Application of small molecule compounds in the preparation of drugs for treating breast cancer

Abstract

This invention discloses the application of small molecule compounds in the preparation of drugs for treating breast cancer. MEN 10207 was screened using molecular docking analysis, and its ability to significantly inhibit the proliferation, migration, and invasion of breast cancer cells was verified in MDA-MB-231 cells, indicating its potential as an inhibitor of breast cancer brain metastasis. Further studies found that MEN 10207 did not exhibit significant toxicity in zebrafish and showed good tolerability in mouse intravenous injection experiments. Treatment of zebrafish transplanted with breast cancer cells with MEN 10207 significantly reduced the survival and migration ability of breast cancer cells in zebrafish; treatment of a mouse model of breast cancer brain metastasis with MEN 10207 also significantly reduced the colonization of breast cancer cells in the mouse brain, confirming its in vivo activity against breast cancer brain metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a small molecule compound in the preparation of a drug for treating breast cancer. Background Technology

[0002] Breast cancer is the second leading cause of brain metastases after lung cancer, with approximately 25% of breast cancer patients experiencing symptomatic brain metastases. Breast cancer brain metastases not only significantly reduce patient survival rates but also trigger a range of severe neurological symptoms, such as headaches, nausea, vomiting, seizures, and even limb paralysis or visual impairment, severely impacting patients' quality of life.

[0003] Currently, the molecular mechanisms of breast cancer brain metastasis are not fully understood, leading to unsatisfactory treatment outcomes and patient prognoses. Clinically, the main treatments for breast cancer brain metastasis include surgical resection, radiotherapy, chemotherapy, and immunotherapy. However, the presence of the blood-brain barrier (BBB) ​​limits the effective penetration of many systemic therapies into brain tissue, thus restricting treatment efficacy. Furthermore, breast cancer brain metastasis exhibits strong heterogeneity and drug resistance, further complicating treatment. Notably, among the various subtypes of breast cancer, triple-negative breast cancer (TNBC) is the most aggressive, with nearly 30% of TNBC patients eventually developing brain metastases. Because TNBC lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), it is difficult to treat effectively with existing targeted therapies. Currently, the available treatment options for TNBC brain metastases are limited, and the prognosis is extremely poor. Therefore, there is an urgent need to explore new drugs for breast cancer brain metastasis to provide novel and more effective treatment strategies. Summary of the Invention

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

[0005] This invention provides the use of small molecule compounds selected from any one of (1) to (3) in the preparation of medicaments for treating breast cancer;

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

[0007] (2) Optical isomers of the compound of formula (I) or their racemates;

[0008] (3) Solvates of compounds of formula (I) or their precursors;

[0009]

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

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

[0012] In some implementations, the transfer is a brain transfer.

[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 selected from MDA-MB-231, MDA-MB-468, and BT549.

[0015] In some embodiments, the patient receiving the drug treatment is a human or a non-human primate.

[0016] In some embodiments, the drug also includes a pharmaceutically acceptable carrier.

[0017] This invention also provides the 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;

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

[0019] (2) Optical isomers of the compound of formula (I) or their racemates;

[0020] (3) Solvates of compounds of formula (I) or their precursors;

[0021]

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

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

[0024] This invention screened out the small molecule compound MEN 10207, which stably binds to LRP8, through molecular docking analysis. Its ability to significantly inhibit the proliferation, migration, and invasion of breast cancer cells was verified in MDA-MB-231 cells, indicating its potential as an inhibitor of breast cancer brain metastasis. Further studies found that MEN 10207 did not exhibit significant toxicity in zebrafish and showed good tolerability in mouse intravenous injection experiments. Treatment of zebrafish transplanted with breast cancer cells with MEN 10207 significantly reduced the survival and migration ability of breast cancer cells in zebrafish; treatment of a mouse model of breast cancer brain metastasis with MEN 10207 also significantly reduced the colonization of breast cancer cells in the mouse brain, confirming its in vivo activity against breast cancer brain metastasis. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

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

[0027] Figure 2The following are the results of the GEO database analysis in Example 1 of this invention; Figure A is a Venn diagram analysis of the intersection of highly expressed genes in breast cancer brain metastasis tissue samples from the GSE100534 and GSE52604 datasets (P<0.01; logFC≥1.5); Figures B-C are heatmaps of 26 commonly highly expressed genes from the GSE 100534 and GSE 52604 datasets, and the hazard ratios of distant metastasis survival in breast cancer patients using these genes are ranked, where BBT: Breast tumor tissue; NNBT: Non-neoplastic breast tissue; BCBMT: Breast cancer brain metastasis tissue; DMSF: Distant metastasis-free survival; HR: Hazard ratio; Figures D-E are Kaplan-Meier survival curve analyses of the relationship between LRP8 expression and overall survival (OS). The relationship between overall survival (OS) and DMFS; the top 25% of the population were defined as the high expression group, and the bottom 75% were defined as the low expression group; Figure F shows the comparison of LRP8 expression between breast cancer patients and normal individuals analyzed using the UALCAN database; Figure G shows the comparison of LRP8 expression in patients with different BC subtypes analyzed using the UALCAN database.

[0028] Figure 3 Figure 1 shows the effect of MEN 10207 on the proliferation, migration, and invasion abilities of MDA-MB-231 cells in this embodiment of the invention; Figure A is a 3D model of the molecular docking between MEN 10207 and LRP8 protein; Figure B shows the effect of MEN 10207 on the viability of MDA-MB-231 cells detected by the CCK-8 assay, IC50 = 19.37 μM; Figure C shows the wound healing ability of MDA-MB-231 cells after MEN 10207 treatment detected by the scratch assay; Scale bar: 200 μm; Figure D shows the effect of MDA-MB-231 cells on the proliferation, migration, and invasion abilities of MEN 10207 detected by the Transwell assay. Migration and invasion capabilities of MDA-MB-231 cells after treatment with 10207 (scale bar: 200 μm); Figure E shows the wound area of ​​MDA-MB-231 cells at 0h and 24h measured using ImageJ software, and the ratio of the 24h area to the 0h area was calculated (n=5); Figures F-G show the number of MDA-MB-231 cells that migrated and invaded the lower chamber (n=4); Data are presented as mean ± SD (n=3), p<0.05 was considered statistically significant (**p<0.01, ***p<0.001, ****p<0.0001), and statistical analysis was performed using a standard t-test.

[0029] Figure 4 Figure 5-6 illustrates the inhibitory effect of MEN 10207 on brain metastasis of breast cancer in zebrafish and mice. Figure A shows the toxicity and safety assessment of MEN 10207 in zebrafish. Figure B shows the toxicity and safety assessment of MEN 10207 in mice. Figure C shows the weight changes in mice before and after MEN 10207 treatment. Figure D is a schematic diagram of the zebrafish xenograft model. Figure E shows the dynamic changes of MDA-MB-231 cells in zebrafish after MEN 10207 treatment using confocal in vivo imaging (scale bar: 100 μm). Figure F shows the ratio of fluorescence grayscale value at 72 hpi to fluorescence grayscale value at 24 hpi (n = 4); hpi: hourspost. 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 is a schematic diagram of the mouse xenograft model; Figure I is a light image of the mouse brain after transparentization treatment, scale bar: 1000μm; Figure J shows the total volume of MDA-MB-231 cells in the mouse brain counted by ImageJ, n=3; Data are presented as mean ± SD, n=3, p<0.05 is considered statistically significant, *p<0.05, ***p<0.001, and statistical analysis was performed using a standard t-test. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[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 Aquatic Biological Resources Center;

[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 As shown. The molecular weight is 1109.24. It has been dissolved in DMSO to prepare a 13.52 mM stock solution, which is stored in a -80°C freezer protected from light.

[0038] Example 1: GEO database analysis to screen key genes for breast cancer brain metastases

[0039] This embodiment analyzes genes related to breast cancer brain metastasis based on the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ). Differential gene expression analysis was performed on datasets GSE100534 and GSE52604 to identify LRP8, a gene highly expressed in breast cancer brain metastasis samples. Subsequently, the prognostic effect of LRP8 in breast cancer patients was assessed using the Kaplan-Meier survival analysis tool (https: / / kmplot.com / analysis). The results showed a negative correlation between LRP8 expression and patient prognosis, suggesting that it may be a risk gene for breast cancer brain metastasis. Figure 2 Subsequently, molecular docking technology was used to screen for small molecule compounds with strong binding ability to LRP8 protein, and finally, compound MEN 10207 was identified, and its three-dimensional docking model was constructed. Figure 3 A).

[0040] Example 2: Effect of MEN 10207 on breast cancer cell proliferation

[0041] This embodiment evaluates the effect of MEN 10207 on breast cancer cell proliferation using a 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 96-well plates, with at least 3 replicates per group. After one day of pre-culture, MDA-MB-231 cells were treated with different concentrations of MEN 107. After culturing the cells for another 24 hours, the CCK-8 assay was performed. To prepare the CCK-8 working solution, 900 μL of serum-free DMEM medium was added to every 100 μL of CCK-8 stock solution, and the mixture was thoroughly mixed. The cell culture medium was aspirated from the 96-well plates, and 100 μL of the CCK-8 working solution was added. The plates were incubated at 37°C for 2 hours, and the absorbance (OD value) was measured at 450 nm.

[0043] Figure 3 Experiment B showed that MEN 10207 treatment significantly reduced the cell viability of MDA-MB-231 cells, and the half-maximal inhibitory concentration (IC50) was calculated to be 19.37 μM.

[0044] Example 3: Effect of MEN 10207 on the migration ability of breast cancer cells

[0045] This embodiment uses a scratch assay to detect the effect of MEN 10207 on the migration ability of breast cancer cells. The specific steps are as follows:

[0046] The day before, MDA-MB-231 cells were seeded into 24-well plates. The next day, the cells filled the entire plate. Using a 200 μl pipette tip, a straight line was drawn perpendicular to the cells on the plate. The cells were washed 2-3 times with PBS and photographed under a microscope. The image was recorded as 0h. After photographing, the cells were divided into two groups: a control group and a MEN 10207 treatment group (9 μM). The cells were then returned to the cell culture incubator for further culture. After 24 hours of cell culture, the images were taken again, and the ratio of the wound area at 24 hours to the wound area at 0 hours was calculated. This ratio represents the healing rate; a lower healing rate indicates stronger cell migration ability.

[0047] Experimental results showed that treatment with MEN 10207 significantly reduced the cell migration ability of MDA-MB-231 cells. Figure 3 C, E).

[0048] Example 4: Effect of MEN 10207 on the invasive ability of breast cancer cells

[0049] This embodiment uses the Transwell assay to evaluate the effect of MEN 10207 on the migration and invasion ability of breast cancer cells. The specific steps are as follows:

[0050] Add 600 μL of 10% FBS medium to each well of a 24-well plate and place the Transwell chambers in the 24-well plates. Then, digest MDA-MB-231 cells and divide them into groups: the control group was resuspended in serum-free medium, while the MEN 10207 treatment group (9 μM) was resuspended in serum-free medium containing MEN 10207. For migration assays, seed the cells at 2 × 10⁶ cells per well. 4 10 cells were placed in the upper chamber of a Transwell chamber, and for invasion experiments, 4 × 10⁶ cells were seeded. 4 Cells were placed in the upper chamber of a Transwell cell culture chamber and cultured in a cell culture incubator for 16 hours. After 16 hours, the cell culture medium was discarded, the chamber was washed twice with sterile PBS, and the unmigrated cells on the upper layer were gently wiped with a cotton swab. The cells were then fixed with methanol for 10 minutes, stained with 0.1% crystal violet for 30 minutes, and finally the crystal violet was recovered. The chamber was then dried and ready for photography.

[0051] Experimental results showed that treatment with MEN 10207 significantly reduced the migration and invasion abilities of MDA-MB-231 cells. Figure 3 D, F, G).

[0052] Example 5: Safety and toxicity assessment of MEN 10207

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

[0054] MEN 10207 was diluted to different concentrations using embryonic water from cultured zebrafish embryos. Embryos 2 days after fertilization were then cultured in embryonic water containing MEN 10207. The number of embryos cultured at each concentration was greater than 10. The survival rate of zebrafish embryos at different concentrations was observed. Fresh embryonic water was changed daily, and the observation continued for 4 days. MEN 10207 was diluted to different concentrations using physiological saline. Each mouse was intravenously injected with different concentrations of MEN 10207 once a day for 5 consecutive days. The survival rate of the mice was observed daily. Three mice were injected at each concentration.

[0055] Experimental results showed that 10 μM MEN 10207 was a safe dose for zebrafish, and injection doses of 1.9 mg / kg and 3.8 mg / kg were well tolerated in mice. Figure 4 (A~C).

[0056] Example 6: In vivo evaluation of the anti-breast cancer brain metastasis activity of MEN 10207 based on a zebrafish model.

[0057] This study established a breast cancer brain metastasis model by transplanting MDA-MB-231 cells into Tg(kdrl:mCherry) zebrafish two days after fertilization. Due to the visualization advantages of zebrafish embryos, this model allows for real-time observation of the dynamic migration changes of MDA-MB-231 cells along blood vessels. After model establishment, the cells were randomly assigned to either a control group or a MEN 10207 treatment group. The therapeutic effect of MEN 10207 was evaluated using confocal in vivo imaging.

[0058] Experimental results showed that after treatment with MEN 10207, the migration ability and cell viability of MDA-MB-231 cells in zebrafish were significantly reduced. Figure 4 D~G).

[0059] Example 7: Validation of the in vivo anti-breast cancer brain metastasis activity of MEN 10207 based on a mouse model

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

[0061] The experimental steps are as follows:

[0062] A mammalian model of breast cancer brain metastasis was established by intracardiac injection of MDA-MB-231 cells into mice. After cell injection, mice were randomly divided into a sham-operated group, a control group, and a MEN 10207 treatment group. MEN 10207 was administered intravenously starting on day 4 post-transplantation, once daily for 5 consecutive days. On day 14 post-transplantation, the mouse brains were dissected and transparentized. Subsequently, light-film imaging was performed to visualize the three-dimensional distribution of MDA-MB-231 cells in the mouse brain.

[0063] Experimental results showed that after treatment with MEN 10207, the distribution and volume of MDA-MB-231 cells in the mouse brain were significantly reduced, indicating that MEN 10207 can inhibit the proliferation and migration of MDA-MB-231 cells in the brain. Figure 4 (H~J). This experiment, combined with a mammalian breast cancer brain metastasis model, further confirms that MEN 10207 has the potential for in vivo therapeutic against breast cancer brain metastasis.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of small molecule compounds in the preparation of drugs for treating breast cancer; The small molecule compound is a compound of formula (I) or a salt thereof; ； （I） The breast cancer mentioned is metastatic breast cancer; The transfer is a brain metastasis.

2. Use according to claim 1, characterized in that, The small molecule compound is the sole active ingredient of the drug for treating breast cancer.

3. Use according to claim 2, characterized in that, The breast cancer cells in question are the MDA-MB-231 breast cancer cell line.

4. Use according to claim 1, characterized in that, The patients receiving the drug treatment are either human or non-human primates.

5. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

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