Method for high-throughput screening of Anti-gastric cancer drugs

Abstract

A method for high-throughput screening of anti-gastric cancer drugs. A small molecule inhibitor that inhibits an interaction between SOS1 and GRB2 is screened on the basis of a fluorescence polarization technique. The small molecule inhibitor that inhibits the interaction between SOS1 and GRB2 has potential for the preparation of a drug for treating gastric cancer. By means of the method for high-throughput screening of anti-gastric cancer drugs, the screening efficiency for the anti-gastric cancer drugs is improved. Digitonin, as a potential candidate for the anti-gastric cancer drugs, is experimentally validated to have an anti-gastric cancer effect and the ability to inhibit the interaction between SOS1 and GRB2.

Description

A high-throughput screening method for anti-gastric cancer drugs Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a high-throughput screening method for anti-gastric cancer drugs. Background Technology

[0002] SOS1 (Gene ID: 6654) and GRB2 (Gene ID: 2885) are key proteins involved in the occurrence and development of gastric cancer. SOS1 is a guanine nucleotide exchange factor that plays an important role in the transition of KRAS (a mutant subtype of RAS protein) from a GDP-loaded "off" state to a GTP-loaded "on" state. Growth factor receptor-binding protein 2 (GRB2) is an adaptor protein that recruits SOS1 from the cytoplasm to the plasma membrane. At the plasma membrane, SOS1 binds to RAS, initiating the RAS-MARK pathway, which participates in the regulation of cell growth and differentiation. This pathway plays a crucial role in various tumors, including gastric cancer.

[0003] Disrupting SOS1 activity, in turn, disrupts KRAS signaling. Therefore, screening for small molecule inhibitors that inhibit the interaction between SOS1 and GRB2 may become a new strategy for treating gastric cancer.

[0004] Currently, reported methods for screening small molecule inhibitors targeting protein interactions mainly include enzyme-linked immunosorbent assay (ELISA), 4T1 cell luciferase reporter gene screening, Drosophila cytogenetics screening, and virtual screening. However, these screening models generally suffer from high false positive rates, long screening cycles, high screening costs, and cumbersome operations, which significantly limit their application in large-scale, high-throughput screening. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a high-throughput screening method for anti-gastric cancer drugs.

[0006] A high-throughput screening method for anti-gastric cancer drugs includes the following steps:

[0007] (1) The fusion proteins GRB2-EGFP and SOS1-GST were obtained by expression, respectively.

[0008] Among them, the fusion protein GRB2-EGFP is a GRB2 protein with EGFP protein fused to its C-terminus; the fusion protein SOS1-GST is an SOS1 protein with GST protein fused to its C-terminus.

[0009] (2) The fusion proteins GRB2-EGFP and SOS1-GST were mixed and incubated, and the drug to be screened was added. The milli-bias value was detected based on fluorescence polarization technology.

[0010] (3) Calculate the inhibition rate of the drug to be screened. The formula for calculating the inhibition rate is as follows:

[0011] Inhibition rate (%) = (mP target compound - mP negative control) / (mP positive control - mP negative control)

[0012] Wherein, mP target compound is the milli-biased value of the drug to be screened; mP negative control is the milli-biased value measured without any drug to be screened; mP positive control is the milli-biased value measured with the positive control drug added.

[0013] Drugs with an inhibition rate >50% are considered candidate anti-gastric cancer drugs.

[0014] Preferably, the amino acid sequence of the fusion protein GRB2-EGFP is shown in SEQ ID No. 1, and the amino acid sequence of the fusion protein SOS1-GST is shown in SEQ ID No. 2.

[0015] The gene sequence encoding the fusion protein GRB2-EGFP is shown in SEQ ID No. 3, and the gene sequence encoding the fusion protein SOS1-GST is shown in SEQ ID No. 4.

[0016] Preferably, in step (1), the fusion proteins GRB2-EGFP and SOS1-GST are both obtained by expression in E. coli and then purification.

[0017] Preferably, in step (2), the final concentration of the fusion protein GRB2-EGFP is 78 nmol·L⁻¹. -1 The final concentration of the fusion protein SOS1-GST was 0.5 μmol·L⁻¹. -1 In step (2), the final concentration of the drug to be screened is 60 μmol·L⁻¹. -1 .

[0018] Preferably, in step (2), before adding the drug to be screened, the fusion proteins GRB2-EGFP and SOS1-GST are mixed and incubated for 30 min; after adding the drug to be screened, incubation continues for 30 min, and then detection is performed. The detection is performed using a multi-functional microplate reader.

[0019] This invention also provides the application of digitalis saponins in the preparation of anti-gastric cancer drugs.

[0020] The present invention also provides an anti-gastric cancer drug, the active ingredient of which is digitoxin.

[0021] The beneficial effects of this invention are:

[0022] This invention constructs a high-throughput screening method for anti-gastric cancer drugs, which is based on fluorescence polarization technology to screen small molecule inhibitors that inhibit the interaction between SOS1 and GRB2. Small molecule inhibitors that inhibit the interaction between SOS1 and GRB2 have the potential to prepare drugs for treating gastric cancer.

[0023] The high-throughput screening method for anti-gastric cancer drugs in this invention improves the screening efficiency. Furthermore, digitalis saponins, as potential candidates for anti-gastric cancer drugs, have been experimentally verified to possess anti-gastric cancer activity. They also exhibit the ability to inhibit the interaction between SOS1 and GRB2. This discovery provides new directions and possibilities for the development of gastric cancer treatments, offering more treatment options for gastric cancer patients. Attached Figure Description

[0024] Figure 1 shows the plasmid maps, where A is the plasmid map of pT7-GRB2-EGFP and B is the plasmid map of pTac-SOS1-GST.

[0025] Figure 2 shows the purification results of proteins GRB2-EGFP and SOS1-GST. In Figure 2, A is the Coomassie Brilliant Blue staining result of the purified protein GRB2-EGFP; and B is the Coomassie Brilliant Blue staining result of the purified protein SOS1-GST.

[0026] Figure 3 shows the properties of proteins EGFP-GRB2 and GRB2-HIS-ATTO488, where A is the concentration-polarization curve of protein EGFP-GRB2; B is the concentration-fluorescence intensity curve of protein EGFP-GRB2; and C is the concentration-polarization curve of protein GRB2-HIS-ATTO488.

[0027] Figure 4 shows the detection of whether the protein SOS1-GST can participate in binding in the fluorescence polarization system, where A is the binding curve of protein SOS1-GST with EGFP-GRB2; and B is the binding curve of protein SOS1-GST with GRB2-HIS-ATTO488.

[0028] Figure 5 shows the high-throughput screening of GRB2 inhibitors using fluorescence polarization.

[0029] Figure 6 shows the detection of the interaction between protein EGFP-GRB2 and digitalis saponins, where A is the result of the cell thermostability experiment, B is the dose-response curve of EGFP-GRB2 and digitalis saponins, and C is the MST trajectory of EGFP-GRB2 and digitalis saponins.

[0030] Figure 7 shows the digitalis saponin concentration-cell viability curve.

[0031] Figure 8 shows the clone formation experiment and its statistical graph.

[0032] Figure 9 shows the scratch test and its statistical chart.

[0033] Figure 10 shows the Transwell experiment and its statistical graph.

[0034] Figure 11 shows a mouse subcutaneous gastric cancer xenograft model.

[0035] Figure 12 shows the weight change curves of mice in each group.

[0036] Figure 13 shows the tumor volume change curves of mice in each group.

[0037] Figure 14 shows the tumor weight change curves of mice in each group. Detailed Implementation

[0038] Example 1

[0039] The amino acid sequence of the fusion protein GRB2-EGFP is shown in SEQ ID No. 1, where 1–239 aa is GRB2 and 240–456 aa is EGFP; the amino acid sequence of the fusion protein SOS1-GST is shown in SEQ ID No. 2, where 1–218 aa is GST and 219–1551 aa is SOS1. The coding gene sequence of the fusion protein GRB2-EGFP is shown in SEQ ID No. 3, and the coding gene sequence of the fusion protein SOS1-GST is shown in SEQ ID No. 4.

[0040] (1) GRB2-EGFP recombinant protein was prepared by prokaryotic expression method of Escherichia coli.

[0041] The codon-optimized GRB2-EGFP gene was ligated into the pT7 expression vector to construct the recombinant plasmid pT7-GRB2-EGFP (Figure 1A). The recombinant plasmid was then transformed into E. coli Rosetta (DE3) competent cells for soluble expression of GRB2-EGFP. The expression product had a His tag at the C-terminus, and the recombinant GRB2-EGFP protein was separated and purified using HisTrap affinity chromatography.

[0042] Specifically, after transforming competent cells with recombinant plasmids whose inserted sequences were identified as correct, bacteria inoculated with the recombinant plasmids were obtained, and the bacterial culture was rejuvenated using the streak plate method. Single colonies were randomly selected and inoculated into LB broth (containing 50 μg / mL of LB broth). -1 Kanamycin), at 0.2 mmol·L⁻¹ -1 Induced with IPTG at 25℃ for 10 h, followed by centrifugation to collect bacterial cells. The cells were then disrupted by sonication, and the supernatant was further processed using HisTrap. TMAffinity column separation and purification were performed. Coomassie brilliant blue staining showed that the induced engineered bacteria exhibited a distinct protein expression band at approximately 50 kDa, and the supernatant contained a large amount of the target protein, indicating that the recombinant human GRB2-EGFP protein was expressed in an intracellular soluble form in *E. coli*. The purified recombinant human GRB2-EGFP protein showed only a single protein band, indicating high purity (Figure 2A). After concentration, dialysis, and BCA quantification, the purified recombinant human GRB2-EGFP protein reached a concentration of 1.5 mg / mL. -1 .

[0043] (2) SOS1-GST recombinant protein was prepared by prokaryotic expression of Escherichia coli.

[0044] The codon-optimized SOS1-GST gene was ligated into the pTac expression vector to construct the recombinant plasmid pTac-SOS1-GST (Figure 1B). The recombinant plasmid was then transformed into E. coli Rosetta (DE3) competent cells for soluble expression of SOS1-GST. The expression product had a GST tag at its C-terminus. The GRB2-EGFP recombinant protein was isolated and purified using Glutathione Magarose Beads (purchased from Tiandi Renhe Co., Ltd., catalog number SM002005).

[0045] Specifically, after transforming competent cells with recombinant plasmids whose inserted sequences were identified as correct, bacteria inoculated with the recombinant plasmids were obtained, and the bacterial culture was rejuvenated using the streak plate method. Single colonies were randomly selected and inoculated into LB broth (containing 50 μg / mL of LB broth). -1 Kanamycin), at 0.2 mmol·L⁻¹ -1 Induced with IPTG at 25℃ for 10 h, bacterial cells were collected by centrifugation. The cells were disrupted by sonication, and the supernatant was purified using Glutathione Magarose Beads. Coomassie brilliant blue staining showed a clear protein expression band at approximately 180 kDa in the induced engineered bacteria, and the supernatant contained a large amount of the target protein, indicating that the recombinant human SOS1-GST protein is expressed in an intracellular soluble form in *E. coli*. The purified recombinant human SOS1-GST protein showed only a single protein band, indicating high purity (Figure 2B). After concentration, dialysis, and BCA quantification, the purified recombinant human SOS1-GST protein had a concentration of 0.6 mg / mL. -1 .

[0046] (3) GRB2-HIS recombinant protein was prepared by prokaryotic expression of Escherichia coli.

[0047] The codon-optimized GRB2 (non-fusion EGFP) was ligated into the pT7 expression vector to construct the recombinant plasmid pT7-GRB2-HIS. The recombinant plasmid was then transformed into E. coli Rosetta (DE3) competent cells for soluble expression of GRB2-HIS. The recombinant GRB2-HIS protein was separated and purified by HisTrap affinity chromatography.

[0048] Example 2

[0049] Construct the GRB2-HIS-ATTO488 fluorescent protein.

[0050] ATTO488 is a water-soluble fluorescent dye. Refer to the ATTO488 conjugation kit (Fast) - (Abcam, catalog number ab269896) Instructions for use: Couple the ATTO488 fluorescent group to the GRB2-HIS protein.

[0051] Example 3

[0052] (1) Determination of the optimal reaction concentration of GRB2-EGFP.

[0053] 2 mmol·L -1 GRB2-EGFP protein was diluted to final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 μmol·L⁻¹. -1 Add 60 μL to each well of a 384-well plate, with 3 replicates per group. The mP value (millipolarization units, mP) is detected using a multi-mode microplate reader.

[0054] Experimental results show that from 50 nmol·L -1 Initially, the mP values ​​of different concentrations of GRB2-EGFP in the experimental system remained relatively consistent with minimal fluctuation, generally staying between 45 and 50 (Figure 3A), and the concentration of GRB2-EGFP protein was directly proportional to the fluorescence intensity (Figure 3B). To maintain good sensitivity and a low background value in the experimental system, the optimal reaction concentration of GRB2-EGFP was selected as 78 nmol·L⁻¹. -1 .

[0055] (2) Determination of the optimal reaction concentration of GRB2-HIS-atto-488.

[0056] 2 mmol·L -1GRB2-HIS-ATTO488 was diluted with fluorescent polarization reaction solution to final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 μmol·L⁻¹. -1 Add 60 μL to each well of a 384-well plate, with 3 replicates per group. The mP value (millipolarization units, mP) is detected using a multi-mode microplate reader.

[0057] Experimental results show that from 10 nmol·L -1 Initially, the mP values ​​of different concentrations of GRB2-HIS-ATTO488 in the experimental system remained relatively consistent with minimal fluctuation, generally staying between 57 and 59 (Figure 3C). To maintain good sensitivity and a low background value in the experimental system, the optimal reaction concentration of GRB2-HIS-ATTO488 was selected as 50 nmol·L⁻¹. -1 .

[0058] Example 4

[0059] (1) Determination of the optimal reaction concentration of SOS1-GST (for GRB2-EGFP).

[0060] 2 mmol·L -1 SOS1-GST serial dilution (starting concentration 16 μmol·L⁻¹) -1 After being diluted 10 times (totaling concentration gradients), 30 μL was added to each well of a 384-well plate, with 3 replicates per group, and incubated at room temperature for 45 min. Then, 156 nmol·L⁻¹ was added... -1 (The final concentration after addition to the system is 78 nmol·L⁻¹) -1 GRB2-EGFP was added sequentially, 30 μL per well, and incubated at room temperature for 30 min. The mP value was detected using a multi-mode microplate reader. The SOS1-GRB2 binding curve was fitted using GraphPad Prism 5.0 software, with an SOS1 concentration of 0.5 μmol·L⁻¹. -1 At this time, the window value of the reaction system is relatively large (Figure 4A), and the detection sensitivity is good.

[0061] (2) Determination of the optimal reaction concentration of SOS1-GST (for GRB2-HIS-ATTO488).

[0062] 2 mmol·L -1 SOS1-GST serial dilution (starting concentration 16 μmol·L⁻¹) -1 After being diluted 10 times (totaling concentration gradients), 30 μL was added to each well of a 384-well plate, with 3 replicates per group, and incubated at room temperature for 45 min. Then, 100 nmol·L⁻¹ was added... -1(The final concentration after addition to the system is 50 nmol·L⁻¹) -1 GRB2-EGFP was added sequentially, 30 μL per well, and incubated at room temperature for 30 min. The mP value was detected by a multi-mode microplate reader. The SOS1-GRB2 binding curve was fitted using GraphPad Prism 5.0 software, and it was found that SOS1-GST did not bind to GRB2-HIS-ATTO488 (Figure 4B).

[0063] Therefore, the subsequent screening used a combination of GRB2-EGFP and SOS1-GST.

[0064] Example 5

[0065] High-throughput screening of lead compounds.

[0066] The TargetMol Natural Compound Library (L6000; approximately 800 units) was diluted with DPBS buffer (Biosharp, catalog number BL1109A) to a concentration of 60 μmol·L⁻¹. -1 This refers to a secondary library.

[0067] First, 312 nmol·L -1 EGFP-GRB2 (final concentration 78 nmol·L⁻¹) -1 Add 7.5 μL to each well of a 384-well plate, followed by the addition of 2 μmol·L⁻¹. -1 SOS1-GST (final concentration 0.5 μmol·L⁻¹) -1 ), 7.5 μL per well, incubate at room temperature for 30 min.

[0068] Then add 60 μmol·L -1 Small molecules (final concentration 30 μmol·L) -1 Add 15 μL to each well of a 384-well plate and incubate at room temperature for 30 min. Detect the mP value using a multi-functional microplate reader and calculate the inhibition rate of the small molecule compound.

[0069] Only 7.5 μL of 312 nmol·L⁻¹ was added to the negative control well. -1 EGFP-GRB2, add 7.5 μL of 312 nmol·L⁻¹ to the positive control well. -1 EGFP-GRB2 and 7.5 μL 2 μmol·L -1 SOS1-GST.

[0070] The formula for calculating the inhibition rate is as follows:

[0071] Inhibition rate (%) = (mP target compound - mP negative control) / (mP positive control - mP negative control)

[0072] Wherein, mP is the target compound's mP value (small molecule compound) to be screened; mP is the negative control, which is the addition of only 7.5 μL of 312 nmol·L⁻¹. -1 The mP value was measured during EGFP-GRB2 testing; the mP positive control was obtained by adding 7.5 μL of 312 nmol·L⁻¹. -1 EGFP-GRB2 and 7.5 μL 2 μmol·L -1 mP value measured during SOS1-GST.

[0073] Initial criteria for identifying a hit compound: a small molecule compound with an inhibition rate >50%.

[0074] The results showed that three compounds were targeted (Figure 5). Here, we selected the one with the highest inhibition rate, digitinoside (CAS No.: 11024-24-1), to further study its anticancer effect in vitro and in vivo.

[0075] Example 6

[0076] Digitalis saponins interact with GRB2 protein.

[0077] The binding between digitoxin and GRB2 in cells was analyzed by cellular thermal shift assay (CETSA). 2 × 10⁻⁶ cells were used in each group. 7 HGC-27 cells were seeded in 10 cm culture dishes. After culturing for 24 h, the cells were pretreated with 10 μM MG132 (proteasome inhibitor) for 1 h, washed with PBS, and collected by trypsin digestion. The samples were centrifuged at 12000 rpm for 2 min at room temperature, gently resuspended in 1 mL PBS, and then centrifuged at 7500 rpm for 3 min at room temperature. The precipitate was resuspended in 1 mL PBS containing 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, and 2 mM benzyl sulfonyl fluoride (PMSF). The samples were then centrifuged at 12000 rpm for 30 min at 4 °C, and the supernatant was transferred to a new PCR tube. For the experimental sample group, digitalis saponins (purchased from Abcam, catalog number ab141501, dissolved in water) were added to a final concentration of 400 nM. For the control sample group, the same volume of carrier solvent (ddH2O) was added. Each group consisted of one control aliquot and one experimental aliquot, heated for 3 minutes at 43℃, 46℃, 49℃, 52℃, 55℃, 58℃, 61℃, or 65℃. Finally, Western blot analysis was performed on the samples. The results showed that the GRB2 protein treated with digitalis saponins exhibited significantly higher thermal stability with increasing temperature compared to the control group (Figure 6A).

[0078] We then used micro-thermal surge (MST) technology to detect the interaction between GRB2-EGFP and digitoxin. 330 μM digitoxin was serially diluted 15 times, with 10 μL of each solution added to PCR tubes. 10 μL of 120 nM GRB2-EGFP was added to each tube. After incubation at room temperature for 20 minutes, the samples were analyzed. Finally, a binding rate-drug concentration curve was plotted based on the relative fluorescence intensity-time curve, and Kd was calculated to be 178 nM (Figures 6B-C).

[0079] Example 7

[0080] Detection of the in vitro and in vivo antitumor activity of digitalis saponins.

[0081] (1) Determination of half-maximal inhibitory concentration.

[0082] A gradient containing 0.0625–4 μM digitalis saponins was set up using a geometric progression (logarithmic 2). HGC-27 gastric cancer cells were seeded at a density of 2000 cells / well in 96-well plates. The survival rate of 2000 cells per well was observed after 24 hours. Cell viability was detected using the CCK8 assay. The absorbance of each well was measured at 490 nm, and the relative cell viability was calculated (relative cell viability % = 100% × (OD)). 待测样本 -OD 空白本底 ) / (OD 对照样本 -OD 空白本底 The half-maximal inhibitory concentration (IC50) of digitalis saponins was found to be 400 nM (Figure 7).

[0083] (2) Detection of clone formation ability.

[0084] To better verify the specificity of digitalis saponins in inhibiting the GRB2 target, we constructed a GRB2 gene knockout HGC27 cell line using lentiviral transfection. WT (non-knockout HGC27 cells) and GRB2-KO (GRB2 gene knockout HGC27 cells) cells were seeded in six-well plates at a density of 2000 cells / well. Different concentrations of digitalis saponins were added to the in vitro cell culture medium. The colony-forming ability of 2000 gastric cancer cells per well was observed in culture environments containing 100 nM, 200 nM, and 400 nM digitalis saponins. Fixation and staining were performed on day 7 of treatment. The results showed that digitalis saponins had a concentration-dependent inhibitory effect on gastric cancer cell colony formation, verifying the tumor-suppressive activity of digitalis saponins (Figure 8).

[0085] (3) Scratch test.

[0086] HGC-27 cells from WT and GRB2-KO were seeded into six-well plates and cultured until the cell confluence in each well reached 95%. Then, using a pipette tip perpendicular to a ruler, scratches were made in each well. Digitalis saponins were added to final concentrations of 0.1, 0.2, and 0.4 μM, respectively. After 48 hours, the scratch healing was observed (Figure 9). We found that the scratch healing of HGC-27 cells from WT was significantly better than that from GRB2-KO, thus demonstrating that digitalis saponins target GRB2 in a concentration-dependent manner to inhibit the migration ability of gastric cancer cells.

[0087] (4) Transwell experiment.

[0088] A cell invasion model was constructed using culture chambers and matrix gel plates. The migration ability of 10,000 HGC-27 gastric cancer cells per chamber was observed in both solvent control and culture environments containing digitalis saponins. Cell attachment at the bottom of the Transwell chambers was observed after 48 hours. Under the interference of digitalis saponins, the migration ability of tumor cells measured by the scratch assay and the invasion ability detected by the Transwell assay were weakened (Figure 10). However, the inhibitory effect of digitalis saponins on cancer cell migration and invasion was poor in both the KO group and the KO plus drug group, and there was no statistically significant difference. This indicates that digitalis saponins exert their anti-cancer effect in vitro by targeting GRB2.

[0089] (5) Mouse experiment.

[0090] A mouse subcutaneous gastric cancer xenograft model was constructed using HGC-27 cell lines of WT and GRB2-KO and divided into four groups. The treatment groups (WT+DIGITONIN group using WT cells, KO+DIGITONIN group using GRB2-KO cells) were administered 8 mg / kg digitalis saponin by gavage every 4 days. Tumor-bearing mice administered an equal volume of physiological saline by gavage served as the negative control group (WT group using WT cells, KO group using GRB2-KO cells). Tumor specimens were collected from each group on day 28 after tumor cell inoculation; the tumor status is shown in Figure 11.

[0091] Figure 12 shows the weight changes of mice in each group over a 20-day period. The weight was recorded every 2 days after tumor formation on day 7. The lower the weight of the mice, the higher the drug toxicity. The results show that there was little difference in weight development among the groups, indicating that the toxic side effects of the administration method were controlled.

[0092] Figure 13 shows the changes in tumor volume in each group over 20 days. The results indicate that there was no significant difference in tumor volume between the KO group and the KO+DIGITONIN group. Compared with the WT group, the tumor volume in the WT+DIGITONIN group was significantly smaller, indicating that digitalis saponins effectively target GRB2 to inhibit tumor growth in vivo. Figure 14 shows the final weight and size of the mouse tumors after 20 days. Smaller tumor volume and lower tumor weight indicate better drug efficacy. The final tumor weight and volume were consistent with the aforementioned treatment effects.

[0093] The above data indicate that digitoxin can exert anti-tumor effects in vitro and in vivo by targeting GRB2.

Claims

1. A high-throughput screening method for anti-gastric cancer drugs, characterized in that, Includes the following steps: (1) The fusion proteins GRB2-EGFP and SOS1-GST were obtained by expression, respectively. Among them, the fusion protein GRB2-EGFP is a GRB2 protein with EGFP protein fused to its C-terminus; the fusion protein SOS1-GST is an SOS1 protein with GST protein fused to its C-terminus. (2) The fusion proteins GRB2-EGFP and SOS1-GST were mixed and incubated, and the drug to be screened was added. The milli-bias value was detected based on fluorescence polarization technology. (3) Calculate the inhibition rate of the drug to be screened. The formula for calculating the inhibition rate is as follows: Inhibition rate (%) = (mP target compound - mP negative control) / (mP positive control - mP negative control) Wherein, mP target compound is the milli-biased value of the drug to be screened; mP negative control is the milli-biased value measured without any drug to be screened; mP positive control is the milli-biased value measured with the positive control drug added. Drugs with an inhibition rate >50% are considered candidate anti-gastric cancer drugs.

2. The high-throughput screening method for anti-gastric cancer drugs according to claim 1, characterized in that, The amino acid sequence of the fusion protein GRB2-EGFP is shown in SEQ ID No. 1, and the amino acid sequence of the fusion protein SOS1-GST is shown in SEQ ID No.

2.

3. The high-throughput screening method for anti-gastric cancer drugs according to claim 1, characterized in that, The gene sequence encoding the fusion protein GRB2-EGFP is shown in SEQ ID No. 3, and the gene sequence encoding the fusion protein SOS1-GST is shown in SEQ ID No.

4.

4. The high-throughput screening method for anti-gastric cancer drugs according to claim 1, characterized in that, In step (1), the fusion proteins GRB2-EGFP and SOS1-GST were both expressed in E. coli and then purified.

5. The high-throughput screening method for anti-gastric cancer drugs according to claim 1, characterized in that, In step (2), the final concentration of the fusion protein GRB2-EGFP is 78 nmol·L⁻¹. -1 The final concentration of the fusion protein SOS1-GST was 0.5 μmol·L⁻¹. -1 .

6. The high-throughput screening method for anti-gastric cancer drugs according to claim 1, characterized in that, In step (2), the final concentration of the drug to be screened is 60 μmol·L⁻¹. -1 .

7. The high-throughput screening method for anti-gastric cancer drugs according to claim 1, characterized in that, In step (2), before adding the drug to be screened, the fusion proteins GRB2-EGFP and SOS1-GST were mixed and incubated for 30 min; after adding the drug to be screened, the incubation continued for 30 min, and then the detection was performed.

8. Application of digitalis saponins in the preparation of anti-gastric cancer drugs.

9. An anti-gastric cancer drug, characterized in that, The active ingredient is digitoxin.

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