Method for screening phenylhydrazine sulfate based on key binding amino acid sites of socs5-cp and application thereof

Abstract

The application belongs to the technical field of molecular biology and drug screening, and provides a method for screening phenylhydrazine sulfate based on a key binding amino acid site of SOCS5-CP and application thereof, which comprises the following steps: key amino acid sites of SOCS5-CP binding are screened through alanine virtual point mutation, and a binding pocket is determined; a ligand molecule is used to perform virtual screening by taking the binding pocket as a docking area, and a compound with the best binding energy is obtained; the obtained drug is screened through AMDET; the selected compound is used for subsequent in-vitro cell experiments, and further screening is performed; the application uses the method of molecular docking and virtual screening in combination with in-vitro experiments for screening, and finds that phenylhydrazine sulfate can be stably combined at the key amino acid sites of SOCS5-CP, in-vitro experiments show that phenylhydrazine sulfate can effectively inhibit the binding of SOCS5-CP, and further down-regulate the protein expression level of HIF1a in hepatoma cells, and also has a significant inhibitory effect on the invasion and migration ability of primary hepatocellular carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and drug screening technology, specifically a method and application for screening phenethylhydrazine sulfate based on the SOCS5-CP key binding amino acid site. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, with consistently high incidence and mortality rates. The treatment of HCC is characterized by multidisciplinary collaboration and the coexistence of various treatment methods. However, recurrence and metastasis remain major obstacles to treatment outcomes. Surgical resection is currently the preferred treatment for HCC stages Ia-IIa, and repeated intraoperative occlusion of the first hepatic hilum significantly reduces the risk of bleeding and is widely used. However, the ischemia, hypoxia, and reperfusion injury during this procedure exacerbate hypoxic stress in HCC, leading to rapid depletion of ATP and oxygen, loss of oxidative phosphorylation substrates, and consequently, mitochondrial dysfunction and the release of large amounts of reactive oxygen species (ROS). Cancer cells adapt to hypoxia by reprogramming their metabolism through gene expression. More importantly, metabolic reprogramming enhances the epithelial-mesenchymal transition and invasive metastatic properties of cancer cells. This suggests that hypoxic stress may be one of the main factors contributing to recurrence and metastasis after hepatectomy, and targeting key oncogenic molecules in hypoxic stress may be a potential therapeutic approach with significant implications for improving survival and prognosis.

[0003] Hypoxia refers to a decrease in oxygen levels, which is prevalent in all solid tumors due to abnormalities in the vascular system. The liver has a rich blood supply, receiving blood from both the portal vein and the hepatic artery; therefore, hypoxia plays a crucial role in the growth of hepatocellular carcinoma. Hepatocellular carcinoma cells can undergo adaptive changes in hypoxic environments, mediating their proliferation, metastasis, and other malignant biological behaviors. Cells adapt to hypoxia through hypoxia-inducible factor (HIF), a heterodimeric protein composed of HIF1a, HIF2a, and HIF1β subunits. In an O2-rich environment, proline hydroxylase (PHD) utilizes O2 to hydroxylate the proline sites of HIF1 / 2a, thereby binding to the E3 ubiquitin ligase von Hippel-Lindau (VHL). VHL drives ubiquitin-mediated proteasome degradation of HIF1a and HIF2a. In O2-deficient environments, HIF1α and HIF2α are stably expressed and translocate to the cell nucleus, where they bind to the constitutively expressed HIF-1β subunit and, together with coactivators, recognize genomic sequences containing hypoxia response elements (HREs), thereby initiating gene transcription mechanisms. HIFs are commonly found in solid tumors and are associated with poor clinical outcomes. However, due to the complexity of the upstream regulatory mechanisms of HIF1α, inhibitors targeting HIF1α are currently not used clinically in the treatment of hepatocellular carcinoma patients. Therefore, in-depth research into the regulatory mechanisms of HIF1α and the development of small molecule drugs to indirectly inhibit HIF1α activity hold significant clinical application potential.

[0004] SOCS5 (Suppressor of Cytokine Signaling 5) belongs to the family of cytokine signaling inhibitors, consisting of eight members including SOCS1-7 and CIS. The basic protein structure of this family consists of three parts: an N-terminal N-region, a central SH2 region, and a C-terminal SOCS box. In previous studies, the research team revealed for the first time that SOCS5 can activate the PI3K / Akt / mTOR signaling pathway to inhibit autophagy, thereby promoting the invasion and metastasis of hepatocellular carcinoma (HCC). Furthermore, they discovered for the first time that in a hypoxic environment, SOCS5 can not only indirectly activate HIF1α activity but also directly bind to ceruloplasmin (CP) to upregulate HIF1α expression, thereby exacerbating HCC invasion and metastasis. Further investigation into the specific regulatory mechanisms of SOCS5-CP and HIF1α in HCC, and the search for drugs that can inhibit SOCS5-CP binding, will provide new targets for HCC treatment and has significant clinical implications.

[0005] Ceruloplasmin (CP) is a copper-containing glycoprotein belonging to the polycopper oxidase family. It is primarily synthesized in the liver and enters the bloodstream. CP plays a crucial role in iron oxidation, transport, and storage and is considered an effective antioxidant. HIF1a target genes include transferrin, transferrin receptor, heme oxygenase-1, and ceruloplasmin, which regulate iron metabolism. HIF1a upregulates these genes, promoting ceruloplasmin synthesis to maintain iron homeostasis and support tumor cell survival and proliferation in hypoxic environments. Studies have shown that HIF1a is frequently activated in the tumor microenvironment, leading to upregulation of ceruloplasmin expression and enhancing the antioxidant capacity and iron metabolism of tumor cells. Therefore, ceruloplasmin and HIF1a may work synergistically in tumor adaptive growth and metastasis. Recent research has found that the positive feedback loop between HIF1a and CP promotes the invasion and metastasis of hepatocellular carcinoma (HCC), consistent with the finding that SOCS5-CP co-regulates HIF1a.

[0006] Phenelzine sulfate is a monoamine oxidase (MAO) inhibitor commonly used to treat depression and anxiety. As an MAO inhibitor, phenelzine can affect neurotransmitter levels in the tumor microenvironment. Studies have shown that MAO plays a crucial role in tumor cell proliferation and invasion, and inhibiting MAO may have a role in suppressing tumor cell proliferation. For example, inhibition of MAO-A and MAO-B is associated with alterations in cancer cell metabolism, particularly in certain tumors such as glioma and prostate cancer. Recent studies have also explored the potential anti-metastatic effects of phenelzine on certain tumor types, such as breast and lung cancer. This may be related to its ability to reduce tumor metastasis by altering processes such as cell adhesion, motility, and angiogenesis. Phenelzine's regulation of neurotransmitters in the tumor microenvironment may help slow tumor cell migration and invasion. Furthermore, some studies have shown that phenelzine can be used in combination with other anti-tumor drugs, especially in chemotherapy. Phenelzine, through its neurotransmitter-modulating effects, may enhance the efficacy of certain chemotherapeutic drugs, such as paclitaxel and cisplatin. For example, phenelzine can enhance the anti-tumor effect by increasing the uptake of drugs by cancer cells or enhancing the cytotoxicity of drugs, but its role in the treatment of primary liver cancer has not been fully studied.

[0007] Therefore, developing a method for screening phenylethylhydrazine sulfate based on key binding amino acid sites of SOCS5-CP is of great scientific significance and application value. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method and application for screening phenylethylhydrazine sulfate based on the SOCS5-CP key binding amino acid site, thereby resolving the issues raised in the background art.

[0009] According to the first aspect of this disclosure, a method and its application for screening phenylethylhydrazine sulfate based on the SOCS5-CP key binding amino acid site are proposed, comprising the following steps:

[0010] S1. Key amino acid sites for SOCS5-CP binding were screened by alanine virtual point mutation to determine the binding pocket;

[0011] S2. Using drugs from the ZINC22 small molecule drug database and FDA-approved drugs as ligand molecules, virtual screening is performed with the binding pocket as the docking region to obtain compounds with the best binding energy.

[0012] S3. Screen the obtained drugs using AMDET;

[0013] S4. Use the selected compounds to conduct subsequent in vitro cell experiments for further screening.

[0014] Preferably, the screening of key binding sites of SOCS5-CP includes: downloading the crystal structure of Ceruloplasmin from the RSCBPDB database, obtaining the SOCS5 structure by AlphaFold modeling, deleting other regions, and leaving only the key binding region SOCS-BOX structure.

[0015] Protein-protein docking was performed using Discovery Studio 2019, with the Angular step size set to 6° and other parameters kept at their default values.

[0016] After docking, Redock optimizes the conformation, selects the highest-scoring conformation, and uses Pymol 2.4 software for visualization analysis.

[0017] Based on the optimal binding conformation, virtual amino acid mutations of alanine were performed to screen key binding sites for SOCS5 and CP.

[0018] Preferably, the key binding sites of SOCS5-CP are determined by COIP experiments through granulation and transfection, immunoprecipitation and Western blotting experiments to verify the key binding sites screened out, and to determine the binding sites for small molecule drug docking. Based on the imaging results, TYR486 and TYR517 are finally determined as key binding sites.

[0019] Preferably, the molecular docking is performed by simulating the interaction between ligand small molecules and receptor biomacromolecules. Discovery Studio 2019 is used to perform molecular docking and virtual screening of key binding sites of small molecule drugs with SOCS5-CP, analyze the binding positions and binding energies, and screen small molecule drugs.

[0020] Preferably, in step S4, the drugs selected in step S3 are further screened according to the ADMET principle to determine that the small molecule drug used for the experiment is phenethylhydrazine sulfate.

[0021] Preferably, the chemical structural formula of the phenylethylhydrazine sulfate is:

[0022]

[0023] According to the second aspect of this disclosure, an application of phenethylhydrazine sulfate screened based on the S0CS5-CP key binding amino acid site in inhibiting the migration of primary liver cancer is proposed.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This invention utilizes molecular docking and virtual screening methods to precisely screen drug molecules capable of binding to key amino acid sites of SOCS5-CP. For the first time, it was discovered that phenelzine sulfate can specifically bind to key amino acid sites of SOCS5-CP. By inhibiting SOCS5-CP binding, it downregulates the expression level of HIF1a protein in liver cancer cells and inhibits the invasion and migration of liver cancer cells, thereby exerting an anti-cancer effect. Based on this mechanism, this study proposes that phenelzine sulfate holds promise as a novel candidate drug for the treatment of primary liver cancer, possessing significant clinical translational potential.

[0026] 2. The method for screening phenelzine sulfate provided by this invention can be widely applied in the drug development of primary liver cancer. As a potential anticancer drug, phenelzine sulfate can provide new drug options and treatment options for the clinical treatment of primary liver cancer. Attached Figure Description

[0027] Figure 1 This is a 3D structural diagram of the SOCS5-CP docking of the present invention;

[0028] Figure 2 This is a 2D structural diagram of the SOCS5-CP docking of the present invention;

[0029] Figure 3 This is a diagram showing the results of COIP in this invention;

[0030] Figure 4 The structural diagrams of the MDL 72527, ethyl nicotinate, and 4-methylpyrazole small molecule drugs of this invention are shown below.

[0031] Figure 5 The diagram shows the structural composition of the L-citrulline and phenethylhydrazine sulfate small molecule drugs of this invention.

[0032] Figure 6 The diagram shows the 3D structure of the five drugs of this invention docked with SOCS5-CP.

[0033] Figure 7 This is a diagram showing the COIP results after drug addition according to the present invention;

[0034] Figure 8 This is a Western blot result diagram of the drug addition in this invention.

[0035] Figure 9 This is a diagram showing the Transweld result of the present invention;

[0036] Figure 10 This is a graph showing the results of the Wounding test of the present invention. Detailed Implementation

[0037] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0038] Example 1: This invention provides a method for screening phenethylhydrazine sulfate based on the SOCS5-CP key binding amino acid site. The chemical structural formula of phenethylhydrazine sulfate is as follows:

[0039]

[0040] Screening method: This invention provides a method for testing potential drugs from a drug compound library based on molecular docking and virtual screening in order to obtain drugs that inhibit SOCS5-CP binding.

[0041] Key amino acid sites for SOCS5-CP binding were identified through virtual point mutation screening of alanine, thus determining the binding pocket. Using drugs from the ZINC22 small molecule drug database and FDA-approved drugs as ligand molecules, the binding pocket was used as the docking region for virtual screening to obtain compounds with optimal binding energies. The obtained drugs were then screened using AMDET. Finally, the selected compounds were used for further screening in in vitro cell experiments. The aim is to identify compounds that can significantly inhibit SOCS5-CP binding through molecular docking and virtual screening. The steps are as follows:

[0042] (1) The crystal structure of Ceruloplasmin was downloaded from the RSCBPDB database, the SOCS5 structure was obtained by AlphaFold modeling, the protein-protein docking was completed by Discovery Studio 2019, and the conformation with the highest score was selected for visualization analysis using Pymol 2.4 software.

[0043] (2) Based on the optimal conformation after docking, alanine virtual amino acid mutation was performed using Discovery Studio 2019 to screen key binding sites for SOCS5 and CP. Subsequently, COIP experiments were performed to verify the key binding sites selected above and determine the binding sites used for small molecule drug docking.

[0044] (3) Molecular docking is a technique that simulates the interaction between small ligand molecules and receptor biomolecules based on the "lock-key principle" of ligand-receptor interactions. Discovery Studio 2019 was used to perform molecular docking and virtual screening of key binding sites of small molecule drugs with SOCS5-CP. A comprehensive analysis of binding sites and binding energies was conducted to screen small molecule drugs.

[0045] (4) Based on the ADMET principle, the drugs selected in the previous step are further screened to determine the small molecule drugs to be used for the experiment.

[0046] (5) Conduct in vitro experiments to determine the inhibitory effect of small molecule drugs and screen for drugs with the best inhibitory effect.

[0047] Mechanism of action of phenelzine sulfate: This invention reveals that phenelzine sulfate can stably bind to key amino acid sites of SOCS5-CP, thereby inhibiting the activity of SOCS5-CP. Further in vitro experiments show that phenelzine sulfate can not only effectively inhibit the binding of SOCS5-CP to its ligands, but also significantly inhibit the migration of primary liver cancer cells.

[0048] Application: Phenylehnialtene sulfate, as a potential drug for treating primary liver cancer, has great application prospects. The method of this invention screened phenylehnialtene sulfate and further verified its inhibitory effect in primary liver cancer, which can provide new drug options and theoretical basis for the clinical treatment of primary liver cancer.

[0049] Example 2: Screening of key binding sites for SOCS5-CP

[0050] The crystal structure of Ceruloplasmin was downloaded from the RSCBPDB database, and the SOCS5 structure was modeled using AlphaFold. Other regions were removed, leaving only the key binding region, the SOCS-BOX structure. Protein-protein docking was performed using DiscoveryStuido 2019, with the angular step size set to 6° and other parameters kept at default. After docking, Redock was used for optimization, selecting the highest-scoring conformation for visualization analysis using Pymol 2.4 software. Based on the optimal binding conformation, alanine virtual amino acid mutations were performed to determine the possible key binding sites for SOCS5-CP binding. The specific docking steps are as follows:

[0051] (1) Using the structure file obtained by modeling with PDB website and AlphaFold, after deleting the irregular curled region and water molecules, leaving the key binding region, protein-protein docking was performed, and the optimal conformation was selected for alanine virtual amino acid mutation.

[0052] (2) In the File Explorer, locate the pdb file in Samples|Tutorials|ReceptorLigand Interaction, double-click to open it and display it in the molecular window.

[0053] (3) In the Hierarchy window (CTRL+H), select Water and press Delete on the keyboard to remove the water of crystallization in the crystal structure.

[0054] (4) Double-click in the Hierarchy window (CTRL+H) <chain>The chain will be renamed Ligand.

[0055] (5) In the Tools Explorer, expand Macromolecules|Prepare Protein, and click Clean Protein to preprocess the protein structure.

[0056] (6) Then, in the Tools Explorer, expand Simulation|ChangeForcefield, click Apply Forcefield, and apply the CHARMm force field to the protein.

[0057] (7) Right-click in the window and set the Atom Display icon in the upper left corner to Line to display each atom in a linear manner.

[0058] (8) After the above processing, select Ligand in the Hierarchy window, and then click Edit|Select in the menu bar.

[0059] (9) In the pop-up window, set Selection Mode to Replace (New Only) and Radius to 3, then click Apply. This will select all amino acids within 3 Å of the ligand as subsequent mutation sites.

[0060] (10) After selecting, right-click the mouse, select Group, and enter mutation in the pop-up dialog box. This will define all amino acids within 3 angstroms of the ligand as a group called mutation.

[0061] (11) Click mutation to select the defined atom, expand Macromolecules|Design Protein in the Tools Explorer, and click Calculate Mutation Energy (Binding).

[0062] (12) Open the corresponding parameters in the parameter browser. Select Single Mutations mode. Next, you need to set the mutation type of amino acid residues in the Mutations parameter bar. Click the pop-up dialog box on the right side of the Mutations parameter bar. Here, mutate all amino acids in the previously defined mutation group to ALA. That is, perform an alanine scan on the amino acids in the mutation group. Use the default settings for the remaining parameters.

[0063] (13) Click the Mutation Energy Terms link in the Results column. This will open a table containing mutation information for all mutation sites. Double-click the Mutation Energy header to sort the mutations from low to high energy. In the Hierarchy window (CTRL+H), select the top 6 amino acids, right-click and select Group, naming it keyresidues.

[0064] (14) In the Tools Explorer, expand Macromolecules|Design Protein, and click Calculate Mutation Energy (Binding). Click the pop-up dialog box on the right side of the Mutation parameters panel, and mutate all the amino acids in the previously defined keyresidues group to the remaining 19 standard amino acids (all amino acids in ALL on the right except ASPH, GLUH, LYSN, ARGN, and HSC). This will saturate the amino acids in the keyresidues group.

[0065] (15) After completing the assignment, double-click the corresponding row in the Jobs Explorer to open the Report page. Click the Mutation Energy Terms link in the Results column. This will open a table containing mutation information for all mutation sites. Double-click the Mutation Energy header to sort the mutations from low to high energy.

[0066] (16) Simultaneous mutation at multiple sites increases the affinity between the enzyme and the substrate. Setting Mutation Sites to TripleMutations will result in a combination of three amino acid mutations in the final output. After completing the job, double-click the corresponding row in the Jobs Explorer to open the Report page. Click the Mutation Energy Terms link in the Results column. This will open a table containing information on the three mutations. Double-clicking the Mutation Energy header will sort the mutations from lowest to highest energy. After simultaneous mutation at three sites, the mutation energy is -1.21, indicating enhanced affinity.

[0067] (17) Virtual amino acid mutation improves thermal stability: In the Tools Explorer, expand Simulation|Change Forcefield, click Apply Forcefield, and apply the CHARMm force field to the protein. In the Hierarchy window, select the previously selected 6 amino acids, right-click and select Group, then name it mutation.

[0068] (18) In the Tools Explorer, expand Macromolecules|Design Protein, and click Calculate Mutation Energy (Stability). The corresponding parameters for the process will be opened in the Parameter Explorer. Click the parameter on the right side of the Mutation parameters panel. In the pop-up dialog box, mutate all amino acids in the previously defined mutation group to the remaining 19 standard amino acids (all amino acids in ALL on the right except ASPH, GLUH, LYSN, ARGN, and HSC). Leave the other parameters at their default settings. Click Run to run the job.

[0069] (19) Click Background and wait for the job to complete. After the job is completed, double-click the corresponding row in the Jobs Explorer to open the Report page. Click the Mutation Energy Terms link in the Results column. This will open a table containing mutation information for all mutation sites. Double-click the Mutation Energy header to sort the mutations from low to high energy.

[0070] Example 3: Identification of key binding sites for SOCS5-CP

[0071] The selected binding sites ARG478, TYR486, ARG494, TYR517, and TYR519 were chosen for COIP experiments. The specific implementation steps are as follows:

[0072] (1) Plasmid transfection: The day before transfection, cells in the culture dish need to be transferred to 2 ml of antibiotic-free medium. During transfection, the cell synthesis rate should be maintained between 70-90%. For each transfection sample, the following preparation of the lipofectamine 3000 complex was prepared: A. Tube 1: Add 6.25 μl of lipofectamine 3000 reagent to 125 μl of Opti-MEM I medium and mix well. B. Tube 2: Add 5 μl of P3000 reagent and 2.5 μg of DNA to 125 μl of Opti-MEM I medium and mix well. Add the solution from tube 1 to tube 2, mix well, incubate at room temperature for 15 min, then add the obtained complex to the cells, gently vortex the plate to ensure the complex is evenly distributed throughout the wells, and after 24 h, replace with complete medium and continue culturing for 48 h / 72 h for subsequent RNA and protein extraction.

[0073] (2) Immunoprecipitation assay: Cells were suspended in NP-40 lysis buffer and incubated on ice for 30 minutes, followed by centrifugation at 12000g for 10 minutes to collect the protein supernatant. For suspended protein A / G magnetic beads, anti-HA magnetic beads, or anti-c-Myc magnetic beads, 10 μL of the beads were placed in a 1.5 mL EP tube. 500 μL of wash buffer was added, ensuring complete suspension. The beads were magnetically separated on a magnetic rack, and the supernatant was discarded. The washing process was repeated twice. The final dilution concentration of IgG antibody was set to 25 μg / mL using binding / wash buffer. Next, 400 μL of diluted IgG antibody was added to the treated magnetic beads, and the mixture was incubated in a flip-flop mixer (30 minutes at room temperature; 2 hours at 4°C), and the beads were collected. 500 μL of cell lysis buffer was added to the magnetic beads, and the mixture was incubated overnight in a flip-flop mixer at 4°C. The magnetic beads needed to be washed three times with washing buffer. 50 μl of 1× protein loading buffer was added to the magnetic beads, and then the mixture was heated at 95°C for 5 minutes. After cooling, magnetic selection was performed, and samples were extracted from the supernatant for Western blot analysis.

[0074] (3) Western blot assay, the specific steps are as follows:

[0075] ①Total protein was extracted from HCC cells. Cells that had undergone each treatment group were collected, and total protein was extracted using NP40 high-efficiency lysis buffer. The cells were then gently pipetted to ensure uniform mixing. Cell lysis was performed on ice for 30 minutes, followed by centrifugation at 12,000 rpm for 15-20 minutes.

[0076] ② Detection of total protein concentration: a. Steps for preparing protein standards: First, take 1 ml of protein diluent, then add it to reagent three for mixing and dissolution, finally obtaining a 1 mg / ml standard. Before use, it needs to be stored on ice. After aliquoting, it should be stored at -20℃.

[0077] b. Preparation method of BCA working solution. Mix BCA solution and copper salt solution evenly at a volume ratio of 50:1, then prepare for use immediately and store the mixed liquid on ice.

[0078] c. Dilute the sample. Add protein sample diluent to the sample to be tested at a ratio of 1:9, then dilute the sample to 10 times its original volume and mix thoroughly before testing.

[0079] d. Preparation of standard wells. A 1.0 mg / ml standard protein was added to the diluent to prepare protein solutions with concentrations of 0 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.9 mg / ml, and 1.0 mg / ml. 20 μl of the protein sample standard was added to each of the corresponding standard wells. 20 μl of diluted sample was added to each well to be tested.

[0080] e. In the upward step, hang each well of the test sample and protein standard vertically, add 200 μl of BCA working solution, then shake to mix for 20 seconds, cover with a membrane, and react at 37°C for 30 minutes.

[0081] f. Place it in a microplate reader and select a wavelength of 562nm to measure the OD value.

[0082] g. Based on the measurement data, a standard curve was plotted, and the protein concentration in the sample to be tested was quantitatively analyzed according to this curve.

[0083] ③ Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis: A. Based on the measured protein concentration, select protein samples of the same mass (20 μg) for each group. Add 5× protein loading buffer at a ratio of protein sample:5×SDS-PAGE protein loading buffer = 4:1. Next, inject DEPC-treated water to ensure that the total volume of each group remains consistent (20 μl). Finally, mix thoroughly and heat to 95°C for 10 minutes.

[0084] B. SDS-PAGE gel electrophoresis: a. Take 10% of the precast gel for subsequent experiments. b. Mix one packet of precast gel electrophoresis solution powder with 500ml of distilled or deionized water until thoroughly mixed. Fix the gel onto the electrophoresis clamp and embed it into the electrophoresis tank, then add the electrophoresis solution. Remove the comb from the precast gel and prepare for sample loading. c. After adding the sample to the well, perform electrophoresis at 80V for 30 minutes, constantly observing the bromophenol blue dye. Once the tip of the dye has passed through the stacking gel, increase the voltage to 120V and continue electrophoresis for about 60 minutes. Stop electrophoresis when the bromophenol blue dye has moved to 0.5cm from the bottom of the precast gel.

[0085] ④ Transfer: a. Cut the PVDF membrane to the same size as the gel, approximately 5×8cm. Immerse the cut PVDF membrane in anhydrous methanol for 1 minute, then rinse it along with the filter paper using transfer buffer. b. Stack the sponge pad, three layers of filter paper, PVDF membrane, gel, three layers of filter paper, and sponge pad in the transfer holder in that order, with the positive electrode facing the negative electrode, ensuring no air bubbles are left between the PVDF membrane and the filter paper. c. Place the prepared transfer holder in the electrophoresis tank. Dilute the 10× electrophoresis buffer to 1× with distilled water, mix thoroughly, and pour into the electrophoresis tank. Embed the electrophoresis tank with crushed ice. Current: 300mA; Transfer time: 2 hours.

[0086] ⑤ Sealing: a. Remove the PVDF membrane from the transfer tank and transfer it to a shaker. Wash the PVDF membrane three times with 1×TBST for 10 minutes each time. b. Mix 5% skim milk powder solution into the PVDF membrane, ensuring the solution completely covers the PVDF membrane. Place it on a shaker at 40 times per minute and seal it at room temperature for 2 hours.

[0087] ⑥ Primary antibody incubation: After removing the blocking buffer, gently rinse the PVDF membrane once with 1×TBST buffer. Then, quickly add the primary antibody, diluted to the appropriate concentration with Western blotting buffer, and incubate overnight at 4°C. After recovering the primary antibody, wash the membrane with 1×TBST buffer for 10 minutes each time, for a total of 3 times.

[0088] ⑦ Incubate the secondary antibody: After aspirating 1×TBST buffer, quickly add the horseradish peroxidase-labeled secondary antibody diluted with 5% skim milk, and then shake on a shaker at room temperature for 1-2 hours. Next, wash the membrane with 1×TBST buffer, ensuring each wash lasts 10 minutes, and repeat this process 3 times.

[0089] ⑧ Development: In a darkroom environment, mix reagents A and B in the ECL luminescent solution in a 1:1 ratio until homogeneous, then evenly coat the PVDF membrane containing the protein. Initiate the reaction at room temperature. Once fluorescence is observed, cover with X-ray film and use a darkroom press for exposure, development, and fixing.

[0090] Based on the imaging results, TYR486 and TYR517 were ultimately identified as key binding sites.

[0091] Example 4: Molecular docking and virtual screening of small molecule drugs that may inhibit SOCS5-CP binding

[0092] Molecular docking and virtual screening were performed on 1.6 million small molecule drugs and 2,203 FDA-approved drugs from the ZINC22 database. The specific steps are as follows:

[0093] (1) Prepare the receptor: The SOCS5-CP binding conformation is pretreated by using the Prepare Protein function in the macromolecule module to remove water and ligand molecules according to the Advanced|Keep Waters and Advanced|Keep Ligands settings.

[0094] (2) Prepare small molecules: Open the Prepare Ligands function in the small molecule module to optimize ligands.

[0095] (3) Define the binding site: Define the binding site based on the ligands that have already been bound.

[0096] (4) In the File Explorer, locate and double-click the Samples|Tutorials|Receptor-Ligand Interactions file. The protein will appear in a new molecular window; the protein has been pre-treated and its active site has been defined.

[0097] (5) In the Tools Explorer, expand Receptor-Ligand Interactions|Define and Edit Binding Site, and then click Show / Hide Residues Outside Sphere and Show / Hide Sphere in sequence. Expand the menu bar View|Transform, and click Fit To Screen to center the protein binding site.

[0098] (6) In the File Explorer, locate and double-click to open the Samples|Tutorials|Receptor-Ligand Interactions|kinase_ligands.sd file. The ligands.sd file contains the ZINC22 database and FDA-approved drugs.

[0099] (7) In the Tools Explorer, expand Receptor-Ligand Interaction|Dock Ligands, and click Dock Ligands (LibDock) to perform molecular docking calculations.

[0100] (8) In the parameter browser, click the Input Receptor parameter and select File > Set Receptor Protein from the drop-down list. Click the Input Ligands parameter and select kinase_ligands:All from the drop-down list to specify the docking ligand. Expand the Docking Preferences parameter, click the Max Hits to Save parameter, and enter the value 10.

[0101] (9) Analyze Molecular Docking Results: After the job is completed, the docking results will automatically open in a new window, containing the protein (only showing residues at the ligand binding site) and all docking conformations. The displayed protein is locked in the window and will become visible as you view all docking conformations. In the newly opened molecular window, ensure that the System View (CTRL+H) and Table View (CTRL+T) are open. In the System View, you can observe the docking results of all 2203 ligand molecules. Click the button in the Table View to observe the docking conformation listed first. Then, you can click the "+" button (or CTRL+Up / CTRL+Down) in the Table View to observe the binding mode of each pose of the ligand molecule to the receptor molecule.

[0102] (10) After LibDock finishes running, ADMET screening is performed, and the screened drugs are precisely matched with CDOCKER.

[0103] (11) Open the ADMET module, select the drug to be docked as described above, leave the program at its default settings, click Run, and after the run is complete, calculate based on the solubility of the aqueous solution,

[0104] Drugs were screened based on five criteria: blood-brain barrier penetration, CYP2D6 binding, hepatotoxicity, and intestinal absorption. The top five drugs that met these criteria were then subjected to CDOCKER.

[0105] (12) In the molecular window, view the three-dimensional structure of the protein with the active site. In the Tools Explorer, expand Receptor-Ligand Interactions|Define and Edit Binding Site, and then click Show / Hide Residues Outside Sphere and Show / Hide Sphere in sequence. Expand the menu bar View|Transform, and click Fit To Screen to display the amino acids of the binding site in the center of the window.

[0106] (13) Locate and double-click to open the Samples|Tutorials|Receptor-Ligand Interactions file. Run CDOCKER. In the Tools Explorer, expand Receptor-Ligand Interactions|Dock Ligands, click Dock Ligands (CDOCKER), and open the corresponding parameter panel. Click the Input SiteSphere parameter, and select the coordinates and radius of the sphere from the drop-down list. Expand the Top Hits parameter and set PoseCluster Radius to 0.5. Set the RMSD threshold to 0.5 angstroms to ensure that the docking conformation is as diverse as possible. Leave the other parameters as default and click Run.

[0107] (14) CDOCKER Result Analysis: After completing the task, double-click the completed molecular docking task in the Jobs Explorer to open the Report interface, click View Results, and open the docking results. Select the top 5 drugs with the best docking conformations. These include MDL 72527, Hexyl nicotinate, Phenelzine sulfate, Fomepizole, and L-Homocitrulline.

[0108] Example 5: Final screening through in vitro experiments

[0109] (1) By purchasing MDL 72527, hexyl nicotinate, phenelzine sulfate, 4-methylpyrazole and L-homocitrulline.

[0110] (2) To verify the inhibitory effect of the five compounds on SOCS5-CP, the following experiment was designed.

[0111] (3) The final concentration of the prepared solution system is as follows:

[0112] ①MDL 72527: 128μM

[0113] ② Hexyl nicotinate: 128 μM

[0114] ③Phenelzine sulfate: 128μM

[0115] ④ 4-Methylpyrazole (Fomepizole): 128 μM

[0116] ⑤ L-Citrulline: 128 μM

[0117] (4) Construct a stable lentiviral strain overexpressing SOCS5. The specific steps are as follows:

[0118] ① Determining the appropriate MOI value: a. To determine the appropriate MOI value, a 96-well plate pre-infection experiment of HEK293T cells was performed. The empty control lentiviral vector and the SOCS5 overexpression lentiviral vector were named LV-vector and LV-SOCS5, respectively. b. On the first day of the 96-well plate, each well was seeded with 5 × 10⁶ cells. 3 Add 100 μl of culture medium to each cell. The cell density should be maintained between 30-50% on the second day, and the cells should be incubated overnight at 37°C. c. On the second day, extract the virus from the -80°C freezer, thaw it in an ice bath, and then prepare three sterile EP tubes. First, add 90 μl of fresh culture medium and then add 10 μl of 5 × 10⁻⁶ cells. 8 Aspirate from the TU / ml viral stock solution and add it to the first tube, mixing thoroughly. Next, extract 10 μl of the mixture from the first tube and add it to the second tube. Repeat the same steps in the third tube to obtain three different concentrations of viral solution: 10 μl of stock solution, a 10-fold dilution, and a 100-fold dilution. First, aspirate 90 μl of culture medium from the culture vessel, then add 90 μl of viral solution with different MOI values ​​to each HEK293T cell. Incubate overnight in a 5% CO2 incubator at 37°C. d. On the third day, change the culture medium: after 24-48 hours, replace the lentivirus-containing culture medium with normal culture medium. e. On the sixth day, assess the infection efficiency: observe the fluorescence phenomenon using an inverted fluorescence microscope and calculate the efficiency of lentivirus infection of target cells to determine the optimal multiplicity of infection (MOI) value.

[0119] ② Formal Infection Experiment: a. On day one, place cells in 6-well plates. The cell density should be approximately 50% by day two. It is recommended to incubate overnight at 37°C. b. On day two, extract the virus from a -80°C freezer and thaw it in an ice bath. Then, according to the pre-set MOI value, dilute the virus to the required concentration using fresh, intact culture medium. First, aspirate the existing culture medium, then dilute the virus solution according to the MOI and add it to the cells. c. Within 48 hours of infection, replace the supernatant with fresh, intact culture medium, and then selectively culture the cells for seven days using 1 μg / ml puromycin.

[0120] (5) Add the prepared solution to the lentivirus stable cell overexpressing SOCS5 and perform COIP experiment. The specific steps are as described in Example 3.

[0121] (6) COIP results are as follows Figure 3 As shown, phenelzine sulfate exhibited the best inhibitory effect on SOCS5-CP. Next, Western blot experiments were conducted to verify the effect of phenelzine sulfate on HIF1a protein expression levels. The specific steps were as described above, and the results are as follows. Figure 8 As shown, the protein expression level of HIF1a decreased after the addition of the drug, indicating that the drug inhibited the expression of HIF1a. This suggests that this drug provides a direct intervention strategy targeting the hypoxia-driven mechanism of postoperative recurrence and metastasis in liver cancer, and has the potential to prolong patient survival.

[0122] (7) Transwell assay was used to verify the effect of phenelzine sulfate on the invasion and migration of liver cancer cells under hypoxic stress. The specific steps are as follows:

[0123] (8) ① When the cells enter the logarithmic growth phase, add the prepared drug. After drug administration, culture the cells at 37°C in a 5% CO2 incubator for 72 hours, followed by 6 hours of hypoxia in a 1% O2 incubator. Digestion can then be performed using 0.25% trypsin solution. Next, wash the cells sequentially with PBS buffer and serum-free culture medium. Immediately afterwards, keep the cells in suspension using serum-free culture medium and count them.

[0124] ② 5×10 4 One cell was suspended in 200 μl of serum-free culture medium and seeded in the upper chamber.

[0125] ③ Transfer 800 μl of culture medium containing 20% ​​FBS to the bottom of the 24-well plate, also known as the lower chamber.

[0126] ④ Continue culturing in a 1% O2 incubator for 24 hours.

[0127] ⑤ Next, carefully remove the top chamber with tweezers, absorb the liquid on it, and then add 4% paraformaldehyde fixative and fix it at room temperature for 30 minutes.

[0128] ⑥ Remove from the small chamber, aspirate the fixative from the upper chamber, and then stain with 0.5% crystal violet at room temperature for 20 minutes.

[0129] ⑦ After several gentle rinsing and soaking operations, the liquid can be effectively extracted from the small chamber and drawn from the upper chamber. Then, carefully remove any remaining cells from the bottom membrane and ensure they are allowed to air dry naturally.

[0130] ⑧ Under a microscope, the number of cells was statistically analyzed in detail from five different perspectives (×40 magnification).

[0131] Transwel l as Figure 9 As shown, after adding the above five drugs, the results showed that phenethylhydrazine sulfate could significantly inhibit the invasion and migration of liver cancer cells in a hypoxic microenvironment, suggesting its potential in the treatment of primary liver cancer.

[0132] (9) The Wounding Test further verified the effect of drug administration on the migration of liver cancer cells under hypoxic stress. The specific steps are as follows:

[0133] Before seeding cells, mark the reverse side of the 6-well plate with horizontal lines using a marker pen. This aids in subsequent image localization. When cells enter the logarithmic growth phase, add the prepared drug. Incubate the cells at 37°C with 5% CO2 for 72 hours, followed by 6 hours of hypoxia in a 1% O2 incubator. Digest the cells using 0.25% trypsin solution and wash twice with phosphate-buffered saline (PBS). Then, count the number of cells using a suspension of fresh, complete culture medium. After counting, seed the cells in 6-well plates to ensure even distribution and good adhesion. After 6-8 hours of cell adhesion, use a pipette tip perpendicular to the wells to create uniformly wide scratches. Remove the culture medium and wash three times with PBS to remove debris caused by the scratches. Immediately after adding fresh, complete culture medium and 24 hours later, record the data using a microscope. Calculate the percentage of scratch area using the following formula: (1 - (24-hour empty area / 0-hour empty area)) × 100.

[0134] Wounding Test, for example Figure 10 As shown, after adding the above five drugs, the results showed that phenethylhydrazine sulfate could significantly inhibit the migration of liver cancer cells in a hypoxic microenvironment, further verifying its potential in the treatment of primary liver cancer.

[0135] It is important to note that the constructions and arrangements of this application shown in several different exemplary embodiments are merely illustrative. Although only a few embodiments are described in detail in this disclosure, those who consult this disclosure will readily understand that many modifications are possible without substantially departing from the novel teachings and advantages of the subject matter described in this application. Other substitutions, changes, and omissions may be made in the design, operation, and arrangement of the exemplary embodiments without departing from the scope of the invention. Therefore, the invention is not limited to the particular embodiments but extends to various modifications that still fall within the scope of the appended claims.

[0136] Furthermore, in order to provide a concise description of exemplary embodiments, not all features of actual embodiments (i.e., those features that are not relevant to the best mode of carrying out the invention as currently considered, or those features that are not relevant to implementing the invention) may be omitted.

[0137] It should be understood that numerous specific implementation decisions can be made during the development of any actual implementation method, and in any engineering or design project. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0138] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.< / chain>

Claims

1. A method for screening phenylhydrazine sulfate based on SOCS5-CP key binding amino acid sites, characterized in that, Comprise the following steps: S1, screening the key amino acid sites of SOCS5-CP binding by alanine virtual point mutation, determining the binding pocket; the determination of the key binding site of SOCS5-CP is carried out by granulation transfection, immunoprecipitation experiment and Western blotting experiment, the key binding site screened by COIP experiment is verified, the binding site for small molecule drug docking is determined, and finally TYR486 and TYR517 are determined as the key binding sites according to the development results; S2, using the drugs in ZINC22 small molecule drug database and the drugs approved by FDA as ligand molecules, and taking the binding pocket as the docking area for virtual screening, the compound with the best binding energy is obtained; S3, screening the obtained drugs by AMDET; S4, using the selected compound for subsequent in vitro cell experiment, further screening; according to the principle of ADMET, the drug selected in step S3 is further screened, and the detected small molecule drug for experiment is phenylhydrazine sulfate; The chemical structural formula of the phenylhydrazine sulfate is: 。 2. The method according to claim 1, wherein the SOCS5-CP key binding amino acid site is screened by phenylhydrazine sulfate. The screening of the key binding site of SOCS5-CP includes: downloading the crystal structure of Ceruloplasmin from RSCBPDB database, obtaining the structure of SOCS5 by AlphaFold modeling, deleting other regions, and only leaving the key binding region SOCS-BOX structure; Protein-protein docking is completed by Discovery stuido 2019, Angular step size is set to 6°, and other parameters remain default; After docking, optimization is carried out by Redock, the conformation with the highest score is selected, and visual analysis is carried out by Pymol2.4 software; According to the optimal conformation of binding, alanine virtual amino acid mutation is carried out to screen the key binding site of SOCS5 and CP.

3. The method according to claim 1, wherein the SOCS5-CP key binding amino acid site is screened by phenylhydrazine sulfate. The molecular docking simulates the interaction between ligand small molecules and receptor biological macromolecules, uses Discovery stuido 2019 to carry out molecular docking and virtual screening of small molecule drugs and the key binding site of SOCS5-CP, analyzes the binding position and binding energy, and screens small molecule drugs.

4. The method of screening phenylthiohydantoin based on SOCS5-CP key binding amino acid sites according to any one of claims 1-3, characterized in that: The method is applied to inhibit the invasion and migration of primary liver cancer.

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