Method for screening medicine for treating tumors

Abstract

The invention relates to a method for screening drugs for treating tumors. According to the method, whether a to-be-tested drug is combined with PTGS2 protein serving as target protein or not is judged, and if the to-be-tested drug is combined with the target protein, the inhibitory activity of the to-be-tested drug on the survival and / or growth of tumor cells is tested. Therefore, the period of screening target spots and candidate drugs with clear anti-tumor mechanisms can be obviously shortened, and a new way and a new method are provided for treating tumors, especially drug-resistant tumors, by taking PTGS2 as a target spot.

Description

Technical Field

[0001] This invention relates to the field of oncology drugs, and in particular to a method for screening drugs for treating tumors. Background Technology

[0002] Antitumor drug screening methods mainly include in vivo screening and in vitro screening. Although in vivo screening can better reflect the in vivo antitumor efficacy of drugs, it is difficult to screen, requires advanced transplantation and culture techniques, has a long experimental cycle, and is difficult to apply on a large scale. Currently, more mature in vitro screening methods include crystal violet staining, clonal assays, and short-term drug sensitivity tests. Although these methods are simpler, require fewer materials, and have a shorter experimental cycle than in vivo screening, the drug targets screened are unclear, and there may be serious toxic side effects. Summary of the Invention

[0003] This invention provides a method for screening drugs for treating or fighting tumors, which determines whether a drug to be tested binds to prostaglandin intraperoxidase 2 (PTGS2) protein, which is a target protein. If the drug to be tested binds to the target protein, then the inhibitory activity of the drug to be tested on tumor cell survival and / or growth is tested.

[0004] In one specific embodiment, the amino acid sequence of the PTGS2 protein is shown in SEQ ID No. 1.

[0005] In one specific embodiment, the target protein originates from tumor cells.

[0006] In one specific embodiment, the tumor is non-small cell lung cancer, and the tumor cells are non-small cell lung cancer cells.

[0007] In one specific embodiment, the tumor is drug-resistant non-small cell lung cancer, and the tumor cells are drug-resistant non-small cell lung cancer cells.

[0008] In one specific embodiment, the tumor is osimertinib-resistant non-small cell lung cancer, and the tumor cells are osimertinib-resistant non-small cell lung cancer cells.

[0009] In one specific embodiment, the method includes the following steps: 1) The test drug is coupled with epoxy-activated agarose microspheres to obtain epoxy-activated agarose-test drug microspheres; 2) Obtain the target protein from the tumor cells; 3) The epoxy-activated agarose-drug microspheres are bound to the target protein to obtain the bound sample; 4) The sample that has undergone the binding treatment is subjected to Western blotting using an antibody against the target protein; 5) If the immunoblotting result of the sample treated with the combination is positive, then the test drug binds to the target protein, and the inhibitory activity of the test drug on tumor cell survival and / or growth is tested.

[0010] In one specific embodiment, a negative control is provided in step 1) using the solvent (e.g., dimethyl sulfoxide or water) used to prepare the test drug solution instead of the test drug solution.

[0011] In one specific embodiment, in step 2), the tumor cells are lysed using a cell lysis buffer and insoluble matter is removed (e.g., by centrifugation) to obtain a solution containing the target protein; in step 3), the epoxy-activated agarose-drug microspheres are bound to the solution containing the target protein to obtain a bound sample.

[0012] In one specific embodiment, the ratio of the volume of the epoxy-activated agarose-drug microspheres to the total protein content in the solution containing the target protein is less than 1 (µL): 2 (µg).

[0013] In one specific embodiment, in step 3), the binding treatment is performed at a temperature of 2 to 8 degrees Celsius (e.g., 4 degrees Celsius) for 12 to 20 hours (e.g., 16 hours), followed by centrifugation, discarding the supernatant, and washing with phosphate buffer at least 5 times to obtain the bound sample.

[0014] In one specific embodiment, the antibody used for immunoblotting is a monoclonal antibody against the target protein.

[0015] In one specific embodiment, in step 5), the tumor cells are treated with the test drug at 10 μmol / L or at least five concentration gradients for at least 24 hours, and then the survival rate of the tumor cells is determined. If the survival rate of the tumor cells is below 50%, the test drug is considered to have good survival-inhibiting activity against the tumor cells. Furthermore, a negative control can be set up using the solvent used to prepare the test drug solution. Further, the IC50 can be calculated based on the negative control and the survival rate of the tumor cells at at least five concentration gradients. 50 Value, if IC 50 If the value is below 10 μmol / L, the test drug is considered to have good survival-inhibiting activity against tumor cells.

[0016] In one specific embodiment, in step 5), the tumor cells are treated with the test drug at a concentration of 1 μmol / L or less for at least 10 days, and then the number of monoclonal cells in the tumor cells is determined. Furthermore, a negative control can be set up using the solvent used to prepare the test drug solution. Further, if the number of monoclonal cells after treatment with the test drug at a concentration of 1 μmol / L or less is significantly lower than that of the negative control, the test drug is considered to have good growth-inhibiting activity against tumor cells.

[0017] The beneficial effects of this invention are as follows: This invention discovers that prostaglandin intraperoxidase 2 (PTGS2) is a binding target for antitumor drugs (such as Fritillaria cirrhosaponin-2). Utilizing this target protein, highly effective antitumor therapeutic drugs that kill tumor cells, especially drug-resistant tumor cells, can be screened, yielding candidate drugs targeting PTGS2 protein against drug-resistant tumors. This significantly shortens the screening cycle for candidate drugs with clear targets and antitumor mechanisms, providing a new approach and method for treating tumors, especially drug-resistant tumors, by targeting PTGS2. Attached Figure Description

[0018] Figure 1 The immunoblotting results show the binding of Fritillaria cirrhosa saponin-2 (TBM-2) to PTGS2.

[0019] Figure 2 An inverted fluorescence micrograph showing the effect of TBM-2 on vacuolization of osimertinib-resistant tumor cells overexpressing the PTGS2 gene.

[0020] Figure 3 The vacuolation rate of osimertinib-resistant tumor cells overexpressing the PTGS2 gene after TBM-2 treatment is shown. *** indicates p <0.001.

[0021] Figure 4 The survival rate of osimertinib-resistant tumor cells overexpressing the PTGS2 gene after TBM-2 treatment is shown. * indicates osimertinib. p <0.05, ** indicates p <0.01.

[0022] Figure 5 An inverted fluorescence micrograph showing the effect of TBM-2 on vacuolization of PTGS2 gene knockout osimertinib-resistant tumor cells.

[0023] Figure 6 The vacuolation rate of osimertinib-resistant PTGS2 gene knockout tumor cells treated with TBM-2 is shown. *** indicates p <0.001.

[0024] Figure 7The survival rate of osimertinib-resistant PTGS2 gene knockout tumor cells treated with TBM-2 is shown. * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001.

[0025] Figure 8 The survival rate of osimertinib-resistant tumor cells treated with TBM-2 is shown.

[0026] Figure 9 The number of monoclonal cells in osimertinib-resistant tumor cells treated with TBM-2 is shown; *** indicates p <0.001. Detailed Implementation

[0027] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0028] Unless otherwise specified, the plasmids and reagents used in the embodiments of this invention can be purchased commercially.

[0029] The amino acid sequence of PTGS2 is shown in SEQ ID No. 1, and the nucleic acid sequence encoding it is shown in SEQ ID No. 2.

[0030] The PTGS2 gene (whose nucleic acid sequence is shown in SEQ ID No. 2) was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0031] RPMI-1640 medium was purchased from Gibco.

[0032] Epoxy-Activated Beads 4FF: The matrix is ​​highly cross-linked 4% agarose microspheres with an active group density of 30 to 40 μmol / mL, a particle size of 45 to 165 μm, a maximum pressure of 0.3 MPa, and a storage buffer of 100% 1,4-dioxane, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0033] Osimertinib solution: Osimertinib is dissolved in dimethyl sulfoxide (DMSO) to prepare an osimertinib solution with a concentration of 10 mmol / L.

[0034] H1975 / OSIR cells: H1975 non-small cell lung cancer cell line (BNCC, Osimertinib IC50) in logarithmic growth phase was cultured in RPMI-1640 medium. 50 Cells at 5 × 10⁻⁶ mcg / L (0.1366 μmol / L) 6Cells were seeded in T25 cell culture flasks. After cell attachment, osimertinib solution was added to a final concentration of 0.05 μmol / L. The flasks were incubated at 37°C. When the cells reached 80% confluence, they were passaged. Again, after cell attachment, osimertinib solution was added to fresh RPMI-1640 medium, with the final concentration of osimertinib increasing sequentially over time to: 0.1 μmol / L, 0.25 μmol / L, 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2.0 μmol / L, 2.5 μmol / L, 3.0 μmol / L, 3.5 μmol / L, and 4.0 μmol / L. After treatment with the final concentration of osimertinib, when the cells reached 80% confluence, the half-maximal inhibitory concentration (IC50) of osimertinib was determined. 50 Increasing the concentration from 0.1366 μmol / L to 5.041 μmol / L resulted in the osimertinib-resistant non-small cell lung cancer cell line H1975 / OSIR, which exhibited a 37-fold increase in resistance index.

[0035] Construction of pcDNA3.1-PTGS2 recombinant plasmid: Using the artificially synthesized PTGS2 gene as a template, PTGS2-F (its nucleic acid sequence is shown in SEQ ID No. 3) and PTGS2-R (its nucleic acid sequence is shown in SEQ ID No. 4) were used as upstream and downstream primers, respectively. The 5' end-containing... Nhe I restriction site, 3' end contains Hind PCR product of the PTGS2 gene with restriction site III was used Nhe I and Hind The purified PCR product was digested with enzyme III to obtain a double-digested PCR fragment; simultaneously, using... Nhe I and Hind III. The pcDNA3.1 vector (Invitrogen) was double-digested to obtain the double-digested pcDNA3.1 vector fragment. The double-digested PCR fragment was then ligated with the double-digested pcDNA3.1 vector fragment using T4 DNA ligase to obtain the recombinant plasmid pcDNA3.1-PTGS2.

[0036] Construction of LentiCRISPR v2-Blast-sgPTGS2 recombinant plasmid: The sequences shown in sgPTGS2-F (as shown in SEQ ID No. 5) and sgPTGS2-R (as shown in SEQ ID No. 6) were annealed to obtain oligoDNA fragments. The oligoDNA fragments were then ligated to the LentiCRISPR v2-Blast plasmid (Miaoling plasmid platform) fragments that had been linearized by BsmBI restriction enzyme using T4 DNA ligase to obtain the recombinant plasmid LentiCRISPR v2-Blast-sgPTGS2.

[0037] sgCtrl lentivirus: Using PEI polyethyleneimine transfection reagent (Polysciences, USA), the empty vector plasmid LentiCRISPR v2-Blast and packaging helper plasmids psPAⅩ2 (Miaoling Plasmid Platform) and pMD2.G (Miaoling Plasmid Platform) were transfected into 293T cells (Beina Biotechnology BNCC). After culturing in DMEM medium at 37°C for 48 h, the supernatant was collected by centrifugation at 4000g for 10 min at 4°C and filtered through a 0.22-micron filter to obtain the sgCtrl lentivirus suspension.

[0038] sgPTGS2 lentivirus: The recombinant plasmid LentiCRISPR v2-Blast-sgPTGS2 and the packaging helper plasmids psPAⅩ2 and pMD2.G were transfected into 293T cells using PEI polyethyleneimine transfection reagent. After culturing in DMEM medium at 37°C for 48 h, the supernatant was collected by centrifugation at 4000g at 4°C for 10 min and filtered through a 0.22-micron filter to obtain the sgPTGS2 lentivirus suspension.

[0039] H1975 / OSIR / sgCtrl cells: H1975 / OSIR cells in logarithmic growth phase were arranged at a ratio of 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates. When the cell confluence reached 50% to 60%, the medium was replaced with fresh RPMI-1640 medium. 1 mL of sgCtrl lentivirus suspension was added to each well. The cells were cultured at 37°C for 16 h. 4 μg / mL of cypermethrin was added. The medium was replaced with fresh RPMI-1640 medium and the same amount of cypermethrin was added every two days. After 14 days, cells resistant to cypermethrin were screened to obtain H1975 / OSIR / sgCtrl cells.

[0040] H1975 / OSIR / sgPTGS2 cells: H1975 / OSIR cells in logarithmic growth phase were arranged at a ratio of 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates. When the confluence of cells reached 50% to 60%, the medium was replaced with fresh RPMI-1640 medium. 1 mL of sgPTGS2 lentivirus suspension was added to each well. The cells were cultured at 37°C for 16 h. 4 μg / mL of cypermethrin was added. The medium was replaced with fresh RPMI-1640 medium and the same amount of cypermethrin was added every two days. After 14 days, cells resistant to cypermethrin were screened to obtain PTGS2 knockout cells H1975 / OSIR / sgPTGS2 cells.

[0041] 10×PBS: 80.0 g NaCl, 2.0 g KCl, 34.84 g Na2HPO4·12H2O, 2.0 g KH2PO4, pH 7.4, and ultrapure water to a final volume of 1000 mL.

[0042] 0.1M pH 4.0 sodium acetate buffer (containing 0.5 M NaCl) was purchased from Shanghai Shangbao Biotechnology Co., Ltd.

[0043] 0.1 M pH 8.0 Tris-HCl buffer (containing 0.5 M NaCl) was purchased from Shenzhen Selma Biotechnology Co., Ltd.

[0044] RIPA lysis buffer was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0045] TBM-2 solution: TBM-2 was dissolved in dimethyl sulfoxide (DMSO) to prepare a TBM-2 solution with a concentration of 10 mmol / L.

[0046] Thiazol blue (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, abbreviated as MTT) solution: Dissolve 0.5 g of thiazolyl blue in 100 mL of phosphate buffer (1×PBS, pH 7.4) and filter through a 0.22 μm filter membrane to obtain a thiazolyl blue solution with a concentration of 5 mg / mL. Example 1: PTGS2 is the drug binding target

[0047] Preparation of TBM-2 coupled epoxy-activated agarose microspheres: Take 1 mL of epoxy-activated agarose microspheres (Epoxy-Activated Beads 4FF), centrifuge at 4000 rpm for 2 min at 4°C, collect the agarose microspheres, wash them 3 times with double-distilled water, gently shaking the agarose microspheres during washing, centrifuge at 4000 rpm for 2 min at 4°C, collect the agarose microspheres; adjust the pH of 1×PBS to 13 with NaOH, wash the agarose microspheres once with 1×PBS at pH 13, centrifuge at 4000 rpm for 2 min at 4°C, discard the supernatant, collect the agarose microspheres, add 80 μL of 10 mM TBM-2 solution to the agarose microspheres, transfer to a 7 mL centrifuge tube, and mix at 40°C for 24 h. Centrifuge at 4000 rpm for 2 min at 4°C, discard the supernatant, and wash the precipitate with 1×PBS (pH 13) to remove excess TBM-2 ligands. Centrifuge again at 4000 rpm for 2 min at 4°C, discard the supernatant, and add 100 μL of 1 M pH 8.0 ethanolamine (blocking solution) to the precipitate. Mix at 4°C and incubate for 8 h to block other sites in the agarose beads not occupied by TBM-2. After blocking, centrifuge at 4000 rpm for 2 min at 4°C, discard the supernatant, and wash the precipitate three times alternately with 0.1 M pH 4.0 sodium acetate buffer (containing 0.5 M NaCl) and 0.1 M pH 8.0 Tris-HCl buffer (containing 0.5 M NaCl). Centrifuge again at 4000 rpm for 2 min at 4°C, discard the supernatant, and obtain Epoxy-TBM-2 microspheres.

[0048] The difference between the Epoxy control microspheres and the Epoxy-TBM-2 microspheres is that 80 μL of 10 mM TBM-2 solution was replaced with 80 μL of DMSO, while the preparation of the Epoxy-TBM-2 microspheres was the same.

[0049] The specific steps of the Epoxy-TBM-2 microsphere binding to protein and the Western blot experiment were as follows: H1975 / OSIR cells were cultured in RPMI-1640 medium at 37°C to the logarithmic growth phase. NP40 lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd.) was added, and cells were collected using a cell scraper. The cells were placed on ice for 30 min, with shaking every 10 min, and centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was collected. The protein concentration in the supernatant was detected using a BCA protein concentration assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). The supernatant was divided into three portions. One 500 μL portion containing 1000 μg of total protein was added to a centrifuge tube containing 500 μL of Epoxy-TBM-2 microspheres. The mixture was gently shaken on a horizontal shaker at 4°C for 16 h, centrifuged at 4000 rpm for 2 min at 4°C, and the supernatant was discarded. The precipitate was treated with pH 7.4. Wash 5-6 times with 1×PBS, centrifuge at 4000 rpm for 2 min at 4°C, discard the supernatant and aspirate the residual liquid; the resulting precipitate is the treatment group sample. Add a second 500 μL supernatant containing 1000 μg total protein to a centrifuge tube containing 500 μL of Epoxy control microspheres, and repeat the same steps as the treatment group sample to prepare the control group sample. Perform polyacrylamide gel electrophoresis on the treatment group samples, control group samples, and the third supernatant, followed by Western blotting detection using PTGS2 monoclonal antibody (dilution 1:1000, Cell Signaling Technology). Results are shown in the figure. Figure 1 .

[0050] Figure 1 The results showed that the treated samples reacted positively with PTGS2 antibody, indicating that TBM-2 and PTGS2 have a direct interaction. Example 2: Effect of TBM-2 on vacuolization of osimertinib-resistant tumor cells overexpressing the PTGS2 gene

[0051] Treatment group-1: H1975 / OSIR cells in logarithmic growth phase were cultured in RPMI-1640 medium at a density of 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured at 37°C for 8 hours. The empty vector plasmid pcDNA3.1 was introduced into H1975 / OSIR cells using Lipofectamine 3000 transfection reagent and cultured at 37°C for 6 hours to obtain H1975 / OSIR / pcDNA3.1 cells. The medium was replaced with fresh RPMI-1640 medium and the same volume of DMSO as TBM-2 in treatment group-2 was added. The cells were then treated at 37°C for 24 hours to obtain treatment group-1.

[0052] Treatment Group-2: H1975 / OSIR cells in logarithmic growth phase were cultured in RPMI-1640 medium at a density of 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured at 37°C for 8 hours. The empty vector plasmid pcDNA3.1 was introduced into H1975 / OSIR cells using Lipofectamine 3000 transfection reagent and cultured at 37°C for 6 hours to obtain H1975 / OSIR / pcDNA3.1 cells. The medium was then replaced with fresh RPMI-1640 medium, and TBM-2 solution was added to bring the final concentration of TBM-2 to 3 μmol / L. The cells were treated at 37°C for 24 hours to obtain treatment group-2.

[0053] Treatment group-3: The empty pcDNA3.1 vector plasmid in treatment group-1 was replaced with the pcDNA3.1-PTGS2 recombinant plasmid and introduced into H1975 / OSIR cells. The cells were cultured at 37 degrees Celsius for 6 h to obtain H1975 / OSIR / pcDNA3.1-PTGS2 cells. All other aspects were the same as in treatment group-1.

[0054] Treatment Group-4: The empty pcDNA3.1 vector plasmid in Treatment Group-2 was replaced with the pcDNA3.1-PTGS2 recombinant plasmid and introduced into H1975 / OSIR cells. The cells were cultured at 37 degrees Celsius for 6 h to obtain H1975 / OSIR / pcDNA3.1-PTGS2 cells. All other operations were the same as in Treatment Group-2.

[0055] Treatment groups 1 to 4 were observed under an inverted fluorescence microscope. Five different fields of view were selected for each group, and 100 cells were selected for each field of view. The number of vacuolated cells was counted and the vacuolation rate of cells was calculated (number of vacuolated cells × 100% / total number of cells). * p <0.05 indicates a statistically significant difference. Results of the inverted fluorescence microscope images are shown below. Figure 2 The results of cell vacuolation rate are shown in [the table below]. Figure 3 .

[0056] Figure 2 and Figure 3 The results showed that overexpression of the PTGS2 gene could promote TBM-2-induced vacuolization of H1975 / OSIR cells, indicating that the PTGS2 protein is a target of TBM-2. Example 3: Effect of TBM-2 on the survival rate of osimertinib-resistant tumor cells overexpressing the PTGS2 gene

[0057] Treatment Group-1: H1975 / OSIR cells in logarithmic growth phase were seeded at 6500 cells per well in 96-well plates using RPMI-1640 medium and cultured at 37°C for 8 h. The pcDNA3.1-PTGS2 recombinant plasmid was then introduced into the H1975 / OSIR cells using Lipofectamine 3000 transfection reagent, and cultured at 37°C for 6 h to obtain H1975 / OSIR / pcDNA3.1-PTGS2 cells. The medium was replaced with fresh RPMI-1640 medium, and TBM-2 solution was added to a final concentration of 2 μmol / L. The cells were incubated at 37°C for 24 h. Then, 20 μL of 5 mg / mL thiazolyl blue solution was added, and the cells were incubated in a 37°C CO2 incubator for 4 h. The medium was discarded, and 100 μL of DMSO was added to each well. The cells were then placed on a horizontal shaker and incubated gently for 5 min at ambient temperature in the dark. After the purple crystals were completely dissolved, the cells were incubated at 570°C. The absorbance of each well was measured using an ELISA reader at nm. Cell viability was calculated using formula (1).

[0058] Cell viability = Treatment group - 1 OD value / Control group - 1 OD value × 100% Formula (1).

[0059] Treatment group-2: Replace the final concentration of TBM-2 in treatment group-1 with 3 μmol / L, replace treatment group-1 in formula (1) with treatment group-2, and everything else is the same as treatment group-1.

[0060] Treatment group-3: Replace the final concentration of TBM-2 in treatment group-1 with 4 μmol / L, replace treatment group-1 in formula (1) with treatment group-3, and everything else is the same as treatment group-1.

[0061] Treatment group-4: Replace the final concentration of TBM-2 in treatment group-1 with 5 μmol / L, replace treatment group-1 in formula (1) with treatment group-4, and all other aspects are the same as treatment group-1.

[0062] Control group-1: The TBM-2 solution in treatment group-4 was replaced with the same volume of DMSO (i.e., the final concentration of TBM-2 was 0), and everything else was the same as in treatment group-4.

[0063] Treatment group-5: Replace the pcDNA3.1-PTGS2 recombinant plasmid in treatment group-1 with the empty pcDNA3.1 vector plasmid to obtain H1975 / OSIR / pcDNA3.1 cells. Replace treatment group-1 in formula (1) with treatment group-5, replace control group-1 with control group-2, and all other conditions are the same as treatment group-1.

[0064] Treatment group-6: Replace the final concentration of TBM-2 in treatment group-5 with 3 μmol / L, replace treatment group-5 in formula (1) with treatment group-6, and all other aspects are the same as treatment group-5.

[0065] Treatment group-7: Replace the final concentration of TBM-2 in treatment group-5 with 4 μmol / L, replace treatment group-5 in formula (1) with treatment group-7, and everything else is the same as treatment group-5.

[0066] Treatment group-8: Replace the final concentration of TBM-2 in treatment group-5 with 5 μmol / L, replace treatment group-5 in formula (1) with treatment group-8, and everything else is the same as treatment group-5.

[0067] Control group-2: The TBM-2 solution in treatment group-8 was replaced with the same volume of DMSO (i.e., the final concentration of TBM-2 was 0), and everything else was the same as in treatment group-8.

[0068] See results Figure 4 .

[0069] Figure 4 The results showed that overexpression of the PTGS2 gene could enhance the inhibitory effect of TBM-2 on the survival of H1975 / OSIR cells and reduce the cell survival rate, indicating that the PTGS2 protein is the target of TBM-2. Example 4: Effect of TBM-2 on vacuolization of osimertinib-resistant tumor cells with PTGS2 gene knockout

[0070] Treatment group-1: H1975 / OSIR / sgCtrl cells in logarithmic growth phase were cultured in RPMI-1640 medium at a rate of 2 × 10⁶ cells per well. 5 One cell was seeded into a 6-well plate and cultured at 37°C for 8 hours. The medium was then replaced with fresh RPMI-1640 medium and DMSO of the same volume as the TBM-2 solution in treatment group-2 was added. The plate was then treated at 37°C for 24 hours to obtain treatment group-1.

[0071] Treatment group-2: H1975 / OSIR / sgCtrl cells in logarithmic growth phase were cultured in RPMI-1640 medium at a rate of 2 × 10⁶ cells per well. 5 One cell was seeded into a 6-well plate and cultured at 37°C for 8 hours. The medium was then replaced with fresh RPMI-1640 medium and TBM-2 solution was added to bring the final concentration of TBM-2 to 3 μmol / L. The plate was then treated at 37°C for 24 hours to obtain treatment group-2.

[0072] Treatment group-3: Replace the H1975 / OSIR / sgCtrl cells in treatment group-1 with H1975 / OSIR / sgPTGS2 cells, and everything else is the same as treatment group-1.

[0073] Treatment Group-4: Replace the H1975 / OSIR / sgCtrl cells in Treatment Group-2 with H1975 / OSIR / sgPTGS2 cells. All other operations are the same as in Treatment Group-2.

[0074] Treatment groups 1 to 4 were observed under an inverted fluorescence microscope. Five different fields of view were selected for each group, and 100 cells were selected for each field of view. The number of vacuolated cells was counted and the vacuolation rate of cells was calculated. * p <0.05 indicates a statistically significant difference. Results of the inverted fluorescence microscope images are shown below. Figure 5 The results of cell vacuolation rate are shown in [the table below]. Figure 6 .

[0075] Figure 5 and Figure 6 The results showed that knocking out the PTGS2 gene could significantly inhibit TBM-2-induced vacuolization of H1975 / OSIR cells, indicating that the PTGS2 protein is a target of TBM-2. Example 5: Effect of TBM-2 on the survival rate of osimertinib-resistant tumor cells with PTGS2 gene knockout

[0076] Treatment Group-1: H1975 / OSIR / sgPTGS2 cells in logarithmic growth phase were seeded at 6500 cells per well into 96-well plates using RPMI-1640 medium and cultured at 37°C for 8 h. TBM-2 solution was added to bring the final concentration of TBM-2 to 2 μmol / L, and the cells were incubated at 37°C for 24 h. Then, 20 μL of 5 mg / mL thiazolyl blue solution was added, and the cells were incubated in a CO2 incubator at 37°C for 4 h. The medium was discarded, and 100 μL of DMSO was added to each well. The cells were then placed on a horizontal shaker and gently shaken at ambient temperature in the dark for 5 min. After the purple crystals were completely dissolved, the absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated according to formula (1) in Example 3.

[0077] Treatment group-2: Replace the final concentration of TBM-2 in treatment group-1 with 3 μmol / L, replace treatment group-1 in formula (1) with treatment group-2, and everything else is the same as treatment group-1.

[0078] Treatment group-3: Replace the final concentration of TBM-2 in treatment group-1 with 4 μmol / L, replace treatment group-1 in formula (1) with treatment group-3, and everything else is the same as treatment group-1.

[0079] Treatment group-4: Replace the final concentration of TBM-2 in treatment group-1 with 5 μmol / L, replace treatment group-1 in formula (1) with treatment group-4, and all other aspects are the same as treatment group-1.

[0080] Control group-1: The TBM-2 solution in treatment group-4 was replaced with the same volume of DMSO (i.e., the final concentration of TBM-2 was 0), and everything else was the same as in treatment group-4.

[0081] Treatment group-5: Replace H1975 / OSIR / sgPTGS2 cells in treatment group-1 with H1975 / OSIR / sgCtrl, replace treatment group-1 in formula (1) with treatment group-5, replace control group-1 with control group-2, and all other conditions are the same as treatment group-1.

[0082] Treatment group-6: Replace the final concentration of TBM-2 in treatment group-5 with 3 μmol / L, replace treatment group-5 in formula (1) with treatment group-6, and all other aspects are the same as treatment group-5.

[0083] Treatment group-7: Replace the final concentration of TBM-2 in treatment group-5 with 4 μmol / L, replace treatment group-5 in formula (1) with treatment group-7, and everything else is the same as treatment group-5.

[0084] Treatment group-8: Replace the final concentration of TBM-2 in treatment group-7 with 5 μmol / L, replace treatment group-5 in formula (1) with treatment group-8, and everything else is the same as treatment group-7.

[0085] Control group-2: The TBM-2 solution in treatment group-8 was replaced with the same volume of DMSO (i.e., the final concentration of TBM-2 was 0), and everything else was the same as in treatment group-8.

[0086] See results Figure 7 .

[0087] Figure 7 The results showed that knocking out the PTGS2 gene could weaken the inhibitory effect of TBM-2 on the survival of H1975 / OSIR cells and increase the cell survival rate, indicating that the PTGS2 protein is the target of TBM-2. Example 6: Inhibitory activity of TBM-2 on the survival of osimertinib-resistant tumor cells

[0088] Treatment Group-1: H1975 / OSIR cells in the logarithmic growth phase were resuspended in RPMI-1640 medium and seeded at 6500 cells per well in a 96-well plate. The cells were cultured at 37°C for 8 hours. TBM-2 solution was added to bring the final concentration of TBM-2 to 0.5 μmol / L. The cells were incubated at 37°C for 24 hours. Then, 20 μL of 5 mg / mL thiazolyl blue solution was added and the cells were incubated in a CO2 incubator at 37°C for 4 hours. The medium was discarded, and 100 μL of DMSO was added to each well. The cells were placed on a horizontal shaker and gently shaken at ambient temperature in the dark for 5 minutes. After the purple crystals were completely dissolved, the absorbance of each well was measured at 570 nm using a microplate reader. The cell viability was calculated according to formula (1) in Example 3, wherein control group-1 in formula (1) was replaced with the control group.

[0089] Treatment group-2: Replace the final concentration of TBM-2 in treatment group-1 with 1 μmol / L, replace treatment group-1 in formula (1) with treatment group-2, and everything else is the same as treatment group-1.

[0090] Treatment group-3: Replace the final concentration of TBM-2 in treatment group-1 with 2 μmol / L, replace treatment group-1 in formula (1) with treatment group-3, and everything else is the same as treatment group-1.

[0091] Treatment group-4: Replace the final concentration of TBM-2 in treatment group-1 with 3 μmol / L, replace treatment group-1 in formula (1) with treatment group-4, and everything else is the same as treatment group-1.

[0092] Treatment group-5: Replace the final concentration of TBM-2 in treatment group-1 with 4 μmol / L, replace treatment group-1 in formula (1) with treatment group-5, and everything else is the same as treatment group-1.

[0093] Treatment group-6: Replace the final concentration of TBM-2 in treatment group-1 with 5 μmol / L, replace treatment group-1 in formula (1) with treatment group-6, and all other aspects are the same as treatment group-1.

[0094] Treatment group-7: Replace the final concentration of TBM-2 in treatment group-1 with 6 μmol / L, replace treatment group-1 in formula (1) with treatment group-7, and everything else is the same as treatment group-1.

[0095] Treatment group-8: Replace the final concentration of TBM-2 in treatment group-1 with 7 μmol / L, replace treatment group-1 in formula (1) with treatment group-8, and all other aspects are the same as treatment group-1.

[0096] Treatment group-9: Replace the final concentration of TBM-2 in treatment group-1 with 8 μmol / L, replace treatment group-1 in formula (1) with treatment group-9, and everything else is the same as treatment group-1.

[0097] Control group: The TBM-2 solution in treatment group-9 was replaced with the same volume of DMSO (i.e., the final concentration of TBM-2 was 0), and everything else was the same as in treatment group-9.

[0098] Statistical analysis was performed on the cell viability of each group, and the results are as follows: Figure 8 . Figure 8 The results showed that TBM-2 reduced H1975 / OSIR cell survival in a concentration-dependent manner, and IC50... 50 With a concentration of 3.280 μmol / L, it can effectively kill osimertinib-resistant non-small cell lung cancer cells. Example 7: Inhibitory activity of TBM-2 on the growth of osimertinib-resistant tumor cells

[0099] Treatment Group-1: H1975 / OSIR cells in logarithmic growth phase were resuspended in RPMI-1640 medium and seeded at 1000 cells per well in 6-well plates. The plates were incubated at 37°C for 8 hours. The culture medium was discarded, and 2 mL of fresh RPMI-1640 medium was added to each well. TBM-2 solution was added to bring the final TBM-2 concentration to 0.3 μmol / L. The plates were then incubated at 37°C, with the TBM-2 concentration of fresh RPMI-1640 medium replaced every 3 days for approximately 10 days, until visible cell clusters (at least 50 cells per cluster) appeared. The culture medium was then discarded, and the cells were washed once with pre-cooled 1×PBS (4°C). 1 mL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 minutes. The paraformaldehyde was discarded, and crystal violet staining solution was added to cover the wells. The plates were stained at room temperature for 3 minutes. min; remove the crystal violet and wash with tap water. After washing, count the cell clusters with more than 50 cells as the number of monoclonal cells and calculate the monoclonal ratio. *** p <0.001 indicates a statistically significant difference.

[0100] Treatment Group-2: Replace the final concentration of TBM-2 in Treatment Group-1 with 0.6 μmol / L, and keep all other parameters the same as in Treatment Group-1.

[0101] Control group: The TBM-2 solution in treatment group-2 was replaced with the same volume of DMSO (i.e., the final concentration of TBM-2 was 0), and everything else was the same as in treatment group-2.

[0102] Monoclonal ratio = number of monoclonal cells in each treatment group × 100% / number of monoclonal cells in the control group.

[0103] The number of monoclonal cells in each group was statistically analyzed, and the results are as follows: Figure 9 , Figure 9The results showed that TBM-2 could significantly reduce the number of monoclonal cells formed by H1975 / OSIR cells and inhibit cell growth in a dose-dependent manner.

Claims

1. A method for screening drugs for treating or treating tumors, wherein the selection is based on whether the test drug binds to the PTGS2 protein as a target protein, and if the test drug binds to the target protein, then the inhibitory activity of the test drug on tumor cell survival and / or growth is tested.

2. The method according to claim 1, characterized in that, The amino acid sequence of the PTGS2 protein is shown in SEQ ID No.

1.

3. The method according to claim 1, characterized in that, The target protein originates from tumor cells; Preferably, the tumor is non-small cell lung cancer, and the tumor cells are non-small cell lung cancer cells; Preferably, the tumor is drug-resistant non-small cell lung cancer, and the tumor cells are drug-resistant non-small cell lung cancer cells; Preferably, the tumor is osimertinib-resistant non-small cell lung cancer, and the tumor cells are osimertinib-resistant non-small cell lung cancer cells.

4. The method according to any one of claims 1 to 3, characterized in that, The method includes the following steps: 1) The test drug is coupled with epoxy-activated agarose microspheres to obtain epoxy-activated agarose-test drug microspheres; 2) Obtain the target protein from the tumor cells; 3) The epoxy-activated agarose-drug microspheres are bound to the target protein to obtain the bound sample; 4) The sample that has undergone the binding treatment is subjected to Western blotting using an antibody against the target protein; 5) If the immunoblotting result of the sample treated with the combination is positive, then the test drug binds to the target protein, and the inhibitory activity of the test drug on tumor cell survival and / or growth is tested.

5. The method according to claim 4, characterized in that, In step 1), a negative control is set up in which the solvent used to prepare the test drug solution is used instead of the test drug solution.

6. The method according to claim 4 or 5, characterized in that, In step 2), the tumor cells are lysed using a cell lysis buffer and insoluble matter is removed to obtain a solution containing the target protein; In step 3), the epoxy-activated agarose-drug microspheres are bound to the solution containing the target protein to obtain a bound sample. Preferably, the ratio of the volume of the epoxy-activated agarose-drug microspheres to the total protein content in the solution containing the target protein is less than 1 (µL): 2 (µg).

7. The method according to claim 6, characterized in that, In step 3), the binding treatment is carried out at a temperature of 2 to 8 degrees Celsius for 12 to 20 hours, then centrifuged, the supernatant is discarded, and the sample is washed with phosphate buffer more than 5 times to obtain the binding-treated sample.

8. The method according to claim 4 or 5, characterized in that, The antibody used for immunoblotting is a monoclonal antibody against the target protein.

9. The method according to any one of claims 4 to 8, characterized in that, In step 5), the tumor cells are treated with the test drug at a concentration of 10 μmol / L or at least 5 concentration gradients for at least 24 hours, and then the survival rate of the tumor cells is determined.

10. The method according to any one of claims 4 to 8, characterized in that, In step 5), the tumor cells are treated with the test drug at a concentration of less than 1 μmol / L for at least 10 days, and then the number of monoclonal cells in the tumor cells is determined.

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