Application of Antrodia camphorata acid A as a small molecule inhibitor targeting ANXA2 in inhibiting angiogenesis

Abstract

The application relates to application of Antcin A as a small-molecule ANXA2 inhibitor in inhibition of angiogenesis, and the application proves for the first time that a natural triterpenoid compound Antcin A has significant in-vitro anti-angiogenesis activity, and for the first time confirms that ANXA2 protein is a direct action target point of the Antcin A, fills the blank of the research of the Antcin A in the field of angiogenesis, and provides a brand-new natural product skeleton for expansion of the structure type of ANXA2 targeted inhibitors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of anotropine A as an inhibitor of the ANXA2 protein target in the preparation of drugs for inhibiting pathological angiogenesis-related diseases. Background Technology

[0002] Angiogenesis refers to the physiological process by which new blood vessels form from an existing vascular network, playing an indispensable role in embryonic development, wound healing, and tissue repair. However, in various pathological conditions, including cancer, atherosclerosis, rheumatoid arthritis, psoriasis, endometriosis, obesity, and infectious diseases, this developmental process is abnormally reproduced and often lacks normal spatiotemporal feedback regulation, thus triggering pathological angiogenesis. Endothelial cells (ECs) are key functional cells in vascular development, covering all vascular luminal surfaces, and their proliferation, migration, and lumen-forming capabilities directly determine the formation of new blood vessels. Therefore, targeted inhibition of pathological angiogenesis has become an important therapeutic strategy for many of the aforementioned diseases.

[0003] Annexin A2 (ANXA2) belongs to the calcium-dependent phospholipid-binding protein family. Its encoding gene is located on human chromosome 15q22.2. The protein consists of 339 amino acids and has a molecular weight of approximately 38 kDa. Its structure comprises an N-terminal tail domain and a C-terminal core domain. The N-terminus contains the S100A10 binding site, phosphorylation site, and nuclear export signal. The C-terminal core domain consists of four repeating units, including an F-actin binding site, an RNA binding site, and a heparin binding site. ANXA2 plays a variety of important biological functions both intracellularly and extracellularly. Intracellularly, it participates in life processes such as endocytosis, exocytosis, apoptosis, cell proliferation, and cell division. Extracellularly, it plays a crucial role in fibrinolysis, cell migration, angiogenesis, and phagocytosis.

[0004] Existing research confirms that ANXA2 plays a crucial role in the regulation of pathological angiogenesis: on the one hand, ANXA2 can directly promote endothelial cell migration, invasion, and lumen formation; on the other hand, ANXA2 can synergistically enhance multiple angiogenesis signals by regulating the production of cell surface plasmin, integrin signaling pathway transduction, and participating in the VEGF signaling pathway. In various solid tumors, retinal neovascularization, and chronic inflammatory tissues, the expression level of ANXA2 is significantly upregulated, and its high expression level is closely related to poor clinical prognosis and resistance to anti-VEGF therapy. Therefore, ANXA2 is considered a highly promising novel anti-angiogenic target.

[0005] Currently, research on inhibitors targeting ANXA2 has made some progress, mainly covering the following four categories: (1) Peptide inhibitors: such as LCKLSL, which can competitively inhibit the binding of tissue plasminogen activator tPA to ANXA2, blocking the generation of plasmin; (2) Small molecule compounds: such as A2ti-1 and A2ti-2, which can specifically block the interaction of the ANXA2-S100A10 heterotetramer complex, used for research on HPV infection and tumor metastasis; (3) Natural products: such as ginsenoside Rg5 and baicalin, which can directly bind to ANXA2 and inhibit downstream pathways such as NF-κB and STAT-3; (4) Antibodies: such as mAb150 and humanized ch2448, which can recognize ANXA2 functional epitopes, block their biological functions and inhibit tumor growth. In addition, siRNA has also been used to indirectly silence the expression of ANXA2.

[0006] However, existing ANXA2 inhibitors have several key technical bottlenecks that severely restrict their clinical translation and application: (1) Insufficient specificity: ANXA2 is widely expressed in normal tissues, and non-targeted inhibition is prone to causing adverse side effects such as bleeding tendency and immune dysfunction; (2) Low biological activity: The half-maximal inhibitory concentration (IC50) of most small molecule inhibitors is relatively high, such as the IC50 of A2ti-2. 50 (2) The in vivo efficacy is difficult to meet clinical needs due to the high concentration of ANXA2 at up to 230 μM; (3) Poor drug-likeness: peptide and antibody drugs have poor stability, low cell membrane permeability, and high production costs; (4) Insufficient target selectivity: existing inhibitors are difficult to effectively distinguish between membrane-bound ANXA2 in pathological states and intracellular ANXA2 in normal physiological states, resulting in a high risk of off-target effects; (5) Limited indication coverage: existing research is mainly focused on the field of oncology. In contrast, research on specific inhibitors for other ANXA2-related diseases such as fibrosis, cardiovascular diseases, and retinal neovascularization is still in its early stages. In summary, existing technologies cannot meet the urgent clinical demand for ANXA2 inhibitors with high specificity, high activity, low toxicity, and good drug-likeness. There is an urgent need to develop novel small molecule ANXA2 targeted inhibitors to overcome the above defects and provide a safe and effective new targeted therapy strategy for diseases such as tumors and fibrosis.

[0007] Antcin A is a triterpenoid natural active compound derived from the precious medicinal fungus Antrodia cinnamomea. It has a molecular weight of 454.64 and the molecular formula C1. 29 H 42O4, CAS number 163597-24-8. Existing literature reports that Antcin A has potential therapeutic value in multiple biological processes: in liver-related disease models, Antcin A can target the NLRP3 inflammasome, alleviating pyroptosis and inflammatory responses associated with non-alcoholic fatty liver disease (NAFLD); in terms of anti-tumor mechanisms, Antcin A can regulate epithelial-mesenchymal transition (EMT) through p53-mediated miR-200c activation, inhibiting the migration and invasion of breast cancer cells; Antcin A can also act as a glucocorticoid receptor agonist, activating microRNA-708 and regulating tumorigenesis and metastasis in breast cancer by downregulating NF-κB signaling; in addition, Antcin A can block the EMT process in HepG2 liver cancer cells by inhibiting the Wnt / β-catenin signaling pathway and can mimic the anti-inflammatory effects of glucocorticoids. However, the effects of Antcin A on angiogenesis and its related molecular mechanisms have not been reported in any domestic or international literature. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a novel application of a natural small molecule ANXA2 targeting inhibitor in the preparation of drugs for inhibiting angiogenesis-related diseases.

[0009] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of this invention provides the use of anotropic acid A in the preparation of drugs that inhibit angiogenesis.

[0011] Furthermore, the inhibition of angiogenesis is achieved by targeting and inhibiting the function of annexin A2.

[0012] Furthermore, the inhibition of angiogenesis includes one or more of the following:

[0013] (1) Inhibits the proliferation of vascular endothelial cells;

[0014] (2) Inhibits the migration of vascular endothelial cells;

[0015] (3) Inhibits the formation of lumen in vascular endothelial cells.

[0016] A second aspect of the present invention provides the use of anotropic acid A in the preparation of medicaments for the prevention and / or treatment of pathological angiogenesis-related diseases.

[0017] Furthermore, the pathological angiogenesis-related diseases are selected from one or more of the following: tumors, retinal neovascularization, atherosclerosis, rheumatoid arthritis, psoriasis, endometriosis, obesity-related vascular lesions, fibrosis-related diseases, and pathological angiogenesis caused by infectious diseases.

[0018] A third aspect of the present invention provides a pharmaceutical composition for inhibiting angiogenesis, the pharmaceutical composition containing a therapeutically effective amount of anotropine A as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0019] It is worth emphasizing that the use of Anticin A to inhibit angiogenesis, protected by this invention, is fundamentally different from and completely independent of its anti-inflammatory, anti-tumor, and EMT uses. The anti-angiogenic effect targets the independent physiological and pathological process of new blood vessel formation from an existing vascular network, involving a series of unique cellular behavioral pathologies such as endothelial cell proliferation, migration, and lumen formation, in order to treat pathological angiogenesis disorders. The beneficial effects of using the above technical solution are as follows:

[0020] 1. This invention is the first to demonstrate that the natural triterpenoid compound Antcin A has significant in vitro anti-angiogenic activity, and the first to confirm that ANXA2 protein is its direct target, filling the gap in the research of Antcin A in the field of angiogenesis, and also providing a new natural product skeleton for the expansion of the structural types of ANXA2 targeted inhibitors.

[0021] 2. This invention employs multiple orthogonal experimental techniques, including cell thermal displacement analysis, drug affinity response target stability technology, and surface plasmon resonance, to confirm the direct and specific binding of Antcin A to ANXA2 protein from various perspectives, such as thermal stability protection, protease resistance enhancement, and real-time binding kinetics. The results are reliable.

[0022] 3. Experimental verification shows that Antcin A can effectively inhibit VEGF-induced endothelial cell proliferation, migration and lumen formation at low concentrations, exhibiting superior targeted inhibitory activity at the cellular level.

[0023] 4. Antcin A is derived from the edible and medicinal fungus *Antrodia camphorata*, which has a long history of medicinal use. It is a natural small molecule compound with cytotoxicity far lower than chemically synthesized small molecule drugs and antibody drugs. Within the concentration range that effectively inhibits angiogenesis, Antcin A has no significant inhibitory effect on the activity of normal endothelial cells and epithelial cells, exhibiting a good therapeutic window of safety.

[0024] 5. Anticin A has a small molecular weight and moderate lipid solubility, with good membrane permeability and oral bioavailability. It can be administered orally, overcoming the disadvantages of peptide and antibody drugs, such as the need for injection, poor stability, and high production costs. It is more in line with the requirements of convenience and economy in clinical drug use. Attached Figure Description

[0025] Figure 1The concentration screening for VEGF-stimulated vascular endothelial cell proliferation and the inhibitory effect of anotropine A on VEGF-induced endothelial cell proliferation activity are shown in the figure.

[0026] Among them, A: Cell viability results of vascular endothelial cells treated with different concentrations of VEGF (*P<0.05, ns indicates no statistical difference); B: Inhibitory effect of anotropine A on VEGF-induced endothelial cell proliferation (***P<0.001).

[0027] Figure 2 To detect the inhibitory effect of anotropine A on VEGF-induced planar migration of vascular endothelial cells using a scratch assay;

[0028] A: Representative microscopic images of vascular endothelial cell scratches at 0 h and 24 h in different treatment groups (scale bar = 50 µm); B: Statistical analysis results of the percentage of cell scratch healing area in each group (*P<0.05, **P<0.01, ***P<0.001).

[0029] Figure 3 To detect the inhibitory effect of anotropine A on VEGF-induced migration of vascular endothelial cells using a scratch assay;

[0030] Wherein, A: Representative microscopic images of vascular endothelial cells passing through the chambers in different treatment groups (scale bar = 100 µm); B: Statistical analysis results of cell migration in each group (*P<0.05, **P<0.01, ***P<0.001).

[0031] Figure 4 To detect the inhibitory effect of anotropine A on VEGF-induced lumen formation ability of vascular endothelial cells in a tube formation assay;

[0032] A: Representative microscopic images of vascular endothelial cell lumen formation in different treatment groups (scale bar = 100 µm); B: Statistical analysis results of the number of lumen junctions in each group;

[0033] Figure 5 A three-dimensional molecular docking model of Anticin A and ANXA2 protein;

[0034] Figure 6 To verify the direct binding of Antcin A to ANXA2 protein in cell thermal displacement experiments;

[0035] Figure 7 To verify the effect of Antcin A binding on ANXA2 protein protease resistance using DARTS;

[0036] Figure 8 To determine the binding affinity between Antcin A and recombinant ANXA2 protein using surface plasmon resonance;

[0037] Figure 9 To verify the knockdown efficiency of ANXA2 gene and protein levels after siRNA transfection;

[0038] In summary, A: qRT-PCR results showed that after transfection with ANXA2 siRNA (Si-ANXA2), the relative expression level of ANXA2 mRNA in vascular endothelial cells was significantly lower than that in the negative control siRNA (Si-Ctrl) group (***P<0.001); B: Western Blot results and protein quantification analysis showed that the expression level of ANXA2 protein in the Si-ANXA2 group was significantly lower than that in the Si-Ctrl group (*P<0.05).

[0039] Figure 10 To verify, through scratch assay, that anotropine A inhibits tumor microenvironment-induced planar migration of vascular endothelial cells via the ANXA2 target;

[0040] Among them, A: Representative microscopic images of vascular endothelial cell scratches at 0 h and 24 h under different treatment conditions (scale bar = 50 µm); B: Statistical analysis results of the percentage of cell scratch healing area in each group;

[0041] Figure 11 To verify, in a tube formation experiment, that anotropine A inhibits tumor microenvironment-induced vascular endothelial cell lumen formation via the ANXA2 target;

[0042] Wherein, A: representative microscopic images of vascular endothelial cell lumen formation under different treatment conditions (scale bar = 100 µm); B: statistical analysis results of the number of lumen connection points in each group. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0044] Source of reagents in this section:

[0045] Antcin A was purchased from Shanghai Taoshu Biotechnology Co., Ltd. (item number: TN7099).

[0046] Human umbilical vein fusion endothelial cells (EA.hy926) were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0047] VEGF recombinant protein was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Catalog No.: rp155965).

[0048] DMEM low-glucose medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0049] Sorafenib was purchased from Shanghai Taoshu Biotechnology Co., Ltd. (item number: T0093L).

[0050] Matrigel was purchased from Xiamen Moji Biotechnology Co., Ltd. (item number: 0827045).

[0051] The CCK-8 kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. (Catalog No.: 40203ES80).

[0052] Example 1: Antcin A inhibits VEGF-induced vascular endothelial cell proliferation.

[0053] 1.1 Experimental Materials

[0054] Human umbilical vein fusion endothelial cells (EA.hy926), recombinant VEGF protein, Antcin A (purity ≥98%, CAS No. 163597-24-8), CCK-8 kit, DMSO, and DMEM low-glucose medium.

[0055] 1.2 Experimental Methods

[0056] (1) Determination of the optimal VEGF concentration for promoting proliferation: EA.hy926 was prepared at a concentration of 5×10⁻⁶. 3 Cells were seeded per well in 96-well plates and cultured overnight. Then, different concentrations of VEGF (0, 5, 10, 20, and 40 ng / mL) were added for 24 hours. 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm, and the relative cell viability of each group was calculated.

[0057] (2) Dose-response relationship of Anticin A inhibiting VEGF-induced proliferation: EA.hy926 cells were seeded in 96-well plates and starved. Cells were stimulated with the determined optimal concentration of VEGF (20 ng / mL) and treated with different concentrations of Anticin A (1, 5, 10 μmol / L) for 24 hours. Cell viability was detected by CCK-8 assay. Sorafenib was used as a positive control.

[0058] 1.3 Experimental Results

[0059] The results of the CCK-8 experiment are as follows: Figure 1A showed that compared with the 0 ng / mL group, 20 and 40 ng / mL VEGF significantly promoted endothelial cell proliferation (P<0.05), with 20 ng / mL being the optimal concentration selected for subsequent experiments. ns indicates no statistical difference. Figure 1 B showed that VEGF significantly promoted endothelial cell proliferation compared with the VEGF-free group (P<0.001). Activated anolyte A inhibited this proliferation effect in a concentration-dependent manner. 10 μmol / L was the optimal inhibitory concentration selected for subsequent experiments. Compared with the VEGF-only treatment group, P<0.001.

[0060] Example 2: Antcin A inhibits VEGF-induced planar migration of vascular endothelial cells.

[0061] 2.1 Experimental Materials

[0062] EA.hy926, VEGF recombinant protein, Antcin A, positive control sorafenib, pipette tip, DMSO, DMEM low-glucose culture medium, fluorescence inverted microscope.

[0063] 2.2 Experimental Methods

[0064] Wound Healing Assay: EA.hy926 cells were seeded in 6-well plates. When cell confluence reached approximately 95%, a 200 μL sterile pipette tip was used to make uniform scratches along a ruler on the monolayer of cells. The cells were washed twice with PBS to remove floating cells, and the medium was replaced with serum-free medium containing VEGF (20 ng / mL) and Antcin A (10 µmol / L). A DMSO blank control group and a sorafenib positive control group were also established. The specific groups were as follows: DMSO blank control group (no VEGF, no drug); VEGF induction group (20 ng / mL VEGF); Antcin A group (20 ng / mL VEGF + 10 µmol / L Antcin A); and sorafenib positive control group (20 ng / mL VEGF + 5 µmol / L Sorafenib). Wound healing was recorded at 0 and 24 hours under an inverted microscope, and the wound healing rate was calculated using ImageJ software.

[0065] 2.3 Experimental Results

[0066] The results of the scratch test are as follows Figure 2 As shown, compared with the control group, VEGF treatment significantly improved the planar migration ability of endothelial cells; however, this trend was significantly reversed after treatment with Antcin A.

[0067] This shows that VEGF can significantly promote the migration of endothelial cells to the scratch area; treatment with anotropine A can significantly inhibit VEGF-induced cell migration, consistent with the effect of the positive control sorafenib, and the difference is statistically significant.

[0068] Example 3: Antcin A inhibits VEGF-induced migration of vascular endothelial cells.

[0069] 3.1 Experimental Materials

[0070] EA.hy926, VEGF recombinant protein, Antcin A, positive control sorafenib, pipette tip, DMSO, DMEM culture medium, Transwell chamber, fluorescence inverted microscope.

[0071] 3.2 Experimental Methods

[0072] Grouping method: control group (no VEGF, no drug), VEGF induction group (20 ng / mL VEGF), VEGF + anotropine A group (20 ng / mL VEGF + 10 µmol / L Antcin A), sorafenib positive control group (20 ng / mL VEGF + 5 µmol / L Sorafenib).

[0073] Transwell migration assay: Serum-free culture medium and cell suspension of each group of reagents were added to the upper chamber of the Transwell chamber (8 μm pore size) according to the group. Complete culture medium was added to the lower chamber as a chemotactic agent. After culturing for 8 hours, the chamber was removed, the cells on the upper chamber membrane were wiped off, and the cells that had passed through the microporous membrane were fixed with 4% paraformaldehyde. After crystal violet staining, the cells were counted and the number of cells that had passed through the membrane was counted under a microscope.

[0074] 3.3 Experimental Results

[0075] Transwell experimental results are as follows: Figure 3 As shown, compared with the control group, the number of endothelial cells that passed through the microporous membrane was significantly increased after VEGF treatment; this phenomenon was significantly reversed after Antcin A treatment.

[0076] This shows that VEGF can significantly promote the migration of endothelial cells; treatment with anotropine A can significantly inhibit VEGF-induced cell migration, consistent with the effect of the positive control sorafenib, and the difference is statistically significant.

[0077] Example 4: Antcin A inhibits VEGF-induced vascular endothelial cell lumen formation.

[0078] 4.1 Experimental Materials

[0079] EA.hy926, recombinant VEGF protein, Antcin A, Matrigel, DMSO, culture medium (DMEM), fluorescence inverted microscope.

[0080] 4.2 Experimental Methods

[0081] Groups: control group (no VEGF, no drug), VEGF treatment group (20 ng / mL VEGF), VEGF + anotropine A group (20 ng / mL VEGF, 10 µmol / L Antcin A), VEGF + sorafenib group (20 ng / mL VEGF, 5 µmol / L Sorafenib, positive control).

[0082] The in vitro tube formation assay was used: 200 μL of Matrigel matrix gel was added to each well of a pre-cooled 24-well plate, and polymerization was carried out at 37°C for 30 minutes to form a gel layer. EA.hy926 (8×10⁻⁶) was then added. 4 Cells (per well) were resuspended in serum-free medium containing the reagents for each group and seeded onto the surface of a Matrigel gel. The cells were incubated at 37°C for 6 hours. Luminous structures were photographed under a fluorescence inverted microscope, and the total luminal length, number of branch points, and number of luminal loops were quantitatively analyzed using the ImageJ software plugin (Angiogenesis Analyzer).

[0083] 4.3 Experimental Results

[0084] Results of extracorporeal lumen formation experiment as follows Figure 4 As shown, compared with the control group, the VEGF-treated group showed a significantly enhanced ability of endothelial cells to form tubular structures, with a significant increase in total lumen length, number of branch points, and number of lumen loops. However, after treatment with Antcin A, the total lumen length, number of branch points, and number of lumen loops were significantly reduced, indicating that Antcin A can effectively inhibit the formation of VEGF-induced in vitro vascular endothelial cell vascular networks.

[0085] Example 5: Small molecule affinity pull-down combined with mass spectrometry target screening based on biotin-labeled probes

[0086] 5.1 Preparation and Activity Verification of Biotin-Labeled Antcin A Probe (Antcin A-biotin)

[0087] A professional chemical synthesis institution was commissioned to introduce biotin labeling into the Antcin A molecule through an appropriate linker to prepare the Antcin A-biotin probe, with a purity verified by HPLC as ≥95%.

[0088] Activity validation: The inhibitory effect of Antcin A-biotin on VEGF-induced endothelial cell proliferation was detected by the CCK-8 assay and compared with that of equimolar concentrations of Antcin A monomer. The results showed that Antcin A-biotin and Antcin A monomer exhibited similar inhibitory effects on VEGF-induced endothelial cell proliferation (P > 0.05), indicating that biotin labeling did not affect the biological activity of Antcin A and can be used for subsequent target-pulling experiments.

[0089] 5.2 Pull-down combined mass spectrometry analysis

[0090] Total protein lysate of EA.hy926 cells was collected. Antcin A-biotin probe (or DMSO control) was incubated with cell lysate at 4°C for 6 hours. Streptavidin magnetic beads were added and incubated for another 12 hours. The magnetic beads were collected using a magnetic rack. After thorough washing, the bound proteins were eluted and separated by SDS-PAGE electrophoresis. After Coomassie brilliant blue staining, the differential bands between the Antcin A-biotin group and the control group (multiple significantly different bands with molecular weights in the range of 30–55 kDa) were excised and identified by liquid chromatography-mass spectrometry (LC-MS / MS).

[0091] 5.3 Stepwise screening of candidate target proteins

[0092] Mass spectrometry analysis yielded 408 candidate proteins, which were then filtered step-by-step according to the following stringent screening criteria: ① Unique peptide count ≥ 9; ② ​​Protein molecular weight range 30–55 kDa. After screening, 16 high-confidence potential target proteins were ultimately identified.

[0093] 5.4 Priority Ranking of Molecular Docking

[0094] Sixteen candidate proteins were selected, and their crystal structures (from the PDB database) were obtained. Molecular docking calculations were performed using AutoDock Vina software to assess the binding energy and key interactions between Antcin A and each candidate protein. Results showed that, based on the number of reliable peptides, ANXA2, ranked second, had a lower molecular docking binding energy (-7.48 kDa) with Antcin A, significantly better than other candidate proteins. The predicted binding site was located in the hydrophobic binding pocket region of ANXA2. The triterpenoid backbone of Antcin A forms hydrogen bonds and hydrophobic interactions with specific amino acid residues of ANXA2, such as... Figure 5 As shown, this suggests that ANXA2 is a possible direct target protein of Antcin A.

[0095] Example 6: Multidimensional in vitro verification of the specific interaction between Antcin A and ANXA2

[0096] 6.1 Cell thermal displacement analysis (CETSA)

[0097] Add 20 μM Antcin A or an equal volume of control DMSO to the lysate of EA.hy926 cells, pre-incubate at 37°C for 2 hours, aliquot into PCR tubes, and incubate at different temperatures (37, 42, 47, 52, 57, 62, 67, 72, 77, 82°C) for 3 minutes. After cooling on ice, centrifuge and use the supernatant for SDS-PAGE and Western blot. Detect the residual amount of ANXA2 protein at each temperature with ANXA2 antibody and perform quantitative image analysis.

[0098] The results are as follows Figure 6 As shown, in the DMSO control group, ANXA2 protein gradually degraded with increasing heating temperature, with significant degradation beginning at 62℃ and almost complete degradation at 82℃. In contrast, the degradation rate of ANXA2 in the Antcin A treatment group was significantly reduced under the same high-temperature conditions, and even at 87℃, its protein residue was still significantly higher than that in the control group. These results indicate that Antcin A can significantly enhance the thermal stability of ANXA2 protein, confirming that Antcin A can directly and specifically bind to ANXA2.

[0099] 6.2 Drug Affinity Response Target Stability Technology (DARTS)

[0100] EA.hy926 cell lysate was incubated with 20 μM Antcin A or control DMSO at room temperature for 2 hours. Different ratios of streptomycin (1:500, 1:250) were added and incubated at room temperature for 30 minutes. SDS-PAGE loading buffer was added to each sample, and the samples were boiled at 100°C for 8 minutes. SDS-PAGE / Western blot was performed to detect the degree of degradation of ANXA2 protein in each group using ANXA2 antibody.

[0101] The results are as follows Figure 7 As shown, compared to the DMSO control group, the degradation rate of ANXA2 in the Antcin A treatment group was significantly slower at the same proteinase K concentration, with more ANXA2 protein retained, and this effect was Antcin A concentration-dependent. The DARTS experiment further confirmed that the specific binding of Antcin A to ANXA2 reduces the sensitivity of ANXA2 to proteases, indicating a high binding affinity between the two.

[0102] 6.3 Surface Plasmon Resonance (SPR)

[0103] Recombinant human ANXA2 protein (purity ≥95%) was coupled and immobilized on the surface of a CM5 sensor chip, with a corresponding reference channel set up as a control. Different concentrations of Antcin A (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 μM) were continuously flowed through the sensor chip, and the binding and dissociation signal change curves (expressed in response units RU) were recorded in real time. The kinetic data were fitted using a 1:1 Langmuir binding model, and the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) were calculated.

[0104] The results are as follows Figure 8 As shown, the response signal of Antcin A binding to the ANXA2 immobilized chip increases with increasing concentration; the binding / dissociation curves exhibit typical rapid binding-rapid dissociation kinetics, and the curves at each concentration fit well, confirming the existence of a specific interaction between the two. Affinity analysis results show that the affinity constant KD value between Antcin A and ANXA2 protein is 43.8 μM, clarifying the affinity strength of their interaction.

[0105] Example 7: siRNA knockdown of ANXA2 to verify the function dependence of Antcin A

[0106] 7.1 siRNA transfection and ANXA2 knockdown efficiency verification

[0107] The specific siRNA sequence Si-ANXA2 targeting human ANXA2 mRNA and the negative control sequence Si-Ctrl were designed and synthesized. The specific double-stranded siRNA sequence targeting ANXA2 and the negative control siRNA double-stranded sequence are as follows:

[0108] Si-ANXA2 sequence:

[0109] Chain of Justice (Sense): 5'-GCAGCAAUGCACAGAGACA / dT / / dT / -3';

[0110] Antisense: 5'-UGUCUCUGUGCAUUGCUGC / dT / / dT / -3';

[0111] Si-Ctrl sequence:

[0112] Sense Chain: 5'-UUCUCCGAACGUGUCACGU / dT / / dT / -3';

[0113] Antisense: 5'-ACGUGACACGUUCGGAGAA / dT / / dT / -3';

[0114] Si-ANXA2 or Si-Ctrl was transfected into EA.hy926 cells using Highgene transfection reagent. After 48 hours of transfection, total RNA and total protein were extracted from the cells.

[0115] ANXA2 mRNA levels were detected by qRT-PCR (using GAPDH as an internal control), and ANXA2 protein levels were detected by Western blot (using β-Tublin as an internal control). Results are as follows: Figure 9 As shown, the levels of ANXA2 mRNA and protein in the Si-ANXA2 treatment group were significantly reduced, while there was no significant difference between the Si-Ctrl group and the blank control group, indicating that siRNA can effectively and specifically silence ANXA2 expression.

[0116] qRT-PCR primer sequence: ANXA2 F: 5´-TGGTTGAACACATTGGCCTC-3´;

[0117] ANXA2 R: 5´-GAGGGGAGATTCAGTGTGGT-3´;

[0118] GAPDH-F:5´-TCAAGAAGGTGGTGAAGCAG-3´;

[0119] GAPDH-R: 5´-GAGGGGAGATTCAGTGTGGT-3´.

[0120] 7.2 Effects of ANXA2 knockdown on endothelial cell migration and lumen formation

[0121] Scratch assay: The assay was performed in EA.hy926 cells transfected with Si-ANXA2 or the negative control Si-Ctrl. The experiment also included a HepG2 tumor conditioned medium (25% HepG2-CM) induction group and an Antcin A treatment group, specifically: ① Control group; ② HepG2-CM induction group; ③ HepG2-CM + Si-ANXA2 knockdown group; ④ HepG2-CM + Si-ANXA2 + Antcin A treatment group.

[0122] The results are as follows Figure 10As shown, compared with the Si-Ctrl control group, HepG2-CM induction significantly promoted endothelial cell migration, while Si-ANXA2 transfection knockdown of ANXA2 significantly inhibited cell migration and reduced scratch healing rate (*P < 0.05). More importantly, in the context of effective ANXA2 knockdown, additional Antcin A treatment did not further inhibit endothelial cell migration (ns, P > 0.05). These results suggest that the inhibitory effect of Antcin A on tumor microenvironment-induced vascular endothelial cell migration depends on ANXA2 expression.

[0123] Lumen formation assay: This assay was performed in EA.hy926 cells transfected with ANXA2-specific siRNA (Si-ANXA2) or negative control siRNA (Si-Ctrl / siNC). The experiment also included a HepG2 tumor-conditioned medium (25% HepG2-CM) induction group and an Antcin A treatment group, specifically: ① Control group; ② 25% HepG2-CM induction group; ③ 25% HepG2-CM + Si-ANXA2 knockdown group; ④ 25% HepG2-CM + Si-ANXA2 + Antcin A treatment group.

[0124] The results are shown in Figure 11: Compared with the control group, HepG2-CM induction significantly promoted endothelial cell lumen formation; while Si-ANXA2 transfection and ANXA2 knockdown significantly inhibited lumen formation and reduced the number of junctional sites (*P < 0.05). More importantly, in the context of effective ANXA2 knockdown, additional Antcin A treatment did not further inhibit endothelial cell lumen formation (ns, P > 0.05). These results demonstrate that the inhibitory effect of Antcin A on tumor microenvironment-induced vascular endothelial cell lumen formation depends on ANXA2 expression.

[0125] The above experimental results consistently demonstrate that the inhibitory effect of Antcin A on vascular endothelial cell migration and lumen formation depends on its target protein ANXA2, exhibiting clear target specificity and ruling out non-specific effects.

[0126] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of anotropine A in the preparation of drugs that inhibit angiogenesis.

2. The application according to claim 1, characterized in that, The inhibition of angiogenesis is achieved by targeting and inhibiting the function of annexin A2.

3. The application according to claim 1, characterized in that, The inhibition of angiogenesis includes one or more of the following: (1) Inhibits the proliferation of vascular endothelial cells; (2) Inhibits the migration of vascular endothelial cells; (3) Inhibits the formation of lumen in vascular endothelial cells.

4. Application of anotropine A in the preparation of drugs for the prevention and / or treatment of pathological angiogenesis-related diseases.

5. The application according to claim 4, characterized in that, The pathological angiogenesis-related diseases are selected from one or more of the following: tumors, retinal neovascularization, atherosclerosis, rheumatoid arthritis, psoriasis, endometriosis, obesity-related vascular lesions, fibrosis-related diseases, and pathological angiogenesis caused by infectious diseases.

6. A pharmaceutical composition for inhibiting angiogenesis, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of anotropine A as the active ingredient, and a pharmaceutically acceptable carrier or excipient.

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