Application of flavonoid natural product Pervianoside I in preparation of medicine for resisting retinoblastoma

Abstract

The invention discloses an application of a flavonoid natural product Peruvianoside I in preparation of an anti-retinoblastoma drug, belongs to the field of medicinal chemistry, and has the following structure: the compound can selectively inhibit LSD1 enzyme activity and can be used as the anti-retinoblastoma drug. Experiments find that the anti-tumor action mechanism is related to inhibition of retinoblastoma cell migration. As a selective histone lysine specific demethylase LSD1 inhibitor, the compound has the potential of developing the retinoblastoma resisting medicine.

Description

Technical Field

[0001] This invention relates to the field of antitumor medicinal chemistry, specifically to histone lysine-specific demethylase LSD1 inhibitors and their application as anti-retinoblastoma drugs, and particularly to the flavonoid natural product Peruvianoside I. Background Technology

[0002] Retinoblastoma (Rb) is a malignant tumor originating from photoreceptor precursor cells. It commonly affects children under 3 years old, has a familial genetic predisposition, and can affect one or both eyes sequentially or simultaneously. It is the most common intraocular malignant tumor in infants and young children, and is rare in adults. Histone lysine-specific demethylase LSD1 is an enzyme involved in the epigenetic control of gene expression. It synergistically regulates the balance of histone lysine methylation status with its corresponding histone lysine methyltransferase. Many diseases, including cancer, may be related to abnormal transcriptional regulation induced by unbalanced histone lysine methylation. There is an urgent clinical need to develop novel inhibitors targeting the anti-tumor target LSD1 with anti-retinoblastoma effects. Summary of the Invention

[0003] Based on the current state of technology and urgent market needs, the purpose of this invention is to provide a selective inhibitor of histone lysine-specific demethylase LSD1 through high-throughput virtual screening, and to apply it in anti-retinoblastoma drugs.

[0004] To achieve the objective of this invention, the technical solution is as follows: This invention has identified Peruvianoside I, a flavonoid natural product, as an inhibitor of the histone lysine-specific demethylase LSD1 through experimental screening. Its structural formula is as follows:

[0005] This invention experimentally confirms that the flavonoid natural product Peruvianoside I, as an inhibitor of histone lysine-specific demethylase LSD1, can selectively inhibit the enzyme activity of LSD1 and has in vitro and in vivo anti-retinoblastoma activity. Its anti-tumor mechanism is related to the inhibition of retinoblastoma cell migration.

[0006] Advantages and innovations of this invention: 1. It was discovered that this compound can selectively inhibit LSD1 enzyme activity, making it a potential drug for treating retinoblastoma. 2. Its anti-tumor mechanism of action was found to be related to the inhibition of retinoblastoma cell migration. It has the potential to be developed into a new anticancer drug, laying the foundation for the development of drugs to treat retinoblastoma. Attached Figure Description

[0007] Figure 1Before and after microscopic comparison (0 μM and 5 μM) of the flavonoid natural product Peruvianoside I inhibiting the migration of SO-RB50 retinoblastoma cells. Detailed Implementation

[0008] To better illustrate the present invention, the following embodiments are provided: Example 1: LSD1 enzyme inhibitory activity and selectivity of Peruvianoside I, a flavonoid natural product The aforementioned flavonoid natural product Peruvianoside I was prepared into test sample solutions of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg using DMSO. In each well of a 96-well plate, 7 μL of LSD1 buffer, 4 μL of LSD1 (0.1 mg / mL), 1 μL of the aforementioned test sample solution, 1 μL of purified water, and 1 μL of transphenylcyclopropanediol were added. At 37°C, 1 μL of histone peptide H3K4me2 (0.5 mM), 6 μL of Cellestial™ Red, and 2 μL of HRP (horseradish peroxidase) were added. Fluorescence was detected at an excitation wavelength of 570 nm and an emission wavelength of 590 nm. The experiment was repeated three times, and the IC50 values ​​of the LSD1 enzyme inhibitory activity of the three compounds were calculated using GraphPad software. Using the same method, LSD1 was replaced with homologous enzymes MAO-A and MAO-B (purchased from Abcam) to determine the inhibitory activities of MAO-A and MAO-B enzymes. The positive control for enzyme activity was LSD1-IN-12 (purchased from MCE, catalog number HY-144673). The enzyme activity results are shown in Table 1.

[0009]

[0010] The experimental results in Table 1 show that the LSD1 inhibitory activity of the flavonoid natural product Peruvianoside I is 0.32 μM, which is stronger than the previously reported LSD1 inhibitor LSD1-IN-12. Therefore, the flavonoid natural product Peruvianoside I is a potent LSD1 inhibitor.

[0011] Meanwhile, the flavonoid natural product Peruvianoside I exhibits 29.6 times and 37.8 times the activity of MAO-A and MAO-B enzymes, respectively, compared to LSD1 enzyme activity. Therefore, the flavonoid natural product Peruvianoside I is also a selective LSD1 inhibitor.

[0012] Example 2: Anti-retinoblastoma cell activity of the flavonoid natural product Peruvianoside I Peruvianoside I, a flavonoid natural product, was prepared into test sample solutions at concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μM using DMSO. Retinoblastoma cells in logarithmic growth phase (SO-RB50, Y79, or WERI-RB1) were seeded into 96-well plates and cultured for 24 h. The culture medium was then discarded, and the test sample solutions were added. After 48 h, 20 μL of MTT was added to each well, and the cells were cultured for another 2 h. The liquid was then aspirated, and 50 μL of DMSO was added. The mixture was shaken thoroughly, and the absorbance was measured at 490 nm using a microplate reader. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - absorbance of the treated group / absorbance of the blank group) × 100%. The IC50 value was calculated using SPSS software. The antitumor drug 5-fluorouracil was used as a control. The experimental results are shown in Table 2.

[0013]

[0014] The experimental results in Table 2 show that the IC50 values ​​of the flavonoid natural product Peruvianoside I in inhibiting retinoblastoma cells are superior to those of the antitumor drug 5-fluorouracil. Therefore, the flavonoid natural product Peruvianoside I is a potent anti-retinoblastoma drug.

[0015] Example 3: Animal-level efficacy experiment against retinoblastoma Approximately 2×10 7 SO-RB50 retinoblastoma cells were subcutaneously inoculated into 21 nude mice, and the tumor volume was increased to 150 mm. 3 At approximately 18 days, the mice were randomly divided into three groups of seven each. The control group received intraperitoneal injections of either the flavonoid natural product Peruvianoside I or the positive control drug 5-fluorouracil, while the control group received distilled water. The drug group received 60 mg / kg every three days, with regular observation for a total of 18 days. Tumor growth and tumor inhibition rate were measured. Tumor inhibition rate = (average tumor volume in the control group - average tumor volume in the drug group) / average tumor volume in the control group × 100%. The results are shown in Table 3. The flavonoid natural product Peruvianoside I effectively inhibited the in vivo tumorigenesis of retinoblastoma cells SO-RB50, with a tumor inhibition rate of 49.09%, significantly better than that of the positive control drug 5-fluorouracil.

[0016]

[0017] Example 4: The flavonoid natural product Peruvianoside I inhibits the migration of SO-RB50 retinoblastoma cells. Peruvianoside I, a flavonoid natural product, was prepared into a 5 μM test solution using DMSO. Retinoblastoma cells (SO-RB50) in logarithmic growth phase were seeded into Transwell plates and cultured for 24 h. The culture medium was then discarded, and the test solution was added. Cells treated with the drug were collected, stained with hematoxylin, and cultured at 37 °C for 20 min. The cells were then observed under a microscope. The experimental results are as follows: Figure 1 As shown, the number of migrating cells in the 5 μM Peruvianoside I treatment group was significantly lower than that in the group without Peruvianoside I, indicating that Peruvianoside I inhibits the migration of SO-RB50 retinoblastoma cells. Therefore, Peruvianoside I, as a selective LSD1 inhibitor, has a unique anti-retinoblastoma mechanism of action, which is related to the inhibition of retinoblastoma cell migration.

Claims

1. The application of Peruvianoside I, a flavonoid natural product with the following chemical structural formula, in pharmaceutical preparation, characterized in that... As an active ingredient, it is used to prepare an inhibitor of histone lysine-specific demethylase LSD1. 。 2. The application of Peruvianoside I, a flavonoid natural product as described in claim 1, in pharmaceutical preparation, characterized in that, It was used to prepare drugs for treating retinoblastoma.

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