was obtained.

[0087] (6) Bismuth bromide induces bicyclic peptide

[0088] Add 10 mg of the peptide obtained above that targets and degrades FOXA1 protein (approximately 5 μmol) to 5 mL of PBS buffer (pH=7.4), mix well, then add 5 mg of bismuth bromide (approximately 10 μmol, 2 molar equivalents) and mix thoroughly. Add DMSO (final concentration ratio of 5%) and react at 25°C for 4 h. After the reaction is complete, use the method in (5) for separation, purification and mass spectrometry identification to obtain the final FOXA1-targeting bicyclic peptide degradation drug FTBPA, and store at -20°C.

[0089] The amino acid sequences of the linear peptide and the bicyclic peptide (FTBPA) targeting the degradation of FOXA1 protein are both shown in SEQ ID NO.1.

[0090] The spatial structure of the bicyclic peptide (FTBPA) for targeted degradation of FOXA1 protein in this invention is shown below. Figure 1 A in the middle.

[0091] 3. Detection of the affinity between the bicyclic peptide FTBPA (targeting FOXA1 protein degradation) and FOXA1 protein.

[0092] The affinity of FTBPA for FOXA1 was determined using isothermal titration calorimetry (ITC). FOXA1 protein was expressed using an *E. coli* expression system and purified by nickel column affinity chromatography. FTBPA was synthesized and prepared using the aforementioned method. In the ITC assay, a 200 μL volume of 20 μM FOXA1 protein solution was first placed in a temperature-controlled sample cell, with both the sample and reference cells under the same external environment. A 40 μL volume of 200 μM FTBPA solution was then added to the syringe. The ITC assay was performed at 25 °C, with an initial injection volume of 0.4 μL, followed by 19 titrations of 2 μL each. As the sample reacted with the titrant, the temperatures of the sample and reference cells changed. The energy of this reaction change was sensitively detected by the ITC analyzer, and a constant temperature was maintained by triggering a thermostat through positive or negative feedback. After the experiment, the heat flow data was integrated using the instrument's software to obtain the corresponding thermodynamic parameters. Based on the endothermic and exothermic curves, the affinity K between FTBPA and FOXA1 is calculated using Formula I. D .

[0093] ΔG=-RT∙LnK D Formula I;

[0094] Where ΔG is the change in free energy, in J / mol; R is a constant, specifically 8.314 J / (mol·K); T represents the absolute temperature, in K; K D It represents the dissociation constant, reflecting the affinity between molecules.

[0095] See results Figure 1 The calculated affinity of FTBPA for FOXA1 protein was 197 nM.

[0096] Example 2: Application of a bicyclic peptide (FTBPA) that targets and degrades FOXA1 protein.

[0097] I. Cellular level experiments with FTBPA

[0098] 1. Cell Culture

[0099] (1) Prostate cancer cells CWR22Rv1 (CRL-2505), C4-2 (CRL-3314), LNCaP (CRL-1740), and VCaP (CRL-2876) were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were changed or passaged as needed depending on their condition.

[0100] (2) Cell passage

[0101] a) Remove the cells from the cell culture incubator and observe them under a microscope. If the adherent cells are observed to be in good condition and the density reaches 80% to 90%, passage the cells according to the experimental purpose.

[0102] b) Open the petri dish lid, carefully aspirate the old culture medium with a negative pressure aspirator, slowly add 5 mL of PBS solution along the wall of the dish to wash away the remaining culture medium, and aspirate it again after washing.

[0103] c) Add 1 mL of trypsin to the culture dish to digest the cells. Gently shake the dish to ensure the trypsin evenly coats the cell surface. Some difficult-to-digest cells need to be placed in an incubator to accelerate the digestion process. During digestion, closely observe the cells under a microscope. Once the cells become rounded and the intercellular connections disappear, digestion is complete.

[0104] d) Add 3 mL of fresh complete culture medium to the cell culture dish where the cells have been completely digested to stop the digestion reaction. Then, pipette the cell suspension evenly and transfer it to a 15 mL centrifuge tube.

[0105] e) Place the centrifuge tubes in a centrifuge and centrifuge at room temperature, 1000 rpm, for 5 min. After centrifugation, discard the supernatant, add 1 mL of fresh complete culture medium to the cell pellet, and gently pipette to resuspend the cells. Seed the cell suspension into new culture dishes according to the requirements of subsequent experiments.

[0106] 2. FTBPA uptake experiment

[0107] (1) Confocal microscopy analysis of FTBPA uptake

[0108] Before analyzing FTBPA uptake using confocal microscopy, FTBPA was preliminarily labeled with fluorescein Cy5. This labeling process involved the reaction of NHS-esterified Cy5 with the amino group of the peptide drug. For drug uptake assays, 2 × 10⁻⁶ FTBPA was pre-labeled with fluorescein Cy5. 5 CWR22Rv1 and 2×10 5C4-2 cells were seeded into glass-bottomed culture dishes. After culturing for 24 hours, 100 nM Cy5-labeled FTBPA was added to each dish, followed by incubation for 1, 3, and 6 hours. The group without 100 nM Cy5-labeled FTBPA was designated as the 0-hour group. After sample collection, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then stained with DAPI. Observation and photography were performed under a NiKon A1R-si confocal microscope. All images were obtained with the same excitation wavelength and detector gain settings (scale bar: 50 μm).

[0109] The results are as follows Figure 1 As shown in Figure C, after 6 hours, most of the FTBPA had been taken up by CWR22Rv1 or C4-2 cells and entered the nucleus.

[0110] (2) Flow cytometry analysis of FTBPA uptake

[0111] When analyzing FTBPA uptake using flow cytometry with the aforementioned fluorescein Cy5-labeled FTBPA, CWR22Rv1 or C4-2 cells were cultured at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured for 24 hours to allow them to adhere. Subsequently, 100 nM Cy5-labeled FTBPA was added to the culture dish, and incubation continued for 1, 3, and 6 hours. A control group without the drug (100 nM Cy5-labeled FTBPA) was also included. After treatment, cells were gently washed twice with pre-chilled PBS, digested with trypsin, and resuspended in PBS to prepare single-cell suspensions. Samples were analyzed using a BD FACSCalibur flow cytometer, with Cy5 fluorescence signal excited by a 633 nm laser and fluorescence intensity collected at 650 nm in the far-red channel. At least 10,000 cells were collected from each sample tube, and statistical analysis was performed using FlowJo software.

[0112] The results are as follows Figure 2 As shown in Figure D, after 6 hours, all FTBPA was taken up by CWR22Rv1 or C4-2 cells.

[0113] 3. Experiment on the ability of FTBPA to inhibit the proliferation of prostate cancer cells

[0114] The Cell Counting Kit-8 assay analyzes the ability of drugs to inhibit the proliferation of prostate cancer cells. The Cell Counting Kit-8 (CCK-8) is a rapid, highly sensitive assay 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. The specific method is as follows:

[0115] In experiments to detect cell viability, CWR22Rv1, C4-2, LNCaP, and VCaP cells were resuspended in RPMI-1640 cell culture medium at a concentration of 5 × 10⁻⁶. 4 Cell suspensions at a density of cells / mL were seeded into 96-well cell culture plates, 100 μL per well. After 24 h of cell adhesion culture, different concentrations of FTBPA (0 nM, 8 nM, 16 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, 2000 nM) were added to the cells as treatment groups. Simultaneously, a complete control group (cells and culture medium only, no other components), a blank group (culture medium only, no cells), and a background group (culture medium only, no cells, with the corresponding concentration of FTBPA) were set up. After 48 h of treatment, 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C for 2 h. After color development, the absorbance of each well was measured at 450 nm and 690 nm using a spectrophotometer. The absorbance of each well was calibrated according to Formula II. Finally, cell viability was calculated according to Formula III. To evaluate the inhibitory effect of FTBPA on the proliferation of prostate cancer cells.

[0116] A = OD 450 -OD 690 Formula II;

[0117] In the formula, A is the calibrated absorbance value; OD 450 Absorbance at 450nm wavelength; OD 690 The absorbance value is at a wavelength of 690nm.

[0118] Cell viability (%) = [A(drug-treated) - A(background)] / [A(complete control) - A(blank)] × 100% (Formula III)

[0119] In the formula, A(drug-added) represents the calibrated absorbance value of the drug-added group; A(background) represents the calibrated absorbance value of the background group; A(complete control) represents the calibrated absorbance value of the complete control group; and A(blank) represents the calibrated absorbance value of the blank group.

[0120] See results Figure 2 In CWR22Rv1, C4-2, LNCaP, and VCaP cells, FTBPA exhibited dose-dependent growth inhibitory effects. The half-maximal inhibitory concentration (IC50) of FTBPA against CWR22Rv1, C4-2, LNCaP, and VCaP prostate cancer cells was also measured. 50 The concentrations were 2.8 μM, 1.3 μM, 1.7 μM, and 3.6 μM, respectively. This indicates that FTBPA exhibited dose-dependent growth inhibition in CWR22Rv1, C4-2, LNCaP, and VCaP cells.

[0121] 4. Analysis of FTBPA's ability to degrade FOXA1

[0122] The degradation ability of FTBPA on FOXA1 was analyzed using Western blotting (WB). The specific experimental procedure was as follows:

[0123] (1) CWR22Rv1, C4-2, LNCaP, and VCaP cells were resuspended in RPMI-1640 cell culture medium to a concentration of 3×10⁻⁶ cells / mL. 5Cell suspensions at a density of cells / mL were seeded into 6-well cell culture plates, with 2 mL of cell suspension added to each well. After culturing the cells in a constant temperature cell culture incubator at 37 °C and 5% CO2 for 24 h, different concentrations of FTBPA (0 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 4000 nM) were added to the 6-well plates for 24 h, or 1000 nM FTBPA was added for 0, 1, 3, 6, 12, and 24 h. After treatment, the culture medium was aspirated and the cells were washed once with PBS. 100 μL of RIPA lysis buffer (Dingguo Biotechnology, WB-0072) containing 1 mmol / L LMSF (Dingguo Biotechnology, WB-0181) was added to each well, and the cells were incubated on ice for 10 min for lysis. The cell lysates were scraped and collected, and centrifuged in a pre-chilled ultracentrifuge at 4 °C and 15,000 rpm for 15 min. After centrifugation, the supernatant was transferred to a new labeled 1.5 mL EP tube. (2) The total protein content in each sample was quantified using the BCA quantitative kit (Dingguo Biotechnology, BCA01), and the protein concentration in each sample was made consistent by adjusting the sample volume. After adjusting the protein concentration, the corresponding volume of 5×SDS protein loading buffer (Dingguo Biotechnology, WB-0091) was added, and the sample was heated in a 100℃ metal bath for 10 minutes to completely denature the protein. Protein samples for immunoblotting experiments were then obtained and stored at -20℃.

[0124] (3) Separate the samples from different groups using SDS-PAGE. Prepare a 10% polyacrylamide separating gel containing SDS and a 5% polyacrylamide stacking gel. Then add the prepared samples and an equal volume of pre-stained protein samples to the sample wells for electrophoretic separation. The electrophoresis conditions are as follows: set the voltage to 80V, separate for about 30 minutes until bromophenol blue reaches the separating gel. Then adjust the voltage to 120V, separate for about 60 minutes until bromophenol blue reaches about 1 cm from the end of the separating gel, and then stop electrophoresis.

[0125] (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, gel, PVDF membrane, and three layers of filter paper. The transfer current was set to 350 mA, and the transfer time was 2 h.

[0126] (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.

[0127] (6) Primary antibody incubation. Dilute the primary antibody using the primary antibody dilution buffer (Saiwell Biotech, G2025) according to the antibody instructions. Anti-Foxa1 antibody (GeneTex, GTX100308) and Anti-Vinculin antibody (Saiwell, GB111328) are prepared at a 1:1000 ratio with the primary antibody dilution buffer. Place the PVDF membrane from the previous step into the prepared primary antibody solution and incubate overnight at 4°C to achieve antibody recognition of the specific antigen.

[0128] (7) Secondary antibody incubation. Prepare species-specific HRP-labeled secondary antibodies (anti-mouse or anti-rabbit) according to the different species of the primary antibody source, and dilute them 1:2000. Then incubate at room temperature for 1 hour.

[0129] (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.

[0130] The results are as follows Figure 2 A~ Figure 2 As shown in Figure D, FTBPA degrades FOXA1 protein in a dose-dependent manner, with the half-maximal degradation concentration (DC) of FTBPA for FOXA1 protein in CWR22Rv1, C4-2, LNCaP, and VCaP prostate cancer cells being the highest. 50 The values ​​were 1.1 μM, 1.8 μM, 1.9 μM, and 1 μM, respectively. For example... Figure 2 F and Figure 2 As shown in G, FTBPA degrades FOXA1 protein in a time-dependent manner.

[0131] 5. The degradation of FOXA1 by FTBPA was determined using cellular immunofluorescence. The specific experimental method is as follows:

[0132] 2×10 5 Two CWR22Rv1 and C4-2 cells were seeded into a glass-bottomed culture dish. After culturing in a constant temperature cell culture incubator at 37°C and 5% CO2 for 24 hours, 1000 nM FTBPA was added, and the cells were treated for 0, 1 h, 3 h, 6 h, and 12 h, respectively, before immunofluorescence detection was performed.

[0133] The cell immunofluorescence detection method is as follows:

[0134] A. Fixation: (1) Rinse the sample twice with PBS pre-cooled at 4°C. (2) Fix the sample for 15 minutes with 4% paraformaldehyde pre-cooled at 4°C. (3) Rinse the sample twice with PBS pre-cooled at 4°C.

[0135] B. Permeability: (1) Treat the sample with 0.1% Triton X-100 for 10 minutes. (2) Rinse the cells 3 times with PBS.

[0136] C. Blocking and Incubation: (1) Incubate cells in PBST containing 1% BSA for 30 minutes to block non-specific antibody binding. (2) Incubate cells with antibody (diluted in PBST containing 1% BSA) overnight at 4°C. (3) Discard the liquid and rinse cells with PBS for 4 minutes. (4) Incubate cells with secondary antibody in 1% BSA at room temperature for 1 hour, protected from light. (5) Discard the secondary antibody solution and rinse cells with PBS for 4 minutes, protected from light.

[0137] D. Counterstaining: (1) Incubate cells with DAPI (DNA staining agent) for 1 minute. (2) Rinse with PBS.

[0138] Observation and photography were performed under a NiKon A1R-si confocal microscope. All images were obtained with the same excitation wavelength and detector gain settings (scale bar: 20 μm).

[0139] The results are as follows Figure 2 As shown in Figure H, after 6 hours of FTBPA treatment, the FOXA1 protein in CWR22Rv1 and C4-2 cells was significantly degraded.

[0140] II. Evaluation of the Application of FTBPA in a Subcutaneous Prostate Cancer Xenograft Model

[0141] 1. Evaluation of the therapeutic effect of FTBPA drugs in vivo

[0142] Twenty 4-week-old male BALB / c nude mice were randomly divided into four groups (n=5 per group), and then 5×10⁶ mice were resuspended in 50 μL of PBS. 6 One CWR22Rv1 cell was mixed 1:1 with 50 μL of matrix gel (Corning, #354234) and subcutaneously injected into the right groin of BALB / c nude mice. When the tumor volume reached 50 mm... 3 Drug treatment began at that time.

[0143] Drug treatment involved administering the same doses of PBS, unconjugated bismuth bromide peptide (Linear Peptide), FTBPA 5 mg / kg, and FTBPA 10 mg / kg to mice every 3 days for 21 consecutive days. Tumor volume was continuously observed and recorded. Tumor volume was calculated using Formula IV. All drugs were injected intraperitoneally every 3 days in 100 μL volumes, with tumor size changes recorded during injection. After 21 days of drug treatment, the mice were euthanized, and the tumor sites and other major organs were removed and subjected to HE or immunohistochemical (IHC) staining. IHC scores were calculated using Formula V.

[0144] Tumor volume (V) = length × width 2 / 2 Formula IV.

[0145] IHC score = weak positive rate × 1 + positive rate × 2 + strong positive rate × 3 Formula V.

[0146] 2. HE staining method

[0147] (1) Tissue fixation: Fresh 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 maintaining the original morphology and structure of the cells. (2) Tissue dehydration: The fixed tissue is trimmed to 25px×25px×5px and rinsed with pure water to remove the fixative. Then, the tissue is gradually replaced with alcohol from low to high concentration. The tissue block is 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 it can be embedded in paraffin. (3) Tissue embedding: The cleared tissue block is placed in melted paraffin and kept warm in a paraffin bath. After the paraffin has completely penetrated the tissue block, it is allowed to cool and solidify into a block. (4) Tissue sectioning: The embedded paraffin block is fixed on a microtome and cut into thin sections about 6μm thick. (5) Staining the sections: Before staining the tissue, the paraffin in the sections needs to be removed again with xylene, then passed through high-concentration to low-concentration alcohol, and finally immersed in pure water to remove the alcohol. Then, staining begins. The sections are stained in hematoxylin solution for 10 minutes. Then, the sections are placed in acid water and ammonia water for color separation 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 in alcohol-eosin staining solution for 2 minutes. (6) Re-dehydration of the sections: The stained sections are dehydrated again in the same way as the tissue dehydration method described above. (7) Mounting: The transparent sections are dripped with resin and covered with a coverslip for mounting. Then, they are observed under a microscope and photographed.

[0148] 3. Ki67 Immunohistochemical Staining Method

[0149] (1) Tissue fixation and sectioning: Same as the HE staining fixation and sectioning method described above.

[0150] (2) Dewaxing and hydration: The 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; rinsed with tap water for 1 minute; rinsed with PBS solution for 3 minutes three times.

[0151] (3) High-pressure heating of citric acid tissue antigen retrieval solution: The pressure cooker is opened and heated to boiling on an induction cooker at high power; the dewaxed and hydrated sections are placed on a high-temperature staining rack and then placed in the pressure cooker; the lid is closed, the pressure valve is closed, and heating continues until steam is released. After timing for 2 minutes, the pressure cooker is removed from the heat source and allowed to cool naturally for 5 minutes; the sections are cooled by rinsing with tap water, the valve is removed and the lid is opened, and the sections are removed after the liquid in the pot has cooled naturally to room temperature; the sections are rinsed with distilled water for 3 minutes × 2 times; and rinsed with PBS solution for 3 minutes × 3 times.

[0152] (4) Blocking endogenous peroxidase: 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.

[0153] (5) Add Ki-67 antibody or blank control reagent: Remove 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 × 3 times.

[0154] (6) Add enzyme-labeled polymer: 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.

[0155] (7) Color development: Remove the PBS solution, add 150 μL of freshly prepared DAB color development solution, incubate for 4 minutes, and observe the staining results under a light microscope for no more than 10 minutes.

[0156] (8) Counterstaining: Rinse with tap water, add 150 μL of hematoxylin somatic staining solution and incubate for 20 seconds. Rinse with PBS solution or tap water to return to blue.

[0157] (9) Dehydration, clearing, and sealing.

[0158] 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 under a microscope and photograph, and perform IHC scoring according to Formula V.

[0159] 4. FOXA1 and AR immunohistochemical staining methods

[0160] Similar to the staining and scoring methods for Ki-67 described above, FOXA1 antibody and AR antibody were used for the corresponding procedures.

[0161] The results are as follows Figure 3 A~ Figure 3 As shown in Figure D, neither PBS nor the uncoupled bismuth bromide peptide (Linear Peptide) inhibited tumor growth. FTBPA 5 mg / kg and FTBPA 10 mg / kg demonstrated high efficacy in the CWR22Rv1 xenograft model and significantly reduced the levels of FOXA1 and AR in tumor tissue. Figure 4 As shown in the model, FTBPA drugs did not exhibit significant toxicity in organs such as the heart, liver, spleen, lungs, and kidneys.

[0162] III. Evaluation of the effectiveness of FTBPA in patient-derived tumor xenograft (PDX) models for prostate cancer

[0163] 1. PDX Model Establishment

[0164] Prostate cancer tissue was obtained from the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China), with written informed consent obtained from the donor patients. To establish a PDX model, fresh tissue was rapidly excised from primary prostate tumors via needle aspiration and collected in DMEM medium containing Primocin. Subsequently, following the standard operating procedure described in the literature (Karkampouna S, La Manna F, Benjak A, et al. Patient-derived xenografts and organoids model therapy response in prostate cancer. Nat Commun. 2021;12(1):1117), these tissue blocks were subcutaneously implanted into anesthetized 4-week-old BALB / c nude mice. After the tumor block reached approximately 1000 mm³ in size, it was dissected and reimplanted into new recipient mice. After three passages, the established PDX model will be used for subsequent animal experiments.

[0165] 2. Drug treatment

[0166] Ten mice were randomly divided into two groups: a control group and an FTBPA 10 mg / kg treatment group. Tumor volume increased to 80 mm. 3Drug treatment began at the specified time. Specifically, the control group received PBS every 2 days, while the FTBPA 10 mg / kg treatment group received 10 mg / kg FTBPA every 2 days. Treatment was administered via intraperitoneal injection, with an injection volume of 100 μL. Tumor size changes were recorded during injection for 14 consecutive days. After 14 days of drug treatment, the mice were euthanized, and the tumor sites and other major organs were removed and processed for HE or immunohistochemical (IHC) staining. Tumor volume and IHC scoring were performed as before.

[0167] 3. HE and IHC treatment

[0168] The treatment methods for HE and IHC are the same as before.

[0169] The results are as follows Figure 5 A~ Figure 5 As shown in Figure I, PBS had no inhibitory effect on tumor growth, while treatment with 10 mg / kg FTBPA showed high efficacy in the prostate cancer PDX model and significantly reduced the levels of FOXA1 and AR in tumor tissue.

[0170] IV. Evaluation of the effectiveness of FTBPA in patient-derived tumor organoid models (PDO) for prostate cancer.

[0171] After extracting prostate cancer tissue from the patient, it was immediately placed in pre-cooled PBS at 4°C. Excess tissue, such as fat and blood vessels, was removed using ophthalmic scissors and forceps. The tumor tissue was cut into tissue blocks with a volume of approximately 2 mm³ and placed in enzymatic digestion solution for digestion. Digestion conditions were 37°C with shaking for 30 minutes. The digestion process was monitored continuously. When a large number of single cells were observed under a microscope, three times the volume of prostate cancer organoid culture buffer was added to stop the digestion. The mixture was filtered through a 100 μm sieve, and the filtrate was collected. The filtrate was centrifuged at 300g for 5 minutes, and the supernatant was removed. The centrifuged cells were resuspended in prostate cancer organoid culture buffer, and the collected tissue volume was observed. 25 times the tissue volume of matrix gel was added to resuspend the cells and plate them. The plated culture plates were placed in a 37°C incubator for about 12 minutes to allow the matrix gel to solidify. Finally, 500 μL of prostate cancer organoid culture medium was added at room temperature for culturing. The culture medium should cover the matrix gel, but avoid adding it directly to the gel droplets. The medium should be completely changed every 2 days to maintain its freshness and sufficient nutrients. Observe the culture of organoids under a microscope and record their growth status and morphological changes.

[0172] To perform drug sensitivity testing, the culture plates were removed from the incubator, photographed under a microscope for archiving, and then placed in a biosafety cabinet. The old culture medium was completely removed, and an appropriate amount of digestion solution was added. The mixture was collected in 15 mL centrifuge tubes, and the cells were dispersed by pipetting. The tubes were then incubated at 37°C for 4 min, followed by the addition of PBS to terminate the digestion. The cells were centrifuged at 500 g for 3 min at 4°C, and the supernatant was discarded. The pellet was resuspended in 1 mL of culture medium, gently mixed by pipetting, and resuspended again by adding an appropriate amount of matrix gel. Cells were then cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded in 5 μL volumes into 96-well plates (operated on an ice box), and incubated for 10 min before adding 90 μL of culture medium. After 3 days of culture, FTBPA at concentrations of 0, 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM, and 10.00 μM were added, and the cells were cultured for another 4 days. CellTiter-GloCTG reagent was then added, and chemiluminescence detection was performed using a microplate reader. Cell viability was then tested according to formula VI.

[0173] PDO activity (%) = [RLU] (药物) -RLU (Blank) ] / [RLU (阴性) -RLU (Blank) ]×100% formula VI;

[0174] Among them, PDO activity represents organoid activity; RLU （药物） RLU represents the luminescence value of cell-containing pores at a specific drug concentration. （阴性） RLU represents the luminescence value of cell-containing pores at a drug concentration of 0. （Blank） The fluorescence value is the value of the blank culture medium without inoculated cells.

[0175] like Figure 5 J represents the results of prostate cancer organoid formation assays on day 0 and day 4 after drug treatment. Figure 5 K represents the viability assay results of prostate cancer organoids treated with FTBPA at concentrations of vehicle (vector control group), 0.32 μM, 0.64 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM. The half-maximal inhibitory concentration (IC50) of FTBPA in the prostate cancer PDO model is also shown. 50 The value is 1.8 μM.

[0176] It should be noted that the solvent used in the preparation of FTBPA drugs of different concentrations in this invention is PBS (pH=7.4, Savill Biotechnology, G4202).

[0177] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0178] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A bicyclic peptide that targets and degrades FOXA1 protein, characterized in that, The bicyclic peptide targeting FOXA1 protein degradation was prepared by bismuth bromide induction of the polypeptide shown in SEQ ID NO.

1. The preparation method was as follows: the polypeptide shown in SEQ ID NO.1 was mixed evenly with PBS, bismuth bromide was added, and the mixture was reacted at 20℃~25℃ for 2h~4h. After purification, the bicyclic peptide targeting FOXA1 protein degradation was obtained.

2. The bicyclic peptide targeting the degradation of FOXA1 protein according to claim 1, characterized in that, The polypeptide shown in SEQ ID NO.1 was synthesized using a solid-phase polypeptide synthesis method.

3. The bicyclic peptide targeting the degradation of FOXA1 protein according to claim 2, characterized in that, In the preparation method, the final concentration of bismuth bromide is 0.5 mg / ml to 2 mg / ml.

4. The bicyclic peptide for targeted degradation of FOXA1 protein according to claim 3, characterized in that, In the preparation method, the final concentration of bismuth bromide is 0.95 mg / mL to 1 mg / mL.

5. The use of the bicyclic peptide of claim 1, which targets and degrades FOXA1 protein, in the preparation of a drug for the treatment and / or prevention of prostate acinar carcinoma.

6. The application according to claim 5, characterized in that, The prostate acinar carcinoma cells are at least one of CWR22Rv1, C4-2, LNCaP, and VCaP cells.

7. The application according to claim 5, characterized in that, The drug uses a bicyclic peptide that targets and degrades the FOXA1 protein as its sole active ingredient.

Images