Application of D724-0491 in preparation of medicine for treating breast cancer

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to application of D724-0491 in preparation of a product for preventing and treating breast cancer. The D724-0491 inhibits the combination of HBXIP and KEAP1, reduces the nuclear entry level of NRF2, further induces oxidative stress of tumor cells and promotes death of cancer cells, is a novel targeted therapeutic drug for breast cancer, and improves the prognosis survival rate of breast cancer patients.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to the application of D724-0491 in preparing products for preventing and treating breast cancer. Background Art

[0002] Tumor is a malignant disease that threatens human life and health. Its various types of cancer have significant distribution differences in various countries and regions around the world. As for breast cancer, its incidence rate has shown an increasing trend year by year worldwide. In addition, researchers' predictions on the global incidence of breast cancer show that in 2040, the number of new breast cancer patients will reach 3 million per year, and the number of deaths will exceed 1 million per year. Unfortunately, the current clinical diagnosis and treatment level of breast cancer cannot fully meet the treatment needs of patients; in addition, the living standards of patients have also been significantly reduced due to traditional treatments such as surgery or chemotherapy. Therefore, in-depth research on the pathogenesis of breast cancer and exploration and development of targeted treatments for breast cancer are of utmost importance.

[0003] Hepatitis B virus X protein binding protein (HBXIP) is a membrane protein located on the surface of human lysosomes. It forms a pentamer regulatory complex with P18 (Lamtor1), P14 (Lamtor2), MP1 (Lamtor3) and C7orf59 (Lamtor4) in the Lamtor family, the latter of which is mainly responsible for activating rapamycin complex 1 (mTORC1); among them, HBXIP-C7orf59 is the core component of the pentamer. In addition, HBXIP has also been found to play a role in insulin regulation and DNA damage repair. In the study of malignant diseases such as tumors, it was found that HBXIP is highly expressed in a variety of malignant tumors including cervical cancer, breast cancer, non-small cell lung cancer, etc., and it usually indicates the stage, metastasis and poor prognosis of the patient's tumor. At present, the research on the carcinogenic mechanism related to HBXIP is constantly deepening. For example, researchers have shown that HBXIP can act as a co-activator of multiple oncogenes to promote the expression of related oncoproteins; in addition, HBXIP has also been shown to be able to regulate the expression of related oncoproteins at multiple levels, such as transcription, post-transcription, and translation, to promote the occurrence and development of tumors. In addition, the latest research has found that HBXIP may be a potential oxidative stress regulator protein in breast cancer. In short, HBXIP has been shown to be able to regulate the malignant progression of tumors through multiple mechanisms, and it is particularly important to find its related targeted drugs.

[0004] The level of reactive oxygen species (ROS) in tumor cells is much higher than that in normal cells, which can promote the malignant progression of tumors by regulating signal pathways, proteins, etc. In order to adapt to high levels of ROS, tumor cells will improve their own antioxidant capacity through various mechanisms. Among them, researchers have found that interfering with the protein-protein interaction between Kelch-like ECH-associated protein (KEAP1) and nuclear transcription factor erythroid 2-related factor 2 (NRF2) is one of the main mechanisms for tumor cells to enhance their antioxidant capacity. Usually, NRF2 is mainly located in the cytoplasm at physiological levels, and its main destination is ubiquitination and degradation by binding to KEAP1; only a small amount of NRF2 enters the cell nucleus and activates related antioxidant elements (ARE) to maintain ROS at normal physiological levels; under the stimulation of pathological conditions such as tumors, NRF2 no longer binds to KEAP1, but enters the cell nucleus in large quantities and acts on downstream ARE, thereby improving the antioxidant capacity of tumor cells. Therefore, in previous studies related to tumors, it is a logical idea to find a small molecule compound that targets KEAP1-NRF2 proteins and protein interactions. The latest research found that there is a group of highly conserved GLNLG motifs on HBXIP, which enables it to actively bind to the KEAP1 protein, and the affinity of HBXIP-KEAP1 binding is higher than that of NRF2; therefore, HBXIP, which is highly expressed in breast cancer, can bind to KEAP1 with its higher affinity, indirectly activate NRF2 to enter the cell nucleus and act on ARE, thereby reducing the accumulation of ROS in breast cancer cells, and ultimately promoting the malignant progression of breast cancer. Interestingly, further studies have found that NRF2 entering the cell nucleus can also act on the promoter sequence of HBXIP, thereby forming a positive feedback and further promoting the occurrence and development of breast cancer. Therefore, finding targeted inhibitors that act on the binding sites of HBXIP and KEAP1, reducing the level of NRF2 entering the cell nucleus and inducing oxidative stress in breast cancer cells has become a potential treatment for breast cancer. Summary of the invention

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

[0006] The present invention provides the use of D724-0491 in preparing a drug for treating breast cancer, wherein the structural formula of D724-0491 is:

[0007]

[0008] In the above technical solution, further, the active ingredient of the breast cancer drug is D724-0491.

[0009] In the above technical solution, further, the only active ingredient of the breast cancer drug is D724-0491.

[0010] In the above technical solution, further, the effective concentration of D724-0491 is 7.5-60 μmol / L, preferably 7.5-30 μmol / L.

[0011] In the above technical solution, further, the breast cancer includes breast cancer cells MCF-M7 and breast cancer cells MDA-MB-231.

[0012] In the above technical solution, further, the drug is an oral preparation or an injectable preparation.

[0013] In the above technical solution, further, the drug inhibits the binding of HBXIP and KEAP1, and reduces the level of NRF2 entering the nucleus.

[0014] In the above technical solution, further, the drug inhibits the proliferation of breast cancer cells.

[0015] In the above technical solution, further, the drug inhibits the invasion of breast cancer cells.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes for the first time the use of compound D724-0491 in the preparation of drugs for treating breast cancer. D724-0491 inhibits the binding of HBXIP and KEAP1, reduces the level of NRF2 entering the nucleus, thereby inducing oxidative stress in tumor cells and promoting cancer cell death. It is a new targeted therapeutic drug for breast cancer and improves the prognosis and survival rate of breast cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Molecular docking schematic diagram showing the action site of D724-0491;

[0019] Figure 2 Cytotoxicity test results; A. The killing effect of D724-0491 on MDA-MB-231 and MCF-7; B. D724-0491 has no toxicity to normal breast epithelial cells and human adrenal epithelial cells; (*P<0.05; **P<0.01; ***P<0.001);

[0020] Figure 3D724-0491 inhibits the proliferation and clone formation of breast cancer cells; A. D724-0491 inhibits the proliferation rate of MDA-MB-231 and MCF-7 breast cancer cells; B. D724-0491 inhibits the size and number of clones formed by MDA-MB-231 and MCF-7 breast cancer cells; C. D724-0491 inhibits the percentage of clones formed by MDA-MB-231 and MCF-7 breast cancer cells (*P<0.05; **P<0.01; ***P<0.001);

[0021] Figure 4 D724-0491 inhibits the invasion and metastasis of breast cancer cells; A. D724-0491 inhibits the migration of MDA-MB-231 and MCF-7 breast cancer cells; B. D724-0491 weakens the invasion of MDA-MB-231 and MCF-7 breast cancer cells; C. The migration rate of MDA-MB-231 and MCF-7 breast cancer cells after the action of D724-0491; D. The number of MDA-MB-231 and MCF-7 breast cancer cells after invasion after the action of D724-0491 (*P<0.05; **P<0.01; ***P<0.001);

[0022] Figure 5 D724-0491 can interfere with the HBXIP-Keap1-Nrf2 signaling pathway and affect the nuclear localization of NRF2; A. After co-precipitation with KEAP, the binding of NRF2 to KEAP1 increased and the binding of HBXIP to KEAP1 decreased in MDA-MB-231 and MCF-7 breast cancer cells treated with D724-0491; B. After the action of D724-0491, the localization of NRF2 in the cell nucleus decreased (*P<0.05; **P<0.01; ***P<0.001);

[0023] Figure 6 D724-0491 affects the Keap-NRF2-related signaling pathway and the expression of HBXIP; A. D724-0491 inhibits the expression of NRF2 and NQO-1 in MDA-MB-231 and MCF-7 breast cancer cells; B. D724-0491 inhibits the expression of HBXIP in MDA-MB-231 and MCF-7 breast cancer cells; C. D724-0491 inhibits the expression rate of NRF2 and NQO-1 in MDA-MB-231 and MCF-7 breast cancer cells; D. D724-0491 inhibits the expression rate of HBXIP in MDA-MB-231 and MCF-7 breast cancer cells (*P<0.05; **P<0.01; ***P<0.001);

[0024] Figure 7 D724-0491 promotes the accumulation of ROS levels in breast cancer cells; A. After the action of D724-0491, the green fluorescence representing ROS increased significantly; B. After the action of D724-0491, the ROS level increased significantly (*P<0.05; **P<0.01; ***P<0.001);

[0025] Figure 8 D724-0491 inhibits breast cancer cell proliferation in vivo; A. Photos of nude mouse corpses treated with Con and D724-0491 groups, the tumor volume in Con group was significantly larger than that in D724-0491 group; B. Comparison of nude mouse tumor tissues in vitro in Con and D724-0491 groups, the tumor volume in Con group was significantly larger than that in D724-0491 group; C. Results of weight changes of nude mice in Con and D724-0491 groups during the administration period; D. Results of volume changes of nude mouse tumor tissues in Con and D724-0491 groups during the administration period; E. Histochemical results, showing the changes in the expression of Ki67 and HBXIP in Con and D724-0491 groups, the expression levels of Ki67 and HBXIP in Con group were significantly higher (*P<0.05; **P<0.01; ***P<0.001). DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0027] Example 1

[0028] Through molecular docking technology, we searched for compounds that interfere with the interaction between Keap1 and NRF2 proteins. The results showed that D724-0491 mainly acts on the hydrophobic region of HBXIP, forming strong hydrogen bond interactions with Gly28 and Asn30; at the same time, the long-chain hydroxyl group of D724-0491 also forms hydrogen bonds with Asp39 and Gly43, respectively, suggesting that hydrogen bonding plays an important role in maintaining the binding of D724-0491 to HBXIP. We found a targeted inhibitor D724-0491 (C 17 H 19 CIN 4 O 2 ),

[0029] 2-(2-{[3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]amino}ethoxy)ethan-1-ol( Figure 1), inhibiting the binding of HBXIP to KEAP1, reducing the level of NRF2 entering the nucleus and inducing oxidative stress in tumor cells. The structural formula of D724-0491 is:

[0030]

[0031] The D724-0491 used in the following examples was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0032] The safety of D724-0491 has been verified.

[0033] Perform MTT cytotoxicity assay:

[0034] 1. Plate laying: Breast cancer cell lines MCF-7 and MDA-MB-231 were inoculated in 24-well plates, with 1×10 5 After the cells are mixed by the cross method, they are observed under a microscope and placed in an incubator for culture. When the cell density reaches about 70%-80%, the drug is administered.

[0035] 2. Dosing: Calculate the required dosage in advance according to the preset dosage concentration gradient. Prepare an equal number of 5mL EP tubes according to the dosage concentration gradient, add an appropriate amount of DMEM complete medium to the tube, add the corresponding volume of drug according to the experimental plan, mix well and set aside. Take out the 24-well plate, use a pipette to suck out the original culture medium, and add 100μL of DMEM complete medium containing the drug to the well. After completion, mix well in the clean bench using the cross method, observe under a microscope and place in an incubator for overnight culture.

[0036] 3. Treatment: The next day, take out the MTT reagent in advance to dissolve. Take out the 24-well plate and add the MTT reagent directly to the well, 20μL per well. After 4 hours, aspirate the MTT and culture medium mixture in the well. Add DMSO, 100μL per well, cut a suitable size of tin foil to wrap the 24-well plate, and then take it to the shaker and incubate at room temperature for 5 minutes. After incubation, use a spectrophotometer at 570nm to detect the absorbance and analyze the data.

[0037] The results showed that at the same treatment concentration, D724-0491 could effectively kill breast cancer cells MCF-7 and MDA-MB-231 ( Figure 2 A), D724-0491 had no cytotoxicity to normal breast epithelial cells 10A and human adrenal epithelial cells 293T ( Figure 2 B). Therefore, we chose D724-0491 as a potential drug for subsequent studies.

[0038] Example 2D724-0491 can inhibit the proliferation and cloning ability of breast cancer cells

[0039] The effect of the inhibitor on the proliferation of breast cancer cells was investigated. After treating breast cancer cells MCF-7 and MDA-MB-231 with D724-0491 at a concentration gradient of 7.5, 15, and 30 μmol / L, the cells were counted for four consecutive days. The experimental verification was as follows:

[0040] 1. Plate laying: Select a 24-well plate for the experiment, with 1×10 5 Each treatment group (different drug concentration treatment group) was made into 3 replicate wells, and after observation under a microscope, the cells were placed in an incubator and cultured overnight.

[0041] 2. Treatment: On the second day, after the cell density reaches the requirements for different treatment purposes, the cells are treated (administered) in a 24-well plate and cultured in an incubator after the treatment is completed.

[0042] 3. Counting: Digest the cells in the corresponding wells every day, resuspend them dozens of times until the cells are no longer stacked and arranged, use abalone counting plate to count the cells, and take an appropriate amount of each group of cell suspension and rinse the pool. Count under a microscope for a total of 4 days.

[0043] The results showed that compared with the control group (cell group without drug administration), the proliferation ability of breast cancer cells treated with inhibitor D724-0491 was significantly inhibited in a dose-dependent manner ( Figure 3 A).

[0044] In addition, cell clone formation experiments were performed:

[0045] 1. Plate laying: Select 6-well plates for the experiment, inoculate breast cancer cells MCF-7 and MDA-MB-231 at a density of 800 cells per well, mix well using the cross-tip method, observe under a microscope and place in an incubator for 24-48 hours.

[0046] 2. Treatment: After the culture time is up, the treatment group cells are given drugs. After the treatment is completed, they are placed in an incubator and cultured for 10-15 days. During this period, the number and size of breast cancer cell clones are observed daily.

[0047] 3. Take photos: When the number and size of breast cancer cell clones reach a certain level, use a pipette pump to remove the old liquid and add 1 mL of PBS along the wall of the culture dish to rinse. Choose the number of rinses according to the situation, usually twice, and add 1 mL of methanol along the wall of the culture dish to fix the cells, and time for 15 minutes. After fixation, discard the methanol, take out 0.1% crystal violet from the 4°C refrigerator, add an appropriate amount of crystal violet to the dish for dyeing, time for 30 minutes, and recover the crystal violet dye after completion. Turn on the faucet and adjust the water flow to a smaller level, place the gaps between the wells of the 6-well plate under the water flow, allow the water flow to gently buffer the remaining dye, and take photos after drying).

[0048] The results of the cell cloning experiment showed that the size and number of breast cancer cell clones also decreased with the increase of D724-0491 concentration ( Figure 3 B, C).

[0049] In summary, it was found that D724-0491 could inhibit the proliferation and clone formation ability of breast cancer cells.

[0050] Example 3D724-0491 can inhibit the invasion and metastasis of breast cancer cells

[0051] The effect of the inhibitor on the invasion and metastasis of breast cancer cells was investigated. The cell scratch experiment was performed as follows:

[0052] 1. Plate laying: First, draw three horizontal lines on the back of the 6-well plate to divide the diameter of the 6-well plate into four equal parts. Then, inoculate breast cancer cells MDA-MB-231 and MCF-7 at appropriate densities, mix well using the cross method, and place in an incubator for culture.

[0053] 2. Treatment: When the cells reach a certain density, use a 10-μl pipette tip to draw a vertical line in the center of each well of the six-well plate, and then suck away the old culture medium. After washing once with PBS, add serum-free culture medium and administer the drug according to the purpose of the experiment. After completion, take a picture under a microscope, and record this time as 0h. Then, take out the six-well plate and take pictures after the culture time reaches 12, 24, and 48h.

[0054] The results showed that the migration ability of breast cancer cells treated with D724-0491 was significantly weakened ( Figure 4 A, C).

[0055] Transwell invasion chamber assay:

[0056] 1. Prepare matrix gel: Corning matrix gel is used to simulate the invasion environment of breast cancer cells. Take it out in advance and melt it overnight. The next day, take out the melted matrix gel and serum-free culture medium from the 4°C refrigerator. Dilute the matrix gel in a ratio of 8:1 serum-free culture medium: matrix gel in a clean bench. According to the purpose of treatment, use tweezers to clamp the appropriate number of chambers (3 parallel wells with different concentrations of the drug group) and place them in a 24-well plate. Then add 100 μL of matrix gel to the upper layer of the chamber and place it in the clean bench overnight to solidify.

[0057] 2. Plate: After digestion of cells, centrifuge and discard the supernatant, add serum-free culture medium to the EP tube to blow away the cell precipitate, and inoculate breast cancer cells MCF-7 and MDA-MB-231 in the chambers, with 1.5×10 5 -2.5×10 5Select a well at a suitable location in the 24-well plate and add 750 μL of DMEM complete medium to it, then transfer the chamber into it, and finally put the 24-well plate into the incubator.

[0058] 3. Treatment: Take out the 24-well plate, and add appropriate amounts of PBS, methanol, and crystal violet to the same number of blank wells according to the number of chambers. Then use tweezers to place the chamber in the well containing PBS and rinse it up and down several times. Then place it in the well containing methanol and fix it for 1 minute. After taking it out, place the chamber in the well containing crystal violet for staining for 30 minutes. After staining is completed, wash away the residual dye with PBS, observe whether there are cells that have not passed through the upper layer of the chamber, if so, gently wipe it with a cotton swab, and then place it under a microscope to take pictures.

[0059] The results also showed that D724-0491 could significantly inhibit the invasion of breast cancer cells in a concentration-dependent manner ( Figure 4 B, D).

[0060] In summary, the above data show that D724-0491 can effectively inhibit the proliferation, invasion and metastasis of breast cancer cells.

[0061] Example 4 D724-0491 can interfere with the HBXIP-Keap1-Nrf2 signaling pathway and affect the nuclear localization of NRF2

[0062] In order to explore whether the changes in the binding of HBXIP, KEAP1 and NRF2 in breast cancer cells after D724-0491 are as expected, Keap1 was first used to immunoprecipitate NRF2 and HBXIP:

[0063] 1. Bead pretreatment: After cutting the tip of the pipette obliquely, draw about 100 microliters of beads into an EP tube, add 400 microliters of ice PBS, blow several times, and centrifuge at 1000g for 3 minutes. After discarding the supernatant PBS, add 400 microliters of PBS again, and repeat the above operation 4 times. After the last wash, add 400 microliters of PBS and store in a 4℃ refrigerator for use. Mix well each time, and add 80 microliters of beads and PBS mixture for every 1ml of protein sample.

[0064] 2. Plating: Add 8 ml of DMEM complete medium and 500 μl of the cell suspension to be used to each large dish, mix well by the cross method, observe under a microscope and place in an incubator. When the cell density reaches about 70%-80%, administer the drug according to the predetermined concentration.

[0065] 3. Drug administration: Calculate the required drug volume according to the established drug concentration, administer the drug directly into the culture dish after protecting from light, shake well and continue culturing.

[0066] 4. Collect cells: Prepare new EP tubes in advance and mark them accordingly, and pipette the cell suspension into the corresponding EP tubes.

[0067] 5. Incubation: Take 40 μl of supernatant from each of the control group and the drug group, add 10 μl of 5× loading buffer, mix thoroughly and boil for 5 minutes to denature, and store in a -20°C refrigerator. Take out the HBXIP antibody from the -20°C refrigerator, calculate the volume of antibody required for the remaining supernatant according to the antibody instructions, add the antibody and incubate at 4°C with rotation for 4 hours. After completion, add 80 μl of the treated beads and PBS mixture and incubate overnight for more than 16 hours.

[0068] 6. Treatment: The next day, after incubation, centrifuge at 5000 rpm for 10 minutes in a low-temperature centrifuge. Discard the supernatant, add 1 ml PBS, wash at 4°C for 10 minutes, then centrifuge at 5000 rpm for 10 minutes in a low-temperature centrifuge, discard the supernatant and repeat twice. After completion, add 8 microliters of 5× loading buffer and boil to denature.

[0069] The results showed that compared with the control group (untreated group), after the action of 30μmol / L D724-0491, the binding of NRF2 to Keap1 increased significantly, while on the contrary, the binding of HBXIP to Keap1 decreased significantly ( Figure 5 A).

[0070] In addition, previous studies have shown that NRF2 dissociated from Keap1 can translocate from the cytoplasm to the nucleus. Therefore, the nuclear translocation of NRF2 after the action of the inhibitor was investigated by immunofluorescence technology, and the experimental verification was as follows:

[0071] 1. Slide: Place the slide in a six-well plate one day in advance and add an appropriate amount of alcohol to sterilize overnight in a clean bench. The next day, discard the alcohol and add PBS to rinse three times before use.

[0072] 2. Plate laying: 1×10 per hole 6 Calculate the required amount of cell suspension according to the cell density, use a pipette to absorb the resuspended cell suspension, and transfer it to the 6-well plate to be used. After mixing by cross-mixing, observe under a microscope and place in an incubator for 24 hours.

[0073] 3. Drug administration: The next day, calculate the required drug volume according to the target drug concentration. After administration, gently mix and place in the incubator for further culturing for 24 hours.

[0074] 4. Treatment: The next day, use a pipette to suck up the original culture medium. Add PBS along the wall of the culture dish and rinse for 5 minutes. Repeat this operation 3 times, and discard the PBS for the last time. Use an appropriate amount of 4% paraformaldehyde to fix the cells, time for 15 minutes, and rinse with PBS 3 times after completion. Add 2mL of 0.5% permeabilization solution (PBS + Triton-100 mixture) along the wall of the culture dish to permeabilize the cells for 10 minutes. After permeabilization, rinse with PBS for 5 minutes, and repeat this operation 3 times.

[0075] 5. Blocking: Add an appropriate amount of normal goat serum to the culture dish to just cover the slide, and block for 30 minutes at room temperature. After completion, rinse with PBS for 5 minutes, and repeat this operation 3 times.

[0076] 6. Incubation: Dissolve the required antibody diluent in advance. Add the NRF2 antibody diluent to the culture dish and place it on a shaker in a 4°C refrigerator overnight. The next day, discard the antibody diluent and rinse with PBS for 5 minutes. Repeat this operation 3 times. Add the secondary antibody, wrap the 6-well plate with tin foil and incubate on a shaker for 1 hour. After completion, rinse with PBS for 5 minutes and repeat three times.

[0077] 7. Staining: Add DAPI dilution solution for staining for 5 minutes in a dark environment. Then rinse with PBS for 5 minutes, and repeat this operation three times.

[0078] 8. Take photos: Turn off the light of the clean bench. Slowly add an appropriate amount of 4% paraformaldehyde along the wall of the culture dish so that it can cover the glass slide, and time it for 15 minutes. After completion, rinse with 1 mL of PBS for 3 minutes, and repeat this operation three times. Then add an appropriate amount of diluted DAPI stain to the glass slide and time it for 5 minutes. Then rinse with PBS for 5 minutes, and repeat this operation 4 times. During this period, prepare an appropriate number of slides and make corresponding marks for use. After the rinsing operation is completed, take out the slide, add an appropriate amount of anti-fluorescence quencher to the slide to be used, and then buckle the slide (containing the cell side) on the slide for sealing. Wrap it with tin foil and take it to a dark room to take pictures with a fluorescence microscope).

[0079] The results showed that after the action of 30μmol / L D724-0491, the NRF2 localized in the cell nucleus decreased significantly ( Figure 5 B).

[0080] Example 5D724-0491 can affect the Keap-NRF2 related signaling pathway and the expression of HBXIP

[0081] Studies have shown that NRF2 entering the cell nucleus can activate the expression of downstream related antioxidant elements ARE such as NQO-1 and other proteins, thereby improving the antioxidant capacity of tumor cells. In addition, it is worth noting that NRF2 in the cell nucleus can also activate the promoter of HBXIP to promote the expression of HBXIP, thereby forming a positive feedback. Therefore, in order to explore the changes in related proteins after the action of D724-0491. Nuclear and cytoplasmic separation of breast cancer cells after the action of the inhibitor was performed, and the experimental verification was as follows:

[0082] 1. Plating: Inoculate breast cancer cells MCF-7 and MDA-MB-231 in a culture dish at an appropriate density, mix well using the cross method in a clean bench, observe under a microscope, and culture in an incubator.

[0083] 2. Drug administration: When the cell density is about 60%-70%, calculate the required dosage according to the target drug concentration. Add the drug to the predetermined culture dish wells and gently mix by the cross method, then place in the incubator for further culture. Collect the cells.

[0084] 3. Prepare reagents: Prepare an ice box in advance, take out the nuclear cytoplasmic separation kit from the -20℃ refrigerator, and place it on the ice box to thaw. Calculate the total amount required based on the requirement of 100μL CEA-A and 50μL NEB for one culture dish, then prepare CEA-A and NEB according to the instructions of the nuclear cytoplasmic separation kit and place on ice for use.

[0085] 4. Treatment: Add 100 μL of CEA-A to the EP tube, vortex for 15 seconds, and then place the EP tube in an ice box for incubation for 10 minutes. After incubation, add 5.5 μL of pre-cooled CEB-B to the EP tube, vortex for a few seconds, and then place it in an ice box for incubation for 1 minute. After completion, vortex for a few seconds and centrifuge at 10,000 x g in a 4°C centrifuge for 5 minutes. During this period, prepare a suitable number of new EP tubes and mark them as cytoplasm. After centrifugation, carefully collect the supernatant into the new EP tube just prepared with a pipette. Then add 500 μL of NEB to the precipitate, vortex for 15 seconds, and then place it in an ice box for incubation for 10 minutes. Repeat the above steps 4 times. After completion, centrifuge at 16,000 rpm for 10 minutes. During this period, prepare a suitable number of new EP tubes and mark them as nuclei. After centrifugation, only aspirate the supernatant and then transfer it to the EP tube marked as nuclei).

[0086] By using Western blot technology, the changes of related proteins were detected:

[0087] 1. Cell treatment: prepare new EP tubes and mark them as treatment group and control group and set them aside for later use. Cell treatment is the same as the previous cell passaging. Pipette the cell suspension into the corresponding EP tube and centrifuge at 800 rpm for 3 minutes. After completion, use a pipette to remove the supernatant. Add 1 mL of PBS to the EP tube to disperse the cell pellet again, centrifuge at 2000 rpm for 3 minutes, and discard the supernatant after completion. Add an appropriate amount of RIPA protein lysis buffer to the EP tube by observing the amount of cell pellet and based on experience, and rotate and lyse on a 4°C rotator for 40 minutes. Then centrifuge at 12,000 rpm in a low-temperature centrifuge for 20 minutes, use a pipette of the corresponding range to aspirate the supernatant and place it in a spare EP tube.

[0088] 2. Protein quantification: Take out the BCA protein quantification kit, 5× loading buffer and standard from the 4°C refrigerator, calculate and prepare the appropriate amount of working solution. Select the appropriate number of wells in the 96-well plate, add the specified volume of protein sample and the above items in turn, and place in the incubator for reaction for about 15 minutes. Measure the absorbance value with a spectrophotometer, and calculate the extracted protein concentration and the amount of 5× loading buffer and PBS required by the standard curve presented by the standard. After adding the above items to the sample, denature at 98°C, and finally place in a -20°C refrigerator for use.

[0089] 3. Electrophoresis: Prepare PAGE gel of the required concentration in advance, and perform electrophoresis for the corresponding time at an appropriate voltage according to the molecular weight of the target protein, with protein samples of equal mass in the set groups.

[0090] 4. Transfer: After electrophoresis is completed, refer to the molecular weight range of the target protein to be cut by Maker, then place it in the transfer tank and transfer it for a certain time at an appropriate voltage according to the molecular weight of the target protein.

[0091] 5. Blocking: After the transfer is completed, take out 5% milk blocking solution from the refrigerator and pour it into the box. Transfer the PVDF into the blocking solution and place it on a shaker at room temperature for one hour.

[0092] 6. Incubate with antibodies: After blocking, recover or discard the blocking solution according to the number of times it has been used. Pour PBST along the box, shake it a few times and discard it, then pour an appropriate amount of PBST and place it on a shaker to wash the membrane for a total of 3 times, 5 minutes each time. During this period, take out the required pre-configured primary antibody dilution and thaw it in water. After the membrane is washed, place the PVDF membrane in the corresponding primary antibody dilution according to the molecular weight and incubate it on a shaker at 4°C overnight. The next day, wash the membrane 3 times with PBST for 15 minutes each time, and recover the antibody. Then add the appropriate secondary antibody to the box, place the PVDF membrane inside, incubate it at room temperature for 1 hour, and then wash the membrane with PBST for 15 minutes, repeating three times.

[0093] 7. Development: Turn on the gel imager 30 minutes in advance to preheat it and enter the working state. At the same time, prepare the luminescent solution according to the ratio of 1:1, and use tin foil to wrap the EP tube to avoid light. After the gel imager is ready, place it on the PVDF membrane and add an appropriate amount of ECL developer for development.

[0094] The results showed that after the action of D724-0491, the content of NRF2 in the cell nucleus decreased significantly; in addition, the expression of NRF2 downstream related proteins such as NQO-1 and HBXIP also decreased ( Figure 6 AD)

[0095] Example 6D724-0491 can promote the accumulation of ROS levels in breast cancer cells

[0096] Based on the above experimental data, it is speculated that D724-0491 can promote the change of ROS levels in breast cancer cells. In order to verify the speculation, flow cytometry was used to verify:

[0097] 1. Plate: Breast cancer cells MCF-7 and MDA-MB-231 were inoculated into six-well plates, 2×10 5 After mixing, observe under a microscope and place in an incubator for culture.

[0098] 2. Treatment: When the cell density is appropriate (about 60%-70%), the drug is administered and then placed in an incubator for further culture.

[0099] 3. Staining: Prepare new EP tubes in advance. The steps for digesting cells are the same as above. Pipette the cell suspension into the spare EP tube and discard the supernatant after centrifugation. After resuspending the cells with DMEM medium (serum-free and without double antibodies), turn off the light tube of the clean bench and add 500μL of DCFH-DA dye to each EP tube. Wrap the EP tube with tin foil and place it in the incubator for reaction for 35 minutes, turning it upside down every 5 minutes. After the reaction is completed, centrifuge to remove the supernatant, resuspend the cells with PBS, wash away the unattached dye and centrifuge again. Repeat this operation three times.

[0100] 4. Detection: During the 35-minute reaction, turn on the flow cytometer in advance and run the self-test program to put the machine in good condition. Take the EP tube to the flow cytometer, resuspend the cells with a 1 mL pipette, filter them, and add them to the flow cytometer detection tube to detect the ROS concentration and record it.

[0101] Immunofluorescence analysis of the changes in ROS levels in breast cancer cells after the action of D724-0491:

[0102] 1. Slide: Place the slide in a six-well plate one day in advance and add an appropriate amount of alcohol to sterilize overnight in a clean bench. The next day, discard the alcohol and add PBS to rinse three times before use.

[0103] 2. Plate laying: breast cancer cells MCF-7 and MDA-MB-231 were inoculated into a spare six-well plate, with (1.2-1.5)×10 6 The cell density was determined by cross-mixing and observed under a microscope, and the cells were cultured in an incubator.

[0104] 3. Treatment: After the cell density reaches the requirement, the treatment group is given medication. After the treatment is completed, the cells are placed in an incubator for further culture.

[0105] 4. Staining: Discard the original culture medium, add 1mL of PBS along the wall of the culture dish, rinse and discard, repeat this operation twice. Take out the DMEM incomplete culture medium, add 1mL to each well along the wall of the dish. Turn off the light of the clean bench, add 1μL of DFHDA to each well, shake gently and place in the incubator for reaction for 35 minutes. After the reaction is completed, rinse with 1mL of PBS for 3 minutes, repeat 3 times, and discard the last PBS.

[0106] 5. Take photos: Turn off the light of the clean bench. Slowly add an appropriate amount of 4% paraformaldehyde along the wall of the culture dish so that it can cover the glass slide, and time it for 15 minutes. After completion, rinse with 1 mL of PBS for 3 minutes, and repeat this operation three times. Then add an appropriate amount of diluted DAPI stain to the glass slide and time it for 5 minutes. Then rinse with PBS for 5 minutes, and repeat this operation 4 times. During this period, prepare an appropriate number of slides and make corresponding marks for standby use. After the rinsing operation is completed, take out the slide, add an appropriate amount of anti-fluorescence quencher to the slide to be used, and then buckle the slide (containing the cell side) on the slide for sealing. Wrap it with tin foil and take it to a dark room to take pictures with a fluorescence microscope.

[0107] The results showed that D724-0491 could significantly increase the level of ROS in breast cancer cells ( Figure 7 AB).

[0108] Based on the above data, it can be concluded that D724-0491 can interfere with the Keap1-NRF2 signaling pathway by targeted inhibition of HBXIP protein function, causing an increase in ROS levels in breast cancer cells and thus achieving a killing effect.

[0109] Example 7D724-0491 is able to inhibit breast cancer cell proliferation in vivo

[0110] In order to further improve the research, a nude mouse model was used to verify whether D724-0491 also has the effect of inhibiting breast cancer in vivo. 10 SPF nude mice (nude mice weighing 17-20g, 4-5 weeks). After being raised in an animal laboratory of the corresponding level for one week, the 10 nude mice were randomly and equally divided into two groups, and Con and D724 were marked on the mouse frames. Inoculation of cell culture: The steps for reviving cells are the same as above. Select MCF-7 cells that have been frozen recently, thaw 4 tubes at a time, and culture and subculture about 30 plates for use. Inoculation: Each nude mouse is inoculated with 1×10 7 cells. The steps for digesting cells are the same as above. Digest and centrifuge 5 plates of cells as a group, resuspend the cells with DMEM for dozens of times, arrange the cells individually in the cell suspension, aspirate 5-10μL of cell suspension and fill it into the cell counting plate, and calculate the required amount of cell suspension. Use a pipette to aspirate the calculated amount of cell suspension into a new EP tube, centrifuge and discard the supernatant, and use serum-free culture medium to blow away the cell sediment again. Add 50μL of matrix gel to every 150μL of cell suspension, mix well and suck it into the syringe, after exhausting the bubbles, inoculate it between the right neck and armpit of the nude mouse. Observation: After tumor inoculation, observe the nude mice in the animal laboratory every day, and prepare to administer the medicine when the tumor size reaches the size of soybeans. At the same time, replace the bedding, water and food every few days. Administration: According to the animal dosage conversion table, the dosage for each nude mouse is calculated to be 50mg / kg, and the medicine is administered every 2 days. When administering the drug, the weights of the nude mice in the Con and D724 groups were weighed and recorded, and the required amount of drug for each mouse was calculated according to the weight of the nude mice in the D724 group and injected intraperitoneally. The control group (untreated group) was given a fixed amount of normal saline. At the same time, the long and short diameters of the tumors of the nude mice in the Con and D724 groups were measured and recorded. Tissue collection: The nude mice were killed by cervical dislocation, and the nude mice were arranged in sequence on white paper and photographed. The tumor tissue was removed and placed according to the original arrangement of the nude mice, and a ruler was placed on top to take a photo. After completion, the tumor tissue was weighed and recorded. Finally, use scissors to cut off part of the tissue and put it in the tissue fixative for subsequent HE staining (1. Passing the cylinder: After taking out the paraffin slices, place them in the dewaxing box for dewaxing for about 40 minutes. After completion, place them at room temperature for 10 minutes. Open the fume hood and then operate according to the cylinder passing sequence. 2. Processing: Add appropriate amounts of hematoxylin staining solution (timed for 3 minutes), hematoxylin differentiation solution (several seconds), and hematoxylin anti-blue solution (several seconds) to the tissue position of the slice in sequence. Before changing the staining solution each time, rinse it from top to bottom under a trace amount of running water. Then, place the slices in 85% ethanol (timed for 5 minutes) and 95% ethanol (timed for 5 minutes) in sequence to complete dehydration. Then add eosin staining to the slices, time for 5 minutes, rinse and pass them again in the opposite direction of the above cylinder passing sequence, and use neutral resin to seal the slices. 3. Take pictures: take pictures and analyze them); the remaining tissues are placed in corresponding marked bags and stored in a -80℃ refrigerator.

[0111] The results showed that the tumor volume of nude mice treated with D724-0491 was significantly smaller than that of the Con group. In addition, there was no significant difference in the weight changes of nude mice between the Con and D724-0491 groups, and the changes were not significant ( Figure 8 AD). Immunohistochemical analysis of tumor tissues in the Con and D724-0491 groups revealed that the expression of the tumor marker Ki67 was significantly decreased in the D724-0491 group ( Figure 8 E); In addition, the expression of HBXIP in the two groups of animals was investigated. The results were the same as expected, and the expression of HBXIP in the drug-treated group was significantly decreased ( Figure 8 E). In summary, it can be concluded that D724-0491 can effectively inhibit the progression of breast cancer in vivo.

Claims

1. Application of D724-0491 in the preparation of a drug for treating breast cancer, wherein the structural formula of D724-0491 is:

2. The use according to claim 1, characterized in that: The active ingredient of the breast cancer drug is D724-0491.

3. The use according to claim 1, characterized in that: The only active ingredient of the breast cancer drug is D724-0491.

4. The use according to claim 1, characterized in that: The breast cancer cells include breast cancer cells MCF-M7 and breast cancer cells MDA-MB-231.

5. The use according to claim 1, characterized in that: The medicine is an oral preparation or an injection preparation.

6. The use according to claim 1, characterized in that: The drug inhibits the binding of HBXIP to KEAP1 and reduces the level of NRF2 entering the nucleus.

7. The use according to claim 1, characterized in that: The drug inhibits breast cancer cell proliferation.

8. The use according to claim 1, characterized in that: The drug inhibits breast cancer cell invasion.

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