Application of small molecule compound in preparation of medicine for treating breast cancer

Abstract

The invention discloses an application of a small molecule compound in preparation of a medicine for treating breast cancer. The MEN 10207 is screened through molecular docking analysis, and the capability of the MEN 10207 for remarkably inhibiting proliferation, migration and invasion of breast cancer cells is verified in MDA-MB-231 cells, so that the MEN 10207 has the potential of serving as a breast cancer brain metastasis inhibitor. A further research discovers that the MEN 10207 does not show obvious toxicity in the zebra fish and also shows good tolerance in a mouse intravenous injection test. After the MEN 10207 treats the zebra fish transplanted with the breast cancer cells, the survival and migration capabilities of the breast cancer cells in the zebra fish body are obviously reduced; after the MEN 10207 treats a mouse model with breast cancer brain metastasis, the colonization of breast cancer cells in the mouse brain is also obviously reduced, which proves that the MEN 10207 has the activity of resisting breast cancer brain metastasis in vivo.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of a small molecule compound in the preparation of a drug for treating breast cancer. Background Art

[0002] Breast cancer is the second most common malignancy leading to brain metastases after lung cancer, and approximately 25% of breast cancer patients will experience symptomatic brain metastases. Breast cancer brain metastases not only significantly reduce the survival rate of patients but also cause a series of severe neurological symptoms, such as headache, nausea, vomiting, epilepsy, and even limb paralysis or visual impairment, seriously affecting the quality of life of patients.

[0003] Currently, the molecular mechanism of breast cancer brain metastases is not fully understood, resulting in unsatisfactory treatment effects and patient prognoses. Clinically, the main treatment methods for breast cancer brain metastases include surgical resection, radiotherapy, chemotherapy, and immunotherapy, etc. However, due to the existence of the blood-brain barrier (BBB), many systemic treatment drugs are difficult to effectively enter the brain tissue, limiting the treatment effect. In addition, breast cancer brain metastases have strong heterogeneity and drug resistance, further exacerbating the treatment difficulty. It is worth noting that among the various subtypes of breast cancer, triple-negative breast cancer (TNBC) is the most aggressive subtype, and nearly 30% of TNBC patients will eventually develop brain metastases. Due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in TNBC, it is difficult to effectively treat it through existing targeted therapies. Currently, the available treatment options for TNBC brain metastases patients are limited and the prognosis is extremely poor. Therefore, there is an urgent need to explore new drugs for breast cancer brain metastases to provide new and more effective treatment strategies for breast cancer brain metastases. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides the use of a small molecule compound in the preparation of a drug for treating breast cancer.

[0005] The present invention provides the use of a small molecule compound selected from any one of (1) to (3) in the preparation of a drug for treating breast cancer;

[0006] (1) the compound of formula (I) or its salt or ester;

[0007] (2) the optical isomer of the compound of formula (I) or its racemate;

[0008] (3) the solvate of the compound of formula (I) or its precursor;

[0009]

[0010] In some embodiments, the small molecule compound is the sole active ingredient of the drug for treating breast cancer.

[0011] In some embodiments, the breast cancer is metastatic breast cancer.

[0012] In some embodiments, the metastasis is brain metastasis.

[0013] In some embodiments, the drug is a drug that inhibits the invasion and metastasis of breast cancer cells by targeting the LRP8 protein in breast cancer cells.

[0014] In some embodiments, the breast cancer cells include at least one breast cancer cell line among MDA-MB-231, MDA-MB-468, and BT549.

[0015] In some embodiments, the patients treated with the drug are humans or non-human primates.

[0016] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0017] The present invention also provides the use of a small molecule compound selected from any one of (1) to (3) in the preparation of a product that targets the LRP8 protein in breast cancer cells;

[0018] (1) The compound of formula (I) or its salt or ester;

[0019] (2) The optical isomer of the compound of formula (I) or its racemate;

[0020] (3) The solvate of the compound of formula (I) or its precursor;

[0021]

[0022] In some embodiments, the breast cancer is metastatic breast cancer.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0024] The present invention screens out the small molecule compound MEN 10207 that stably binds to LRP8 through molecular docking analysis, and verifies its ability to significantly inhibit the proliferation, migration and invasion of breast cancer cells in MDA-MB-231 cells, indicating its potential as an inhibitor of breast cancer brain metastasis. Further research finds that MEN 10207 does not show obvious toxicity in zebrafish and also shows good tolerance in the intravenous injection test of mice. After treating zebrafish transplanted with breast cancer cells with MEN 10207, the survival and migration abilities of breast cancer cells in zebrafish are significantly reduced; after treating a mouse model of breast cancer brain metastasis with MEN 10207, the colonization of breast cancer cells in the mouse brain is also significantly reduced, confirming its anti-breast cancer brain metastasis activity in vivo. Brief Description of the Drawings

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0026] Figure 1 It is the 2D compound structural formula of MEN 10207 of the present invention.

[0027] Figure 2Analysis results of the GEO database in Example 1 of the present invention; Figure A is a Venn diagram analyzing the intersection of highly expressed genes in breast cancer brain metastasis tissue samples in the GSE100534 and GSE52604 datasets (P<0.01; logFC≥1.5); Figures B - C are heatmaps of 26 co - highly expressed genes in the GSE 100534 and GSE 52604 datasets, and the hazard ratios of distant metastasis survival rates of these genes in breast cancer patients are used for ranking, where BBT: Breast tumor tissue, breast cancer tissue; NNBT: Non - neoplastic breast tissue, non - tumor breast tissue; BCBMT: Breast cancer brain metastasis tissue, breast cancer brain metastasis tissue; DMSF: Distant metastasis - free survival, distant metastasis - free survival rate; HR: Hazard ratio: risk ratio; Figures D - E are Kaplan - Meier survival curve analyses of the relationship between the high or low expression of LRP8 and OS (Overall survival, overall survival rate) and DMFS; the top 25% of the population is defined as the high - expression group, and the bottom 75% of the population is defined as the low - expression group; Figure F is the comparison of LRP8 expression between breast cancer patients and normal people analyzed by the UALCAN database; Figure G is the comparison of LRP8 expression among different BC subtypes analyzed by the UALCAN database.

[0028] Figure 3 Results diagram of the effects of MEN 10207 on the proliferation, migration and invasion abilities of MDA - MB - 231 cells in Example of the present invention; Figure A is a 3D molecular docking pattern diagram of MEN 10207 and LRP8 protein; Figure B is a CCK - 8 experiment to detect the effect of MEN 10207 on the viability of MDA - MB - 231 cells, IC50 = 19.37 μM; Figure C is a scratch experiment to detect the wound - healing ability of MDA - MB - 231 cells after treatment with MEN 10207; Scale bar: 200 μm; Figure D is a Transwell experiment to detect the migration and invasion abilities of MDA - MB - 231 cells after treatment with MEN 10207, Scale bar: 200 μm; Figure E is to measure the wound area of MDA - MB - 231 cells at 0 h and 24 h using ImageJ software, calculate the ratio of the area at 24 h divided by the area at 0 h, n = 5; Figures F - G are to count the number of MDA - MB - 231 cells migrating and invading into the lower chamber of the chamber, n = 4; The data are expressed as mean ± SD, n = 3, p<0.05 is significantly different, **p<0.01, ***p<0.001, ****p<0.0001, and statistical analysis is performed using the standard t - test.

[0029] Figure 4 Inhibition effects of MEN 10207 on breast cancer brain metastasis in zebrafish and mice in Examples 5-6 of the present invention; Figure A shows the toxicity and safety evaluation of MEN 10207 on zebrafish. Figure B shows the toxicity and safety evaluation of MEN 10207 on mice; Figure C shows the body weight changes of mice before and after treatment with MEN 10207; Figure D shows the schematic diagram of the zebrafish xenograft model; Figure E shows the dynamic changes of MDA-MB-231 cells in zebrafish after treatment with MEN 10207 tracked by confocal in vivo imaging, scale bar: 100 μm; Figure F shows the calculation of the ratio of the fluorescence gray value at 72 hpi to the fluorescence gray value at 24 hpi, n = 4; hpi: hours post injection, hours after injection; Figure G shows the migration distance of MDA-MB-231 cells along the posterior cerebral vein of zebrafish calculated by ImageJ, n = 4; Figure H shows the schematic diagram of the mouse xenograft model; Figure I shows the light sheet imaging after mouse brain clearing, scale bar: 1000 μm; Figure J shows the total volume of MDA-MB-231 cells in the mouse brain counted by ImageJ, n = 3; The data are expressed as mean ± SD, n = 3, p < 0.05 indicates significant difference, *p < 0.05, ***p < 0.001, and statistical analysis was performed using the standard t-test. Detailed implementation manners

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0032] Experimental materials:

[0033] 8-week-old BALB / c Nude mice: Beijing Vital River Laboratory Animal Technology Co., Ltd.;

[0034] Tg(kdrl:mCherry) zebrafish: National Center for Aquatic Biological Resources;

[0035] MEN 10207: MCE, #HY-151413;

[0036] MDA-MB-231(GFP+) cells: Derived from the First Affiliated Hospital of Anhui Medical University;

[0037] The 2D compound structure of MEN 10207 is as follows Figure 1 shown. Its molecular weight is 1109.24. It has been dissolved in DMSO to prepare a stock solution of 13.52 mM and stored in a -80 °C refrigerator protected from light.

[0038] Example 1 Screening of key genes for breast cancer brain metastasis by GEO database analysis

[0039] In this example, genes related to breast cancer brain metastasis were analyzed based on the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ). Datasets GSE100534 and GSE52604 were selected for differential gene expression analysis, and the gene LRP8 highly expressed in breast cancer brain metastasis samples was screened out. Subsequently, the prognostic situation of LRP8 in breast cancer patients was evaluated using the Kaplan-Meier survival analysis tool (https: / / kmplot.com / analysis). The results showed that the expression of LRP8 was negatively correlated with the prognosis of patients, suggesting that it might be a risk gene for breast cancer brain metastasis ( Figure 2 ). Subsequently, molecular docking technology was used to screen small molecule compounds with strong binding ability to the LRP8 protein, and finally the compound MEN 10207 was determined, and its three-dimensional docking model was constructed ( Figure 3 A).

[0040] Example 2 Effect of MEN 10207 on the proliferation of breast cancer cells

[0041] In this example, the effect of MEN 10207 on the proliferation of breast cancer cells was evaluated by CCK-8 cell viability assay. The specific steps are as follows:

[0042] CCK-8 assay: MDA-MB-231 cells were seeded at 2500 cells / well in a 96-well plate, with at least 3 replicates in each group. After pre-culturing for one day, different concentrations of MEN 107 were added to act on MDA-MB-231 cells. After the cells were cultured for another 24 hours, the CCK-8 assay was performed; the CCK-8 working solution was prepared by adding 900 μL of serum-free DMEM medium to 100 μL of CCK-8 stock solution and mixing well; the cell culture medium in the 96-well plate was aspirated, 100 μL of the CCK-8 working solution was added, and it was placed in a 37 °C cell culture incubator for 2 h. Finally, the absorbance (OD value) was measured at 450 nm.

[0043] Figure 3 The experimental results in B showed that after treatment with MEN 10207, the cell viability of MDA-MB-231 decreased significantly, and the half maximal inhibitory concentration (IC50) was calculated to be 19.37 μM by analysis.

[0044] Effect of Example 3 MEN 10207 on the Migration Ability of Breast Cancer Cells

[0045] In this example, the effect of MEN 10207 on the migration ability of breast cancer cells was detected by a scratch assay. The specific steps are as follows:

[0046] MDA-MB-231 cells were plated the day before. An appropriate amount of cells was inoculated into a 24-well plate, and the next day the cells grew to cover the entire well. A 200 μl pipette tip was used to draw a straight line perpendicular to the cells on the well plate. The cells were washed 2 - 3 times with PBS and photographed using a microscope, which was recorded as 0 h. After taking the photos, the cells were grouped. One group was the control group, and the other group was the MEN 10207 treatment group (9 μM). Then the cells were returned to the cell incubator for further culture. After 24 h of cell culture, photos were taken again. The ratio of the wound area at 24 h to the wound area at 0 h was calculated. This ratio can represent the healing rate. The smaller the healing rate, the stronger the cell migration ability.

[0047] The experimental results showed that after treatment with MEN 10207, the cell migration ability of MDA-MB-231 decreased significantly ( Figure 3 C, E).

[0048] Example 4 Effect of MEN 10207 on the Invasion Ability of Breast Cancer Cells

[0049] In this example, a Transwell assay was used to evaluate the effect of MEN 10207 on the migration and invasion abilities of breast cancer cells. The specific steps are as follows:

[0050] 600 μL of medium containing 10% FBS was added to each well of a 24-well plate, and a Transwell chamber was placed in the 24-well plate. Subsequently, MDA-MB-231 cells were digested and grouped. In the control group, the cells were resuspended in serum-free medium, while in the MEN 10207 treatment group (9 μM), the cells were resuspended in serum-free medium containing MEN 10207. For the migration experiment, 2×10 4 cells were seeded onto the upper chamber of the Transwell chamber, and for the invasion experiment, 4×10 4 cells were seeded onto the upper chamber of the Transwell chamber. The cells were then placed in the cell incubator and cultured for 16 h. After 16 h, the cell medium was discarded, and the chamber was washed twice with sterile PBS. The non-migrated cells on the upper layer were gently wiped with a cotton swab and then fixed with methanol for 10 min. Then, the cells were stained with 0.1% crystal violet for 30 min. Finally, the crystal violet was recovered, and the chamber was air-dried for photographing.

[0051] The experimental results showed that after treatment with MEN 10207, both the migration and invasion abilities of MDA-MB-231 cells decreased significantly ( Figure 3 D, F, G).

[0052] Safety and Toxicity Evaluation of MEN 10207 in Example 5

[0053] This example evaluates the safety and tolerance of MEN 10207 in zebrafish and mouse models.

[0054] Dilute MEN 10207 to different concentrations with embryo water from cultured zebrafish embryos, and then use the embryo water containing MEN 10207 to culture embryos at 2 days post-fertilization. The number of embryos cultured at each concentration is greater than 10. Observe the survival rate of zebrafish embryos at different concentrations, change the fresh embryo water daily, and continuously observe for 4 days; Dilute MEN 10207 to different concentrations with normal saline, and intravenously inject different concentrations of MEN 10207 into each mouse once a day for 5 consecutive days. Observe the survival rate of mice daily, and the number of mice injected at each concentration is 3.

[0055] The experimental results show that 10 μM MEN 10207 is a safe dose for zebrafish, and the injection doses of 1.9 mg / kg and 3.8 mg / kg are both tolerable for mice ( Figure 4 A - C).

[0056] Example 6 Evaluation of the Anti-Breast Cancer Brain Metastasis Activity of MEN 10207 in Vivo Based on the Zebrafish Model

[0057] In this example, a breast cancer brain metastasis model is constructed by transplanting MDA-MB-231 cells into Tg(kdrl:mCherry) zebrafish embryos at 2 days post-fertilization. Due to the advantage of zebrafish embryo visualization, this model can observe the dynamic migration changes of MDA-MB-231 cells on blood vessels in real time. After the model is constructed, it is randomly divided into a control group and a MEN 10207 treatment group, and the therapeutic effect of MEN 10207 is evaluated by confocal in vivo imaging.

[0058] The experimental results show that after treatment with MEN 10207, both the migration ability and cell survival ability of MDA-MB-231 cells in zebrafish are significantly decreased ( Figure 4 D - G).

[0059] Example 7 Verification of the Anti-Breast Cancer Brain Metastasis Activity of MEN 10207 in Vivo Based on the Mouse Model

[0060] In this example, an intracardiac injection method is used to construct a breast cancer brain metastasis model in mice for further evaluation of the therapeutic effect of MEN 10207 on breast cancer brain metastasis.

[0061] The experimental steps are as follows:

[0062] A breast cancer brain metastasis model of mammals was constructed by intracardiac injection of MDA-MB-231 cells into mice. After injection of the cells, the mice were randomly divided into a sham operation group (Sham), a control group, and a MEN 10207 treatment group. Starting from the 4th day after transplantation, MEN 10207 was intravenously injected once a day for 5 consecutive days. At 14 days after transplantation, the mouse brains were dissected and subjected to brain clearing, and then light sheet imaging was performed to photograph the three-dimensional distribution of MDA-MB-231 cells in the mouse brains.

[0063] The experimental results showed that after treatment with MEN 10207, the distribution and volume of MDA-MB-231 cells in the mouse brains were significantly decreased, indicating that MEN 10207 could inhibit the proliferation and migration of MDA-MB-231 cells in the brains ( Figure 4 H–J). Combining with the breast cancer brain metastasis model of mammals, this experiment further confirmed that MEN 10207 has in vivo therapeutic potential against breast cancer brain metastasis.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a small molecule compound selected from any one of (1) to (3) in the preparation of a drug for treating breast cancer; (1) a compound of formula (I) or a salt or ester thereof; (2) an optical isomer of the compound of formula (I) or a racemate thereof; (3) a solvate of the compound of formula (I) or a precursor thereof; 2. The use according to claim 1, characterized in that: The small molecule compound is the only active ingredient of the drug for treating breast cancer.

3. The use according to claim 1, characterized in that: The breast cancer is metastatic breast cancer.

4. The use according to claim 3, characterized in that: The metastasis is brain metastasis.

5. The use according to claim 1, characterized in that: The drug is a drug that inhibits the invasion and metastasis of breast cancer cells by targeting the LRP8 protein in breast cancer cells.

6. The use according to claim 5, characterized in that: The breast cancer cells include at least one breast cancer cell line selected from the group consisting of MDA-MB-231, MDA-MB-468 and BT549.

7. The use according to claim 1, characterized in that: The patient to be treated with the drug is a human or a non-human primate.

8. The use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

9. Use of a small molecule compound selected from any one of (1) to (3) in the preparation of a product targeting LRP8 protein in breast cancer cells; (1) a compound of formula (I) or a salt or ester thereof; (2) an optical isomer of the compound of formula (I) or a racemate thereof; (3) a solvate of the compound of formula (I) or a precursor thereof; 10. The use according to claim 9, characterized in that: The breast cancer is metastatic breast cancer.

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