A method for screening capsaicin targeting SOCS5-RBMX protein interaction and its application

Abstract

This invention belongs to the field of molecular biology and drug screening technology, and provides a method for screening capsaicin targeting SOCS5-RBMX protein interactions and its application. The method involves analyzing the structure of the SOCS5-RBMX protein complex to determine the SOCS5-RBMX binding domain and key binding sites; verifying the inhibitory effect on protein binding through point mutations at these key sites; identifying the binding pocket; using drugs from the ZINC22 small molecule drug database and FDA-approved drugs as ligand molecules, and performing virtual screening with the binding pocket as the docking region to obtain compounds; screening the obtained compounds using AMDET to identify capsaicin as the drug inhibiting SOCS5-RBMX binding; and further screening and verification using capsaicin in in vivo and in vitro experiments. This invention, through the analysis of the SOCS5-RBMX protein complex structure to determine the binding domain and key sites, and then using this as a basis for virtual screening and experimental verification, can accurately screen for drugs inhibiting SOCS5-RBMX binding, improving the accuracy and efficiency of drug screening.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and drug screening technology, specifically a method for screening capsaicin targeting SOCS5-RBMX protein interaction and its application. Background Technology

[0002] Primary liver cancer is the fourth leading cause of cancer-related deaths worldwide, with hepatocellular carcinoma (HCC) being the most common type. Due to the lack of specific and highly sensitive early diagnostic methods, HCC patients often do not receive timely treatment. Therefore, further understanding of the pathogenesis of HCC and more diagnostic and treatment methods is crucial. Altered lipid metabolism is one of the most significant metabolic changes in cancer. Metastasis, as the most prominent feature of tumors, is often the leading cause of death. Due to acquired resistance to existing treatments in metastatic tumors, clinically significant metastases remain largely incurable, with only a few exceptions. Enhanced lipid synthesis or uptake can enhance the invasive and metastatic capabilities of tumor cells through multiple pathways. While some studies are currently exploring the role of abnormal lipid metabolism in tumor metastasis, such as how abnormal lipid metabolism reprogramming can promote invasive metastasis, cell stemness, and drug resistance in liver cancer, research is ongoing.

[0003] Higher SOCS5 expression in hepatocellular carcinoma (HCC) tissues is associated with poorer patient prognosis, and SOCS5 can promote HCC invasion and metastasis. A lipid-rich HCC cell model was successfully constructed using overexpressed SOCS5. Staining with lipid droplet-specific fluorescent dyes BODITY and Oil Red O revealed significant lipid droplet accumulation in the cytoplasm, confirming that SOCS5 is indeed a crucial driver gene for lipid metabolism abnormalities in SBC-HCC. Mass spectrometry (MS) identified proteins that may interact with SOCS5, and co-immunoprecipitation (CO-IP) confirmed that RNA-binding modified protein (RBMX) interacts with SOCS5. RBMX is a transcriptional regulator of steroid regulatory element-binding protein-1 (SREBP1), a key transcription factor specifically expressed in the liver and regulating lipase. RBMX can indirectly activate SREBP1. We also investigated the regulatory role of SOCS5-RBMX on SREBP1, demonstrating that the SOCS5-RBMX-SERBP1 axis can promote HCC metastasis by inducing lipid synthesis.

[0004] In recent years, molecular targeted therapy has emerged as a novel treatment option for cancer. Compared to traditional therapies, molecular targeted therapy improves targeting, specifically killing tumor cells while reducing damage to normal tissues. It also has a lower resistance rate, making it safer and more tolerable for patients. Molecular targeted therapy works by selecting specific inhibitors to target overexpressed cell receptors, key genes, and certain marker molecules on tumor cells, inhibiting tumor growth, progression, and metastasis. The principle behind molecular targeted therapy is to design small molecule inhibitors that target key genes and signaling pathways in tumorigenesis, or proto-oncogenes, tumor suppressor genes, suicide genes, etc., reversing the biological behavior of tumor cells at the molecular level, thereby inhibiting tumor cell proliferation and metastasis. Furthermore, with the widespread adoption of immunotherapy, liver cancer patients have new treatment options. However, not all patients are sensitive to immunotherapy; therefore, improving the efficacy of targeted drugs and immunotherapy is of great significance.

[0005] Capsaicin is a substance mainly found in chili peppers. my country is one of the earliest countries to use chili peppers as medicine; traditional Chinese medicine uses chili peppers to treat stomach cold, rheumatism, and other ailments. Modern research shows that capsaicin has anti-inflammatory, analgesic, anesthetic, and detoxifying effects, and has significant therapeutic effects on postherpetic neuralgia, trigeminal neuralgia, diabetic neuropathy, rheumatoid arthritis, osteoarthritis, psoriasis, and alopecia. In addition, capsaicin can inhibit the occurrence of malignant tumors and has special effects on treating skin diseases and weight loss.

[0006] To this end, those skilled in the art have proposed a capsaicin screening method targeting SOCS5-RBMX protein interaction and its application, aiming to screen out drugs that inhibit SOCS5-RBMX binding, namely capsaicin, and to apply capsaicin to lipid-rich liver cancer. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a capsaicin screening method targeting SOCS5-RBMX protein interaction and its application, thereby resolving the issues raised in the background art.

[0008] According to a first aspect of this disclosure, a method for screening capsaicin targeting SOCS5-RBMX protein interaction is proposed, comprising the following steps:

[0009] S1. Analyze the structure of the SOCS5-RBMX protein complex to determine the SOCS5-RBMX binding domain and key binding sites.

[0010] S2. Verify the effect of inhibiting protein binding by point mutations at key sites;

[0011] S3. Determine the binding pocket. Using drugs from the ZINC22 small molecule drug database and FDA-approved drugs as ligand molecules, the binding pocket is used as the docking region for virtual screening to obtain the compound with the optimal binding energy.

[0012] S4. The obtained compounds were screened by AMDET, and capsaicin was identified as the drug that inhibits SOCS5-RBMX binding.

[0013] S5. Use capsaicin in in vitro cell experiments for further screening and verification.

[0014] Preferably, in step S1, the key binding site for SOCS5 and RBMX binding is determined by self-consistent calculation using DFTB embedded in QuantumATK and by molecular dynamics simulation of the complex, predicting four configurations to obtain the alanine virtual point mutant of SOCS5-SH2 as the key binding site.

[0015] Preferably, in step S2, point mutation and CO-IP verification are performed on the alanine virtual point mutant of SOCS5-SH2 to determine the site with the most significant inhibitory effect on SOCS5-RBMX binding.

[0016] Preferably, in step S3, based on the site with the most significant inhibitory effect on SOCS5-RBMX binding determined in step S2, virtual screening is performed using Discovery Studio software in the FDA-approved drug library and the ZINC22 small molecule drug library. The drugs are then finally screened and determined based on docking scores and ADMET pharmacokinetic methods. Capsaicin is found to be the drug that specifically blocks the binding of SOCS5 and RBMX, and its blocking effect on SOCS5-RBMX is verified by CO-IP.

[0017] Preferably, the effect of capsaicin on blocking the binding of SOCS5 and RBMX protein was verified by CO-IP, lipid-rich hepatocellular carcinoma cell lines were constructed using SOCS5-overexpressing lentiviruses, the inhibitory effect of capsaicin on hepatocellular carcinoma invasion and migration was verified by Transweld, and the effect on lipid droplet formation was verified by Oil Red O staining.

[0018] Preferably, in step S5, capsaicin is administered to mice via gavage, and blood samples are collected for testing, including ALT, AST, creatinine, and urea levels, to verify the safety of capsaicin.

[0019] According to a second aspect of this disclosure, the application of capsaicin selected from the first aspect of this disclosure in the preparation of a drug for inhibiting metastasis of lipid-rich liver cancer is proposed.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention analyzes the structure of the SOCS5-RBMX protein complex to determine the binding domain and key sites, and then uses this as a basis for virtual screening and experimental verification. This allows for the precise screening of drugs that inhibit SOCS5-RBMX binding, improving the accuracy and efficiency of drug screening and avoiding the blindness of traditional drug screening.

[0022] 2. This invention has conducted multi-level and multi-angle experimental verification, from point mutation verification at key sites to AMDET screening after virtual screening of compounds, and then to in vitro cell experiments and animal experiments, to ensure that the screened drugs have good inhibitory effects and safety.

[0023] 3. This invention discovers that capsaicin has the effect of inhibiting SOCS5-RBMX binding, providing a new potential drug for the treatment of liver cancer and related diseases, broadening the treatment ideas and drug selection range, and experimentally verifying the inhibitory effect of capsaicin on the invasion and migration of liver cancer cells and lipid droplet formation. Attached Figure Description

[0024] Figure 1 The above is a prediction map of the docking binding sites of SOCS5 and RBMX molecules in this invention, wherein the predicted sites for the left and right sides of a1 and a2 are R424, the predicted site for the left side of a3 is F467, the predicted sites for the left and right sides of a4 are M466, the predicted site for the right side of a2 is Y422, and the predicted site for the right side of a4 is D443.

[0025] Figure 2 This is a diagram showing the binding of SOCS5 and RBMX after point mutation according to the present invention;

[0026] Figure 3 This is a diagram showing the migration and lipid synthesis capabilities of lipid-rich liver cancer cells after point mutation according to the present invention.

[0027] Figure 4 This is a diagram of drugs that may block the binding of SOCS5 and RBMX based on the binding site screening of the present invention;

[0028] Figure 5 This is a diagram showing the binding of SOCS5 and RBMX after the application of the drug according to the present invention;

[0029] Figure 6 This is a diagram showing the migration and lipid synthesis capacity of lipid-rich liver cancer cells after the application of the drug according to the present invention;

[0030] Figure 7 This is a graph showing liver and kidney function in mice after using capsaicin according to the present invention;

[0031] Figure 8 This is a graph showing the migration and lipid synthesis capacity of lipid-rich liver cancer in mice after the use of capsaicin according to the present invention; Detailed Implementation

[0032] 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.

[0033] Example 1: Based on the mechanism by which the SOCS5-RBMX protein complex promotes lipid-rich liver cancer, capsaicin, a drug that inhibits SOCS5-RBMX binding, was screened. To obtain a drug that inhibits SOCS5-RBMX binding, the structure of the SOCS5-RBMX protein complex was first analyzed, identifying the SOCS5-RBMX binding domain and key binding sites. The inhibitory effect on protein binding was verified through point mutations at these key sites. The binding pocket was determined, and drugs from the ZINC22 small molecule drug database and FDA-approved drugs were used as ligand molecules. Virtual screening was performed using the binding pocket as the docking region 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. Through molecular docking and virtual screening, compounds that significantly inhibit SOCS5-RBMX binding were identified.

[0034] The specific steps are as follows:

[0035] The key binding site for SOCS5 and RBMX was determined by self-consistent calculations using DFTB embedded in QuantumATK and molecular dynamics simulations of the complex, predicting four configurations and identifying the alanine virtual point mutant of SOCS5-SH2 as the key binding site.

[0036] Point mutations and CO-IP validation were performed on the aforementioned virtual sites to identify the sites with the most significant inhibitory effect on SOCS5-RBMX binding. Functional experiments were then conducted to verify the effects of these point mutations on the migration and lipid synthesis capabilities of liver cancer cells.

[0037] Based on the aforementioned sites, virtual screening was conducted using Discovery Studio software in the FDA-approved drug database and the ZINC22 small molecule drug database. Final screening was performed based on docking scores and the ADMET pharmacokinetic method to identify drugs that potentially and specifically block the binding of SOCS5 to RBMX. Their blocking effect on SOCS5-RBMX was verified using CO-IP.

[0038] CO-IP validation revealed the drug's effect on blocking the binding of SOCS5 to RBMX protein. Then, lipid-rich hepatocellular carcinoma cell lines (lipid-rich Huh7) were successfully constructed using SOCS5-overexpressing lentiviruses. Transweld assays were used to validate the drug's inhibitory effect on hepatocellular carcinoma invasion and migration, and Oil Red O staining was used to verify its effect on lipid droplet formation.

[0039] Mice were administered the drug via gavage, and blood samples were collected to detect indicators such as ALT, AST, creatinine, and urea to verify the drug's safety. Lipid-rich hepatocellular carcinoma cells were implanted in situ into the livers of nude mice to explore the number of intrahepatic metastases after drug treatment.

[0040] Example 2: Tight-binding calculation method DFTB. SOCS5 and RBMX monomers, as well as their complex systems, contain 96, 86, and 182 amino acids respectively. For the complex systems, molecular docking was used to prepare four most probable configurations (complex I, II, III, and IV). First, a quasi-Newton algorithm was used to optimize the structure, with the force convergence criterion being... For calculations of binding energy, molecular energy spectrum, projected density of states, and charge density for optimized configurations, a single k-point integral is performed over the Brillouin zone centered at Γ using a cutoff energy of 60Ry. Both methods are based on the density functional-based tight-binding method DFTB embedded in QATK, using Dirichlet-type boundary conditions and a mio⁻¹⁻¹ potential as the parameter set.

[0041] The tight-binding computation method DFTB is a hybrid approach that combines density functional theory (DFT) and tight-binding models, aiming to improve computational efficiency and accuracy.

[0042] The DFTB method significantly reduces computational complexity while maintaining high accuracy by expanding the electron wavefunction into a linear combination of atomic orbitals and using density functional theory to calculate the energies of these orbitals. The specific computational steps are as follows:

[0043] Preliminary preparation of the structural model: Create or import a structural model, such as graphene or carbon nanotubes, in Materials Studio.

[0044] To set up the electron transport calculation task: In the DFTB+Calculation dialog box, select the ElectronTransport task and set the relevant calculation parameters, such as the transfer function and potential charge density.

[0045] Molecular docking is based on the laws of physicochemical processes. By calculating the interactions between atoms or molecules, it simulates the binding process of drugs to target proteins, thereby predicting the binding mode and binding affinity of small molecules to target proteins. Specifically, molecular docking involves placing molecules from a known three-dimensional structure database one by one at the active site of the target molecule. By continuously optimizing the position, conformation, dihedral angles of rotatable bonds within the receptor compound, and the amino acid residue side chains and backbone of the receptor, it predicts the binding mode, affinity, and selects the ligand with the best affinity to the receptor that closely approximates the native conformation using a scoring function. The molecular docking process includes the following steps:

[0046] Prepare ligand structure files: Preprocess the ligand structure, including structure transformation and optimization.

[0047] Prepare the protein crystal structure file: Preprocess the protein crystal structure, including structure repair and adjustment. If a protein crystal structure is unavailable, structural modeling using the complete amino acid sequence is required.

[0048] Active site prediction: Identify the approximate location of the docking pocket of the protein.

[0049] Create the docking box: Create the docking box with the center of the predicted pocket location as the origin of the coordinate system. The shape and size are set according to the project requirements.

[0050] Set docking parameters: including docking accuracy and other relevant parameters.

[0051] Start the docking program: After checking the structure file and parameter file, call the server resources to start the docking program.

[0052] Example 3: Virtual Screening

[0053] 1. Receptor-based virtual screening

[0054] The method employed is receptor-based virtual screening, which studies the characteristic properties of the target protein binding site and its interaction mode with small molecule compounds based on the three-dimensional structure of the SOCS5-RBMX complex. This approach uses molecular docking technology to evaluate the binding ability of proteins and small molecule compounds based on an affinity scoring function related to binding energy. Ultimately, compounds with reasonable binding modes and high prediction scores are selected from a large pool of molecules for subsequent bioactivity testing.

[0055] 2. Ligand-based virtual screening

[0056] Ligand-based virtual screening utilizes known active small molecule compounds and searches a compound database for matching chemical structures based on shape similarity or pharmacophore models. This method compares the structural characteristics of known active compounds, searches the database for structurally similar compounds, and then conducts experimental screening studies on these selected compounds.

[0057] The screening criteria and process for ADMET drugs mainly include the following aspects:

[0058] Definition and Importance of ADMET: ADMET (absorption, distribution, metabolism, excretion, and toxicity) is a crucial concept in drug development, encompassing the behavior and effects of drugs in the body. ADMET studies are essential in drug design and screening, significantly improving the success rate of drug development, reducing development costs, and minimizing toxicity and side effects.

[0059] ADMET's five main aspects:

[0060] Absorption: The process by which a drug enters the systemic circulation from the site of administration. The rate and extent of absorption directly affect the effective concentration of the drug in the body and its therapeutic effect.

[0061] Distribution: The process by which a drug is transported in the body determines its coverage and intensity of action in different tissues and organs.

[0062] Metabolism: The process by which drugs undergo structural changes in the body due to the action of enzyme systems, affecting the efficacy and potential side effects of drugs.

[0063] Excretion: The process by which drugs and their metabolites are excreted from the body through excretory organs, affecting the duration of drug residence in the body and its therapeutic effect.

[0064] Toxicity: The toxic effects of a drug on the body, and the assessment of drug safety.

[0065] ADMET screening process:

[0066] Computer simulation and prediction: Using computer simulation and artificial intelligence models, the ADMET parameters of drugs are predicted starting from molecular structure. This method can rapidly evaluate a large number of compounds, improving screening efficiency.

[0067] Experimental Example: CO-IP Experiment:

[0068] Cells meeting experimental conditions were collected using trypsin (TE) cell culture. After centrifugation at a constant speed of 1000 rpm for approximately 5 minutes, a precipitate was observed on the tube wall. The cells were resuspended in culture medium and diluted to an appropriate concentration. The cells were then added to each well of a 6-well plate, ensuring a density of approximately 70% and containing approximately 1 ml of culture medium per well. The cell culture plate was placed in an incubator, and the medium was changed regularly, with observation of cell growth. Transfection with the target protein was initiated when the density reached 90%. After 24 hours of transfection, if the cell condition met the requirements for the next experiment, the cells were lysed to harvest the protein. The wells were repeatedly washed with 1 ml of PBS, which was then aspirated and discarded. This process was gentle and could be repeated multiple times. Commercially available IP lysis buffer was purchased and, with the protease inhibitor added at a ratio of 1:80 to 1:120, 1 ml of which was added to each well. The cell culture plate was gently shaken or pipetteed to ensure the lysis buffer thoroughly wetted every area of ​​the wells. After ensuring each cell is lysed, collect the cell lysis buffer into a new EP tube, invert and mix to ensure full contact and lysis, and place on ice for 30 minutes. While waiting for lysis, pre-cool the EP tube to 4°C. After the required time, place the EP tube containing the lysis buffer and protein into a centrifuge, balance it, and centrifuge at a constant speed of 12000 rpm for 25-35 minutes. Aspirate 50 μL of the supernatant as the positive control group (Input group), which contains all existing protein interactions. Preheat the water bath to 98°C before centrifugation. Add the appropriate volume of 5*Loading buffer, mix well, and boil in a water bath for 10 minutes, ensuring the EP tube is tightly sealed during the process. Label this as the Input group for later use. Next, divide the remaining supernatant equally into two new EP tubes, labeled as the IP group and the negative control group, respectively. Add 50 μL of beads to each tube. Add 1 μL of the corresponding species' IgG antibody to the negative control group, and add Flag (or HA, GFP, etc.) tag antibody to the IP group. Place the EP tubes on a rotary shaker in a 4°C refrigerator, mix thoroughly, and incubate overnight. The next day, centrifuge the negative control group and the IP group at a constant speed of 2500 rpm for 3 minutes. After discarding the supernatant, add 1 ml of NP-40 to the precipitate for washing, then discard the supernatant again. This washing process can be repeated 2-3 times for better experimental results. The water bath can be preheated to 98°C before use. Finally, add 40 μl of 1*Loading buffer, mix well, and boil in a water bath for 10 minutes, ensuring the EP tubes are tightly sealed during the process. Label these tubes as the IP group and the influence control group for subsequent use.

[0069] Transwell assay: Prepare 24-well plates and Transwell chambers in advance. Add cell culture medium containing 10% serum to each well of the lower 24-well plate, and serum-free cell suspension to the upper chamber. Treat cells with the drug for 48 hours, then collect cells by trypsin digestion. Centrifuge at a constant speed of 800 rpm for approximately 5 minutes; a precipitate will be visible on the tube wall. Resuspend the cells in serum-free culture medium, count them using a cell counting chamber, and dilute the cells to 2*10⁻⁶. 5 After determining the concentration, aspirate 100 μL of the suspension and add it to the Transwell chamber. Collect the suspension after 12-16 hours, remove the Transwell chamber, discard the culture medium, wash once with PBS, then add 4% paraformaldehyde and fix for approximately 15-20 minutes. Wash twice with PBS, 5 minutes each time. Finally, gently scrape away any remaining cells from the chamber with a cotton swab. After fixing with crystal violet for 15 minutes, cells stained with crystal violet can be observed in the lower layer of the chamber. Take photographs of the chamber under a microscope and import the images into ImageJ image analysis software for quantitative analysis.

[0070] Oil Red O staining:

[0071] Preparation of Oil Red O dry solution: Add 0.4g of Oil Red O dry powder to 10mL of isopropanol (analytical grade), seal, and heat in a 37℃ water bath to fully dissolve. The dry solution can be stored at room temperature for a long time. Preparation of Oil Red O staining solution: Mix the dry solution with triple-distilled water in a 3:2 ratio, filter, and use the filtrate as fresh staining solution within 2 hours. After cell treatment, wash three times with PBS, absorb excess water with filter paper, fix with 50% isopropanol for 1 min or 10% neutral formaldehyde for 10 min, stain with Oil Red O staining solution for 10-15 min. Cover during staining to prevent isopropanol evaporation and dye precipitation; wash away excess staining solution with 60% isopropanol, rinse three times with triple-distilled water; counterstain with Mayer's hematoxylin for 5-10 min, separate with 1% hydrochloric acid and return to blue, then rinse with triple-distilled water for 10 min. Observe and photograph under an inverted microscope.

[0072] Detection of biochemical parameters in mouse blood:

[0073] A sterile 1mL syringe used for blood collection draws 1mL of anticoagulant from the vial into the syringe, and then the entire amount of anticoagulant is returned to the vial. This means the syringe is "perfused" with anticoagulant before blood collection. The method for mixing the blood with the anticoagulant after collection is as follows: Add no more than 150μL of anticoagulant to a 1.5mL centrifuge tube. Centrifuge the blood sample at a relative centrifugation force of 14000g for 10-15 minutes. Use a pipette to aspirate the separated plasma and transfer it to a clean centrifuge tube. The plasma sample can be further centrifuged at 14000g for 2-3 minutes to separate any remaining red blood cells. Transfer the separated plasma to a clean centrifuge tube. A fully automated biochemical analyzer is an instrument that measures specific chemical components in serum or plasma based on the principle of photoelectric colorimetry, and is used to detect liver and kidney function in mice.

[0074] Mouse orthotopic implantation model of liver cancer:

[0075] Hepatocellular carcinoma cells were prepared into a cell suspension and subcutaneously inoculated into the backs of nude mice to establish a BALB / c nude mouse subcutaneous hepatocellular carcinoma tumor model. Animal in vivo imaging technology was used to periodically monitor the growth of the primary tumor using GFP markers carried by the tumor cells. Observations included subcutaneous tumor volume, subcutaneous tumor weight, and mouse body weight. Mice bearing tumors were euthanized by cervical dislocation when the tumor diameter reached 1–1.5 cm. Tumor tissue was harvested from the subcutaneous tissue on the back and placed in 0.85% sodium chloride containing penicillin and streptomycin. The fibrous capsule was removed, and well-grown, pale red, fish-flesh-like tumor tissue was selected and cut into 1 mm × 1 mm × 1 mm pieces. These pieces were then inoculated subcutaneously into another nude mouse using a large-bore needle. The tumor was passaged four times per passage, and this process was repeated until the sixth generation, serving as the tumor source for orthotopic transplantation. Tumors were harvested subcutaneously from the backs of passaged mice. The fibrous capsule was removed, and well-grown, pale red, fish-flesh-like tumor tissue was selected and placed in 0.85% sodium chloride solution. The tissue was then cut into 1mm × 1mm × 1mm pieces for later use. The experimental animals were fasted for 12 hours preoperatively, anesthetized intraperitoneally with 0.4% sodium pentobarbital, and the skin was disinfected. An incision was made below the xiphoid process to access the abdomen, exposing the left lobe of the liver. A 1mm incision was gently made in the liver with a sterile scalpel, and the tumor piece was implanted into the incision. One to two drops of medical OB biological adhesive were applied to cover the tumor surface. After approximately 40 seconds of solidification, the liver was returned to the peritoneal cavity, and the peritoneum and skin were sutured together with silk sutures. The abdomen was closed. Strict aseptic technique was followed throughout the entire procedure. Postoperatively, the overall condition, movement, and abdominal signs of all mice were observed daily. Four nights later, the experimental and control groups were each divided into two subgroups for subsequent ischemia-reperfusion model construction and control.

[0076] 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 herein, and 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.

[0077] 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.

[0078] 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.

Claims

1. A method for screening capsaicin targeting SOCS5-RBMX protein interaction, characterized in that, Includes the following steps: S1. Analyze the structure of the SOCS5-RBMX protein complex to determine the SOCS5-RBMX binding domain and key binding sites. S2. Verify the effect of inhibiting protein binding by point mutations at key sites; S3. Determine the binding pocket. Using drugs from the ZINC22 small molecule drug database and FDA-approved drugs as ligand molecules, the binding pocket is used as the docking region for virtual screening to obtain the compound with the optimal binding energy. S4. The obtained compounds were screened using ADMET, and capsaicin was identified as the drug that inhibits SOCS5-RBMX binding. S5. Use capsaicin in in vitro cell experiments for further screening and verification; In step S1, the key binding site of SOCS5 and RBMX is calculated using DFTB embedded in QuantumATK and molecular dynamics simulation of the complex to predict four configurations, resulting in the alanine virtual point mutant of SOCS5-SH2 as the key binding site. In step S2, point mutations and CO-IP verification are performed on the alanine virtual point mutant of SOCS5-SH2 to determine the site with the most significant inhibitory effect on SOCS5-RBMX binding. In step S3, based on the site with the most significant inhibitory effect on SOCS5-RBMX binding determined in step S2, virtual screening was performed in the FDA-approved drug library and the ZINC22 small molecule drug library using Discovery Studio software. The drugs were finally screened and determined based on docking scores and ADMET pharmacokinetic methods. Capsaicin was found to be the drug that specifically blocks the binding of SOCS5 and RBMX, and its blocking effect on SOCS5-RBMX was verified by CO-IP. The effect of capsaicin on blocking the binding of SOCS5 and RBMX protein was verified by CO-IP. Lipid-rich hepatocellular carcinoma cell lines were constructed using SOCS5-overexpressing lentiviruses. The inhibitory effect of capsaicin on hepatocellular carcinoma invasion and migration was verified by Transwell assay, and the effect on lipid droplet formation was verified by Oil Red O staining. In step S5, capsaicin was administered to mice via gavage, and blood samples were collected to detect ALT, AST, creatinine, and urea levels, verifying the safety of capsaicin.

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