New application of ginkgetin in treatment of lung cancer

Abstract

The invention discloses a novel application of ginkgo biflavone in lung cancer treatment, belongs to the technical field of medicines, and researches show that ginkgo biflavone can target CDC7 and is a novel targeted medicine. It is confirmed for the first time that ginkgo biflavone regulates and controls the thermal stability and enzymatic degradation of ginkgo biflavone through targeting CDC7 kinase (binding free energies-8.3 kcal / mol), directly intervenes in the DNA replication process, and provides a natural drug lead compound for development of a novel CDC7 small-molecule inhibitor. The compound has great significance in preventing and treating malignant tumors, and has a wide prospect in the aspect of preparing medicines for treating lung tumors.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a new application of ginkgetin in the treatment of lung cancer. Background Art

[0002] Traditional treatment strategies for lung cancer include surgical resection, radiotherapy, chemotherapy, traditional Chinese medicine treatment, etc. With the development of modern technology, the treatment of lung cancer has entered the era of precision medicine. Targeted therapy and immunotherapy have greatly improved the clinical efficacy of advanced lung cancer, but they are only applicable to some patients. Regardless of the progress of emerging treatment methods and drug research and development, the global burden of lung cancer is constantly increasing. Therefore, studying new and effective anti-cancer molecules and related pathways to address the occurrence, development, extend the life expectancy, and improve the quality of life of lung cancer patients has become a current hotspot.

[0003] Existing literature [Suski, J.M., Ratnayeke, N., Braun, M. et al. CDC7-independent G1 / S transition revealed by targeted protein degradation. Nature 605, 357–365 (2022)] has shown that simultaneously targeting CDC7 and CDK1 may be an effective cancer treatment strategy.

[0004] Existing patent CN106166149A provides a new application of biflavonoids. Biflavonoids (especially ginkgetin) have the effect of preventing and treating lung cancer (especially non-small cell lung cancer). Experimental results show that ginkgetin has strong cytotoxic effects on A549 non-small cell lung cancer, can significantly increase the reduction rate of mitochondrial membrane potential and the production of reactive oxygen species, increase the expression of apoptosis-related proteins and key autophagy proteins, and induce apoptosis of A549 non-small cell lung cancer; it can significantly inhibit the tumor growth of A549 tumor-bearing nude mice, has good anti-tumor activity against A549 lung cancer-bearing nude mice, and has no obvious toxicity; when ginkgetin is combined with cisplatin, a commonly used drug for treating lung cancer clinically, it can increase the anti-cancer effects of cisplatin and ginkgetin alone and reduce the toxicity of cisplatin. Biflavonoids (especially ginkgetin) can be used to prepare drugs or health products for preventing and treating lung cancer (especially non-small cell lung cancer).

[0005] However, the above patent has not conducted in-depth research on the specific anti-cancer mechanism of ginkgetin, nor has it revealed the specific mechanism of action of ginkgetin. Based on this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a new application of ginkgetin in the treatment of lung cancer to determine the specific mechanism of action of biflavonoids in the treatment of lung cancer.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] New application of ginkgetin in the treatment of lung cancer.

[0009] Furthermore, ginkgetin targets CDC7 to combat lung cancer.

[0010] Furthermore, the ginkgetin is used as one of the active pharmaceutical ingredients for treating lung cancer.

[0011] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0012] Furthermore, the pharmaceutical dosage form is at least one of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections, inhalants or sprays.

[0013] Furthermore, the administration concentration of the ginkgetin is 10 - 20 μM.

[0014] Principle and beneficial effects of this technical solution:

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] As a core kinase in the initial stage of DNA replication, the overexpression of CDC7 is closely related to the abnormal proliferation of the tumor cell cycle. The present invention for the first time demonstrates that ginkgetin regulates its thermal stability and enzymatic degradation by targeting the CDC7 kinase (binding free energy -8.3 kcal / mol), directly intervening in the DNA replication process, providing a natural drug lead compound for the development of novel CDC7 small molecule inhibitors. This is of great significance for the prevention and treatment of malignant tumors and has broad prospects in the application of preparing drugs for treating lung tumors. Description of the Drawings

[0017] Figure 1 Volcano plot of differentially expressed proteins upregulated or downregulated after non-small cell lung cancer cell line A549 was treated with ginkgetin (0 μM, 20 μM) for 48 h.

[0018] Figure 2 GO enrichment analysis (biological process, cellular component, and molecular function) of differentially expressed proteins after non-small cell lung cancer cell line A549 was treated with ginkgetin (0 μM, 20 μM) for 48 h. The metabolic pathways involved in the differentially expressed proteins were analyzed using the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG).

[0019] Figure 3Gene set enrichment analysis (GSEA) of differentially expressed proteins in non-small cell lung cancer cell line A549 after treatment with ginkgetin (0 μM, 20 μM) for 48 h.

[0020] Figure 4 Unpaired sample expression difference T-test analysis of the targets enriched by GSEA between lung adenocarcinoma and adjacent normal tissues. The results are expressed as mean ± standard deviation. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical significance.

[0021] Figure 5 The mRNA levels of differentially expressed genes in DIA proteomics were verified by qPCR after treatment with different concentrations of ginkgetin (0 μM, 5 μM, 10 μM, 20 μM) for 48 h. The results are expressed as mean ± standard deviation. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical significance.

[0022] Figure 6 The representative bands and semi-quantitative statistical results of the protein levels of differentially expressed genes in DIA proteomics were verified by Western blot after treatment with different concentrations of ginkgetin (0 μM, 5 μM, 10 μM, 20 μM) for 48 h. The results are expressed as mean ± standard deviation. Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no statistical significance.

[0023] Figure 7 (A) Molecular docking to simulate the binding potential of ginkgetin to CDC7 kinase; (B) CETSA experiment to detect the effect of ginkgetin on the thermal stability of CDC7 protein at different temperatures; (C) DARTS experiment to detect the effect of different concentrations of ginkgetin on the degradation of CDC7 protein by Pronase E. Detailed implementation manners

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and implementation manners:

[0025] The effects of 7 cycad small molecule compounds (catechin, naringenin, sciadopitysin, hinokiflavone, bilobetin, amentoflavone, ginkgetin) at different doses on the proliferation viability of A549 cells after treatment for 24 h and 48 h were detected by CCK-8 method. Ginkgetin showed the best inhibitory activity against the proliferation of lung adenocarcinoma cells after administration for 24 h and 48 h, and the inhibitory ability increased with the increase of concentration. Therefore, ginkgetin was used as the main small molecule for research, and the specific implementation process is as follows.

[0026] I. DIA Proteomics Reveals Potential Targets and Mechanisms of Action of Ginkgo Biloba Flavones in Inhibiting the Growth of Non-Small Cell Lung Cancer

[0027] The human non-small cell lung cancer A549 cell line was treated with 0 μM (control group) and 20 μM ginkgo biloba flavones for 48 hours, and then whole proteome analysis was performed using DIA proteomics technology. By setting the differential expression screening criteria (fold change in expression > 2.0 and P value < 0.05), a total of 732 significantly differentially expressed proteins were identified, including 315 up-regulated proteins and 417 down-regulated proteins, as Figure 1 shown.

[0028] The gene ontology (GO) database was used to perform multi-dimensional functional annotation analysis of the differentially expressed proteins, covering three major categories: biological processes, cellular components, and molecular functions. At the same time, pathway enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database to systematically reveal the biological pathway network involved in the differential proteins. The results showed that it might be closely related to extracellular matrix remodeling, ECM-receptor interaction, Wnt signaling pathway, PI3K-Akt signaling pathway, etc., as Figure 2 shown in A, 2B, 2C, 2D.

[0029] The GSEA 3.0 software (http: / / www.gsea-msigdb.org / gsea / index.jsp) was used to perform GSEA enrichment analysis on the differentially expressed data set, and 6 key pathways were significantly enriched, including NABA_MATRISOME, WP_PI3KAKT_SIGNALING_PATHWAY, KEGG_PATHWAYS_IN_CANCER, WP_TGFBETA_SIGNALING_PATHWAY, KEGG_CELL_CYCLE, REACTOME_SUMOYLATION, etc., as Figure 3 shown.

[0030] Independent sample t-tests were performed on the target proteins in the above GSEA enrichment pathways in the TCGA-LUAD database and the TCGA_GTEx-LUAD database. Through statistical analysis, it was found that there were significant differences in the expression of 72 targets between lung adenocarcinoma tissues and normal tissues (P < 0.05), as Figure 4 shown.

[0031] II. Verification of DIA Proteomics of Ginkgo Biloba Flavones Treated A549 Cells

[0032] To verify the results of DIA proteomics, real-time fluorescence quantitative PCR was first used to verify the targets enriched by GSEA at the mRNA level. The results showed that after ginkgetin intervention, the mRNA expressions of S100A3, CDC25B, CDC7, CCND3, SCMH1, NCOR2, etc. were decreased, while the mRNA expressions of CTGF (CCN2), CRIM, etc. were increased. Among them, 10 μM and 20 μM were the most significant, as Figure 5 shown.

[0033] Then, combined with the results of DIA protein quantitative analysis, bioinformatics differential expression analysis and GSEA analysis results, three differentially expressed genes (DEGs: CDC7, CDC25B, CTGF) were further selected for verification by Western blot. The results showed that only CDC7 showed a consistent trend with mRNA and had significant differences (P < 0.05). Therefore, it can be considered that CDC7 is a key target for ginkgetin to exert its anti-lung cancer effect, as Figure 6 shown.

[0034] III. Molecular mechanism of ginkgetin targeting and binding to CDC7 kinase to regulate its thermal stability and enzymatic degradation

[0035] The molecular docking results showed that ginkgetin could stably bind to the active pocket of CDC7 protein through multi-modal molecular interactions. Key amino acid residues such as GLN-215, ARG-100, ARG-373, ALA-374, GLY-375, etc. participated in the interaction. The molecular docking calculation showed that the binding free energy was -8.3 kcal / mol, indicating strong binding stability, as Figure 7 shown in A.

[0036] The expression of CDC7 protein in the DMSO treatment group and the ginkgetin treatment group was investigated at different temperatures (40℃ - 65℃). The CETSA results showed that compared with the DMSO control group, the Ginkgetin treatment group still maintained a significant CDC7 protein band intensity under high temperature conditions, while the control group showed obvious degradation at 60℃. This result confirmed that Ginkgetin could significantly enhance the thermal stability of CDC7 protein, as Figure 7 shown in B.

[0037] After treatment with different concentrations of ginkgetin (0 - 160 μM) at a fixed concentration of Pronase E (1 μg / ml), the expression of CDC7 protein was detected. Using GAPDH as an internal reference, the results showed that with the increase of the ginkgetin administration concentration, the intensity of the CDC7 protein band gradually weakened, suggesting that ginkgetin might enhance the degradation of CDC7 protein by Pronase E, which provided evidence for the mechanism study of ginkgetin acting on CDC7, asFigure 7 as shown in C

[0038] The present invention provides a potential new anti-tumor drug for the treatment of lung cancer, and it targets CDC7, providing a new targeted drug for anti-cancer. It is of great significance for the prevention and treatment of malignant tumors and has broad prospects in the application of preparing drugs for treating lung tumors. There is no reported public literature yet.

[0039] The above are only embodiments of the present invention. Specific technical solutions and / or common knowledge such as characteristics known to the public are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect and practicality of the present invention.

Claims

1. New application of ginkgetin in the treatment of lung cancer.

2. The application according to claim 1, wherein: Ginkgetin targets CDC7 to treat lung cancer.

3. The application according to claim 2, wherein: The ginkgetin is used as one of the active pharmaceutical ingredients to treat lung cancer.

4. The application according to claim 3, wherein: The drug includes a pharmaceutically acceptable carrier.

5. The application according to claim 4, wherein: The dosage form of the drug is at least one of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections, inhalants or sprays.

6. Any of the applications according to claims 1-5, characterized in that: The administration concentration of the ginkgetin is 10 - 20 μM.

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