Screening method of small molecule inhibitors targeting CD276 and its anti-tumor application

Abstract

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

Description

Technical Field

[0001] This application relates to screening methods for CD276 small molecule inhibitors and their anti-tumor applications, belonging to the field of biomedicine. Background Technology

[0002] Prostate cancer is one of the most common urogenital tumors in clinical practice. Globally, it is the second leading cause of cancer death in men and has consistently ranked first in male cancer incidence for many years, with a gradually increasing trend. Tumor immunotherapy, as a cancer treatment strategy, exerts its anti-tumor effect 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 application. Targeted drugs against the immune checkpoint CD276 mainly include monoclonal antibodies, ADCs, and ADCCs; small molecule inhibitors also show promising application prospects due to their advantages such as low cost, ease of modification, good permeability, and low likelihood of inducing immunogenicity and adverse host reactions. However, screening for effective CD276 inhibitors from numerous large databases is a laborious and time-consuming task. If the key binding sites on CD276 can be identified, targeted screening can be conducted, thereby greatly improving the efficiency of obtaining targeted inhibitors. Summary of the Invention

[0003] To address the aforementioned technical problems, this application provides a method for screening CD276 inhibitors, including screening inhibitors that bind to amino acids at positions 217, 218, and / or 339 of CD276; the amino acid sequence of CD276 is referenced in UniProtKB / Swiss-Prot:Q5ZPR3.1.

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

[0005] In some embodiments, the screening method described above further includes obtaining structural data of the CD276 protein and candidate inhibitors, as well as the spatial structure of the complexes formed after they come into contact.

[0006] This application also provides the use of amino acids at positions 217, 218, and / or 339 of CD276 for screening CD276 inhibitors or anticancer drugs.

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

[0008] This application also provides the use of a CD276 inhibitor in the preparation of a medicament for the prevention and / or treatment of tumors, wherein the CD276 inhibitor is bound to amino acids at positions 217, 218, and / or 339 of CD276.

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

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

[0011] This application also provides the non-diagnostic or non-therapeutic use of flavonoids in the inhibition of CD276.

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

[0013] Advantages of this application: The method of this application can efficiently screen or pre-evaluate drugs, compounds, compositions or formulations that are expected to inhibit CD276, and can effectively predict the inhibitory effect of candidate inhibitors based on key binding sites on CD276, which greatly saves time and effort in random blind selection. Attached Figure Description

[0014] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application and to make other features, objectives and advantages of this application more apparent.

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

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

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

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

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

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

[0021] Figure 7 Quercetagetin inhibits castration and resists tumor growth.

[0022] Figure 8 Quercetagetin has good biocompatibility.

[0023] Figure 9 Chemical structural formulas of Quercetin, Quercetagetin, and Patuletin. Detailed Implementation

[0024] Embodiments of this application will now be described with reference to the accompanying drawings. Elements and features described in one drawing or embodiment of this application may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that, for clarity, representations and descriptions unrelated to this application and known to those skilled in the art have been omitted from the drawings and description. The application will now be further described with reference to the accompanying drawings.

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

[0026] In some implementations, the screening method includes contacting a candidate inhibitor with CD276, detecting the binding site of the candidate inhibitor to CD276, and selecting the candidate inhibitor if the binding site contains amino acids at positions 217, 218, and / or 339 of CD276.

[0027] In some embodiments, the screening method includes obtaining structural data of the CD276 protein and candidate inhibitors, as well as the spatial structure of the complex formed after they come into contact, determining the binding sites of the complex, and using the binding of amino acids 217, 218, and / or 339 of CD276 as screening criteria to screen for candidate inhibitors with high affinity for CD276.

[0028] This application also provides the use of an inhibitor of the 217th, 218th, and / or 339th amino acids of CD276 in the preparation of a medicament, compound, composition, or formulation targeting CD276.

[0029] This application also provides the use of a CD276 inhibitor in the preparation of a medicament for the prevention and / or treatment of tumors, wherein the CD276 inhibitor is bound to amino acids at positions 217, 218, and / or 339 of CD276.

[0030] This application also provides the non-diagnostic or non-therapeutic use of flavonoids in the inhibition of CD276.

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

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

[0033] definition

[0034] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0035] As used in the specification and the accompanying claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

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

[0037] "CD276 inhibitor" refers to a substance that can inhibit the transcription or translation of the CD276 gene, or inhibit the expression or activity of the CD276 protein, or 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, peptides, 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 large molecule. In some embodiments, the inhibitor is a drug, compound, composition, or formulation. In some embodiments, the CD276 inhibitor may or may not include flavonoids (i.e., non-flavonoids). In some specific embodiments, the flavonoid is one or more of quercetagetin, quercetin, or patientetin. In some embodiments, the flavonoid is not quercetagetin, quercetin, or patientetin, but other substances.

[0038] "Flavones" refers to a group of compounds consisting of two benzene rings connected by three carbon atoms, that is, a class of compounds with a C6-C3-C6 structure.

[0039] A “composition” (such as a pharmaceutical composition) may contain pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are those that, when properly administered to animals or humans, do not produce adverse, allergic, or other adverse reactions.

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

[0041] "Cell" can be an animal cell or a human cell, and can be an isolated cell, an in vitro cell, or an in vivo cell; optionally, it can be a tumor cell (cancer cell) or a metastatic cancer cell, such as breast cancer cells, pancreatic cancer cells, prostate cancer cells, etc. In some embodiments, the cell is a metastatic castration-resistant prostate cancer (mCRPC) cell.

[0042] Cell proliferation is the basis of an organism's growth, development, reproduction, and heredity. Abnormal cell proliferation may lead to the formation of tumors.

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

[0044] The methods described in this application can be in vitro or in vivo; they can be diagnostic or therapeutic methods or non-diagnostic or non-therapeutic methods; and they can be used for drug screening or preparation in humans or non-human animals.

[0045] Example

[0046] Example 1 Analysis of Combined Pocket

[0047] The alphafold prediction structure of human CD276 was selected and downloaded from the Uniprot database website (https: / / www.uniprot.org / Q5ZPR3). The SiteFinder module of MOE software was used to analyze the alphafold structure of CD276, which contains three binding pockets: Site1, Site2, and Site3 (see...). Figure 1Among 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. Studies have found that the CD276 gene undergoes genome duplication, resulting in tandem repeats of the immunoglobulin-like V and C domains (VC domain). The dominant human CD276 isoform contains tandemly repeated VC domains.

[0048] Since the drug-like properties of Site 1 and Site 2 pockets are relatively good, we selected Site 1 and Site 2 for initial screening. However, the later screening results of Site 2 were not good (few compounds were bound and the scores were not high). Therefore, we focused on Site 1 as the binding pocket for further study.

[0049] Example 2: Screening for compounds with the best affinity

[0050] We use a library of natural product monomer compounds as candidate inhibitors to illustrate the screening method of this application, but this should not be considered a limitation of this application. By contacting the candidate inhibitors with CD276, based on the analysis in Example 1, focusing on the affinity score information between the compounds and CD276, we obtained 194 and 65 compounds, respectively, that could directly interact with the CD276 structure at Site1 and Site2. Ultimately, we found that the compound Quercetagetin has a significantly high affinity for the CD276 protein. Subsequently, we fully validated the high affinity of Quercetagetin for the CD276 protein using SPR, MST, and CETSA experiments. Specifically:

[0051] 2.1 SPR affinity experiment:

[0052] The CD276 recombinant protein (His Tag) was dissolved to a concentration of 0.25 mg / mL. 80 μL of this solution was mixed with 120 μL of sodium acetate buffer (PH4.0) to induce a positive protein response. 100 μL of LEDC and NHS, and 140 μL of ethanolamine were prepared for chip-coupled protein administration. Subsequently, different concentration gradients of compounds were prepared (half-dilution method), including 0 μM plus an intermediate concentration. The samples were placed sequentially in the sample holder and then placed in the instrument. The program was set to begin the experiment.

[0053] 2.2 MST affinity experiment:

[0054] (1) Mix 100 nM Red-NHS647 dye with protein of a previously determined concentration at a volume ratio of 1:1, incubate at room temperature in the dark for 30 min, centrifuge at 12000 rpm for 10 min, take the supernatant and label it.

[0055] (2) Prepare 16 200 μL low adsorption EP tubes in sequence. Add 5 μL of PBST solution to each tube in sequence. Add 5 μL of the compound Quercetagetin to the top of the cap of tube 1. Centrifuge, vortex and centrifuge again. Draw 5 μL of solution from tube 1 to the top of the cap of tube 2. Dilute in this way. Draw 5 μL from the last tube as well.

[0056] (3) Add 5 μL of labeled protein to the top of each of the 16 EP tube caps, centrifuge, vortex and 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 tube in sequence to aspirate the mixture. Place them on the detection tray for testing.

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

[0059] 2.3 Cell thermal displacement (CETSA) assay for the affinity of quercetagetin for CD276 protein:

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

[0061] (2) Cell heat treatment: After 2 hours of drug treatment, the culture medium was discarded, the cells were washed three times with PBS buffer, the cells were digested with 0.25% trypsin and collected, and the cells were resuspended with PBS buffer. The two groups of cells were divided into 6 parts each, and the 12 groups of cells were treated in a water bath at 40℃, 45℃, 50℃, 55℃, 60℃ and 65℃ for 90 seconds.

[0062] (3) Protein preparation: After cell heat treatment, the cells were allowed to return to room temperature for 90 seconds, then placed in liquid nitrogen for 3 seconds. After thawing at room temperature, the cells were vortexed thoroughly. This process was repeated 5 times to lyse the cells. The supernatant was collected at 12000 rpm and 4℃ for 20 minutes and stored at -80℃. The effect of quercetagetin on the thermostability of CD276 protein was subsequently detected by Western blot.

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

[0064] Example 3: Identification of key binding sites on CD276

[0065] First, use Molecular docking of the compound Quercetagetin with the CD276 protein revealed that Quercetagetin forms stable hydrogen bonds with amino acids at positions T217, Y218, and / or T339 of CD276 via its flavonoid backbone. Figure 3 This gives Quercetagetin and CD276 a strong affinity for each other.

[0066] Therefore, those skilled in the art can foresee that flavonoids, such as quercetin, will... Figure 9-1 Quercetagetin (hexahydroxyflavone) Figure 9-2 ), or Patuletin ( Figure 9-3 All of these can bind stably to the CD276 protein through the same flavonoid backbone.

[0067] Secondly, to verify this result, we constructed mutant cell lines containing CD276 mutated from 217T / 218Y / 339T to 217R / 218R / 339R, and used MST assays to detect the affinity of quercetagetin for wild-type and mutant CD276 proteins.

[0068] (1) Extraction of membrane proteins: This step was performed in accordance with the instructions of the Membrane Protein and Plasma Protein Extraction Kit (Catalog No.: P0033) from Shanghai Beyotime Biotechnology Co., Ltd.

[0069] (2) Adjusting the fluorescence intensity of membrane proteins: First, since EGFP protein is green fluorescent, blue light was selected for subsequent operations after the MST detection instrument was turned on. After setting all the instrument parameters, 10 μL of wild-type and mutant membrane proteins were taken and drawn into the instrument using the capillary tube provided with the instrument. The fluorescence value of each sample was adjusted to 600 based on the detected fluorescence value.

[0070] (3) The compound Quercetagetin was serially diluted from 50 μM to 16 concentrations. Then, 5 μL of each concentration was mixed with 5 μL of the fluorescence-adjusted membrane protein and analyzed. The detection data were calculated and evaluated using MO.Affinity Analysis software.

[0071] result: Figure 4 The results indicate that wild-type CD276 protein maintains a high affinity for quercetagetin, while the T217 / Y218 double mutation reduces the affinity between CD276 protein and quercetagetin by approximately 7-fold; the T339 single mutation reduces the affinity by approximately 4-fold; and the 217T / 218Y / 339T triple mutant completely impairs the affinity between CD276 protein and quercetagetin. It can be seen that the T217, Y218, and / or T339 sites play a crucial role in the binding of CD276 protein to quercetagetin and are key sites for efficient CD276 binding. Therefore, amino acids at positions T217, Y218, and / or T339 of CD276 can be widely used as key binding sites for the rapid screening of any CD276 inhibitors or anticancer drugs.

[0072] Example 4: Anticancer activity of quercetagetin

[0073] 4.1 MTT cell proliferation assay:

[0074] (1) Cell seeding: LNCaP, PC-3, Myc-CaP, and RM-1 prostate cancer cells in logarithmic growth phase and in good condition were collected. After digestion with 0.25% trypsin, the cells were counted, and then the cell concentration was adjusted using medium containing 10% FBS. The concentrations of LNCaP and PC-3 cells were adjusted to 1.5 × 10⁴ cells / mL, and the concentrations of Myc-CaP and RM-1 cells were adjusted to 4 × 10³ cells / mL. Cells were seeded into 96-well plates with a final volume of 200 μL per well, with 6 replicates for each cell type. The cells were sealed in the wells with 200 μL of PBS.

[0075] (2) Drug treatment: Accurately weigh an appropriate amount of the compound Quercetagetin using a balance, prepare a 10mM stock solution with DMSO, dissolve the compound Quercetagetin in the stock solution with medium containing 10% FBS to a concentration of 50μM, and then dilute it twice; after the cells have completely adhered to the wall, add medium containing different concentrations of the compound Quercetagetin to the experimental group, and add an equal amount of DMSO to the control group without adding the compound Quercetagetin; set up a blank group, which only has medium and does not inoculate cells, and adds the corresponding amount of the compound Quercetagetin and DMSO as a control.

[0076] (3) MTT treatment: After treatment with the compound Quercetagetin for 48 h, 20 μL of MTT (5 mg / mL) solution was added to each well under dark conditions and incubated in an incubator for 4 h in the dark.

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

[0078] 4.2 Effects on mouse lung metastasis model

[0079] (1) Group 1 (G1) is the shCtrl group, referring to the control stable cell line group of RM-1 cells infected with Control shRNA lentivirus and selected. Group 2 (G2) is the shCtrl inhibitor group, referring to the control stable cell line group of RM-1 cells infected with Control shRNA lentivirus and selected, plus Quercetagetin treatment. Group 3 (G3) is the shCD276 group, referring to the CD276 knockdown stable cell line group of constructed RM-1 cells infected with CD276 shRNA lentivirus and selected. RM-1-shCtrl and RM-1-shCD276 cells in logarithmic growth and good condition were collected, digested into cell suspension, and the cell density was adjusted to 5×10⁻⁶. 6 The single-cell suspension was prepared at 1 cell / mL and placed on ice for later use.

[0080] (2) Using a 1 mL syringe, 200 μL of RM-1-shCtrl and RM-1-shCD276 cell suspensions were drawn and injected into mice via the tail vein. Specifically, one week after tumor bearing, mice injected with RM-1-shCtrl were re-randomized and began daily intraperitoneal injection of 200 μL of 2 mg / kg Quercetagetin or 2% DMSO saline, while mice injected with RM-1-shCD276 were treated daily with daily intraperitoneal injection of 200 μL of 2% DMSO saline.

[0081] (3) After 12 days of administration, stop administration for 2 days. Take one lung from each group for fixation and HE staining. Use picric acid staining solution to irrigate the lungs of the rest. Take photos and record them after removal.

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

[0083] (1) Collect RM-1-shCtrl and RM-1-shCD276 cells that are in good logarithmic growth condition, digest them into a cell suspension, and adjust the cell density to 5×10⁻⁶. 6 The single-cell suspension was prepared at 1 cell / mL and placed on ice for later use.

[0084] (2) Using a 1mL syringe, 200μL of RM-1-shCtrl and RM-1-shCD276 cell suspension was injected into the right dorsal groin area. The injection continued until the tumor grew to approximately 250mm. 3 At that time, Degareli was administered subcutaneously at a dose of 25 mg / kg for castration treatment.

[0085] (3) After about a week of regression, the tumors regenerated. Mice injected with RM-1-shCtrl were re-randomized and treated with daily intraperitoneal injection of 200 μL of 2 mg / kg Quercetagetin or 2% DMSO saline. Mice injected with RM-1-shCD276 were treated with daily intraperitoneal injection of 200 μL of 2% DMSO saline.

[0086] (4) After 12 days of administration, the administration was stopped. One animal from each group was used to fix the heart, liver, spleen, lungs and kidneys for HE staining. At the same time, the tumor tissue was photographed.

[0087] (5) During the administration period, the weight of the mice was measured and recorded every two days to plot a weight change curve and observe the effect of the drug on the weight of the mice. At the same time, the length (a) and width (b) of the tumor on the back of each mouse were measured using calipers. The tumor volume was calculated according to the formula: Tumor volume = 0.5 × a × b × b, and the tumor growth curve was plotted.

[0088] 4.4 Results:

[0089] ① The MTT assay demonstrated that quercetagetin significantly inhibited the proliferation of prostate cancer cells, and this inhibitory effect was closely related to the expression level of CD276. In human cells, compared to PC-3 cells, quercetagetin showed a more significant inhibitory effect on cell proliferation in LNCaP cells with higher CD276 expression levels, as evidenced by a lower IC50 value in LNCaP cells than in PC-3 cells. In mouse cells, compared to Myc-CaP cells, RM-1 cells had a higher CD276 expression level, and quercetagetin showed a more significant inhibitory effect on their cell proliferation, as evidenced by a lower IC50 value in RM-1 cells than in Myc-CaP cells. Figure 5 This indicates that Quercetagetin inhibits cancer cell proliferation by inhibiting CD276. ② Quercetagetin significantly inhibits prostate cancer metastasis. Compared with the control group, the compound Quercetagetin significantly reduced the burden of tumor lung metastases. Figure 6 ③Quercetagetin significantly inhibited the proliferation of prostate cancer cells. Based on tumor growth curves and the weight of ex vivo tumors, compared to the control group, the compound Quercetagetin significantly inhibited tumor growth (…). Figure 7④ Quercetagetin exhibits good biocompatibility. Tests revealed no significant changes in routine blood tests and blood biochemical indicators in mice. H&E staining of major organs such as the heart, liver, spleen, lungs, and kidneys also showed that Quercetagetin had no toxic side effects on these organs. Figure 8 ).

[0090] The foregoing description of the specific embodiments of this application discloses the technical details of this application in detail and illustrates the technical concept of this application with examples, aiming to meet the authorization requirements of the patent law, but should not be regarded as a limitation on the scope of protection of this application. Those skilled in the art can make various changes or modifications based on this application and in combination with the knowledge and technology at that time, as long as they do not depart from the core concept and spirit of this application, they should all fall within the protection scope of the appended claims.

Claims

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

1.

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

3. The screening method as described in claim 1 or 2, characterized in that, It also includes obtaining structural data on the CD276 protein and candidate inhibitors, as well as the spatial structure of the complexes formed after they come into contact.

4. The use of amino acids 217, 218, and / or 339 of CD276 for screening CD276 inhibitors; including screening inhibitors that bind to amino acids 217, 218, and / or 339 of CD276, the amino acid sequence of which is referenced in UniProtKB / Swiss-Prot: Q5ZPR3.

1.

5. Use of hexahydroxyflavone in the preparation of drugs, compounds, compositions or formulations targeting CD276.

6. Use of hexahydroxyflavone in the preparation of drugs for treating prostate cancer.

7. Non-diagnostic or non-therapeutic uses of hexahydroxyflavone in the inhibition of CD276.

Images