Application of protopanaxidine B in the preparation of PD-L1 protein inhibitors

Abstract

This invention discloses the application of catarrhalpinx B in the preparation of PD-L1 protein inhibitors. Catarrhalpinx B or its pharmaceutically acceptable salt can be used as a PD-L1 protein inhibitor in the preparation of antitumor drugs. For the first time, surface plasmon resonance (SPR) experiments have confirmed that catarrhalpinx B can specifically target and bind to OTUB1, the deubiquitinating enzyme of PD-L1 protein. By inhibiting the deubiquitinating enzyme activity of OTUB1, it promotes the degradation of PD-L1 protein through the ubiquitin-proteasome pathway, thereby reducing the expression stability of PD-L1 at the post-translational level and inhibiting the growth of osteosarcoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of cataboloid B in the preparation of PD-L1 protein inhibitors. Background Technology

[0002] Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, known for its rapid growth, strong local invasiveness, and high metastatic potential, especially in the lungs, leading to a persistently high mortality rate. Although the comprehensive treatment model of "neoadjuvant chemotherapy + surgery + adjuvant chemotherapy" (with core drugs including methotrexate, doxorubicin, cisplatin, and ifosfamide) has significantly improved patient prognosis over the past few decades, current treatment still faces two major challenges: first, the severe toxic side effects of chemotherapy drugs and the development of drug resistance in tumor cells; second, the complex molecular mechanisms of its pathogenesis, with no universally applicable driver genes and effective molecular targets yet identified, resulting in slow progress in targeted drug development. Therefore, the search for novel drugs that can effectively inhibit osteosarcoma growth with fewer side effects is urgently needed.

[0003] To overcome treatment bottlenecks, research is advancing in multiple directions. Among these, modulating the tumor microenvironment to increase the infiltration of anti-tumor immune cells has emerged as a promising solution, aiming to transform "cold" tumors into "hot" tumors, thereby utilizing the body's own immune system to attack cancer cells. Simultaneously, the search for highly effective and low-toxicity therapies from natural compounds has also shown great potential. Plant-derived natural compounds not only possess extremely high structural diversity and biological activity, but also exhibit unique advantages in inhibiting proliferation, inducing apoptosis, and preventing metastasis. Summary of the Invention

[0004] The purpose of this invention is to provide a drug that can effectively treat osteosarcoma, especially osteosarcoma with high and normal expression.

[0005] To achieve the above objectives, the present invention provides the use of cataboloid B or a pharmaceutically acceptable salt thereof in the preparation of a therapeutic antitumor drug comprising a PD-L1 protein inhibitor.

[0006] Optionally, the cataboloid B has the structure shown in Formula I: Formula I.

[0007] Optionally, the tumor includes at least one of osteosarcoma, liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, or cervical cancer.

[0008] Optionally, the PD-L1 protein inhibitor is a substance that inhibits PD-L1 protein expression or promotes PD-L1 protein degradation.

[0009] Optionally, the PD-L1 protein inhibitor promotes the degradation of PD-L1 protein by targeting the PD-L1 deubiquitinating enzyme OTUB1 and reducing its deubiquitinating enzyme activity.

[0010] Optionally, the antitumor drug may further comprise a CTLA4 antibody.

[0011] This invention also provides the use of cataboloid B or a pharmaceutically acceptable salt thereof in the preparation of products for treating immunosuppression-related diseases.

[0012] Optionally, the immunosuppression-related disease is caused by overexpression of the PD-L1 protein.

[0013] Optionally, the immunosuppression-related diseases include at least one of metabolic diseases, osteoporosis, neurodegenerative diseases, respiratory diseases, cardiovascular diseases, and brain injuries.

[0014] This invention also provides the use of cytosine B or a pharmaceutically acceptable salt thereof in the preparation of products that promote the infiltration of immune cells, wherein the immune cells are CD8+. + T cells.

[0015] Compared to the prior art, the beneficial effects of the present invention include at least the following: This invention is the first to discover that vincetoxicoside B (VB) can act as a PD-L1 protein inhibitor, providing a novel therapeutic candidate compound for tumors with high PD-L1 expression (especially osteosarcoma). Surface plasmon resonance (SPR) experiments have demonstrated for the first time that vincetoxicoside B can specifically target and bind to OTUB1, the deubiquitinating enzyme of PD-L1 protein. By inhibiting the deubiquitinating enzyme activity of OTUB1, it promotes the degradation of PD-L1 protein via the ubiquitin-proteasome pathway, thereby reducing the expression stability of PD-L1 at the post-translational level and inhibiting the growth of osteosarcoma. Vincetoxicoside B of this invention provides a therapeutic approach different from monoclonal antibody drugs by degrading PD-L1 rather than blocking its binding to the receptor. Attached Figure Description

[0016] Figure 1 This illustrates the expression of PD-L1 in osteosarcoma according to the present invention. A: IHC staining in clinical osteosarcoma samples, scale bar 30 μm. Tumor samples from all three patients showed high PD-L1 expression; B: Correlation curves between PD-L1 expression levels and survival time in sarcoma patients in public databases, logrank P=0.012.

[0017] Figure 2This invention relates to the screening of cataboloid B, a drug targeting PD-L1, from a natural compound library. Specifically, A: A brief scheme targeting PD-L1 was screened from a natural compound library; B: Analysis of PD-L1 expression levels in four human and four murine osteosarcoma cells; C: PD-L1 expression levels after treating SJSA cell lines with 322 compounds screened by Western blotting; the figure shows the heatmap statistics. D: Molecular structural formula of cataboloid B; E: When three human osteosarcoma cell lines were treated with cataboloid B at different time points, the PD-L1 expression level decreased in a gradient with increasing treatment time. The figure shows the WB plot and its statistical plot. F: After treating three human cell lines with different concentrations of cataboloid B, the expression level of PD-L1 decreased with increasing concentration. The figure shows the WB plot and its statistical plot. G: Flow cytometry, different concentrations of cataboloid B were used to treat two types of cells. The surface PD-L1 level decreased with increasing treatment depth. The figure shows the histogram and its statistical graph. H: Immunofluorescence green represents PD-L1. Catastrophin B can reduce PD-L1 on the surface of tumor cells in a dose-dependent manner. I: Different concentrations of cataboloid B were used to treat osteosarcoma cell lines SJSA and HOS, and EDU staining showed no significant differences.

[0018] Figure 3 The present invention utilizes procyanidin B to degrade PD-L1 via the ubiquitin-proteasome pathway. A: Tumor cells were treated with cytoplasmic reticulin B at different concentration and time gradients, and q-PCR verification showed no significant change in PD-L1 DNA levels. B: Treatment of tumor cells with actinomycin and cytosine B can significantly reduce the half-life of PD-L1; C: After treatment with MG132, Western blot analysis of total protein showed that it could significantly restore the degradation of prostaglandin B. DE: After treatment with CQ and 3MA, Western blot analysis of total protein showed that neither treatment significantly restored the degradation of prostaglandin B. FH: After treatment with MG132, CQ, and 3MA, flow cytometry was used to verify cell surface proteins. MG132 significantly restored the degradation of cataboloid B. I: Transcriptomics testing, listing the top 20 pathways; J: When VB is present, the ubiquitination level of PD-L1 is significantly increased; K: In protein isolation experiments, after treatment with cataboloid B, PD-L1 was mainly located in the endoplasmic reticulum and nucleus.

[0019] Figure 4 This invention provides an immune-dependent inhibition of osteosarcoma development in mice using procytoside B. AD: Tumor cells, after being co-cultured with effector T cells and stained with crystal violet, showed that the presence of cataboloid B enhanced the killing ability of T cells. E: Schematic diagram of the in vivo experimental procedure. BALB / c mice were inoculated with K7M2 cells, followed by intraperitoneal injection of drugs. The experiment was observed until day 16. F: Actual images of mouse tumors, showing tumor samples from mice in each group after treatment with different doses of cataboloid B; G: Line graph of tumor volume change, showing the trend of tumor volume change in mice after treatment with different doses of VB. H: Scatter plot of tumor weight, statistics of tumor weight in mice after treatment with different doses of VB; I: Detection of PD-L1 protein expression in tumor tissues after treatment with different doses of VB; J: Immunohistochemical staining results of Ki67, Cl-Cas-3, CD8, and PD-L1 in tumor tissue after VB treatment; K: Schematic diagram of in vivo experimental procedure. Nude mice were inoculated with K7M2 cells and injected with drugs intraperitoneally. The experiment was observed until day 13. L: Actual image of mouse tumors; tumor samples from each group of mice after VB processing; M: Line graph of tumor volume change, showing the trend of tumor volume change in mice after treatment with different doses of VB; N: Scatter plot of tumor weight, statistics of tumor weight in mice after treatment with different doses of VB.

[0020] Figure 5 This invention demonstrates the inhibitory effect of cytosine B in combination with a drug on tumor growth in mice. Among other things, A: Schematic diagram of subcutaneous tumor model. BALB / c mice were inoculated with K7M2 cells, followed by intraperitoneal injection of drugs. The experiment was observed until day 16. B: Showing tumor samples from mice in different groups after drug treatment; C: The trend of tumor volume in mice after treatment with different groups of drugs as of the number of days after treatment; D: Tumor weight statistics of mice after drug treatment in different groups; EG: The expression levels of Gzmb, CD8, CD4 and Foxp3 in tumors after different treatment groups were detected by flow cytometry. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, all experimental methods described in this invention are conventional experimental methods in the art, and all experimental materials not specifically described in this invention are commercially available materials commonly used in the art.

[0022] In this invention, the term "immunosuppression" refers to a negative regulatory mechanism of the body's immune response. It actively inhibits or weakens the activation, proliferation, and function of immune cells (such as effector T cells and B cells) by activating inhibitory immune cells such as regulatory T cells (Tregs) or by using inhibitory receptors (such as CTLA-4 and PD-L1) to transmit negative signals, thereby maintaining immune tolerance, preventing excessive immune damage, or achieving therapeutic immune intervention.

[0023] In this invention, the term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds existing in the form of a free base or free acid can be converted into their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid according to methods known to those skilled in the art. Salts of the compounds of this invention can be converted into their free base or acid forms using standard techniques.

[0024] In this invention, pharmaceutically acceptable salts of cataboloid B include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange, and containing an amino group. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Other pharmaceutically acceptable salts include those formed by the quaternization of amines using suitable electrophilic agents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0025] In this invention, a "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, meaning it can be mixed with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0026] In this invention, the amino acid sequence of the PD-L1 protein is shown in SEQ ID NO.1: SEQ ID No. 1: MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET.

[0027] I. High expression of PD-L1 in osteosarcoma tissue Take 4 μm paraffin sections and bake them in an oven at 60℃ for 30 min. Dewax and hydrate them sequentially with xylene I-IV (5 min each), anhydrous ethanol I-II (3 min each), 95% ethanol (2 min), 85% ethanol (2 min), and distilled water (2 × 2 min).

[0028] Antigen retrieval was performed using citrate buffer (pH 6.0, Ki67 / PD-L1) or EDTA buffer (pH 9.0, Cl-Cas3 / CD8). The sections were immersed in the buffer, boiled in an autoclave for 2 minutes, then allowed to cool naturally for 20 minutes, and rinsed three times with distilled water. Endogenous peroxidase was blocked with 3% H2O2-methanol for 10 minutes, followed by washing with PBS 3×5 minutes each time. Blocking with 5% BSA at room temperature for 30 minutes was then performed. Primary antibodies (Ki67 1:200, Cl-Cas3 1:100, CD8 1:100, PD-L1 1:100) were incubated overnight at 4°C, followed by warming at 37°C for 45 minutes the next day, and then washing with PBS 3×8 minutes each time. HRP goat anti-rabbit / mouse IgG secondary antibody (1:500) was incubated at room temperature for 30 minutes, followed by washing with PBS 3×8 minutes each time.

[0029] Freshly prepared DAB solution (1 mL DAB buffer + 50 μL 30% H2O2) was used for staining for 5-10 min (controlled to a brownish-yellow color under a microscope), followed by washing with distilled water for 3 × 5 min. Hematoxylin was used for counterstaining for 2 min, followed by washing with tap water for 5 min, rapid differentiation with hydrochloric acid and alcohol, rinsing with tap water for 5 min, and dehydration with a gradient of ethanol (70%, 85%, 95%, and 100% for 2 min each), followed by clearing with xylene for 2 × 5 min, and mounting with neutral resin. ImageJ software was used to analyze the percentage of DAB-positive area or the average OD value of 5 representative fields / sections (n=6 mice / group, 3 sections per mouse).

[0030] Experimental results: such as Figure 1 As shown, IHC staining revealed that PD-L1 is generally highly expressed in osteosarcoma clinical samples, therefore PD-L1 can be considered as an important marker protein for osteosarcoma development.

[0031] II. Screening for low-toxicity drugs targeting PD-L1, such as cataboloid B, from a natural compound library.

[0032] The cataboloid B of the present invention has the structure of Formula I as follows: Formula I.

[0033] Cell proliferation assay: Adherent cells in the logarithmic growth phase were collected by trypsin digestion, centrifuged, and resuspended in fresh culture medium to prepare a single-cell suspension. Cells were counted using a hemocytometer. The cell suspension concentration was adjusted to 10^4 cells / mL. Using a multichannel pipette, 100 μL of cell suspension was added to each well of a 96-well plate. The suspension was gently mixed during addition to ensure uniform cell density in each well. After 24 hours, cell adhesion and morphology were observed under an inverted microscope. The old culture medium was carefully aspirated. Then, 100 μL of fresh culture medium containing the pre-set drug concentration was added. The 96-well plate was returned to a 37°C, 5% CO2 incubator and cultured for 6–72 hours according to the experimental design. After adding 10% CCK8 reagent and incubating for 2 hours, the plate was read at 475 nm. SJSA, 143B, and HOS cells were treated with leukopoietic acid B and analyzed by flow cytometry at specified time points. Cells were then collected and incubated with PE-conjugated PD-L1 antibody in the dark at 4°C for 30 minutes. After washing twice with PBS, the cells were analyzed using a BD Accuri C6 flow cytometer.

[0034] Immunofluorescence staining: Place sterile round cell slides into 24-well plates. Coat the slides with poly-L-lysine beforehand to prepare a single-cell suspension and count the cells. Stain each well with 2 × 10⁶ cells. 4 Seed cells at a density of [number] cells / well in 24-well plates. Incubate overnight at 37°C with 5% CO2. Remove the 24-well plates and discard the old culture medium. Slowly add pre-chilled 1× PBS along the well walls and wash 2-3 times, 3 minutes each time. Add 500 μL of 4% paraformaldehyde fixative to each well and incubate at room temperature for 15 minutes. After fixation, wash very gently 3 times with PBS on a decolorizing shaker for 5 minutes each time. Add 0.5% Triton X-100 solution to each well and incubate at room temperature for 15 minutes. After fixation, wash 3 times with PBS for 5 minutes each time.

[0035] Add PBS solution containing 10% BSA to each well and block at room temperature for 60 minutes. After blocking, discard the blocking solution directly. Dilute the primary antibody 1:200 with 1% BSA-PBS according to the antibody instructions and add overnight. The next day, remove the slides and place them back into the 24-well plate. Wash three times with PBS, 5 minutes each time. Add the secondary antibody and let it sit at room temperature for 1 hour. Place the slides back into the plate and wash three times with PBS, 5 minutes each time. Add 50 μL of DAPI working solution to each well and incubate at room temperature in the dark for 5-10 minutes. Wash four times with PBS, 5 minutes each time. Remove excess DAPI. Mount the slides. Place one drop of anti-fluorescence quenching mounting solution in the center of a clean glass slide. Use pointed forceps to pick up the slide and gently touch the edges to absorb excess PBS on absorbent paper. Then, with the cell side down, slowly place the slide at a 45-degree angle onto the mounting solution, avoiding air bubbles as much as possible. Incubate at room temperature in the dark for 15-30 minutes to allow the mounting solution to partially solidify. It can then be observed under a fluorescence microscope or a laser confocal microscope.

[0036] Western blots: Seed cells in 6cm dishes. When confluence reaches 50%-60%, proceed with the appropriate treatment: wash twice with pre-chilled PBS, add approximately 100μL of RIPA lysis buffer, vortex on ice for 30 minutes, centrifuge at 14000g, 4℃ for 15 minutes, collect the supernatant, quantify using the BCA method, add 5×SDS loading buffer, and boil at 100℃ for 10 minutes. Stack the gel at 80V for 20 minutes. Separate the gel at a constant voltage of 110V until the bromophenol blue front reaches 1cm below the lower edge. After soaking the PVDF membrane in methanol for 10 seconds, transfer it to the membrane. Equilibrate with buffer for 10 minutes, then rapidly transfer at 300 mA for 70 minutes (4°C ice bath). Prepare TBST (0.1% Tween-20) with 5% skim milk powder. Block on a shaker at room temperature for 1 hour. For phosphorylated proteins, use 5% BSA. To avoid interference from the milk powder on the phosphorylation signal, discard the blocking buffer. Wash with TBST 3 x 5 minutes. Dilute the primary antibody with universal diluent, add it, and incubate overnight at 4°C with shaker. Wash with TBST 3 x 5 minutes. Shake on a shaker at room temperature for 1 hour, wash with TBST 3 x 5 minutes. Mix ECL substrate in equal proportions, add evenly to the membrane surface, and let stand for 1-3 minutes. Collect data using a chemiluminescence analyzer.

[0037] Experimental results: such as Figure 2As shown, this invention identifies drugs with cytotoxic effects against osteosarcoma from a library containing 179 natural compounds. First, drugs that downregulate PD-L1 are screened by Western blotting (WB), and the effects of all drugs on cell proliferation are assessed using CCK8 assay. Programmed death-ligand receptor 1 (PD-L1) is a T-cell inhibitory checkpoint molecule 1 structurally expressed on the surface of tumor cells. The interaction between PD-L1 and its receptor can reduce the cytotoxic effect of tumor-infiltrating immune cells expressing cell surface programmed death-ligand-1 (PD-1), allowing tumor cells to evade immune surveillance. Combining the two screening methods, drugs that significantly downregulate PD-L1 and have no significant effect on proliferation are selected. In other words, the drugs exert their effects by influencing the immune system and have no significant cellular toxicity; ultimately, the drug cataboloid B was selected.

[0038] III. Procytosine B degrades PD-L1 via the ubiquitin-proteasome pathway. qPCR experiment: Total RNA was extracted from cells using TRIzol. RNA integrity was detected by 1.5% agarose gel electrophoresis. Take 1 μg of total RNA and reverse transcribe it using the PrimeScript™ RT Reagent Kit (TaKaRa, Japan) according to the manufacturer's instructions to synthesize cDNA. Dilute the reverse transcription product 5-fold with nuclease-free water and store at -20°C for later use. qPCR was performed using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa) kit. The total reaction volume was 20 μL, containing: 10 μL 2× SYBR Green premix, 0.8 μL forward primer, 0.8 μL reverse primer, 2 μL cDNA template, and 6.4 μL enzyme-free water. Each sample was tested in triplicate. The reaction program was: 95°C pre-denaturation for 30 s; followed by 40 cycles of 95°C denaturation for 5 s, 60°C annealing / extension for 30 s (fluorescence signal acquisition was performed here). After cycling, melting curve analysis (65°C to 95°C, heating rate 0.1°C / s) was performed to verify the specificity of the amplified product.

[0039] CoIP experiment: Collect cells and wash them twice with pre-chilled PBS. Add an appropriate amount of pre-chilled non-denaturing cell lysis buffer and lyse on ice for 30 min. Centrifuge at 14,000 × g for 15 min at 4°C, and transfer the supernatant to a new centrifuge tube. Denature the cells at 98°C for 5 min using 5× protein loading buffer, and store the resulting solution at -20°C for later use. Add equal amounts of total protein to two centrifuge tubes: (1) Experimental group: Add the target protein-specific antibody; (2) Negative control group: Add normal IgG from the same species. Make up the volume to 500 μL with lysis buffer and incubate overnight at 4°C to allow the antibody to fully bind with the target protein to form an immune complex. Add the magnetic beads to a 1.5 mL centrifuge tube and wash them 2-3 times with pre-chilled lysis buffer. Add the antibody-protein mixture to the pretreated magnetic beads and incubate at 4°C for 2-4 h. After incubation, place the centrifuge tubes on a magnetic rack to separate the magnetic beads, and carefully discard the supernatant. Resuspend the magnetic beads in pre-chilled lysis buffer, gently invert to mix and wash, and discard the supernatant after magnetic separation; repeat washing 3-5 times. Heat at 95°C for 10 min for denaturation and elution. Collect the supernatant after magnetic separation, and use Western blot to detect the target protein and its potential interacting proteins (prey proteins). Input samples and IP samples were analyzed together by SDS-PAGE electrophoresis and Western blotting.

[0040] Experimental results: such as Figure 3 As shown, qPCR results proved that it was not related to the transcriptional level. CHX experiments demonstrated that protein degradation accelerated after drug addition, presumably related to post-translational modifications. Testing with classical pathway inhibitors revealed a significant recovery in protein degradation after MG132 addition, presumably related to the ubiquitin-proteasome pathway. Flow cytometry further validated these results. Transcriptomics analysis revealed enrichment of the ubiquitin-proteasome pathway. CoIP experiments confirmed a significant increase in PD-L1 ubiquitination after drug addition. Finally, cytoplasmic separation revealed PD-L1 enrichment in the endoplasmic reticulum, further confirming the preceding process of degradation via the ubiquitin-proteasome pathway.

[0041] IV. Immunodependent inhibition of osteosarcoma development in mice by procytoside B PBMC cell isolation, activation, and culture: Collect whole blood from volunteers using anticoagulant tubes and dilute it 1:1 with PBS. Add an equal volume of Ficoll-Paque separation buffer to a 15 mL centrifuge tube. Slowly add diluent (1:2 volume ratio) to the top layer of the separation buffer, being careful to prevent mixing. Centrifuge at 800 g for 30 min (acceleration set to minimum). After centrifugation, the liquid levels in the tube, from top to bottom, should be: diluted plasma layer, PBMC layer, separation buffer layer, and red blood cell layer. Discard the plasma layer and carefully aspirate the PBMC layer (i.e., the white membrane layer) and transfer it to a 15 mL centrifuge tube. Resuspend the cells in 10 mL of 1× dilution wash buffer, centrifuge at 250 g for 10 min at room temperature, and discard the supernatant. Repeat this step 1 to 2 times before use in subsequent experiments.

[0042] Before isolating PBMCs, 2 μg / mL anti-IL-3 was added to 24-well plates overnight and incubated at 4°C overnight. The cells were washed twice with PBS and resuspended in complete culture medium to a concentration of 1.0 × 10⁶ cells / well. 6 Cells / mL, add anti-human CD28 (2 μg / mL) and IL-2 (50 U / mL), 1 mL / well, and seed into 24-well plates. Incubate in an incubator for 48 h. After activation, change the medium every 2-3 days, 1.0 × 10⁻⁶ cells / mL. 6 Seeds were prepared at a rate of 1 mL / well in 24-well plates, avoiding excessive pipetting during medium changes. A partial medium change method was used for both medium changes and amplification.

[0043] In vitro T cell killing assay: 143B or K7M2 cells were cultured at 3 × 10⁶ cells per well. 6 -6×10 6 Cells were seeded at a density of [number] cells per well in 12-well plates. After 24 hours of exposure to VB, PBMCs activated with PHA (1 mg / mL) / PMA (50 ng / mL) and overexpressing PD-1 were added and co-cultured for 24 hours at a 9:1 ratio of effector cells to target cells. Viable tumor cells were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and imaged using a Bio-Rad Cytation 5 instrument.

[0044] Subcutaneous tumor model: Four-week-old female BALB / c mice were subcutaneously injected with 3 × 10⁻⁶ mol / L saturates. 6 K7M2 osteosarcoma cells were collected. Tumors formed after one week. Mice were randomly assigned to groups and injected intraperitoneally with different concentrations of cataboloid B every two days, while tumor diameter was measured using calipers. Sixteen days later, mice were sacrificed and tumors were harvested. The tumor tissue was weighed.

[0045] Immunohistochemical (IHC) analysis: Tumor specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded tissue sections were dewaxed in xylene and treated with a series of fractionated alcohols and distilled water. Endogenous peroxidase activity was blocked by boiling at 95°C in 10 mmol / L citrate buffer (pH 6.0) and incubation with 5% hydrogen peroxide, and antigen was recovered. 5-micron thick sections were stained overnight at 4°C for PD-L1 (1:200), CD8 (1:200), c-caspase-3 (1:200), and Ki-67 (1:200). Horseradish peroxidase-labeled secondary antibody (1:1000) and DAB were used for detection, and sections were reverse stained with hematoxylin. Images were taken using a Leica (DM4 B, USA).

[0046] Immunoblotting: Mouse tumors were lysed using a 4-degree lysing device, RIPA was used for lysis, and after boiling with SDS for 10 minutes, PD-L1 expression levels were detected by Western blotting.

[0047] Experimental results: such as Figure 4 As shown, to demonstrate that cytosine B can inhibit tumor cell growth through cytotoxic T cells, a T cell co-culture system was used. The addition of cytosine B enhanced the cytotoxic ability of T cells. Simultaneously, in vivo experiments were conducted. Subcutaneous tumors were constructed in mice, and the mice were treated with intraperitoneal injection of cytosine B for two weeks. The growth rate and volume of bone tumors were significantly inhibited. Western blot analysis of proteins extracted from lysed mouse tumors showed that cytosine B significantly reduced the level of PD-L1 within the tumor, a result confirmed by immunohistochemistry. Immunohistochemistry simultaneously stained CD8, c-caspase-3, and Ki67, markers of CD8 cytotoxic T cells. Cytosine B significantly increased CD8 levels, promoting tumor apoptosis and inhibiting tumor proliferation. However, when the same cell line was used to construct subcutaneous tumors in immunodeficient mice and treated with cytosine B, it was found that tumor proliferation and growth were not altered. This demonstrates that the tumor-inhibiting ability of cytosine B is dependent on the presence of immune cells.

[0048] In some embodiments, the tumor comprises at least one of osteosarcoma, liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, or cervical cancer.

[0049] It is understood that the cataboloid B of the present invention, or a pharmaceutically acceptable salt thereof, can be used to prepare products for treating immunosuppression-related diseases. These immunosuppression-related diseases are caused by overexpression of the PD-L1 protein. These immunosuppression-related diseases include at least one of metabolic diseases, osteoporosis, neurodegenerative diseases, respiratory diseases, cardiovascular diseases, and brain injury.

[0050] V. Synergistic effect of procytoside B and PD-1 antibody to inhibit tumor growth in mice Tumor cells were collected, lysed into single cells, and resuspended in pre-chilled flow cytometry staining buffer (1% BSA in PBS) to prepare a single-cell suspension. To eliminate interference from dead cells, live cell staining was performed using LIVE / DEAD. Approximately 1 × 10⁶ cells were then used for live cell staining. 6 Cells were incubated with the corresponding fluorescein-conjugated monoclonal antibody at 4°C for 1 hour in the dark. The cells were washed twice with PBS containing 1% BSA to remove unbound antibodies. After resuspending the samples in an appropriate amount of buffer, they were analyzed using a Fortessa (BD USA) flow cytometer. All data were analyzed using FlowJo v10.8.1 software. Target cell populations were initially delineated using forward and side-scattering, and non-specific staining was excluded based on single staining. Finally, the expression levels of target molecules were analyzed.

[0051] Experimental results: such as Figure 5 As shown, compared with the control group, tumor growth was significantly inhibited in the groups treated with either catarrhalpinx B or anti-PD-1 alone. After combined treatment with catarrhalpinx B and anti-PD-1, tumor growth, tumor volume, and tumor weight were further improved. Flow cytometry revealed that the expression level of GzmB was significantly increased after the combined treatment with catarrhalpinx B and PD-1 antibody, confirming increased activity of cytotoxic T cells. Furthermore, the number of Treg cells was significantly reduced in the catarrhalpinx B and PD-1 antibody combined treatment group, indicating that catarrhalpinx B shifted the immune microenvironment from a suppressed state to an activated state. These results suggest that the combined use of catarrhalpinx B and PD-1 antibody can exert a better anti-tumor effect in vivo.

[0052] In summary, this invention provides the application of catarrhalpinx B in the preparation of PD-L1 protein inhibitors. Catarrhalpinx B or its pharmaceutically acceptable salt can be used as a PD-L1 protein inhibitor in the preparation of antitumor drugs. Surface plasmon resonance (SPR) experiments have demonstrated for the first time that catarrhalpinx B can specifically target and bind to OTUB1, the deubiquitinating enzyme of PD-L1 protein. By inhibiting the deubiquitinating enzyme activity of OTUB1, it promotes the degradation of PD-L1 protein through the ubiquitin-proteasome pathway, thereby reducing the expression stability of PD-L1 at the post-translational level and thus inhibiting the growth of osteosarcoma.

[0053] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The use of cataboloid B or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs, characterized in that, The anti-tumor drug includes a PD-L1 protein inhibitor.

2. The application as described in claim 1, characterized in that, The catabolone B has the structure shown in Formula I: Formula I.

3. The application as described in claim 1, characterized in that, The tumor includes at least one of osteosarcoma, liver cancer, pancreatic cancer, stomach cancer, intestinal cancer, or cervical cancer.

4. The application as described in claim 1, characterized in that, The PD-L1 protein inhibitor is a substance that inhibits PD-L1 protein expression or promotes PD-L1 protein degradation.

5. The application as described in claim 4, characterized in that, The PD-L1 protein inhibitor promotes the degradation of PD-L1 protein by targeting the PD-L1 deubiquitinating enzyme OTUB1 and reducing its deubiquitinating enzyme activity.

6. The application as described in claim 1, characterized in that, The anti-tumor drug also contains a CTLA4 antibody.

7. Use of cytosine B or a pharmaceutically acceptable salt thereof in the preparation of products for the treatment of immunosuppression-related diseases.

8. The application as described in claim 7, characterized in that, The immunosuppression-related disease is caused by overexpression of the PD-L1 protein.

9. The application as described in claim 7, characterized in that, The immunosuppression-related diseases include at least one of the following: metabolic diseases, osteoporosis, neurodegenerative diseases, respiratory diseases, cardiovascular diseases, and brain injuries.

10. The use of cytosine B or a pharmaceutically acceptable salt thereof in the preparation of products that promote immune cell infiltration, characterized in that, The immune cells are CD8. + T cells.

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