Application of compound U37i as a small molecule inhibitor targeting UCH37 and UCHL3

Abstract

The present invention provides the use of compound U37i as a small molecule inhibitor targeting UCH37 and UCHL3. Experimental results show that U37i can inhibit the deubiquitinase activity of UCH37, cause an increase in the level of polyubiquitinated proteins in tumor cells, and promote the degradation of UCH37 substrate β-catenin in cells. Although U37i also inhibits UCHL3, which belongs to the UCH family, it does not inhibit another member of the family, UCHL1, indicating that U37i has a certain specificity. Similar to UCH37, UCHL3 is also associated with the occurrence of various cancers, including pancreatic cancer, cervical cancer, ovarian cancer and lung cancer. U37i can further cause tumor cell cycle arrest and cell proliferation cessation, promote tumor cell apoptosis, inhibit clone formation and migration. U37i's simultaneous inhibition of UCH37 and UCHL3 makes it possible to be used for a wider spectrum of cancers or to achieve dual-target treatment of specific cancers. In addition, the small molecule compound U37i described in the present invention may also effectively inhibit the inflammatory phenotype of autoimmune diseases and acute colitis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the use of compound U37i as a small molecule inhibitor targeting UCH37 and UCHL3, and more specifically relates to the use of compound U37i as a small molecule inhibitor targeting UCH37 and UCHL3 in the treatment of diseases such as tumors. Background Art

[0002] The ubiquitin-proteasome pathway is the main pathway responsible for protein degradation in human cells. There are different types of proteasomes in the human body, such as 26S, PA28 and PA200, which are composed of dozens of subunits. Among them, the 26S proteasome that degrades ubiquitinated substrates is composed of 20S catalytic particles and 19S regulatory particles located at one or both ends. The barrel-shaped structure composed of 20S catalytic particles can hydrolyze substrate proteins and release peptides, while 19S regulatory particles mainly recognize and bind ubiquitinated substrate proteins, and then deubiquitinate, unfold and transport the substrates. At present, there are many clinically used proteasome inhibitors for the treatment of multiple myeloma and mantle cell lymphoma. However, these inhibitors all target the catalytic subunits of 20S particles, still have large side effects, and are prone to drug resistance. Therefore, targeted intervention of different types of proteasomes or subunits is very important for improving the efficacy of these cancers and for the treatment of other cancers or diseases.

[0003] The 19S particles of the 26S proteasome have three deubiquitinases (DUBs), among which UCH37 belongs to the UCH ubiquitin carboxyl-terminal hydrolase family. UCH37 binds to the 19S particles by interacting with the proteasome ubiquitin receptor subunit Rpn13, and removes ubiquitin chain branches or deubiquitinates ubiquitinated substrate proteins, thereby promoting substrate degradation. However, UCH37 protects certain specific substrate proteins, such as β-catenin, NFRKB, COPS5, etc., thereby protecting these substrates from proteasomal degradation. UCH37 is highly expressed in many cancers such as cervical cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma, and epithelial ovarian cancer, and may serve as a potential cancer therapeutic drug target. In addition, UCH37 is involved in regulating the degradation of key infection immune factors such as inducible nitric oxide synthase (iNOS) and IκB, and may also be associated with autoimmune diseases and acute colitis. In addition to UCH37, there are three other members in the UCH deubiquitinase family: UCHL1, UCHL3, and BAP1. All UCH DUBs have a UCH deubiquitinase domain of about 230 amino acids at the N-terminus, and have different ring structures outside the UCH active site to control different substrate specificities. UCHL1 is a special one in the UCH family. It is expressed in large quantities in the brain and has been reported to be highly associated with Alzheimer's disease and Parkinson's syndrome. UCHL3 is a protein with an amino acid sequence that is highly similar to UCHL1. While the molecular size is similar, there is 53% similarity in the amino acid sequence. However, UCHL1 and UCHL3 have huge differences in enzymatic properties. For example, UCHL1 has a unique deubiquitinase activity among UCH family members, while UCHL3 has 200 times the activity of UCHL1 for ubiquitin fluorescent substrates. Because of the important role of UCHL3 in DNA damage repair, the overexpression of UCHL3 is highly correlated with the poor effect of radiotherapy / chemotherapy in breast cancer cells, making UCHL3 a potential anticancer drug target. To date, there are still no specific UCH37 or UCHL3 inhibitors. Summary of the invention

[0004] The present invention is dedicated to discovering highly effective and selective UCH37 / UCHL3 small molecule inhibitors against human UCH. The discovery of inhibitors that can act on human UCH37 / UCHL3 can provide new ideas and methods for the treatment of diseases such as tumors, autoimmune diseases and acute colitis.

[0005] The object of the present invention is to provide a new medical use of compound U37i or its stereoisomers, tautomers and pharmaceutically acceptable salts, hydrates and solvates, prodrugs or deuterated derivatives or pharmaceutical compositions containing the same.

[0006] The novel medical use of the compound U37i or its stereoisomers, tautomers and pharmaceutically acceptable salts, hydrates and solvates, prodrugs or deuterated derivatives or pharmaceutical compositions comprising the same provided by the present invention is the use of the compound U37i or its stereoisomers, tautomers and pharmaceutically acceptable salts, hydrates and solvates, prodrugs or deuterated derivatives or pharmaceutical compositions comprising the same in the preparation of any of the following products:

[0007] 1) UCH37 and / or UCHL3 inhibitors;

[0008] 2) Drugs for treating tumors;

[0009] 3) Drugs that inhibit tumor metastasis;

[0010] 4) Drugs for the treatment of autoimmune diseases;

[0011] 5) Drugs for the treatment of acute colitis.

[0012] The tumor is a benign tumor or a malignant tumor.

[0013] More specifically, the tumors include digestive system tumors, respiratory system tumors, reproductive system tumors, blood circulation system tumors, nervous system tumors, endocrine system tumors, circulatory system tumors and urinary system tumors.

[0014] Preferably, the tumor is cancer, including cervical cancer, multiple myeloma, liver cancer, osteosarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, kidney cancer, gastric cancer, colorectal cancer and lung cancer.

[0015] Further preferably, the cancer includes cervical cancer, multiple myeloma, liver cancer and osteosarcoma.

[0016] Specifically, the above application is achieved by inhibiting UCH37 and / or UCHL3.

[0017] More specifically, compound U37i achieves anti-tumor function by inhibiting UCH37 and / or UCHL3 to inhibit cancer cell proliferation, cancer cell clone formation, and cancer cell migration.

[0018] The CAS number of compound U37i is 1135138-09-8, and its structural formula is:

[0019]

[0020] The chemical formula is C 30 H 35ClN4O5S2·HCl, the full name is Benzamide,4-[(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]-N-(4,7-dimethoxy-2-benzothiazolyl)-N-[3-(dimethylamino)propyl]-,hydrochloride(1:1).

[0021] In the application, compound U37i can be prepared into a variety of dosage forms, including tablets, injections, and capsules.

[0022] Compound U37i has an inhibitory effect on the proliferation, cell clone formation and in vitro migration ability of cervical cancer, multiple myeloma, liver cancer and osteosarcoma cells. It is a highly effective and selective small molecule inhibitor targeting UCH37 / UCHL3 with great potential, and can provide an important basis and basis for the development of new anti-tumor drugs.

[0023] The small molecule compound U37i may also effectively inhibit the inflammatory phenotype of autoimmune diseases and acute colitis, and will most likely provide new drug development guidance for the clinical treatment of these diseases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the inhibition curve of compound U37i on UCH37.

[0025] Figure 2 This is the inhibition curve of compound U37i on UCHL1 and UCHL3.

[0026] Figure 3 Compound U37i inhibits the binding of Ub-VS to His-UCH37.

[0027] Figure 4 Compound U37i treatment induces accumulation of polyubiquitinated proteins in HeLa cells; (A) polyubiquitinated protein levels in HeLa cells; (B) a bar graph drawn using β-actin as a standard and statistical analysis of relevant data.

[0028] Figure 5 Compound U37i promotes the degradation of UCH37 substrate β-catenin; (A) β-catenin levels after treatment with CHX under fixed conditions and U37i for different time periods; (B) bar graph drawn using β-actin as the standard and statistical analysis of relevant data.

[0029] Figure 6The compound U37i has an inhibitory effect on the cell activity of HeLa, RPMI 8226, U2OS, and HepG2 cancer cells.

[0030] Figure 7 Compound U37i causes HeLa cell cycle arrest at the G2 / M phase; (A) HeLa cells are treated with different concentrations of U37i, and the proportions of HeLa cell cycle G2 / M, S, and G0 / G1 phases are detected by flow cytometry; (B) statistical analysis of relevant data is performed, and finally a bar graph is drawn based on the relevant data.

[0031] Figure 8 Compound U37i induces apoptosis of HeLa cells; (A) HeLa cells were treated with U37i and stained with PI and Annexin V-FITC, and the apoptosis level of HeLa cells was detected by flow cytometry; (B) The differences in the proportions of early apoptotic Q4-4 and late apoptotic Q4-2 cells induced by U37i treatment and the control group were statistically analyzed, and the relevant data were statistically analyzed to draw a bar graph.

[0032] Fig. 9 Compound U37i reduces the cloning ability of HeLa cells; wherein, (A) the number level of clones formed after HeLa cells are treated with different concentrations of U37i and continued to be cultured; (B) statistical analysis of relevant data is performed, and finally a bar graph is drawn based on the relevant data.

[0033] Fig.10 Compound U37i reduces the wound healing ability of HeLa cells; (A) HeLa cells form a monolayer of intercellular "wounds" and are then treated with U37i, and photographs are taken at different times to record the "wound area", with the scale bar representing 200 μm; (B) The "wound" area is calculated using ImageJ, and the relevant data are statistically analyzed to produce a bar graph. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1: U37i inhibits the Ub-AMC hydrolysis activity of purified protein UCH37

[0037] Experimental methods:

[0038] Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC, Boston Biochem, U-550) was used as a substrate for the cysteine ​​isopeptidase activity of UCH37. His-UCH37 protein expressed and purified from E. coli strain BL21 can cleave Ub-AMC, releasing a detectable fluorescent signal. This reaction can be monitored in real time on a Flexstation3 multifunctional microplate reader (Molecular Devices, Flexstation3) using a black 96-well plate (Corning, 3925).

[0039] The reaction was carried out using HTS buffer: 20mM HEPES-Na (BioFROXX, 1112GR500) pH 7.5, 150mM NaCl (Sinopharm, 10019318), 0.05% Tween-20 (McLean, T818927), 1mM EDTA (Sinopharm, 10009717), 2mM DTT (Solarbio, D8220). Deubiquitinase and fluorescent substrate Ub-AMC were first diluted with HTS buffer, and the final concentrations in the reaction system were UCH371 nM and Ub-AMC500 nM.

[0040] First, add 75 μL of purified protein of deubiquitinase UCH37 to a black 96-well plate, then add 1 μL of small molecule U37i of different concentrations dissolved in DMSO (Sigma, D1435), and mix at room temperature for 30 minutes on a shaker. Finally, add substrate Ub-AMC, and immediately use Flexstation3 multifunctional microplate reader to measure the curve of AMC fluorescence signal growth over time for 30 minutes. The excitation wavelength of AMC fluorescence is 380nm, and the emission wavelength is 460nm.

[0041] Experimental results:

[0042] The results showed that U37i could inhibit the activity of purified protein UCH37. The percentage of UCH37 activity was plotted against the logarithmic concentration of U37i, and nonlinear fitting was performed using Graphpad Prism 7.0 software to give the half inhibitory concentration (IC 50 ), and the IC of U37i for UCH37 is obtained 50 =2.9μM( Figure 1 ).

[0043] Example 2: U37i inhibits the deubiquitination activity of UCHL3

[0044] Experimental methods:

[0045] Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC, Boston Biochem, U-550) was used as a substrate for the UCHL1 and UCHL3 cysteine ​​isopeptidase activity. His-UCHL1 and His-UCHL3 proteins expressed and purified from E. coli strain BL21 can cleave Ub-AMC, releasing a detectable fluorescent signal. This reaction can be monitored in real time on a Flexstation3 multi-function microplate reader (Molecular Devices, Flexstation3) using a black 96-well plate (Corning, 3925).

[0046] The reaction was carried out using HTS buffer: 20 mM HEPES-Na (BioFROXX, 1112GR500) pH 7.5, 150 mM NaCl (Sinopharm, 10019318), 0.05% Tween-20 (McLean, T818927), 1 mM EDTA (Sinopharm, 10009717), 2 mM DTT (Solarbio, D8220). Deubiquitinase and fluorescent substrate Ub-AMC were first diluted with HTS buffer, and the final concentrations in the reaction system were UCHL1 / UCHL37.5 / 0.25 nM, and Ub-AMC 500 nM.

[0047] First, add 75 μL of purified protein of deubiquitinase UCHL1 or UCHL3 to a black 96-well plate, add 1 μL of small molecule U37i of different concentrations dissolved in DMSO (Sigma, D1435), and mix at room temperature for 30 minutes on a shaker. Finally, add substrate Ub-AMC, and immediately use Flexstation3 multifunctional microplate reader to measure the curve of AMC fluorescence signal growth over time for 30 minutes. The excitation wavelength of AMC fluorescence is 380nm, and the emission wavelength is 460nm.

[0048] Experimental results:

[0049] The results showed that U37i could not effectively inhibit the activity of purified protein UCHL1, but could inhibit the activity of purified protein UCHL3. The percentage of UCHL1 or UCHL3 activity was plotted against the logarithmic concentration of U37i, and nonlinear fitting was performed using the software Graphpad Prism 7.0 to give the half inhibitory concentration (IC 50 ), the half inhibitory concentrations of U37i for UCHL1 and UCHL3 were IC 50 >25μM and IC50 =3.2μM( Figure 2 ). Thus, U37i can effectively inhibit UCHL3 with an IC 50 Similar to inhibition of UCH37.

[0050] Example 3: Qualitative verification of U37i combined with UCH37

[0051] Experimental methods:

[0052] Ubiquitin vinyl sulfone (Ub-VS, Boston Biochem, U-212) is a Ub derivative that specifically binds to cysteine ​​DUBs and can spontaneously and irreversibly form a covalent bond with the active site of cysteine ​​DUBs, thereby abolishing their deubiquitinase activity. Purified His-UCH37 protein was incubated with different concentrations of U37i and the previously reported UCH37 small molecule inhibitor b-AP15 (Biyuntian, SG0020) at 37°C, and then Ub-VS was added to bind to the His-UCH37 protein whose active site was not occupied by the small molecule inhibitor, and the sample was prepared for western blotting experiments.

[0053] Use Ub-VS binding buffer: 25mM Tris-HCl, pH 7.5, 100mM NaCl, 5mM DTT to dilute His-UCH37 protein to 100nM. Dilute small molecule U37i to a specific concentration with DMSO, add to the diluted His-UCH37 in the previous step, mix thoroughly, and incubate at 37℃ for 1h. Add Ub-VS to a final concentration of 500nM, mix thoroughly, and incubate at 37℃ for 30min. Add 4×SDS to all liquids and detect the results by protein immunoblotting.

[0054] Experimental results:

[0055] It can be seen that after the addition of Ub-VS, part of the His-UCH37 protein covalently binds to it to form His-UCH37-Ub-VS with a molecular weight increase of 8kD. The small molecule U37i or the positive control b-AP15 with increasing concentration gradient can reduce the production of His-UCH37-Ub-VS, which qualitatively proves that U37i binds to the UCH37 protein like the positive control b-AP15 ( Figure 3 ).

[0056] Example 4: U37i induces accumulation of polyubiquitinated proteins in HeLa cells

[0057] Experimental methods:

[0058] HeLa cells were cultured in DMEM medium (HyClone, SH30243.01) supplemented with 100 U / mL penicillin (Gibco, SV30010) and 100 μg / mL streptomycin (Gibco, SV30010) and 10% fetal bovine serum (Sijiqing, 11011-8611) at 37°C and 5% CO2. Cells in the exponential growth phase were obtained for the next analysis.

[0059] HeLa cells (from ATCC) were treated with U37i gradient concentrations for 12 h, or U37i 4 μM for gradient time, and the cells were collected for western blotting experiments. Ubiquitin (Ub) antibody (Cell Signaling Technology, 3936S) was used to detect the accumulation of cellular polyubiquitinated proteins.

[0060] Experimental results:

[0061] The small molecule U37i, as an inhibitor of the proteasome 19S deubiquitinase UCH37, can also induce the accumulation of polyubiquitinated proteins in cells. The results showed that U37i can increase the accumulation of polyubiquitinated proteins in cells by increasing the concentration gradient or prolonging the treatment time ( Figure 4 ), and the amount of polyubiquitinated bands in cells was counted by ImageJ, and calculated relative to the internal reference protein β-actin (antibody from Sigma, A5441), and repeated three times independently. It can be determined that U37i induces a significant increase in the accumulation of polyubiquitinated proteins in HeLa cells.

[0062] Example 5: U37i inhibits UCH37 in cells and promotes the degradation of its substrate β-catenin

[0063] Experimental methods:

[0064] HeLa cells were treated with cycloheximide (CHX, Sigma, C7698) for 4, 8, and 12 hours to inhibit nascent protein synthesis, making protein levels completely dependent on their degradation. They were also treated with 4 μM U37i for 12 hours, and the cells were collected for western blotting and β-catenin levels were detected with BD antibody (610154).

[0065] Experimental results:

[0066] The results showed that U37i treatment promoted the degradation of β-catenin in HeLa cells ( Figure 5). This indicates that unlike traditional 20S proteasome inhibitors that inhibit β-catenin degradation, U37i inhibits UCH37, which protects β-catenin, thereby significantly promoting the degradation of β-catenin; this proves that U37i works in cells by inhibiting UCH37.

[0067] Example 6: U37i inhibits cell activity of different cancer cells

[0068] Experimental methods:

[0069] The cells were digested with trypsin (HyClone, 25200-072) and counted, and diluted to 5×10 4 / mL, add 100 μL to the 96-well plate with a dispenser. Continue to culture the cells for 24 hours. Carefully aspirate the DMEM medium in the wells and add DMEM mixed with U37i to treat the cells.

[0070] After the U37i treatment, carefully remove the DMEM, carefully add 100 μL DMEM medium mixed with 10 μL CCK-8 (Biyuntian, C0037) to each well, and continue to culture at 37°C for 2 hours. The medium turns yellow. The absorbance at 450 nm is measured using a Flexstation3 multifunctional microplate reader.

[0071] The inhibition curve was fitted nonlinearly using Graphpad Prime 7, and the half-inhibitory concentration of U37i on the activity of various cancer cells (HeLa, HepG2, U2OS were all from ATCC; RPMI8226 was from the National Biomedical Experimental Cell Resource Bank) was obtained.

[0072] Experimental results:

[0073] As shown in the results, the half-inhibitory concentration IC of U37i for HeLa, RPMI 8226, U2OS and HepG2 50 They are: 3.3μM, 1.0μM, 4.6μM and 4.2μM ( Figure 6 ), showing the inhibitory activity of U37i against different cancer cells.

[0074] Example 7: U37i causes HeLa cell cycle arrest at G2 / M phase

[0075] Experimental methods:

[0076] HeLa cells treated with 2, 4, and 6 μM U37i for 12 h were stained with propidium iodide (PI, Beyotime, C1052-2), and then the cell cycle was analyzed by flow cytometry.

[0077] For drug-treated cells, the culture medium supernatant and PBS and trypsin used for cell digestion were collected during the cell digestion process. After the cells were just digested, the cells were gently blown off with DMEM, and the cells and the above collection were centrifuged together at 500g for 5 minutes at room temperature.

[0078] After centrifugation, remove the supernatant, gently resuspend the cell pellet with PBS, and transfer to a 1.5mL EP tube. Centrifuge again and remove PBS. In this step, you can leave 50μL of liquid to avoid removing the cells.

[0079] The cells were gently resuspended with pre-cooled 70% ethanol (Jingchun, JC-AR20140714) and fixed at 4°C overnight.

[0080] Aspirate the ethanol in the same way, wash the cells once with pre-cooled PBS, and aspirate the PBS.

[0081] Prepare the staining solution with the formula of 500 μL staining buffer, 25 μL propidium iodide staining solution, and 10 μL RNase A (Biyuntian, C1052-3) for each sample and mix well. Gently resuspend the cells with the staining solution and place them in a 37°C water bath for 30 minutes in the dark.

[0082] The samples were placed on ice to avoid light. The cells were gently resuspended before loading. The adherent cells were removed by filtration using a cell flow cytometer (Corning, 352235) and the cell cycle was analyzed using a NovoCyte flow cytometer.

[0083] Experimental results:

[0084] The results showed that compared with the control, 4 and 6 μM U37i reduced the proportion of cells in the G0 / G1 phase from 50.3% to 37.9% and 31.6%, respectively; the proportion of cells in the S phase increased from 35.6% to 39.4% and 37.3%, respectively; the proportion of cells in the G2 / M phase increased from 13.8% to 20.0% and 29.3% ( Figure 7 ). 4.6μM U37i significantly increased the number of HeLa cells in the G2 / M phase, slightly increased the number of cells in the S phase, and correspondingly reduced the proportion of cells in the G0 / G1 phase, and the degree of change was positively correlated with the concentration gradient of U37i. Based on the above results, it can be concluded that the main effect of U37i on the HeLa cell cycle is to block it in the G2 / M phase, and then inhibit the proliferation of HeLa cells.

[0085] Example 8: U37i induces apoptosis in HeLa cells

[0086] Experimental methods:

[0087] HeLa cells treated with 4 μM U37i for 12 h were stained with Annexin V-FITC (Biyuntian, C1062) and propidium iodide (PI), and then flow cytometry was performed to analyze cell apoptosis.

[0088] For drug-treated cells, the culture medium supernatant and PBS and trypsin used for cell digestion were collected during the cell digestion process. After the cells were just digested, the cells were gently blown off with DMEM, and the cells and the above collection were centrifuged together at 500g for 5 minutes at room temperature.

[0089] After the supernatant was removed, the cells were resuspended in PBS and counted. 1×10 5 The cells were placed in EP tubes and centrifuged to remove PBS.

[0090] Gently resuspend the cells in 195 μL Annexin V-FITC binding solution for each sample, and add 5 μL Annexin V-FITC staining solution and 10 μL propidium iodide staining solution in sequence, and mix gently. The samples were incubated at room temperature in the dark for 20 minutes, during which the cells were gently resuspended 3 times. After the binding process is completed, the samples were placed on ice in the dark. In order to calibrate the parameters of the NovoCyte flow cytometer, it is necessary to set up Annexin V-FITC and propidium iodide single-staining tubes and double-staining tubes for apoptosis positive controls.

[0091] Gently resuspend the cells before loading, filter through a cell flow cytometer to remove adherent cells, and then use a NovoCyte flow cytometer to analyze cell apoptosis.

[0092] Experimental results:

[0093] The results showed that the treatment of HeLa cells with 4 μM U37i for 12 hours significantly increased the proportion of early apoptotic cells in Q4 from 0.9% in the control group to 6.7% in the U37i-treated group; the proportion of late apoptotic cells in Q2 significantly increased from 0.8% in the control group to 8.3% in the U37i-treated group. Figure 8 ). The total percentage of apoptotic cells was calculated, which increased from 1.7% in the control group to 15% in the group treated with 4 μM U37i, proving that U37i can induce apoptosis in HeLa cells.

[0094] Example 9: U37i reduces the colony-forming ability of HeLa cells

[0095] Experimental methods:

[0096] HeLa cells were digested with trypsin and counted, and after multiple dilutions with DMEM 1:10, a cell density of 200 / mL was reached. The diluted HeLa cells were added to a 12-well plate (Corning, 3513), 1 mL per well, i.e., 200 cells, and gently shaken and cultured for 24 hours.

[0097] The cells were treated with drugs for 12 h, and the culture medium was replaced every 3 days after treatment for 10 to 12 days.

[0098] The culture medium was removed by aspiration, and the cells were washed twice with PBS. Anhydrous methanol (Jingchun, JC-AR20140713) was added to fix the cells and incubated at room temperature for 20 min.

[0099] The anhydrous methanol was removed, the sample was washed twice with deionized water, 0.5% crystal violet stain (Biyuntian, C0121) was added, and the sample was incubated at room temperature for 5 min.

[0100] Wash several times with deionized water until excess dye is removed and cells are clearly stained.

[0101] Invert the tube on filter paper, air-dry overnight, and then count the number of cell clones with more than 50 cells under a bright field microscope.

[0102] Experimental results:

[0103] The results showed that 2 and 3 μM U37i could significantly reduce the number of HeLa cell colonies ( Fig. 9 ). This demonstrates the inhibitory effect of U37i on the colony-forming ability of HeLa cells.

[0104] Example 10: U37i reduces the migration ability of HeLa cells

[0105] Experimental methods:

[0106] Place the surface-dried Ibidi two-chamber (Ibidi, 81176) insert into the center of the 6-well plate and fit it tightly.

[0107] HeLa cells were digested with trypsin and counted, then diluted to 7.0 × 10 5 / mL. Diluted HeLa cells were evenly added to the insert, with 100 μL added to each chamber. The cells were cultured for 24 hours to form a uniform monolayer of densely packed cells.

[0108] The insert was carefully removed, carefully washed three times with PBS to remove the remaining serum and cell debris in the culture medium, and photographed under a bright field microscope with a 10× objective lens, with 4 images taken for each scratch.

[0109] Pictures were taken after 12 h, 24 h, and 36 h of treatment with 4 μM U37i in serum-free DMEM medium, and the scratch area was analyzed and statistically analyzed using ImageJ.

[0110] Experimental results:

[0111] The experimental results showed that the treatment of HeLa cells with 4 μM U37i significantly reduced the area of ​​"wound" recovery of HeLa cells at 12h, 24h, and 36h ( Fig.10 ), indicating that U37i has a significant inhibitory effect on the healing ability of HeLa cells, proving that U37i can inhibit the cell migration ability of HeLa cells.

[0112] In summary, the present invention obtained a small molecule inhibitor U37i of the proteasome deubiquitinase UCH37 through drug screening. Although U37i also inhibits the non-proteasome deubiquitinase UCHL3 belonging to the UCH family, it cannot effectively inhibit another member of the family, UCHL1, indicating that U37i has a certain specificity. Similar to UCH37, UCHL3 is also associated with the occurrence of various cancers, including pancreatic cancer, cervical cancer, ovarian cancer and lung cancer. The simultaneous inhibition of UCH37 and UCHL3 by U37i makes it possible to be used for a wider spectrum of cancers or to achieve dual-target treatment of specific cancers. These results provide useful ideas and an important basis for the further development of specific UCH37 or UCHL3 inhibitors for cancer treatment.

[0113] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of compound U37i or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same in the preparation of a drug for treating tumors; The structural formula of compound U37i is: The tumor is selected from: cervical cancer, multiple myeloma, liver cancer or osteosarcoma.

2. The use according to claim 1, characterized in that: In the application, the compound U37i achieves anti-tumor function by inhibiting UCH37 and / or UCHL3 to inhibit cancer cell proliferation, cancer cell clone formation, and cancer cell migration.

3. The use according to claim 1, characterized in that: In the application, compound U37i is prepared into a variety of dosage forms selected from tablets, injections or capsules.

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