Application of small molecule medicine Esculin in preparation of medicine for treating tumors

Abstract

The invention discloses an application of a small molecular medicine Esculin in preparation of a medicine for treating tumors, and belongs to the technical field of biological medicines. The invention discloses an application of a small molecule drug Esculin in preparation of drugs for tumor treatment / oxaliplatin sensitization and the like. Results show that the small molecule drug Esculin forms hydrogen bonds with Asp-70, Ser-158, Asp-221, Arg-318, Glu-360 and Trp-422 residues of GRWD1 by targeting an interaction structural domain of GRWD1 and NPM1, inhibits the combination of GRWD1 with NPM1, RPL11 and p53, inhibits the degradation of p53, activates a p53 signal channel, and finally enhances the stability of p53, inhibits liver cancer cell proliferation, promotes liver cancer cell apoptosis and improves the sensitivity to OXA; therefore, the oxaliplatin can be used for preparing medicines for treating tumors, enhancing sensitivity of oxaliplatin and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of small molecule drug Esculin in the preparation of drugs for treating tumors. Background Art

[0002] The small molecule drug Esculin, also known as horse chestnut bark glycoside, aesculin, etc., has a molecular formula of C 15 H 16 O9·xH2O, molecular weight varies according to the crystal water, the structure is as follows Figure 1 As shown, CAS No.531-75-9. It usually exists in the form of white needle-shaped crystals or powder, has a high melting point, and is soluble in hot alcohol, methanol and other solvents. The small molecule drug Esculin has anti-inflammatory, antibacterial, anticoagulant, and analgesic activities. It can also inhibit the aldose reductase in the lens of rat eyes, is a growth inhibitor of Bacillus subtilis, and has an inhibitory effect on chemical carcinogenesis. In addition, the small molecule drug Esculin can also improve venous return, brain function, and achieve the effect of reducing swelling. The small molecule drug Esculin is widely used in the medical field and can be used to treat discomfort symptoms caused by diseases such as dysentery, lumbar disc herniation, cervical spondylosis, and encephalitis.

[0003] A key feature of malignant tumors is uncontrolled cell proliferation. As one of the important tumor suppressor genes, the p53 gene plays a vital role in regulating the cell cycle, DNA repair, and apoptosis. Studies have shown that the p53 gene is mutated or deleted in more than 50% of human cancers, and even in cancers without p53 mutations, the p53 downstream signaling pathway often loses its activity. Therefore, restoring the activity of p53 in tumors has always been an important direction for the development of drugs targeting the p53 pathway.

[0004] MDM2 (Mouse Double Minute 2) is an E3 ubiquitin ligase that can degrade p53 through ubiquitination, thereby inhibiting its tumor suppressor function. In many tumors, overexpression of MDM2 leads to the functional inactivation of p53. In the prior art, inhibiting the interaction between MDM2 and p53 to stabilize and restore the activity of p53 has become an important strategy for the development of anticancer drugs.

[0005] NPM1 (Nucleophosmin 1) is another protein closely related to p53 regulation. The localization and function of NPM1 in the nucleus have an important influence on the stability and activity of p53. Mutations or abnormal expression of the NPM1 gene can affect the function of p53 and are closely related to the occurrence and development of various tumors. However, research on regulators for NPM1 in the prior art is still insufficient.

[0006] GRWD1 (Glutamate-Rich WD Repeat-Containing 1) is a protein containing multiple WD40 repeat domains, which form a paddle-like ring structure and provide multiple binding sites for interaction with other proteins or molecules. GRWD1 is an important negative regulator of p53 in cells, affecting the activity of p53 protein at the protein level by direct or indirect interactions between proteins. For example, in melanoma, GRWD1 directly interacts with p53 to inhibit the expression of p53 downstream genes (melanoma); GRWD1 can also competitively inhibit the interaction between RPL11 and MDM2, promote the ubiquitination and degradation of p53 by MDM2, inhibit the activation of the p53 signaling pathway, and thus lead to tumor development and treatment resistance (PMID: 27856536).

[0007] Existing studies have shown that 16 ribosomal proteins (RPs), including NPM1, RPL23, and RPL5, can directly bind to MDM2, inhibit MDM2-mediated p53 degradation, and activate p53 (Deisenroth C, Franklin DA, Zhang Y. The Evolution of the Ribosomal Protein-MDM2-p53 Pathway. Cold Spring Harb Perspect Med. 2016 Dec 1;6(12):a026138.). The inactivation of this RP-MDM2-p53 signaling pathway is closely related to the occurrence of various tumors. Among them, NPM1 plays an important role in p53 activation and ribosome stress sensing by binding to the C4 zinc finger region of MDM2.

[0008] However, the prior art lacks a small molecule drug compound that can block the interaction between GRWD1 and NPM1, RPL11, and p53, inhibit p53 degradation, and activate the p53 signaling pathway, thereby significantly inhibiting tumor cell proliferation and causing tumor cell death both in vitro and in vivo. Summary of the invention

[0009] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of drugs for treating tumors, and for preparing drugs for treating liver cancer. Another technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of oxaliplatin sensitizing drugs. Another technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of drugs for inhibiting the proliferation of liver cancer cells. Another technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of drugs that promote tumor cell death. Another technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of drugs or agents that induce cell cycle arrest. Another technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of drugs or agents that induce tumor cell death. Another technical problem to be solved by the present invention is to provide the use of the small molecule drug Esculin in the preparation of drugs that mediate tumor cell death through p53 or drugs that induce tumor cell death independently of p53.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0011] Application of the small molecule drug Esculin in the preparation of drugs for treating tumors.

[0012] The small molecule drug Esculin interacts with hydrogen bonds of ASP-70, SER-158, ASP-221, ARG-318, GLU-360, and TRP-422 residues of GRWD1, thereby blocking the binding of GRWD1 with NPM1, RPL11, and p53.

[0013] Application of the small molecule drug Esculin in the preparation of oxaliplatin sensitizing drugs.

[0014] The small molecule drug Esculin interacts with hydrogen bonds of ASP-70, SER-158, ASP-221, ARG-318, GLU-360, and TRP-422 residues of GRWD1, blocks the binding of GRWD1 with NPM1, RPL11, and p53, and promotes the improvement of the sensitivity of liver cancer cells to oxaliplatin.

[0015] Application of the small molecule drug Esculin in the preparation of drugs for inhibiting the proliferation of liver cancer cells.

[0016] Application of the small molecule drug Esculin in the preparation of drugs that promote tumor cell death.

[0017] Application of the small molecule drug Esculin in the preparation of drugs or reagents for inducing cell cycle arrest.

[0018] Application of the small molecule drug Esculin in the preparation of drugs or reagents for inducing tumor cell death.

[0019] The small molecule drug Esculin is used at a concentration of 10 μM-1000 μM.

[0020] Application of the small molecule drug Esculin in the preparation of drugs that mediate tumor cell death through p53 or drugs that induce tumor cell death independently of p53.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The small molecule drug Esculin disclosed in the present invention is used in the preparation of drugs for treating liver cancer / preparing oxaliplatin sensitizing drugs / preparing drugs for inhibiting liver cancer cell proliferation / preparing drugs for promoting tumor cell death / preparing drugs or reagents for inducing cell cycle arrest / preparing drugs or reagents for inducing tumor cell death / preparing drugs for mediating tumor cell death through p53 or drugs that do not rely on p53 to induce tumor cell death. The results show that the small molecule drug Esculin targets the interaction domain of GRWD1 and NPM1, forms hydrogen bonds with Asp-70, Ser-158, Asp-221, Arg-318, Glu-360, and Trp-422 residues of GRWD1, inhibits the binding of GRWD1 with NPM1, RPL11, and p53, inhibits p53 degradation, activates the p53 signaling pathway, and ultimately enhances the stability of p53, inhibits liver cancer cell proliferation, promotes liver cancer cell apoptosis, and improves sensitivity to OXA; therefore, it is used to prepare drugs for treating tumors / oxaliplatin sensitization, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the structural diagram of the small molecule drug Esculin;

[0024] Figure 2 The following is the expression analysis of GRWD1 gene in pan-cancer in TCGA (https: / / portal.gdc.cancer.gov) (A) (statistical method is Wilcoxon rank sum test. *, ** and *** represent p < 0.05, p < 0.01, p < 0.005, respectively); the expression analysis of GRWD1 in liver cancer in TCGA database (B) (374 cancer tissues and 50 adjacent tissues); the expression of GRWD1 gene in 50 paired samples in the public database (C);

[0025] Figure 3(A) Typical example of GRWD1 immunohistochemistry in liver cancer and adjacent tissues (A) (scale bar 100 μm); Overall survival (OS), disease-free survival (Disease Specific Survival) and progression-free survival (Progression Free Interval) of HCC patients in the TCGA database (B); Kaplan-Meier curve analysis of 190 patients (C); Overall survival (OS) analysis of 74 HCC patients who received TACE (containing oxaliplatin) after hepatocellular carcinoma resection (D);

[0026] Figure 4 Figure 2 shows the expression of GRWD1 protein in SK-Hep-1, HepG2, Huh7 and Hep3B cells (A); the expression of GRWD1 mRNA in the four cell lines (B); (C-F) the protein intervention effect (left) and mRNA intervention effect (right) after knocking down GRWD1 in HepG2 cells (C); the protein intervention effect (left) and mRNA intervention effect (right) after overexpressing GRWD1 in SK-Hep-1 cells (D); the protein intervention effect (left) and mRNA intervention effect (right) after knocking down GRWD1 in Huh7 cells (left) and mRNA intervention effect (right) (E); (F) protein intervention effect (left) and mRNA intervention effect (right) after overexpression of GRWD1 in Hep3B cells; (G) IC50 value results after adding different concentrations of oxaliplatin to HepG2 and Huh7 cells with knockdown of GRWD1 and cells overexpressing SK-Hep-1 and Hep3B as well as the corresponding control cells; (H) Proteins were collected at 0, 6, 12 and 24 hours after stimulating the four cell lines with 15 μM oxaliplatin, and the protein expression of p53 and p21 was detected by Western blot (GAPDH was used as the internal reference); (I) The protein expression of the corresponding molecules was detected by the same method after the four cell lines and control group cells were treated with the same drug concentration after intervention in GRWD1 (*, ** and *** represent p < 0.05, p < 0.01, p < 0.005, respectively, and ns represents no statistical significance);

[0027] Figure 5 Figure 2 is the change of cell proliferation ability after knocking down GRWD1 in HepG2 and Huh7 cells and overexpressing GRWD1 in SK-Hep-1 and Hep3B cells (A); Figure 2 is the change of cell colony formation ability after knocking down GRWD1 in HepG2 and Huh7 cells and overexpressing GRWD1 in SK-Hep-1 and Hep3B cells (B) (*p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no statistical significance);

[0028] Figure 6 Cell line apoptosis diagram (left 2), corresponding statistical diagram (middle), and detection diagram of apoptosis-related molecules after intervention with GRWD1 in Hep3B (A), SK-Hep-1 (B) liver cancer cells overexpressing GRWD1, and Huh7 (C), HepG2 (D) liver cancer cell lines with knockdown of GRWD1 after 12 hours of stimulation with 20 μM oxaliplatin (*p < 0.05, **p < 0.01, ns indicates no statistical significance);

[0029] Figure 7 Figure 2 is the endogenous interaction diagram of GRWD1 and NPM1 (A); the interaction diagram of GRWD1 and NPM1 in HepG2 and SK-Hep-1 cells before and after 12 hours of oxaliplatin (20 μM) treatment (B); the interaction diagram of exogenous GRWD1 and NPM1 in 293T cells (C); the ubiquitination results of p53 protein detected by immunoprecipitation with anti-p53 antibody in GRWD1 silenced and GRWD1 overexpressed cells after MG132 (20 μM) treatment for 4 hours (D); the p53 levels in GRWD1 silenced HepG2 cells and GRWD1 overexpressed SK-Hep-1 cells treated with CHX (100 μg / mL) at the specified time intervals, and their control group (E); the p53 protein levels in GRWD1 silenced and GRWD1 overexpressed cells transfected or not with MDM2 siRNA (F); the p53 protein levels in the same cells transfected or not with NPM1 siRNA (G);

[0030] Figure 8 Docking mode diagram of GRWD1 with NPM1, p53 and RPL11 (A); overall 3D structure diagram of GRWD1 complex (left), close-up diagram of NPM1 active site and peptide binding (right) (B); interaction diagram of Esculin and GRWD1 amino acid residues (C); RMSD diagram of GRWD1 and Esculin during molecular dynamics simulation (D); RMSF diagram of GRWD1 and Esculin during molecular dynamics simulation (E); interaction diagram of Esculin and GRWD1 amino acid residues (F); effect of Esculin combined with oxaliplatin on cell viability in two liver cancer cell lines (Huh7 and HepG2) (G). DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific examples. In the following examples, operations not described in detail are all routine biological experimental operations, which can be performed with reference to molecular biology experimental manuals and existing published journals, or according to the operating steps in the instructions of the kit.

[0032] Example 1 Expression of GRWD1 in liver cancer cells and tissues and its correlation with clinical prognosis

[0033] 1. Immunohistochemical staining

[0034] Drying: Preheat the temperature of the drying machine to 85°C, then take out the prepared liver cancer tissue chip (stored in Nantong Cancer Hospital) and dry it on the drying machine for 10 minutes.

[0035] Dewaxing of tissue sections: Place the baked tissue sections into the xylene solution in tanks No. 1 and No. 2 and soak for 15 minutes each.

[0036] Hydration: Soak the liver cancer tissue sections in the following concentrations: anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol, for 2 minutes each.

[0037] Heat repair of antigens on liver cancer sections: Fill a small stainless steel basin with EDTA antigen repair solution, place the sections in the slice rack at intervals, and immerse them in the small basin. Fill a pressure cooker with an appropriate amount of clean water, then place the small basin in the pressure cooker and heat it for 3 minutes after the pressure cooker sprays. After the pressure cooker cools down, take out the small basin, cool it to room temperature, and then gently rinse it with PBS 3 times to ensure that there is no antigen repair solution residue on the sections.

[0038] Block endogenous peroxidase with hydrogen peroxide: shake off excess liquid and draw a circle around the tissue with a tissue pen. Add 3% hydrogen peroxide to the circle and cover the tissue for 20 minutes. Wash with PBS for 5 minutes for a total of 3 times.

[0039] 1. Secondary antibody incubation: Gently shake the tissue chip, carefully drop the diluted GRWD1 antibody into the tissue circle, and gently use the gun tip to make the diluted antibody evenly cover the tissue. Carefully place the liver cancer tissue slices into the antibody incubation box and place them in the refrigerator at 4°C overnight. The next day, take the tissue slices out and warm them to room temperature, shake them on a PBS shaker for 5 minutes for a total of 3 times, shake dry, add secondary antibody and incubate for 20 minutes, and then wash them with PBS for 5 minutes for a total of 3 times.

[0040] DAB color development and nuclear staining: Prepare DAB color development solution, drop it on the slice to evenly cover the tissue, and wash it immediately when brown color appears. Restain with hematoxylin for half a minute, and wash it again with clean water to remove excess dye.

[0041] Dehydration and sealing: Dehydrate in alcohol according to the concentration gradient (the opposite order of hydration). After air-drying, immerse in two bottles of xylene for 8 minutes each. Seal the slides with neutral resin, taking care to prevent bubbles.

[0042] Scoring: Two professional pathologists will evaluate the results. The percentage of positive cells is scored as follows: 0, none; 1, <25%; 2, 25% to 50%; 3, 50% to 75%; 4, >75%. The staining intensity is scored as follows: 0, none; 1, weak; 2, intermediate; 3, strong. Finally, the strength of molecular expression is judged by the calculation formula of the percentage of positive cells multiplied by the staining intensity, and the results are used for the next step of analysis.

[0043] 2. Western Blot

[0044] Protein extraction: Place the cell culture dish on an ice basin, add the prepared lysis buffer, fully lyse for 5 minutes, scrape the protein into a 1.5mL EP tube and mark it, boil it in a 100℃ dry incubator for 10 minutes, and then put it on ice to cool for later use.

[0045] Gel electrophoresis: Set up the cleaned glass plate and check for leakage with clean water. If there is no leakage, wipe the plate with filter paper and prepare 10% gel, and then prepare fresh electrophoresis solution according to the recipe. After installing the glass plate, fill the inner tank with the prepared electrophoresis solution until it overflows to the scale line of the outer tank, take out the Bio-rad comb and add the sample, set the voltage to 80V and start running the gel. When the protein runs from the compression gel to the separation gel in a straight line, the voltage can be adjusted to 120V until the end.

[0046] Transfer: Polarize the PVDF membrane in methanol for 1 minute, depolarize it in ddH2O, and finally place it in transfer solution for later use. Remove the glass plate, cut off the excess gel, and then place it in the assembled transfer clip in the form of sponge pad-filter paper-PVDF membrane-gel-filter paper-sponge pad. Insert the assembled transfer clip into the groove, install it and place it in an ice basin, and cover it with enough ice. Transfer the membrane at a constant voltage of 100V for 60 minutes. Block the membrane in blocking buffer (5% skim milk) for 1 hour.

[0047] Antibody incubation: Dilute GRWD1, NPM1, p53 and other antibodies according to the respective ratios in the instructions, cover them on the PVDF membrane, and place them in the refrigerator at 4°C overnight. The next day, take them out and warm them up at room temperature, and shake them gently on a TBST shaker for 5 minutes for a total of 3 times. Dilute the secondary antibody, incubate at room temperature for 2 hours, and shake them gently on a TBST shaker for 10 minutes for a total of 3 times. Finally, use the developer to develop.

[0048] 3. Expression of GRWD1 in liver cancer cells and tissues and its correlation with clinical prognosis

[0049] Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad software Inc.) and R (4.2.1) software. The differences between the groups were analyzed using the chi-square test or Student's test. The Kaplan-Meier method was used to calculate the overall survival (OS) after surgery. Cox stepwise multivariate regression analysis was performed for prognostic factors. P values ​​less than 0.05 were considered statistically significant. The TCGA database was used to analyze the expression of GRWD1 in pan-cancer and liver cancer-related datasets.

[0050] The results are as follows Figure 2 As shown in Figure 2, GRWD1 is upregulated in most cancer tissues, and its expression in liver cancer tissues is significantly higher than that in adjacent tissues ( Figure 2 A. Figure 2 B), and 50 paired tissues in the public data also showed that GRWD1 was highly expressed in cancer ( Figure 2 C).

[0051] The results are as follows Figure 3 As shown in the figure, a total of 190 liver cancer tissues from patients stored in Nantong Cancer Hospital were immunohistochemically stained, and typical images of immunohistochemical staining chips were displayed ( Figure 3 A). The Kaplan-Meier curve shows the relationship between GRWD1 and prognosis and survival. The results show that in the TCGA data, the higher the expression of GRWD1, the shorter the patient's overall survival (OS), disease-free survival (Disease Specific Survival) and progression-free survival (Progression Free Interval) ( Figure 3 B); Kaplan-Meier curve analysis of 190 patients also showed that high expression of GRWD1 indicated shorter overall survival and poor prognosis ( Figure 3 C). In addition, 74 HCC patients who received TACE (including oxaliplatin) after liver cancer resection in Nantong Cancer Hospital and Nantong University Affiliated Hospital were collected for Kaplan-Meier curve analysis. The results showed that patients with high expression of GRWD1 had a poor prognosis, which also indicated that GRWD1 was related to the patient's local chemotherapy ( Figure 3 D).

[0052] Example 2 GRWD1 promotes liver cancer cell proliferation, inhibits liver cancer cell apoptosis and reduces sensitivity to OXA

[0053] 1. Cell culture and transfection

[0054] Four liver cancer cell lines (HepG2, Huh7, SK-Hep-1 and Hep3B cells, all purchased from Wuhan Pronocell) were selected. HepG2 and Huh7 were cultured in DMEM medium, SK-Hep-1 and Hep3B were cultured in MEM medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, and placed in an incubator at 37°C with a CO2 concentration of 5%. GRWD1shRNA and GRWD1 overexpression virus were purchased from Genetech. Virus transfection was carried out according to the instructions, and 5×10 cells were placed in a 12-well cell culture plate. 4 Hepatocellular carcinoma cells were used, and the virus dosage was calculated according to the virus titer (MOI of 50). The medium was changed after 14-16 hours of infection, and the cell protein was collected 2-3 days later for western blot detection of efficiency. The GRWD1 overexpression plasmid was purchased from Weizhen Company, the NPM1 plasmid was purchased from Shandong Weizhen Biological Company, and the NPM1 and MDM2 siRNAs were purchased from Shanghai Heyuan Company. The transfection reagent for plasmids and siRNAs was jetPRIME®polyplus transfection reagent, and transfection was performed according to the instructions. The culture medium was changed 4-6 hours after transfection. The cells were collected 48 hours after plasmids and 72 hours after siRNAs for subsequent experiments.

[0055] 2. Cell proliferation assay and cell colony formation assay

[0056] The transfected or treated cells were cultured at 4×10 3 The cells were inoculated in 96-well plates at a density of 500 cells / well and incubated for 1-5 days. Three replicate wells were set up for each group. Cell viability was detected using a cell counting kit (CCK-8). For the cell colony formation experiment, the treated liver cancer cells were evenly inoculated in a 6-well plate at a density of 500 cells / well. After 2 weeks, the cell colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.5% crystal violet (diluted with PBS) for 15-20 min.

[0057] 3. Apoptosis

[0058] Digest the prepared cells with EDTA-free trypsin, centrifuge at 1000 rpm for 5 min to collect the cells. Add an appropriate amount of PBS to resuspend the cells, centrifuge at 1000 rpm for 5 min to collect the cells again. Dilute 10×binding buffer to 1×binding buffer with ddH2O, add 300μL buffer and gently blow the cells. Add 5μL of FITCAnnexin V solution, mix well and protect from light, incubate at room temperature for 15 min. Add 5μL of PI solution and add 200μL buffer before using the flow cytometer for 5 min.

[0059] 4. Real-time quantitative reverse transcription polymerase chain reaction (q-rtPCR)

[0060] Total RNA was extracted using an RNA extraction kit (Gene Gene, Shanghai, China), and reverse transcribed complementary DNA was synthesized by reverse transcription using a reverse transcription kit (Vazyme, Nanjing, China). cDNA was amplified using SYBR green-based qRT-PCR performed on a Bio-Rad CFX96 real-time PCR system. The conditions were set as follows: pre-denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. GAPDH was used as an internal control for mRNA quantification. The relative expression of GRWD1 was calculated using the 2-ΔΔCt method. The primer sequences for GRWD1 and GAPDH primers are as follows:

[0061] GRWD1-sense: 5'-CCATCTTCTCCTTCGCTGGACA-3';

[0062] GRWD1-anti-sense: 5'-AGGTGTCCAGAGGTGGATGTTC-3';

[0063] GAPDH-sense: 5'-CATGAGAAGTATGACAACAGCCT-3';

[0064] GAPDH-anti-sense: 5'-AGTCCTTCCACGATACCAAAGT-3'.

[0065] The results are as follows Figure 4 As shown, the protein expression of GRWD1 in SK-Hep-1, HepG2, Huh7 and Hep3B cells was detected by Western blot ( Figure 4 A), qRT-PCR was used to detect the mRNA expression of GRWD1 in the corresponding cells ( Figure 4 B), the results showed that the expression levels of HepG2 and Huh7 cells were relatively high, while the expression levels of SK-Hep-1 and Hep3B cells were relatively low, so the former two were selected for knockdown treatment, and the latter two were selected for expression treatment, and protein immunoblotting was used respectively ( Figure 4 CF, left) and PCR (3C-F, right) were used to detect the intervention efficiency. Different concentrations of oxaliplatin were added to HepG2 and Huh7 cells with knockdown of GRWD1, SK-Hep-1 and Hep3B cells overexpressing GRWD1, and the corresponding control cells. The proliferation activity of cells was detected using the CCK-8 kit, and the IC50 value was calculated ( Figure 4G), the results showed that the IC50 value of the control group in HepG2 cells was 16.46μM, and the IC50 value decreased to 11.65μM after knocking down GRWD1; the IC50 value of the control group in SK-Hep-1 cells was 11.48μM, and it increased to 14.30μM after overexpression; the detection value of the control group in Huh7 cells was 13.78μM, which was slightly reduced to 13.10μM after knocking down; finally, the value in the Hep3B control group cells was 10.36μM, and the increase after overexpression was not obvious, which was 11.48μM. It is known that HepG2 and SK-Hep-1 are p53 wild-type cells, while Huh7 and Hep3B are p53 mutant and deletion cells, respectively. In summary, GRWD1 can significantly reduce the sensitivity of wild-type p53 liver cancer cells to oxaliplatin, and it also has an effect in mutant and deletion p53 cells, but it is not significant. It is known that NPM1 mediates cell death after drug stimulation by stabilizing p53. Subsequently, the protein expression levels of p53 and its downstream effector molecule p21 in four cell lines at different time points after oxaliplatin stimulation were detected ( Figure 4 H), the results showed that the expression of these two molecules in the four cell lines (except Hep3B, where p53 protein was not detected) showed a step-by-step upward trend over time, and the expression was most obvious at 12 hours. After intervention with GRWD1, the same method was used to detect the expression of p53 (except for the deletion cells, where p53 was not detected) and p21 proteins in the control group and the intervention group at different time points in the four cell lines ( Figure 4 I), the results showed that, in the intervention groups of SK-Hep-1 and Hep3B cells overexpressing GRWD1, the expression of p21 protein was decreased compared with that in the control group, but it was more obvious in SK-Hep-1, and the expression of p53 protein in SK-Hep-1 cells was also decreased compared with that in the control group; in Huh7 and HepG2 cells, knockdown of GRWD1 upregulated the expression of p53 and p21 proteins compared with that in the control group, and it was more obvious in HepG2 cells.

[0066] The results are as follows Figure 5 As shown in the figure, the proliferation and colony formation ability of HepG2 cells with knockdown of GRWD1 expression level was significantly reduced, while that of Huh7 cells was slightly reduced. After overexpression of GRWD1 in the other two liver cancer cell lines, the proliferation and colony formation ability of SK-Hep-1 tumor cells was significantly increased, but the enhancement effect was not obvious in Hep3B cells ( Figure 5 A, B).

[0067] The results are as follows Figure 6As shown, four pairs of cell lines were overexpressed and knocked down in Hep3B, SK-Hep-1, HepG2, and Huh7 liver cancer cell lines before and after oxaliplatin stimulation. All cells were collected 12 hours later and stained with FITC kit in the dark, and then flow cytometry was used to detect cell apoptosis ( Figure 6 The left side of AD is the apoptosis diagram, and the bar graph is the statistical result). Western blot was also used to detect the expression of apoptosis-related proteins Bcl-2, Bax and EI24. It is known that oxaliplatin stimulates cells to produce nucleolar stress, and nucleolar stress induces cytotoxicity through the p53 pathway, so the expression of p53 protein was detected. Bcl-2, Bax and EI24 are all downstream effector molecules of p53 ( Figure 6 AD, right). The results showed that GRWD1 could inhibit the expression of p53, Bax and EI24, and enhance the expression of Bcl-2 anti-apoptotic gene, but its effect was more obvious in p53 wild-type cells (HepG2, SK-Hep-1) than in p53 mutant cells (Huh7) and p53 null cells (Hep3B). The apoptosis detection results also showed that GRWD1 inhibited oxaliplatin-induced apoptosis, and it was more obvious in p53 wild-type liver cancer cells.

[0068] Example 3 GRWD1 interacts with NPM1 to reduce the stability of p53

[0069] The above results indicate that the effect of GRWD1 is more obvious in p53 wild-type cells, so in order to further explore the mechanism of action of GRWD1 in liver cancer, HepG2 and SK-Hep-1 cells were selected for subsequent experiments.

[0070] 1. Protein co-immunoprecipitation (CO-IP)

[0071] First, wash the magnetic beads 3 times with 400μL detergent (0.1% TritonX-100, diluted in PBS) by vortexing, dilute the corresponding antibody according to the instructions, add the magnetic beads and place them in a 1.5mL EP tube, mark it, place it in a rotating mixing rack at 4℃ for 4-6 hours, place the EP tube on a magnetic rack to absorb the antibody, add 400μL detergent, place it on an ice box and shake it on a shaker for 5min×3 times, and place it on a magnetic rack again to remove the liquid. Collect the cells and lyse them into protein solution with the prepared lysis buffer, then add an equal amount of protein solution to the EP tube, and then place it in a rotating mixing rack overnight. On the second day, take out the EP tube and place it on a magnetic rack to remove the protein solution, and wash it with detergent for 5min in the same way for a total of 3 times. Finally, remove the detergent, add 30μL of IP-specific lysis buffer containing loading and shake and heat at 95℃ for 5min, and then perform Western blot to detect the corresponding molecules.

[0072] 2. Half-life and ubiquitination

[0073] 1) Half-life

[0074] Western blot was used to detect the p53 levels in GRWD1 silenced or overexpressed cells after CHX (100 μg / mL) treatment.

[0075] 2) Ubiquitination

[0076] The cells were treated with the proteasome inhibitor MG132 (20 μM) for 4 h, and then whole-cell lysates were immunoprecipitated with anti-p53 antibody and analyzed by immunoblotting with anti-ubiquitin antibody.

[0077] The results are as follows Figure 7 As shown, the results of co-immunoprecipitation experiments showed that GRWD1 interacted with NPM1 endogenously ( Figure 7 A). After oxaliplatin stimulation, GRWD1 can bind to more endogenous NPM1 protein ( Figure 7 B). Furthermore, 293T cells were used to confirm the existence of exogenous interaction between the two ( Figure 7 C). The expression and ubiquitination level of p53 protein before and after GRWD1 intervention were detected by half-life, ubiquitination experiments and immunoblotting. The results showed that GRWD1 inhibited the stability of p53 by enhancing its ubiquitination level ( Figure 7 D. Figure 7 E). E3 ubiquitin protein ligase MDM2 is a classic enzyme that degrades p53. MDM2 siRNA was transfected or not in the viral cell lines that interfered with GRWD1, and then the protein expression of p53 was detected ( Figure 7 F) The results showed that knocking down MDM2 could inhibit the negative regulation of p53 protein by overexpression of GRWD1. Figure 7 G ) The results showed that knockdown of NPM1 enhanced the inhibitory effect of GRWD1 on p53.

[0078] Example 4 Small molecule drug Esculin inhibits the interaction between GRWD1 and NPM1 and restores the sensitivity of liver cancer cells to OXA

[0079] 1. Preparation of compound library

[0080] The compound library for this docking is mainly from the Selleck compound entity library (FDA drug library: 3113, preclinical and clinical drug library: 3408, https: / / www.selleck.cn / ), and the LigPre module in the Maestro11.9 platform is used to protonate and minimize the energy of all compounds, and the force field is selected as OPLS3e.

[0081] 2. Preparation of GRWD1 target protein structure

[0082] The GRWD1 target protein structure was processed on the Maestro11.9 platform, including removal of water and ions (retaining Ni ions in the active site), protonation, addition of missing atoms, completion of missing groups, protein energy minimization, and energy optimization. The force field was selected as OPLS3e.

[0083] 3. Molecular docking (protein-protein docking)

[0084] The HDOCK server predicts binding complexes between two molecules such as proteins and nucleic acids by using a hybrid docking strategy. Using the HDOCK software, each protein was set to rigid, the docking contact site was set to the full surface, the conformations generated after docking were set to 100, and the docking score was calculated based on the knowledge-based iterative scoring function ITScorePP. A lower docking score means a more likely binding model. This time, the conformation with the most negative energy was selected using the scoring function and optimized using the Minimization module in the Schrödinger 2019.1 software platform to solve the unreasonable spatial structure contacts that may exist in rigid docking. The force field selected for energy minimization is OPLS3e, and the solvation model selects water molecules. The optimization methods are divided into two-step steepest descent method (Steepest Descent) and conjugate gradient (Conjugate Gradient), and the maximum number of iterations is 5000. The optimization results are then used for visualization and mapping analysis models using Pymol 2.1 software.

[0085] 4. Docking screening of protein and small molecule drug Esculin

[0086] Virtual screening processing and optimization were performed by the Glide module in Schrödinger Maestro software. Protein processing was performed using the Protein Preparation Wizard module. Receptors were preprocessed, optimized, and minimized (constrained minimization using the OPLS3e force field). All compounds were prepared using the default settings of the LigPre module. For screening in the Glide module, the prepared receptors were imported to specify the appropriate positions in the receptor grid generation. The docking site was determined based on protein-protein interactions and used as the center of mass of the 15 Å box. The ligands were first redocked to confirm the feasibility of the choice of docking method. The data set was then screened using SP docking, which is a template suitable for screening compounds in large quantities. Finally, the XP docking template was used to screen the high-scoring pre-ligands determined by the SP method. The XP method is designed to provide a better correlation between good poses and scores.

[0087] MD simulations of protein-complexes were performed using Desmond version 2020. Here, the molecular force field selected for MD simulations was OPLS3e, and the system was solvated using the TIP3 water model. The system charge was neutralized by adding ions. The energy minimization of the entire system was achieved using the OPLS4e force field (all-atom type force field). The geometry of the water molecules, the bond lengths and bond angles of the heavy atoms were constrained using the SHAKE algorithm. The continuum system was simulated by applying periodic boundary conditions, and long-range electrostatics were maintained by the particle mesh Ewald method. The system was equilibrated using the NPT ensemble at a temperature of 300 K and a pressure of 1.0 bar. The coupling of the temperature-pressure parameters was performed using the Berendsen coupling algorithm. During the post-preparation of the system, a 20 ns run was performed with a time step of 1.2 fs, and trajectories were recorded every 100 ps for a total of 20,00 frames. The RMSD (root mean square deviation) of the backbone atoms was calculated and graphically analyzed to understand the nature of the protein-ligand interaction.

[0088] 5. Combination medication

[0089] Human liver cancer Huh7 and HepG2 cells were used, and cell viability was determined according to the instructions of CCK8 reagent.

[0090] The results are as follows Figure 8 As shown, the binding sites of the two proteins form a cavity, and the SER-88, LEU-89, GLY-90, GLY-91, and PHE-92 (NPM1 sequence, as shown in SEQ ID NO.1) of the target protein are inserted into the GRWD1 protein cavity, forming strong hydrogen bonds with the cavity and hydrophobic interactions ( Figure 8A). In order to further confirm the binding site, the target protein sequence (GRWD1 sequence, as shown in SEQ ID NO.2) located in the GRWD1 protein cavity was intercepted (peptide) as an active site reference and feasibility verification was performed ( Figure 8 B), the results showed that the peptide could form strong hydrogen bond interactions with key residues ARG-73 and ARG-318, and the hydrophobic residues of the peptide could also form good hydrophobic interactions with amino acids such as ARG-73, PRO-271, PRO-224, TRP-159, ARG-318, PRO-363, and ARG-319. These interactions play an important role in stabilizing the ligand.

[0091] The docked data was scored according to energy, and the top 314 compounds were screened out. 35 compounds were finally obtained by combining energy scoring and evaluation of key residues in the active site. The compound Esculin stood out because of its high docking score and effective GRWD1 inhibitory activity. Molecular dynamics simulation of the protein and the small molecule drug Esculin complex was performed for 200ns; RMSD analysis indicates whether the simulation has reached equilibrium. For normal globular proteins, RMSD changes of the order of 1-3 Å are completely acceptable. However, changes much larger than this indicate that the protein is undergoing huge conformational changes during the simulation. Simulation convergence is also important—the RMSD value stabilizes near a fixed value. If the RMSD of the protein is still increasing or decreasing on average at the end of the simulation, the system has not yet reached equilibrium and the simulation time may not be sufficient for rigorous analysis. Similarly, RMSD also reflects the stability of the protein and the small molecule drug Esculin. The larger the RMSD, the more unstable the protein. The results are as follows Figure 8 As shown in DE, the average RMSD of GRWD1 protein is less than 1.8Å, and the complex reaches dynamic equilibrium in a short time (6ns), which indicates that the small molecule drug Esculin matches the protein target well and can form a stable complex with the protein. In addition, there is no obvious fault problem in the conformational change of the complex, which also indicates that the small molecule drug Esculin can bind well to the protein and will not leave the active pocket of the protein. Figure 8As shown in C and F, the small molecule drug Esculin has strong hydrogen bond interactions with ASP-70, SER-158, ASP-221, ARG-318, GLU-360, and TRP-422, and the hydrogen bond distance exists for more than 50% of the time during the entire molecular dynamics simulation process, which indicates that the small molecule drug Esculin has a sustained and effective interaction at the protein site, and also shows that the hydrogen bond has an important contribution to stabilizing the small molecule drug Esculin in the protein pocket. The results of the combined use of Esculin and OXA in liver cancer cell lines Hun7 and HepG2 showed that Esculin and OXA have good synergistic effects ( Figure 8 G).

Claims

1. Application of the small molecule drug Esculin in the preparation of drugs for treating tumors.

2. The use according to claim 1, characterized in that: The small molecule drug Esculin interacts with hydrogen bonds of ASP-70, SER-158, ASP-221, ARG-318, GLU-360, and TRP-422 residues of GRWD1, thereby blocking the binding of GRWD1 with NPM1, RPL11, and p53.

3. Application of the small molecule drug Esculin in the preparation of oxaliplatin sensitizing drugs.

4. The use according to claim 2, characterized in that: The small molecule drug Esculin interacts with hydrogen bonds of ASP-70, SER-158, ASP-221, ARG-318, GLU-360, and TRP-422 residues of GRWD1, blocks the binding of GRWD1 with NPM1, RPL11, and p53, and promotes the improvement of the sensitivity of liver cancer cells to oxaliplatin.

5. Application of the small molecule drug Esculin in the preparation of drugs for inhibiting the proliferation of liver cancer cells.

6. Application of the small molecule drug Esculin in the preparation of drugs that promote tumor cell death.

7. Application of the small molecule drug Esculin in the preparation of drugs or reagents that induce cell cycle arrest.

8. Application of the small molecule drug Esculin in the preparation of drugs or reagents for inducing tumor cell death.

9. The use according to any one of claims 1 to 8, characterized in that: The small molecule drug Esculin is used at a concentration of 10 μM-1000 μM.

10. Use of the small molecule drug Esculin in the preparation of drugs that mediate tumor cell death through p53 or drugs that induce tumor cell death independently of p53.

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