Ferroptosis-inducing bisphenol compound and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a bisphenol compound for inducing ferroptosis and application thereof. According to the invention, HCC cells are treated by TMBPS, and lipid peroxide, Fe < 2 + >, malondialdehyde, reduced glutathione or oxidized glutathione, and mitochondrial morphology and potential in the HCC cells are detected. Target spots of TMBPS are predicted through a molecular docking method, and verification is carried out through quantitative real-time polymerase chain reaction, western blot and cell heat transfer experiments. The result of the embodiment shows that the TMBPS can induce ferroptosis of the HCC cells by increasing the levels of lipid peroxide, Fe < 2 + > and malondialdehyde in the cells and reducing the ratio of reduced glutathione or oxidized glutathione and the mitochondrial potential at the same time, and furthermore, the cell death triggered by the TMBPS in the HCC cells is related to ferroptosis and is mediated by inhibiting SLC7A11 expression.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a bisphenol compound that induces ferroptosis and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is characterized by high recurrence rate, high progression rate and high mortality rate. At present, liver resection and liver transplantation are the preferred traditional treatments for early HCC patients, but due to the limitation of early diagnosis level, most patients are diagnosed in the late stage, thus losing the best surgical period. For patients who cannot undergo surgery, systemic treatment (chemotherapy) of drugs is still the most important treatment method. Commonly used chemotherapy drugs include lenvatinib, regorafenib and sorafenib, which can effectively delay the tumor progression of patients. However, they also have disadvantages, including high toxicity, drug resistance and late complications of tumors. Therefore, the quality of life and five-year survival rate of patients with advanced HCC are still very low. Therefore, there is an urgent need for an innovative drug chemotherapy drug to develop a new drug chemotherapy drug with high efficiency, low toxicity and high tolerance for the treatment and improvement of the quality of life of HCC patients.

[0003] Among the evolving cancer therapies, iron mutagenesis is a prominent mode of programmed cell death (PCD) caused by dysregulated intracellular redox balance and elevated levels of lipid reactive oxygen species (ROS), as elucidated by seminal studies by Wei et al. and Chen et al., which have sparked intense interest in exploiting iron mutagenesis as a new strategy to target tumor cells and prompted us to explore its role in HCC treatment.

[0004] TMBPS is a bisphenol molecule with a sulfone structure and is one of the raw materials for making aromatic polyimides. It includes alternating block copolymer carbonates containing polysulfone component groups. Studies have shown that TMBPS can significantly inhibit tumor growth in HCC cell lines and mouse xenograft models, while having no obvious toxicity to normal liver cells L0-2.

[0005] However, how it induces tumor death has not been well studied. Therefore, it is urgent to further study the relationship between TMBPS inhibition of HCC proliferation and ferroptosis. Summary of the invention

[0006] The object of the present invention is to provide a bisphenol compound TMBPS that induces ferroptosis. The bisphenol compound TMBPS provided by the present invention can induce ferroptosis by inhibiting the expression of SLC7A11.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a bisphenol compound inducing ferroptosis, wherein the bisphenol compound is TMBPS.

[0009] The present invention also provides a ferroptosis inducer, wherein the active ingredient of the inducer includes the bisphenol compound.

[0010] The present invention also provides the use of the bisphenol compound in inducing ferroptosis of cancer cells.

[0011] Preferably, in said use, cancer cell death can be reversed by a ferritin inhibitor.

[0012] Preferably, the cancer cells include lung cancer cells, preferably HepG2 or Huh7 cells of the hepatocellular carcinoma cell line.

[0013] Preferably, the bisphenol compound can inhibit the expression of SLC7A11 protein.

[0014] The present invention also provides the above-mentioned bisphenol compound in increasing the lipid peroxide, Fe 2+ or malondialdehyde levels.

[0015] The present invention also provides a drug for treating diseases related to ferroptosis, wherein the active ingredients of the drug include the above-mentioned bisphenol compound and pharmaceutically acceptable excipients, and the dosage form of the drug is tablets, granules, powders, emulsions, capsules or injections.

[0016] The present invention also provides a pharmaceutical composition for inhibiting the expression of SLC7A11, wherein the composition comprises the above-mentioned bisphenol compound and a pharmaceutically acceptable excipient.

[0017] The present invention also provides the use of the above-mentioned bisphenol compound in the preparation of a medicine for treating diseases associated with ferroptosis.

[0018] Beneficial effects of the present invention:

[0019] The present study verified that Ferrostatin-1 (Fer-1) can effectively reverse TMBPS-induced HCC cell death. TMBPS treatment leads to typical ferroptosis features, including intracellular lipid peroxides, Fe 2+ The results showed that TMBPS-induced cell death in HCC cells was associated with ferroptosis and was mediated by the inhibition of SLC7A11 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is a graph showing the effect of TMBPS on the growth of HepG2 and Huh7 cell lines as determined by MTT colorimetric assay. The data are shown as the mean ± SD of three different experiments (compared with the control group with 0 μM TMBPS addition, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0022] Figure 2 Figure 1 shows the effect of TMBPS on the proliferation of HepG2 and Huh7 cells evaluated by EdU staining experiment (magnification 10 times);

[0023] Figure 3 The figure shows the effect of TMBPS on the expression of PCNA in HepG2 and Huh7 cells. The data are shown as the mean ± SD of three different experiments (compared with the control group with 0 μM TMBPS addition, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0024] Figure 4 The figure shows the effect of TMBPS on the iron ion concentration in HepG2 and Huh7 cells and the effect of TMBPS-induced iron ion concentration in liver cancer cells (magnified 20 times). The data are shown as the mean ± SD of three different experiments (compared with the control group with 0 μM TMBPS addition, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0025] Figure 5 The effect of TMBPS on lipid peroxidation in HepG2 and Huh7 cells, the effect of TMBPS on MDA in HepG2 and Huh7 cells, and the effect of TMBPS on GSH / GSSG levels in HepG2 and Huh7 cells are shown in the figure. The data are shown as the mean ± SD of three different experiments (compared with the control group with 0 μM TMBPS addition, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0026] Figure 6TMBPS inhibits the mitochondrial membrane potential of liver cancer cells. A shows the effect of TMBPS on the mitochondrial morphology of HepG2 and Huh7 cells (scale bar 1:500nm). B shows the effect of TMBPS on the mitochondrial membrane potential of HepG2 and Huh7 cells (magnification 10 times).

[0027] Figure 7 The effect of TMBPS on the key proteins of the main pathways in HepG2 and Huh7 mRNA levels is shown in the figure. The data are shown as the mean ± SD of three different experiments (compared with the control group with 0 μM TMBPS addition, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0028] Figure 8 The effect of TMBPS on the expression levels of SL7A11 and GPX4 in HepG2 and Huh7 cells is shown in the figure. The data are shown as the mean ± SD of three different experiments (compared with the control group with 0 μM TMBPS addition, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0029] Fig. 9 is the structural diagram of bis(4-hydroxy-3,5-dimethylphenyl)sulfone;

[0030] Fig.10 To show that TMSBP can reduce the expression of SLC7A11 and GPX4 in liver cancer cells, Discovery Studio used DS to process the two-dimensional model of seven iron mutant protein domains (SLC7A11, TFRC, IREB2, ACSL4, HSPB1, FTH1, GPX4) binding to TMBPS and used PyMOL to visualize the three-dimensional interaction between SLC7A11 and TMBPS at the residue level.

[0031] Fig.11 For CETSA to demonstrate SLC7A11 target engagement of TMBPS in Huh7 cells, protein stability was quantified and plotted as relative remaining amount versus temperature;

[0032] Fig.12TMBPS inhibits SLC7A11 and induces iron allergy in HCC. (AB) Expression levels of SLC7A11 mRNA and protein in HepG2 and Huh7 cells with overexpression of SLC7A11. (CE) Effects of TMBPS on cell viability, SLC7A11 mRNA and protein expression in HepG2 and Huh7 cells with overexpression of SLC7A11. (F) Effects of TMBPS on GPX4 protein expression in HepG2 and Huh7 cells with overexpression of SLC7A11. (GH) Effects of TMBPS on lipid peroxidation and iron ion concentration in HepG2 and Huh7 cells after overexpression of SLC7A11. Data are shown as mean ± SD of three different experiments (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control h-NC).

[0033] Fig.13 The effect of fer-1 and TMBPS combined treatment on liver cancer cells; A is a diagram showing the reversal effect of fer-1 on TMBPS-induced liver cancer cell activity inhibition by MTT colorimetry; B is a diagram showing the effect of fer-1 on TMBPS-induced lipid peroxidation in liver cancer cells, and the data are shown as the mean ± SD of three different experiments (compared with the control, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0034] Fig.14 The effect of fer-1 on the iron ion concentration in TMBPS-induced hepatoma cells (magnified 20 times). DETAILED DESCRIPTION

[0035] The present invention provides a bisphenol compound TMBPS for inducing ferroptosis. The bisphenol compound promotes intracellular lipid peroxides, Fe 2+ The increase in MDA levels simultaneously reduced the GSSH / GSH ratio and mitochondrial potential, thereby inducing ferroptosis in cancer cells.

[0036] The present invention uses TMBPS to treat hepatocellular carcinoma cell lines, preferably HepG2 and Huh7 cells. The cell viability, cell proliferation ability, intracellular lipid peroxides, reduced glutathione or oxidized glutathione, ions and malondialdehyde content of the cancer cell lines are detected over time. The experimental results show that after TMBPS treatment, as the TMBPS concentration increases, the lipid peroxides, Fe 2+The levels of reduced glutathione or oxidized glutathione and mitochondrial potential were significantly increased, while the ratio of reduced glutathione to oxidized glutathione and mitochondrial potential were significantly decreased. The mitochondria of the cells showed obvious iron mutation morphology, with reduced or even disappeared mitochondrial ridges, smaller mitochondria, and increased double membrane density, indicating that TMBPS destroyed the function of cancer cell mitochondria and participated in ferroptosis.

[0037] After treating cells with different doses of TMBPS, the present invention detected the expression levels of related genes using qRT-PCR and found that only the mRNA levels of SLC7A11 and GPX4 showed a significant concentration-dependent decrease. In addition, the present invention also used Western blotting to verify the results of qRT-PCR. The results showed that after TMBPS treatment, the levels of SLC7A11 and GPX4 in HCC cells were significantly decreased (P<0.01). These findings indicate that SLC7A11 and GPX4, important proteins in the system Xc signaling pathway, are potential targets for TMBPS-induced iron oxidation in HCC cells.

[0038] The present invention uses molecular docking experiments, and the results show that the binding affinity of TMBPS to SLC7A11 is much stronger than that to GPX4, and GPX4 is downstream of the membrane protein SLC7A11 located on the cell membrane and is regulated by it, thus further indicating that TMBPS can directly interact with SLC7A11. In order to further verify this interaction, the present invention performs CETSA detection on TMBPS and SLC7A11. The CETSA results show that in Huh7 cells treated with TMBPS, SLC7A11 physically interacts with TMBPS and is more stable in thermal changes, indicating that TMBPS-induced hepatocellular carcinoma cell ferroptosis response is related to its inhibition of SLC7A11.

[0039] The present invention uses an iron allergy inhibitor Ferrostatin-1 (Fer-1) and TMBPS to treat cells together, which will lead to changes in intracellular lipid peroxide and iron ion levels, indicating that Fer-1 can significantly inhibit Fe 2+ Furthermore, it was shown that Fer-1 could inhibit TMBPS-induced cell death, and the inhibition of TMBPS on the proliferation of liver cancer cells was related to its induced cell iron mutation.

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] TMBPS powder was purchased from Yunnan Xili Technology Co., Ltd., China; Fer-1, Z-VAD-FMK, Necrostatin-1 (Nec-1), MHY1485, and BAY 11-7082 powders were purchased from MedChem Express Co., USA; β-actin, SLC7A11, and GPX4 were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Proliferating cell nuclear antigen (PCNA) was provided by Abcam, Cambridge, UK; hepatocellular carcinoma cell lines HepG2 and Huh7 were from the Chinese University of Hong Kong; DMEM was from (HyClone, Logan, UT, United States.), inactivated fetal bovine serum was purchased from (Thermo Fisher Scientific, Green Island, NY, USA); penicillin-streptomycin was purchased from (HyClone, Logan, UT, United States).

[0042] EdU was purchased from (Beyotime Biotechnology, Cat.: C0078S); MTT was purchased from (Beyotime Biotechnology, Cat.: ST316); C11 BODIPY 581 / 591 probe was purchased from (ABclonal, Catalog: RM02821); FerroOrange was purchased from (Dojindo, F374); Beyotime MDA detection kit was purchased from (Dojindo, M496); GSH / GSSG detection kit was purchased from (Beyotime Biotechnology, Cat.: S0053); mitochondrial membrane potential detection kit (JC-1) was purchased from (Beyotime Biotechnology, Cat.: C2006); RIPA was purchased from (Beyotime Biotechnology, Cat.: P0013B); BCA protein assay kit was purchased from (Beyotime Biotechnology, Cat.: P0010); PVDF membrane was purchased from (Merck Millipore); Lipo6000 TM The transfection reagent was purchased from Bio-Tech Biotechnology Co., Ltd.

[0043] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0044] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0045] Example 1 Material preparation and cell culture

[0046] 1. Materials

[0047] TMBPS powder with a purity of ≥98% (determined by HPLC).

[0048] TMBPS and Fer-1 were dissolved in dimethyl sulfoxide (DMSO) to prepare 100 mM stock solutions with a final DMSO concentration of less than 0.1% and stored at -80°C.

[0049] 2. Cell Lines and Cell Culture

[0050] Hepatocellular carcinoma cell line cells were cultured in DMEM at 37°C in a 5% carbon dioxide incubator supplemented with 10% (v / v) inactivated fetal bovine serum and 1% penicillin-streptomycin.

[0051] Example 2 Cell Viability Analysis

[0052] To explore the effect of TMBPS on cell growth, the MTT assay was used to evaluate the cell viability of two human HCC cell lines (i.e., HepG2 and Huh7) after treatment with different doses of TMBPS.

[0053] 5×10 per well 3 HepG2 cells and Huh7 cells were seeded in 96-well plates and treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h, 48 h and 72 h. After adding 10% MTT to each well, the cells were cultured at 37°C for 4 hours. The absorbance of the cells was measured at a wavelength of 490 nm.

[0054] The results are as follows Figure 1 As shown, the results showed that TMBPS significantly inhibited the growth of lung cancer cells HepG2 and Huh7 cells in a time- and dose-dependent manner.

[0055] Example 3 EdU fluorescence staining experiment

[0056] EdU detection of cell proliferation experiment is a commonly used cell proliferation detection method, which evaluates the cell proliferation ability by detecting the binding of EdU to DNA.

[0057] 2×10 5 HepG2 and Huh7 cells were seeded at a cell density of 100 cells / mL / well in 12-well plates and treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h. The cells were then labeled with EdU, fixed, washed and permeabilized, and the nuclei were stained with DAPI, followed by fluorescence detection.

[0058] The results of EdU staining experiments are shown in Figure 2 As shown in Figure 3, after TMBPS treatment, the red fluorescence reflecting the cell proliferation level was weakened. Figure 3 As shown in the figure, the expression level of proliferating cell nuclear antigen (PCNA) also decreased with the increase of TMBPS concentration (P<0.01). These data indicate that TMBPS can inhibit the development of HCC cells.

[0059] Example 4 Determination of intracellular lipid peroxide content and iron ion content

[0060] The C11 BODIPY 581 / 591 probe was used to detect the level of lipid peroxides in the cell membrane.

[0061] 2×10 5 HepG2 and Huh7 cells were inoculated in 12-well plates at a cell density of 100 μg / mL / well. After overnight attachment, they were treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h, and the cells were cultured with C11-BODIPY581 / 591 (1 μM) in growth medium at 37 °C for 30 min. Next, the cells were collected and loaded with lipid peroxidation fluorescent probes for flow cytometry detection. FerroOrange, a divalent iron ion detection probe, was loaded for iron ion content determination, and detected by flow cytometry and fluorescence microscopy, respectively.

[0062] The results are as follows Figure 4 As shown, Fe in HCC cells measured by flow cytometry after 24 h of TMBPS treatment 2+ The level of β-catenin increased (P<0.01) and was positively correlated with the concentration of MBPS. Immunofluorescence staining also obtained similar results.

[0063] Iron poisoning in cells is mainly caused by the cumulative effect of lipid peroxides, which is dependent on Fe 2+ existence.

[0064] The oxidative stress indicators related to ferroptosis were detected in HCC cells treated with TMBPS. Lipid peroxides, the main indicator of ferroptosis, increased significantly after 24 hours of TMBPS treatment (P<0.01).

[0065] These results suggest that TMBPS promotes Fe 2+ Accumulation and increased levels of lipid peroxides induce iron allergy.

[0066] Example 5 Determination of intracellular MDA content and reduced glutathione (GSH) / oxidized glutathione (GSSG) content

[0067] Malondialdehyde (MDA) is a compound formed after the decomposition of lipid peroxides. Therefore, MDA is one of the most common indicators for detecting lipid peroxides in cells and tissues.

[0068] 2×10 5 Cells were inoculated in a 12-well plate at a cell density of 100 μL / well. After overnight attachment, cells were treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h. The experimental steps were performed according to the instructions of the MDA detection kit, and the fluorescence intensity was detected by a fluorescence microplate reader (Ex: 540 nm, Em: 590 nm). The MDA concentration in the sample was calculated using the MDA standard curve.

[0069] Use the GSH / GSSG detection kit for detection and quantification according to the instructions. Prepare cell samples and mix with reagents in a 96-well plate and immediately measure using an ELISA reader. GSH = Total Glutathione-GSSG × 2.

[0070] The results are as follows Figure 5 As shown, malondialdehyde (MDA), the end product of oxidative stress, also showed a significant increase in the TMBPS 10μM and 20μM groups (P<0.01). The levels of lipid peroxides and MDA in cancer cells treated with TMBPS were higher than those in cancer cells not treated with TMBPS, and were dose-dependent with TMBPS. Glutathione (GSH) is an important intracellular antioxidant that can scavenge lipid peroxides and inhibit ferroptosis through glutathione peroxidase. However, the ratio of GSH / GSSG decreased significantly with the increase of TMBPS concentration. (P<0.01).

[0071] Example 6 Determination of intracellular mitochondrial membrane potential

[0072] Mitochondria are multifaceted regulators of cell death, including necroptosis, ferroptosis, pyroptosis, and programmed cell death. Mitochondria undergo changes during ferroptosis that are different from other programmed cell death processes. Transmission electron microscopy and a mitochondrial membrane potential assay were used to evaluate the effect of TMBPS on mitochondrial damage in HCC cells.

[0073] 2×10 5 The cells were inoculated at a density of cells / 2 mL / well in a 12-well plate. After overnight attachment, they were treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h. The cells were treated according to the mitochondrial membrane potential detection kit (JC-1), and the changes in mitochondrial morphology were observed under an electron microscope, and the mitochondrial membrane potential was observed under a fluorescence microscope.

[0074] The results are as follows Figure 6 As shown in the figure, after 24 hours of TMBPS treatment, the mitochondria of HCC cells showed obvious iron mutation morphology, such as the reduction or even disappearance of mitochondrial ridges, the smaller mitochondria, and the increased density of double membranes. As the concentration of TMBPS in HCC cells increased, the membrane potential of mitochondria also increased.

[0075] These results suggest that TMBPS disrupts mitochondrial function in HCC cells and is involved in the initiation of ferroptosis.

[0076] Example 7 Real-time quantitative PCR (qRT-PCR)

[0077] 2×10 5 Cells were seeded in 12-well plates at a cell density of 100 μg / 2 mL / well. After overnight attachment, they were treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h. RNA was extracted from the cancer cell lines (Trizol, AG) and reversed into cDNA. Then the qPCR program was performed. β-actin was used as an internal reference, and the relative expression of different genes in the samples was analyzed by the 2-ΔΔCt relative quantitative method. Table 1 lists the primers for the seven major proteins in the ferritin sedimentation regulation pathway used in this study.

[0078] Table 1 Primer sequences used for qRT-PCR

[0079]

[0080]

[0081] qRT-PCR was used to detect the expression levels of related genes. Figure 7 Only the mRNA levels of SLC7A11 and GPX4 showed a significant concentration-dependent decrease (P<0.01).

[0082] Example 8 Western blotting experiment

[0083] Next, the results of qRT-PCR were verified by Western blotting.

[0084] 2×10 5 Cells were seeded in 12-well plates at a cell density of 100 μg / well / 2 mL / well. After overnight attachment, cells were treated with different concentrations of TMBPS (0, 5, 10, 20 μM) for 24 h, and then the collected cells were lysed in RIPA on ice for 30 minutes. After centrifugation at 10,000 rpm, the supernatant was collected and the protein concentration was quantified by the BCA protein assay kit. Equal amounts (12 μg) of total protein were separated in 15% SDS-PAGE and then transferred to PVDF membranes. The membrane containing the protein was blocked, washed, incubated with antibodies, and finally detected by enhanced chemiluminescence.

[0085] Western blotting was used to verify the results of qRT-PCR. Figure 8 As shown in the results, after TMBPS treatment, the levels of SLC7A11 and GPX4 in HCC cells were significantly decreased (P<0.01), indicating that SLC7A11 and GPX4, important proteins in the system Xc signaling pathway, are potential targets of TMBPS-induced iron oxidation in HCC cells.

[0086] Example 9 Molecular Docking Verification

[0087] The two-dimensional structure of TMBPS (PubChem CID: 13288-70-5) is shown in Fig. 9 The ligands were obtained from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) in SDF format, optimized using the “PrepareLigands” and “Full Minimization” modules of Discovery Studio 2019, and designated as ligands in all docking systems.

[0088] The protein structures of seven genes associated with iron mutations, SLC7A11 (PDB: 7P9V), GPX4 (PDB: 5H5Q), TFRC (PDB: 6OKD), IREB2 (PDB: 6VCD), HSPB1 (PDB: 6GJH), and FTH1 (PDB: 6J4A), were obtained from the RCSB PDB protein database (http: / / www.rcsb.org / ). The ACSL4 (Alpha Fold: AF-O60488-F1) protein structure was retrieved from the UniProt database (https: / / www.uniprot.org / ), which was predicted based on the Alpha Fold structure database (https: / / alphafold.com / ).

[0089] After removing water molecules, hydrogenation, and optimizing energy for each protein, molecular docking was performed using CDOCKER (Discovery Studio 2019), a grid-based molecular docking method that uses the CHARMm force field. For proteins with original ligands (HSPB1, TFRC, SLC7A11), CDOCKER docked TMBPS to the original ligand position according to the default settings. For proteins without original ligands, the receptor cavity module of Discovery Studio 2019 was used to predict the binding site, and then LibDock was used to select the best binding site according to the default settings. The site with the highest binding energy was then selected as the docking site for CDOCKER.

[0090] During the docking process, the top 10 conformations of TMBPS were generated based on the priority of docking scores. The binding mode was verified by evaluating the interactions between TMBPS and the binding site residues. To explore the interaction mode, the binding affinity of all docked complexes was analyzed using the CDOCKER_ENERGY scoring function. Lower docking affinity values ​​indicate stronger interactions between the active chemical and the target.

[0091] The docking results are as follows Fig.10 As shown in Table 2, it indicates that TMBPS has good binding potential with these proteins. The molecular docking results are shown in Table 2, showing that the binding affinity of TMBPS to SLC7A11 is much stronger than that to GPX4; CDOCKer energy: -34.3846 kcal mol -1 vs -14.3613 kcal mol -1 . It shows that SLC7A11 has the strongest binding energy with TMBPS.

[0092] Therefore, it was shown that TMBPS can directly interact with SLC7A11.

[0093] Table 2 Docking ability of TMBPS with ferroptosis-related core proteins

[0094]

[0095]

[0096] Example 10 Cell thermal transfer assay

[0097] Considering that the molecular docking results showed that the binding affinity of TMBPS to SLC7A11 was much stronger than that to GPX4, and GPX4 is downstream of and regulated by the membrane protein SLC7A11 located on the cell membrane, TMBPS is more likely to interact directly with SLC7A11. To further verify this interaction, CETSA was performed on TMBPS and SLC7A11. The cellular thermal shift assay (CETSA) is used to evaluate the stabilizing effect of compound interactions on targets in cells.

[0098] Human hepatocellular carcinoma cell line Huh7 cells in logarithmic growth phase were cultured at 1×10 6 When the cell confluence reached 60%, the cells were harvested with PBS solution containing 1% protease inhibitor (cocktail).

[0099] The supernatant was collected after repeated freezing and thawing three times at -37°C in liquid nitrogen and centrifuged for 20 minutes (4°C, 10,000 rpm) to detect the binding affinity of TMBPS to SLC7A11 under different temperature and concentration conditions.

[0100] To examine the effect of supernatant at different temperatures, DMSO and 10 μM TMBPS were added to the supernatant and incubated at room temperature for 30 min. The sample was then placed in a temperature range of 40–60 °C for 3 min and then rapidly cooled in an ice bath. Subsequently, 5× loading buffer was added and heated at 100 °C for 5 min.

[0101] The CETSA results are shown in Figure 11, indicating that in Huh7 cells treated with TMBPS, SLC7A11 physically interacted with TMBPS and was more stable during thermal changes.

[0102] Example 11 Overexpression of SLC7A11 reverses TMBPS-induced ferroptosis

[0103] To determine whether TMBPS specifically targets SLC7A11, HCC cells overexpressing SLC7A11 were constructed.

[0104] The SLC7A11 overexpression glycerol plasmid (h-SLC7A11) and control glycerol plasmid (h-NC) were designed and constructed by GeneCare Genetics.

[0105] The glycerol bacteria were cultured overnight at 37°C on a constant-temperature high-speed shaker. The plasmid was extracted according to the instructions of the Acres plasmid DNA extraction kit, and then lysed with Lipo6000. TM Transfection reagent for transfection.

[0106] The results are as follows Fig.12 As shown:

[0107] like Fig.12 As shown in AB, overexpression of SLC7A11 reversed the decrease in intracellular SLC7A11 mRNA and protein levels (P<0.01) and restored SLC7A11 to normal.

[0108] To evaluate the effect of TMBPS on the viability of HCC cells after SLC7A11 overexpression, MTT assay was performed.

[0109] like Fig.12 As shown in C, TMBPS suppressed the expression of SLC7A11 compared with the control group, and this inhibitory effect persisted even after SLC7A11 overexpression.

[0110] like Fig.12 Similar results were observed using Western blotting and qRT-PCR, further demonstrating that TMBPS inhibited the expression of SLC7A11 in hepatoma cells.

[0111] The effect of TMBPS on GPX4 protein expression in SLC7A11-overexpressing HepG2 and Huh7 cells was investigated.

[0112] like Fig.12 As shown in F, TMBPS can inhibit the expression of GPX4 compared with the control group, and this inhibitory effect on GPX4 expression still exists after SLC7A11 overexpression and addition of TMBPS. These results are also consistent with the role of SLC7A11 as an upstream regulator of GPX4 expression.

[0113] like Fig.12 As shown in G, compared with the h-NC group, overexpression of SLC7A11 reduced the level of intracellular lipid peroxidation (P<0.01), and after TMBPS treatment, the lipid peroxidation level in the overexpression group was also significantly lower than that in the h-NC-TMBPS group (P<0.01).

[0114] like Fig.12 As shown in Figure 2, similar results were obtained when measuring intracellular iron ion levels (P<0.01).

[0115] The above research results indicate that TMBPS-induced iron allergy in HCC cells is related to its inhibition of SLC7A11.

[0116] Example 12 Fer-1 reverses TMBPS-induced ferroptosis in HCC cells

[0117] In order to restore the inhibitory effect of TMBPS on HCC, an iron allergy inhibitor (Fer-1) was used. Cancer cells were co-treated with Fer-1 and TMBPS, and the results showed that co-treatment of cells with Fer-1 and TMBPS led to changes in intracellular lipid peroxide and iron ion levels.

[0118] like Fig.13 As shown, Fer-1 can significantly reduce the inhibitory effect of TMBPS on HCC cell viability in a concentration-dependent manner, and also significantly reduce the increase of intracellular lipid peroxides induced by TMBPS.

[0119] Immunofluorescence experiments Fig.14 As shown, in the Fer-1 and TMBPS combination group, Fer-1 could significantly inhibit the 2+ The results further showed that Fer-1 could effectively reverse TMBPS-induced cell death, and the inhibition of TMBPS on the proliferation of liver cancer cells was related to its induced cell iron mutation.

[0120] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bisphenol compound that induces ferroptosis, characterized in that: The bisphenol compound is TMBPS.

2. A ferroptosis inducing agent, characterized in that The active ingredient of the inducer includes the bisphenol compound according to claim 1.

3. Use of the bisphenol compound according to claim 1 in inducing ferroptosis of cancer cells.

4. The use according to claim 3, characterized in that: In such applications, cancer cell death can be reversed by ferritin inhibitors.

5. The application according to claim 3, characterized in that: The cancer cells include lung cancer cells.

6. The use according to claim 3, characterized in that: The bisphenol compound can inhibit the expression of SLC7A11 protein.

7. The bisphenol compound of claim 1 is effective in increasing the activity of lipid peroxides, Fe 2+ or malondialdehyde levels.

8. A drug for treating a disease associated with ferroptosis, characterized in that: The active ingredients of the drug include the bisphenol compound according to claim 1 and pharmaceutically acceptable excipients, and the dosage form of the drug is tablets, granules, powders, emulsions, capsules or injections.

9. A pharmaceutical composition for inhibiting the expression of SLC7A11, characterized in that: The composition comprises the bisphenol compound according to claim 1 and pharmaceutically acceptable excipients.

10. Use of the bisphenol compound according to claim 1 in the preparation of a drug for treating diseases associated with ferroptosis.

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