Compound with activity of resisting high-expression AGTRAP breast cancer and application thereof

Abstract

The invention provides a compound with high-expression AGTRAP breast cancer resisting activity and application thereof, and belongs to the technical field of biological medicine. The structural formula of the compound Necrocide 1 is shown as a formula (I), and a Pull-down experiment shows that the compound Necrocide 1 can be specifically combined with AGTRAP protein; through cell proliferation and protein expression quantity detection experiments, the compound Necrocide 1 shows that the compound Necrocide 1 has high inhibitory activity on proliferation of breast cancer cells with high expression of AGTRAP and has no toxic or side effect on cells with low expression of AGTRAP, so that the compound Necrocide 1 can be applied to preparation of a novel medicine for resisting breast cancer with high expression of AGTRAP, and the compound has a good development prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a compound having anti-high-expression AGTRAP breast cancer activity and its application. Background Art

[0002] Cancer has become a major threat to human health globally. The occurrence and development of cancer are affected by multiple factors, including genetics, environment, and lifestyle, etc. In recent years, researchers have gradually realized that the abnormal expression of proteins plays a crucial role in the occurrence and progression of cancer.

[0003] AGTRAP (angiotensin II receptor-associated protein), as a key regulatory protein, is a protein that interacts with the angiotensin II type 2 receptor (AT2R), and is mainly involved in regulating various physiological functions of the angiotensin system in the body, such as blood pressure control, cell proliferation, anti-inflammation, anti-fibrosis, etc. The abnormal expression of AGTRAP protein has been proven to be related to the occurrence of various diseases. Especially during tumorigenesis, its high expression is closely related to characteristics such as the proliferation, migration, and invasion of cancer cells (Zhou Wenjianlong, et al. Mechanism of angiotensin II receptor antagonist regulating the tumor microenvironment of breast cancer brain metastasis to inhibit tumor proliferation [J]. Chinese Journal of Neurosurgery, 2023, 39(8): 819-825.; Qiu Sijie. Research on the possible mechanism of angiotensin II regulating the progression of breast cancer [D]. Jiangsu University, 2020.).

[0004] Breast cancer is one of the most common malignant tumors in women globally. Breast cancer includes Luminal A type, Luminal B type, HER-2 positive type, and triple-negative breast cancer according to the classification (Dai, X., et al . Breast Cancer Cell Line Classification and Its Relevance with Breast Tumor Subtyping. Journal of Cancer [J], 2017, 8(16): 3131-3141.). Some studies have shown that AGTRAP protein is a driver gene of breast cancer. Its abnormal high expression in breast cancer cells promotes the aggressiveness of breast cancer and is positively correlated with poor prognosis (Wang, D., et al . USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT / mTOR Signaling. International Journal ofBiological Sciences [J], 2022, 18(6), 2452–2471.). Although there are currently targeted therapeutic drugs for breast cancer on the market, the clinical treatment effect for triple-negative breast cancer is very limited (Zhao Shinan, et al. Current Status and Progress in the Treatment of Triple-Negative Breast Cancer [J]. Advances in Clinical Medicine, 2024, 14(4): 1797-1800.). Therefore, in view of the key role of AGTRAP in breast cancer, developing new therapeutic drugs for triple-negative breast cancer with high expression of AGTRAP has important clinical significance and broad application prospects.

[0005] According to the literature report, compound Necrocide 1, namely (3S)-3-cycloheptyl-3-(4-hydroxyphenyl)-6-methoxy-7-methyl-2,3-dihydro-1H-indol-2-one, showed certain proliferation inhibitory activity in the Luminal A type MCF-7 cell line (Christensen, M. K., et al . Synthesis and antitumor effect in vitro and in vivo of substituted 1,3-dihydroindole-2-ones. Journal of Medicinal Chemistry [J], 2010, 53(19): 7140-7145.), but its target is unknown. Further, the analysis of cell protein expression levels showed that the expression level of AGTRAP in the Luminal A type MCF-7 cell line was very low, only similar to that of normal mammary epithelial cells MCF-10A (Wang, D., et al . USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT / mTOR Signaling. International Journal of Biological Sciences [J], 2022, 18(6), 2452-2471.). There is currently no study showing that this compound has an inhibitory effect on triple-negative breast cancer with high expression of AGTRAP. Summary of the Invention

[0006] The object of the present invention is to provide a compound that has an inhibitory effect on the proliferation of breast cancer cells with high expression of AGTRAP, and apply it to the development of new anticancer drugs for treating breast cancer with high expression of AGTRAP.

[0007] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides the use of compound Necrocide 1 in the preparation of a medicament for treating breast cancer with high expression of AGTRAP. The structural formula of the compound Necrocide 1 is shown in formula (I), and its Chinese name is (3S)-3-cycloheptyl-3-(4-hydroxyphenyl)-6-methoxy-7-methyl-2,3-dihydro-1H-indol-2-one, with the CAS number 1247028-61-0. (I).

[0008] The research of the present invention shows that the action target of compound Necrocide 1 is the AGTRAP protein in breast cancer cells. There is a strong negative correlation between the expression level of the AGTRAP protein in breast cancer cell lines and the logarithm of the IC 50 value of its anti-proliferative activity, and the Pearson correlation coefficient r is -0.98. The Pull-down experiment shows that compound Necrocide 1 can specifically bind to the AGTRAP protein. Therefore, compound Necrocide 1 can be applied to the development of medicaments for anti-breast cancer with high expression of AGTRAP.

[0009] Furthermore, the expression level of the AGTRAP protein in breast cancer cells with high expression of AGTRAP is significantly higher than that in the human mammary epithelial cell line MCF-10A. Research shows that compared with the human mammary epithelial cell line MCF-10A, the AGTRAP protein is significantly highly expressed in the human triple-negative breast cancer cell lines HCC-70, SUM-149PT, and the human non-triple-negative breast cancer cell lines MDA-MB-453, T-47D, and compound Necrocide 1 shows significant inhibitory activity on the proliferation of these breast cancer cells.

[0010] Furthermore, the breast cancer with high expression of AGTRAP is triple-negative breast cancer with high expression of AGTRAP.

[0011] The present invention provides a pharmaceutical composition for treating breast cancer with high expression of AGTRAP, and the pharmaceutical composition includes an effective dose of compound Necrocide 1 with the structural formula shown in formula (I).

[0012] In the pharmaceutical combination provided by the present invention, compound Necrocide 1 can be used as the only active ingredient that exerts the inhibitory effect on the proliferation of breast cancer cells, or can be compounded with other active ingredients with the same effect. The effective dose refers to the amount of the compound that is sufficient to take effect in the treatment of this disease when administered to a subject for treating the disease. The effective dose can vary with the severity of the disease, as well as the physical condition, age, weight, gender, etc. of the subject to be treated.

[0013] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver the active substance in an effective dose of the present invention, does not interfere with the biological activity of the active substance, and is non-toxic and has no side effects on the host or subject.

[0014] The present invention uses compound Necrocide 1 as the main active ingredient, adds a pharmaceutically acceptable carrier, and is prepared into a preparation according to the preparation method recorded in pharmacy.

[0015] Furthermore, the pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.

[0016] Furthermore, the preparation form of the pharmaceutical composition is an injection preparation or an oral preparation.

[0017] The present invention also provides an application of the pharmaceutical composition in the preparation of a drug for treating triple-negative breast cancer with high expression of AGTRAP.

[0018] Beneficial effects of the present invention: The present invention for the first time reveals that compound Necrocide 1 has high inhibitory activity on the proliferation of breast cancer cell lines with high expression of AGTRAP. In particular, for triple-negative breast cancer cell lines SUM-149PT and HCC-70, as well as non-triple-negative breast cancer cell lines MDA-MB-453 and T-47D, it shows significant inhibitory activity; and at the IC 50 concentration that has a killing effect on breast cancer cells with high expression of AGTRAP, it has no toxic and side effects on cells with low expression of AGTRAP. Therefore, this compound can be applied to the preparation of a new anti-cancer drug for treating breast cancer with high expression of AGTRAP, which has important clinical significance. Description of the Drawings

[0019] Figure 1 It is the LC-MS data of compound Necrocide 1.

[0020] Figure 2 It is of compound Necrocide 1 1 1H-NMR data.

[0021] Figure 3 It is the result of the pull-down experiment and immunoblot analysis of AGTRAP protein.

[0022] Figure 4 It is the experimental result of detecting the expression level of AGTRAP protein in different breast cancer cell lines.

[0023] Figure 5Cell proliferation assay results of T-47D, MDA-MB-453, SUM-149PT, and HCC-70 breast cancer cell lines with high expression of AGTRAP treated with different concentrations of Necrocide 1 compound.

[0024] Figure 6 Cell proliferation assay results of AU-565, BT-474, and MDA-MB-231 breast cancer cell lines with low expression of AGTRAP treated with different concentrations of Necrocide 1 compound. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0026] The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0027] The experimental materials used in the following embodiments: 1. Cell lines The standard cell lines used in this experiment are: human mammary epithelial cell line MCF-10A, human non-triple-negative breast cancer cell line BT-474, human non-triple-negative breast cancer cell line AU-565, human non-triple-negative breast cancer cell line MDA-MB-453, human non-triple-negative breast cancer cell line T-47D, human triple-negative breast cancer cell line MDA-MB-231, human triple-negative breast cancer cell line HCC-70, and human triple-negative breast cancer cell line SUM-149PT, human cervical cancer cell line HeLa, all purchased from Wuhan Procell Life Science Co., Ltd.

[0028] 2. Compounds (3S)-3-Cycloheptyl-3-(4-hydroxyphenyl)-6-methoxy-7-methyl-2,3-dihydro-1H-indol-2-one (Necrocide 1): CAS No. 1247028-61-0, purchased from MedChemExpress. The LC-MS data and 1 1H-NMR data of compound Necrocide 1 are shown in Figure 1 and Figure 2 .

[0029] The probe modified with Necrocide 1 was synthesized by commissioning a biological company. The probe contains a covalent modification group (photoactivatable diazirine group) and a detection group (bioorthogonal alkyne handle).

[0030] 3. Antibody The primary antibody against AGTRAP (product number: A12178) was purchased from Abbkine Scientific Co., Ltd.

[0031] Example 1: Pull-down experiment and immunoblot analysis of AGTRAP protein Experimental principle: First, a pull-down experiment based on a live cell culture system was carried out. That is, the Necrocide 1 compound and the probe modified with Necrocide 1 were sequentially incubated with the cells. The probe, as a competitor of the Necrocide 1 compound, covalently binds to the target protein. Magnetic beads were used to capture and separate the target protein AGTRAP. Finally, immunoblot analysis was used to prove the binding between the compound Necrocide 1 and AGTRAP. This experiment was repeated twice in parallel.

[0032] Experimental steps: 1) After washing the cultured Hela cells twice with sterile PBS, the medium was replaced with serum-free DMEM medium. 1 mL of Necrocide 1 gradient concentration solutions (0 μM, 30 μM, 100 μM) were added to 3 identical culture systems respectively and incubated with sufficient mixing for 1 hour. Then 1 mL of Necrocide 1 probe solution (10 μM) was added to each system and incubated with sufficient mixing for 1 hour. Subsequently, the cells were pre-cooled to 4 °C and irradiated under 365 nm light for 10 minutes.

[0033] 2) The cultured cells were washed twice with pre-cooled PBS, scraped and collected on ice, and immediately lysed with lysis buffer (1×PBS, 1% Triton X-100 (v / v), 0.5% SDS (w / v), 1× protease inhibitor mixture). After lysis, the BCA protein quantification kit (catalog number C503021, Sangon Biotech) was used to measure the protein concentration. The protein solution concentration was diluted to 2 mg / mL, and 1 mg of protein was aliquoted into the lysis buffer for further processing.

[0034] 3) In each 500 µL sample, premix and add according to the following formula: 10 µL of THPTA (5 mM, dissolved in DMSO, final concentration 100 µM), 2.5 µL of CuSO 4(200 mM, dissolved in distilled water, final concentration 1 mM), 2.5 µL sodium ascorbate (400 mM, dissolved in distilled water, final concentration 2 mM), and 1 µL TAMRA-Azide (50 mM, dissolved in DMSO, final concentration 100 µM). After mixing the samples, incubate at room temperature for 3 hours.

[0035] 4) Boil the magnetic beads in 5×Laemmli sample buffer for 10 minutes to elute the enriched proteins. The eluted proteins were then separated using a 10% SDS-PAGE gel and transferred onto a PVDF membrane.

[0036] 5) Block the PVDF membrane with TBST buffer containing 5% non-fat milk powder, then add the primary antibody and incubate overnight at 4°C. Wash the membrane three times with TBST for 5 minutes each time. Then add the secondary antibody and incubate for 1 hour at room temperature. Before imaging, gently shake the membrane with 1 mL Beyo ECL Plus solution for 2 minutes at room temperature. Immunoblotting was imaged using a Bio-Rad ChemiDoc Imaging System and quantitatively analyzed using ImageJ 1.54 software.

[0037] The experimental results are as Figure 3 shown. In the Hela cell culture system, as the concentration of the Necrocide 1 compound increased, the labeling efficiency of the chemical probe for the AGTRAP protein gradually decreased, and the binding rate decreased significantly. This indicates that the compound Necrocide 1 can specifically bind to the AGTRAP protein.

[0038] Example 2: Detection of the expression level of AGTRAP protein in cell lines Experimental principle: The detection of the expression level of AGTRAP protein in cell lines uses Western Blot experiment. This experiment is based on the specific binding of antigen and antibody, and mainly realizes the quantitative analysis of AGTRAP protein by separating proteins, transferring them to a support membrane and detecting the AGTRAP protein signal. This experiment was repeated in parallel three times.

[0039] Experimental steps: 1) Wash the cultured cells twice with sterile PBS, then lyse them on ice for 30 minutes with RIPA lysis buffer containing 1% PMSF, with vortex mixing and sonication every 10 minutes.

[0040] 2) Centrifuge the lysed cells at 12000 rpm for 30 minutes (4°C) and collect the supernatant. Use a BCA protein quantification kit to measure the protein concentration of the supernatant.

[0041] 3) Add loading buffer to the supernatant and heat it in a 95 °C water bath for 10 minutes to denature the proteins.

[0042] 4) Electrophoretically separate the protein samples on a 12% SDS-PAGE gel. Transfer the separated proteins to a PVDF membrane. Block the membrane with protein-free rapid blocking buffer for 30 - 50 minutes.

[0043] 5) Incubate the PVDF membrane with the primary antibody against AGTRAP overnight at 4 °C. Subsequently, incubate with the secondary antibody for 2 hours at room temperature.

[0044] 6) Visualize the immunoblot images using an Odyssey infrared imaging system and perform quantitative analysis with ImageJ 1.54 software.

[0045] Experimental results: The experimental results are as Figure 4 shown. Compared with the human mammary epithelial cell line MCF-10A, the AGTRAP protein was significantly highly expressed in the human triple-negative breast cancer cell lines HCC-70, SUM-149PT, and the human non-triple-negative breast cancer cell lines MDA-MB-453, T-47D, while the expression levels of the AGTRAP protein were lower in the human triple-negative breast cancer cell line MDA-MB-231 and the human non-triple-negative breast cancer cell lines BT-474, AU-565.

[0046] Example 3: Cell proliferation assay Experimental principle: The CCK-8 assay is based on the reduction reaction of the water-soluble tetrazolium salt (WST-8) in living cells. The optical density OD value at 450 nm is measured using a microplate reader to reflect the number of living cells, thereby evaluating the ability of compounds to inhibit the proliferation of tumor cells.

[0047] Experimental procedure: 1) Collect cells in the logarithmic phase, adjust the cell suspension concentration, add 100 μL to each well, and plate to make the density of the cells to be tested 10,000 cells / well (fill the edge wells with sterile PBS).

[0048] 2) Place the 96-well plate in a 5% CO 2 , 37 °C incubator and incubate for 24 hours, then add drugs with a concentration gradient (in all cell culture systems, the DMSO concentration is less than 0.1%). Set 8 gradients for the drugs, 5 μL per well, and set 3 parallel wells. Incubate in a 5% CO 2 , 37 °C incubator for 48 hours.

[0049] 3) Add 20 μL of CCK-8 solution to each well and continue to culture for 1 - 3 hours.

[0050] 4) Absorbance values were recorded at 450 nm using a Varioskan LUX multimode microplate reader.

[0051] 5) The IC 50 value of Necrocide 1 was calculated using GraphPad Prism 8.0 software.

[0052] Experimental results: The experimental results are shown in Figure 5 and Figure 6 . The experimental results showed that compound Necrocide 1 exhibited significant inhibitory activity against the proliferation of human triple-negative breast cancer cell lines HCC-70, SUM-149PT with high AGTRAP expression, and human non-triple-negative breast cancer cell lines MDA-MB-453, T-47D. The IC 50 values were 17.13, 9.94, 9.53, and 4.91 nM, respectively ( Figure 5 ).

[0053] Compound Necrocide 1 showed lower inhibitory activity against the proliferation of human triple-negative breast cancer cell line MDA-MB-231 with low AGTRAP expression and human non-triple-negative breast cancer cell lines BT-474, AU-565. The IC 50 values were 32.10, 64.80, and 17.03 μM, respectively ( Figure 6 ).

[0054] In addition, through the correlation analysis of the AGTRAP expression level in the cell lines and the anti-cell proliferation activity of Necrocide 1, as shown in Table 1, a strong negative correlation was found between the AGTRAP protein expression level in the cell lines and the logarithm of the IC 50 value of the compound's anti-proliferation activity. The Pearson correlation coefficient r was -0.98.

[0055] Table 1. AGTRAP protein expression level in cell lines and the corresponding anti-proliferation activity of compound Necrocide 1 Cell line AGTRAP expression level compared with MCF-10A <![CDATA[Necrocide 1 Anti - cell Proliferation Activity (IC 50 , nM)]]> <![CDATA[Log 10 (IC 50 )]]> MCF-10A 1.0 ± 0.098 - - BT-474 0.265 ± 0.048 64800 4.812 AU-565 1.208 ± 0.204 17030 4.231 MDA-MB-453 2.961 ± 0.293 9.532 0.979 T-47D 3.376 ± 0.242 4.911 0.691 MDA-MB-231 0.398 ± 0.135 32100 4.507 HCC-70 2.954 ± 0.047 17.13 1.234 SUM-149PT 3.667 ± 0.185 9.937 0.997 These results indicate that compound Necrocide 1 exhibits significant inhibitory effects on the proliferation of triple-negative breast cancer cells with high AGTRAP expression and has good prospects for the development of anti-high-AGTRAP triple-negative breast cancer drugs. From the above results, it can be seen that compound Necrocide 1 has no effect on cells with low AGTRAP expression at the IC 50 concentration that kills tumor cells with high AGTRAP expression. This compound is expected to become a candidate molecule for a new type of high-AGTRAP triple-negative breast cancer treatment drug.

Claims

1. Use of the compound Necrocide 1 in the preparation of a drug for treating breast cancer with high AGTRAP expression, characterized in that: The structural formula of the compound Necrocide 1 is shown in formula (I), and its target is the AGTRAP protein in breast cancer cells; (Ⅰ)。 2. The use according to claim 1, characterized in that The expression level of AGTRAP protein in the breast cancer cells of the AGTRAP-highly expressing breast cancer is significantly higher than the expression level of AGTRAP protein in the human breast epithelial cell line MCF-10A.

3. The use according to claim 2, characterized in that The breast cancer with high AGTRAP expression is triple-negative breast cancer with high AGTRAP expression.

4. A pharmaceutical composition for treating breast cancer with high AGTRAP expression, characterized in that: The pharmaceutical composition comprises an effective dose of Necrocide 1, a compound having a structural formula as shown in formula (I).

5. The pharmaceutical composition according to claim 4, characterized in that Also included are pharmaceutically acceptable carriers.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of an injection preparation or an oral preparation.

8. Use of the pharmaceutical composition according to claim 4 in the preparation of a drug for treating triple-negative breast cancer with high AGTRAP expression.

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