Application of virtual screening-based African swine fever virus targeting micromolecule in resisting African swine fever virus

Abstract

The invention discloses an application of an African swine fever virus targeting micromolecule based on virtual screening in resisting African swine fever virus. A natural product with relatively strong binding capacity with the African swine fever virus g5Rp protein is obtained through virtual screening analysis, one small molecule, namely 9 ''-salvianolic acid B monomethyl ester, is used for treating counteracting cells, and an experimental result shows that the 9''-salvianolic acid B monomethyl ester can inhibit the expression of the African swine fever virus g5Rp protein so as to inhibit the replication of the African swine fever virus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a drug or a lead compound for preventing and controlling African swine fever from natural products, in particular to a small molecule for preventing and controlling African swine fever by virtual screening and challenge experiment. BACKGROUND

[0002] African swine fever virus (ASFV) is a nucleocytosolic complex DNA virus that can infect domestic pig and wild pig populations, thereby causing huge economic losses to the pig industry. At present, in addition to developing vaccines and immunomodulatory agents (see: CN111690055A, CN111620954A, etc.), developing small molecule drugs (see: CN116832044A, etc.) is also a potential way to prevent and control ASFV infection.

[0003] Decapping enzymes in DNA viruses all contain a conserved sequence GX5EX7REUXEEXGU (usually U is a larger side chain hydrophobic amino acid, and X is any amino acid), so it is also called Nudix hydrolase. This kind of enzyme mainly acts on substrates containing nucleotides, such as nucleotide diphosphate, triphosphate, etc., by hydrolyzing the phosphate ester bond of nucleotides, releasing nucleotides or similar molecules, participating in RNA metabolism, DNA repair, energy metabolism, signal transduction and other processes. The g5Rp protein is the only decapping enzyme encoded by the ASFV genome, which contains a single Nudix motif. When it binds to mRNA, it will hydrolyze the RNA cap structure and release m7GDP as a product. Removing the 5' cap on the cell mRNA through the g5Rp protein is conducive to the early gene expression of viral infection and accumulation during the entire ASFV infection process. In “Structural biology research on mRNA decapping protease g5Rp of African swine fever virus”, the g5Rp protein is used as a potential drug target for inhibiting ASFV infection. Through protein structure surface charge distribution analysis, substrate binding site identification, g5Rp binding (interacting residues: Gln6, Lys8, Lys94, Lys98, Lys133) and enzyme activity experiment, the action of g5Rp inhibitor Ins P6 is verified (the action mechanism is that Ins P6 competitively binds to g5Rp with RNA substrate).

[0004] Virtual screening is an analysis method of compound screening relying on small molecule database, which can quickly screen out compounds with potential for drug use from tens to millions of molecules. Compared with traditional experimental screening methods, virtual screening can quickly evaluate the binding ability and affinity between compounds and target points, and provide certain clues and research direction for subsequent experiments. Although the use of virtual screening technology can improve the efficiency of drug research and development and reduce experimental costs, the results of virtual screening analysis of protein targets are mainly protein activity inhibitors or activators. Therefore, in the absence of pharmacological experimental evidence, compounds obtained by virtual screening analysis cannot be directly used for clinical trials of related drugs (see: CN109461474A, "Virtual screening and molecular dynamics simulation verification of natural product inhibitors of cholesteryl ester transfer protein", etc.). At present, the structure and function of ASFV key proteins are not fully understood, which limits the accuracy of virtual screening; at the same time, the virtual screening research of ASFV is still in its infancy, and there are still many challenges in the accurate analysis of ASFV targets, the optimization of virtual screening algorithm, and the accuracy and reliability of screening results, and there has been no report of anti-ASFV drugs obtained by virtual screening and verified. SUMMARY

[0005] The purpose of the present application is to provide the use of African swine fever virus targeting small molecules for anti-African swine fever virus based on virtual screening.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect, the application provides the use of a compound targeting African swine fever virus g5Rp protein in the preparation of an anti-African swine fever virus drug.

[0007] Preferably, the site of interaction between the compound and the African swine fever virus g5Rp protein includes one or more amino acid residues in the following African swine fever virus g5Rp protein structure: Ser10, Lys32, Ser39, His45, Lys131, Lys133.

[0008] Preferably, the compound is selected from any one of 9''-methyl salvianolate B, β-cyclodextrin, and Hosenkoside K.

[0009] Preferably, the 9''-hyperoside monomethyl ester reduces the expression level of the African swine fever virus g5Rp protein in a host (mainly pigs) or host cells (i.e., the 9''-hyperoside monomethyl ester can inhibit the expression of the African swine fever virus g5Rp protein), thereby inhibiting the replication of the African swine fever virus in the host or host cells.

[0010] Preferably, the medicine comprises 9''-hyperoside monomethyl ester or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

[0012] Preferably, the medicine can adopt any one of the following dosage forms: tablets, sprays, granules, capsules, oral liquids, and injections.

[0013] In a second aspect, a method for virtually screening a medicine against the African swine fever virus by targeting the African swine fever virus g5Rp protein is provided, comprising the following steps: 1) Scoring of docking According to the crystal structure of the prokaryotic expressed African swine fever virus g5Rp protein, the target information is determined, and the compounds to be screened are docked with the target to form a protein complex, and the scoring of the compounds docked is obtained. 2) Ranking and screening Referring to the ranking results of the scoring of the compounds docked, the compounds for experimental verification against the African swine fever virus (e.g., inhibiting the replication of the virus) are selected from the compounds.

[0014] Preferably, the amino acid sequence of the African swine fever virus g5Rp protein is shown in SEQ. ID. NO. 1.

[0015] Preferably, the high-quality (prokaryotic expression and high-purity protein obtained by separation and purification) African swine fever virus g5Rp protein crystal harvested by sitting-drop vapor diffusion is subjected to X-ray diffraction, and the obtained crystal X-ray diffraction data is analyzed and processed to obtain the high-resolution 3D protein structure of the crystal, i.e., the crystal structure of the prokaryotic expressed African swine fever virus g5Rp protein.

[0016] Preferably, the target information comprises a reference active pocket for positioning the interaction interface between the African swine fever virus g5Rp protein and the substrate (the substrate is a compound to be screened) in molecular docking; the reference active pocket is the IHP crystal small molecule ligand bound on the 7DNU of the crystal structure of the African swine fever virus g5Rp protein.

[0017] Preferably, the compound to be screened is a natural product.

[0018] Preferably, in step 1, the compound to be screened is docked to the binding pocket on the target interaction interface.

[0019] Preferably, in step 2, the top-ranked compounds are selected by scoring the post-docking data; and the compounds stably bound to the African swine fever virus g5Rp protein are determined according to the scoring of the binding free energy and the evaluation of the key residues in the active site of the African swine fever virus g5Rp protein for binding to the corresponding compound, and are used for subsequent experimental verification.

[0020] Preferably, through experimental verification, the compound targeting the African swine fever virus g5Rp protein obtained through virtual screening (for example, 9''-Hydroxysafflor Yellow A monomethyl ester) has the effect of inhibiting the replication of the African swine fever virus, that is, the anti-African swine fever virus compound is obtained through virtual screening.

[0021] The beneficial effects of the present application are embodied in: The present application first discovers and verifies the compound capable of resisting the African swine fever virus by targeting the African swine fever virus g5Rp protein, provides a basis for the research and development of drugs for preventing and treating the African swine fever, and has important significance for meeting the urgent needs of accelerating the establishment of the drug prevention and control approach for the African swine fever.

[0022] Further, the present application determines the crystal structure of the prokaryotic expressed African swine fever virus g5Rp protein, and performs virtual screening on the African swine fever virus g5Rp protein, and obtains the compound (including 9''-Hydroxysafflor Yellow A monomethyl ester) having good binding with the African swine fever virus g5Rp protein and high matching degree; the experimental results (taking 9''-Hydroxysafflor Yellow A monomethyl ester as an example) show that the compound can inhibit the expression of the African swine fever virus g5Rp protein, and further inhibit the replication of the African swine fever virus. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is the purification diagram of the prokaryotic expressed African swine fever virus g5Rp protein: A is an exemplary result of small-scale induction expression (lane M is a protein marker, lane 1 is uninduced whole bacteria, lane 2 is induced whole bacteria, lane 3 is induced supernatant, and lane 4 is induced precipitate); B is SDS-PAGE verification of the target protein after elution from a 250 m nickel column (lane 1); C is SDS-PAGE verification of the collected target protein by taking the corresponding protein eluate according to the UV monitoring peak diagram of the molecular sieve elution (lanes 1, 2, 3, and 4 correspond to eluate volumes of 60, 70, 80, and 90 mL, respectively).

[0024] Figure 2 The figure is the crystal diagram of the prokaryotic expressed African swine fever virus g5Rp protein.

[0025] Figure 3Crystal structure model of prokaryotic expression of African swine fever virus g5Rp protein core protein.

[0026] Figure 4-1 Molecular docking results of prokaryotic expression of African swine fever virus g5Rp protein and 9''-Methyl salvianolate B, β-Cylodextrin, Hosenkoside K.

[0027] Figure 4-2 Structural formula of 9''-Methyl salvianolate B, β-Cylodextrin, Hosenkoside K.

[0028] Figure 5 Surface plasmon resonance results of prokaryotic expression of African swine fever virus g5Rp protein and 9''-Methyl salvianolate B.

[0029] Figure 6-1 Effects of 9''-Methyl salvianolate B on cell activity (CCK-8). 50 ) Figure 6-2 Effects of 9''-Methyl salvianolate B on cell activity (CCK-8).

[0030] Figure 7 Effects of 9''-Methyl salvianolate B on expression of African swine fever virus g5Rp protein in cells attacked by virus (immunofluorescence staining).

[0031] Figure 8 Effects of 9''-Methyl salvianolate B on expression of African swine fever virus g5Rp protein in cells attacked by virus (western blotting).

[0032] Figure 9 Effects of 9''-Methyl salvianolate B on replication of African swine fever virus in cells attacked by virus. DETAILED DESCRIPTION

[0033] The application will be further described below in conjunction with the accompanying drawings and examples, which are illustrative of the application and should not be construed as limiting the scope of the application.

[0034] (I) Target of anti-African swine fever virus drug Previous studies have shown that g5Rp protein plays an important role in the replication of African swine fever virus (ASFV). Specifically, overexpression of g5Rp protein promotes the replication of African swine fever virus, and the replication level of African swine fever virus decreases after inhibition of g5Rp protein expression by interference technology. Therefore, it is considered to take g5Rp protein as a target and screen candidate drugs against African swine fever virus through molecular docking.

[0035] (II) Prokaryotic expression and purification of African swine fever virus g5Rp protein 2.1 Molecular cloning The full-length gene of g5Rp protein of African swine fever virus strain China / LN / 2018 / 1 (GenBank: OP856591.1) was downloaded from NCBI and submitted to Shenguo Bioengineering Company for E. coli codon optimization and g5Rp gene sequence synthesis. The synthesized g5Rp gene sequence is used to express the amino acid sequence of African swine fever virus g5Rp protein (i.e. SEQ. ID. NO. 1) as shown below: MDTAMQLKT S IGLITCRMNTQNNQIETILVQ K RYSLAF S EFIHC H YSINANQGHLIKMFNNMTINERLLVKTLDFDRMWYHIWIETPVYELYHKKYQKFRKNWLLPDNGKKLISLINQAKGSGTLLWEIP K G K PKEDESDLTCAIREFEEETGITREYYQILPEFKKSMSYFDGKTEYKHIYFLAMLCKSLEEPNMNLSLQYENRIAEISKISWQNMEAVRFISKRQSLNLEPIIGPAFNFIKNYLRYKH.

[0036] The following primers were used to PCR amplify the fragment containing the g5Rp gene sequence: F: 5`- GAATTC ATGGATACTGCCATGCAGCT-3` ; R: 5`- CTCGAG AGTGCTTGTATCGTAAATC-3` 。

[0037] The pET-32a vector (available from the company) and the obtained PCR product were double-digested (EcoRI and Eco R I and XhoI) After the experiment, the g5Rp gene sequence was ligated with the pET-32a skeleton at 16°C for 1 h, and then immediately incubated with DH5a E. coli competent cells on ice for 30 min, followed by 42°C heat shock for 90 s and then incubation on ice for 5 min. After that, 600 μL of liquid LB medium was added, and the mixture was incubated in a shaker at 37°C for 60 min. After the incubation, the mixture was plated on solid LB medium containing 100 μg / mL ampicillin, and then inverted in a 37°C incubator overnight. The next day, single colonies were picked for double enzyme digestion verification. After verification, the plasmid was extracted and sent to the company for sequencing.

[0038] The plasmid with completely correct sequencing results and no mutation sites (i.e., the constructed pET-32a-g5Rp) was transformed, and the specific steps were as follows: 1 μL of the sequenced plasmid was added to 25 μL of BL21(DE3) E. coli competent cells, immediately ice-bathed for 10 min, then heat-shocked in a 42°C water bath for 90 s, immediately placed on ice for continued ice-bathing for 5 min, then 200 μL of liquid LB medium was added, and the mixture was incubated in a shaker at 37°C for 30 min. After the incubation, the mixture was plated on solid LB medium containing 100 μg / mL ampicillin, and then inverted in a 37°C incubator overnight. The next day, different single colonies (recombinant E. coli) were picked and added to 5 mL of liquid LB medium containing 100 μg / mL ampicillin, and incubated at 37°C for 8 h. Then, 600 μL of the bacterial solution was mixed with 400 μL of 50% glycerol, and stored in a -20°C refrigerator for subsequent large-scale protein expression. In addition, 200 μL of the bacterial solution was taken as a sample before expression induction. The remaining bacterial solution was added with 1 mM IPTG, and then incubated at 36°C for 6 h. Then, 200 μL of the bacterial solution was taken as a sample after expression induction. All samples were ultrasonically broken and centrifuged at 12000 rpm for 1 min. The whole bacteria, supernatant and precipitate were taken, 30 μL of H2O and 10 μL of loading buffer were added, and the samples were boiled at 100°C for 10 min. Then, SDS-PAGE analysis was performed.

[0039] According to the analysis results (see Figure 1 A) The recombinant E. coli with obvious changes in the expression amount of the target protein (about 44 kD) before and after induction was used for subsequent large-scale protein expression.

[0040] 2.2 Protein expression The recombinant E. coli was first cultured in 5 mL liquid LB medium containing 100 μg / mL ampicillin at 37°C overnight, and then transferred into 1 L liquid LB medium for culture at 37°C the next day. When the OD value at 600 nm was 0.8, 1 mM IPTG was added, and then the target protein expression was induced at 16°C overnight. The bacterial cells were collected and centrifuged at 4000 rpm for 25 min, and the supernatant was discarded. The bacterial cell pellet was suspended with buffer A (formula: 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 20 mM imidazole, and 5% glycerol).

[0041] 2.3 Protein purification (1) Nickel column purification The recombinant E. coli containing the target protein was first lysed by high-pressure homogenization and then broken by an ultrasonic cell crusher. Subsequently, the mixture was centrifuged at 4°C and 15000 rpm for 1 h in a refrigerated centrifuge. The supernatant was incubated with Ni medium and then subjected to affinity chromatography purification. The specific steps are as follows: Nickel column equilibration: On the AKTA protein purification system, the nickel column was first equilibrated with at least 5 column volumes of buffer A to ensure that the column was filled with buffer A; Loading: The above supernatant was pumped into an affinity chromatography column filled with Ni SepharoseTM 6 Fast Flow packing; Primary impurity removal: The nickel column was washed with buffer A to remove unbound impurities and cell debris; Further impurity removal: A solution containing a certain proportion of buffer B (containing a higher concentration of imidazole compared with buffer A, such as an imidazole concentration of 250 mM) was used for impurity removal. Usually, a solution containing 30% buffer B was used to remove more non-specifically bound impurities; Target protein elution: A solution containing a higher proportion (such as 50%) of buffer B was used for elution to release the target protein from the nickel column; Collection and analysis: The eluate was collected in sections, and SDS-PAGE was used to analyze each section of the eluate to confirm the presence and purity of the target protein. Figure 1 B).

[0042] (2) Further purification by gel filtration chromatography Molecular sieve equilibration: The AKTA protein purification system was turned on, and the molecular sieve column was connected. The molecular sieve column was equilibrated with a molecular sieve buffer (formula: 0.05 M NaPO4 and 0.15 M NaCl; pH 7.2) at a flow rate of 1.6 mL / min, and two column volumes were washed until the baseline was stable; Sample preparation: 4 mL of protein sample purified by nickel column was concentrated to about 1 mL using ultrafiltration concentration tube with a molecular weight cut-off of 10 kDa; the concentrated sample was centrifuged at high speed to remove air bubbles; Sample loading: the concentrated protein sample was injected into the sample loop using a syringe and air bubbles were avoided; the molecular sieve purification program was started and the sample was automatically separated by the molecular sieve column; Elution and collection: elution was performed with molecular sieve buffer and the eluate containing the target protein was collected; UV absorbance was monitored to determine the elution position of the target protein; according to the position of the UV detection peak, a small amount of protein eluate was treated with 5x loading buffer and subjected to SDS-PAGE to detect the content and purity of the target protein; according to the electrophoresis results Figure 1 C) The protein sample with good purity was collected and concentrated to the required concentration at 4°C and 5000 r / min using a 10 kDa concentration tube; it was aliquoted and stored frozen; System cleaning and maintenance: after completion of the purification, the molecular sieve column and the sample loop were removed and the AKTA protein purification system was turned off.

[0043] Finally, the target protein (i.e. prokaryotic expression of African swine fever virus g5Rp protein) with high purity and good uniformity was obtained and its concentration was adjusted to 10 mg / mL and 5 mg / mL for crystallography experiments.

[0044] (Three) Crystallography experiments 3.1 Initial screening of protein crystallization conditions using micro vapor diffusion sitting drop method (1) First, use the gun to add 100 μL of initial screening buffer to the mother pool of the 96-well crystal sitting drop plate.

[0045] (2) Then take 1 μL of prokaryotic expression of African swine fever virus g5Rp protein and 1 μL of initial screening buffer in the mother pool and add it to the crystallization well next to the mother pool.

[0046] (3) Finally, seal with transparent tape and incubate at 16°C in the dark.

[0047] (4) Regularly count the buffer conditions that can crystallize and observe the growth of the crystals; when taking pictures for observation, be careful to handle them gently; if there are suspected crystals, identify them under a UV lamp.

[0048] 3.2 Optimization of protein crystallization conditions (1) According to the original conditions, prepare the crystallization buffer; optimize the concentration of precipitants (salts, PEG), the concentration of the protein, the temperature, the pH, etc.; for pure salt crystallization, mainly prepare a gradient of the maximum concentration of salt; for crystallization conditions containing PEG, first consider setting a gradient of PEG concentration.

[0049] (2) Seed crystal optimization: Carefully crush the crystals with crystal tweezers, suck up the broken crystals with a pipette tip and dilute them 10 times, and add 1 / 10 volume of crystal seeds of different dilutions to the crystallization wells on a new 24-well crystal culture plate.

[0050] (3) Optimization of cryoprotectant reagents.

[0051] (4) After setting up the gradient buffer, add it to the mother pool of a 24-well crystal culture plate (500 μL per well) and mark it.

[0052] (5) Then, 1 μL of prokaryotic expression of African swine fever virus g5Rp protein and 1 μL of buffer in the mother pool were mixed on the dropper.

[0053] (6) Seal with transparent tape, incubate at a constant temperature of 16°C, and observe the crystal growth periodically.

[0054] Finally, in a 16°C crystal incubator, the prokaryotic expression of African swine fever virus g5Rp protein core protein was grown by the sitting drop method at 5 mg / mL, and protein crystals were obtained in a pool solution (0.2M sodium phosphate monobasic monohydrate and 20% (w / v) polyethylene glycol 3350; pH 9.1) ( Figure 2 The obtained crystals were stored in a reservoir solution containing 20% ​​glycerol and snap-frozen in liquid nitrogen before being transported to the Shanghai Synchrotron Radiation Facility to obtain protein crystal diffraction data.

[0055] (IV) Crystal diffraction data of prokaryotic expression of African swine fever virus g5Rp protein X-ray diffraction data were collected at the BL02U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF), and all diffraction data were analyzed and processed using HKL3000 software.

[0056] (V) Virtual screening 5.1 Protein structure analysis and preparation Based on the target being g5Rp, the source being ASFV, the UniProt ID being P32092, and the sequence length being 250 AA, two crystal structures were selected from the PDB database (https: / / www.rcsb.org / ), of which 7DNU had the highest resolution and was bound to a small molecule ligand IHP, with a resolution of 2.25Å.

[0057] For the analyzed crystal diffraction data, the crystal structure 7DNU in PDB was used as a model for molecular replacement, and the structure of the core protein of African swine fever virus g5Rp protein was analyzed and corrected using coot and PHENIX (Table 1). After structure analysis and correction, the crystal structure of African swine fever virus g5Rp protein was drawn using Pymol2.1 software, as shown in Figure 3 The crystal shows a dimeric structure.

[0058] Table 1. Diffraction data collection and correction

[0059] Table 1. Diffraction data collection and correction (continued)

[0060] The crystal structure preparation of the internal g5Rp protein was performed using the Protein Preparation Wizard module of Schrödinger software (2023-2), which included the following steps: Based on the crystal structure shown in Figure 3 (or Table 1), the bond order assignment, hydrogen addition, etc. were performed, and if there were missing side chains, the Prime module was used to complete them; then the water molecules and cofactors in the protein were removed; then the PROPKA method was used to optimize the hydrogen bond network of the protein amino acid residues at pH=7.0; finally, the OPLS_4 force field was used for protein energy minimization, only the hydrogen atoms were optimized, and the RMSD converged to 0.3 Å.

[0061] 5.2 Preparation of substrate structure The 2D sdf file was imported into Schrödinger software using the L6000 natural product library (L6000-Targetmol-Natural Product Library for HTS--4320cpds) provided by Tao Technology Company, and the 3D structure of each substrate (i.e. each natural product in the above natural product library as a compound to be screened) was prepared and the coordinates were determined based on the OPLS_4 force field using the Ligprep module; wherein the compounds to be screened were prepared according to the default settings of the Ligprep module and used for later molecular docking, including de-duplication processing, conformation optimization, charge assignment, hydrogen atom addition. All possible stereoisomers and related protonation states were determined using the Epik module.

[0062] 5.3 Generation of docking grid According to the literature reports, K8, K94, K95, K98, K175, R221 and K243 of g5Rp are the key amino acid residues binding to mRNA, therefore, when a compound (such as a natural product small molecule) interacts with these amino acid residues, it can inhibit the enzymatic activity of g5Rp. The small molecule ligand of 7DNU is bound to the binding site of K8, K94, K95, K98 and K175, and forms hydrogen bond and salt bridge interactions with K8. Superimposing the coordinates of 7DNU with the internal g5Rp protein crystal structure, the RMSD is only 0.364 Å, which shows that the two crystal structures are very similar, and therefore the crystallized small molecule ligand in the structure of 7DNU is used as the reference active pocket. Based on the pocket site of the small molecule ligand, the docking grid center is set to the crystallized ligand, the size of the outer box is set to be similar to that of the crystallized ligand, and the size of the inner box is set to 10 Å 3 , and structure-based virtual screening is performed on the binding pocket set.

[0063] 5.4 Docking scoring and ranking screening Molecular docking is performed using the Virtual Screening Workflow module in Schrödinger software. The data set for screening is prepared by the Ligprep module from each natural product 3D structure. The data set is first screened by a standard docking method, and then docked by standard precision. The specific process is as follows: (1) HTVS (high throughput screening): retain all stereoisomeric states of each ligand, and select only one conformation for each stereoisomeric state of the ligand. After docking is completed, the top 50% of compounds with the best scores are retained for the next step of screening.

[0064] (2) SP (standard precision): only retain the stereoisomeric state with the best score for each ligand, and select only one conformation for each stereoisomeric state of the ligand. After docking is completed, the top 20% of compounds with the best scores are retained for the next step of screening.

[0065] (3) XP (extra precision): only retain the stereoisomeric state with the best score for each ligand, and select only one conformation for each stereoisomeric state of the ligand. After docking is completed, the top 100 compounds are retained for the next step.

[0066] (4) MM / GBSA (binding free energy): re-score the retained 100 compounds using MM / GBSA.

[0067] 5.5 Virtual screening results Taking into account factors such as Glide-XP molecular docking scores and MM / GBSA binding free energy predictions, a preliminary screening yielded 43 compounds. After clustering and conformational analysis, and by positional alignment with existing crystalline small molecule ligands, 22 compounds were further selected. Molecular docking studies showed that the top three hit compounds among the 22 compounds obtained had similar binding cavities and interacted with key residues in the pockets. Figure 4-1 As shown (Ser6, Lys28, Ser35, His41, Lys127, and Lys129 shown in the figure are given in order based on the recoding results of the amino acid sequence of the protein structure in PyMol. If these sites are mapped back to the protein amino acid sequence, they should be Ser10, Lys32, Ser39, His45, Lys131, and Lys133, i.e., S10, K32, S39, H45, K131, and K133); and the main forces between the g5Rp protein (specifically referring to the crystal structure of the internal g5Rp protein) and these three compounds are conventional hydrogen bonds, hydrophobic interactions, salt bridges, and π-π stacking forces. These forces reduce the binding energy and increase the affinity, thereby achieving close binding of the corresponding compounds to the g5Rp protein and forming a stable complex.

[0068] Finally, the compounds 9''-methyl salvianolate B, β-Cyclodextrin, and Hosenkoside K were identified as the best hit molecules (see Table 2 for details), and the natural product small molecule 9''-methyl salvianolate B was selected for subsequent experimental verification.

[0069] Table 2. Screening of compounds targeting African swine fever virus g5Rp protein

[0070] (6) Molecular dynamics simulation Molecular dynamics simulation (MD) is a computational method based on the principles of classical mechanics that tracks the motion trajectories of particles to understand and predict the dynamic behavior and thermodynamic properties of matter at the microscopic level.

[0071] 6.1 System construction based on MD simulation The protonation states of the protein amino acid residues were calculated using PROPKA 3.0. All initial crystallographic water molecules from the selected protein crystal structure were retained. The initial all-atom complex model was constructed using the SystemBuilder module of Schrödinger software. The OPLS_4 force field was used to parameterize the protein and small molecule, and TIP3P was used to solvate the water model with a boundary thickness of 10.0 Å. The g5Rp protein-small molecule complex was solvated in a cubic water box, and the box volume was minimized. The entire system was neutralized by adding sodium or chloride ions.

[0072] 6.2 Simulation process Molecular Dynamics was used to set simulation parameters. The system's energy was first minimized for 50,000 steps using the steepest descent minimization method. Then, the positions of heavy atoms were constrained to run NVT and NPT equilibration for 50,000 steps. The system temperature was maintained at 300 K and the system pressure was maintained at 1 atm. After completing the two equilibration stages, unrestricted dynamics simulations were performed for 50 ns. The energy and coordinates of the trajectory were saved every 10 ps. After the trajectory simulation, Schrödinger software was used to draw interactive patterns and dynamic trajectory animations. After the 50 ns molecular dynamics simulation, the RMSD, RMSF, Rog, and hydrogen bond analysis of the g5Rp protein-small molecule complex were calculated throughout the entire process. The MM / GBSA (binding free energy) and energy decomposition were also calculated for the final frame of equilibrium for the g5Rp protein-small molecule system.

[0073] 6.3 Simulation results The results are shown in Table 3, where the binding free energy between the g5Rp protein and the small molecule 9''-methyl salvianolate B is -70.6523 kcal / mol. The results indicate that the small molecule binds well to the g5Rp protein after reaching a steady state.

[0074] Table 3. Parameters calculated by molecular dynamics simulation

[0075] Surface Plasmon Resonance (SPR) was used to detect the interaction between g5Rp protein and small molecules. 7.1 Methods (1) Chip preparation The activator was prepared immediately before injection by mixing 400 mM EDC and 100 mM NHS. The CM5 sensor chip was activated with the activator for 420 seconds at a flow rate of 10 μL / min.

[0076] (2) Ligand immobilization Prokaryotically expressed African swine fever virus g5Rp protein was diluted to 20 μg / mL in immobilization buffer and injected into the sample channel Fc2 at a flow rate of 10 μL / min. Immobilization levels of 12,600 RU were typically achieved. No ligand immobilization step was required for the reference channel Fc1. The CM5 sensor chip was activated with 1 M ethanolamine hydrochloride at a flow rate of 10 μL / min for 420 seconds.

[0077] (3) Multi-cycle method to run analytes The analyte, 9''-methyl salvianolate B, was diluted to eight concentrations (1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.3nM, 15.6nM, and 0) using the same analyte buffer; the analyte at 0 was diluted in analyte buffer. The various concentrations of 9''-methyl salvianolate B were then injected into channels Fc1-Fc2 at a flow rate of 20μL / min. The association phase lasted 100s, followed by a 180s dissociation phase. Both the association and dissociation phases were performed in analyte buffer. Eight cycles were repeated in ascending order of analyte concentration. The chip was regenerated after each cycle of interaction analysis. 7.2 Results The results are as follows Figure 5 As shown, the binding curve of the interaction between g5Rp protein and 9''-methyl salvianolate B shows obvious saturation within a certain concentration range and a smooth dissociation process. The results indicate that the binding energy between g5Rp protein and 9''-methyl salvianolate B is strong. (8) Effect of 9''-methyl salvianolate B on African swine fever virus (ASFV) replication 8.1 Testing the Cytotoxicity of 9''-methyl salvianolate B Detection of CC of 9''-methyl salvianolate B using 3D4 / 21 cells 50 (ConcentrationCausing 50% Inhibition), the results are as follows Figure 6-1 As shown, CC 50 It is 16.22μM.

[0078] CCK-8 assay (Cell Counting Kit-8) was used to detect the activity of 3D4 / 21 cells treated with 9''-methyl salvianolate B. The results showed that after treating 3D4 / 21 cells with different concentrations of compounds (i.e. 9''-methyl salvianolate B), the maximum concentration of 9''-methyl salvianolate B used was 10 μM without affecting cell viability Figure 6-2 ). And under this concentration treatment condition, the cell viability could still be maintained above 90% after 24h of action.

[0079] 8.2 Challenge experiment (1) Inoculation of cells: 3D4 / 21 cells were inoculated in 24-well plates and cultured at 37°C, 5% CO2 until the cells reached 70%-80% confluence. Prepare a certain concentration (TCID 50 or PFU / mL) of ASFV (strain China / LN / 2018 / 1) virus solution for infection in subsequent experimental steps; perform small-scale virus infection test to determine the optimal ASFV infection dose (the determined optimal dose is 1 MOI).

[0080] (2) Dilution of small molecules: 9''-methyl salvianolate B was diluted to a working solution of 10 μM (weigh 9''-methyl salvianolate B about 7.33 mg, dissolved in DMSO, adjust the volume to 10 mL to get 1 mM stock solution; take 100 µL from the stock solution, add to 9900 µL of culture medium, mix well to get it).

[0081] (3) Virus infection: The pre-titrated optimal dose of virus solution was added to the cells treated with 10 μM of 9''-methyl salvianolate B (incubated with 10 μM working solution for 6 hours in advance) and cells without 9''-methyl salvianolate B treatment, and incubated at 37°C, 5% CO2 for 1-2 hours, during which the treatment group was gently shaken to promote virus binding to cells.

[0082] (4) Washing: After incubation, the culture medium was discarded and the cells were washed twice with PBS to remove unbound viruses.

[0083] (5) Add fresh culture medium: After washing, add fresh culture medium and continue to culture the cells for 24 to 96 hours.

[0084] (6) Immunofluorescence staining and Western blotting Immunofluorescence staining: After 24 hours of infection with 1 MOI of African swine fever virus (ASFV), the cells were fixed with 4% paraformaldehyde and permeabilized with 0.15% Triton X-100 for 20 minutes. Subsequently, the cells were incubated with mouse anti-g5Rp antibody (laboratory-made, reference: Bioinformatics analysis of D250R protein of African swine fever virus and preparation of polyclonal antibody. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4745-4755; 1:200) at 37°C for 1 hour and washed with PBS three times. Then, the cells were incubated with FITC-labeled goat anti-mouse IgG antibody (D110105, Shanghai Gener Biotech Co., Ltd.; 1:1000) at 37°C for 30 minutes. After washing with PBS three times again, the cells were treated with 4', 6-diamidino-2-phenylindole (DAPI) at 37°C for 15 minutes. Finally, images were acquired using a laser scanning confocal microscope (Leica TCS SP8, Germany).

[0085] Western blotting: After 24 hours of infection with 1 MOI of African swine fever virus (ASFV), the cells were lysed with RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) at room temperature for 30 minutes, and then centrifuged at 12000 rpm for 15 minutes to separate the proteins. Next, the extracted proteins were mixed with 5 times the concentration of sodium dodecyl sulfate buffer and boiled for 10 minutes for denaturation, followed by electrophoretic separation. The separated proteins were transferred to a nitrocellulose membrane, which was blocked with 5% skim milk powder for 2 hours to prevent non-specific binding. Then, the membrane was incubated with mouse anti-g5Rp antibody overnight at 4°C, and washed with TBST containing 0.1% Tween 20 three times. Next, the secondary antibody labeled with horseradish peroxidase (C650201, Shanghai Gener Biotech Co., Ltd.) was incubated at room temperature, and washed with TBST three times again. Finally, the membrane was visualized using an electrochemiluminescence imager, and the signal of the target protein was observed.

[0086] Experimental results: Immunofluorescence staining was performed 24 hours after infection with 1 MOI of African swine fever virus, and the results are shown in Figure 7 Compared with the group of cells treated with 10 μM of 9''-methyl salvianolate B for 6 hours, the group of cells treated with 9''-methyl salvianolate B had a lower g5Rp fluorescence intensity; at the same time, Western blotting detected that 9''-methyl salvianolate B treatment reduced the expression of African swine fever virus g5Rp protein in cells Figure 8 ).

[0087] (7) By adding the pre-titrated optimal dose of virus liquid into the cells treated with 10 μM of 9''-methylsalvianolate B (pre-incubation for 6 hours) and the cells without 9''-methyl salvianolate B treatment, respectively, the results after culture showed that 9''-methyl salvianolate B can effectively inhibit the replication of African swine fever virus (ASFV) (Fig. 6). Figure 9 ; p <0.01, compared with the Mock group).

[0088] In summary, the compound obtained by virtual screening (taking 9''-methyl salvianolate B as an example) can not only form a stable protein-small molecule complex with the viral target, i.e. African swine fever virus g5Rp protein, in molecular docking, but also inhibit the replication of African swine fever virus, so as to be expected to be applied in the development of drugs for preventing and / or treating African swine fever.

Claims

1. Use of a compound targeting the g5Rp protein of African swine fever virus in the preparation of drugs against African swine fever virus.

2. The use according to claim 1, characterized in that: The sites where the compound interacts with the African swine fever virus g5Rp protein include one or more of Ser10, Lys32, Ser39, His45, Lys131, and Lys133.

3. The use according to claim 1, characterized in that: The compound is selected from any one of 9''-salvianolic acid B monomethyl ester, β-cyclodextrin, and impatiens tetraol glycoside K.

4. The use according to claim 3, characterized in that: The 9''-salvianolic acid B monomethyl ester reduces the expression level of African swine fever virus g5Rp protein in a host or a host cell.

5. The use according to claim 3, characterized in that: The 9''-salvianolic acid B monomethyl ester inhibits the replication of African swine fever virus in a host or a host cell.

6. Use of a compound targeting the g5Rp protein of African swine fever virus in the preparation of a drug for preventing and / or treating African swine fever.

7. A method for virtual screening of drugs against African swine fever virus, characterized in that: The following steps are involved: The target information is determined based on the crystal structure of the prokaryotic expressed African swine fever virus g5Rp protein. The compounds to be screened are respectively docked with the target to form protein complexes and scores for the molecular docking compounds are obtained. Then, the compounds that stably bind to the African swine fever virus g5Rp protein are determined based on the ranking results of the scores of each compound.

8. The method for virtual screening of drugs against African swine fever virus according to claim 7, characterized in that: The amino acid sequence of the African swine fever virus g5Rp protein is shown in SEQ.ID.NO.

1.

9. The method for virtual screening of drugs against African swine fever virus according to claim 7, characterized in that: The target information includes a reference active pocket for locating the interaction interface between African swine fever virus g5Rp protein and substrate in molecular docking.

10. The method for virtual screening of drugs against African swine fever virus according to claim 7, characterized in that: The compound to be screened is a natural product.

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