Small molecule compounds and their use as drugs to inhibit bisphenol s-induced breast cancer metastasis and use of d2 dopamine receptors in drug screening

Abstract

The present application relates to the technical field of cancer treatment drugs, in particular to small molecule compounds and their application as drugs for inhibiting bisphenol S-induced breast cancer metastasis and the application of D2 dopamine receptors in drug screening. The present application first discovers the role of D2 dopamine receptors in the accelerated development of bisphenol S-induced breast cancer, and uses D2 dopamine receptors as a drug screening target to screen small molecule drugs, and from 100,000 candidate compounds, two compounds ZDA6 and ZDA9 are established. The two compounds bind to D2 dopamine receptors and effectively inhibit the enhanced migration ability of breast cancer cells caused by bisphenol S exposure and the accelerated development of breast cancer, and antagonize the breast cancer progression effect caused by bisphenol S exposure. The technical scheme can solve the technical problem that there is a lack of drugs that can inhibit bisphenol S-induced breast cancer metastasis in the prior art, create conditions for treating diseases related to environmental pollutants, and have ideal application value.

Description

Technical Field

[0001] This invention relates to the field of cancer treatment drug technology, specifically to small molecule compounds and their application as drugs to inhibit bisphenol S-induced breast cancer metastasis and the application of D2 dopamine receptor in drug screening. Background Technology

[0002] In 2020, breast cancer (BC) surpassed lung cancer to become the most common cancer in women, with an estimated 2.3 million new cases. Globally, despite significant advances in breast cancer detection and treatment, mortality rates remain high. Chinese women account for approximately 18% of global breast cancer deaths, and the high mortality rate is primarily attributed to distant metastases, such as those to the lungs, bones, liver, and lymph nodes.

[0003] It has been reported that the development and progression of breast cancer are closely related to exposure to endocrine disruptors (EDCs), particularly bisphenol A (BPA) and its alternative, bisphenol S (BPS). BPA is an endocrine disruptor widely used in the production of consumer products such as beverages, adhesives, cigarette filters, and food plastic containers. BPA exposure is linked to obesity, diabetes, reproductive disorders, and cancer. Increasing evidence of the negative health effects of BPA has prompted its removal from consumer products. As a BPA alternative, the bisphenol analog BPS has been produced for various applications. To date, BPS has become a widely used industrial product globally, including in polycarbonate plastics, epoxy resins, and thermally conductive paper. Studies have shown that BPS, as a xenoestrone, can promote the development and progression of breast cancer. Epidemiological studies have shown that BPS is widely detectable in human blood and urine, and that it can increase the risk of breast cancer. As an emerging environmental pollutant, humans can be exposed to BPS through inhalation and ingestion in various ways, promoting the progression of breast cancer, particularly with studies indicating a strong effect in promoting breast cancer metastasis. Therefore, it is urgent to conduct a thorough study on the mechanism by which BPS promotes the occurrence and development of breast cancer, in order to discover key molecular targets, screen related drugs based on these targets, and ultimately obtain candidate small molecule compounds that can effectively reverse the metastatic effect of bisphenol S on breast cancer, thereby obtaining potential drugs that can be used to control the occurrence and development of breast cancer. Summary of the Invention

[0004] The present invention aims to provide the use of small molecule compounds in the preparation of medicaments for treating bisphenol S-induced breast cancer, in order to solve the technical problem of the lack of medicaments in the prior art that can inhibit bisphenol S-induced breast cancer metastasis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of small molecule compounds in the preparation of medicaments for treating bisphenol S-induced breast cancer, wherein the small molecule compounds are used to inhibit bisphenol S-induced breast cancer metastasis.

[0007] Bisphenol S (BPS), an emerging environmental pollutant, can be exposed to humans through inhalation and ingestion in various ways, promoting the progression of breast cancer. If breast cancer is affected by BPS exposure during its development, its progression will be accelerated, including accelerated metastasis and invasion. This study discovered small molecule compounds that can effectively inhibit BPS-induced breast cancer metastasis, thus producing a therapeutic effect on breast cancer affected by BPS exposure. This technical approach investigates treatment methods for cancer under the influence of specific environmental pollutants, considering not only general cancer treatment but also the impact of environmental factors on cancer development and progression. The compounds in this study have an effect of mitigating exposure damage to the specific environmental pollutant BPS, particularly in delaying the accelerated progression of breast cancer caused by BPS exposure, thereby prolonging the survival time of such patients.

[0008] Furthermore, the small molecule compound is used to bind to the D2 dopamine receptor.

[0009] Furthermore, the D2 dopamine receptor is a transmembrane protein; the binding site of the small molecule compound to the D2 dopamine receptor is located in the extracellular region of the D2 dopamine receptor;

[0010] Furthermore, the small molecule compound is used to bind to the Site2 small molecule drug binding pocket in the extracellular region of the D2 dopamine receptor; the Site2 small molecule drug binding pocket is located at amino acid positions 41-416 of the D2 dopamine receptor.

[0011] Furthermore, the small molecule compound interacts with aspartic acid residue 114 of the D2 dopamine receptor. Studies have found that bisphenol S promotes the metastasis and invasion of breast cancer cells through the D2 dopamine receptor (DRD2). The compounds screened using this technique inhibit the induction of breast cancer metastasis by bisphenol S through interaction with DRD2. Small molecule drug binding sites are screened based on the protein structure of human DRD2. There are two binding sites: Site1, located in the intracellular region, and Site2, located in the extracellular region. As a transmembrane protein, DRD2 uses the extracellular region as the binding site for small molecule compounds. Small molecule drugs can directly interact with the extracellular portion of DRD2, without necessarily needing to enter the cell to exert their effect. Selecting the Site2 small molecule drug binding pocket as the target for drug screening can improve drug bioavailability, increase therapeutic efficacy, and reduce the difficulty of drug administration.

[0012] Site2, the small molecule drug binding pocket, is located at amino acid positions 41-416 of the D2 dopamine receptor. The key amino acids in Site2 are: LEU41, VAL91, LEU94, GLU95, GLY98, GLU99, TRP100, LYS101, PHE102, SER103, ARG104, CYS107, PHE110, VAL111, ASP114, VAL115, CYS118, THR119, ALA122, GLU181, CYS182, ILE184, ALA185, PHE189, VAL190, SER193, SER194, SER197, PHE198, PHE382, TRP386, PHE389, PHE390, HIS393, PRO405, TYR408, SER409, THR412, TRP413, and TYR416. Regarding the aforementioned key sites, small molecule compounds interact with Asp114 at a relatively high frequency, while the interaction frequencies of the remaining amino acids are relatively uniform. All small molecule compounds screened using this method can interact with aspartic acid, the 114th amino acid residue of the D2 dopamine receptor.

[0013] Furthermore, the small molecule compound is as shown in Formula I or Formula II;

[0014]

[0015] This technical solution also provides the application of a small molecule compound in the preparation of a drug that resists bisphenol S exposure damage, wherein the bisphenol S exposure damage is bisphenol S-induced increased migration and / or invasion of breast cancer cells, or bisphenol S-induced accelerated progression of breast cancer.

[0016] The small molecule compound interacts with aspartic acid, the 114th amino acid residue of the D2 dopamine receptor, as shown in Formula I or Formula II.

[0017]

[0018] Furthermore, bisphenol S-induced migration of breast cancer cells includes metastasis of breast cancer cells to the liver and lungs.

[0019] Furthermore, the bisphenol S exposure is breast cancer cells exposed to a bisphenol S environment of 0.01-1 μM, or the bisphenol S exposure is bisphenol S exposure caused by drinking water containing 50 μg / L of bisphenol S.

[0020] The small molecule compounds are used to inhibit the metastasis of bisphenol S-exposed breast cancer cells MCF-7 and / or BT474; the working concentration of the small molecule compounds is 5-80 μM.

[0021] The screening of novel small molecule drugs for this protocol (for the treatment of breast cancer and / or to combat damage caused by bisphenol S exposure) is based on the following principles and beneficial effects:

[0022] In the drug design process, the binding pocket of small molecule drugs was predicted based on the protein structure of the D2 dopamine receptor. Using 100,000 structurally diverse compounds, drug screening was conducted based on the Site2 site of the D2 dopamine receptor. Finally, 205 compounds binding to the Site2 site were selected, and the top 10 were chosen for further experimental validation. Of the 10 small molecule compounds used in experimental studies, only two compounds simultaneously and significantly inhibited the migration ability of two breast cancer cell lines in a bisphenol A (BPA)-exposed environment. Some candidate compounds did not show significant inhibitory effects in one or two cell lines. Both screened small molecule compounds possessed the ability to bind to the Asp114 site of the D2 dopamine receptor. The D2 dopamine receptor is a key molecule influencing the progression of BPA in breast cancer and a target for drug therapy; this is a first discovery in this technical approach. Two small molecule compounds can inhibit the accelerated migration of breast cancer cells induced by bisphenol S (BPS) exposure, and could be used as drugs to treat BPS-exposed breast cancer. BPS-exposed breast cancer is characterized by accelerated cancer cell metastasis and invasion, faster disease progression, and increased malignancy, unlike typical breast cancer. In addition, these two small molecule compounds can also be used to antagonize the damage caused by BPS exposure, acting as antagonistic drugs against an environmental pollutant.

[0023] This technical solution also provides the application of the D2 dopamine receptor in screening drugs that resist bisphenol S exposure damage or drugs for treating breast cancer.

[0024] Furthermore, amino acid residues 41-416 of the D2 dopamine receptor are used for computer-aided molecular docking with candidate small molecule drugs.

[0025] Furthermore, the 114th amino acid residue of the D2 dopamine receptor, aspartic acid, is used for computer-aided molecular docking with candidate small molecule drugs.

[0026] Based on the discovery of new drug screening targets, the principle and beneficial effects of this technical solution are as follows:

[0027] Molecular structure analysis revealed that the Site2 small molecule drug binding pocket score of the D2 dopamine receptor molecule was much higher than that of other sites. Furthermore, the Site2 small molecule drug binding pocket contains sites such as Asp114, which can bind to small molecule drugs, thereby enabling the regulation of breast cancer development by small molecule drugs under bisphenol S exposure. More specifically, the drug screening targets the small molecule drug-binding pocket formed by amino acids at positions 41-416 of the D2 dopamine receptor protein. More specifically, these are the following amino acid residues: LEU41, VAL91, LEU94, GLU95, GLY98, GLU99, TRP100, LYS101, PHE102, SER103, ARG104, CYS107, PHE110, VAL111, ASP114, VAL115, CYS118, THR119, ALA122, GLU181, CYS182, ILE184, ALA185, PHE189, VAL190, SER193, SER194, SER197, PHE198, PHE382, TRP386, PHE389, PHE390, HIS393, PRO405, TYR408, SER409, THR412, TRP413, and TYR416. Specifically, this refers to the Asp114 site in the small molecule drug binding pocket. Using these drug screening sites, molecular docking, molecular interaction mode analysis (interaction sites and force types), and drug property analysis can be performed to screen drugs that resist bisphenol S exposure damage or treat breast cancer.

[0028] The above screening process can be achieved through conventional computer simulations using existing technologies. By analyzing the binding affinity between the various active amino acid residues of the D2 dopamine receptor protein and candidate drugs in a small molecule compound library, and through drug performance analysis, potential drugs can be obtained. These potential drugs can then be compared and selected as preferred candidates through experimental screening studies. This technical solution, through the above-mentioned drug screening targets, has obtained two drugs (ZDA6 and ZDA9) as shown in Formula I or Formula II. Therefore, the key to achieving drug screening lies in the precise identification of the drug screening target (D2 dopamine receptor molecule). Using the D2 dopamine receptor molecule as a drug screening target can effectively obtain drugs for resisting bisphenol S exposure damage or for treating breast cancer. In particular, it is especially suitable as a drug screening target for breast cancer that is accelerated in its development and progression due to exposure to the environmental pollutant bisphenol S, leading to metastasis and / or increased invasion of cancer cells.

[0029] In summary, the overall beneficial effects of this technical solution are as follows:

[0030] (1) The mechanism by which bisphenol S induces the development and progression of breast cancer was discovered, and the D2 dopamine receptor, a potential target for drug design, was also discovered.

[0031] (2) Based on the study of the protein structure of the D2 dopamine receptor, potential drugs that can be used to antagonize bisphenol S toxicity and treat bisphenol S-related breast cancer were discovered through molecular docking and other techniques.

[0032] (3) Through experimental studies on screened drugs, two small molecule compounds, ZDA6 and ZDA9, were identified that can bind to the D2 dopamine receptor. These two compounds can effectively inhibit the increased migration ability of breast cancer cells caused by bisphenol S exposure, thereby achieving a therapeutic effect on bisphenol S-related breast cancer. Attached Figure Description

[0033] Figure 1 The results of the experimental study on BPS promoting lung and liver metastases of breast cancer in MMTV-Erbb2 mice in Example 1 are shown below (A: Schematic overview of the BPS-induced BC model; B: Kaplan-Meier plot of tumor-free survival in the control and BPS groups, n=10; C: Typical images of HE-stained lungs from control mice and BPS-exposed mice, scale bar: 1000 μm original; D: Number of mice with or without lung metastases; E: Typical images of HE-stained livers from control mice and BPS-exposed mice, scale bar: 1000 μm original; F: Number of mice with or without liver metastases).

[0034] Figure 2 The results of the study on the effect of BPS on metastasis-related proteins of mammary tumors in MMTV-Erbb2 mice in Example 1 (A, B: IHC staining shows the protein expression of Vimentin and Snail in mammary tumors of MMTV-Erbb2 mice, n=6, compared with the control group, ***p<0.001, scale bar: 100μm).

[0035] Figure 3 The results of the study on the effect of low-dose BPS on the proliferation of breast cancer cells in vitro in Example 1 (A: MCF-7 cells; B: BT474 cells; treated with different concentrations of 0.01, 0.1, and 1 μM BPS for 24, 48, and 72 hours, and cell viability was determined by the CCK8 assay, n=3; *p<0.05, **p<0.01, ***p<0.001).

[0036] Figure 4The results of the study on the effects of low-dose BPS on the migration and invasion of mammary cells in Example 1 are as follows (A, B: Cell migration was measured in Transwell and the number of cells in each image was quantified by ImageJ, n=3; C, D: Cell invasion was measured in Transwell coated with Matrigel and the number of cells in each image was quantified by ImageJ, n=3; E, G: Expression of migration-related proteins FN, Vimentin, and Snail was measured by Western blot; F, H: Protein expression levels were normalized according to GAPDH levels, *p<0.05, **p<0.01, ***p<0.001).

[0037] Figure 5 Transcriptomic analysis results of potential target genes for BPS-induced breast cancer cell metastasis in Example 2 (A: Volcano plot of DEGs between the control and BPS groups; B: Clustering heatmap of DEGs between the control and BPS groups; C: GO enrichment analysis showing significant enrichment of differentially expressed genes in G protein-coupled signaling pathways; D: Venn diagram showing differentially expressed genes intersecting gene sets in five G protein-coupled pathways; E: mRNA levels of three downregulated genes in MCF-7 and BT474 cells treated with or without 0.01 μM BPS for 72 h; F, G: Western blot analysis of DRD2 and ADRA2A protein expression in MCF-7 and BT474 cells treated with or without 0.01, 0.1, and 1 μM BPS for 72 h, n=3, protein expression levels normalized according to GAPDH levels, *p<0.05, **p<0.01, ***p<0.001).

[0038] Figure 6 To illustrate the reduction of DRD2 expression in mammary tumors of MMTV-Erbb2 mice by BPS in Example 2 (A: IHC staining showing DRD2 protein expression in mammary tumors of MMTV-Erbb2 mice, n=6, p<0.001 compared with the control group, scale bar: 100μm).

[0039] Figure 7The results of the experiment in Example 3 on the inhibition of BPS-induced breast cancer cell metastasis by DRD2 overexpression are as follows: (AC: MCF-7 and T474 cells were transfected with the specified OE-DRD2 plasmid, and the overexpression efficiency was determined by qRT-PCR and Western blot, n=3; D, E: migration of MCF-7 and BT474 cells, n=3, scale bar: 100 μm; F, G: invasion of MCF-7 and BT474 cells, n=3, scale bar: 100 μm; HK: expression of metastasis-related proteins FN, Vimentin and Snail was detected by Western blot, and the protein expression level was normalized according to GAPDH level; compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the BPS-exposed group, #p<0.05, ##p<0.01, ###p<0.001).

[0040] Figure 8 The results of the study on the mechanism of action of BPS in in vitro inducing breast cancer metastasis by DRD2 activation of AKT in Example 4 are as follows: (A: Based on the BPS-related gene dataset in the CTD database, the BPS-related dataset was significantly enriched in the PI3K / AKT signaling pathway by KEGG pathway prediction analysis; B, C: MCF-7 cells were treated with different concentrations of 0.01, 0.1, and 1 μM BPS for 72 h, and then the total expression and phosphorylated AKT and GSK3β expression were measured by Western blot. The protein expression level was normalized according to GAPDH level; D, E: MCF-7 cells were treated with different concentrations of 0.01, 0.1, and 1 μM BPS for 72 h, and then the total expression and phosphorylated AKT and GSK3β expression were measured by Western blot. The protein expression level was normalized according to GAPDH level; FI: MCF-7 and BT474 cells were treated with or without OE-DRD2 plasmid, and then with or without 0.01 μM BPS for 72 h. The results were measured by Western blot.) Blot analysis was performed to measure the expression of phosphorylated AKT and GSK3β proteins, and protein expression levels were normalized according to GAPDH levels. Statistical results are expressed as mean ± SD of three independent experiments. Compared with the control group, *p < 0.05, **p < 0.01, and ***p < 0.001; compared with the BPS group, #p < 0.05, ##p < 0.01, and ###p < 0.001).

[0041] Figure 9 The results are from the virtual screening experiment in Example 5.

[0042] Figure 10The results of the small molecule compound screening for cytotoxicity in Example 6 are shown (compared with the control group, *p<0.05, **p<0.01, ***p<0.001).

[0043] Figure 11 The results of screening for the effect of the small molecule compound in Example 6 on BPS-promoted breast cancer metastasis (compared with the control group, **p<0.01; compared with the BPS-exposed group, #p<0.05, ##p<0.01, ##p<0.001).

[0044] Figure 12 The detection spectrum of the small molecule compound ZDA6 in Example 6 is shown.

[0045] Figure 13 The detection spectrum of the small molecule compound ZDA9 in Example 6 is shown.

[0046] Figure 14 This is a schematic diagram showing the molecular binding of the small molecule compound ZDA6 with DRD2 in Example 7.

[0047] Figure 15 The results of the study on the inhibitory effect of the small molecule compound ZDA6 in Example 7 on BPS-promoted breast cancer metastasis (AD: representative immunoblot images and quantitative analysis results of tumor metastasis-related proteins FN, Vimentin and Snail in MCF-7 and BT474 cells; compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the BPS-exposed group, #p<0.05, ##p<0.01, ###p<0.001).

[0048] Figure 16 The results of the study on the inhibitory effect of the small molecule compound ZDA6 in Example 7 on BPS-induced activation of the AKT / GSK3β pathway (AD: representative immunoblot images and quantitative analysis results of AKT / GSK3β pathway-related proteins p-AKT and AKT in MCF-7 and BT474 cells; compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the BPS-exposed group, #p<0.05, ##p<0.01, ###p<0.001).

[0049] Figure 17 This is a schematic diagram showing the molecular binding of the small molecule compound ZDA9 with DRD2 in Example 7.

[0050] Figure 18The results of the study on the inhibitory effect of the small molecule compound ZDA9 on BPS-promoted breast cancer metastasis in Example 7 are as follows (AD: representative immunoblot images and quantitative analysis results of tumor metastasis-related proteins FN, Vimentin and Snail in MCF-7 and BT474 cells; compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the BPS-exposed group, #p<0.05, ##p<0.01, ###p<0.001).

[0051] Figure 19 The results of the study on the inhibitory effect of the small molecule compound ZDA9 on BPS-induced activation of the AKT / GSK3β pathway in Example 7 are as follows (AD: representative immunoblot images and quantitative analysis results of AKT / GSK3β pathway-related proteins p-AKT and AKT in MCF-7 and BT474 cells; compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the BPS-exposed group, #p<0.05, ##p<0.01, ###p<0.001). Detailed Implementation

[0052] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0053] Example 1: Study on the biological effects of low-dose BPS exposure

[0054] This embodiment of the experiment found that low-dose BPS not only promotes the migration and invasion of breast cancer cells, but also promotes the metastasis of breast cancer to the liver and lungs in MMTV-Erbb2 mice.

[0055] (1) Animal experiments

[0056] The transgenic MMTV-Erbb2-induced breast cancer (BC) mouse model was purchased from the Jackson Laboratory in Sacramento, California, USA. Mice were bred, maintained, and genotyped according to standard procedures. Semi-heterozygous, unmated female mice were selected for subsequent experiments. To investigate the effect of BPS on promoting BC metastasis, female MMTV-Erbb2 mice (6 weeks old) were divided into two groups: (i) a control group (n=10) and (ii) a BPS group (n=10). Mice in the BPS group received drinking water containing 50 μg / L BPS for 19 weeks, while mice in the control group received plain water for the same duration. Tumor development was assessed by palpation. After palpable tumors appeared in the mouse mammary glands, the size of the mammary tumors was measured and recorded every week using calipers. Tumor volume was calculated using the following formula: Volume = Length × Width 2 / 2.

[0057] The experiment was terminated when mice were euthanized at 25 weeks of age or when the tumor diameter reached 1.5 cm. Mice were dissected on a clean workbench. Fresh organs, including the liver and lungs, were harvested promptly and fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China, E672002-0500). These organs were then dehydrated in ethanol. The organ samples were then embedded in paraffin and 5 μm thick sections were prepared for hematoxylin and eosin (H&E) staining and histopathological observation of tumor metastases.

[0058] Hematoxylin and eosin staining and immunohistochemistry were performed according to standard procedures using existing techniques.

[0059] (2) Cell experiments

[0060] MCF-7 and BT474 cells were purchased from Procell Life Science and Technology Co., Ltd., Wuhan, China. These BC cells were supplemented with 10% fetal bovine serum (AUSGENEX; Molendinal, Australia; FBS 500-S) and 1% (v / v) penicillin / streptomycin (Gibco, NY, USA; 15140-122), and DMEM (Gibco, NY, USA; C11995500BT). The MCF-7 and BT474 cell lines were maintained at 37°C in a humid environment containing 5% CO2. BPS (Sigma, USA, 80-09-1) was dissolved in DMSO. To evaluate the effect of BPS exposure on the proliferation and metastasis of MCF-7 and BT474 cells, they were then exposed to different concentrations of BPS (0, 0.01, 0.1, and 1 μM) and cultured in a flat culture for 72 h.

[0061] Cell viability was assessed using the standard CCK-8 assay, and cell migration and invasion were assessed using the Transwell assay. Western blotting was used to detect the expression of relevant proteins.

[0062] (3) Statistical Analysis

[0063] Data were analyzed using GraphPad Prism 9.0 software (GraphPad, USA), and are expressed as mean ± SEM. Unpaired two-sided t-tests were used for comparisons between two experimental groups, and one-way ANOVA was used for comparisons among multiple groups. Each experiment was repeated at least three times, and *p < 0.05 was defined as statistically significant.

[0064] (4) Experimental Results

[0065] Since BPA is known to be an environmental factor involved in the development of hormone-dependent breast cancer, and BPS is a known alternative to BPA, we exposed MMTV-Erbb2 mice to a 50 μg / L concentration of BPS in their drinking water for 19 weeks to assess the effects of chronic environmental doses of BPS on the development and progression of breast cancer in mice. Figure 1 A) The number of mice developing breast cancer and the size of tumors were recorded weekly. When the tumor diameter reached 1.5 cm, it was surgically removed. In this longitudinal study, palpable breast tumors were detected in 9 mice aged 19-21 weeks exposed to BPS; palpable tumors were detected in 8 mice aged 20-22 weeks in the control group (when the maximum diameter of the tumor in the BPS group reached 1.5 cm, no tumors were found in the two mice in the control group). Figure 1 B). The median time to detection of breast tumors in the BPS group was significantly shorter (median time: 20.5 weeks in the BPS group and 21.5 weeks in the control group, p < 0.05), indicating that BPS exposure promoted the earlier onset of breast cancer in MMTV-Erbb2 mice. After 19 weeks of water exposure, MMTV-Erbb2 mice were sacrificed, and liver and lung tissues were collected, fixed with paraformaldehyde, and prepared for pathological sections. After HE staining, the number of lung metastases and liver metastases was counted. In the control group, 4 MMTV-Erbb2 mice (4 / 8) developed lung metastases, while all mice in the BPS-exposed group (8 / 8) developed lung metastases. Figure 1 C, Figure 1 D). Consistent with the lung metastasis results, 4 MMTV-Erbb2 mice (4 / 8) in the control group developed breast cancer liver metastasis, while all mice in the BPS exposure group (8 / 8) developed breast cancer liver metastasis. Figure 1 E, Figure 1 F). Immunohistochemistry was used to assess changes in the expression of the metastasis-related proteins Vimentin and Snail markers in the BC tissues of MMTV-ERBB2 mice after BPS exposure. Figure 2 A, Figure 2 B) Increased protein expression of Vimentin and Snail was found in the BPS-treated group. Therefore, BPS promotes the metastatic ability of breast cancer in MMTV-Erbb2 mice. These results indicate that chronic low-dose BPS exposure significantly promotes the metastasis of breast cancer to the liver and lungs in MMTV-Erbb2 mice and significantly increases the malignancy after metastasis.

[0066] We used the CCK-8 assay to detect the effect of BPS on the viability of MCF-7 and BT474 breast cancer cells cultured in vitro. The results confirmed that after treating breast cancer cells with gradient concentrations (0, 0.01, 0.1, and 1 μM) of BPS for 72 h, we found no change in the proliferation of breast cancer cells. Figure 3 A, Figure 3 B). However, besides proliferative capacity, another indicator of increased malignancy in breast cancer cells is decreased adhesion, increased motility, and associated enhanced invasiveness. Therefore, we first investigated the effect of BPS on the migration ability of MCF-7 and BT474 cells using the Transwell-migration method. The Transwell-migration results showed that, compared to the control group, treatment with gradient concentrations of BPS significantly increased the number of breast cancer cells that crossed the Transwell chambers. Figure 4 (AB). BPS treatment enhances the migration ability of breast cancer cells. Then, to assess the invasive ability of breast cancer cells, we used the Transwell-invasion method, where diluted matrix gel was pre-spread evenly in Transwell chambers, and cell penetration through the matrix gel and the inner membrane of the chamber was measured to evaluate the effect of BPS exposure on the invasive ability of MCF-7 and BT474 cells. Our experiments showed that after treatment with gradient concentrations of BPS, the number of breast cancer cells reaching the surface of the lower chamber of the Transwell chamber significantly increased (AB). Figure 4 C, Figure 4 D). To understand the promoting effect of BPS on breast cancer cell migration and invasion, we examined tumor metastasis-related proteins at the molecular level. Western blot results showed that BPS exposure significantly promoted MCF-7 (…). Figure 4 E and Figure 4 F) and BT474 cells ( Figure 4 G, Figure 4 Increased expression of tumor metastasis-related proteins such as FN, Vimentin, and Snail was observed in H). In summary, BPS exposure had no effect on breast cancer cell proliferation, but it increased their migration and invasion abilities, thereby increasing their malignancy.

[0067] Example 2: Transcriptomics study reveals potential target genes for BPS-induced breast cancer metastasis

[0068] This embodiment uses transcriptomics analysis to discover that DRD2 is a potential target gene for BPS-induced breast cancer metastasis.

[0069] (1) Eukaryotic transcriptome sequencing with reference

[0070] Control group MCF-7 cells and experimental group MCF-7 cells were exposed to 0.01 μM BPS for 72 h. After treatment, routine total RNA extraction was performed, and mRNA was purified from the total RNA using the polyA structure specific to mRNA. Then, mRNA fragmentation, BPSNA synthesis, PCR enrichment of library fragments, and finally sequencing were performed. RNA was extracted and purified from the sample samples, and libraries were constructed. The sample libraries were then sequenced using the Illumina HiSeq sequencing platform.

[0071] Eukaryotic reference transcriptome sequencing was subjected to routine quality control using existing techniques before eukaryotic reference transcriptome sequencing analysis. Gene expression levels were standardized using FPKM, with FPKM > 1 used to determine gene expression. Gene structure annotation information was then read, and the alignment results were compared with the gene structures, and the results were statistically analyzed. When a read partially covers a gene region or partially covers an intron region, the Union and Intersection_nonempty protocols identified the read as belonging to that gene, while the Intersection_nonempty protocol identified the read as not belonging to any gene. When a read fully covers one gene and partially covers another gene, the Union protocol identified the read as belonging to both genes, while the Intersection_strict and Intersection_nonempty protocols identified the read as belonging to the first gene. FPKM density distribution maps were plotted, and saturation analysis, sample correlation tests, differential expression detection (P < 0.05, |log2FC| > 2), and cluster analysis were performed.

[0072] (2) Functional enrichment analysis

[0073] Differentially expressed genes were analyzed using Gene Ontology (GO). The proteome was annotated using the UniProt-GOA database (http: / / www.ebi.ac.uk / GOA / ). First, the identified protein IDs were converted to UniProt IDs, and then mapped to GO IDs. If some identified proteins were not annotated in the UniProt-GOA database, their GO functions were annotated using InterProScan software based on protein sequence alignment. Then, the proteins were categorized into three main groups: biological processes, cellular components, and molecular functions.

[0074] (3) Perform fluorescence quantitative PCR analysis on the target gene. The total RNA extraction, reverse transcription and qRT-PCR processes are performed in accordance with existing conventional techniques.

[0075] Cell migration and invasion capacity assessment, Western blot, immunohistochemistry, and statistical analysis were performed as described in Example 1.

[0076] (4) Experimental Results

[0077] Analysis was performed using data from the control group and the BPS group with a fold change (FC) > 4, p < 0.05, and volcano plots were used. Figure 5 A) 853 genes were upregulated and 763 genes were downregulated. (Heatmap) Figure 5 B) showed significant differences between the control and treatment groups. Then, a GO enrichment plot was created using differentially expressed genes with downregulated FC > 4-fold. Figure 5 C), after assessing enrichment scores and significance, G protein-coupled receptor pathways involving four biological processes (BP) and one molecular function (MF) were identified as the most significantly enriched pathways. Venn diagram ( Figure 5 D) represents the intersection genes from differentially expressed gene datasets across five G protein-coupled receptor pathways. DRD2, CHRM1, and ADRA2A were identified as candidate genes for further investigation. RT-qPCR experiments were performed in MCF-7 and BT474 cells to verify the mRNA expression levels of ADRA2A, CHRM1, and DRD2 in the control and BPS groups, respectively. The results showed that the mRNA expression levels of ADRA2A and DRD2 were consistently decreased in both MCF-7 and BT474 cells. Figure 5 E). Western blotting showed decreased DRD2 protein expression in the BPS-treated group. Figure 5 F), the protein expression level of ADRA2A remained unchanged. Figure 5 G). Simultaneously, immunohistochemistry assessed changes in DRD2 in breast cancer tissues of MMTV-Erbb2 mice after BPS exposure, observing a decrease in DRD2 protein expression levels. Figure 6Therefore, DRD2 was identified as the final target gene for this study. More specifically, the D2 dopamine receptor (DRD2) is a key member of the dopamine receptor family and one of the main types of D2-like receptors. The gene IDs of DRD2 are 1813 (human) and 13489 (mouse).

[0078] Example 3: Study on the effect of DRD2 overexpression (OE-DRD2) on BPS-induced metastasis of breast cancer cells

[0079] The DRD2 gene expression vector was constructed using conventional techniques and transfected into MCF-7 and BT474 cells to create a DRD2 overexpression cell model. Cells were analyzed using RT-qPCR, cell migration and invasion assessments, and Western blot analysis. Experimental and statistical methods are described in Example 1.

[0080] Before examining the effect of OE-DRD2 on breast cancer cell function, the transfection efficiency of the OE-DRD2 plasmid was first tested in both cell lines to ensure the effectiveness of OE-DRD2 plasmid transfection. RT-qPCR and WB results showed that after transfection of the OE-DRD2 plasmid into MCF-7 and BT474 cells, the mRNA and protein expression levels of DRD2 were significantly increased, and the differences were statistically significant. Figure 7 (AC). Experimental results showed that the OE-DRD2 plasmid had high transfection efficiency in MCF-7 and BT474 cells, proving that the overexpression plasmid OE-DRD2 could be used for further experiments. Breast cancer cell lines were divided into: control group, BPS alone group, OE-DRD2 alone group, and BPS+OE-DRD2 combined treatment group for experiments. Transwell-migration and transwell-invasion experiments showed that overexpression of DRD2 could reverse the ability of BPS-induced increased migration and invasion in MCF-7 and BT474 cells. Figure 7 DG). Through WB experiment ( Figure 7 HK observed that overexpression of DRD2 could inhibit the expression of proteins related to BPS-induced breast cancer cell metastasis. In summary, these results indicate that increased DRD2 receptor expression can inhibit environmental bisphenol S-induced breast cancer cell metastasis.

[0081] Example 4: Study on the mechanism of action of BPS

[0082] Using CTD database prediction and online KEGG analysis, this study found that BPS induces breast cancer cell metastasis in vitro by activating AKT via DRD2.

[0083] To explore the potential molecular mechanisms by which bisphenol S (BPS) promotes breast cancer metastasis, we performed bioinformatics analysis using the Comparative Toxicogenomics Database (CTD). The CTD database integrates a large amount of data on chemical substances and gene interactions, enabling the prediction of disease-related environmental exposures and potential drug mechanisms of action. Using the CTD database, we predicted a set of genes associated with BPS and performed KEGG pathway analysis, which revealed a close correlation between bisphenol S and the PI3K / AKT signaling pathway. Figure 8 A).

[0084] Signaling molecules such as threonine protein kinase (AKT) and glycogen synthase kinase (GSK3β) are known to be involved in tumor metastasis. Therefore, we analyzed the expression of these proteins. Western blot results showed that BPS significantly induced phosphorylation of AKT and GSK3β in MCF-7 cells. Figure 8 B, Figure 8 C). Simultaneously, BPS significantly induced phosphorylation of AKT and GSK3β in BT474 cells ( Figure 8 D, Figure 8 E). Additionally, to examine whether overexpression of DRD2 could inhibit AKT / GSK3β activation, Western blotting showed that overexpression of DRD2 could inhibit BPS-induced activation of the AKT / GSK3β pathway. Figure 8 These results provide strong evidence for BPS inducing breast cancer metastasis in vitro by activating AKT via DRD2.

[0085] Example 5: Screening for small molecule compounds that may inhibit BPS-induced breast cancer metastasis based on DRD2 binding sites

[0086] Based on the protein structure of human DRD2, small molecule drug binding sites were screened. According to MOE software prediction, there are two binding sites: Site1 located in the intracellular region and Site2 located in the extracellular region. Site2 was selected for subsequent molecular docking studies. For details on the predicted DRD2 molecular structure and the predicted binding pocket diagram for small molecule compounds, please refer to [link to relevant documentation]. Figure 9 .

[0087] Molecular docking experiments were conducted on approximately 100,000 compounds with structural diversity. (Software). The key amino acids for Site 2 are: LEU41, VAL91, LEU94, GLU95, GLY98, GLU99, TRP100, LYS101, PHE102, SER103, ARG104, CYS107, PHE110, VAL111, ASP114, VAL115, CYS118, THR119, ALA122, GLU181, CYS182, ILE184, ALA185, PHE189, VAL190, SER193, SER194, SER197, PHE198, PHE382, TRP386, PHE389, PHE390, HIS393, PRO405, TYR408, SER409, THR412, TRP413, TYR416. The 41st to 416th amino acid residues of the D2 dopamine receptor are used for molecular docking with candidate small molecule drugs, preferably the amino acid residues listed above. Docking experiments showed that 17,870 compounds from the D3100 library bound to the Site2 region, with their affinity ranging from -13.1492 kcal / mol to -7.5052 kcal / mol. Due to the varying affinity distribution within the Site2 region, 713 compounds with affinity values ​​less than -10 kcal / mol were selected.

[0088] Next, molecular interaction mode analysis was performed. The Protein Ligand Interaction Fingerprints (PLIF) method was used to analyze the interaction sites and force types (hydrogen bonding, ionic bonding, etc.) between 713 compounds and the DRD2 structure. Small molecule compounds interacted with Asp114 more frequently, while the interaction frequencies of the remaining amino acids were relatively uniform. Details of the amino acid sites of the 710 compounds involved in the binding and the frequencies of these amino acid sites interacting with small molecule compounds can be found in [link to relevant documentation]. Figure 9 Then, the drug-like properties of the small molecule compounds were studied (ADME pharmacokinetics), and 291 compounds were screened from 710 compounds. Further structural diversity analysis narrowed down the selection to 205 compounds. The compounds with the highest scores from the virtual screening were selected for subsequent experimental studies.

[0089] The aforementioned computer-aided virtual screening process can all be outsourced to relevant biotechnology companies. Specifically, the process of screening 205 compounds from 100,000 compounds targeting amino acid residues of DRD2, including the Asp114 site, through molecular docking, affinity data analysis, and druggability analysis, is a conventional method in existing technology and can be outsourced. The key to drug screening lies in the identification of the drug screening target, specifically determining that DRD2 is a regulatory target for BPS in inducing breast cancer metastasis. BPS induces breast cancer metastasis by affecting DRD2. It is precisely because of the discovery of the drug screening target that subsequent drug screening can be implemented, thus obtaining the potential drugs in this embodiment.

[0090] Example 6: Experimental screening of small molecule compounds that inhibit breast cancer metastasis by binding to the DRD2 site

[0091] Breast cancer cells (MCF-7 and BT474 cells) were selected as in vitro models of breast cancer. MCF-7 and BT474 cells were cultured in DMEM / H (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (AUSGENEX, FBS500-S) and 1% penicillin / streptomycin (Beyotime, CO222) at 37°C and 5% CO2. BPS (Sigma, USA, 80-09-1) was dissolved in DMSO and diluted with culture medium.

[0092] Cytotoxicity assay of the compounds: Cell viability was assessed using the CCK-8 (Cell Counting Kit-8) cell proliferation-toxicity assay kit from Tokyo Dojin. This kit is based on the fact that the orange-yellow formazan dye generated by the oxidation-reduction of WST-8 by intracellular dehydrogenases is soluble in the culture medium, and the amount of formazan generated is directly proportional to the number of viable cells. MCF-7 and BT474 cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 / hole and 8×10 4Cells were seeded in 96-well plates and incubated overnight. Three replicates were made for each experimental group. When cells reached 50% confluence, small molecule compounds (0, 5, 10, 20, 40, 80 μM) were added to each well for 72 h. After treatment, the culture medium was removed, and 100 μL of well-mixed medium containing 10% CCK-8 reagent was added to each well. Three additional wells were added as blank wells to avoid air bubbles interfering with readings. The 96-well plates were incubated for 30-45 min. Absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability = [(At-Ab) / (Ac-Ab)]; At: Absorbance of treatment wells (medium containing CCK-8, cells under different treatments), Ac: Absorbance of control wells (medium containing CCK-8, control group cells), Ab: Absorbance of blank wells (medium containing CCK-8, cell-free).

[0093] To examine the effect of the compound on cell migration ability: the cell density was adjusted to MCF-7 cells: 2.5 × 10⁻⁶. 5 / mL, BT474 cells: 4×10 5 / mL. Seed the cell suspension (200 μL) into the upper chamber of a pre-prepared Transwell chamber. Add 800 μL of 0.01 μM BPS solution to the lower chamber, with or without the addition of small molecule compounds (maximum safe dose for each compound). Incubate the Transwell plate in an incubator for 72 h, fix with anhydrous methanol for 15 min, stain with crystal violet for 8 min, and then wash. Wipe away any unpenetrated cells from the upper layer, place the chamber in a clean 24-well plate, and observe under a microscope at 100x magnification. Take photos and count cells from three randomly selected fields of view.

[0094] The CCK-8 assay demonstrated the toxicity of 10 small molecule compounds (obtained from screening in Example 5, compound information is detailed in Table 1) at different concentrations (0, 5, 10, 20, 40, 80 μM) to breast cancer cells MCF-7 and BT474, in order to determine the safe concentration range for each compound in MCF-7 and BT474 cells. Figure 10 A, 10B). Based on the results, we selected the maximum dose concentrations of 10 small molecule compounds that could be simultaneously applied to two cell lines (see Table 2). Then, in the BPS-treated cell model, the 10 small molecule compounds were added or not added according to the selected doses, and the exposure was continuous for 72 hours. Transwell-Migration was used to observe the inhibitory effects of the small molecule compounds on BPS-induced breast cancer metastasis. The screening results showed that compounds 6 and 9 had the most significant inhibitory effects on BPS-induced breast cancer metastasis. Figure 11AD) showed significant effects on both cell lines and is worthy of further research.

[0095] Table 1: Compound Information

[0096]

[0097]

[0098] Table 2: Maximum Safe Concentration of Small Molecule Compounds

[0099] name Maximum safe concentration ZDA1 20μM ZDA2 20μM ZDA3 10μM ZDA4 20μM ZDA5 5μM ZDA6 40μM ZDA7 20μM ZDA8 10μM ZDA9 40μM ZDA10 20μM

[0100] We named compound 6 ZDA6 (Cas: 685844-11-5) and compound 9 ZDA9 (Cas: 423737-47-7) for further studies on their effects. The structural formula of ZDA6 is shown in Formula I, and the corresponding detection spectrum is detailed in [reference needed]. Figure 12 The structural formula of ZDA9 is shown in Formula II, and the corresponding detection spectrum is detailed in [reference needed]. Figure 13 Both small molecule compounds can bind to DRD2, thereby inhibiting BPS-promoted (induced) breast cancer cell metastasis and invasion, and have a therapeutic effect on breast cancer affected by environmental pollutants.

[0101]

[0102] Example 7: Study on the binding sites and biological effects of ZDA6 and ZDA9

[0103] The affinity data of the screened compounds were analyzed using the MOE (Mean Exchange Electron) module. Then, the PLIF (Protein Ligand Interaction Fingerprint) module was selected in the MOE to statistically analyze the interactions between the compounds and amino acid residues. Molecular docking analysis was applied to examine the binding site of ZDA6 and DRD2. The results showed that D114 is an effective binding site for ZDA6 and DRD2. ZDA6 can inhibit breast cancer metastasis by binding to the D114 site on Site2. Figure 14 Cells were then treated with 40 μM ZDA6 to detect the effect of ZDA6 on the expression of tumor metastasis-related proteins. The specific experimental procedure was as follows: MCF-7 and BT474 cells were treated with 5 × 10⁻⁶ ZDA6 at different rates. 4 / hole and 8×10 4 / wells were seeded in 96-well plates and cultured overnight in an incubator; when the cells grew to 50% confluence, breast cancer cells with and without BPS (0.01 μM, 72 h) exposure were treated with ZDA6 (40 μM, 72 h). Western blot analysis showed that treatment with 40 μM ZDA6 in BPS-exposed MCF-7 and BT474 cell models inhibited the expression of BPS-promoted tumor metastasis-related proteins. Figure 15 AD), and ZDA6 can inhibit the expression of p-AKT pathway-related proteins (AD), Figure 16 Therefore, ZDA6 can reverse BPS-induced breast cancer metastasis promoted by DRD2.

[0104] For the DRD2 binding site analysis of ZDA9, see [link to relevant documentation]. Figure 17 For details of the study on how ZDA9 can inhibit the expression of BPS-promoted tumor metastasis-related proteins, please refer to [link to study]. Figure 18 AD and Figure 19 Alternatively, molecular docking analysis was used to examine the binding site of ZDA9 to DRD2. The results showed that D114 is the effective binding site for ZDA9 to DRD2 (its binding affinity to other amino acid residues T412 and F198 is relatively weak). ZDA9 can inhibit breast cancer metastasis by binding to the D114 site of Site2. Subsequently, we treated cells with 40 μM ZDA9 to examine its effect on the expression of tumor metastasis-related proteins. Western blot analysis revealed that treatment with 40 μM ZDA9 in BPS-exposed MCF-7 and BT474 cell models inhibited the expression of BPS-promoted tumor metastasis-related proteins, and also inhibited the expression of p-AKT pathway-related proteins. Therefore, ZDA9 can reverse BPS-induced breast cancer metastasis promoted by DRD2.

[0105] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Use of a small molecule compound for the preparation of a medicament for the treatment of a breast cancer affected by bisphenol S, characterized in that: The small molecule compound is used for inhibiting bisphenol S-induced breast cancer metastasis. The small molecule compound is shown as formula I or formula II. Formula I Formula II.

2. Use of a small molecule compound according to claim 1 for the manufacture of a medicament for the treatment of a breast cancer affected by bisphenol S, characterized in that, The small molecule compound is used for binding with D2 dopamine receptor.

3. Use of a small molecule compound according to claim 2 for the manufacture of a medicament for the treatment of a breast cancer affected by bisphenol S, characterized in that, The D2 dopamine receptor is a transmembrane protein; the binding site of the small molecule compound with the D2 dopamine receptor is located in the extracellular region of the D2 dopamine receptor.

4. Use of a small molecule compound according to claim 3 for the manufacture of a medicament for the treatment of a breast cancer affected by bisphenol S, characterized in that, The small molecule compound is used for binding with the Site2 small molecule drug binding pocket of the extracellular region of the D2 dopamine receptor; the Site2 small molecule drug binding pocket is located at the amino acids at positions 41-416 of the D2 dopamine receptor.

5. Use of a small molecule compound according to claim 4 for the manufacture of a medicament for the treatment of a breast cancer affected by bisphenol S, characterized in that, The small molecule compound interacts with the amino acid residue aspartic acid at position 114 of the D2 dopamine receptor.

6. Use of a small molecule compound for the preparation of a medicament for protection against injury from bisphenol S exposure, characterized in that: The bisphenol S exposure damage is bisphenol S-induced aggravation of migration and / or invasion of breast cancer cells, or bisphenol S-induced acceleration of the progression of breast cancer. The small molecule compound interacts with the amino acid residue aspartic acid at position 114 of the D2 dopamine receptor, which is shown as formula I or formula II. Formula I Formula II.

7. Use of a small molecule compound according to claim 6 for the manufacture of a medicament for protection against damage by bisphenol S exposure, characterized in that: The migration of bisphenol S-induced breast cancer cells includes breast cancer cell metastasis to the liver and lung.

8. Use of a small molecule compound according to claim 7 for the manufacture of a medicament for protection against damage by bisphenol S exposure, characterized in that: The bisphenol S exposure is breast cancer cell exposure to an environment containing 0.01-1 μM of bisphenol S, or the bisphenol S exposure is bisphenol S exposure produced by using drinking water containing 50 μg / L of bisphenol S; The small molecule compound is used for inhibiting the metastasis of breast cancer cells MCF-7 and / or BT474 under bisphenol S exposure; the working concentration of the small molecule compound is 5-80 μM.

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