Targeted BRD7 tumor suppressor protein stabilizing agent and application thereof in preparation of solid tumor treatment drugs

Abstract

The invention belongs to the field of tumor treatment medicines, and particularly relates to a stabilizing agent aiming at BRD7 tumor suppressor protein, which is a free compound with a structure # imgabs0 # as shown in a formula 1, pharmaceutically acceptable salt, crystal and solvate. The invention also comprises application of the stabilizing agent of the targeted BRD7 tumor suppressor protein in preparation of drugs for treating solid tumors. Researches show that the compound in the formula 1 has an effect of stabilizing BRD7 protein, and can realize efficient inhibition on solid tumors such as breast cancer, nasopharynx cancer and lung cancer based on a mechanism of stabilizing BRD7 tumor suppressor protein.

Description

Technical Field

[0001] The present invention belongs to the field of medical treatment, and specifically relates to the field of anti-solid tumor drugs. Background Art

[0002] Malignant tumors are one of the major public health issues that seriously endanger human life and health. Tumors are the main cause of death and an important obstacle to extending life expectancy in countries around the world. The occurrence of tumors is usually driven by abnormal activation of tumor genes or abnormal reduction or inactivation of tumor suppressor genes. With the development of molecular biology, pharmacy and other disciplines, the functional genes closely related to the occurrence and development of tumors have gradually become clear, and targeted therapies targeting these genes or proteins have become an important molecular strategy for tumor treatment. Tumor suppressor genes are a class of key functional genes that can inhibit the occurrence, development, and metastasis of tumors. Among them, the reduced expression or stability of tumor suppressor proteins is an important molecular mechanism leading to tumor drive and malignant progression. However, almost all of the approximately 80 targeted drugs currently used in clinical practice target tumor proteins, so the development of drugs that target and activate tumor suppressor proteins is urgent.

[0003] According to the origin and characteristics of the tissue, tumors are divided into solid tumors and blood tumors. Solid tumors refer to solid tissues from various parts of the body, including breast cancer, nasopharyngeal cancer, lung cancer, liver cancer, colorectal cancer, etc. Blood tumors mainly originate from hematopoietic organs such as bone marrow and lymph nodes, such as leukemia and lymphoma. Traditional surgery, chemotherapy, and radiotherapy are still the main methods for treating solid tumors, but the formation of resistance to radiotherapy and chemotherapy leads to tumor recurrence and treatment failure. Therefore, targeted therapy is an indispensable and important method for the treatment of mid- and late-stage solid tumors.

[0004] BRD7 is a conserved protein containing a single bromodomain. It is a member of the SWI / SNF chromatin remodeling complex and has been confirmed to be an important transcription factor. The previous work of the research group and the literature showed that BRD7 is downregulated in a variety of tumors such as nasopharyngeal carcinoma, breast cancer, lung cancer, ovarian cancer, osteosarcoma and liver cancer, and is negatively correlated with the clinical progression and poor prognosis of patients. It has also been confirmed that BRD7 can inhibit the proliferation, migration, invasion, chemotherapy resistance of tumor cells, and the growth and metastasis of tumors in vivo, thereby playing the role of a tumor suppressor gene in tumors. At the same time, it has been confirmed that BRD7 is a highly unstable protein, and its reduced protein stability may be an important molecular mechanism leading to tumorigenesis, malignant progression and the formation of treatment resistance. Therefore, targeted activation or stabilization of BRD7 protein may be a very effective clinical targeted treatment strategy for tumors. However, there has been no report on the development or clinical application of activating drugs for BRD7 protein, so the development of drugs targeting activation or stabilization of BRD7 protein is still urgent. Summary of the invention

[0005] The first purpose of the present invention is to provide a stabilizer (BRD7-St) that can stabilize BRD7 protein (also known as BRD7 tumor suppressor protein), aiming to provide a small molecule compound that can stabilize BRD7 protein.

[0006] The second purpose of the present invention is to provide the use of the stabilizer targeting BRD7 tumor suppressor protein in the preparation of drugs for activating and stabilizing BRD7 protein and treating solid tumors.

[0007] The third object of the present invention is to provide a drug for treating solid tumors comprising the stabilizer targeting the BRD7 tumor suppressor protein.

[0008] A stabilizer targeting BRD7 tumor suppressor protein, which is a free compound, a pharmaceutically acceptable salt, a crystal and a solvate having a structure of formula 1;

[0009]

[0010] R1 and R2 are independently H or C1-C6 alkyl.

[0011] The innovative research of the present invention shows that the compound of formula 1 can specifically target and activate the BRD7 protein and can improve its stability.

[0012] In the present invention, the compound of formula 1 may further be of formula 1-A, which is a compound of formula 1 wherein R1 and R2 are methyl groups.

[0013] The present invention also provides a use of the stabilizer targeting the BRD7 tumor suppressor protein in the preparation of a drug for activating and increasing the stability of the BRD7 protein.

[0014] The innovative research of the present invention shows that the formula 1 can specifically target and activate the BRD7 protein.

[0015] The application of the present invention is to use it as an active ingredient for preparing a drug for treating solid tumors that activates and increases the stability of the BRD7 protein.

[0016] The innovative research of the present invention shows that the BRD7-St can specifically target and activate the BRD7 protein to achieve the pharmacological efficacy of highly effective treatment of solid tumors.

[0017] In the present invention, the solid tumor is a solid tumor with low expression of BRD7, for example, including at least one of breast cancer, nasopharyngeal carcinoma, lung cancer, etc.

[0018] In the present invention, the breast cancer cells include at least one of MDA-MB-231 and MCF7; the nasopharyngeal cancer cells include at least one of CNE2 and 5-8F.

[0019] The application of the present invention combines the stabilizer targeting the BRD7 tumor suppressor protein with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable drug dosage form.

[0020] The application of the present invention, the dosage form is an oral dosage form and / or an injection dosage form. It can further be a powder injection, an injection, a tablet, a pill, a capsule, a spray, a dispersion, etc. The dosage of the drug BRD7-St is a pharmaceutically acceptable dosage, and the administration method includes but is not limited to the tumor, vein, artery, abdominal cavity, oral, etc.

[0021] The present invention also provides a drug for treating solid tumors, which comprises a pharmaceutically effective amount of the stabilizer targeting the BRD7 tumor suppressor protein.

[0022] The drug for treating solid tumors of the present invention also includes other known solid tumor inhibitors.

[0023] The drug for treating solid tumors of the present invention preferably comprises a pharmaceutically acceptable excipient;

[0024] Preferably, the drug is in a pharmaceutically acceptable dosage form.

[0025] Beneficial Effects

[0026] The present invention shows that formula 1 BRD7-St can increase the stability of BRD7 protein in a time- and dose-dependent manner in tumor cells such as breast cancer and nasopharyngeal carcinoma, and can inhibit tumor cell proliferation, cloning and tumor growth in vivo in a BRD7-dependent manner, thereby playing an anti-tumor role in malignant tumors such as breast cancer and nasopharyngeal carcinoma, and has no obvious toxic side effects in mice. Therefore, BRD7-St is expected to become a potential anti-tumor drug targeting BRD7 protein.

[0027] The present invention's research shows that the small molecule drug of formula 1 has a binding affinity with the BRD7 protein in vitro, and the binding affinity is 59.9 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Study on the protein expression level of BRD7 in breast cancer patient tissues and adjacent tissues, and the stability of BRD7 protein in breast cancer cells

[0029] A: Six pairs of clinical biopsy tissues and their paired adjacent tissues from breast cancer patients were collected, and the expression level of BRD7 protein was detected by Western blot;

[0030] B: After co-treatment with CHX or CHX+MG132 for different time periods (0h, 1h, 2h, 4h), the protein expression level of BRD7 in breast cancer cells was detected by Western blot.

[0031] Figure 2 Screening of small molecule compound BRD7-St formula 1-A and the results of ion surface resonance experiment (SPR) of its binding with BRD7 protein in vitro

[0032] A: Molecular docking simulation of the binding of small molecule compound formula 1-A to BRD7 protein;

[0033] B: Surface plasmon resonance (SPR) experiment results of in vitro binding of Formula 1-A to BRD7 protein.

[0034] Figure 3 Detection of BRD7 protein expression level in breast cancer and nasopharyngeal carcinoma cells after BRD7-St-1-A was treated with cells at different times and different doses

[0035] A: After treating breast cancer cells (MDA-MB-231, MCF7) with different concentrations of Formula 1-A, the expression level of BRD7 protein was detected by Western blot;

[0036] B: After treating nasopharyngeal carcinoma cells (CNE2, 5-8F) with different concentrations of Formula 1-A, the expression level of BRD7 protein was detected by Western blot;

[0037] C: After breast cancer cells (MDA-MB-231, MCF7) were treated with Formula 1-A for different time periods, the expression level of BRD7 protein was detected by Western blot.

[0038] D: After treating breast cancer cells (MDA-MB-231) with formula 1-A analogs such as acalabrutinib, zanubrutinib and rizarutinib for 24 h, the expression level of BRD7 protein was detected by Western blot.

[0039] Figure 4 Detection of the killing effect of BRD7-St 1-A on breast cancer and nasopharyngeal cancer cells

[0040] A: IC50 values ​​of CCK8 detection after treating breast cancer cells MDA-MB-231 with different concentrations of Formula 1-A for different time periods (24h and 48h);

[0041] B: IC50 values ​​of CCK8 detection after treating breast cancer cells MCF7 with different concentrations of Formula 1-A for different time periods (24h and 48h);

[0042] C: IC50 values ​​of CCK8 detection after treating nasopharyngeal carcinoma cell CNE2 with different concentrations of Formula 1-A for different time periods (24h and 48h);

[0043] D: IC50 values ​​of CCK8 detection after treating nasopharyngeal carcinoma cell 5-8F with different concentrations of Formula 1-A for different time periods (24h and 48h);

[0044] Data are presented as mean ± SEM, and three independent experiments were performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no statistical difference.

[0045] Figure 5 BRD7-St1-A increases BRD7 protein stability by reducing BRD7 ubiquitination level

[0046] A: Breast cancer cells (MDA-MB-231, MCF7) were treated with formula 1-A, and the ubiquitination level of BRD7 was detected by Western blot;

[0047] B: Nasopharyngeal carcinoma cells (CNE2, 5-8F) were treated with formula 1-A, and the level of BRD7 ubiquitination was detected by Western blot;

[0048] Figure 6 The killing effect of BRD7-St 1-A on breast cancer and nasopharyngeal cancer cells depends on its targeted activation of BRD7 protein

[0049] A: After breast cancer cells (MDA-MB-231, MCF7) were treated with Formula 1-A for different time periods, the IC50 values ​​of the DMSO group, Formula 1-A group, and Formula 1-A group + shBRD7 group were detected by CCK8;

[0050] B: After treating nasopharyngeal carcinoma cells (CNE2, 5-8F) with Formula 1-A for different time periods, the IC50 values ​​of the DMSO group, Formula 1-A group, and Formula 1-A group + shBRD7 group were detected by CCK8;

[0051] Figure 7 The inhibitory effect of BRD7-St 1-A on breast cancer cell clone formation depends on its targeted activation of BRD7 protein

[0052] A: DMSO group, Formula 1-A group, Formula 1-A group + shBRD7 group were set up, and breast cancer cells MDA-MB-231 were treated with Formula 1-A (8 μM, 12 μM) to detect the effect of Formula 1-A on the clone formation ability of cells in different groups;

[0053] B: DMSO group, Formula 1-A group, Formula 1-A group + shBRD7 group were set up, and breast cancer cells MCF7 were treated with Formula 1-A (8 μM, 12 μM) to detect the effect of Formula 1-A on the clone formation ability of cells in different groups;

[0054] Data are presented as mean ± SEM, and three independent experiments were performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no statistical difference.

[0055] Figure 8 Inhibitory effect of BRD7-St 1-A on tumor growth in breast cancer-bearing mice

[0056] A: Photos of tumor-bearing mice in the DMSO group and Formula 1-A group;

[0057] B: After the mice were killed, the transplanted tumors of the DMSO group and the Formula 1-A group were peeled off and compared. The tumor of the Formula 1-A group was smaller;

[0058] C: The size of the transplanted tumor was measured every 2 days, and the mouse tumor growth curve was drawn. It was found that Formula 1-A could significantly inhibit the tumor growth rate.

[0059] D: The transplanted tumors in the DMSO group and the formula 1-A group were weighed, and it was found that the transplanted tumors in the formula 1-A group were lighter;

[0060] E: IHC was used to detect the expression levels of BRD7, Ki67, cleaved-PARP, CDK4 and P21 in the tumors of the DMSO group and Formula 1-A group;

[0061] F: The hearts, livers, spleens, lungs and kidneys of the mice in the DMSO group and the Formula 1-A group were taken out, HE staining was performed and the tissue status of each organ was observed.

[0062] The data are presented as mean ± standard error, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no statistical difference. DETAILED DESCRIPTION

[0063] The present invention is further described below in conjunction with specific embodiments, but is not intended to limit the present invention.

[0064] As an illustrative embodiment, in the following cases, the small molecule drug BRD7-St is represented by Formula 1-A.

[0065] Formula 1-A may inhibit the binding of E3 ubiquitinase to BRD7 protein and increase its protein stability by competing with E3 ubiquitin ligase to bind to the highly ubiquitinated modification site outside the bromodomain pocket of BRD7. These potential binding sites include K173, K182, K184, K186 or K197.

[0066] The small molecule drug BRD7-St formula 1-A exerts its anti-tumor function in vitro by targeting and activating the BRD7 tumor suppressor protein, and can be used to intervene in the proliferation and growth of tumor cells in vitro.

[0067] The anti-tumor effect of the small molecule drug Formula 1-A is dependent on the BRD7 protein. When BRD7 is knocked down, the anti-tumor effect of the drug is significantly reversed.

[0068] The tumor or tumor cell is a solid tumor or tumor cell that lowly expresses BRD7 protein, including solid tumors such as breast cancer, nasopharyngeal carcinoma, lung cancer, ovarian cancer, etc. in which BRD7 plays a tumor suppressor function.

[0069] The small molecule drug BRD7-St formula 1-A has a significant inhibitory effect on tumor cell proliferation and clone formation in breast cancer and nasopharyngeal carcinoma, and its 48-hour IC50 values ​​are 12.74 μM (MDA-MB-231), 8.035 μM (MCF7), 7.376 μM (CNE2) and 8.573 μM (5-8F), respectively.

[0070] As a preferred embodiment of the present invention, the small molecule drug formula 1-A can inhibit or delay tumor growth in mice and can be used to prepare anti-tumor targeted drugs for preventing, inhibiting and / or delaying tumor occurrence and growth.

[0071] The small molecule drug formula 1-A has the function of inhibiting tumor growth in vivo in breast cancer cell nude mouse transplanted tumors, and has no obvious toxic side effects on the internal organs of mice.

[0072] The dosage form of the drug formula 1-A is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection solution, tablet, pill, capsule, spray, dispersion, etc.

[0073] The dosage of the drug of Formula 1-A is a pharmaceutically acceptable dosage, and the modes of administration include but are not limited to tumor, intravenous, intraperitoneal, oral, etc.

[0074] Formula 1-A of the present invention can target and activate BRD7 protein, has a strong tumor growth inhibitory effect on breast cancer and nasopharyngeal cancer cells, and can be used to prepare drugs for preventing and / or treating breast cancer and nasopharyngeal cancer malignant tumors. The present invention shows through in vitro experiments that Formula 1-A can inhibit the proliferation of breast cancer and nasopharyngeal cancer cells; the results of in vivo experiments show that Formula 1-A can inhibit the growth of breast cancer cells in vivo, and has good therapeutic potential and application prospects. The drug formula 1-A provided by the present invention for the targeted activation of BRD7 protein to prevent and / or treat malignant tumors such as breast cancer and nasopharyngeal cancer can be subsequently prepared into a pharmaceutical dosage form that is convenient for clinical use and can exert the best therapeutic effect, and has a high clinical application value.

[0075] The MDA-MB-231, MCF7, 5-8F, and CNE2 cell lines used in the present invention are all preserved in the Molecular Genetics Laboratory of the Tumor Research Institute of Central South University. The cell culture conditions are: DMEM liquid culture medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin and streptomycin), and adherent growth in a constant temperature incubator at 37° C. and 5% CO2 concentration.

[0076] Example 1: In breast cancer cells, BRD7 was confirmed to be an unstable protein, and the ubiquitin-proteasome system may be involved in the regulation of BRD7 protein stability.

[0077] 1.1 Experimental plan:

[0078] Six pairs of clinical breast cancer patient tumor tissues and adjacent normal tissues were collected, and the tissues were ground for protein extraction. The expression levels of BRD7 in breast cancer patient tissues and adjacent normal tissues were detected by Western Blot. In breast cancer cells MDA-MB-231 and MCF7, a cycloheximide (CHX) treatment group (50μM) and a CHX (50μM) and proteasome pathway inhibitor MG132 (20μM) co-treatment group were set up; then, cells were collected at different time points (0, 1, 2, 4h) and protein extraction was performed immediately. The protein expression level of BRD7 was detected by Western Blot technology.

[0079] 1.2 Experimental results:

[0080] Compared with adjacent normal tissues, the expression of BRD7 in breast cancer tissues was significantly decreased ( Figure 1 A). In addition, the protein expression level of BRD7 gradually decreased with the extension of CHX treatment time, and the protein half-life of BRD7 was about 1-2h. The protein expression level of BRD7 in the CHX and MG132 co-treated group was partially restored, and the half-life of BRD7 was prolonged ( Figure 1 B). This indicates that BRD7 is lowly expressed in breast cancer tissues and is confirmed to be an unstable protein in breast cancer cells. The proteasome pathway may be involved in the regulation of BRD7 protein stability.

[0081] Example 2, in vitro binding experiment of small molecule compound BRD7-St formula 1-A targeting and activating BRD7 protein and BRD7 protein

[0082] 2.1 Experimental plan:

[0083] Based on the structural characteristics of BRD7 protein, potential small molecule activating drugs that can bind to the outside of the bromodomain pocket of BRD7 protein and whose stability is greater than that of the pocket region were screened. The docking of small molecules with BRD7 protein was simulated by computer simulation, and the mutual binding between small molecules and BRD7 protein was confirmed by surface plasmon resonance (SPR) technology.

[0084] 2.2 Experimental results:

[0085] Based on the structure and instability characteristics of BRD7 protein, it was found that K173, K182, K184, K186 and K197 are potential high ubiquitination modification sites outside the active pocket of BRD7 bromodomain (brd), and the small molecule compound formula 1-A can bind to the high ubiquitination site enriched region outside the pocket of BRD7 protein. The virtual binding mode diagram of formula 1-A and BRD7 is shown in Figure 2 A. And the SPR experiment confirmed that the small molecule activator BRD7-St formula 1-A binds to BRD7 protein in vitro, with an affinity coefficient of 59.9μM ( Figure 2 B).

[0086] Example 3, BRD7-St Formula 1-A can target and activate BRD7 protein expression in a time- and dose-dependent manner in breast cancer and nasopharyngeal cancer cells

[0087] 3.1 Experimental plan:

[0088] Different concentrations of Formula 1-A were used to treat breast cancer cells MDA-MB-231 (0μM, 4μM, 8μM, 12μM) and MCF7 (0μM, 2μM, 4μM, 8μM), nasopharyngeal carcinoma cells CNE2 (0μM, 0.5μM, 1μM, 2μM, 4μM) and 5-8F (0μM, 0.5μM, 1μM, 2μM 4μM), and the cells were collected after 24h of treatment and protein extraction was performed immediately. The protein expression level of BRD7 was detected by Western Blot technology. The same concentration of Formula 1-A was used to treat breast cancer cells MDA-MB-231 (8μM) and MCF7 (4μM), and the cells were collected after 0h, 6h and 24h of treatment and protein extraction was performed immediately. At the same time, similar small molecule compounds such as Acalabrutinib, ZAnubrutinib and Rilzabrutinib were used to treat breast cancer MDA-231 cells, and the effects of these compounds with similar structures to Formula 1-A, such as Acalabrutinib, ZAnubrutinib and Rilzabrutinib, on the expression of BRD7 protein were explored by Western Blot technology.

[0089] 3.2 Experimental results:

[0090] The protein expression level of BRD7 in breast cancer cells and nasopharyngeal carcinoma cells gradually increased with the increase of the concentration of Formula 1-A treatment ( Figure 3 AB), and the protein expression level of BRD7 in breast cancer cells gradually increased with the extension of the treatment time of Formula 1-A ( Figure 3 C). It shows that Formula 1-A can target and activate the protein expression level of BRD7 in breast cancer and nasopharyngeal cancer cells, and shows obvious time and dose dependence. At the same time, it was found that small molecule compounds with similar structures to Formula 1-A, such as acalabrutinib, zanubrutinib and rizarutinib, could not significantly increase the expression or stability of BRD7 protein, indicating that BRD7-St Formula 1-A relies on its specific structure to achieve specific stabilization of BRD7 protein, and has relatively good original innovation in stabilizing BRD7 protein.

[0091] Example 4, BRD7-St Formula 1-A can kill breast cancer and nasopharyngeal cancer cells in vitro, thereby showing anti-tumor activity

[0092] 4.1 Experimental plan:

[0093] (1) Different types of breast cancer cells (MDA-MB-231, MCF7) and nasopharyngeal carcinoma cells (CNE2, 5-8F) were inoculated at 4000 cells / well in a 96-well plate. The blank group was DMEM medium (containing serum) but without cells.

[0094] (2) Different drug concentrations were set to treat breast cancer cells MDA-MB-231 (0 μM, 3 μM, 9 μM, 27 μM, 81 μM) and MCF7 (0 μM, 3 μM, 9 μM, 27 μM, 81 μM), nasopharyngeal cancer cells CNE2 (0 μM, 3 μM, 9 μM, 27 μM, 81 μM) and 5-8F (0 μM, 3 μM, 9 μM, 27 μM, 81 μM), respectively. Each concentration gradient was repeated 5 times, and the control group was the DMSO treatment group.

[0095] (3) After treatment for 24 h and 48 h, 10% CCK8 solution was added to each well and incubated in a 37°C incubator for 2 h. The absorbance was measured using an ELISA reader (wavelength 450 nm).

[0096] (4) Data analysis: Calculate the IC50 value of formula 1-A for tumor cell lines. The formula is as follows:

[0097] The cell survival rate (cell viability) of the tumor cell line % = (absorbance value of the drug-added group - absorbance value of the blank group) / (absorbance value of the control group - absorbance value of the blank group) × 100%.

[0098] 4.2 Experimental results:

[0099] The results of CCK8 experiments showed that BRD7-St formula 1-A showed good antitumor effects in breast cancer cells and nasopharyngeal carcinoma cells, with IC50 values ​​of MDA-MB-231 (IC50-24 h: 18.34 μM, IC50-48 h: 12.74 μM), MCF7 (IC50-24 h: 8.035 μM, IC50-48 h: 4.68 μM), CNE2 (IC50-24 h: 18.99 μM, IC50-48 h: 7.376 μM) and 5-8F (IC50-24 h: 12.01 μM, IC50-48 h: 8.573 μM) ( Figure 4 AD). It shows that formula 1-A can significantly inhibit the growth of tumor cells in vitro and exhibits good anti-tumor effect.

[0100] Example 5, BRD7-St Formula 1-A improves the protein stability of BRD7 by reducing the ubiquitination level of BRD7

[0101] 5.1 Experimental plan:

[0102] Breast cancer cells (MDA-MB-231, MCF7) and nasopharyngeal carcinoma cells (CNE2, 5-8F) were treated with 8 μM BRD7-St formula 1-A for 24 hours, and then treated with 20 μM proteasome inhibitor MG132 for 4 hours, and then the cells were collected and protein extracted immediately. The ubiquitination level of BRD7 was detected by Western Blot technology.

[0103] 5.2 Experimental results:

[0104] The experimental results show that in breast cancer cells ( Figure 5 A) and nasopharyngeal carcinoma cells ( Figure 5 In B), the ubiquitination level of BRD7 was significantly reduced after treatment with BRD7-St Formula 1-A, indicating that Formula 1-A improves the protein stability of BRD7 by reducing the ubiquitination level of BRD7 in breast cancer cells and nasopharyngeal carcinoma cells.

[0105] Example 6, knocking down BRD7 expression can reverse the inhibitory effect of BRD7-St formula 1-A on the cell viability of breast cancer cells and nasopharyngeal carcinoma cells in vitro

[0106] 6.1 Experimental plan:

[0107] (1) Well-growing breast cancer cells (MDA-MB-231, MCF7) and nasopharyngeal carcinoma cells (CNE2, 5-8F) were seeded in 12-well plates, and when the cell density reached 60-80%, shBRD7 was transfected using Polyplus.

[0108] (2) 24 h after transfection, cells were counted and plated in a 96-well plate at 4000 cells / well. The blank group was DMEM medium (containing serum) but without cells.

[0109] (3) The same drug concentrations, MDA-MB-231 (8 μM), MCF7 (8 μM), CNE2 (4 μM), and 5-8F (4 μM) were used, and 5 replicates were set for each concentration gradient. The control group was the DMSO-treated group.

[0110] (4)) Six time points were set: 0d, 1d, 2d, 3d, 4d, and 5d. Then, 10% CCK8 solution was added to each well and incubated in a 37°C incubator for 2 h. The absorbance value was measured using an enzyme reader (wavelength 450 nm).

[0111] (5) Data analysis: Calculate the IC50 value of the tumor cell line using the following formula:

[0112] The cell survival rate (cell viability) of the tumor cell line % = (absorbance value of the drug-added group - absorbance value of the blank group) / (absorbance value of the control group - absorbance value of the blank group) × 100%.

[0113] 6.2 Experimental results:

[0114] The results of CCK8 experiments showed that compared with the DMSO group, BRD7-St formula 1-A treatment could significantly inhibit the growth of breast cancer cells (MDA-MB-231, MCF7) and nasopharyngeal carcinoma cells (CNE2, 5-8F). After knocking down BRD7 expression in advance with shBRD7, the growth inhibitory effect of formula 1-A on tumor cells was partially or completely reversed, indicating that formula 1-A inhibited the growth of tumor cells in vitro by targeting and activating the stability of BRD7 protein ( Figure 6 AB).

[0115] Example 7, knocking down BRD7 expression can reverse the inhibitory effect of BRD7-St formula 1-A on the clone formation ability of breast cancer cells and nasopharyngeal carcinoma cells

[0116] 7.1 Experimental plan:

[0117] (1) Well-growing breast cancer cells (MDA-MB-231, MCF7) were seeded in a 12-well plate, and when the cell density reached 60-80%, shBRD7 was transfected using Polyplus.

[0118] (2) 24 hours after transfection, the cells were counted and plated in a 12-well plate at 800 cells / well. After small clones grew, the tumor cells were co-cultured with Formula 1-A for 3 days, and then replaced with complete medium for continued culture for 10-14 days. The concentration of Formula 1-A in each well of the experimental group was 8 μM and 12 μM, and the control group was a DMSO-treated group.

[0119] (3) Discard the culture medium and wash the cells three times with 1× PBS for 5 minutes each time. Then, add 4% paraformaldehyde and fix the cells at room temperature for 1 hour.

[0120] (4) Aspirate the fixative and wash the wells three times with 1× PBS for 5 minutes each time. Then add crystal violet stain to each well and stain for 30 minutes at room temperature.

[0121] (5) Aspirate the crystal violet stain and rinse with distilled water until no crystal violet remains. After drying, use a scanner to scan and record the image.

[0122] (6) Count the number of effective cell clones in each group and analyze the data.

[0123] 6.2 Experimental results:

[0124] The results of the cloning experiment showed that compared with the DMSO group, Formula 1-A treatment could significantly inhibit the cloning ability of MDA-MB-231 and MCF7, and after knocking down BRD7 expression in advance, the inhibitory effect of BRD7-St Formula 1-A on the cloning ability of tumor cells was reversed, confirming that Formula 1-A targeted activation of BRD7 protein stably inhibited the cloning ability of tumor cells in vitro ( Figure 7 AB).

[0125] Example 8, in vivo experiments confirmed that BRD7-St formula 1-A can significantly inhibit the growth of breast cancer transplanted tumors in vivo without obvious tissue and organ toxicity

[0126] 8.1 Experimental plan:

[0127] Sixteen 4-6-week-old female BALB / C nude mice weighing 16±2 g were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. All nude mice passed the quality inspection. Animal breeding and related operations were completed under specific pathogen-free (SPF) conditions at the Department of Experimental Animals of Central South University.

[0128] (1) Prepare breast cancer MCF7 cells in good growth condition, collect the cells and wash them three times with pre-cooled physiological saline.

[0129] (2) Prepare 4×10 6 150 μl of the cell suspension was inoculated into the subcutaneous area above the axilla of the right forelimb of nude mice.

[0130] (3) Starting from the fourth day after injection, the length (L) and width (W) of the subcutaneous tumor of nude mice were measured every 2 days with a vernier caliper, the results were recorded and the tumor volume was calculated using the following formula: Volume = L × W 2 / 2.

[0131] (4) When the tumor volume of mice reaches 30-50 mm 3 Afterwards, the mice were randomly divided into 2 groups, 8 in each group, where each mouse in the experimental group was injected with 10 mg / kg of BRD7-St Formula 1-A peritumorally, and the mice in the control group were injected with DMSO, once every 2 days, for a total of 3 injections.

[0132] (5) After the mice were fed for another 4 days, they were euthanized by cervical dislocation, and the tumors were removed, photographed, and the tumor volume and weight were recorded.

[0133] (6) Part of the tumor tissue in each group was dehydrated, fixed, paraffin-embedded, and sectioned to detect the expression of proliferation, cycle, and apoptosis markers.

[0134] (7) Three mice were randomly selected from each group. The heart, liver, spleen, lung, and kidney were removed, dehydrated, fixed, paraffin-embedded, sectioned, and stained with HE to observe the tissue status of each organ.

[0135] 8.2 Experimental results:

[0136] The results of the in vivo nude mouse transplant tumor model showed that compared with the control group, the tumor growth of mice in the BRD7-St formula 1-A treatment group was significantly inhibited and the tumor weight was significantly reduced ( Figure 8 AD), indicating that Formula 1-A can significantly inhibit tumor growth in vivo, thereby exhibiting an in vivo anti-tumor effect. In addition, the expression of BRD7, cell proliferation-related molecule Ki67, cell cycle-related molecules CDK4 and P21, and apoptosis-related molecule cleaved-PARP in the above-mentioned nude mouse tumor tissues was detected by immunohistochemistry (IHC). The results showed that compared with the control group, the expression of BRD7 in the BRD7-St Formula 1-A treatment group was significantly increased, and the expression levels of cleaved-PARP and P21 were increased, while the expression levels of Ki67 and CDK4 were decreased ( Figure 8 E), indicating that after nude mice transplanted tumors were treated with Formula 1-A, BRD7 protein was activated, cell proliferation and cell cycle were significantly inhibited, and cell apoptosis was initiated. According to the HE staining results, there was no obvious damage to the various organs and tissues of mice in the Formula 1-A treatment group, indicating that Formula 1-A has a certain in vivo biosafety ( Figure 8 F). The above results show that BRD7-St formula 1-A can significantly inhibit the growth of breast cancer cells without obvious organ toxicity.

Claims

1. A stabilizer targeting BRD7 tumor suppressor protein, characterized in that: A free compound, a pharmaceutically acceptable salt, a crystal and a solvate having the structure of Formula 1; R1 and R2 are independently H or C1-C6 alkyl.

2. Use of the stabilizer targeting BRD7 tumor suppressor protein according to claim 1 in the preparation of a drug for activating and increasing the stability of BRD7 protein.

3. The use according to claim 2, characterized in that The invention is used as an active ingredient for preparing a drug for treating solid tumors by activating and increasing the BRD7 tumor suppressor protein.

4. The use according to claim 3, characterized in that The solid tumor is a solid tumor with low BRD7 expression, preferably at least one of breast cancer, nasopharyngeal carcinoma and lung cancer.

5. The use according to claim 4, characterized in that The breast cancer cells include at least one of MDA-MB-231 and MCF7; The nasopharyngeal carcinoma cancer cells include at least one of CNE2 and 5-8F.

6. The use according to any one of claims 2 to 5, characterized in that: The stabilizer targeting the BRD7 tumor suppressor protein is combined with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable drug dosage form.

7. The use according to claim 6, characterized in that The dosage form is an oral dosage form and / or an injection dosage form.

8. A drug for treating solid tumors, characterized in that: A stabilizer targeting the BRD7 tumor suppressor protein according to claim 1 is included in a pharmaceutically effective amount.

9. The drug for treating solid tumors according to claim 8, characterized in that: Other solid tumor inhibitors are also included.

10. The drug for treating solid tumors according to claim 8 or 9, characterized in that: Also contains pharmaceutically acceptable excipients; Preferably, the drug is in a pharmaceutically acceptable dosage form.

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