Application of inhibitor for targeted inhibition of UHRF1 in preparation of tumor prevention and treatment medicament

Abstract

The invention belongs to the technical field of biological medicine, virtual screening based on molecular docking is carried out on a small molecule compound library according to UHRF1 representative conformation, four small molecule compounds capable of inhibiting UHRF1 enzyme activity in vitro are obtained, cell experiments show that F913-0023 inhibits esophageal squamous cell carcinoma proliferation and clone formation by inhibiting UHRF1 enzyme activity, and the F913-0023 has a good application prospect. The compound can be developed and applied to preparation of anti-esophageal cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of small molecule inhibitors targeting UHRF1 in anti-tumor drugs. Background Art

[0002] E3 ubiquitin-protein ligase UHRF1 (UHRF1) is an important epigenetic regulatory protein, which is widely present in various tissue cells throughout the body and is expressed in all periods. UHRF1 is highly expressed in a variety of tumor cells, and knockdown of UHRF1 can significantly inhibit the occurrence and development of tumors. Previous studies have shown that UHRF1 is an independent prognostic factor for esophageal squamous cell carcinoma (ESCC), is highly expressed in esophageal squamous carcinoma cells, and down-regulation of UHRF1 can significantly inhibit the proliferation of esophageal squamous carcinoma cells. These studies indicate that UHRF1 may be a potential therapeutic target for ESCC. Therefore, the development of UHRF1 inhibitors has extremely important clinical significance and application potential for the treatment of ESCC.

[0003] It is reported that the PHD domain and TTD domain of UHRF1 cooperate to recognize the tail of histone H3, thereby promoting DNA methylation. Specifically, the PHD domain recognizes the N-terminus of histone H3 (H3R2), and TTD recognizes histone H3K9me2 / me3. When the PHD domain mutates, it will disrupt the binding of TTD to H3K9me3. Conversely, the mutation of TTD does not affect the binding of PHD to H3R2, which indicates that the PHD domain plays a key role in the process of UHRF1 recognizing H3K9me3. Therefore, in the present invention, based on the crystal structure of the PHD domain of UHRF1, molecular docking and pharmacophore-based virtual screening methods are used to search for small molecule compounds that can interact with the PHD domain and inhibit its binding to H3K9me3. Summary of the Invention

[0004] The object of the present invention is to screen specific inhibitors of UHRF1. The binding of small molecule inhibitors to UHRF1 and the effects of the inhibitors on the proliferation, cloning, and migration abilities of esophageal squamous carcinoma cells are verified by SPR experiments, drug affinity reaction target stability experiments, and cellular thermal shift, and then the effects of small molecule inhibitors on esophageal squamous carcinoma cells in vivo are explored.

[0005] To achieve the object of the present invention, the technical solution of the present invention is as follows:

[0006] Virtual Screening (VS) is the screening of active compounds based on the development of small molecule databases. By using virtual screening based on small molecule docking, it is possible to quickly screen out active compounds with drug-like properties from dozens to millions of molecules, greatly reducing the number of compounds screened experimentally, shortening the research cycle, and reducing the cost of drug development. The inventors first constructed the complex structure of UHRF1 and a known small molecule inhibitor, and then obtained a suitable conformation of the binding of UHRF1 and the inhibitor through energy minimization and long-time scale molecular dynamics simulations. A virtual screening based on molecular docking was performed on the small molecule compound library, and the hit compounds obtained were tested for their inhibitory activity against UHRF1 through in vitro enzyme activity experiments. On this basis, 4 representative compounds with better activity were used on esophageal squamous cell carcinoma cells or colorectal cancer cells to detect the toxicity of the compounds to esophageal squamous cell carcinoma or colorectal cancer cells. Finally, a novel UHRF1 inhibitor, namely F913-0023, was obtained, which can target UHRF1 to inhibit the proliferation and colony formation of esophageal squamous cell carcinoma cells.

[0007] The present invention provides the use of 4 small molecule compounds such as F913-0023 in the preparation of inhibitors of UHRF1. That is, the present invention has discovered the inhibitory effect of 4 small molecule compounds such as F913-0023 on UHRF1, and thus can be used in the preparation of UHRF1 inhibitors.

[0008] The present invention has found that: F913-0023 inhibits the proliferation and colony formation of esophageal squamous cell carcinoma by inhibiting the enzyme activity of UHRF1, and promotes the apoptosis of esophageal squamous cell carcinoma cells. The IC50 of F913-0023 against esophageal squamous cell carcinoma cell line KYSE30 is 17.62 μM, and the IC50 against esophageal squamous cell carcinoma cell line KYSE150 is 16.98 μM.

[0009] The present invention also provides the use of F913-0023 in the preparation of anti-tumor drugs. F913-0023 can inhibit the proliferation and colony formation of esophageal squamous cell carcinoma cell lines KYSE30 and KYSE150 in the concentration range of 10 - 20 μM.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0011] (1) The present invention has experimentally found that: the inhibitory effect of F913-0023 on tumors is related to its inhibition of UHRF1 enzyme activity, and this discovery will be of great significance for the treatment of tumor patients with abnormal expression of UHRF1;

[0012] (2) Taking the present invention as an example, F913-0023 can be used as a UHRF1 enzyme inhibitor in the prevention and treatment of tumors. In the present invention, F913-0023 can inhibit the activity of UHRF1 enzyme and the proliferation and clone formation of esophageal cancer cells, and thus can be used in the prevention and treatment of tumors. Brief Description of the Drawings

[0013] Figure 1 Structural diagrams of the 4 compounds obtained from this screening;

[0014] Figure 2 Toxic effects of the 4 compounds on esophageal squamous carcinoma cells (KYSE30 and KYSE150) and colorectal carcinoma cells (HCT116 and DLD-1). After treating the above cells with different concentrations of the compounds for 48 h, the cell viability was detected by CCK-8;

[0015] Figure 3 (A) SPR detection of the binding of F913-0023 to the UHRF1 PHD domain; (B) Molecular docking simulation diagram of F913-0023 and the UHRF1 PHD domain;

[0016] Figure 4 Inhibitory effect of F913-0023 on the proliferation of esophageal squamous carcinoma cells. F913-0023 can inhibit the proliferation of esophageal squamous carcinoma cells KYSE30 and KYSE150 at concentrations of 10 and 20 μM. In this figure, the control group was taken as 100%, and the cell activities of other groups with different drug concentrations were calculated by comparing them with the control group. *p<0.05, **p<0.01, ***p<0.001;

[0017] Figure 5 Inhibitory effect of F913-0023 on the clone formation of esophageal squamous carcinoma cells. Among them, F913-0023 can inhibit the clone formation of esophageal squamous carcinoma cells KYSE30 and KYSE150 at concentrations of 10 and 20 μM. In this figure, the control group was taken as 100%, and the cell activities of other groups with different drug concentrations were calculated by comparing them with the control group. *p<0.05, **p<0.01, ***p<0.001;

[0018] Figure 6 Promotion of apoptosis of esophageal squamous carcinoma cells by F913-0023. F913-0023 can promote the apoptosis of esophageal squamous carcinoma cells KYSE30 and KYSE150 at concentrations of 10 and 20 μM. *p<0.05, **p<0.01, ***p<0.001;

[0019] Figure 7Effect of F913-0023 on the growth of esophageal squamous cell carcinoma CDX tumors. (A) Tumor pictures of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) (B) Statistical chart of tumor volume of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) (C) Statistical chart of tumor weight of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) and calculation of tumor growth inhibition rate (TGI) (D) Body weight change curves of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) during F913-0023 treatment. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0020] Figure 8 Effect of F913-0023 on the growth of esophageal squamous cell carcinoma PDX tumors. (A) Tumor pictures of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) (B) Statistical chart of tumor volume of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) (C) Statistical chart of tumor weight of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) and calculation of tumor growth inhibition rate (TGI) (D) Body weight change curves of mice in the control group and the F913-0023 administration groups (25 mg / kg or 75 mg / kg) during F913-0023 treatment. *p<0.05, **p<0.01, ***p<0.001;

[0021] Figure 9 Schematic diagram of HE staining of mouse organs for F913-0023 in mice. Compared with the control group, administration at 25 mg / kg or 75 mg / kg in the administration group showed no toxicity to mouse organs. (A) HE staining map of organs of CDX tumor mice (B) HE staining map of organs of PDX tumor mice. Scale bar: 100 μm. Detailed implementation manners

[0022] To better illustrate the present invention, the following examples are given:

[0023] Example 1. Cytotoxicity experiment (CCK-8 method)

[0024] Specifically through the following steps:

[0025] (1) Preparation of cell suspension: Take out KYSE30 and KYSE150 cells. When the cell status is observed to be good under the microscope and the confluence reaches 80%-90%, subsequent experiments can be carried out. First, use a negative pressure suction pump to discard the old culture medium in the culture dish, wash it 2-3 times with 1×PBS, then add 800 μl - 1000 μl of 0.25% trypsin to a 10 cm cell culture dish to digest the cells. Gently shake the dish left and right to let the trypsin in the dish soak the cells and evenly cover the entire cell culture dish. Place it in a 37°C cell incubator for digestion for 3-5 minutes, then take it out, gently pat the outer side of the culture dish, and terminate digestion when the cells are no longer adherent and the cell morphology becomes round under the microscope. Transfer it to a 10 ml centrifuge tube, centrifuge for 3 minutes, discard the supernatant, and add 10 ml of complete medium to pipette the cells into a dispersed cell suspension.

[0026] (2) Cell counting: Turn on the power of the cell counter, and first wash it several times with the dielectric fluid until the displayed number is small. Add 9.5 ml of cell counting fluid to the counting cup (the counting cup is soaked in acid for disinfection before use), then add 500 μl of cell suspension and mix well. Count on the automatic cell counter and repeat three times to calculate the average value. Multiply the average value by 40 to obtain the cell concentration of the original suspension.

[0027] (3) Seeding: Prepare the required amount of cell suspension according to the cell concentration of 3×10 3 / well, place it in a disposable pipette trough, and pipette to make the cells evenly distributed in the pipette trough. Take out the 96-well plate, make marks, and use a multi-channel pipette to evenly seed the cells in the 96-well plate (during the seeding process, intermittently pipette the cells in the pipette trough with the multi-channel pipette to prevent cell precipitation and uneven cell seeding quantity). Add 100 μl / well of PBS solution to the four sides of the 96-well plate to prevent the evaporation of cell moisture in the 96-well plate during incubation in the incubator.

[0028] (4) Treat the cells with drugs: After the cells adhere for 16 h, discard the original culture medium, and add 100 μl / well of culture medium containing different drug concentrations. A total of seven drug concentrations of 0.001, 0.01, 0.1, 1, 10, 50, and 100 μM are set, and three replicate wells are set for each drug concentration.

[0029] (5) Detection with CCK-8 kit: Set the drug addition time to 0 h. After 48 h, take out the 96-well plate with corresponding labels from the incubator, discard the original culture medium, and add 100 μL of CCK-8 reagent per well using a multi-channel pipette (the ratio of culture medium to CCK-8 reagent is 100:1, pay attention to avoiding light when preparing CCK-8 reagent). Put the 96-well plate with added CCK-8 detection reagent into the cell incubator and continue to culture for 2 - 4 h. Turn on the multifunctional microplate reader. After the culture time is up, take out the 96-well plate and place it in the microplate reader. Use the CCK-8 standard detection program in the microplate reader to detect the absorbance value at 450 nm and record the data. After 48 h, take out the 96-well plate with corresponding labels and repeat the above operations to record the experimental data.

[0030] (6) Draw a line graph of the cytotoxicity experiment: Draw a line graph at the 48 h time point, with the set drug concentration as the abscissa and the absorbance value at the corresponding time point as the ordinate.

[0031] The experimental results are as Figure 2 , and the results show that:

[0032] When F913 - 0023 acts on esophageal squamous cell carcinoma cells for 48 h, it has a toxic effect on esophageal squamous cell carcinoma cells. At the same action time, with the increase of drug concentration, the toxic effect on esophageal squamous cell carcinoma cells becomes more obvious.

[0033] Example 2. Cell growth experiment (CCK-8 method)

[0034] Specifically through the following steps:

[0035] (1) Prepare cell suspension: Take KYSE30 and KYSE150 cells respectively. When the cell state is observed to be good under the microscope and has grown to 80 - 90%, start the experiment. The digestion and centrifugation steps are the same as before. After centrifugation, discard the supernatant, and add an appropriate amount of complete culture medium to the centrifuge tube to make a single-cell suspension.

[0036] (2) Cell counting: Dilute the cell suspension 5 times, and slowly pipette 10 μL along the edge and add it to the counting chamber for counting. Set the concentration of KYSE30 cells to 3×10 3 cells / mL, and the concentration of KYSE150 cells to 3×10 3 cells / mL.

[0037] (3) Plating: Take out a 96-well plate and mark 5 time points: 0 h, 24 h, 48 h, 72 h, 96 h. Prepare the cell suspension according to the above concentrations, mix well and inoculate it in the 96-well plate, 100 μL per well, and set 3 replicates. Finally, add PBS to the periphery of the 96-well plate and place it in the incubator for culture.

[0038] (4) Treat the cells with drugs: After 16 - 18 h, discard the old culture medium, re - prepare the drug - containing culture medium at the corresponding concentration, 100 μL per well, and record this time as 0 h.

[0039] (5) Detect the absorbance by CCK8: Discard the old culture medium from the 96 - well plate marked at 0 h, add CCK8 (CCK8 reagent: complete culture medium = 1:9, pay attention to light protection when preparing and adding CCK8), 100 μL per well. After completion, put the 96 - well plate into the incubator for 2 h. Then detect its absorbance, save and analyze the data. Repeat the above steps for the corresponding 96 - well plates at 24 h, 48 h, 72 h, and 96 h time points respectively, and save and analyze the data.

[0040] (6) Draw a line graph of cell proliferation: Draw a line graph of the experimental data measured in 5 days at the corresponding time points. The vertical coordinate is the absorbance value, and the horizontal coordinate is the time point. Observe the proliferation of esophageal cancer cells by the drug and make a statistical analysis.

[0041] The experimental results are as Figure 4 , and the results show that:

[0042] Within the same time, as the concentration of F913 - 0023 drug increases, the growth of cells is inhibited, and the higher the drug concentration, the more obvious the inhibitory effect.

[0043] Example 3: Colony formation assay in vitro

[0044] Specifically, it is carried out through the following steps:

[0045] (1) Prepare the cells: Take KYSE30 and KYSE150 cells respectively. When the cell state is good and they grow to 80 - 90% under the microscope, start the experiment. The method of obtaining the cell suspension is the same as before.

[0046] (2) Cell counting: Dilute the cell suspension by 5 times, draw 10 μL and slowly add it along the edge to the counting plate for counting. Calculate the concentration of each type of cell and record it. The concentration of KYSE30 cells inoculated per well is 800 cells / mL, and the concentration of KYSE150 cells inoculated per well is 800 cells / mL.

[0047] (3) Plating: After thoroughly mixing the cell suspension, inoculate it into a 6 - well plate, set 3 replicates, shake it in a "cross" shape to make the cells evenly distributed, and place it in the cell incubator for culture.

[0048] (4) Change the culture medium: Change the drug - containing culture medium for the cells treated with drugs and continue the culture.

[0049] (5) When the size and quantity of cell clones are appropriate under the microscope, pour out the old culture medium, wash twice with 1×PBS, then fix it with 4% paraformaldehyde for 30 min, wash twice with 1×PBS again, add crystal violet to stain the cells, incubate in the dark at room temperature for 15 min. After the staining is completed, slowly rinse off the excess crystal violet with tap water and air-dry at room temperature.

[0050] (6) After air-drying, take pictures for counting and analyze the results for plotting.

[0051] The experimental results are as Figure 5 , and the results show that:

[0052] F913-0023 can inhibit the clone size and number of esophageal squamous carcinoma cells. This indicates that F913-0023 can inhibit the clone formation ability of esophageal squamous carcinoma cells.

[0053] Example 4: Cell apoptosis experiment

[0054] Specifically, it is carried out through the following steps:

[0055] (1) Prepare cells: Take KYSE30 and KYSE150 cells respectively. When the cell state is good and they grow to 80-90% under the microscope, start the experiment. The method for obtaining the cell suspension is the same as before.

[0056] (2) Transfer the cells into 3 six-well plates respectively. After the cells adhere to the wall, perform drug treatment. Divide each group of cells into a non-drug group, a 10 μM group, and a 20 μM group.

[0057] (3) After the drug acts for 48 h, wash twice with PBS, then digest the cells with trypsin digestion solution without EDTA, centrifuge at 1000 rpm for 3 min, and collect the cells. Wash twice with PBS again and centrifuge at 1000 rpm for 3 min.

[0058] (4) Resuspend the cells with 1 ml of PBS, count 1-5x10 6 cells, centrifuge at 2000 rpm for 5 min at 4 °C, and then resuspend the cells with 1xBinding Buffer.

[0059] (5) Take 100 μL of the cell suspension into a 5 mL flow tube, add 5 μL of AnnexinV / FITC and 5 μL of propidium iodide solution (PI), mix well, incubate in the dark at room temperature for 15 min, add 400 μL of 1xBinding Buffer, pipette and mix well, and immediately perform flow cytometry detection.

[0060] (6) Experimental design:

[0061] Blank tube: Cells of the negative control group, without AnnexinV / FITC and PI, used to adjust the voltage.

[0062] Single-stained tube: Cells of the positive control group, with only AnnexinV / FITC added, used to adjust compensation.

[0063] Detection tube: Treated cells, with AnnexinV / FITC and PI added. After adjusting the voltage compensation with the blank tube and the single-stained tube, the required flow cytometry data is obtained.

[0064] (7) Analysis of experimental results: Analyzed using flowjo v 10.8.1 software, with FITC as the abscissa and PI as the ordinate. Data analysis and statistics were performed using Graphpad Prism 8.0.

[0065] The experimental results are as Figure 6 , and the results show that:

[0066] F913-0023 acts on esophageal squamous cell carcinoma cells and can promote the apoptosis of esophageal squamous cell carcinoma cells KYSE30 and KYSE150. Example 5, Western blot experiment

[0067] Specifically through the following steps:

[0068] (1) Extraction of cell proteins from KYSE30 and KYSE150 treated with inhibitors

[0069] ① Seed the cells of each cell line in a culture dish. Take 4.5×10 6 cells and seed them in a 10 cm cell culture dish, and place them in a cell culture incubator for 16 - 18 h until the cells adhere and grow well.

[0070] ② After the cells adhere and grow, prepare complete media with corresponding drug concentrations and add them to each cell line, replacing the media in the original cell culture dish (wash the cells 2 - 3 times with 1×PBS solution before replacement). Add 8 ml of drug-containing media to each 10 cm culture dish.

[0071] ③ Place the culture dishes with the replaced media back into the cell culture incubator and continue to culture for 24 h. Use a cell brush to collect the cells into a 1.5 ml EP tube, then centrifuge at 4°C and 5000 rpm for 5 min, and discard the supernatant. Add an appropriate amount of freshly prepared RIPA cell lysate to the lower layer precipitate according to the cell amount, vortex to mix the cells and the RIPA lysate thoroughly, and then shake on a shaker for 40 - 60 min to fully lyse the cells (the cell lysate is prepared freshly and used immediately).

[0072] ④ After the cells are lysed, centrifuge at 16,000 rpm for 30 min at 4°C. The middle liquid is drawn out as the protein solution. Place the protein solution in another new EP tube, place it on ice and label it with the protein name.

[0073] (2) Measuring protein concentration by BCA method

[0074] ① Add 1×PBS to the 5 mg / ml standard protein solution to dilute it to 0.5 mg / ml. After aliquoting, store it at -20°C for later use.

[0075] ② Add PBS solution to the prepared 0.5 mg / ml standard protein solution in turn to prepare protein standards with concentrations of 0 mg / ml, 0.025 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, and 0.5 mg / ml. Prepare the BCA working solution according to the ratio of Solution A: Solution B = 50:1 (the amount of BCA working solution used is 200 μl for one sample to be measured). Let it stand at room temperature for later use.

[0076] ③ Mark a 96-well plate, and set two duplicate wells for each protein sample. First, add the prepared protein standards to the 96-well plate in order of concentration, 10 μl per well. Then add the sample to be measured, 2 μl of protein sample and 8 μl of lysis buffer per well. Finally, add 200 μl of the prepared BCA working solution to each well. Place the 96-well plate in an incubator at 37°C for 30 min.

[0077] ④ After the time is up, take out the 96-well plate from the incubator, place it on a multifunctional microplate reader, select 562 nm in the program, detect the absorbance value at this wavelength. After calculating the average absorbance value of the experimental wells, draw a standard curve with the absorbance value of the standard protein as the x-axis coordinate and the standard protein concentration as the y-axis coordinate, calculate the concentration of the protein to be measured, and determine the loading volume for the subsequent experiment (the protein sample to be measured is diluted 5 times when added to the 96-well plate, pay attention to multiplying the concentration value by 5 when calculating).

[0078] (3) Measuring protein concentration by BCA method

[0079] ① Prepare special glass plates for preparing gels, and prepare 10% separating gel and 5% stacking gel for SDS-PAGE.

[0080] ② Prepare protein samples: Mix 50 μg of the target protein sample with RIPA lysis buffer to make a volume of 20 μl in an Ep tube. Then add 6×Loading Buffer reagent to the Ep tube to form a 24 μl system. After heating in a 100°C water bath for 5 min, immediately take it out and cool it on ice for 2 - 3 min. Centrifuge briefly before loading to prevent liquid residue on the tube wall.

[0081] ③ Loading samples: Place the prepared gel in the electrophoresis tank, and pull out the lane comb in the upper layer of the gel, taking care not to skew it. Prepare 1×Running buffer and add it to the electrophoresis tank and the lane wells for loading samples. The sample order is 1×Loading Buffer, Protein Ladder, and protein samples in sequence. Try to avoid excessive bubbles during the loading process to ensure consistent loading amounts of each protein sample.

[0082] ④ Electrophoresis: Perform SDS-PAGE electrophoresis. Set the electrophoresis instrument to constant current, 25 mA / block, for 60 min.

[0083] ⑤ Transfer: Prepare the transfer buffer 1×Trans Buffer in advance and pre-cool it at 4°C. Cut a PVDF membrane of the same size as the gel and activate it in methanol solution. Place the transfer cassette in 1×Trans Buffer. After wetting the sponges in the transfer cassette, place them in the upper and lower layers of the transfer cassette respectively. Wet 5 - 6 layers of filter paper and place them in the upper and lower layers respectively, then lay 1 clean filter paper on the lower layer. Drive out the air bubbles between the filter papers in the lower layer. Remove the gel and place it on the clean filter paper in the lower layer. Place the PVDF membrane face up on top of the gel (cut a corner of the PVDF membrane to make a mark indicating the loading order). Cover the gel with a new wet filter paper on top, and drive out the air bubbles between the filter paper, gel, and PVDF membrane again (be careful to prevent damage to the gel). Seal the transfer cassette, place it in the transfer tank, and fill it with 1×Trans Buffer. Ensure that the positive electrode of the transfer tank is aligned with the positive electrode of the transfer instrument, and the negative electrode of the transfer tank is aligned with the negative electrode of the transfer instrument. Adjust the transfer instrument to constant voltage of 90 V for 120 min.

[0084] ⑥ Blocking: After the transfer process, use 5% BSA solution to block the protein bands. Place the protein bands on a horizontal shaker at room temperature at a rotation speed of 10 rpm. After blocking for 60 min, recover the blocking solution (for reuse next time), and wash the protein bands 4 times with 1×TBST reagent, 5 min each time.

[0085] ⑦ Incubating with primary antibody: Prepare the primary antibody at the required concentration with 5% BSA reagent (according to the antibody instruction manual), place it at 4°C, and incubate on a shaker at 10 rpm overnight (13 h - 15 h) to allow the protein to bind to the antibody. After overnight incubation with the primary antibody, recover it and wash 4 times with 1×TBST reagent at room temperature, 5 min each time.

[0086] ⑧ Incubating with secondary antibody: Place the bands and the secondary antibody on a shaker at 10 rpm and incubate at room temperature for 2 h, then recover. Wash the bands 4 times with 1×TBST reagent, 5 min each time.

[0087] ⑨ Exposure: Prepare the ECL luminescent solution (prepare it immediately before use) and mix it in a 1:1 ratio. After the protein bands are soaked in the luminescent solution, expose them in a chemiluminescence instrument, save the image results after exposure, and store the bands in a refrigerator at 4°C.

[0088] The experimental results are as Figure 6 , and the results show that:

[0089] The Western blot results showed that F913-0023 could increase the pro-apoptotic protein Bax, decrease the anti-apoptotic protein Bcl-2, and for Caspase enzymes: Caspase3 decreased while Active-Caspase3 increased, indicating that F913-0023 could promote the apoptosis of esophageal squamous carcinoma cells.

[0090] Example 6, Human Tumor Cell Line Xenograft (CDX) Experiment

[0091] Specifically, it is carried out through the following steps:

[0092] (1) Experimental animals: Purchase 6-week-old immunodeficient mice BALB / Cnude from Beijing Vital River Laboratory Animal Technology Co., Ltd., raise them for one week, and then prepare for the experiment.

[0093] (2) Prepare cells: Take out KYSE30 cells, inoculate 4×10 6 cells / 100 μL of KYSE30 cells per mouse, and prepare cells according to the number of mice. When the cell status is observed to be good under the microscope and the cell number reaches the required amount, prepare the cell suspension. The preparation method of the cell suspension is the same as before, and store it on ice for later use.

[0094] (3) Inoculate cells: Disinfect the mouse skin with alcohol, draw the corresponding cell suspension with a syringe for inoculation, and inoculate the corresponding cell suspension on the upper right side. All are inoculated subcutaneously. During the inoculation process, inject slowly and gently rotate and pull out the needle. Observe the mouse status after inoculation.

[0095] (4) Monitoring of tumor growth: Observe the mouse status and tumor size every day. ① When the average tumor volume of the mouse control group reaches 100 mm 3 , start measuring the mouse body weight and tumor volume, and measure it with a vernier caliper every 3 days. When the average tumor volume of the control group reaches 800 mm 3 , the experiment can be terminated. ② When the average tumor volume of the mice reaches 100 mm 3 , evenly divide the mice into three groups according to the tumor volume size, namely the control group, F913-0023: 25 mg / kg, and F913-0023: 75 mg / kg. Administer drugs to the mice by intraperitoneal injection every day, and measure with a vernier caliper every 3 days. When the average tumor volume of the control group reaches 800 mm 3When the time came, the mice were sacrificed by cervical dislocation. Tumor volume calculation method: Tumor volume = length × width × width / 2.

[0096] (5) The tumor tissues were taken out from the subcutaneous part of the mice for photographing and weighing, and the experimental data were sorted out and analyzed for graphing. All animal experiments complied with the ethical regulations of the Life Science Ethics Review Committee of Zhengzhou University.

[0097] The experimental results are as Figure 7 , and the results show that:

[0098] F913-0023 inhibited the growth of esophageal squamous cell carcinoma cells in vivo. After treating the mice with 25 mg / kg and 75 mg / kg of F913-0023, compared with the control group, there was no significant effect on the body weight of the mice, indicating that 25 mg / kg and 75 mg / kg of F913-0023 had no obvious toxic effect on the mice.

[0099] Example 7, PDX xenograft tumor model

[0100] Specifically through the following steps:

[0101] (1) Experimental grouping:

[0102] CB17 / SCID mice (about 13 - 16 g) at 4 - 5 weeks old were purchased from Beijing Vital River Laboratories Co., Ltd. and raised in an SPF - level barrier system. They were randomly divided into a control group, a 25 mg / kg drug - treated group, and a 75 mg / kg drug - treated group, with 9 mice in each group; two weeks before the experiment, the mice were pre - raised to adapt to the environment.

[0103] (2) Experimental process:

[0104] Esophageal squamous cell carcinoma tissue from 1 clinical patient, numbered LEG397, was transplanted near the axilla on the back of the mice to construct a human - derived PDX mouse model. After continuous tumor passage 4 times, the experiment began.

[0105] Tumor tissues with a volume of about 1 cm x 1 cm and a mass of about 0.1 g - 0.2 g were inoculated subcutaneously in 27 mice. When the subcutaneous tumors grew to about 100 mm 3 (about 9 days after tumor inoculation), they were divided into 3 groups, and each group was given the corresponding solvent or drug. The solvent was corn oil containing 10% DMSO. The administration method and dose were as follows:

[0106] ① Control group: Intraperitoneal injection of 100 μL of solvent

[0107] ② 25 mg / kg drug - treated group: Intraperitoneal injection of 100 μL of drug solution

[0108] ③ 75 mg / kg drug - treated group: Intraperitoneal injection of 100 μL of drug solution

[0109] Administer the drug for 2 days, pause for 1 day, and continuously administer for three weeks. Measure the long and short diameters of the tumor with a vernier caliper every other day, observe the tumor growth and the survival of the mice, and at the same time monitor the changes in the body weight and body temperature of the mice.

[0110] After the experiment, the mice were sacrificed by cervical dislocation, the tumor volume and weight were measured, a part of the tumor tissue was lysed and subjected to WB detection, and another part of the tumor tissue was fixed, dehydrated, paraffin-embedded, sectioned, and subjected to immunohistochemical staining. The mice were dissected, and the internal organs (heart, liver, spleen, lung, kidney) were taken out and weighed to calculate the organ index: the organs were fixed, dehydrated, paraffin-embedded, sectioned, and subjected to HE staining.

[0111] The tumor volume was calculated according to the following formula:

[0112] Tumor volume (mm 3 ) = (tumor long diameter) × (tumor short diameter) 2 x 0.5.

[0113] The experimental results are as Figure 8 , and the results show that:

[0114] F913-0023 inhibits the growth of esophageal squamous cell carcinoma in vivo. After treating mice with 25 mg / kg and 75 mg / kg of F913-0023, compared with the control group, there was no significant effect on the body weight of the mice, indicating that 25 mg / kg and 75 mg / kg of F913-0023 have no obvious toxic effect on mice.

[0115] Example 8, HE staining

[0116] Specifically through the following steps:

[0117] (1) Baking the slices: Insert the tissue sections into the immunohistochemical staining rack and bake them in an incubator at 65 °C for 2 h.

[0118] (2) Gradient dewaxing: 100% xylene I (15 min), 100% xylene II (15 min), 100% alcohol I (5 min), 100% alcohol II (5 min), 95% alcohol (5 min), 70% alcohol (5 min), 50% alcohol (5 min).

[0119] (3) Dropwise add hematoxylin and stain for 5 min, terminate the staining in tap water and rinse for 3 min.

[0120] (4) Put the sections into hydrochloric acid alcohol for differentiation for 2 s, and rinse with tap water for 15 min (blue return).

[0121] (5) Take out the sections, blot the liquid around the tissue with blotting paper, add eosin staining solution to the sections and stain for a certain period of time (the staining time varies for different tissues), and terminate the staining in tap water. Note: Eosin is highly soluble in water, and if the time is too long, all the red color will be removed. The staining situation should be observed under the microscope at any time.

[0122] (6) Gradient dehydration: 50% alcohol (2 s), 70% alcohol (2 s), 95% alcohol (2 s), 100% alcohol I (2 s), 100% alcohol II (2 s), 100% xylene I (5 min), 100% xylene II (5 min).

[0123] (7) Sealing the sections: Let the sections dry naturally, drop neutral resin in the middle of the tissue, cover with a coverslip, gently squeeze to expel air bubbles, and complete the section sealing. Scan with a panoramic scanner and analyze the results.

[0124] The experimental results are as Figure 9 , and the results show that:

[0125] F913-0023 acts in vivo, inhibits the growth of esophageal squamous cell carcinoma, but has no toxic effect on the heart, liver, spleen, lungs, and kidneys.

[0126] The experimental results

[0127] 1. The IC50 of F913-0023 in inhibiting esophageal squamous cell carcinoma cell line KYSE30 is 17.62 μM, and the IC50 of inhibiting esophageal squamous cell carcinoma cell line KYSE150 is 16.98 μM.

[0128] 2. When F913-0023 acts on esophageal squamous cell carcinoma cells for 48 h, it has a toxic effect on esophageal squamous cell carcinoma cells. At the same action time, with the increase of the drug concentration, the toxic effect on esophageal squamous cell carcinoma cells becomes more obvious.

[0129] 3. Within the same time, with the increase of the F913-0023 drug concentration, the growth of cells is inhibited, and the higher the drug concentration, the more obvious the inhibitory effect.

[0130] 4. F913-0023 can inhibit the clone size and number of esophageal squamous cell carcinoma cells. This indicates that F913-0023 can inhibit the clone formation ability of esophageal squamous cell carcinoma cells.

[0131] 5. When F913-0023 acts on esophageal squamous cell carcinoma cells, it can promote the apoptosis of esophageal squamous cell carcinoma cell lines KYSE30 and KYSE150.

[0132] 6. F913-0023 inhibits the growth of esophageal squamous cell carcinoma in vivo. After treating mice with 25 mg / kg and 75 mg / kg of F913-0023, there is no significant effect on the body weight of the mice compared with the control group, indicating that 25 mg / kg and 75 mg / kg of F913-0023 have no obvious toxic effect on mice.

[0133] 7. F913-0023 acts in vivo to inhibit the growth of esophageal squamous cell carcinoma, but has no toxic effect on the heart, liver, spleen, lungs, and kidneys.

[0134] In summary, it can be seen that: as a small molecule compound, F913-0023 can inhibit the activity of UHRF1 enzyme and is a UHRF1 enzyme inhibitor; it can inhibit the proliferation and colony formation of esophageal squamous cell carcinoma cells KYSE30 and KYSE150 and promote the apoptosis of esophageal squamous cell carcinoma cells. F913-0023 can inhibit the growth of esophageal squamous cell carcinoma by inhibiting the activity of UHRF1 enzyme.

Claims

1. Application of F913-0023 in the preparation of UHRF1 inhibitors.

2. The use according to claim 1, characterized in that The IC50 of F913-0023 for esophageal squamous cell carcinoma cell KYSE30 is 17.62 μM, and the IC50 for esophageal squamous cell carcinoma cell KYSE150 is 16.98 μM.

3. The use according to claim 2, characterized in that Application of F913-0023 in the preparation of anti-esophageal squamous cell carcinoma drugs.

4. The use according to claim 3, characterized in that F913-0023 can inhibit the proliferation and clone formation of esophageal squamous cell carcinoma cells in the concentration range of 10-20μM.

5. The use according to claim 4, characterized in that F913-0023 inhibits the proliferation and clonogenicity of esophageal squamous cell carcinoma cells by targeting UHRF1.

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