Use of a separating enzyme inhibitor for antitumor purposes

Abstract

The application discloses application of a separating enzyme inhibitor in preparation of an antitumor drug, and the separating enzyme inhibitor is toxoflavin and a derivative thereof. The compound can inhibit, at a molecular level, cleavage activity of an important proteinase, namely separating enzyme, on one subunit Scc1 of a mucin; at a cellular level, the toxoflavin derivative Walrycin B can inhibit proliferation of cancer cells such as human cervical cancer (HeLa), liver cancer (HepG2), breast cancer (MCF-7) and prostate cancer (PC3), and can arrest the cell cycle of the cancer cells at a G2 / M phase; and at an allogeneic transplantation tumor animal model, the Walrycin B can significantly inhibit growth of a mouse tumor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to an application of a separase inhibitor in anti-tumor. BACKGROUND

[0002] The proliferation of eukaryotic cells is realized through mitosis. It has been proved that the intervention of the mitosis process of cells is a direct and effective strategy for inhibiting the growth of cancer cells. The anti-mitotic agents currently used in clinic, such as Paclitaxel and Ixabepilone, have been widely applied in the treatment of various cancers, such as breast cancer, ovarian cancer, lung cancer and prostate cancer. The direct target of these drugs in vivo is tubulin. Tubulin is responsible for the migration of chromosomes during mitosis, and loses the migration ability after being combined with drugs such as Paclitaxel, so that the sister chromatids cannot be normally separated, resulting in the inhibition of cell division and apoptosis.

[0003] Separase is a member of the CD subfamily of cysteine proteases, which is distributed in eukaryotes from fungi to human, and is an essential gene for cell division in eukaryotes. Separase has high specificity for substrates and is only activated during mitosis. During most of the cell cycle, separase is in an inactive state combined with its inhibitory chaperone, securin, until the cell is in the process of moving from metaphase to anaphase, and securin is degraded by the proteasome mediated by the anaphase-promoting complex or cyclosome (APC / C), and then separase is released and activated. The activated separase can specifically recognize and cut the Scc1 subunit of cohesin to make it fall off from the chromosome, and the sister chromatids are released and enter two daughter cells under the traction of spindle fibers at both ends of the cell, and the cell is thus divided into two. Therefore, the drug discovery and drug design targeting separase can open up a new way for the development of new anti-tumor drugs with low toxicity and side effects. SUMMARY

[0004] The present application aims to provide an application of a separase inhibitor in anti-tumor.

[0005] To achieve the above object, the present application adopts the following technical scheme:

[0006] The present application protects the application of toxoflavin and its derivatives as a key protease in the mitosis process of human cells, separase, in the preparation of anti-tumor drugs.

[0007] Further, the toxoflavin derivatives are Walrycin B, 3-Methyltoxoflavin, 3-Phenyltoxoflavin. The structural formulas of each compound are as follows:

[0008] .

[0009] The present application has the following advantages:

[0010] At the molecular level, the toxoflavin and its derivatives can inhibit the cleavage activity of human decatenation enzyme on one subunit Scc1 of cohesin; at the cellular level, the toxoflavin derivative Walrycin B can inhibit the proliferation of human cervical cancer (HeLa), liver cancer (HepG2), breast cancer (MCF-7) and prostate cancer (PC3) cells, and can arrest the cell cycle at the G2 / M phase; in the xenograft tumor animal model, Walrycin B can significantly inhibit the growth of mouse tumors. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 It is a polyacrylamide gel electrophoresis (SDS-PAGE) diagram of the human decatenation enzyme protein purified in Example 1.

[0012] Figure 2 It is a schematic diagram of the principle of the fluorescence polypeptide probe method for determining the activity of decatenation enzyme in Example 2 (upper diagram) and the inhibition curve of toxoflavin and its derivatives on human decatenation enzyme (lower diagram).

[0013] Figure 3 It is the Western blot method for determining the inhibition activity of toxoflavin and its derivatives on decatenation enzyme in Example 2.

[0014] Figure 4 It is the cycle distribution state diagram of HeLa and HepG2 cells after treatment with toxoflavin derivative Walrycin B in Example 3.

[0015] Figure 5 It is the cell activity diagram of HeLa, HepG2, MCF-7 and other cells after treatment with toxoflavin derivative Walrycin B in Example 4.

[0016] Figure 6A: Schematic diagram of the administration time and administration method of animal experiment, the experimental mice were divided into 4 groups, 5 mice in each group; B: After the experiment, the tumor entity taken out from the dissection mouse; C: Inhibition curve of Walrycin B on the growth of mouse tumor volume; D: Effect of Walrycin B on the weight of tumor in mice; E: Effect of Walrycin B on the weight of mice (**P < 0.01%; ***P < 0.001%). DETAILED DESCRIPTION

[0017] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described below in combination with specific embodiments, but the application is not limited thereto.

[0018] Example 1 Purification of Separase Protein

[0019] The Securin 138-202 The gene covalently combined with full-length human separase was constructed into an insect cell expression vector pFastBac, and then the recombinant plasmid was transformed into a DH10bac strain to extract a baculovirus shuttle vector Bacmid carrying the separase gene. The Bacmid was transfected into sf9 insect cells to prepare baculovirus. The sf9 cells were infected with the baculovirus to recombinantly express the separase protein in the cells. The sf9 cells expressing the separase protein were collected, resuspended with a lysis buffer containing 20 mM HEPES pH 7.2, 500 mM NaCl, 5% glycerol and 1 mM DTT, broken, centrifuged at 18000 rpm for 30 min at 4°C to collect the supernatant, purified through a StrepTactin affinity column, aliquoted and stored at -80°C for standby.

[0020] Figure 1 Polyacrylamide gel electrophoresis diagram of the purified human separase protein. From left to right: marker, purified human separase protein, purified human separase protein Figure 1 It can be seen that the purified separase has high purity and can be used for subsequent research.

[0021] Example 2 Inhibition activity determination of toxoflavin and its derivatives on separase

[0022] (1) Fluorescent polypeptide probe method: the substrate polypeptide Ac-MDDREIMR-AMC is cut by the separase to produce free AMC, which can be detected by an enzyme label instrument at an excitation wavelength of 345 nm and an emission wavelength of 445 nm, so as to determine the cutting activity of the separase. The higher the cutting activity of the separase, the higher the fluorescence value.

[0023] Specifically, 500 nM of the enzyme was pre-incubated with a series of concentrations of flavin and its derivatives in an activity buffer (50 mM HEPES pH 7.2, 20 mM NaCl, and 1 mM DTT) at room temperature for 15 min. Then, the substrate peptide Ac-MDDREIMR-AMC was added, and the reaction was carried out at 37°C for 1 h. The fluorescence value of AMC was detected using a multi-functional microplate reader at an excitation wavelength of 345 nm and an emission wavelength of 445 nm (each concentration was repeated three times). The obtained data were used to fit a curve using Graphpad to obtain the IC50 values ​​of flavin and its derivatives. 50 Value, result as Figure 2 As shown.

[0024] Depend on Figure 2 It is evident that toxin and its derivatives have a significant inhibitory effect on the cleavage activity of human isosorbide dinitrate, with an IC50 value of [missing information]. 50 The values ​​are shown in Table 1.

[0025] Table 1. IC50 of the inhibitory effect of toxin-flavin derivatives on human isolating enzyme activity 50 value

[0026]

[0027] (2) Western blot method: 500 nM dissociation enzyme and toxoflavin and its derivatives were pre-incubated in an activity buffer (50 mM HEPES pH 7.2, 20 mM NaCl, and 1 mM DTT) at room temperature for 15 min. Simultaneously, the Myc-tagged Scc1 fusion protein was expressed using a protein translation kit (Promega). The Myc-Scc1 protein was added to the mixture of dissociation enzyme and compound, and the reaction was carried out at 37°C for 1 h. The reactants were separated by SDS-PAGE, transferred to a membrane, and then detected by Western blot using an anti-Myc antibody. The results are shown below. Figure 3 As shown.

[0028] Depend on Figure 3 It is evident that toxin and its derivatives have a significant inhibitory effect on human isolating enzyme.

[0029] Example 3: Effects of the toxin derivative Wallycin B on the cell cycle progression of cancer cells.

[0030] Cells have different DNA content at different cell cycle stages. Normally, cells have diploid DNA content (2N) at G1 / G0 stage, tetraploid DNA content (4N) at G2 / M stage, and DNA content between diploid and tetraploid at S stage. Propidium iodide (PI) can bind to DNA, and its fluorescence intensity is positively correlated with the DNA content in cells. Therefore, when detecting the DNA content in cells by flow cytometry PI staining method, the G1 / G0 stage, S stage and G2 / M stage of cell cycle can be distinguished. The flow cytometry histogram obtained can be used to calculate the percentage of cells at each phase of the cell cycle by FlowJo software.

[0031] Inhibition of the separation enzyme activity will cause the sister chromatid separation to be blocked, thus causing abnormal cell cycle progression. First, Hela and HepG2 cells were synchronized by Thymidine, and then released for 20 h, while incubated with 0, 5.0, 10.0, 20.0 μM of the thymine derivative Walrycin B. Then the distribution state of the cell cycle after 20 h was determined by flow cytometry, and the results are shown in Figure 4 .

[0032] From Figure 4 It can be seen that Thymidine treatment can synchronize most of the cells to the G1 / G0 stage (0 h), and in the cells treated with the thymine derivative Walrycin B, the proportion of G2 / M stage cells significantly increases with the increase of drug concentration, indicating that Walrycin B significantly interferes with the cell cycle of Hela and HepG2 cells, and blocks them at the G2 / M stage.

[0033] Example 4 Determination of the anti-tumor activity of the thymine derivative Walrycin B at the cell level

[0034] MTT method was used to determine the inhibitory activity of the thymine derivative Walrycin B on tumor cell proliferation. Human cervical cancer (HeLa), liver cancer (HepG2), breast cancer (MCF-7) and prostate cancer (PC3) cells were inoculated in 96-well plates, 100 μL of cell suspension per well, with 3 repeated holes. After incubation in a 37℃, 5% CO2 incubator until the cells adhered, a series of 2-fold diluted thymine derivative Walrycin B (10 μL) was added to the cell culture solution, and then incubated in a 37℃, 5% CO2 incubator for 72 h. 0.5 mg / mL MTT solution was added, and incubated at 37℃, 5% CO2 for 4 h. Finally, the supernatant was discarded, 100 μL of DMSO was added and incubated for about 30 min, and then the light absorption value was determined at a wavelength of 570 nm, and the results are shown in Figure 5 .

[0035] Depend on Figure 5 It is evident that the toxin derivative Wallycin B exhibits significant inhibitory activity against the proliferation of HeLa, HepG2, MCF-7, PC3, and mouse breast cancer cells 4T1, with an IC50 value of [missing information]. 50 The values ​​were 5.02 μM, 1.93 μM, 1.95 μM, 0.21 μM, and 5.8 μM, respectively.

[0036] Example 5: Determination of the antitumor activity of the toxoflavone derivative Wallycin B at the animal level

[0037] Establishment of mouse tumor model and drug treatment: approximately 10 6 4T1 mouse breast cancer cells were transplanted subcutaneously into the back of BALB / c mice until the tumor grew to approximately 50 mm. 3 The drug was then administered. Mice were divided into a control group (solvent group) and a treatment group, with five mice in each group. Based on previous drug toxicity evaluations, a safe dose that did not significantly affect the overall physiological indicators of the mice was selected. The drugs were administered via tail vein injection, once every three days, for five consecutive days. Mice died suddenly on day 15 and were then autopsied for analysis. Results are as follows... Figure 6 As shown.

[0038] Depend on Figure 6 It is evident that the toxin derivative Wallycin B significantly and dose-dependently inhibits tumor growth in mice, and the weight of mice in the treatment group was not significantly affected compared with the control group, indicating that Wallycin B has a certain degree of safety.

[0039] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. The use of a separating enzyme inhibitor as the sole active ingredient in the manufacture of an antitumor medicament, characterized in that, The separation enzyme inhibitor is Walrycin B; ​ The tumor is human cervical cancer, liver cancer, breast cancer and prostate cancer.

Images