Application of BF4 in preparation of medicine for treating gastric cancer

Abstract

The invention provides application of BF4 in preparation of a medicine for treating gastric cancer. The anti-gastric cancer mechanism of the medicine is mainly targeted microtubulin which causes the depolymerization of microtubules in cells; meanwhile, NQO2 protein is targeted, and the anti-gastric cancer effect of the compound BF4 can be further enhanced by inhibiting the activity of the NQO2 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a new dual-target gastric cancer treatment drug, especially the use of BF4 in the preparation of a drug for treating gastric cancer. Background Art

[0002] Gastric cancer is one of the common health problems faced by all mankind and has become one of the key issues of medical and scientific research in various countries. Although in recent years, humans have made some new progress in the treatment of gastric cancer. However, the heterogeneity of gastric cancer cells, resistance to chemotherapeutic drugs, and adverse reactions in patients during treatment have brought severe challenges to the clinical treatment of gastric cancer. With the development of research, researchers have begun to pay attention to the impact of multi-molecular interactions in cells on the occurrence and development of cancer. Among them, the connection and effect between NQO2 (NAD(P)H quinone oxidoreductase 2) protein and microtubule depolymerizing agents (such as colchicine) have attracted people's attention. The connection between the two and the synergistic effect in cancer treatment provide a new research direction for the treatment of cancer, especially the development of new therapies and multi-molecular combined treatment strategies.

[0003] Microtubules are an important cytoskeleton, assembled from α / β tubulin. Microtubules have dynamic structural characteristics, can rapidly depolymerize and polymerize, and maintain dynamic balance. In cells, microtubules play a huge role, such as maintaining cell morphology and structure, participating in cell movement, transferring intracellular substances, cell division, etc. In the process of cell mitosis, microtubules play an indispensable role. The depolymerization and polymerization of microtubules ensure the normal progress of mitosis. If microtubules cannot polymerize and depolymerize normally, it will affect the normal progress of cell mitosis, causing cell cycle arrest and cell death. Tumor cell proliferation is in a highly active state, so microtubule-targeted drugs are also used to treat malignant tumors. By inhibiting the tubulin of tumor cells, tumor cell proliferation can be effectively blocked, tumor cell death can be induced, and the role of treating cancer can be played.

[0004] Common microtubule-targeted drugs can be divided into two categories. One category is tubulin stabilizers represented by paclitaxel, whose mechanism of action is to promote tubulin polymerization and stabilize microtubules to prevent their depolymerization; the other category is tubulin destabilizers represented by colchicine and vincristine, whose mechanism of action is to promote tubulin depolymerization and prevent its polymerization. Both types of drugs can affect the normal polymerization or depolymerization of tubulin, affect the formation of microtubules, thereby inhibiting the normal formation of spindles, causing cell cycle arrest and inducing cell death. There are currently six main binding sites on microtubules that have been discovered, namely, paclitaxel site, colchicine site, vinblastine site, maytansine site, laulimalide / pelorusideA site and pironetin site.

[0005] NAD(P)H benzoquinone dehydrogenase 2 (NQO2) is an enzyme present in organisms, which plays an important role in the detoxification of quinones and the reduction of oxides. NQO2 participates in many processes in the body, such as detoxification, participation in metabolism in the body, and maintaining the balance of redox in the body. With the deepening of research, the relationship between NQO2 and cancer has also attracted people's attention. At present, some studies have shown that in some cancers, the prognosis of patients is often related to the expression of NQO2. The higher the expression of NQO2, the worse the prognosis of patients. The reason may be that NQO2 increases the ability of tumor cells to resist treatment. On the contrary, low expression of NQO2 can increase the sensitivity of cells to drugs. In addition, some studies have shown that NQO2 also affects the microenvironment of tumors and may affect the growth and metastasis of tumor cells.

[0006] In recent years, the interaction between NQO2 and microtubule depolymerizing agents has also attracted the attention of some researchers. The upregulation of NQO2 may enhance the resistance of cancer cells to microtubule depolymerizing agents, resulting in poor chemotherapy effects. For example, when some cancer cells are treated with microtubule depolymerizing agents, the activity of NQO2 is enhanced, thereby improving the antioxidant capacity of the cells and reducing drug-induced cell apoptosis. Conversely, inhibiting the expression or activity of NQO2 may make tumor cells more sensitive to microtubule depolymerizing agents, thereby improving the therapeutic effect. Based on this relationship, researchers began to explore the possibility of combining NQO2 inhibitors with microtubule depolymerizing agents to treat cancer. By comprehensively regulating the redox balance and the dynamic balance of microtubules in cancer cells, it may be possible to improve the effectiveness of cancer treatment and overcome the problem of drug resistance.

[0007] In the previous work, the inventors used gastric cancer cells and gastric cancer stem cells derived from gastric cancer patients as models to screen the activity of the drug library (2106 compounds). During the screening process, the inventors found that compound BF4 can inhibit the growth of gastric cancer cells and gastric cancer stem cells derived from gastric cancer patients, causing the death of tumor cells. In further mechanism studies, the inventors found that compound BF4 is a dual-target drug. The anti-gastric cancer mechanism of BF4 mainly stems from BF4's targeted binding to microtubules and NQO2 in cells. On the one hand, in cancer cells, BF4 can bind to microtubules in cells, interfere with the polymerization of microtubules, inhibit the formation of microtubules, and cause tumor cell mitosis to be unable to proceed normally, causing cell cycle arrest, and ultimately causing tumor cell death. On the other hand, BF4 can specifically bind to the NQO2 protein in cells and inhibit the activity of the NQO2 protein, which to a certain extent enhances the anti-gastric cancer effect of BF4.

[0008] At present, the anti-gastric cancer effect and mechanism of compound BF4 have been basically elucidated. It is expected that BF4 can be developed into a new drug for the treatment of gastric cancer, providing new high-efficiency and low-toxic drugs for the treatment of gastric cancer, reducing patients' adverse reactions and creating greater social value. Summary of the invention

[0009] The purpose of the present invention is to provide the use of BF4 in preparing medicine for treating gastric cancer.

[0010] The present invention can be implemented by the following scheme:

[0011] The BF4 is used as an active ingredient of a drug for treating gastric cancer, and the drug for treating gastric cancer also contains pharmaceutically acceptable excipients.

[0012] Preferably, in the drug for treating gastric cancer, the BF4 targets both tubulin and NQO2 protein.

[0013] Preferably, the drug for treating gastric cancer is used to cause the depolymerization of tubulin in gastric cancer cells and gastric cancer stem cells derived from gastric cancer patients, and has a significant killing effect on gastric cancer cells and gastric cancer stem cells.

[0014] Preferably, in the drug for treating gastric cancer, the drug induces cell cycle G2 / M phase arrest in gastric cancer cells and gastric cancer stem cells, thereby inhibiting the proliferation of gastric cancer cells and tumor stem cells derived from gastric cancer patients, promoting cell death, and having obvious concentration dependence.

[0015] Preferably, the drug for treating gastric cancer is used as a microtubule destabilizing agent in the treatment of gastric cancer, and is also used in the treatment of other tumors.

[0016] Preferably, the drug for treating gastric cancer can specifically bind to the NQO2 protein in cells and inhibit the activity of the NQO2 protein.

[0017] The anti-gastric cancer mechanism of the drug of the present invention is mainly to target tubulin, causing the depolymerization of intracellular microtubules; at the same time, it targets NQO2 protein, and inhibiting the activity of NQO2 protein can further enhance the anti-gastric cancer effect of compound BF4. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , schematic diagram of the structures of compounds BF4 and BF4-Biotin.

[0019] Figure 2 , detect the activity of compound BF4-Biotin in HGC27, GT112 and AGS cells.

[0020] Figure 3. Detection of the specific band of the probe BF4-Biotin pull-down by pull-down experiment, gel electrophoresis and silver staining. Using Biotin as the control group, different concentrations of BF4-Biotin were designed for pull-down and different concentrations of BF4 were designed for competition. The red box indicates the location of the specific band of BF4-Biotin pull-down.

[0021] Figure 4. Validation of target proteins by pull-down experiments and Western blot experiments. The selected cells are HGC27, AGS, and GT112. In Vitro refers to pull-down experiments performed in cell lysate, and In Situ refers to pull-down experiments performed in cells.

[0022] Figure 5. Effect of compound BF4 on the activity of NQO2. The X-axis is the concentration of compound BF4, and the Y-axis is the activity of NQO2.

[0023] Figure 6. Protein thermal shift experiment reflects the relationship between protein stability and temperature. Fig. 6A For the compound BF4 group, Figure 6B As the temperature increased, the degradation of NQO2 protein in the compound BF4 group decreased compared with the DMSO group, and there was no significant difference in the degradation of the internal reference protein Actin.

[0024] Figure 7 , molecular docking model of compound BF4 and NQO2 protein.

[0025] Figure 8 , Western blot results of WT-NQO2 and MT-MQO2 pull down in GT112 cells.

[0026] Figure 9. Immunofluorescence staining results. The cells used were GT112 cells, green fluorescence was Tubulin protein staining, and blue fluorescence was cell nucleus staining.

[0027] Fig.10 , the effect of compound BF4 on microtubule polymerization in vitro. The X-axis is time, and the Y-axis is fluorescence intensity, reflecting the polymerization of microtubules. Paclitaxel (microtubule polymerizer) and colchicine (microtubule depolymerizer) were used as controls.

[0028] Fig.11 , Cell cycle flow cytometry. The X-axis is the amount of DNA in the cell, and the Y-axis is the number of cells.

[0029] Fig.12, Western blot experimental results, detecting the relationship between the expression of cell cycle protein CyclinB1 and compound BF4 in HGC27 and GT112 cells.

[0030] Fig. 13. Molecular docking model of compound BF4 and tubulin dimer. Fig.13A For a three-dimensional structure diagram, Fig. 13B It is a two-dimensional plane diagram.

[0031] Fig.14 , gastric cancer tumor cells HGC27, AGS and gastric cancer tumor stem cells GT112 cells, small molecule interfering RNA was used to determine the relationship between the expression level of NQO2 protein and the anti-gastric cancer effect of compound BF4. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings:

[0033] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0034] Specific implementation methods

[0035] 1. Effects of BF4-Biotin on HGC27, GT112, and AGS cells

[0036] In the experiment, gastric cancer stem cells GT112 and gastric cancer cell lines HGC27 and AGS in the logarithmic growth phase were inoculated in 96-well culture plates. After 24 hours of cell culture, the cells were treated with the compound BF4-Biotin, and the concentrations of the compound BF4-Biotin were set to 10μM, 20μM, and 40μM. After another 24 hours of culture, the cell morphology was observed, photographed, and recorded.

[0037] 2. Explore the target protein of BF4 through pulldown experiments

[0038] In the experiment, gastric cancer cell line HGC27 in the logarithmic growth phase was inoculated in 10 cm 2 The cells were cultured in the culture dish in the incubator for 48 hours before the experiment. The specific method is: remove the culture medium in the culture dish, wash twice with room temperature PBS solution, add IP lysis solution containing protease inhibitors and phosphatase inhibitors after removing PBS, collect the cells and sonicate to fully lyse the cells, and obtain the cell protein sample after centrifugation. The sample was divided into 3 parts, namely:

[0039] ①Control group: add 1μM Biotin, ②BF4-Biotin group: add 1μM BF4-Biotin, ③Competition group: add 1μM BF4-Biotin and 10μM BF4 at the same time. After the addition of the drug, place the three samples on a rotating mixer at the same time and incubate at 4°C overnight. After incubation, use streptavidin-coupled magnetic beads to bind BF4-Biotin, pull down the target protein, and perform gel electrophoresis after elution. After silver staining, determine the position of the differential bands between BF4-Biotin and Biotin. Cut out the differential bands, perform protein spectrum detection, and determine the target protein.

[0040] 3. Verify target protein in cells

[0041] In the experiment, gastric cancer stem cells GT112 and gastric cancer cell lines HGC27 and AGS in the logarithmic growth phase were inoculated in 10 cm 2 In a culture dish. After the cells were cultured in the incubator for 24 hours, the drugs were administered and divided into a control group and a BF4-Biotin group, containing 20 μM Biotin and BF4-Biotin, respectively. The experiment was carried out after continuing to culture for 24 hours. The specific method is: remove the culture medium in the culture dish, wash twice with a PBS solution at room temperature, add IP lysis buffer containing protease inhibitors and phosphatase inhibitors after removing the PBS, collect the cells and sonicate to fully lyse the cells, and obtain the cell protein sample after centrifugation. Use streptavidin-coupled magnetic beads to bind BF4-Biotin, pull down the target protein, and after elution, perform gel electrophoresis, transfer, block, incubate with primary antibody, incubate with secondary antibody, and develop to determine the target protein.

[0042] 4. Effect of BF4 on NQO2 activity

[0043] The activity of NQO2 was determined by continuous spectrophotometry, with menadione as substrate and NQO2 enzyme cofactor EPR as cosubstrate. The activity of NQO2 was detected by detecting the reduction of the dye diphenyltetrazolium bromide (MTT) at 590nm. The specific method is as follows: at 30°C, each group of samples to be tested is 1ml of reaction solution, which contains Tris-hydrochloric acid buffer (25mM, pH=7.5), 0.01% Tween-20, 0.18mg / ml BSA, 1μM FAD, 100μM menadione, 0.134mg / ml MTT and 500μM CHP, 100ng recombinant NQO2, inhibitor BF4 (5-8 gradients). An equal amount of DMSO was added to the control group. After mixing, it was added to a 96-well plate, and the absorbance of the sample wells at 590nm was detected using an ELISA reader preheated to 30°C. GraphPad prism 9.0 software was used for analysis. Each group of samples was tested in triplicate and repeated three times.

[0044] 5. Protein thermal shift experiment

[0045] In the experiment, gastric cancer cell line HGC27 in the logarithmic growth phase was inoculated in 10 cm 2 In the culture dish, the cells were cultured in the incubator for 48 hours before the experiment. The specific method is: remove the original culture medium in the culture dish, add room temperature PBS to wash twice, add trypsin, digest and collect the cells after centrifugation. Resuspend the cells with room temperature PBS, divide the cell suspension into two parts, add 1μM compound BF4 to one part, and add an equal amount of DMSO to the other part. After the cell suspension is incubated in the incubator for another 4 hours, take eight tubes and divide each group of cell suspensions into 8 parts, heat them at different temperatures (40°C to 65°C) for 3 minutes, and then cool them at room temperature for 3 minutes. After that, the cells were repeatedly frozen and thawed three times with liquid nitrogen, centrifuged, and the supernatant was taken for gel electrophoresis analysis.

[0046] 6. Verification of the binding site between compound BF4 and NQO2 protein

[0047] In order to determine the binding site of compound BF4 and NQO2 protein, Huh7 cells that can stably express WT-NQO2 (wild type) and MT-NQO2 (W106A / N162D) were constructed. In the experiment, Huh7 gastric cancer stem cells in the logarithmic growth phase were inoculated in 10 cm 2 The cells were cultured in the culture dish in an incubator for 48 hours before the experiment. The specific method was as follows: remove the culture medium in the culture dish, wash twice with room temperature PBS solution, add IP lysis buffer containing protease inhibitors and phosphatase inhibitors after removing PBS, collect the cells and sonicate to fully lyse the cells, and obtain cell protein samples after centrifugation. 1μM compound BF4 was added to each group of samples, and pull-down experiments and Western blot experiments were performed after incubation.

[0048] 7. Immunofluorescence Experiment

[0049] 7.1 The immunofluorescence experiment was divided into DMSO group, compound BF4 group, colchicine group, and paclitaxel group. GT112 cells treated with drug compounds were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes. After washing with PBS, 0.5% Triton-X 100 was added and incubated for 15 minutes. After washing with PBS, PBS containing 5% BSA was added and blocked at room temperature for 1 hour. The cells were incubated with the primary antibody at 4°C overnight. After washing, the cells were incubated with the secondary antibody labeled with Alex Flour 488 at room temperature for 1 hour. After washing with PBS, the cell nuclei were stained with DAPI solution for 1 minute. Finally, the cells were photographed using a fluorescence microscope.

[0050] 7.2GT112 cells were first incubated at 4°C for 1 hour to cause the depolymerization of peripheral microtubules, and then switched to 37°C for another 1 hour in the presence or absence of compounds. The cells were then fixed, blocked, and stained as described in 7.1, and photographed using a fluorescence microscope.

[0051] 8. Effect of BF4 on in vitro polymerization of tubulin protein

[0052] Tubulin polymerization detection was performed using the Cytoskeleton (#BK011P) tubulin polymerization detection kit. Four groups were set up, namely DMSO group, paclitaxel group, colchicine group, and compound BF4 group. The reaction solution was prepared and transferred to a 96-well plate. The detection was performed using a multifunctional microplate reader preheated to 37°C. The excitation wavelength was set to 360nm and the emission wavelength was set to 420nm. Each detection interval was 1 minute, and repeated 60 times.

[0053] 9. Flow cytometry to detect the effect of BF4 on cell cycle

[0054] In the experiment, gastric cancer stem cells GT112 and gastric cancer cell lines HGC27 and AGS in the logarithmic growth phase were inoculated in 6 cm 2 In a culture dish. After the cells were cultured in the incubator for 24 hours, the drugs were administered and divided into a control group and a BF4 group. The BF4 concentration was set to 0.125μM, 0.25μM, and 0.5μM. After further culture for 16 hours, the cell cycle was detected using a flow cytometer and a cell cycle detection kit.

[0055] 10. Western blot detection of the relationship between Cyclin B1 protein expression and compound BF4

[0056] In the experiment, gastric cancer cells HGC27 and gastric cancer stem cells GT112 in the logarithmic growth phase were inoculated in 6 cm 2 The cells were cultured in the incubator for 24 hours before administration and divided into a control group and a BF4 group. The BF4 concentrations were set at 0.125 μM, 0.25 μM, and 0.5 μM. After 16 hours of continuous culture, cell proteins were extracted for Western blot analysis.

[0057] 11. Effect of NQO2 protein expression on the anti-gastric cancer effect of BF4

[0058] In the experiment, gastric cancer stem cells GT112 and gastric cancer cell lines HGC27 and AGS in the logarithmic growth phase were inoculated in 96-well culture plates. After 24 hours of cell culture, the cells were transfected with small interfering RNA (SiRNA), and after another 24 hours of culture, the medium was changed to the medium containing the compound BF4. After another 24 hours of culture, the cell morphology was observed, photographed, and recorded.

[0059] 12. Statistical analysis

[0060] The data are expressed as mean ± standard deviation (SD) for each experimental result. The t-test is used to determine the statistically significant difference. When the P value is less than 0.05, the data results are significantly different. All line graphs and bar graphs in this experiment were drawn using GraphPad Prism 9 software.

[0061] Experimental Results

[0062] 1. The compound BF4-Biotin exhibits similar activity against gastric cancer cells and gastric cancer stem cells as BF4

[0063] like Figure 2 As shown in the figure, after BF4-Biotin treated HGC27, GT112, and AGS cells for 24 hours, it was observed that BF4-Biotin had a significant killing effect on gastric cancer cells and showed a concentration-dependency. In the BF4-Biotin group, a large number of cells became obviously rounded, the intercellular space was significantly enlarged, the cell body shrank, and fell off the culture dish and lost the normal cell morphology.

[0064] 2. Pull down experiments using the compound BF4-Biotin to pull down specific proteins

[0065] The compound BF4-Biotin was used to perform pull-down experiments in HGC27 cells. Figure 3A As shown, the silver staining results showed that at 25kd, compared with Biotin, the compound BF4-Biotin had an obvious specific band, and the specific band could be obviously competed by 10-fold concentration of BF4.

[0066] Pull down experiments were performed in HGC27 cells using different concentrations of BF4-Biotin. Figure 3B As shown, the silver staining results showed that at 25kd, compared with Biotin, as the concentration of BF4-Biotin increased, the intensity of this specific band also increased.

[0067] Competition experiments were performed in HGC27 cells using different concentrations of BF4. Figure 3C As shown, the silver staining results showed that at 25kd, compared with Biotin, BF4-Biotin had a specific band, and as the concentration of BF4 competition increased, the intensity of the band gradually weakened.

[0068] 3. Validation of Extracellular and Intracellular Target Proteins

[0069] Figure 4A:Western blot analysis showed that BF4-Biotin specifically bound to NQO2 protein in the cell lysates of HGC27, GT112 and AGS cells. In the presence of BF4, the binding of BF4-Biotin to NQO2 protein could be significantly competed by BF4.

[0070] Figure 4B : Western blot analysis showed that BF4-Biotin specifically bound to NQO2 protein in HGC27, GT112 and AGS cells. In the presence of BF4, the binding of BF4-Biotin to NQO2 protein could be significantly competed by BF4.

[0071] 4. BF4 inhibits the activity of NQO2 protein in a concentration-dependent manner

[0072] The effect of compound BF4 on NQO2 activity was evaluated using continuous spectrophotometry. In the presence of the coenzyme factor EPR, NQO2-mediated reduction of menadione will cause an increase in the absorbance of MTT at 590nM. The effect of compound BF4 on NQO2 activity is reflected by detecting the change in absorbance. Figure 5A As shown, the concentration of BF4 was set to 0, 0.01μM, 0.1μM, 1μM, and 10μM to determine the effect of BF4 on the activity of NQO2. The results showed that compound BF4 inhibited the activity of NQO2. Figure 5B As shown, the concentration of BF4 was set to 1nM, 25nM, 50nM, 100nM, 200nM, 400nM, 800nM, and 1600nM. The results showed that compound BF4 inhibited the activity of NQO2. The calculated IC50 was 358.2nM.

[0073] 5.CETSA experiments show that BF4 can increase the thermal stability of NQO2 protein

[0074] The cell thermal migration assay is a method to verify the binding of small molecule inhibitors to targets. Generally, small molecule inhibitors will cause changes in protein function and stabilization of protein spatial conformation after binding to proteins. Therefore, by analyzing whether compound BF4 can bind to NQO2 protein in cells and stabilize NQO2 protein, it can be used to determine whether the target of compound BF4 is NQO2 protein. As shown in Figure 6, the results show that after pre-incubation of HGC27 cells with BF4, compared with the control group, it was found that pre-incubation with BF4 can effectively stabilize NQO2 protein from temperature-dependent degradation.

[0075] 6. Molecular docking model of compound BF4 and NQO2 protein

[0076] like Figure 7As shown, the molecular docking simulation of compound BF4 and NQO2 protein showed that the binding of BF4 to NQO2 protein mainly involved hydrogen bond polar interactions formed with Asn161 residue and π-π stacking hydrophobic interactions formed with Trp105 residue.

[0077] 7.Trp-106 and Asn-162 residues are essential for the binding of BF4 to NQO2

[0078] In order to speculate the binding site of compound BF4 to NQO2 protein, we mutated tryptophan-106 to alanine and asparagine-162 to aspartic acid in NQO2 protein. Figure 8 As shown, the pulldown results showed that WT-NQO2 protein can be specifically bound by BF4-Biotin, and the MT-NQO2 group lost the ability to bind to BF4-Biotin. Supplementing WT-NQO2 in cells expressing MT-NQO2 protein can restore the ability to bind to BF4-Biotin. These results show that compound BF4 can specifically bind to NQO2 protein, and Trp-106 and Asn-162 residues are the key sites for BF4 to bind to NQO2 protein.

[0079] 8. Compound BF4 can promote the depolymerization of tubulin

[0080] Fig. 9A In order to explore whether the compound BF4 affects microtubule dynamics, GT112 cells were phenotyped by immunofluorescence analysis, and the polymerization of cell microtubules was observed by staining with β-Tubulin. The results showed that treatment with 0.125μM BF4 caused the depolymerization of cell microtubules and induced the appearance of multi-stage spindles. Treatment with 0.125μM colchicine significantly induced the depolymerization of cell microtubules. Paclitaxel at 0.125μM promoted the polymerization of intracellular microtubules.

[0081] Fig. 9B In order to explore whether compound BF4 affects microtubule dynamics, GT112 cells were phenotypically analyzed using immunofluorescence analysis, and the polymerization of cell microtubules was observed by staining results of β-Tubulin. The cells were first incubated at 4°C for 1 hour to cause the depolymerization of peripheral microtubules, and then transferred to 37°C in the presence or absence of compounds and continued to be incubated for 1 hour to cause the repolymerization of microtubules. The results showed that compared with the control group and paclitaxel, incubation with compound BF4 or colchicine significantly enhanced the depolymerization of peripheral microtubules of cells, indicating that compound BF4 exhibits microtubule destabilization activity.

[0082] 9. Compound BF4 can reduce tubulin polymerization kinetics

[0083] like Fig.10 As shown, in order to determine whether compound BF4 would directly affect the polymerization of microtubules, an in vitro tubulin polymerization assay was performed. The results showed that compound BF4 significantly reduced the kinetics of tubulin polymerization, showing the same trend as the control colchicine group (microtubule depolymerizing agent). The control paclitaxel group (microtubule polymerizing agent) significantly increased the kinetics of tubulin polymerization.

[0084] 10. Compound BF4 can induce G2 / M phase arrest in the cell cycle

[0085] Microtubule depolymerizing agents affect the normal progress of cell mitosis by affecting the dynamic balance of microtubules, causing G2 / M phase arrest in the cell cycle. Therefore, the effect of BF4 on the cell cycle was further studied. Fig.11 As shown, in HGC27, AGS, and GT112 cells, BF4 induced G2 / M phase arrest of the cell cycle in a dose-dependent manner.

[0086] 11. Compound BF4 can upregulate the expression of CyclinB1 protein in HGC27 and GT112 cells

[0087] like Fig.12 As shown, in HGC27 cells and GT112 cells treated with compound BF4, the expression level of CyclinB1 protein was significantly upregulated and showed concentration dependence with compound BF4, further proving that compound BF4 can induce G2 / M phase arrest in gastric cancer cell cycle.

[0088] 12. Molecular docking model of compound BF4 and tubulin dimer

[0089] like Fig.13A , 13B As shown in the figure, the molecular docking simulation of compound BF4 and NQO2 protein shows that compound BF4 shows a strong fit with the colchicine binding pocket of tubulin dimer, can bind under natural conditions, and has good binding activity between the two. In addition, the small molecule forms a hydrogen bond polar interaction with the GLU-77 amino acid residue in the protein. At the same time, the protein can make non-bonded contact with the small molecule, forming forces represented by electrostatic potential energy and van der Waals force.

[0090] 13. Interfering with the expression of NQO2 protein in cells can enhance the killing effect of BF4 on gastric cancer cells

[0091] like Fig.14 As shown, after knocking down the expression level of NQO2 protein in HGC27, AGS, and GT112 cells, the killing effect of compound BF4 on gastric cancer cells was enhanced compared with the NC group.

[0092] in conclusion

[0093] Compound BF4 is a dual-target drug. On the one hand, compound BF4 can specifically bind to NQO2 protein in cells and inhibit the activity of NQO2 protein. On the other hand, compound BF4 can affect the dynamic balance of microtubules, causing the depolymerization of microtubules, affecting the normal progress of cell mitosis, causing cell cycle arrest and cell death. Further studies have shown that the inhibitory effect of compound BF4 on NQO2 protein can synergistically enhance the killing effect of BF4 on gastric cancer cells and gastric cancer stem cells. Therefore, the compound can be used as a new NQO2 protein inhibitor and a new microtubule depolymerization drug.

[0094] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. Application of BF4 in the preparation of drugs for treating gastric cancer.

2. The use of BF4 according to claim 1 in preparing a drug for treating gastric cancer, characterized in that: The BF4 is used as an active ingredient of a drug for treating gastric cancer, and the drug for treating gastric cancer also contains pharmaceutically acceptable excipients.

3. The use of BF4 according to claim 1 in preparing a drug for treating gastric cancer, characterized in that: In the drug for treating gastric cancer, the BF4 targets both tubulin and NQO2 protein.

4. The use of BF4 according to claim 1 in preparing a drug for treating gastric cancer, characterized in that: The drug for treating gastric cancer is used to cause the depolymerization of microtubules in gastric cancer cells and gastric cancer stem cells derived from gastric cancer patients, and has a significant killing effect on gastric cancer cells and gastric cancer stem cells.

5. The use of BF4 according to claim 1 in preparing a drug for treating gastric cancer, characterized in that: The drug for treating gastric cancer induces cell cycle G2 / M phase arrest in gastric cancer cells and gastric cancer stem cells, thereby inhibiting the proliferation of gastric cancer cells and tumor stem cells derived from gastric cancer patients, promoting cell death, and having obvious concentration dependence.

6. The use of BF4 according to claim 1 in preparing a drug for treating gastric cancer, characterized in that: The drug for treating gastric cancer is used as a microtubule destabilizing agent in the treatment of gastric cancer, and is also used in the treatment of other tumors.

7. The use of BF4 according to claim 1 in preparing a drug for treating gastric cancer, characterized in that: The drug for treating gastric cancer can specifically bind to the NQO2 protein in cells and inhibit the activity of the NQO2 protein.

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