Screening method and application of candidate compound for reducing docetaxel drug resistance of prostate cancer patient

Abstract

The invention relates to the technical field of tumor biology, and particularly discloses a screening method and application of a candidate compound for reducing docetaxel drug resistance of a prostate cancer patient. The screening method comprises the following steps: detecting the influence of a compound on the binding of a B lymphocytic tumour-2 protein and a UBR family protein IV, and when the compound promotes the binding of the B lymphocytic tumour-2 protein and the UBR family protein IV, the compound is a candidate compound. According to the application, the candidate compound for reducing the docetaxel drug resistance of the prostatic cancer patient is screened by detecting the influence of the compound on the combination of the B lymphocytic neoplasm 2 protein and the UBR family protein IV, and the inherent and adaptive DTX drug resistance of TGF-beta in PCa bone metastasis induction is overcome.

Description

Technical Field

[0001] This application relates to the field of tumor biology technology, and in particular to a method and application for screening candidate compounds that reduce docetaxel resistance in prostate cancer patients. Background Technology

[0002] According to global cancer incidence and mortality data from 2007 to 2023, prostate cancer (PCa) has the highest incidence rate among male cancers worldwide and is the second leading cause of cancer death in men globally. Most prostate cancers remain androgen-dependent, at least in their initial stages, and androgen deprivation therapy (ADT) has been the primary treatment for metastatic prostate cancer. Over time, the patient's response to ADT weakens, and the cancer progresses to castration-resistant prostate cancer (CRPC). CRPC accounts for the majority of prostate cancer deaths, therefore, CRPC has been a focus of basic research and drug development in recent years.

[0003] Patients with CRPC initially benefited from docetaxel (DTX) as first-line treatment. DTX continuously activates microtubule depolymerization in proliferating cells by binding to β-tubulin, inducing cytotoxicity. Studies have shown that adding DTX to ADT prolongs the life expectancy of patients with metastatic hormone-sensitive prostate cancer (10–13.6 months), indicating that more patients will receive chemotherapy, prolonging survival time and improving survival rates. However, most patients treated with DTX eventually develop resistance.

[0004] Therefore, new treatment strategies are needed to treat DTX-resistant prostate cancer. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this application is to provide a method and application for screening candidate compounds to reduce docetaxel resistance in prostate cancer patients, with the aim of screening candidate compounds to solve the problem of poor treatment effect caused by docetaxel resistance in prostate cancer patients.

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

[0007] A first aspect of this application provides a method for screening candidate compounds to reduce docetaxel resistance in prostate cancer patients, comprising the steps of: detecting the effect of the compound on the binding of B-lymphoma-2 protein to UBR family protein IV, wherein when the compound promotes the binding of B-lymphoma-2 protein to UBR family protein IV, the compound is a candidate compound.

[0008] Preferably, the screening method uses NanoBRET technology to detect the binding of B lymphoma-2 protein to UBR family protein IV.

[0009] Preferably, the step of detecting the effect of the compound on the binding of B-lymphoma-2 protein to UBR family protein IV includes: expressing a fusion protein containing B-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; co-culturing the first and second cells and treating them with the compound to obtain a sample to be tested; and detecting the sample to be tested using NanoBRET technology to determine the effect of the compound on the binding of B-lymphoma-2 protein to UBR family protein IV.

[0010] Preferably, a first plasmid containing a gene encoding B lymphoma-2 protein and a gene encoding HaloTag protein is constructed, and the first plasmid is transferred into engineered cells 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 engineered cells 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 compounds in the Taoshu Biometabolism Library L7000 or compounds in the FDA Anticancer Metabolism Library.

[0013] Preferably, the step of detecting the sample to be tested includes: adding a substrate to the sample to be tested, and measuring the absorbance values ​​(ODs) of the sample with the added substrate at 460 nm and 618 nm, respectively. 460 and OD 618 .

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

[0015] A second aspect of this application provides the use of a candidate compound obtained by the above-described screening method for reducing docetaxel resistance in prostate cancer patients in the preparation of a medicament for reducing docetaxel resistance in prostate cancer patients.

[0016] Preferably, the candidate compounds are selected from proflavin sulfate, triptolide, atorvastatin calcium, epirubicin hydrochloride, lometapate, HMN-214, Ret tyrosine kinase inhibitors, nordihydroguaiac acid, Src family kinase inhibitors, and dimethoxyestradiol.

[0017] The beneficial effects of this application are:

[0018] Existing technologies have demonstrated 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. Furthermore, Bcl-2, as an apoptosis inhibitor, mediates chemotherapy resistance in various malignancies. This application further discovers through experiments that one pathway by which DTX induces Bcl-2 degradation is regulated by increasing the binding of Bcl-2 to the ligase domain of UBR4. Therefore, this application targets Bcl-2 to screen for compounds that can enhance the binding of Bcl-2 to UBR4, thereby identifying candidate compounds that can reduce docetaxel resistance in prostate cancer patients and overcome TGF-β-induced intrinsic and adaptive DTX resistance in PCa bone metastases. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0020] Figure 1 A protein screening analysis diagram for binding to Bcl-2 protein provided for embodiments of this application:

[0021] A is a schematic diagram of the screening process; B and C are t-test analysis charts; D shows the change in the binding ability of UBR4 with Bcl-2 under different conditions; E is the result of principal component analysis.

[0022] Figure 2 A graph showing the expression level of UBR4 in tumor cells, provided for embodiments of this application:

[0023] A is a graph showing the protein level analysis of UBR4 in various tissues from the Human Protein Atlas database; B is a graph showing the expression level analysis of UBR4 in tumor cells from the TCGA database; C is a graph showing the expression level analysis of UBR4 in DTX-resistant cells detected by qPCR.

[0024] Figure 3 A test diagram illustrating the regulatory effect of UBR4 on Bcl-2 expression provided in an embodiment of this application:

[0025] A shows the change in Bcl-2 expression after overexpression of UBR4 in 293FT cells; B shows the change in Bcl-2 expression after overexpression of UBR4 in DU145 cells; C shows the change in Bcl-2 expression after overexpression of UBR4 in HeLa cells; D shows the effect of TGF-β and DTX on the binding of UBR4 and Bcl-2.

[0026] Figure 4 A diagram illustrating the binding domains of UBR4 and Bcl-2 during the degradation of Bcl-2 by UBR4, provided in an embodiment of this application:

[0027] A is a schematic diagram of the structure of the UBR4 truncated form; B and C are the expression levels of Bcl-2 in 293FT cells overexpressing different UBR4 truncated forms, analyzed by Western blot.

[0028] Figure 5 Verification diagram of Bcl-2 binding to the ligase domain of UBR4 during Bcl-2 degradation in DTX degradation, provided in the embodiments of this application:

[0029] A shows the analysis of protein docking between EZH2 and UBR4 using Gramm; B shows the analysis of protein docking between Bcl-2 and UBR4 using Gramm; C shows the expression level of Bcl-2 in 293FT cells that simultaneously overexpress Bcl-2 and the ligase domain using Western blot; D shows the binding ability of Bcl-2 and the ligase domain in 293FT cells using immunoprecipitation; E shows the binding ability of Bcl-2 and the ligase domain in 293FT cells after treatment with DTX.

[0030] Figure 6 An analysis diagram illustrating the binding of Bcl-2 and the ligase domain in UBR4 provided for embodiments of this application:

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

[0032] Figure 7 This is an analytical diagram showing the effect of different compounds provided in the embodiments of this application on the binding of Bcl-2 and UBR4;

[0033] Figure 8The pHTC plasmid map provided in the embodiments of this application;

[0034] Figure 9 The PNLF1-C plasmid map provided for the embodiments of this application. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0036] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0037] During chemotherapy for prostate cancer, docetaxel can enhance the binding of Bcl-2 to the ligase domain of the E3 ubiquitin ligase UBR4, thereby inducing Bcl-2 degradation via the ubiquitin-proteasome pathway. However, the presence of transforming growth factor-β (TGF-β) in the bone metastasis microenvironment effectively inhibits this binding, protecting Bcl-2 from degradation and causing apoptosis escape, thus leading to secondary chemotherapy resistance. Venetoclax, a treatment for chronic lymphocytic leukemia and acute myeloid leukemia, can target and inhibit Bcl-2, but it readily induces neutropenia. Therefore, a better target for Bcl-2 in prostate cancer needs to be found.

[0038] Based on this, a first aspect of the present application provides a method for screening candidate compounds to reduce docetaxel resistance in prostate cancer patients, comprising the steps of: screening candidate compounds by detecting the effect of the compound on the binding of B lymphoma-2 protein to UBR family protein IV, wherein when the compound inhibits the binding of B lymphoma-2 protein to UBR family protein IV, the compound is a candidate compound.

[0039] B-cell lymphoma-2 (Bcl-2) is an apoptosis inhibitor that binds to the pro-apoptotic protein bax, inhibiting changes in mitochondrial outer membrane permeability. Bcl-2 has also been reported to mediate chemotherapy resistance in various malignancies, including lung, lymphoma, and thyroid tumors. Bcl-2 is primarily degraded via the ubiquitin-proteasome system (UPS), and both parkin and F-box proteins can mediate Bcl-2 ubiquitination, thereby reducing its half-life. Furthermore, DTX can induce Bcl-2 phosphorylation, preventing it from binding to bax and leading to Bcl-2 inactivation. Due to its anti-apoptotic effect, high expression of Bcl-2 in prostate cancer can also lead to decreased sensitivity to DTX.

[0040] In some implementations, the binding of B-lymphoma-2 protein to UBR family protein IV is detected using NanoBRET technology. NanoBRET is a modified bioluminescent fluorescence resonance transfer technology that enables real-time monitoring of protein interactions within living cells.

[0041] Furthermore, the screening method specifically includes the following steps:

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

[0043] In some embodiments, a first plasmid containing a gene encoding B-lymphoma-2 protein and a gene encoding HaloTag protein is constructed, and the first plasmid is transferred into engineered cells 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 engineered cells to obtain the second cell. Preferably, in the first plasmid, the gene encoding Bcl-2 protein precedes the gene encoding HaoTag protein, and in the second plasmid, the gene encoding UBR4 protein precedes the gene encoding NanoLuc protein. The construction methods of the expression vectors for the fusion proteins (the first plasmid and the second plasmid) are conventional techniques in the art and will not be described in detail.

[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-culture the first and second cells and treat them with the compound to obtain the sample to be tested.

[0046] In some embodiments, the compounds are selected from compounds in the Taoshu Biometabolism Library L7000 or compounds in the FDA Anticancer Metabolism Library.

[0047] S3. Detect the sample to be tested to determine the effect of the compound on the binding of B lymphoma-2 protein to UBR family protein IV.

[0048] In some embodiments, step S3 may specifically include: the step of detecting the sample to be tested includes: adding a substrate to the sample to be tested, and measuring the absorbance values ​​(ODs) of the sample with added substrate at 460 nm and 618 nm, respectively. 460 and OD 618 Calculate the OD of the sample to be tested. 460 and OD 618 The ratios were sorted by numerical value, and the compounds in the top ten test samples were considered to promote the binding of B lymphoma-2 protein to UBR family protein IV, and were thus considered candidate compounds.

[0049] In this process, the substrate reacts with the NanoLuc protein to generate a donor light signal, while the HaloTag protein acts as the energy acceptor for this donor light signal. When UBR family protein 4 and B-lymphoma-2 protein interact, the donor light signal from NanoLuc couples to the HaloTag protein, resulting in superposition of spectral signals. The binding status of B-lymphoma-2 protein to UBR family protein IV is determined by measuring the absorbance values ​​of the test sample at 460 nm and 618 nm.

[0050] In a second aspect of this application, a candidate compound is provided obtained using the screening method described above for reducing docetaxel resistance in prostate cancer patients.

[0051] In some embodiments, the candidate compounds are selected from proflavin sulfate, triptolide, atorvastatin calcium, epirubicin hydrochloride, lometapate, HMN-214, Ret tyrosine kinase inhibitors, nordihydroguaiac acid, Src family kinase inhibitors, and dimethoxyestradiol.

[0052] The following specific examples provide further illustration. Unless otherwise specified, the reagents, instruments, and testing methods used in the examples are conventional in the art.

[0053] Example 1

[0054] Proteins were extracted from DU 145 cells, purified, and enriched. The protein samples were then digested enzymatically to break them down into smaller peptides. These peptides were then analyzed by mass spectrometry to determine their mass and charge. Mass spectrometry analysis allows us to obtain the mass spectra of the peptides and further infer the protein sequence and structure. The screening process is as follows: Figure 1 As shown in Figure A, the binding of the protein and Bcl-2 was analyzed by mass spectrometry.

[0055] The results are as follows Figure 1 Figures B, C, D, and E are shown in the diagram. Mass spectrometry analysis of the binding of 946 proteins to Bcl-2 revealed that 386 proteins bound to Bcl-2 were detected. Of these 386 proteins, the binding of 87 proteins to Bcl-2 was affected by DTX treatment, and the binding of 71 proteins differed between the DTX-treated group and the DTX / TGF-β combined treatment group. Taking the intersection of these differences, 33 proteins were selected. Among them, UBR4, an E3 ligase, showed increased binding to Bcl-2 after DTX treatment, while the binding in the DTX / TGF-β combined treatment group decreased compared to DTX treatment; it was the only protein that met the expected target protein criteria.

[0056] Example 2

[0057] In the Human Protein Atlas (https: / / www.proteinatlas.org / ENSG00000127481-UBR4 / tissue), the protein levels of UBR4 vary in different tissues. Among them, the UBR4 protein level in the prostate is among the highest, making it a good target. Figure 2 (As shown in Figure A). Data analysis from the TCGA database shows that in prostate cancer, the expression level of UBR4 in tumors is lower than in normal tissues (as shown in Figure A). Figure 2 (As shown in Figure B). The expression level of UBR4 in DTX-resistant cells was detected using qPCR, and the results are shown below. Figure 2 As shown in Figure C, the result that the UBR4 level in drug-resistant cells is lower than that in wild-type cells coincides with the higher Bcl-2 protein level in tumors. It can be inferred that the decrease in UBR4 level leads to the reduction of Bcl-2 protein degradation, thereby causing apoptosis escape.

[0058] Example 3

[0059] To verify the regulatory role of UBR4 on Bcl-2, the Bcl-2 and UBR4 genes were overexpressed to different degrees in 293FT, DU145, and HeLa cells. Specifically, in 293FT, DU145, and HeLa cells, equal amounts of Bcl-2 were simultaneously overexpressed, and UBR4 was overexpressed in ascending order of concentration. Western blot analysis was used to detect the expression levels of Bcl-2 and UBR4, and the results are as follows: Figure 3 As shown in Figures A, B, and C. Combined with... Figure 3 The results from A, B, and C show that the level of Bcl-2 in the three cell types exhibits a UBR4 concentration-dependent decreasing trend.

[0060] The binding of UBR4 to Bcl-2 was verified by immunoprecipitation assay, and the results are as follows: Figure 3 As shown in Figure D, where C represents the negative control, D indicates treatment with 10 nM DTX for 48 h, T indicates treatment with 10 ng / ml TGF-β for 48 h, and TD indicates combined treatment with 10 ng / ml TGF-β and 10 nM DTX for 48 h. Figure 3 As can be seen from the results, the binding of the two increased after treatment with 10 nM DTX for 48 h. However, the binding decreased after combined treatment with 10 ng / ml TGF-β and 10 nM DTX.

[0061] Example 4

[0062] UBR4, as a protein in the UBR family, possesses the UBR-box domain common to the UBR family, but lacks the classic substrate-binding domains of E3, such as the HECT or RING domains. UBR4 has a ligase domain at 4367-5163, which has been shown to mediate the degradation of UBR4 substrates. Based on this, this embodiment constructed a series of truncated versions of UBR4, including FL (containing the full length of UBR4), Δbox (removing the UBR-box), NTD (retaining only the N-terminus from 0-3184), CTD (retaining only the C-terminus from 3185-5163), ΔLD (removing the ligase domain), and LD (retaining only the ligase domain), specifically as follows: Figure 4 As shown in Figure A.

[0063] Subsequently, the truncated form of UBR4 was overexpressed in 293FT cells, and Bcl-2 expression was detected by Western blot. The results are as follows: Figure 4 As shown in B and C. From Figure 4As shown in B and C, only the FL group (containing the full-length UBR4) showed a significant decrease in Bcl-2 expression. This means that only the full-length UBR4 can promote Bcl-2 degradation. Neither the absence of the UBR-box nor the absence of the ligase domain can promote Bcl-2 degradation, indicating that both the UBR-box and the ligase domain play important roles in the degradation of Bcl-2 by UBR4.

[0064] Example 5

[0065] UBR family proteins require N-terminal recognition rules to recognize substrates. The UBR-box can recognize the N-terminal residues exposed after substrate cleavage, including type I N-degrons phenylalanine, tryptophan, tyrosine, leucine, and isoleucine, and type II N-degrons arginine, lysine, histidine, aspartic acid, glutamic acid, and cysteine. According to other literature, Bcl-2 can be cleaved by caspase-3, exposing A32 and V35. However, neither alanine nor valine are N-degrons recognized by the UBR-box; therefore, other cleavage sites for Bcl-2 may exist and require further investigation.

[0066] Due to the complexity of the N-terminal rule, the ligase domain of UBR4 was prioritized for investigation. Studies have reported that the ligase domain of UBR4 can mediate the degradation of EZH2. Therefore, protein docking was performed using Gramm (https: / / gramm.compbio.ku.edu / ), and the docking results were analyzed (e.g., Figure 5 As shown in Figures A and B, it was found that EZH2 binds to the ligase domain through its N-terminus, and Bcl-2 can also bind to the ligase domain through its N-terminus, indicating that the binding of Bcl-2 to the ligase domain is somewhat similar to that of EZH2.

[0067] Subsequently, in 293FT cells uniformly overexpressing Bcl-2, ligase domains were transiently transfected using a gradient method, and intracellular Bcl-2 expression was detected by qPCR. The results are as follows: Figure 5 As shown in C. From Figure 5 As can be seen from Figure C, the protein level of Bcl-2 decreases with the increase of ligase domain, which is the same as the result of the group that overexpressed the full-length gene of UBR4 in Example 4.

[0068] Furthermore, the binding of Bcl-2 to the ligase domain was detected by immunoprecipitation assay. Bcl-2-Myc and LD-Flag were simultaneously overexpressed in 293FT cells, and LD-Flag was bound by Anti-FLAG Nanobody Magarose Beads. The results are as follows: Figure 5 As shown in Figure D. Next, 293FT cells overexpressing Bcl-2-Myc and LD-Flag were treated with 10 mM DTX, and the results are shown in Figure E. Figure 5 As can be seen from the results, after treatment with 10 nM DTX, the binding degree of Bcl-2 to the ligase domain was significantly higher than that of the group not treated with DTX, indicating that DTX-induced Bcl-2 degradation was caused by increasing its binding to the ligase domain of UBR4.

[0069] Example 6

[0070] (1) The NanoBRET system was used for testing. By constructing fusion proteins with HaloTag and NanoLuc respectively, when the two groups are close enough, using the NanoLuc fusion protein as the energy donor and the fluorescently labeled HaloTag fusion protein as the energy acceptor, a 618 nm spectral signal could be detected, and the signal increased as the distance decreased. The principle is as follows: Figure 6 As shown in Figure A.

[0071] HaloTag protein was expressed using the p53-HT plasmid, and NanoLuc protein was expressed using the NL-MDM2 plasmid, serving as the control group. The Bcl-2-HT plasmid (pHTC plasmid containing the Bcl-2 gene) and LD-NL (pNLF1-C plasmid containing the ligase domain) were used as experimental groups, and the results were analyzed using the NanoBRET system. When the two groups were sufficiently close together, the NanoLuc fusion protein was used as the energy donor, and the fluorescently labeled HaloTag protein was used as the energy acceptor. The spectral signal at 618 nm was detected. Results are as follows: Figure 6 As shown in B. From Figure 6 As can be seen from Figure B, the spectral signals of LD-NL and Bcl-2-HT are higher than those of the control group, which proves the binding of the ligase domain and Bcl-2.

[0072] (2) Cells expressing LD-NL and Bcl-2-HT were treated with DTX and TGF-β, respectively, and in combination. The results are as follows: Figure 6 As shown in C. From Figure 6As can be seen from Figure C, the DTX-treated group did not enhance the spectral signal, but the TGF-β-treated group reduced the spectral signal. This indicates that TGF-β treatment can reduce the binding of Bcl-2 and the ligase domain, proving that the reason why TGF-β protects Bcl-2 from degradation is due to the reduced binding of the ligase domain.

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

[0074] Example 7

[0075] The materials used included: 293FT cells, pHTN plasmid (a vector capable of expressing the HaloTag protein tag), pNLF1-N plasmid (a plasmid capable of expressing the NanoLuc fusion protein), and Bcl-2-HT plasmid (a pHTC plasmid carrying the Bcl-2 gene, such as...). Figure 8 As shown), LD-NL (pNLF1-C plasmid with ligase domain, such as...) Figure 9 (As shown), the liposome transfection reagent, NanoBRET. TM Substrate NanoBRET TM 618 Ligand, Costar 3917 white 96-well plate, metabolite library: Taoshu Bio L7000, containing a total of 385 compounds.

[0076] The specific steps are as follows:

[0077] (1) Seed 293FT cells in 10cm culture dishes, with 3.2 × 10⁻⁶ cells per dish. 6 Each sample was cultured overnight in a low-serum medium free of penicillin and streptomycin.

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

[0079] (3) Take 293FT cells from the culture dish in (2) and spread them into a white 96-well plate, with 2.2 × 10⁻⁶ cells per well. 4The experimental group (Bcl-2-HT+LD-NL) was seeded in 59 wells, and the control group (pHTN+pNLF1-N) was seeded in 1 well. Each well contained 50 μL of LDMEM medium and 0.05 μL of 1:1000 618 ligand. 100 μL of PBS buffer was added to the wells surrounding the wells containing the test sample, and the mixture was incubated overnight.

[0080] (4) In the 96-well plate containing the test sample in (3), add 1 μL of metabolite drug diluted 40 times to the first 58 wells, and add 1 μL of DMSO diluted 40 times to the remaining experimental group well and blank control group well, and incubate for 24 hours.

[0081] (5) Add 12.5 μL of NanoBRET substrate diluted 100 times to the experimental wells (60 wells in total) of (4). TM Substrate, react for ten minutes, remove the liquid from the well, and use a spectrophotometer to measure the absorbance values ​​(OD) at 460 nm and 618 nm. 460 and OD 618 And with OD 460 / OD 618 The values ​​are compared as results, and the results are as follows: Figure 7 As shown, according to Figure 7 Based on the data in Table 1, we obtain the OD. 460 / OD 618 The metabolite with the highest value was proflavine hemisulfate. And OD... 460 / OD 618 The top ten metabolites by value include proflavin sulfate, 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 <![CDATA[(Treated OD 460 / OF 618 ) / (DMSO OD 460 / OF 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, this application further discovered through experiments that one of the pathways by which DTX induces Bcl-2 degradation is regulated by increasing the binding of Bcl-2 to the ligase domain of UBR4. Therefore, this application screens for compounds that can enhance the binding of Bcl-2 to UBR4 by targeting Bcl-2, thereby screening for candidate compounds that can reduce docetaxel resistance in prostate cancer patients and overcome the intrinsic and adaptive DTX resistance induced by TGF-β in PCa bone metastases.

[0085] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

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 the 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, characterized in that The binding of B-lymphocytoma-2 protein to UBR family protein IV was detected by 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 lymphocyte tumor-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 samples to be tested are 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, characterized in that 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, characterized in that 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 lymphocyte tumor-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 candidate compounds that can promote the binding of B lymphocytoma-2 protein to UBR family protein IV.

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 drug for reducing docetaxel resistance in prostate cancer patients.

10. The use according to claim 9, characterized in that: The candidate compound is selected from the group consisting of proflavine sulfate, celastrol, atorvastatin calcium, epirubicin hydrochloride, lomitapide, HMN-214, Ret tyrosine kinase inhibitor, nordihydroguaiaretic acid, Src family kinase inhibitor, and dimethoxyestradiol.

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