A screening method for a candidate compound for reducing docetaxel resistance of prostate cancer patients and application thereof

Abstract

The application relates to the technical field of tumor biology, and particularly discloses a screening method of a candidate compound for reducing docetaxel resistance of prostate cancer patients and application. The screening method comprises the following steps: detecting the influence of a compound on the combination of B-cell lymphoma-2 protein and UBR family protein IV; and when the compound promotes the combination of B-cell lymphoma-2 protein and UBR family protein IV, the compound is a candidate compound. The application screens the candidate compound for reducing docetaxel resistance of prostate cancer patients by detecting the influence of the compound on the combination of B-cell lymphoma-2 protein and UBR family protein IV, so that the intrinsic and adaptive DTX resistance induced by TGF-beta in PCa bone metastasis is overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor biology, in particular to a screening method for a candidate compound for reducing docetaxel resistance of prostate cancer patients and application thereof. BACKGROUND

[0002] According to the global cancer incidence and mortality from 2007 to 2023, prostate cancer (PCa) ranks first in the global male cancer incidence and is the second leading cause of death from male cancer in the world. Most prostate cancers remain androgen-dependent at least in the initial stage, and androgen deprivation therapy (ADT) has been the main treatment for metastatic prostate cancer. With time, the patient's response to ADT weakens, and the cancer progresses to castrate-resistant prostate cancer (CRPC). CRPC accounts for most of the deaths from prostate cancer, so CRPC has been the focus of basic research and drug development in the past few years.

[0003] CRPC patients initially benefit from docetaxel (DTX) as first-line treatment, DTX actively depolymerizes microtubules in proliferating cells by binding to β-tubulin, inducing cytotoxicity. Studies have shown that the addition of DTX on the basis of ADT prolongs the life expectancy of patients with metastatic hormone-sensitive prostate cancer (10-13.6 months), indicating that more patients will receive chemotherapy, prolong survival time, and improve survival rate. However, most patients receiving DTX treatment eventually develop resistance.

[0004] Therefore, new treatment strategies are needed to treat DTX-resistant prostate cancer. SUMMARY

[0005] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide a screening method for a candidate compound for reducing docetaxel resistance of prostate cancer patients and application thereof, which aims to screen candidate compounds to solve the problem of poor treatment effect of prostate cancer patients due to docetaxel resistance.

[0006] The technical solution of the present application is as follows:

[0007] In a first aspect of the present application, a screening method for a candidate compound for reducing docetaxel resistance of prostate cancer patients is provided, comprising the step of: detecting the effect of a compound on the binding of B-cell lymphoma-2 protein to UBR family protein IV, wherein when the compound promotes the binding of B-cell lymphoma-2 protein to UBR family protein IV, the compound is a candidate compound.

[0008] Preferably, the screening method detects the binding of B lymphocytoma-2 protein to UBR family protein IV by NanoBRET technology.

[0009] Preferably, the step of detecting the effect of the compound on the binding of B-lymphocytoma-2 protein to UBR family protein IV comprises: expressing a fusion protein containing B-lymphocytoma-2 protein and HaloTag protein in a first cell, and expressing a fusion protein containing UBR family protein IV and NanoLuc protein in a second cell; co-culturing the first cell and the second cell, and treating them with the compound to obtain a sample to be tested; and detecting the sample to be tested using the NanoBRET technique to determine the effect of the compound on the binding of B-lymphocytoma-2 protein to UBR family protein IV.

[0010] Preferably, a first plasmid containing a gene encoding B lymphocytoma-2 protein and a gene encoding HaloTag protein is constructed, and the first plasmid is transferred into an engineered cell to obtain the first cell; a second plasmid containing a gene encoding UBR family protein IV and a gene encoding NanoLuc protein is constructed, and the second plasmid is transferred into an engineered cell to obtain the second cell.

[0011] Preferably, the engineered cells are selected from 293FT cells and C4-2B cells.

[0012] Preferably, the compound is selected from the compounds in the Taoshu biological metabolite library L7000 or the compounds in the FDA anticancer metabolite library.

[0013] Preferably, the step of detecting the sample to be detected comprises: adding a substrate to the sample to be detected, and measuring the absorbance OD of the sample to be detected at 460nm and 618nm respectively. 460 and OD 618 .

[0014] Preferably, the step of determining the effect of the compound on the binding of B lymphocytoma-2 protein to UBR family protein IV comprises: calculating the OD of the sample to be tested. 460 and OD 618 The ratios are sorted by numerical value, and the compounds in the top ten tested samples are considered to promote the binding of B lymphocytoma-2 protein to UBR family protein IV and are candidate compounds.

[0015] The second aspect of the present application provides a use of a candidate compound obtained by the above-mentioned method for screening candidate compounds that reduce docetaxel resistance in prostate cancer patients in the preparation of a drug for reducing docetaxel resistance in prostate cancer patients.

[0016] Preferably, the candidate compound is selected from proflavine sulfate, triptolide, atorvastatin calcium, epirubicin hydrochloride, lomitapide, HMN-214, Ret tyrosine kinase inhibitor, nor-dihydroguaiaretic acid, Src family kinase inhibitor, dimethoxyestradiol.

[0017] Advantages of the present application:

[0018] The prior art proves that B lymphoma-2 protein (Bcl-2) is degraded after docetaxel (DTX) treatment, TGF-β protects Bcl-2 from docetaxel-induced protein degradation in a ubiquitin-dependent manner, and Bcl-2, as an apoptosis inhibitor, mediates the chemotherapeutic resistance of various malignant tumors. It is further found in the present application that one of the pathways of DTX-induced Bcl-2 degradation is regulated by increasing the binding of Bcl-2 to the ligase domain of UBR4, and then the present application screens for compounds that can enhance the binding of Bcl-2 to UBR4 by targeting Bcl-2, and further screens for candidate compounds that can reduce the docetaxel resistance of prostate cancer patients, overcoming the intrinsic and adaptive DTX resistance induced by TGF-β in PCa bone metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows.

[0020] Figure 1 The protein screening analysis diagram provided by the embodiments of the present application for Bcl-2 protein binding:

[0021] A is a screening flowchart; B and C are T test analysis diagrams; D is the change of the binding ability of UBR4 to Bcl-2 under different conditions; E is the principal component analysis result;

[0022] Figure 2 The UBR4 expression level test diagram provided by the embodiments of the present application in tumor cells:

[0023] A is the protein level analysis diagram of UBR4 in each tissue in the Human Protein Atlas database; B is the expression level analysis diagram of UBR4 in tumor cells in the TCGA database; C is the qPCR detection UBR4 expression amount analysis diagram in cells resistant to DTX;

[0024] Figure 3 The UBR4 expression regulation test diagram provided by the embodiments of the present application:

[0025] A is a diagram showing the change in the expression level of Bcl-2 after overexpression of UBR4 in 293FT cells; B is a diagram showing the change in the expression level of Bcl-2 after overexpression of UBR4 in DU145 cells; C is a diagram showing the change in the expression level of Bcl-2 after overexpression of UBR4 in Hela cells; D is a test of the effect of TGF-β and DTX on the binding of UBR4 and Bcl-2;

[0026] Figure 4 A diagram showing the analysis of the binding domain of UBR4 and Bcl-2 in the process of degradation of Bcl-2 by UBR4 provided in the embodiments of the present application:

[0027] A is a structural diagram of a UBR4 truncation; B and C are analyses of the expression level of Bcl-2 in 293FT cells overexpressing different UBR4 truncations by Western blot;

[0028] Figure 5 A diagram showing the verification of the binding of Bcl-2 and the ligase domain of UBR4 in the degradation of Bcl-2 by DTX provided in the embodiments of the present application:

[0029] A is an analysis of the protein docking of EZH2 and UBR4 by Gramm; B is an analysis of the protein docking of Bcl-2 and UBR4 by Gramm; C is a Western blot detection of the expression level of Bcl-2 in 293FT cells overexpressing Bcl-2 and the ligase domain; D is an immunoprecipitation detection of the binding ability of Bcl-2 and the ligase domain in 293FT cells; E is the binding ability of Bcl-2 and the ligase domain in 293FT cells overexpressing Bcl-2 and the ligase domain after treatment with DTX;

[0030] Figure 6 A diagram showing the analysis of the binding of Bcl-2 and the ligase domain of UBR4 provided in the embodiments of the present application:

[0031] A is a schematic diagram of the NanoBRET system; B is an analysis of the binding of the ligase domain and Bcl-2; C is an analysis of the binding of the ligase domain and Bcl-2 after treatment of cells overexpressing the ligase domain and Bcl-2 with DTX and / or TGF-β;

[0032] Figure 7 A diagram showing the analysis of the effect of different compounds on the binding of Bcl-2 and UBR4 provided in the embodiments of the present application;

[0033] Figure 8A pHTC plasmid map provided for the embodiments of the present application is shown in FIG. 1.

[0034] Figure 9 A PNLF1-C plasmid map provided for the embodiments of the present application is shown in FIG. 2. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings and embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. The embodiments below and the features in the embodiments can be combined with each other without conflict.

[0036] It should be noted that if the description of "first", "second" and the like is involved in the embodiments of the present application, the "first", "second" and the like are only for the purpose of description, and cannot be understood as indicating or implying the relative importance and implicitly indicating the number of the indicated technical features. Therefore, the features with "first", "second" can explicitly or implicitly include at least one of the features. In addition, the technical solutions of each embodiment can be combined with each other, but the combination of the technical solutions must enable the person skilled in the art to realize the basis, and when the combination of the technical solutions is contradictory or cannot be realized, it should be considered that the combination of the technical solutions does not exist, and is not within the protection scope claimed by the present application.

[0037] In the chemotherapy of prostate cancer, docetaxel can enhance the binding of Bcl-2 and E3 ubiquitin ligase UBR4 ligase domain, thereby inducing the degradation of Bcl-2 through the ubiquitin proteasome pathway. However, due to the presence of transforming growth factor-β (TGF-β) in the microenvironment of bone metastasis, it can effectively inhibit the binding of the two, protect Bcl-2 from degradation, cause apoptosis escape, and thus cause secondary chemotherapy resistance. Venetoclax, as a therapeutic drug for chronic lymphocytic leukemia and acute myeloid leukemia, although it can target the inhibition of Bcl-2, it is very easy to cause neutropenia. Therefore, a better target for Bcl-2 in prostate cancer needs to be found.

[0038] Based on this, the first aspect of the embodiments of the present application provides a screening method for a candidate compound for reducing docetaxel resistance of prostate cancer patients, comprising the steps of: detecting the effect of the compound on the binding of B-cell lymphoma-2 protein and UBR family protein IV, and screening the candidate compound. When the compound inhibits the binding of B-cell lymphoma-2 protein and UBR family protein IV, the compound is a candidate compound.

[0039] B-cell lymphoma-2 gene (Bcl-2) is an apoptosis inhibitor that binds to pro-apoptotic protein bax to inhibit mitochondrial outer membrane permeability change Bcl-2. Bcl-2 is also reported to mediate the chemoresistance of various malignancies, including lung, lymphoid and thyroid tumors. Bcl-2 is mainly degraded by ubiquitin-proteasome system (UPS), and parkin protein and F-box protein can mediate Bcl-2 ubiquitination, thereby reducing its half-life. In addition, DTX can induce Bcl-2 to be phosphorylated, so that it cannot bind to bax protein, resulting in inactivation of Bcl-2. Due to the anti-apoptotic effect of Bcl-2, its high expression in prostate cancer also leads to reduced sensitivity to DTX.

[0040] In some embodiments, the binding of B-cell lymphoma-2 protein and UBR family protein IV is detected by NanoBRET technology. NanoBRET technology is a modified bioluminescence fluorescence resonance transfer technology that can realize real-time monitoring of protein interaction in living cells.

[0041] Further, the screening method specifically comprises the following steps:

[0042] S1, expressing a fusion protein containing B-cell lymphoma-2 protein and HaloTag protein in a first cell, and expressing a fusion protein containing UBR family protein IV and NanoLuc protein in a second cell. The fusion protein containing B-cell lymphoma-2 protein and HaloTag protein is the expression product of the recombination of the gene encoding Bcl-2 protein and the gene encoding HaoTag protein, and the fusion protein containing UBR family protein IV and NanoLuc protein is the expression product of the recombination of the gene encoding UBR4 protein and the gene encoding NanoLuc protein.

[0043] In some embodiments, a first plasmid containing a gene encoding B-cell lymphoma-2 protein and a gene encoding HaloTag protein is constructed, the first plasmid is transformed into an engineering cell to obtain the first cell; a second plasmid containing a gene encoding UBR family protein IV and a gene encoding NanoLuc protein is constructed, and the second plasmid is transformed into an engineering cell to obtain the second cell. Preferably, the gene encoding Bcl-2 protein in the first plasmid is before the gene encoding HaoTag protein, and in the second plasmid, the gene encoding UBR4 protein is before the gene encoding NanoLuc protein. The construction method of the expression vector of the fusion protein (the first plasmid and the second plasmid) is a conventional technical solution in the art, which is not described here.

[0044] In some embodiments, the engineered cells are selected from 293FT cells and C4-2B cells. The engineered plasmids used to construct the first and second plasmids are selected from pHTC plasmids or pNLF1-C plasmids. Preferably, the engineered plasmid used to construct the first plasmid is selected from pHTC plasmids, and the engineered plasmid used to construct the second plasmid is selected from pNLF1-C plasmids.

[0045] S2. Co-culturing the first cell and the second cell, and treating them with the compound to obtain a sample to be tested.

[0046] In some embodiments, the compound is selected from the compounds in the Taoshu Biometabolite Library L7000 or the compounds in the FDA Anticancer Metabolite Library.

[0047] S3. Testing the sample to be tested to determine the effect of the compound on the binding between B lymphocytoma-2 protein and UBR family protein IV.

[0048] In some embodiments, the specific step of S3 may be: the step of detecting the sample to be detected includes: adding a substrate to the sample to be detected, and measuring the absorbance OD of the sample to be detected at 460nm and 618nm respectively. 460 and OD 618 Calculate the OD of the sample to be tested 460 and OD 618 The ratios are sorted by numerical value, and the compounds in the top ten tested samples are considered to promote the binding of B lymphocytoma-2 protein to UBR family protein IV and are candidate compounds.

[0049] The substrate reacts with the NanoLuc protein to generate a donor light signal, and the HaloTag protein acts as an energy acceptor for the donor light signal. When UBR family protein 4 and B-lymphocytoma-2 protein interact, the NanoLuc donor light signal couples to the HaloTag protein, resulting in a superposition of spectral signals. The absorbance of the sample at 460nm and 618nm is then measured to determine the binding status of B-lymphocytoma-2 protein and UBR family protein IV.

[0050] In a second aspect of the embodiments of the present application, a candidate compound obtained by the above-mentioned method for screening candidate compounds that reduce docetaxel resistance in prostate cancer patients is provided.

[0051] In some embodiments, the candidate compound is selected from proflavine sulfate, triptolide, atorvastatin calcium, epirubicin hydrochloride, lomitapide, HMN-214, Ret tyrosine kinase inhibitors, nordihydroguaiaretic acid, Src family kinase inhibitors, and dimethoxyestradiol.

[0052] The following is further described by way of specific examples. Unless otherwise specified, the reagents, instruments, and test methods used in the examples are all conventional in the art.

[0053] Example 1

[0054] Proteins are extracted from DU 145 cells, purified, and enriched. The protein samples are then digested with enzymes to break them down into small peptides. The digested peptides are then subjected to mass spectrometry analysis to determine their mass and charge. Through mass spectrometry analysis, we can obtain the mass spectrum of the peptides and further infer the sequence and structure of the protein. The screening process is as follows: Figure 1 As shown in Figure 5A, the binding of the protein to Bcl-2 was analyzed by mass spectrometry.

[0055] The results are as follows Figure 1 As shown in Figures B, C, D, and E. Mass spectrometry analysis of 946 proteins binding to Bcl-2 revealed that 386 proteins were found to bind to Bcl-2. Of these 386 proteins, 87 showed reduced Bcl-2 binding after DTX treatment, while 71 showed differential Bcl-2 binding between the DTX-treated group and the combined DTX and TGF-β-treated group. Using this intersection, a total of 33 proteins were selected. Among these, UBR4, an E3 ligase, showed increased Bcl-2 binding after DTX treatment, but decreased binding after combined DTX and TGF-β treatment compared to DTX treatment. This was the only target protein that met expectations.

[0056] Example 2

[0057] In the Human Protein Atlas (https: / / www.proteinatlas.org / ENSG00000127481-UBR4 / tissue), the protein levels of UBR4 in various tissues are different. Among them, the UBR4 protein level in the prostate is in the first tier, making it a good target ( Figure 2 As shown in Figure A). Data analysis from the TCGA database showed that in prostate cancer, the expression level of UBR4 in tumors was lower than that in normal tissues ( Figure 2 The expression of UBR4 in DTX-resistant cells was detected by qPCR. Figure 2 As shown in C, it can be seen that the UBR4 level in resistant cells is lower than that in wild-type cells. This result coincides with the higher Bcl-2 protein level in tumors. It can be inferred that it is the reduction in UBR4 level that causes the reduced degradation of Bcl-2 protein, thereby causing apoptosis escape.

[0058] Example 3

[0059] To verify the regulation of UBR4 on Bcl-2, Bcl-2 gene and UBR4 gene were overexpressed in 293FT, DU145 and Hela cells at different levels. Among them, equal amount of Bcl-2 was overexpressed in 293FT, DU145 and Hela cells, and UBR4 was overexpressed in increasing concentrations. The expression levels of Bcl-2 and UBR4 were detected by Western blot, and the results are shown in Figure 3 A, B and C of FIG. 6. It can be seen from the results of A, B and C of FIG. 6 that in the three cells, the level of Bcl-2 showed a concentration-dependent decrease in UBR4. Figure 3

[0060] The binding of UBR4 and Bcl-2 was verified by co-immunoprecipitation experiment, and the results are shown in Figure 3 D of FIG. 7, in which C represents a negative control, D represents treatment with 10 nM DTX for 48 h, T represents treatment with 10 ng / ml TGF-β for 48 h, and TD represents combined treatment with 10 ng / ml TGF-β and 10 nM DTX for 48 h. It can be seen from Figure 3 D of FIG. 7 that when treated with 10 nM DTX for 48 h, the binding of the two increased. When treated with 10 ng / ml TGF-β and 10 nM DTX, the binding of the two decreased again.

[0061] Example 4

[0062] UBR4 is a UBR family protein, which has a UBR-box domain common to UBR family proteins, but lacks the E3 classic substrate binding domain, such as HECT or RING domain. UBR4 has a domain named ligase domain at 4367-5163, which is proved to mediate the degradation of UBR4 substrate. Based on this, a series of UBR4 truncations were constructed in this embodiment, including FL (containing the full length of UBR4), Δbox (UBR-box removed), NTD (only retaining the N-terminal 0-3184), CTD (only retaining the C-terminal 3185-5163), ΔLD (ligase domain removed) and LD (only retaining the ligase domain), as shown in Figure 4 A of FIG. 8.

[0063] Subsequently, the above-mentioned UBR4 truncations were overexpressed in 293FT cells, and the expression of Bcl-2 was detected by Western blot, and the results are shown in Figure 4 B and C of FIG. 9. It can be seen from Figure 4 ​As shown in the middle B and C, only the group of FL (containing the full length of UBR4) can significantly reduce the expression of Bcl-2, that is, only the full length of UBR4 can promote the degradation of Bcl-2, and neither the lack of UBR-box nor the lack of ligase domain can promote the degradation of Bcl-2, indicating that UBR-box and ligase domain both play an important role in the degradation of Bcl-2 by UBR4.

[0064] Example 5

[0065] The UBR family protein needs to meet the N-terminal rule to recognize the substrate. The UBR-box can recognize the N-terminal residues exposed after the substrate is cut, including the type I N-degron phenylalanine, tryptophan, tyrosine, leucine and isoleucine, the type II N-degron arginine, lysine, histidine, aspartic acid, glutamic acid and cysteine. According to the report of other literatures, Bcl-2 can be cut by caspase-3 to expose A32 and V35. However, neither alanine nor valine is the N-degron recognized by UBR-box, so there may be other cleavage sites of Bcl-2 to be explored.

[0066] Due to the complexity of the N-terminal rule, the ligase domain of UBR4 is preferentially studied. It has been reported that the ligase domain of UBR4 can mediate the degradation of EZH2. Therefore, protein docking is performed by Gramm (https: / / gramm.compbio.ku.edu / ) to analyze the docking results (as shown in the middle A and B). Figure 5 As shown in the middle A and B, EZH2 binds to the ligase domain through the N-terminal, and Bcl-2 can also bind to the ligase domain through the N-terminal, indicating that the binding of Bcl-2 to the ligase domain has certain similarity with EZH2.

[0067] Subsequently, in the 293FT cells overexpressing Bcl-2, the ligase domain was transiently transfected in gradient, and the expression of Bcl-2 in the cells was detected by qPCR, and the results are shown in the middle C. Figure 5 As shown in the middle C, the protein level of Bcl-2 decreases with the increase of the ligase domain, which is the same as the result of the group of overexpressing the full length gene of UBR4 in Example 4. Figure 5

[0068] ​Further, the binding of Bcl-2 and ligase domain was detected by co-immunoprecipitation experiment. Bcl-2-Myc and LD-Flag were co-expressed in 293FT cells, and LD-Flag was combined by Anti FLAG Nanobody Magarose Beads, and the results are shown in Fig. D. Then, 293FT cells overexpressing Bcl-2-Myc and LD-Flag were treated with 10 mM DTX, and the results are shown in Fig. E. As can be seen from Fig. E, after treatment with 10 nM DTX, the binding of Bcl-2 and ligase domain was significantly higher than that of the group without DTX treatment, indicating that DTX-induced degradation of Bcl-2 was caused by increasing its binding to the ligase domain of UBR4. Figure 5 Figure 5

[0069] Example 6

[0070] (1) The NanoBRET system was used for testing. By constructing fusion proteins with HaloTag and NanoLuc respectively, when the distance between the two groups is close enough, the NanoLuc fusion protein is used as the energy donor, and the fluorescently labeled HaloTag fusion protein is used as the energy acceptor, and the spectral signal at 618 nm can be detected, and the signal is enhanced with the shortening of the distance, as shown in Fig. A. Figure 6

[0071] The p53-HT plasmid was used to express the HaloTag protein, and the NL-MDM2 plasmid was used to express the NanoLuc protein as a control group; the Bcl-2-HT plasmid (pHTC plasmid with Bcl-2 gene) and LD-NL (pNLF1-C plasmid with ligase domain) were used as experimental groups, and the NanoBRET system was used for testing. When the distance between the two groups is close enough, the NanoLuc fusion protein is used as the energy donor, and the fluorescently labeled HaloTag protein is used as the energy acceptor, and the spectral signal at 618 nm is detected. The results are shown in Fig. B. As can be seen from Fig. B, the spectral signal of LD-NL and Bcl-2-HT is higher than that of the control group, proving the binding of ligase domain and Bcl-2. Figure 6 Figure 6

[0072] (2) DTX and TGF-β were used to treat cells expressing LD-NL and Bcl-2-HT respectively, and the results are shown in Fig. C. As can be seen from Fig. C, the spectral signal of LD-NL and Bcl-2-HT treated with DTX and TGF-β was higher than that of the control group, proving the binding of ligase domain and Bcl-2. Figure 6 Figure 6 ​​​​​​As can be seen from FIG. 6, the DTX treatment group did not enhance the spectral signal, but the TGF-β treatment group was able to reduce the spectral signal, indicating that TGF-β treatment can reduce the binding of Bcl-2 and ligase domain, proving that the reason why TGF-β protects Bcl-2 from degradation is that the ligase domain binding is reduced.

[0073] In combination with the results of Examples 1 to 6, it can be concluded that small molecule drugs can be screened by targeting Bcl-2, enhancing the binding of Bcl-2 and UBR4, and thus overcoming the intrinsic and adaptive DTX resistance induced by TGF-β in PCa bone metastasis.

[0074] Example 7

[0075] The materials used include: 293FT cells, pHTN plasmid (vector capable of expressing HaloTag protein tag), pNLF1-N plasmid (plasmid capable of expressing NanoLuc fusion protein), Bcl-2-HT plasmid (pHTC plasmid with Bcl-2 gene, as shown in FIG. 2), LD-NL (pNLF1-C plasmid with ligase domain, as shown in FIG. 3), Lipofectamine 3000 transfection reagent, NanoBRET Figure 8 substrate, Costar 3917 white 96-well plate, metabolite library: Tao Zhu Biology L7000, containing a total of 385 compounds. Figure 9 TM Substrate, NanoBRET TM 618 Ligand, Costar 3917 white 96-well plate, metabolite library: Tao Zhu Biology L7000, containing a total of 385 compounds.

[0076] The specific steps are as follows:

[0077] (1) 293FT cells were plated in 10 cm dishes, with a cell amount of 3.2 x 10 6 cells per dish, and cultured overnight in low serum medium without penicillin and streptomycin.

[0078] (2) 8 μg of Bcl-2-HT plasmid and 0.8 μg of LD-NL plasmid were transfected into the dishes of (1), respectively. At the same time, 8 μg of pHTN plasmid and 0.8 μg of pNLF1-N plasmid were transfected into another dish of (1) as a blank control group. After 6 h, the medium was changed to complete medium and cultured overnight.

[0079] (3) The 293FT cells in the dishes of (2) were plated in a white 96-well plate, with a cell amount of 2.2 x 10 4 ​One. Among them, 59 holes were laid in the experimental group (Bcl-2-HT+LD-NL), and 1 hole was laid in the control group (pHTN+pNLF1-N). 50 μL of DMEM medium and 0.05 uL of 618 ligand diluted 1:1000 were added to each hole. 100 μL of PBS buffer was added to the holes outside the periphery of the holes added with the test sample, and incubated overnight.

[0080] (4) In the holes containing test samples in the 96-hole plate in (3), 1 μL of metabolite drug diluted 40 times was added to the first 58 holes, and 1 μL of DMSO diluted 40 times was added to the remaining one experimental group hole and blank control group hole, and incubated for 24 hours.

[0081] (5) 12.5 μL of substrate NanoBRET TM Substrate was added to the experimental holes (a total of 60 holes) in (4), and after 10 minutes of reaction, the liquid in the holes was removed and the absorbance values OD at 460 nm and 618 nm were detected using a spectrophotometer 460 and OD 618 . And OD 460 / OD 618 value as the result for comparison, the results are shown in Figure 7 , according to Figure 7 and the data in Table 1, the metabolite with the highest OD 460 / OD 618 value is Proflavine hemisulfate. And the top ten metabolites with OD 460 / OD 618 value. The metabolites include Proflavine hemisulfate, Tripterin, Atorvastatin hemicalcium salt, Epirubicin hydrochloride, Lomitapide, HMN-214, Ret tyrosine kinase inhibitor (RPI-1), Nordihydroguaiaretic acid, Src family kinase inhibitor (SU6656), and 2-Methoxyestradiol.

[0082] Table 1, metabolite OD 460 / OD 618 value

[0083] Metabolite name (Treated OD 460 / OD 618 ) / (DMSO OD 460 / OD 618 )]]> Proflavine Hemisulfate 2.129179 Tripterin 1.959312 Atorvastatin hemicalcium salt 1.506338 Epirubicin hydrochloride 1.493866 Lomitapide 1.32556 HMN-214 1.26023 RPI-1 1.250511 Nordihydroguaiaretic acid 1.248209 SU6656 1.21975 2-Methoxyestradiol 1.216466

[0084] In summary, the present application further finds through experiments that one of the pathways of DTX-induced Bcl-2 degradation is regulated by increasing the binding of Bcl-2 and UBR4 ligase domain, and then the present application screens out a compound capable of enhancing the binding of Bcl-2 and UBR4 by targeting Bcl-2, and then screens out a candidate compound capable of reducing the docetaxel resistance of prostate cancer patients, overcoming the intrinsic and adaptive DTX resistance induced by TGF-β in PCa bone metastasis.

[0085] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the claims attached to the present application.

Claims

1. A method for screening candidate compounds for reducing docetaxel resistance in prostate cancer patients, characterized in that: The method comprises the steps of detecting the effect of a compound on the binding between B lymphocytoma-2 protein and UBR family protein IV; when the compound promotes the binding between B lymphocytoma-2 protein and UBR family protein IV, the compound is a candidate compound.

2. The screening method according to claim 1, wherein The binding of B-lymphocytoma-2 protein to UBR family protein IV was detected using NanoBRET technology.

3. The screening method according to claim 2, characterized in that The step of detecting the effect of the compound on the binding between B lymphocytoma-2 protein and UBR family protein IV comprises: A fusion protein containing B lymphocytoma-2 protein and HaloTag protein is expressed in a first cell, and a fusion protein containing UBR family protein IV and NanoLuc protein is expressed in a second cell; co-culturing the first cell and the second cell, and treating them with the compound to obtain a sample to be tested; The sample to be tested is tested by NanoBRET technology to determine the effect of the compound on the binding of B lymphocytoma-2 protein and UBR family protein IV.

4. The screening method according to claim 3, wherein constructing a first plasmid containing a gene encoding B lymphocytoma-2 protein and a gene encoding HaloTag protein, and transferring the first plasmid into an engineered cell to obtain the first cell; A second plasmid containing a gene encoding UBR family protein IV and a gene encoding NanoLuc protein is constructed, and the second plasmid is transferred into the engineered cell to obtain the second cell.

5. The screening method according to claim 4, characterized in that The engineered cells are selected from 293FT cells and C4-2B cells.

6. The screening method according to claim 3, wherein The compound is selected from the compounds in the Taoshu biological metabolite library L7000 or the compounds in the FDA anticancer metabolite library.

7. The screening method according to claim 3, characterized in that The step of detecting the sample to be detected includes: adding a substrate to the sample to be detected, and measuring the absorbance OD of the sample to be detected at 460nm and 618nm respectively. 460 and OD 618 .

8. The screening method according to claim 7, characterized in that The step of determining the effect of the compound on the binding between B lymphocytoma-2 protein and UBR family protein IV comprises: calculating the OD of the sample to be tested. 460 and OD 618 The ratios are sorted by numerical value, and the compounds in the top ten tested samples are considered to be able to promote the binding of B lymphocytoma-2 protein to UBR family protein IV and are candidate compounds.

9. Use of a candidate compound obtained by the method for screening a candidate compound for reducing docetaxel resistance in prostate cancer patients according to any one of claims 1 to 8 in the preparation of a medicament for reducing docetaxel resistance in prostate cancer patients; The candidate compound is proflavine sulfate.

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