Small molecule compound and application thereof as drug for inhibiting bisphenol S induced breast cancer metastasis and application of D2 dopamine receptor in drug screening

Abstract

The invention relates to the technical field of cancer treatment drugs, in particular to a small molecule compound and application thereof as a drug for inhibiting bisphenol S induced breast cancer metastasis and application of a D2 dopamine receptor in drug screening. According to the scheme, the effect of the D2 dopamine receptor in acceleration of the development process of bisphenol S induced breast cancer is found for the first time, the D2 dopamine receptor is used as a drug screening target for small molecule drug screening, and two compounds ZDA6 and ZDA9 are determined from 100,000 candidate compounds. The two compounds are combined with a D2 dopamine receptor, effectively inhibit enhancement of migration ability of breast cancer cells and acceleration of a breast cancer process caused by bisphenol S exposure, and antagonize a breast cancer progress promoting effect generated by bisphenol S exposure. According to the technical scheme, the technical problem that drugs capable of inhibiting bisphenol S-induced breast cancer metastasis are lacked in the prior art can be solved, conditions are created for treating diseases related to environmental pollutants, and ideal application value is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer therapeutic drugs, and 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. Background Art

[0002] Female breast cancer (BC) has surpassed lung cancer to become the most common cancer in 2020, with an estimated 2.3 million new cases. Globally, despite significant advances in breast cancer detection and treatment, the mortality rate remains high. Chinese female breast cancer accounts for approximately 18% of breast cancer deaths worldwide, and the high mortality rate of breast cancer patients is mainly attributed to distant metastases, such as lung, bone, liver, and lymph node metastases.

[0003] It has been reported that the occurrence and development of breast cancer is closely related to exposure to endocrine disrupting chemicals (EDCs), especially exposure to bisphenol A (BPA) and its substitute bisphenol S (BPS). BPA is an environmental endocrine disrupting chemical widely used in the production of consumer products, such as beverages, adhesives, cigarette filters, and plastic food containers. BPA exposure is associated with obesity, diabetes, reproductive disorders, and cancer. As more and more evidence shows the negative effects of BPA on human health, it has prompted its removal from consumer products. As a substitute for BPA, the bisphenol analog BPS has been produced for various applications. So far, BPS has become a widely used industrial product worldwide, including polycarbonate plastics, epoxy resins, and thermal conductive paper. Studies have shown that BPS, as a xenoestrogen, can promote the occurrence and development of breast cancer. Epidemiological studies have shown that BPS can be widely detected in human blood and urine, and BPS can increase the risk of breast cancer. As an emerging environmental pollutant, humans can be exposed to BPS in a variety of different ways through inhalation and ingestion, which promotes the progression of breast cancer. In particular, studies have shown that BPS has a strong effect in promoting the metastasis of breast cancer. Therefore, it is urgent to fully study the mechanism by which BPS promotes the occurrence and development of breast cancer, and then discover the key molecular targets, and screen related drugs based on this target, and finally obtain candidate small molecule compounds that can effectively reverse the effect of bisphenol S in promoting the metastasis of breast cancer, and then obtain potential drugs that can be used to control the occurrence and development of breast cancer. Summary of the invention

[0004] The present invention is intended to provide an application of a small molecule compound in the preparation of a drug for treating breast cancer affected by bisphenol S, so as to solve the technical problem in the prior art of lacking a drug that can inhibit bisphenol S-induced breast cancer metastasis.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] The invention discloses an application of a small molecule compound in preparing a drug for treating breast cancer affected by bisphenol S. The small molecule compound is used for inhibiting the metastasis of breast cancer induced by bisphenol S.

[0007] As an emerging environmental pollutant, bisphenol S can be exposed to humans in a variety of ways through inhalation and ingestion, which promotes the progression of breast cancer. If the development of breast cancer is affected by bisphenol S exposure, its progression will be accelerated, including accelerated metastasis and invasion. The small molecule compounds discovered in this study can effectively inhibit the metastasis of breast cancer induced by bisphenol S, and thus have a therapeutic effect on breast cancer affected by bisphenol S exposure factors. This technical solution studies the treatment of cancer under the influence of specific environmental pollutants. It is not only a treatment for general cancers, but also takes into account the impact of environmental factors on the development of cancer. The compounds of this scheme have the effect of alleviating exposure damage to the specific environmental pollution factor bisphenol S exposure, especially in delaying the accelerated development of breast cancer caused by bisphenol S 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 and 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 the amino acids 41-416 of the D2 dopamine receptor;

[0011] Furthermore, the small molecule compound interacts with aspartic acid, the 114th amino acid residue 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 out by this technical solution interact with DRD2, thereby inhibiting the induction of bisphenol S on breast cancer metastasis. The small molecule drug binding sites were screened according to the protein structure of human DRD2. There are two binding sites, Site 1 is located in the intracellular region, and Site 2 is 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 part of DRD2, and small molecule compounds do not have to enter the cell to work. Selecting the Site2 small molecule drug binding pocket as a target for drug screening can improve the bioavailability of the drug, increase the therapeutic effect, and reduce the difficulty of medication.

[0012] The Site2 small molecule drug binding pocket is located at amino acids 41-416 of the D2 dopamine receptor. The key amino acids of Site2 are: LEU41 VAL91 LEU94 GLU95 GLY98 GLU99 TRP100 LYS101 PHE102SER103 ARG104 CYS107 PHE110 VAL111 ASP114 VAL115 CYS118 THR119 ALA122 GLU181CYS182 ILE184 ALA185 PHE189 VAL190 SER193 SER194 SER197 PHE198 PHE382 TRP386PHE389 PHE390 HIS393 PRO405 TYR408 SER409 THR412 TRP413 TYR416. For the above key sites, the frequency of interaction between small molecule compounds and Asp114 is high, and the interaction frequencies of the remaining amino acids are relatively uniform. The small molecule compounds screened in this scheme can all interact with the 114th amino acid residue of D2 dopamine receptor, aspartic acid.

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

[0014]

[0015] The technical scheme also provides an application of a small molecule compound in the preparation of a drug for resisting bisphenol S exposure damage, wherein the bisphenol S exposure damage is an aggravation of migration and / or invasion of breast cancer cells induced by bisphenol S, or an acceleration of the progression of bisphenol S-induced breast cancer;

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

[0017]

[0018] Furthermore, BPS-induced breast cancer cell migration included metastasis to the liver and lungs.

[0019] Further, the bisphenol S exposure is exposure of breast cancer cells to a 0.01-1 μM bisphenol S environment, or the bisphenol S exposure is exposure to bisphenol S using drinking water containing 50 μg / L bisphenol S;

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

[0021] The principle and beneficial effects of the screening of new small molecule drugs in this scheme (for the treatment of breast cancer and / or resistance to damage caused by bisphenol S exposure) are as follows:

[0022] In the process of new drug design, the small molecule drug binding pocket is predicted based on the protein structure of the D2 dopamine receptor, and 100,000 compounds with structural diversity are used for drug screening based on the Site2 site of the D2 dopamine receptor. Finally, 205 compounds that bind to the Site2 site were selected, and 10 of them with high scores were selected for subsequent experimental verification. Among the 10 small molecule compounds used in experimental research, only two compounds can significantly inhibit the migration ability of two breast cancer strains in the environment of bisphenol A exposure at the same time. Some candidate compounds do not have significant inhibitory effects in one or two cell lines. The two small molecule compounds screened have the ability to bind to Asp114 of the D2 dopamine receptor. The D2 dopamine receptor is a key molecule for bisphenol S to affect the progression of breast cancer, and it is a target for drug treatment. This is the first discovery of this technical solution. The two small molecule compounds can inhibit the increased migration of breast cancer cells caused by bisphenol S exposure, and can be used as a drug to treat breast cancer affected by bisphenol S exposure. Breast cancer affected by bisphenol S exposure has the characteristics of accelerated metastasis and invasion of cancer cells, accelerated course of disease, and increased malignancy of cancer, which is different from general breast cancer. In addition, the two small molecule compounds can also be used to antagonize the damage caused by bisphenol S exposure, and are antagonists of environmental pollutants.

[0023] The technical solution also provides an application of a D2 dopamine receptor in screening drugs that resist damage caused by bisphenol S exposure or drugs for treating breast cancer.

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

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

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

[0027] Through molecular structure analysis, it was found that the Site2 small molecule drug binding pocket score of the D2 dopamine receptor molecule was much higher than other sites, and the Site2 small molecule drug binding pocket contained Asp114 sites, etc., which can bind to small molecule drugs, thereby achieving the regulation of breast cancer development under bisphenol S exposure by small molecule drugs. More specifically, the target of drug screening is the small molecule drug binding pocket formed by the amino acids at positions 41-416 of the D2 dopamine receptor protein. More specifically, the following amino acid residues: LEU41 VAL91 LEU94 GLU95 GLY98 GLU99 TRP100 LYS101PHE102 SER103ARG104 CYS107 PHE110 VAL111 ASP114 VAL115 CYS118 THR119 ALA122GLU181 CYS182 ILE184ALA185 PHE189 VAL190 SER193 SER194 SER197 PHE198 PHE382TRP386 PHE389 PHE390 HIS393PRO405 TYR408 SER409 THR412 TRP413 TYR416. In particular, it is the Asp114 site in the small molecule drug binding pocket. Through the above drug screening sites, molecular docking, molecular interaction mode analysis (interaction site and force type), and drug-forming property analysis can be performed to screen drugs that resist bisphenol S exposure damage or drugs for treating breast cancer.

[0028] The above screening process can be realized by conventional computer simulation of the prior art. By analyzing the binding ability between each active amino acid residue site of the D2 dopamine receptor protein and the candidate drugs in the small molecule compound library, and by analyzing the drug properties, etc., potential drugs can be obtained. These potential drugs can be compared to obtain preferred candidate drugs through experimental screening studies. This technical solution screened and obtained two drugs (ZDA6 and ZDA9) as shown in Formula I or Formula II through the above drug screening targets. Therefore, the key point to achieve drug screening lies in the confirmation of the exact drug screening target (D2 dopamine receptor molecule). Using the D2 dopamine receptor molecule as a target for drug screening can effectively obtain drugs for resisting bisphenol S exposure damage or drugs for treating breast cancer. In particular, breast cancer with accelerated cancer metastasis and / or aggravated invasion caused by exposure to the environmental pollutant bisphenol S is particularly suitable as a target for drug screening.

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

[0030] (1) The mechanism of action of bisphenol S in inducing the occurrence and development of breast cancer was discovered, and the D2 dopamine receptor, a potential target for drug design, was discovered.

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

[0032] (3) Through experimental studies on drug screening, two small molecule compounds, ZDA6 and ZDA9, were identified that can bind to the D2 dopamine receptor. These two compounds can effectively inhibit the enhanced migration ability of breast cancer cells caused by bisphenol S exposure, thereby achieving a therapeutic effect on bisphenol S-related breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are the results of an experimental study on the promotion of lung and liver metastasis of breast cancer in MMTV-Erbb2 mice by BPS in Example 1 (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 metastasis; 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 metastasis).

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

[0035] Figure 3 These are the results of a study on the effect of low-dose BPS of Example 1 on the proliferation of breast cancer cells in vitro (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 method, n=3; *p<0.05, **p<0.01, ***p<0.001).

[0036] Figure 4These are the research results of the effect of low-dose BPS on the migration and invasion of breast cells in Example 1 (A, B: cell migration was measured in Transwell, and the number of cells in each image was quantified using ImageJ, n=3; C, D: cell invasion was measured in Transwell coated with Matrigel, and the number of cells in each image was quantified using ImageJ, n=3; E, G: the expression of metastasis-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 The transcriptomic analysis results of the potential target genes of BPS in promoting breast cancer cell metastasis in Example 2 (A: volcano plot of DEGs between the control group and the BPS group; B: cluster heat map of DEGs between the control group and the BPS group; C: GO enrichment analysis showed that the differentially expressed genes were significantly enriched in the G protein-coupled signaling pathway; D: Venn diagram showed the differentially expressed genes at the intersection of gene sets on five G protein-coupled pathways; E: mRNA levels of three down-regulated 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, 1 μM BPS for 72 hours, n=3, protein expression levels were normalized according to GAPDH levels, *p<0.05, **p<0.01, ***p<0.001).

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

[0039] Figure 7The experimental study results of Example 3 on the inhibition of BPS-induced metastasis of breast cancer cells by overexpression of DRD2 (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: 100μm; F, G: invasion of MCF-7 and BT474 cells, n=3, scale bar: 100μm; HK: Western blot was used to detect the expression of metastasis-related proteins FN, Vimentin and Snail, and the protein expression levels were normalized according to the GAPDH level; compared with the control group, *p<0.05, **p<0.01, ***p<0.001; compared with the BPS exposure 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 inducing breast cancer metastasis in vitro by activating AKT through DRD2 in Example 4 (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 through 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 determined by western blot, and the protein expression levels were standardized according to the 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 determined by western blot, and the protein expression levels were standardized according to the GAPDH level; FI: MCF-7 and BT474 cells were treated with or without OE-DRD2 plasmid, and then treated with or without 0.01 μM BPS for 72 h, and then the protein expression levels were standardized according to the GAPDH level. The expression of phosphorylated AKT and GSK3β proteins was measured by blot, and the protein expression levels were normalized according to the GAPDH level; the 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] Fig. 9 This is the virtual screening experiment result of Example 5.

[0042] Fig.10These are the results of the cytotoxic concentration screening of the small molecule compounds in Example 6 (compared with the control group, *p<0.05, **p<0.01, ***p<0.001).

[0043] Fig.11 This is the screening result of the small molecule compound of Example 6 for the effect 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] Fig.12 This is the detection spectrum of the small molecule compound ZDA6 in Example 6.

[0045] Fig.13 This is the detection spectrum of the small molecule compound ZDA9 in Example 6.

[0046] Fig.14 Schematic diagram of the molecular binding between the small molecule compound ZDA6 of Example 7 and DRD2.

[0047] Fig.15 These are the research results of 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 exposure group, #p<0.05, ##p<0.01, ###p<0.001).

[0048] Fig.16 These are the research results of the inhibitory effect of the small molecule compound ZDA6 in Example 7 on the activation of the AKT / GSK3β pathway induced by BPS (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 exposure group, #p<0.05, ##p<0.01, ###p<0.001).

[0049] Fig.17 Schematic diagram of the molecular binding between the small molecule compound ZDA9 of Example 7 and DRD2.

[0050] Fig.18These are the research results of the inhibitory effect of the small molecule compound ZDA9 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 exposure group, #p<0.05, ##p<0.01, ###p<0.001).

[0051] Fig.19 These are the research results of the inhibitory effect of the small molecule compound ZDA9 in Example 7 on the activation of the AKT / GSK3β pathway induced by BPS (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 exposure group, #p<0.05, ##p<0.01, ###p<0.001). DETAILED DESCRIPTION

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

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

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

[0055] (1) Animal experiments

[0056] The transgenic MMTV-Erbb2-induced breast cancer (BC) mouse model was purchased from Jackson Laboratory, Sacramento, California, USA. The breeding, maintenance and genotyping of mice were performed according to conventional methods in the prior art. Semi-heterozygous unmated female mice were selected for subsequent experiments. In order to study the effect of BPS on promoting BC metastasis, female MMTV-Erbb2 mice (6 weeks old) were divided into two groups: (i) control group (n=10) and (ii) BPS group (n=10). Mice in the BPS group drank drinking water containing 50 μg / L BPS for 19 weeks, and mice in the control group were given boiled water for the same duration. Tumor development was evaluated by palpation. After a palpable tumor appeared in the mouse mammary gland, the size of the mouse mammary tumor was measured and recorded every 1 week with a vernier caliper, and the tumor volume was calculated according to the following formula: Volume = length × width 2 / 2.

[0057] The experiment was terminated and mice were euthanized at 25 weeks of age or when the tumor diameter reached 1.5 cm. Mice were dissected on a clean bench. Fresh organs, including liver and lungs, were harvested in time and fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China, E672002-0500). These organs were then dehydrated in an ethanol solution. 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 metastasis.

[0058] Hematoxylin and eosin staining and immunohistochemistry were performed according to conventional methods.

[0059] (2) Cell experiments

[0060] MCF-7 cells and BT474 cells were purchased from Procell Life Science and Technology Co, Wuhan, China. These BC cells were supplemented with 10% fetal bovine serum (AUSGENEX; Molendiner, Australia; FBS 500-S) and 1% (v / v) penicillin / streptomycin (Gibco, NY, USA; 15140-122), DMEM (Gibco, NY, USA; C11995500BT). MCF-7 and BT474 cell lines were maintained at 37°C in a humidified atmosphere 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, MCF-7 and BT474 cells were then exposed to different concentrations of BPS (0, 0.01, 0.1 and 1 μM) and cultured flat for 72 h.

[0061] The cell viability was evaluated by conventional CCK-8 method, the cell migration and invasion ability was evaluated by Transwell method, and the expression of related proteins was detected by Western Blotting method.

[0062] (3) Statistical analysis

[0063] Data were analyzed using GraphPad Prism 9.0 software (GraphPad, USA) and expressed as mean ± SEM. Unpaired two-sided t-test was used for comparison between two experimental groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. Each experiment was repeated at least 3 times, and *p < 0.05 was defined as statistically significant.

[0064] (4) Experimental results

[0065] Since BPA is an environmental factor known to be involved in the development of hormone-dependent breast cancer, BPS was used as a substitute for BPA. To evaluate the effect of chronic environmental doses of BPS on the development of breast cancer in mice, we exposed MMTV-Erbb2 mice to 50 μg / L BPS in drinking water for 19 weeks ( Figure 1 A), the number of mice with breast cancer and tumor size were recorded weekly, and tumors were surgically removed when they grew to 1.5 cm in diameter. In this longitudinal study, palpable mammary 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, two mice in the control group had no tumors) ( Figure 1 B). The median time of breast tumor detection 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). The results showed that BPS exposure promoted the early onset of breast cancer in MMTV-Erbb2 mice. The MMTV-Erbb2 mice that had been exposed to drinking water for 19 weeks were killed and their liver and lung tissues were removed. After fixation with paraformaldehyde, pathological sections were made and the number of lung metastases and liver metastases were counted after HE staining. There were 4 MMTV-Erbb2 mice with lung metastasis of breast cancer in the control group (4 / 8), and all of them in the BPS exposure group developed lung metastasis (8 / 8) ( Figure 1 C, Figure 1 D). Consistent with the results of lung metastasis, 4 MMTV-Erbb2 mice (4 / 8) in the control group developed breast cancer liver metastasis, while all mice (8 / 8) in the BPS exposure group developed breast cancer liver metastasis ( Figure 1 E, Figure 1 F). Immunohistochemical evaluation of changes in the expression of metastasis-related proteins Vimentin and Snail markers in BC tissues of MMTV-ERBB2 mice after BPS exposure ( Figure 2 A, Figure 2 B), it was found that the protein expression of Vimentin and Snail increased in the BPS-treated group. Therefore, BPS promotes the metastatic ability of breast cancer in MMTV-Erbb2 mice. The above results show that chronic low-dose BPS exposure significantly promotes the metastasis of breast cancer in MMTV-Erbb2 mice to the liver and lungs, and significantly increases the malignancy after metastasis.

[0066] We used CCK-8 method to detect the effect of BPS on the viability of breast cancer cells MCF-7 and BT474 cells cultured in vitro. The results confirmed that after treating breast cancer cells with gradient concentrations of BPS (0, 0.01, 0.1 and 1 μM) for 72 h, we found that the proliferation of breast cancer cells did not change ( Figure 3 A, Figure 3 B). However, in addition to proliferation ability, another indicator of increased malignancy of breast cancer cells is decreased adhesion, increased motility and increased invasion ability. Therefore, we first used the Transwell-migration method to study the effect of BPS on the migration ability of MCF-7 cells and BT474 cells. The results of the Transwell-migration experiment showed that compared with the cells in the control group, the number of breast cancer cells passing through the Transwell chamber increased significantly after treatment with gradient concentrations of BPS ( Figure 4 AB). BPS treatment can enhance the migration ability of breast cancer cells. Then, for the invasion ability of breast cancer cells, we used the Transwell-invasion method, that is, the diluted matrix gel was evenly spread in the Transwell chamber in advance, and then the cells were tested for penetrating the matrix gel and the inner membrane of the chamber to evaluate the effect of BPS exposure on the invasion ability of MCF-7 cells and BT474 cells. The experiment found 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 increased significantly ( Figure 4 C, Figure 4 D). To understand the promoting effect of BPS on breast cancer cell migration and invasion, we detected tumor metastasis-related proteins at the molecular level. Immunoblotting results showed that BPS exposure significantly promoted MCF-7 ( Figure 4 E and Figure 4 F) and BT474 cells ( Figure 4 G, Figure 4 H) The expression of tumor metastasis-related proteins such as FN, Vimentin and Snail increased. In summary, BPS exposure had no effect on the proliferation of breast cancer cells, but increased their migration and invasion abilities, thereby increasing their malignancy.

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

[0068] In this example, transcriptomic analysis revealed that DRD2 is a potential target gene for BPS-induced breast cancer metastasis.

[0069] (1) Eukaryotic transcriptome sequencing

[0070] MCF-7 cells in the control group and the experimental group were exposed to 0.01 μM BPS for 72 hours. After the treatment, conventional total RNA extraction was performed, and the mRNA in the total RNA was purified through the unique polyA structure of mRNA. Then mRNA fragmentation, BPSNA synthesis, PCR enrichment of library fragments were performed, and finally sequencing was performed. The sample RNA was extracted and purified and the library was constructed. The sample library was sequenced accordingly on the Illumina HiSeq sequencing platform.

[0071] The conventional quality control of the eukaryotic transcriptome sequencing was performed, and then the eukaryotic transcriptome sequencing analysis was performed. The gene expression was standardized using FPKM, and FPKM>1 was used to determine whether the gene was expressed. The gene structure annotation information was then read, and the alignment results were compared with the gene structure and the results were statistically analyzed. When only a part of a read was covered on the gene region or one part was covered on the intron region of the gene, the Union scheme and the Intersection_nonempty scheme determined that the read belonged to the gene, while the Intersection_nonempty scheme determined that the read did not belong to any gene. When a read was fully covered on one gene and partially covered on another gene, the Union scheme determined that the read belonged to both genes, and the Intersection_strict scheme and the Intersection_nonempty scheme determined that the read belonged to the first gene. The FPKM density distribution map was drawn, and saturation analysis and sample correlation test were performed, and then differential expression detection (P<0.05, |log2FC|>2) and cluster analysis were performed.

[0072] (2) Functional enrichment analysis

[0073] GO analysis of differentially expressed genes was performed. The gene ontology (GO) annotated protein group was derived from the UniProt-GOA database (http: / / www.ebi.ac.uk / GOA / ). First, the identified protein ID was converted to UniProt ID, and then mapped to GOID by protein ID. If some of the identified proteins were not annotated by the UniProt-GOA database, InterProScan software was used to annotate the GO function of the protein based on the protein sequence alignment method. Then, the protein was annotated with gene ontology based on three categories: biological process, cellular component and molecular function.

[0074] (3) Fluorescence quantitative PCR analysis is performed on the target gene, wherein the processes such as total RNA extraction, reverse transcription and qRT-PCR are performed according to conventional methods in the prior art.

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

[0076] (4) Experimental results

[0077] The data results of differential gene multiples (FC) > 4 and p < 0.05 between the control group and the BPS group were used for analysis. The volcano plot ( Figure 5 A) 853 up-regulated genes and 763 down-regulated genes were observed. Heat map ( Figure 5 B) shows that the control group and the treatment group are significantly different. Then, the GO enrichment analysis diagram was made using the differential gene set with down-regulated FC>4 times ( Figure 5 C), after evaluating the enrichment score and significance, the G protein-coupled receptor pathway of four biological processes (BP) and one molecular function (MF) became the most significantly enriched pathways. Venn diagram ( Figure 5 D) is the intersection gene of the differential gene data set within the five G protein coupled receptor pathways, and DRD2, CHRM1 and ADRA2A were determined as candidate genes for further research. RT-qPCR experiments verified the expression levels of ADRA2A, CHRM1 and DRD2 mRNA in the control group and BPS group in MCF-7 and BT474 cells, respectively, and found that the mRNA expression levels of ADRA2A and DRD2 were consistently reduced in MCF-7 and BT474 cells ( Figure 5 E). WB experiments observed that DRD2 protein expression was decreased in the BPS treatment group ( Figure 5 F), the protein expression level of ADRA2A did not change ( Figure 5 G). At the same time, immunohistochemistry was used to evaluate the changes of DRD2 in breast cancer tissues of MMTV-Erbb2 mice after BPS exposure, and a decrease in DRD2 protein expression was observed ( Figure 6). Therefore, DRD2 was determined as the final research target gene. More specifically, 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 ID of DRD2 is 1813 (human) and 13489 (mouse).

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

[0079] The DRD2 gene expression vector was constructed according to conventional methods in the prior art, and transfected into MCF-7 cells and BT474 cells to construct a DRD2 overexpression cell model. The cells were subjected to RT-qPCR, cell migration and invasion ability assessment, Western blot and other analyses. The experimental methods and statistical methods are shown in Example 1.

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

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

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

[0083] In order to explore the potential molecular mechanism by which BPS promotes breast cancer metastasis, we conducted bioinformatics analysis using the Comparative Toxicogenomics Database (CTD). The CTD database integrates a large amount of data on the interaction between chemicals and genes, and can predict disease-related environmental exposure factors and potential drug mechanisms. We used the CTD database to predict the gene set related to BPS and performed KEGG pathway analysis, and found that bisphenol S is closely related to 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. WB results showed that BPS significantly induced the phosphorylation of AKT and GSK3β in MCF-7 cells ( Figure 8 B, Figure 8 C). Meanwhile, BPS significantly induced the phosphorylation of AKT and GSK3β in BT474 cells ( Figure 8 D, Figure 8 E). In addition, we tested whether overexpression of DRD2 could inhibit the activation of AKT / GSK3β. WB experiments showed that overexpression of DRD2 could inhibit the activation of AKT / GSK3β pathway induced by BPS ( Figure 8 FI). These results provide solid evidence that BPS induces breast cancer metastasis in vitro through activation of AKT by 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, we screened for small molecule drug binding sites. According to the prediction of MOE software, there are two binding sites, Site 1 is located in the intracellular region and Site 2 is located in the extracellular region. Site 2 was selected for subsequent molecular docking studies. For details on the prediction of DRD2 molecular structure and the prediction of DRD2 small molecule compound binding pocket, please see Fig. 9 .

[0087] About 100,000 compounds with structural diversity were selected for molecular docking experiments ( Software). The key amino acids of Site2 are: LEU41 VAL91 LEU94 GLU95 GLY98 GLU99 TRP100 LYS101 PHE102SER103ARG104 CYS107 PHE110 VAL111 ASP114 VAL115 CYS118 THR119 ALA122 GLU181CYS182 ILE184ALA185 PHE189 VAL190 SER193 SER194 SER197 PHE198 PHE382 TRP386PHE389 PHE390 HIS393PRO405 TYR408 SER409 THR412 TRP413 TYR416. The amino acid residues 41 to 416 of the D2 dopamine receptor are used for molecular docking with candidate small molecule drugs, and the amino acid residues listed above are more preferably used for molecular docking. The docking experiment results showed that 17,870 compounds in the D3100 compound library bound to the Site2 region, and their affinities were distributed between -13.1492 kcal / mol and -7.5052 kcal / mol. Due to the difference in the affinity distribution of binding to the Site2 region, a total of 713 compounds with an affinity value of less than -10 kcal / mol were selected for binding to the Site2 region.

[0088] Next, we conducted a molecular interaction pattern analysis, using the Protein Ligand Interaction Fingerprints (PLIF) method to analyze the interaction sites and interaction force types (hydrogen bonding, ionic bonding, etc.) between 713 compounds and the DRD2 structure. The frequency of small molecule compounds interacting with Asp114 was high, and the interaction frequencies of the remaining amino acids were relatively uniform. The amino acid sites of the 710 compounds involved in binding and the frequency of amino acid sites involved in binding small molecule compounds are detailed in Fig. 9 Then, the drug properties of small molecule compounds were studied (ADME pharmacokinetics), and 291 compounds were screened out from 710 compounds. After structural diversity analysis, 205 compounds were screened out. For the compounds screened out virtually, the compounds with the highest scores were selected for subsequent experimental studies.

[0089] The above-mentioned computer-assisted virtual screening process can be entrusted to relevant biotechnology companies, that is, for the amino acid residues including the Asp114 site of DRD2, the process of screening and obtaining 205 compounds from 100,000 compounds through molecular docking, affinity data analysis, and drugability analysis is a conventional method of the prior art and can be entrusted to complete. The key point to achieve drug screening is mainly the confirmation of drug screening targets, that is, determining that DRD2 is the role and regulation target of BPS in inducing breast cancer metastasis, and BPS induces breast cancer metastasis by affecting DRD2. It is precisely because of the discovery of drug screening targets that subsequent drug screening can be implemented, thereby obtaining the potential drugs in this embodiment.

[0090] Example 6: Experimental screening of small molecule compounds that inhibit breast cancer metastasis by acting on DRD2 binding sites

[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, C0222), and cultured at 37°C and 5% CO2. BPS (Sigma, USA, 80-09-1) was dissolved in DMSO and diluted with culture medium.

[0092] Detection of cytotoxicity of compounds: CCK-8 (Cell Counting Kit-8) cell proliferation-toxicity detection kit from Tokyo Tongjin was used to detect cell viability. This is a yellow-orange formazan dye generated by the oxidation and reduction of WST-8 by intracellular dehydrogenases. It can be dissolved in the culture medium, and the amount of formazan generated is proportional to the number of living cells. MCF-7 and BT474 cells were cultured at 5×10 4 / hole and 8×10 4 / well were inoculated in a 96-well plate and cultured overnight in an incubator. According to the experimental grouping, 3 replicates were inoculated in each group. When the cells grew to a density of 50%, small molecule compounds (0, 5, 10, 20, 40, 80 μM) were added and treated for 72 hours. After the treatment, the culture medium was removed, 100 μL of mixed culture medium containing 10% CCK-8 reagent was added to each well, and 3 additional wells were added as blank wells to avoid bubbles interfering with the reading. The 96-well plate was placed in an incubator and incubated for 30-45 minutes. The absorbance was detected at 450 nm using an enzyme reader. Cell viability was calculated using the following formula: Cell viability = [(At-Ab) / (Ac-Ab)]; At: absorbance of the treated well (culture medium containing CCK-8, cells with different treatments), Ac: absorbance of the control well (culture medium containing CCK-8, cells in the control group), Ab: absorbance of the blank well (culture medium containing CCK-8, no cells).

[0093] To test the effect of compounds on cell migration ability: the cell density was adjusted to MCF-7 cells: 2.5×10 5 / mL, BT474 cells: 4×10 5 / mL. The cell suspension (200 μL) was inoculated into the upper chamber surface of the pre-prepared Transwell chamber, and a 0.01 μM BPS solution (800 μL) was added to the lower chamber, with or without small molecule compounds (maximum safe dose of each compound). The Transwell culture plate was placed in an incubator for 72 hours, fixed with anhydrous methanol for 15 minutes, stained with crystal violet for 8 minutes, and then washed. The unpenetrated cells on the upper layer were wiped off, and the chamber was placed in a clean 24-well plate. It was observed under a microscope at 100 times, and 3 fields of view were randomly selected for photographing and counting.

[0094] The CCK-8 experiment showed the toxic effects of 10 small molecule compounds (screened in Example 5, compound information is shown in Table 1) at different concentrations (0, 5, 10, 20, 40, 80 μM) on breast cancer cells MCF-7 and BT474, in order to determine the safe concentration range of each compound applied to MCF-7 and BT474 cells ( Fig.10 A, 10B). According to the results, we selected 10 small molecule compounds, and the corresponding maximum dose concentrations that can be applied to both cell lines are shown in Table 2. Then, in the cell model with BPS added, 10 small molecule compounds were added or not added according to the selected dose, and the exposure lasted for 72 hours. Transwell-Migration was used to observe the inhibitory effect of small molecule compounds on breast cancer metastasis induced by BPS exposure. The screening results showed that compounds No. 6 and No. 9 had the most significant effect in inhibiting BPS-induced breast cancer metastasis ( Fig.11AD), which had significant effects on both cell lines and is worthy of further study.

[0095] Table 1: Compound information

[0096]

[0097]

[0098] Table 2: Maximum safe concentrations 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 No. 6 ZDA6 (Cas: 685844-11-5) and compound No. 9 ZDA9 (Cas: 423737-47-7) for further study of their effects. The structural formula of ZDA6 is shown in Formula I, and the corresponding detection spectrum is shown in Fig.12 ; The structural formula of ZDA9 is shown in Formula II, and the corresponding detection spectrum is shown in Fig.13 Both small molecule compounds can bind to DRD2, thereby inhibiting the metastasis and invasion of breast cancer cells promoted (caused) by BPS, 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] MOE was used to analyze the affinity data of the compounds in the screening results, and then the PLIF module (Protein Ligand Interaction Fingerprint) was selected in MOE to statistically analyze the interaction between the compounds and amino acid residues. Molecular docking was used to analyze the interaction between ZDA6 and DRD2 binding sites. The results showed that D114 was found to be an effective binding site for ZDA6 and DRD2. ZDA6 can inhibit breast cancer metastasis by binding to the D114 site of Site2 ( Fig.14 ). Then, the cells were treated with 40 μM ZDA6 to detect the effect of ZDA6 on the expression of tumor metastasis-related proteins in cells. The specific experimental operation was as follows: MCF-7 and BT474 cells were cultured at 5×10 4 / hole and 8×10 4 / well were inoculated in a 96-well plate and cultured overnight in an incubator; when the cells grew to a density of 50%, we used ZDA6 (40μM, 72h) to treat breast cancer cells with or without BPS (0.01μM, 72h) exposure. Western blot analysis showed that ZDA6 at a concentration of 40μM treated BPS-exposed MCF-7 and BT474 cell models, and ZDA6 could inhibit the expression of tumor metastasis-related proteins promoted by BPS ( Fig.15 AD), and ZDA6 can inhibit the expression of p-AKT pathway-related proteins ( Fig.16 AD). Therefore, ZDA6 can reverse BPS-induced breast cancer metastasis promoted by DRD2.

[0104] Analysis of DRD2 binding sites for ZDA9 can be found in Fig.17 , the research results of ZDA9 inhibiting the expression of tumor metastasis-related proteins promoted by BPS are detailed in Fig.18 AD and Fig.19 AD. Molecular docking was used to analyze the interaction between ZDA9 and DRD2 binding sites. The results showed that D114 was found to be an effective binding site for ZDA9 and DRD2 (the binding force with other amino acid residues T412 and F198 was relatively weak). ZDA9 can inhibit breast cancer metastasis by binding to the D114 site of Site2. We then treated the cells with 40 μM ZDA9 to detect the effect of ZDA9 on the expression of tumor metastasis-related proteins in cells. Western blot analysis showed that ZDA9 treated BPS-exposed MCF-7 and BT474 cell models with 40 μM ZDA9. We found that ZDA9 could inhibit the expression of tumor metastasis-related proteins promoted by BPS, and ZDA9 could inhibit the expression of proteins related to the p-AKT pathway. Therefore, ZDA9 can reverse BPS-induced breast cancer metastasis promoted by DRD2.

[0105] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Use of a small molecule compound in the preparation of a drug for treating breast cancer affected by bisphenol S, characterized in that: The small molecule compound is used for inhibiting bisphenol S-induced breast cancer metastasis.

2. Use of the small molecule compound according to claim 1 in the preparation of a drug for treating breast cancer affected by bisphenol S, characterized in that: The small molecule compound is used to bind to the D2 dopamine receptor.

3. Use of the small molecule compound according to claim 2 in the preparation of a drug for treating 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 and the D2 dopamine receptor is located in the extracellular region of the D2 dopamine receptor; Preferably, 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 acids 41-416 of the D2 dopamine receptor; Preferably, the small molecule compound interacts with aspartic acid, the 114th amino acid residue of the D2 dopamine receptor.

4. Use of the small molecule compound according to claim 3 in the preparation of a drug for treating breast cancer affected by bisphenol S, characterized in that: The small molecule compound is as shown in Formula I or Formula II; 5. Use of a small molecule compound in the preparation of a drug for resisting damage caused by exposure to bisphenol S, characterized in that: The bisphenol S exposure damage is that the migration and / or invasion of breast cancer cells induced by bisphenol S is aggravated, or the progression of breast cancer induced by bisphenol S is accelerated; The small molecule compound interacts with the 114th amino acid residue aspartic acid of the D2 dopamine receptor, as shown in Formula I or Formula II; 6. Use of the small molecule compound according to claim 5 in the preparation of a drug for resisting damage caused by exposure to bisphenol S, characterized in that: Bisphenol S-induced breast cancer cell migration includes metastasis to the liver and lungs.

7. Use of the small molecule compound according to claim 6 in the preparation of a drug for resisting damage caused by exposure to bisphenol S, characterized in that: The bisphenol S exposure is that the breast cancer cells are exposed to a 0.01-1 μM bisphenol S environment, or the bisphenol S exposure is bisphenol S exposure produced by using drinking water containing 50 μg / L bisphenol S; The small molecule compound is used to inhibit the metastasis of breast cancer cells MCF-7 and / or BT474 exposed to bisphenol S; the working concentration of the small molecule compound is 5-80 μM.

8. Application of D2 dopamine receptor in screening drugs that resist damage caused by bisphenol S exposure or drugs for treating breast cancer.

9. Use of the D2 dopamine receptor according to claim 8 in screening drugs for resisting damage caused by bisphenol S exposure or drugs for treating breast cancer, characterized in that: The amino acid residues at positions 41 to 416 of the D2 dopamine receptor are used for computer-assisted molecular docking with candidate small molecule drugs.

10. Use of the D2 dopamine receptor according to claim 9 in screening drugs for resisting damage caused by bisphenol S exposure or drugs for treating breast cancer, characterized in that: The aspartic acid at position 114 of the D2 dopamine receptor is used for computer-assisted molecular docking with candidate small molecule drugs.

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