Screening method of targeted CD276 small-molecule inhibitor and anti-tumor application of targeted CD276 small-molecule inhibitor

Abstract

The invention relates to a screening method of a targeted CD276 small-molecule inhibitor and anti-tumor application of the targeted CD276 small-molecule inhibitor, three sites with the strongest binding effect on CD276 are determined through indexes such as affinity, structural diversity and binding modes, and it is verified that the three binding sites are key sites for screening the high-affinity targeted CD276 inhibitor through mutation of the three binding sites.

Description

Technical Field

[0001] The present application relates to a screening method for small molecule inhibitors targeting CD276 and its anti-tumor application, belonging to the field of biomedicine. Background Art

[0002] Prostate cancer is one of the common clinical urogenital system tumors. Worldwide, it is the second leading cause of cancer death in men, has ranked first in the incidence of male tumors for many consecutive years, and shows a gradually increasing trend. Tumor immunotherapy, as a tumor treatment strategy, exerts anti-tumor effects by activating the host's innate and adaptive immune systems. Immune checkpoint blockade is one of the most important immunotherapies and has achieved remarkable results in clinical applications. Among them, the targeted drugs for the immune checkpoint CD276 mainly include monoclonal antibodies, ADCs, ADCCs, etc.; small molecule inhibitors also have good application prospects due to their advantages such as low cost, easy modification, good permeability, and less likely to cause immunogenic responses and host adverse reactions. However, screening for effective CD276 inhibitors in a variety of large databases is a very laborious and time-consuming task. If the key binding sites on CD276 can be known, screening can be carried out targeted, thus greatly improving the efficiency of obtaining the target inhibitor. Summary of the Invention

[0003] To solve the above technical problems, the present application provides a screening method for CD276 inhibitors, including screening inhibitors that bind to the 217th, 218th, and / or 339th amino acids of CD276; the amino acid sequence of CD276 refers to UniProtKB / Swiss-Prot: Q5ZPR3.1.

[0004] In some embodiments, the above screening method further includes contacting a candidate inhibitor with CD276 and detecting the binding site of the candidate inhibitor to CD276; if the binding site contains the 217th, 218th, and / or 339th amino acids of CD276, then the candidate inhibitor is selected.

[0005] In some embodiments, the above screening method further includes obtaining the structural data of CD276 protein and candidate inhibitor and the spatial structure of the complex formed after their contact.

[0006] The present application also provides the use of the 217th, 218th, and / or 339th amino acids of CD276 for screening CD276 inhibitors or anti-cancer drugs.

[0007] The present application also provides the use of an inhibitor that binds to the 217th, 218th, and / or 339th amino acids of CD276 in the preparation of a drug, compound, composition, or preparation targeting CD276.

[0008] The present application also provides the use of a CD276 inhibitor in the preparation of a drug for preventing and / or treating tumors, wherein the CD276 inhibitor binds to the 217th, 218th, and / or 339th amino acid of CD276.

[0009] In some embodiments, the drug is a drug that inhibits the proliferation or metastasis of cancer cells expressing CD276; or the drug is used to treat prostate cancer (especially castration-resistant prostate cancer and lung metastasis of prostate cancer), small cell lung cancer, osteosarcoma, solid tumors (such as non-small cell lung cancer, urothelial carcinoma, etc.), glioblastoma, breast cancer, and / or cervical cancer and other diseases.

[0010] In some embodiments, the CD276 inhibitor refers to a substance that can inhibit the transcription or translation of the CD276 gene, or can inhibit the expression or activity of the CD276 protein, or can inhibit the binding of CD276 to its receptor. In some embodiments, the CD276 inhibitor includes or does not include flavonoids (such as one or more of quercetagetin, quercetin or patuletin). In some embodiments, the CD276 inhibitor is a non-flavonoid compound.

[0011] The present application also provides the non-diagnostic or non-therapeutic use of flavonoids in inhibiting CD276.

[0012] In some embodiments, the flavonoids include one or more of quercetagetin, quercetin or patuletin.

[0013] Advantages of the present application: The method of the present application can efficiently screen or pre-evaluate drugs, compounds, compositions or preparations that are expected to inhibit CD276, and effectively predict the inhibitory effect of candidate inhibitors based on the key binding sites on CD276, greatly saving the time and effort of random blind selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which form a part of the present application, are used to provide a further understanding of the present application, and make other features, objects and advantages of the present application more obvious.

[0015] Figure 1 Three druggable pockets of the CD276 protein.

[0016] Figure 2 Quercetagetin has a high affinity for the CD276 protein.

[0017] Figure 3Quercetagetin forms stable hydrogen bonds with the amino acids at positions T217, Y218, and / or T339 of the CD276 protein through its flavone backbone.

[0018] Figure 4 The amino acids at positions T217, Y218, and / or T339 of the CD276 protein are the key binding sites for screening CD276 inhibitors.

[0019] Figure 5 The expression levels of CD276 in human prostate cancer cells LNCaP and PC-3, murine prostate cancer cells Myc-CaP and RM-1, and the association between CD276 and the anti-cancer activity of Quercetagetin.

[0020] Figure 6 Quercetagetin significantly inhibits prostate cancer metastasis.

[0021] Figure 7 Quercetagetin inhibits the growth of castration-resistant tumors.

[0022] Figure 8 Quercetagetin has good biosafety.

[0023] Figure 9 The chemical structural formulas of Quercetin, Quercetagetin, and Patuletin. Detailed implementation manners

[0024] The embodiments of the present application will be described below with reference to the accompanying drawings. The elements and features described in one drawing or one embodiment of the present application can be combined with the elements and features shown in one or more other drawings or embodiments. It should be noted that for the sake of clarity, the representations and descriptions unrelated to the present application and known to those of ordinary skill in the art are omitted in the drawings and the description. The present application will be further described below with reference to the accompanying drawings.

[0025] The present application provides a method for screening CD276 inhibitors, including screening for inhibitors that bind to CD276 using the amino acids at positions 217, 218, and / or 339 of CD276 as binding sites. The sequence of CD276 refers to UniProtKB / Swiss-Prot: Q5ZPR3.1.

[0026] In some embodiments, the screening method includes contacting a candidate inhibitor with CD276 and detecting the binding site of the candidate inhibitor with CD276. If the binding site contains the amino acids at positions 217, 218, and / or 339 of CD276, then the candidate inhibitor is selected.

[0027] In some embodiments, the screening method includes obtaining the structural data of CD276 protein and a candidate inhibitor, as well as the spatial structure of the complex formed after their contact, determining the binding site of the complex, and using the binding to the 217th, 218th, and / or 339th amino acids of CD276 as the screening basis, so as to screen out a candidate inhibitor with high affinity for CD276.

[0028] The present application also provides the use of an inhibitor that binds to the 217th, 218th, and / or 339th amino acids of CD276 in the preparation of a drug, compound, composition, or formulation targeting CD276.

[0029] The present application also provides the use of a CD276 inhibitor in the preparation of a drug for preventing and / or treating tumors, wherein the CD276 inhibitor binds to the 217th, 218th, and / or 339th amino acids of CD276.

[0030] The present application also provides the non-diagnostic or non-therapeutic use of flavonoids in inhibiting CD276.

[0031] In some embodiments, flavonoids inhibit CD276 by binding to the 217th, 218th, and 339th amino acids of CD276.

[0032] In some embodiments, the drug is a drug that inhibits the proliferation or metastasis of cancer cells expressing CD276. In some embodiments, the drug is an anti-tumor drug. In some specific embodiments, the drug is a drug for treating prostate cancer (especially castration-resistant prostate cancer and lung metastasis of prostate cancer), small cell lung cancer, osteosarcoma, solid tumors (such as non-small cell lung cancer, urothelial cancer, etc.), glioblastoma, breast cancer, cervical cancer, etc.

[0033] Definitions

[0034] In the description of the embodiments of the present application, the term "and / or" is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this article generally represents an "or" relationship between the associated objects before and after.

[0035] As used in the specification and the appended claims, unless clearly specified in the context, the singular forms "a", "an", and "the" include plural referents.

[0036] "CD276", also known as B7-H3, is a type I transmembrane glycoprotein consisting of 534 amino acids (amino acid sequence referring to UniProtKB / Swiss-Prot: Q5ZPR3.1), belonging to the immune checkpoint molecules of the B7-CD28 family. Under normal circumstances, the protein expression level of CD276 is tightly regulated and maintained at a low level. However, CD276 is overexpressed in various tumors. For example, in cancers such as prostate cancer, pancreatic cancer, and breast cancer, the high expression of CD276 is positively correlated with the development of cancer and negatively correlated with the survival period of patients.

[0037] "CD276 inhibitor" refers to a substance that can inhibit the transcription or translation of the CD276 gene, or can inhibit the expression or activity of the CD276 protein, or can inhibit the binding of CD276 to its receptor. Preferably, the CD276 inhibitor is selected from one or more of nucleic acid molecules, small molecules (chemical drugs), antibody drugs, polypeptides, proteins, nucleic acid constructs, interfering lentiviruses, interfering adeno-associated viruses, and gene editing systems. In some embodiments, the inhibitor is a small molecule or a macromolecule. In some embodiments, the inhibitor is a drug, compound, composition, or preparation. In some embodiments, the inhibitor of CD276 includes or does not include flavonoid compounds (i.e., non-flavonoid compounds). In some specific embodiments, the flavonoid compound is one or more of quercetagetin, quercetin, or patuletin. In some embodiments, the flavonoid compound is not quercetagetin, quercetin, or patuletin, but other substances.

[0038] "Flavonoid compounds" generally refer to a series of compounds in which two benzene rings are connected to each other through three carbon atoms, that is, a general term for a class of compounds with a C6-C3-C6 structure.

[0039] "Composition" (such as a pharmaceutical composition) may contain pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients refer to substances that do not produce adverse, allergic, or other adverse reactions when the drug is appropriately administered to animals or humans.

[0040] "Treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0041] The "cell" is an animal cell or a human cell, and can be a separated cell, an in vitro cell or an in vivo cell; optionally, it is a tumor cell (cancer cell) or a metastatic cancer cell, for example, breast cancer cell, pancreatic cancer cell, prostate cancer cell, etc. In some embodiments, the cell is a metastatic Castration Resistant Prostate Cancer (mCRPC) cell.

[0042] "Cell proliferation" is the basis for the growth, development, reproduction and heredity of organisms. Abnormal cell proliferation may lead to the occurrence of tumors.

[0043] "Cell metastasis" refers to the invasion of tumor cells from their primary site into lymphatic vessels, blood vessels or body cavities. The tumor cells are carried by the blood flow or lymph flow to another site or organ to continue growing, forming a tumor of the same type as the primary tumor.

[0044] The method of the present application can be an in vitro method or an in vivo method; it can be a diagnostic method or a therapeutic method, or a non-diagnostic method or a non-therapeutic method; it can be used for drug screening or preparation for humans or non-human animals.

[0045] Examples

[0046] Example 1 Analyze the binding pocket

[0047] Select the Alphafold predicted structure of human CD276 and download its structure from the official website of the Uniprot database (https: / / www.uniprot.org / Q5ZPR3). Use the SiteFinder module of the MOE software to analyze the Alphafold structure of CD276, which contains three binding pockets, namely Site1, Site2 and Site3 (see Figure 1)。Among them, the Site1 region (ASP180, GLY181, GLN182, VAL210, VAL211, LEU212, GLY213, ALA214, ASN215, GLY216, THR217, TYR218, ILE237, GLN286, LYS291, THR339, PHE341) and the Site2 region (ARG156, PRO157, GLY158, ASP159, SER242, PRO243, ALA279, GLN280, LEU281, ASN282, THR298, GLU299, SER343, ILE344, ARG345, ASP346) are located between the Ig-like V-type and Ig-like C2-type functional domains. The Site3 region (PRO398GLY399GLN400VAL428VAL429LEU430GLY431ASN433GLY434THR435TYR436) is located in the Ig-like C2-type 2 functional domain. It was found that genomic duplication of the CD276 gene led to tandem duplication of the immunoglobulin-like V and C domains (VC domains). The dominantly expressed human CD276 isoform contains tandemly duplicated VC domains.

[0048] Since the druggability of the Site1 and Site 2 pockets is relatively good, we selected Site1 and Site2 for preliminary screening. However, the later results of the screening for Site2 were not good (few compounds bound and the scores were not high). Therefore, Site1 was taken as the binding pocket for key research.

[0049] Example 2 screened out the compound with the best affinity

[0050] Taking the natural product monomer compound library as a candidate inhibitor as an example to illustrate the screening method of this application, but it should not be considered as a limitation of this application. Contacting the candidate inhibitor with CD276, based on the analysis in Example 1 and focusing on the affinity score information of the compound with CD276, we obtained 194 and 65 compounds that can form direct interactions with Site1 and Site2 of the CD276 structure respectively. Finally, a compound Quercetagetin was found to have a significantly high affinity for the CD276 protein. Subsequently, we fully verified the high affinity of Quercetagetin for the CD276 protein through SPR, MST, and CETSA experiments. Specifically:

[0051] 2.1 SPR affinity experiment:

[0052] Dissolve the CD276 recombinant protein (His Tag) to 0.25 mg / mL. Take 80 μL and mix it with 120 μL of sodium acetate buffer solution at pH 4.0 to make the protein positive. Prepare 100 μL of EDC and NHS and 140 μL of ethanolamine for chip coupling of the protein respectively. Subsequently, set different concentration gradients of the compound (half-dilution method), including 0 μM plus an intermediate concentration. Place the samples in order on the sample rack and then put them into the machine, and set the program to start the experiment.

[0053] 2.2 MST affinity experiment:

[0054] (1) Mix 100 nM Red-NHS647 dye with the protein whose concentration has been previously explored at a volume ratio of 1:1. After incubating in the dark at room temperature for 30 min, centrifuge at 12000 rpm for 10 min, and take the supernatant for labeling.

[0055] (2) Prepare 16 200-μL low-binding EP tubes in sequence. Add 5 μL of PBST solution to each tube in turn. Add 5 μL of the compound Quercetagetin above the lid of tube 1, centrifuge, vortex and then centrifuge again. Draw 5 μL of the solution from tube 1 to above the lid of tube 2, and dilute in this way in sequence. Draw 5 μL from the last tube as well.

[0056] (3) Add 5 μL of the labeled protein to above the lids of the 16 EP tubes respectively, centrifuge, vortex and then centrifuge again, and incubate at room temperature for 5 min.

[0057] (4) Take 16 MST detection capillaries and insert them into the bottom of the EP tubes in sequence, suck out the mixed solution, and place them above the detection tray for on-machine detection.

[0058] (5) Use the MO.Affinity Analysisn software to calculate and evaluate the detection data.

[0059] 2.3 Detection of the affinity between Quercetagetin and CD276 protein by Cellular Thermal Shift Assay (CETSA):

[0060] (1) Cell drug treatment: Weigh an appropriate amount of Quercetagetin accurately on the balance, prepare a stock solution of 10 mM with DMSO, and dilute the stock solution to a working solution with a concentration of 6.25 μM with the medium containing 10% FBS. When the LNCaP cells are in good growth state and in the logarithmic growth phase, replace the original medium with the working solution, and add the medium containing an equal amount of DMSO to the control group.

[0061] (2) Heat treatment of cells: After 2 h of drug treatment, the culture medium was discarded, and the cells were washed three times with PBS buffer. The cells were digested with 0.25% trypsin and collected, and then resuspended in PBS buffer. The two groups of cells were each divided into six portions, and the 12 groups of cells were treated in a water bath at 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, and 65 °C for 90 s.

[0062] (3) Protein preparation: After the heat treatment of the cells was completed, the cells were allowed to recover at room temperature for 90 s, then placed in liquid nitrogen for 3 s, dissolved at room temperature, and vortexed thoroughly on a vortex mixer. The operation was repeated 5 times to break the cells. The supernatant was collected at 12,000 rpm, 4 °C for 20 min and stored at -80 °C. Subsequently, Western blot was used to detect the effect of Quercetagetin on the thermal stability of CD276 protein.

[0063] Results: SPR, MST, and CETSA experiments showed that Quercetagetin had high affinity for CD276 protein (see Figure 2 A, Figure 2 B, Figure 2 C).

[0064] Example 3 Determination of the key binding site on CD276

[0065] First, molecular docking of the compound Quercetagetin with CD276 protein showed that Quercetagetin formed stable hydrogen bonds with the amino acids at positions T217, Y218, and / or T339 of CD276 through its flavone skeleton ( ), making Quercetagetin and CD276 have a strong affinity for each other. Figure 3 )

[0066] Therefore, those skilled in the art can foresee that flavonoids such as Quercetin ( Figure 9-1 ), Quercetagetin ( Figure 9-2 ), or Patuletin ( Figure 9-3 ) can all stably bind to CD276 protein through the same flavone skeleton.

[0067] Secondly, to verify this result, we constructed mutant cell lines containing the mutations of 217T / 218Y / 339T of CD276 to 217R / 218R / 339R, and detected the affinity of Quercetagetin for the wild-type and mutant proteins of CD276 by MST experiment. Specifically:

[0068] (1) Extraction of membrane proteins: This step was carried out according to the instructions of the Membrane Protein and Cytoplasmic Protein Extraction Kit (Cat. No.: P0033) from Shanghai Beyotime Biotechnology Co., Ltd.

[0069] (2) Adjustment of membrane protein fluorescence intensity: First, since the EGFP protein emits green fluorescence, blue light was selected for subsequent operations after the MST detector was turned on. After setting the parameters of the instrument, 10 μL of the obtained wild-type and mutant membrane proteins were respectively taken, aspirated into the capillary provided with the instrument, and then detected on the machine. According to the detected fluorescence values, the fluorescence values of each sample were adjusted to 600.

[0070] (3) The compound Quercetagetin was serially diluted from 50 μM to 16 concentrations, and then 5 μL of each concentration was mixed evenly with 5 μL of the membrane protein with adjusted fluorescence and detected on the machine. The MO.Affinity Analysisn software was used to calculate and evaluate the detection data.

[0071] Results: Figure 4 It was shown that the wild-type CD276 protein still maintained a high affinity with Quercetagetin, while the double mutation of T217 and Y218 decreased the affinity between the CD276 protein and Quercetagetin by about 7-fold; the single mutation of T339 decreased the affinity between the CD276 protein and Quercetagetin by about 4-fold; the triple mutant of 217T, 218Y, and 339T completely impaired the affinity between the CD276 protein and Quercetagetin. It can be seen that the T217, Y218, and / or T339 sites play a very important role in the binding of the CD276 protein to Quercetagetin and are the key sites for efficient binding to CD276. Therefore, the amino acids at positions T217, Y218, and / or T339 of CD276 can be used as key binding sites for the rapid screening of any CD276 inhibitor or anti-cancer drug.

[0072] Example 4 Anti-cancer activity of Quercetagetin

[0073] 4.1 MTT cell proliferation assay:

[0074] (1) Cell seeding: Collect prostate cancer cells LNCaP, PC-3, Myc-CaP, and RM-1 cells in the logarithmic growth phase with good cell status. After digestion with 0.25% trypsin, perform cell counting, and then adjust the cell concentration using a medium containing 10% FBS. Adjust the cell concentrations of LNCaP and PC-3 cells to 1.5×104 cells / mL, and the cell concentrations of Myc-CaP and RM-1 cells to 4×103 cells / mL, respectively. Inoculate the cells into a 96-well plate with a final volume of 200 μL per well. Set 6 replicates for each type of cell, and seal the wells around the cells with 200 μL of PBS.

[0075] (2) Drug treatment: Accurately weigh an appropriate amount of the compound Quercetagetin using a balance, prepare a stock solution of 10 mM in DMSO, dissolve the compound Quercetagetin in a medium containing 10% FBS to a concentration of 50 μM, and then perform a two-fold serial dilution; after the cells are completely adherent, add the medium containing different concentrations of the compound Quercetagetin to the experimental group, and add an equal amount of DMSO without adding the compound Quercetagetin to the control group; set a blank group that only contains the medium, without inoculating cells, and add the corresponding amounts of the compound Quercetagetin and DMSO for comparison.

[0076] (3) MTT treatment: After adding the compound Quercetagetin and treating for 48 h, add 20 μL of MTT (5 mg / mL) solution to each well under light protection, and place it in an incubator for 4 h of incubation in the dark.

[0077] (4) Detection: Preheat the multifunctional microplate reader (CLARIOstar), take out the 96-well plate, discard the medium, add 150 μL of DMSO to each well, place it on a shaker and incubate for 10 min at room temperature in the dark to fully dissolve the formazan crystals, detect the absorbance value (OD value) of the samples at a wavelength of 490 nm, and normalize the detection data under different concentration treatments using the detection data of the DMSO control group as the standard, and calculate the inhibition rate of the compound Quercetagetin on cell growth.

[0078] 4.2 Effects on the mouse lung metastasis model

[0079] (1) Group1 (G1) is the shCtrl group, referring to the control stable transfected cell line group of RM-1 cells infected with Control shRNA lentivirus and screened. Group2 (G2) is the shCtrl inhibitor group, referring to the control stable transfected cell line of RM-1 cells infected with Control shRNA lentivirus and screened plus the group treated with Quercetagetin. Group3 (G3) is the shCD276 group, referring to the stable transfected cell line group with knocked-down CD276 of RM-1 cells constructed by infecting with CD276 shRNA lentivirus and screened. RM-1-shCtrl and RM-1-shCD276 in logarithmic growth and in good condition were collected, digested into cell suspensions, and the cell density value was adjusted to 5×10 6 cells / mL, and the single-cell suspension was placed on ice for standby.

[0080] (2) A 1 mL syringe was used to aspirate 200 μL of RM-1-shCtrl and RM-1-shCD276 cell suspensions, and the cells were injected into the mice through the tail vein. Specifically, one week after tumor-bearing, the mice injected with RM-1-shCtrl were randomly re-grouped, and started to be intraperitoneally injected with 200 μL of 2 mg / kg compound Quercetagetin or normal saline containing 2% DMSO every day for treatment, and the mice injected with RM-1-shCD276 were intraperitoneally injected with 200 μL of normal saline containing 2% DMSO every day for treatment.

[0081] (3) After 12 days of drug administration, drug administration was stopped for 2 days. One mouse from each group was taken to fix the lungs for HE staining, and the remaining were perfused with picric acid staining solution, and photographed after removal.

[0082] 4.3 Effects on the subcutaneous tumor-bearing mouse model

[0083] (1) RM-1-shCtrl and RM-1-shCD276 in logarithmic growth and in good condition were collected, digested into cell suspensions, and the cell density value was adjusted to 5×10 6 cells / mL, and the single-cell suspension was placed on ice for standby.

[0084] (2) A 1 mL syringe was used to aspirate 200 μL of RM-1-shCtrl and RM-1-shCD276 cell suspensions and injected them at the right dorsal inguinal area. When the tumor grew to about 250 mm 3 , subcutaneous injection of 25 mg / kg dose of Degareli was given for castration treatment.

[0085] (3) After about one week of regression reaction, the tumors showed regrowth. The mice injected with RM-1-shCtrl were randomly re-grouped and started receiving intraperitoneal injection of 200 μL of 2 mg / kg compound Quercetagetin or normal saline containing 2% DMSO daily, and the mice injected with RM-1-shCD276 were given intraperitoneal injection of 200 μL of normal saline containing 2% DMSO daily.

[0086] (4) After 12 days of drug administration, the drug was stopped for 1 day. One mouse from each group was sacrificed to fix the heart, liver, spleen, lungs and kidneys for HE staining, and at the same time, the tumor tissues were photographed.

[0087] (5) During the drug administration period, the body weights of the mice were weighed and recorded every two days to draw the body weight change curve for observing the effect of the drug on the body weight of the mice in the later stage. At the same time, the length (a) and width (b) of the tumors on the backs of each mouse were measured using vernier calipers. The tumor volume was calculated according to the formula: tumor volume = 0.5 × a × b × b, and the growth curve of the tumors was drawn.

[0088] 4.4 Results:

[0089] ① The MTT assay demonstrated that Quercetagetin significantly inhibited the proliferation of prostate cancer cells, and the inhibitory effect of Quercetagetin on the proliferation of prostate cancer cells was closely related to the expression level of CD276. In human cells, compared with PC-3, in LNCaP cells with a higher CD276 expression level, the inhibition of cell proliferation by Quercetagetin was more obvious, manifested as a lower IC50 value of Quercetagetin in LNCaP cells than in PC-3 cells; in murine cells, compared with Myc-CaP, RM-1 cells had a higher CD276 expression level, and the inhibitory effect of Quercetagetin on their cell proliferation was more obvious, manifested as a lower IC50 value of Quescetagetin in RM-1 cells than in Myc-CaP cells ( Figure 5 ), indicating that Quercetagetin inhibited the proliferation of cancer cells by inhibiting CD276. ② Quercetagetin significantly inhibited the metastasis of prostate cancer. Compared with the control group, the compound Quercetagetin significantly reduced the tumor lung metastasis burden ( Figure 6 ). ③ Quercetagetin significantly inhibited the proliferation of prostate cancer cells. From the tumor growth curve and the weight of the excised tumors, compared with the control group, the compound Quercetagetin inhibited tumor growth ( Figure 7) ④Quercetagetin has good biosafety. Detection found that there were no obvious changes in the blood routine and blood biochemical indexes of mice, and H&E staining of major organs such as the heart, liver, spleen, lungs, and kidneys also showed that Quercetagetin had no toxic or side effects on the major organs of mice( Figure 8 )

[0090] The above description of the specific implementation manners of the present application discloses in detail the technical details of the present application and illustrates the technical idea of the present application, aiming to meet the authorization requirements of the Patent Law. However, it should not be construed as a limitation on the protection scope of the present application. Those skilled in the art can make various changes or deformations according to the present application, in combination with the knowledge and technology at that time, as long as they do not depart from the core idea and spirit of the present application, they shall fall within the protection scope of the appended claims of the present application.

Claims

1. A method for screening CD276 inhibitors, comprising screening inhibitors that bind to the 217th, 218th, and / or 339th amino acids of CD276; the amino acid sequence of CD276 refers to UniProtKB / Swiss-Prot:Q5ZPR3.

1.

2. The screening method according to claim 1, characterized in that The method also includes contacting the candidate inhibitor with CD276 and detecting the binding site between the candidate inhibitor and CD276; if the binding site comprises amino acids 217, 218, and / or 339 of CD276, the candidate inhibitor is selected.

3. The screening method according to claim 1 or 2, characterized in that It also includes obtaining the structural data of CD276 protein and candidate inhibitors and the spatial structure of the complex formed after their contact.

4. The use of the 217th, 218th, and / or 339th amino acids of CD276 for screening CD276 inhibitors or anti-cancer drugs.

5. Use of a CD276 inhibitor that binds to the 217th, 218th, and / or 339th amino acid of CD276 in the preparation of a drug, compound, composition or preparation targeting CD276.

6. Use of a CD276 inhibitor in the preparation of a drug for preventing and / or treating tumors, wherein the CD276 inhibitor binds to the 217th, 218th, and / or 339th amino acid of CD276.

7. The use according to any one of claims 4 to 6, characterized in that The drug is used to treat prostate cancer (especially castration-resistant prostate cancer and prostate cancer lung metastasis), small cell lung cancer, osteosarcoma, solid tumors (such as non-small cell lung cancer, urothelial carcinoma, etc.), glioblastoma, breast cancer, and / or cervical cancer.

8. The screening method according to any one of claims 1 to 3 or the use according to any one of claims 4 to 7, characterized in that: The inhibitor is a substance that inhibits the transcription or translation of the CD276 gene, or a substance that inhibits the expression or activity of the CD276 protein, or a substance that inhibits the binding of CD276 to its receptor.

9. Non-diagnostic or non-therapeutic uses of flavonoids in inhibiting CD276.

10. The use according to claim 9, characterized in that The flavonoid compound includes one or more of Quercetagetin, Quercetin or Patuletin.

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