Application of brucein A in preparation of DNA damage reaction inhibitor drug

Abstract

The invention discloses an application of brucein A in preparation of a DNA damage reaction inhibitor drug, and relates to the technical field of biological medicines. The inhibitor drug is based on brucein A and simultaneously inhibits homologous recombination repair and non-homologous end ligation in cells. In drug screening, it is found for the first time that the brucein A can effectively inhibit DNA damage response of cells and can inhibit two main DNA damage repair approaches, namely homologous recombination repair and non-homologous end connection; in addition, the bruceine A is combined with a chemotherapeutic drug for use, so that the sensitivity of the triple negative breast cancer cell MDA-MB-231 to the chemotherapeutic drug cis-platinum can be remarkably enhanced.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, and particularly relates to application of a calanolide A in preparation of a DNA damage response inhibitor drug. BACKGROUND

[0002] DNA damage response (DDR) is a highly conserved defense mechanism in cells, which plays a core regulatory role in response to DNA damage caused by exogenous environmental factors and endogenous metabolic stress. Through precise regulation of the coordination between DNA repair system and cell cycle checkpoint network, the mechanism effectively blocks the replication and transmission of damaged DNA, thereby protecting the intergenerational inheritance of genome integrity. Notably, the functional defects of the DDR pathway not only directly drive tumor occurrence and development, but also profoundly affect the clinical treatment effect and patient prognosis, which makes the DDR regulatory network an important target system for tumor research. In-depth analysis of the dynamic regulation mechanism of DDR signals and their tissue specificity not only provides theoretical support for revealing the evolution rules of tumors, but also lays a molecular foundation for the development of targeted intervention strategies.

[0003] Based on the above biological significance, targeting DDR pathway has become a frontier field in the research and development of anti-tumor drugs, and its therapeutic value mainly lies in three aspects: first, inhibiting key nodes of DDR can significantly enhance the sensitivity of traditional chemotherapy and reverse drug resistance; second, based on the "synthetic lethality" effect, it provides a precise treatment strategy for DDR-deficient tumors; third, inhibiting DDR can remodel the tumor immune microenvironment, significantly enhancing the efficacy of tumor immunotherapy and providing new ideas for the development of combination therapy strategies. PARP1 (poly (ADP-ribose) polymerase) is a key DNA repair enzyme that plays an important role in base excision repair (BER) and single-strand break (SSBs) repair. In 1975, the first PARP inhibitor was found to significantly enhance the cytotoxicity of DNA damaging agent dimethyl sulfate, which laid the foundation for the subsequent development of PARP inhibitors. In 2005, two pioneering studies by Ashworth and Helleday laboratories confirmed that BRCA1 / 2 gene-deficient cancer cells are highly sensitive to PARP inhibition, which provided an important theoretical basis for the clinical application of PARP inhibitors. Four years later, olaparib showed significant anti-tumor activity in BRCA mutation carriers, and was approved by FDA in 2014, becoming the first PARP inhibitor for the treatment of BRCA mutation-carrying advanced breast cancer and ovarian cancer. The success of olaparib has promoted the rapid development of PARP inhibitor research and development. So far, there are 6 approved PARP inhibitors on the market.

[0004] Despite the great success of PARP inhibitors, there are still many problems to be solved, such as drug resistance, lack of biomarkers, limited expansion of indications, and challenges of combination therapy. The clinical breakthrough of PARP inhibitors in homologous recombination repair (HR) defective tumors based on the "synthetic lethality" strategy fully confirms the translational medicine value of targeting DNA damage repair (DDR) pathways, and greatly promotes the development process of DDR targeted drugs. Currently, multiple key node proteins of DDR pathways have become research hotspots in the field of tumor treatment, including ATM / ATR, CHK1 / CHK2, DNA-PK, WRN, USP1, POLQ and WEE1, etc. Among them, the research progress of WRN inhibitors is particularly eye-catching. Recently, two independent studies reported a new WRN inhibitor that selectively kills microsatellite instability (MSI) tumors through the "synthetic lethality" strategy. However, the development of DDR pathway inhibitors still faces many challenges: the candidate drugs currently in clinical research generally have significant toxic side effects, lack of target specificity, lack of reliable biomarkers, and other key problems, resulting in no DDR inhibitor targeting new targets being successfully launched so far. Therefore, developing new DNA damage response inhibitors with higher selectivity and lower toxicity to meet the urgent needs of clinical treatment is still an important research direction in the field of tumor treatment that needs to be broken through.

[0005] Natural products are a key source of innovative drug development due to their complex structures and wide range of biological activities, particularly in oncology and antibacterial therapy. Brucea javanica is a traditional Chinese medicinal material with the functions of clearing heat and resolving toxins, relieving swelling and pain, and is often used to treat diseases such as malaria, dysentery, and cancer. Bruceine A, as one of its main active ingredients, has various biological activities, including anti-tumor, anti-inflammatory, and anti-bacterial activities. Extensive research has demonstrated the efficacy of Bruceine A against various cancers, including pancreatic cancer, triple-negative breast cancer, colon cancer, and liver cancer. In recent years, with the deepening of research on natural medicines, the pharmacological effects and potential clinical application value of Bruceine A have gradually been revealed. However, the role of Bruceine A in DNA damage response has not been reported. SUMMARY

[0006] The purpose of the present application is to solve the problems existing in the prior art and provide an application of Bruceine A in the preparation of a DNA damage response inhibitor drug.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: Use of a acutin A in the preparation of a DNA damage response inhibitor drug, the inhibitor drug is based on acutin A inhibits the formation of 53BP1 and RAP80 foci in cells after DNA damage; the cell is osteosarcoma cell.

[0008] Preferably: the osteosarcoma cell is human osteosarcoma cell U2OS.

[0009] Use of a acutin A in the preparation of a DNA damage response inhibitor drug, the inhibitor drug is based on acutin A simultaneously inhibits homologous recombination repair and non-homologous end joining in cells.

[0010] Use of a acutin A in the preparation of a drug for enhancing the sensitivity of tumor cells to chemotherapeutic drugs, the tumor cells are breast cancer cells.

[0011] Preferably: the breast cancer cell is human triple negative breast cancer cell MDA-MB-231.

[0012] Preferably: the chemotherapeutic drug is a platinum-based chemotherapeutic drug.

[0013] Preferably: the platinum-based chemotherapeutic drug is cisplatin.

[0014] A pharmaceutical composition for treating or assisting in the treatment of breast cancer, comprising a therapeutically effective amount of acutin A as an active ingredient, and a pharmaceutically acceptable carrier.

[0015] The beneficial effects of the present application are: 1. The present application first found in drug screening that acutin A can effectively inhibit the DNA damage response of cells, and can also simultaneously inhibit two main DNA damage repair pathways, namely homologous recombination repair and non-homologous end joining; in addition, acutin A combined with chemotherapeutic drugs can significantly enhance the sensitivity of triple negative breast cancer cells MDA-MB-231 to the chemotherapeutic drug cisplatin.

[0016] 2. The present application uses acutin A as an active ingredient to prepare a drug with DNA damage response inhibition effect and a drug with anti-breast cancer effect and a drug with the effect of enhancing the sensitivity of breast cancer to chemotherapeutic drugs, which has important application value for obtaining a new type of DNA damage response inhibitor and for the field of tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is the experimental result graph of example 1 of the present application; Figure 2 It is the experimental result graph of example 2 of the present application; Figure 3 It is the experimental result graph of example 3 of the present application. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0019] In the following examples, U20S cells and MDA-MB-231 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). U20S cells and MDA-MB-231 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S), and passaged and all experiments were performed at 37°C in a humidified incubator containing 5% CO2 and 95% air. In the attached figure, *P < 0.05, **P < 0.01, and ***P < 0.001.

[0020] For ease of description, crotonin A will be abbreviated as BA in some places in the following embodiments and in the accompanying drawings.

[0021] Example 1: Caulis crocin A inhibits the formation of 53BP1 foci after DNA damage. Cell imaging and statistical analysis were performed using a high-content screening system. 5000 U20S-GFP-Tudor / U2OS-GFP-RAP80 cells were seeded per well in black 384-well plates and cultured for 12-24 hours. After cell attachment, the cells were pretreated for 1 hour with crotonin A (10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003 uM) / ATMi (KU-55933, 10 uM). Then, 2 uM bleomycin was added to each well to induce DNA damage. After 2 hours, high-content images were taken, and the data were analyzed using the "ImageAnalysis" module. One-way ANOVA was then performed using GraphPad Prism9 to compare different groups.

[0022] The effect of crocin A (30 nM) on the formation of exogenous 53BP1 foci was investigated using cell smear immunofluorescence assay. First, smears matching the well size were placed in 24-well plates, and U2OS cells were spaced at 5 × 10⁶ cells per well. 4Inoculation, culture for 12-24 hours, wait for cell adhesion, add drug treatment in groups, blank control group (no treatment), positive drug control group (KU-55933 pretreatment for 1 hour, then add 2uM Bleomycin), experimental group (Kuwanons A pretreatment for 1 hour, then add 2uM Bleomycin), 2 hours later, sample collection for immunofluorescence experiment. First, discard the DMEM culture medium, wash three times with PBS, use 4% paraformaldehyde to fix at room temperature for 10 minutes, discard the fixing solution, wash three times with PBS, add PBS containing 0.2% Triton X-100, slowly punch on the shaker for 15 minutes at room temperature, discard, then wash three times with PBS, 10 minutes each time, then use PBS containing 5% BSA to slowly block on the shaker for 1 hour, after blocking, wash three times with PBS, then discard the blocking solution, dilute 53BP1 with 5% BSA (1:1000) and incubate overnight at 4°C, the next day, discard the primary antibody, wash three times with PBS, 10 minutes each time, in the dark, dilute the fluorescent secondary antibody with 5% BSA and incubate at room temperature for 1 hour, then dilute DAPI (1:1000) with PBS and stain the nucleus for 10 minutes at room temperature in the dark, discard, wash the slide with PBS slowly on the shaker three times, 10 minutes each time. Finally, carefully remove the slide from the well plate, let it dry, use anti-fluorescence quenching mounting medium to mount the slide, observe under a fluorescence microscope and collect images, and analyze the foci formation and results.

[0023] The results are shown in Figure 1 As shown in the results, the half-inhibitory concentration of Kuwanons A inhibiting Tudor foci formation is 12.63uM, and the inhibitory effect of Kuwanons on Tudor foci shows a concentration-dependent effect. Cell slide immunofluorescence experiments also confirmed that 30nM Kuwanons A can effectively inhibit the formation of endogenous 53BP1 foci, and can also inhibit the formation of RAP80 foci in a concentration-dependent manner.

[0024] Example 2: Effect of Kuwanons A on the two main DNA damage repair pathways of HR and NHEJ U2OS cells of DR-GFP and EJ5-GFP reporter system were inoculated at 5×10 5 Cells were inoculated in a 6-well plate, five wells per cell, cultured for 12-24 hours, and waited for cell adhesion. 2ug endonuclease I-SceI was transfected per well using PEI transfection reagent, and at the same time, Kuwanons A was added for treatment (0, 10, 30, 100nM). 6-8 hours later, the transfection solution was changed while maintaining the Kuwanons A drug treatment. 48 hours later, the cells were collected and detected using a BD flow cytometer.

[0025] The results are shown in Figure 2The statistical results of three independent repeated experiments are shown. Compared with the blank group, the fluorescence increased after transfection of endonuclease I-SceI, indicating successful transfection. After treatment with acutunine A, the fluorescence intensity decreased significantly and showed a concentration-dependent manner, indicating that acutunine A treatment affected both homologous recombination and non-homologous end joining repair pathways. This further proves the effect of acutunine A on DNA damage repair.

[0026] Example 3: Acutunine A can enhance the sensitivity of triple-negative breast cancer to cisplatin; Since the enhancement of DNA damage repair ability is one of the important mechanisms of tumor cells to produce chemotherapy resistance, small molecules that inhibit the DNA damage repair pathway are expected to overcome chemotherapy resistance and improve the efficacy of chemotherapy. Therefore, by combining acutunine A with the chemotherapy drug cisplatin, the effect of acutunine A as a DNA damage inhibitor was further demonstrated. The effect of acutunine A on the cell viability of MDA-MB-231 cells was detected using CellTiter-Glo® 2.0 cell viability assay kit, and the effect of 30 nM acutunine A combined with cisplatin on the cell viability of MDA-MB-231 cells was detected compared with cisplatin alone. 400 cells per well were seeded in a 96-well plate and cultured for 12-24 hours to allow the cells to adhere. When detecting the cell viability of MDA-MB-231 cells treated with acutunine A (0.0015, 0.0045, 0.0137, 0.0411, 0.1234, 0.3703, 1.1111, 3.3333, 10 uM) for 6 days, the CellTiter-Glo® 2.0 Reagent and cells were equilibrated to room temperature. 100 uL of CellTiter-Glo® 2.0 Reagent was added to each well, the contents were mixed to induce cell lysis, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. Then the absorbance was measured using a microplate reader to evaluate the cell viability. The relative cell viability was calculated according to the optical density value. Then the IC50 value was calculated according to the relative cell viability using GraphPad Prism 8 (GraphPad Software, Inc.). When detecting the cell viability of MDA-MA-231 cells treated with cisplatin alone (0.0050, 0.0152, 0.0457, 0.1371, 0.4115, 1.2345, 3.7037, 11.1111, 33.3333, 100 uM) for 6 days, the combination group used 30 nM acutunine A combined with cisplatin.

[0027] The results are shown in Figure 3 The CellTiter-Glo® 2.0 cell viability detection results showed that the IC50 of acutunine A acting on MDA-MB-231 cells was 59.76 0.03nM, based on this, in the treatment of cisplatin combination, select 30nM BA and cisplatin combination treatment MDA-MB-231 cells, the results observed, compared with cisplatin monotherapy, combined with 30nM BA treatment significantly enhanced the sensitivity of MDA-MB-231 cells to cisplatin. Based on these findings, we conclude that the effective DNA damage response inhibitor, is expected to provide a combination of drug resistance to solve chemotherapy.

[0028] The above, only for the preferred embodiments of the present application, but the scope of protection of the present application is not limited to this, any skilled in the art of the technical personnel in the technical range of the present application disclosed, according to the technical scheme of the present application and the inventive concept of equivalent replacement or change, should be covered within the scope of protection of the present application.

Claims

1. Use of a acutubine A in the preparation of a medicament for inhibiting DNA damage response, characterized in that, The inhibitor drug is based on the inhibition of 53BP1 and RAP80 foci formation in cells after DNA damage by acetylbaccatin A.

2. The use of avarol in the preparation of a DNA damage response inhibitor agent according to claim 1, characterized in that, The cell is an osteosarcoma cell.

3. The use of avarol in the preparation of a DNA damage response inhibitor agent according to claim 2, characterized in that, The osteosarcoma cell is human osteosarcoma cell U2OS.

4. Use of a acutubine A for the manufacture of a medicament for inhibiting DNA damage response, characterized in that, The inhibitor drug is based on the inhibition of homologous recombination repair and non-homologous end joining in cells by acetylbaccatin A.

5. Use of acetylbaccatin A in the preparation of a medicament for enhancing the sensitivity of tumor cells to chemotherapeutic drugs.

6. Use according to claim 5, characterized in that, The tumor cell is a breast cancer cell.

7. Use according to claim 6, characterized in that, The breast cancer cell is human triple negative breast cancer cell MDA-MB-231.

8. Use according to claim 6, characterized in that, The chemotherapeutic drug is a platinum-based chemotherapeutic drug.

9. Use according to claim 8, characterized in that, The platinum-based chemotherapeutic drug is cisplatin.

10. A pharmaceutical composition for treating or adjuvant treatment of breast cancer, characterized in that, A pharmaceutical composition comprising a therapeutically effective amount of acetylbaccatin A as an active ingredient, and a pharmaceutically acceptable carrier.

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