Application of glycoside compounds in prevention and treatment of plant pathogenic viruses

Abstract

The invention discloses application of glycoside compounds in prevention and treatment of plant pathogenic viruses, and belongs to the technical field of pesticide antivirus. According to the invention, targeting tobacco mosaic virus capsid protein is taken as an example, and small molecule picroside I (A0) and ZINC85593196 (B0) containing sugar rings are screened through virtual screening, molecular dynamics simulation, molecular mechanics and MM / GBSA free energy calculation. On the basis of further optimization, six compounds of 4-methylumbelliferone-beta-D-xyloside (A1), geniposide (A2), 4-methylumbelliferone acyl-beta-D-glucopyranoside (A3), 4-methylumbelliferone acyl-beta-D-glucopyranoside (A4), catalpol (A5) and aesculin (sesquihydrate) (A6) which are similar to the A0 in structure are obtained, and in-vivo and in-vitro biological activity evaluation is carried out. The active component screened by the method has a good inhibition effect on plant viruses.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide antiviral technology, in particular to the application of glycoside compounds in preventing and controlling plant pathogenic viruses. Background Art

[0002] Animals, plants, bacteria, fungi and other microorganisms can all be infected by corresponding viruses. Viruses seriously threaten human health, threaten the growth of crops, and seriously reduce crop yield and quality. Typical agricultural viruses, such as tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato virus Y (PVY), etc., can infect hundreds of species and show difficult-to-control viral symptoms during growth, causing considerable economic threats to agricultural production. Chemical control, as an efficient and labor-saving control method, has been widely used in pesticide production. However, some chemical agents have obvious limitations. Therefore, there is an urgent need to develop economically feasible and accessible chemical preparations with multiple modes of action to ensure the sustainable development of medical and agricultural systems.

[0003] The traditional drug discovery process is characterized by high risk, time-consuming, and costly shortcomings. Computer-aided drug design has revolutionized target identification, virtual screening, molecular docking, kinetic simulation, and ADME property prediction, significantly accelerating drug development, reducing costs, and improving efficiency and success rates. It also provides valuable insights into the underlying mechanisms of receptor-ligand interactions. Therefore, multiple screening strategies based on cross-validation can effectively improve hit accuracy. Drug design based on pathogen functional proteins or enzymes is an effective approach for developing targeted pesticides. Natural products offer advantages such as systemicity, unique functionality, high efficiency, low toxicity, and environmental friendliness. Due to their abundant resources and excellent performance, natural molecules have long been widely used in drug development and disease prevention. Therefore, the development of chemotoxicants based on glycosylated natural products is a valuable and fruitful avenue.

[0004] The TMV capsid protein, composed of 158 amino acids (17.5 kDa), is a structural and functional protein translated from subgenomic RNA that self-assembles with TMV RNA to form a complete, mature virus. The TMV capsid protein plays a crucial role in protecting nucleic acids from degradation by external factors, facilitating long-distance transport of TMV, maintaining its infectious capacity, and participating in nucleic acid replication. These essential biological functions make the TMV capsid protein a promising pesticide target. To efficiently and accurately identify potential inhibitors of TMV capsid protein and their potential resistance to viral physiological functions, a library of sugar-modified natural molecules was constructed, the TMV capsid protein was used as the receptor target, and the optimal structure of picroside I (A0) was used as a reference. Six compounds with similar structures to picroside I (A0) were further screened: 4-methylumbelliferyl-β-D-xyloside (A1), geniposide (A2), 4-methylumbelliferyl-β-D-pyranoside (A3), 4-methylumbelliferyl-β-D-glucopyranoside (A4), catalpol (A5), and esculin (sesquihydrate) (A6). The anti-TMV bioactivity of these compounds was further confirmed by microthermophoresis (MST), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and in vivo validation. Summary of the Invention

[0005] The present invention aims to provide the application of glycoside compounds in the prevention and treatment of plant pathogenic viruses to solve the problems existing in the above-mentioned prior art. Taking the agricultural disease tobacco mosaic virus as an example, the present invention specifically describes the screening of small molecule inhibitors targeting TMV-CP.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a method for screening small molecule inhibitors targeting TMV-CP, comprising the following steps:

[0008] (1) Pre-processing of TMV capsid protein structure;

[0009] (2) Download the original virtual natural molecules from the ZINC database and select compounds with a molecular weight of 350-500 as the screening library; import the screening library into Discovery Studio for filtering using the intelligent sugar ring format to obtain a natural product ligand screening library containing sugar rings;

[0010] (3) Virtual screening based on TMV shell protein using Discovery Studio, AutodockVina, Sybyl, and Schrodinger to select the top 100 ligands with the highest affinity scores;

[0011] (4) Using a Venn diagram to present co-ligands, a preliminary screening of lead compounds with strong binding ability to TMV-CP was conducted, and similarity searches were performed based on the lead compounds to obtain molecules with similar structures to the lead compounds and obtain secondary lead compounds;

[0012] (5) The lead compound and secondary lead compound are further screened to obtain the small molecule inhibitor through molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis.

[0013] The second technical solution of the present invention is a small molecule inhibitor obtained by screening using the method. The small molecule inhibitor is a glycoside compound, including A0 to A6, and its structural formula is as follows:

[0014]

[0015] The third technical solution of the present invention is the use of the small molecule inhibitor in preventing and controlling plant pathogenic viruses.

[0016] A fourth technical solution of the present invention is a drug for preventing and treating plant pathogenic viruses, comprising the small molecule inhibitor.

[0017] The fifth technical solution of the present invention is a method for preventing and controlling plant pathogenic viruses, which uses the small molecule inhibitor or the drug for prevention and control in agricultural production.

[0018] Based on the above technical solution, the present invention has the following technical effects:

[0019] 1. Through virtual screening, the number of experimental screening compounds can be reduced, shortening the R&D cycle and saving costs;

[0020] 2. Through experiments, it was confirmed that the virtual screening hit compounds have significant TMV-CP activity and have the effect of preventing and treating agricultural viruses;

[0021] 3. The virtual screening hit compounds have novel skeletons and can be used to further develop new TMV-CP inhibitor-type antiviral agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a virtual screening and experimental evaluation process for TMV-CP inhibitors based on molecular docking.

[0024] Figure 2 RMSD diagrams of A0-capsid, B0-capsid and NNM-capsid complexes.

[0025] Figure 3 RMSF (A) and MM-GBSA (B) plots of A0-capsid, B0-capsid and NNM-capsid compounds.

[0026] Among them, A is the RMSF graph of A0-capsid, B0-capsid and NNM-capsid, and B is the MM-GBSA graph of A0-capsid, B0-capsid and NNM-capsid.

[0027] Figure 4 Figures are from a microscale thermophoresis (MST) experiment. A is a schematic diagram of the MST procedure, B is an MST analysis of A0 and TMV protein, C is an MST analysis of A1 and TMV protein, D is an MST analysis of A2 and TMV protein, E is an MST analysis of A3 and TMV protein, F is an MST analysis of A4 and TMV protein, G is an MST analysis of A5 and TMV protein, and H is an MST analysis of A6 and TMV protein.

[0028] Figure 5 This is a similarity search graph using picroside I (A0) as a template. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0035] The present invention provides a method for screening small molecule inhibitors targeting TMV-CP, comprising the following steps:

[0036] (1) Pre-processing of TMV capsid protein structure;

[0037] (2) Download the original virtual natural molecules from the ZINC database and select compounds with a molecular weight of 350-500 as the screening library; import the screening library into Discovery Studio for filtering using the intelligent sugar ring format to obtain a natural product ligand screening library containing sugar rings;

[0038] (3) Virtual screening based on TMV shell protein using Discovery Studio, AutodockVina, Sybyl, and Schrodinger to select the top 100 ligands with the highest affinity scores;

[0039] (4) Using a Venn diagram to present co-ligands, a preliminary screening of lead compounds with strong binding ability to TMV-CP was conducted, and similarity searches were performed based on the lead compounds to obtain molecules with similar structures to the lead compounds and obtain secondary lead compounds;

[0040] (5) The lead compound and secondary lead compound are further screened to obtain the small molecule inhibitor through molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis.

[0041] In some specific embodiments, the method for pre-processing the TMV capsid protein structure is as follows: removing all crystal water and non-polar hydrogen atoms in the Autodock tool, determining the TMV capsid protein potential active site coordination in the box (x=0.363, y=-25.381, z=4.010), and setting the grid spacing to

[0042] In some specific embodiments, the grid size is 18×22×20 xyz points.

[0043] In some specific embodiments, the smart glycosyl ring is ((#8)1-(#6)(-(#6)[#8])——(#6)(-[#8])(#6)(-[#8])(#6)(-[#8])(#6)1-[#8]-[#6].

[0044] In some specific embodiments, the further screening of the lead compound and secondary lead compound to obtain the small molecule inhibitor by molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis includes:

[0045] Optimize the docking prediction binding mode of the screened compounds and simulate each receptor-ligand complex to further evaluate the binding stability of the ligand-receptor complex;

[0046] The root mean square fluctuation of each residue was calculated to further evaluate the binding stability of the ligand-receptor complex;

[0047] The binding free energy of the receptor-ligand complex was calculated by MM / GBSA free energy calculation, and the mmpbsa module in Amber12 was used to perform MM / GBSA free energy decomposition analysis to study the detailed binding interaction between TMV-CP and inhibitors.

[0048] In some specific embodiments, the method further comprises the step of verifying the small molecule inhibitors obtained by in vivo screening.

[0049] The present invention also provides a small molecule inhibitor obtained by screening using the method. The small molecule inhibitor is a glycoside compound, including A0 to A6, and has the following structural formula:

[0050]

[0051] The embodiments of the present invention also provide the use of the small molecule inhibitor in preventing and controlling plant pathogenic viruses.

[0052] In some specific embodiments, the plant pathogenic viruses include tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato virus Y (PVY), and tomato spotted wilt virus (TSWV).

[0053] An embodiment of the present invention further provides a drug for preventing and treating plant pathogenic viruses, comprising the small molecule inhibitor.

[0054] In some specific embodiments, the dosage form of the drug is selected from any one of emulsifiable concentrate, powder, wettable powder, granule, aqueous solution, suspension, ultra-low volume spray, soluble powder, microcapsule, smoke agent, aqueous emulsion or water-dispersible granule.

[0055] An embodiment of the present invention further provides a method for preventing and controlling plant pathogenic viruses, wherein the small molecule inhibitor or the drug is used for prevention and control in agricultural production.

[0056] The design concept of the present invention is as follows: First, compounds in a natural product screening library are filtered using an intelligent format sugar ring to obtain a library of natural product sugar ring molecules containing sugar rings. Using TMV-CP as the target protein, a molecular docking program is used for virtual screening to select molecules with high scores. Taking drug availability and cost into consideration, secondary lead compounds are rapidly obtained through molecular similarity searches. These compounds are then verified through biological activity assays to identify lead compounds for TMVCP preparations with antiviral activity. This invention provides a rapid screening method for antiviral lead compounds.

[0057] Example 1

[0058] Molecular docking-based virtual screening: The discovery and identification of active natural precursors is a time-consuming and complex undertaking. Intelligent screening strategies combined with bioactivity assessment are a smart and time-saving approach that can provide leads for the discovery of therapeutics against difficult-to-treat plant pathogens.

[0059] 1. Experimental methods:

[0060] ① The crystal structure of TMV capsid protein (TMV-CP, PDB code: 1EI7) was obtained from the RCSB database (https: / / www.rcsb.org / );

[0061] ② Before molecular docking, the TMV capsid protein was pretreated by removing all crystal water and non-polar hydrogen atoms using the Autodock tool (1.5.7);

[0062] ③ Then, determine the potential active site coordination of TMV capsid protein in the box (x=0.363, y=-25.381, z=4.010), and the grid spacing is set to (grid size: 18×22×20xyz ponts);

[0063] ④ Download original virtual natural molecules from the ZINC database (https: / / zinc15.docking.org / substances / subsets / natural-products / ), which contains more than 80,617 natural products as the screening library;

[0064] ⑤ The screening library was introduced into Discovery Studio (2019) for filtration using the intelligent format sugar ring ((#8)1-(#6)(-(#6)[#8])——(#6)(-[#8])(#6)(-[#8])(#6)(-[#8])(#6)1-[#8]-[#6] to obtain a natural product ligand screening library containing sugar rings;

[0065] ⑥ Based on virtual screening of TMV shell protein using four mainstream docking software, Discovery Studio (2019), AutodockVina (1.2.1), Sybyl (2.0), and Schrodinger (2021), the top 100 ligands with the highest affinity scores were selected.

[0066] Example 2

[0067] MD simulations and MM / GBSA free energy calculations.

[0068] 1. Experimental methods:

[0069] ① Use GROMACS software (2020) for MD simulation, and use the CHARMM36 force field and TIP3P water model recommended by the tutorial to simulate complex systems;

[0070] ② ZINC390822436 (picroside I, denoted as A0), ZINC85593196 (B0), and commercial Ningnanmycin (NNM) topologies were generated in the CHARMM official general force field server (https: / / cgenff.umaryland.edu / ) and combined with the TMV capsid protein topology;

[0071] The structural formulas of A0 and B0 are as follows:

[0072]

[0073] ③ Place the complex appropriately in a simulation box 1 nm away from the box edge, and give the system 4 sodium ions to neutralize the system charge;

[0074] ④ Minimize the system using the steepest descent method and balance it using a combination of NVT and NPT (100 ps, ​​50,000 steps);

[0075] ⑤ MD simulations were performed within 100 ns (300 K) to evaluate the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of the receptor-ligand complex. The g_mmpbsa tool (molecular mechanics Poisson-Boltzmann surface area) was used to calculate the binding energy of the protein-ligand complex.

[0076] The results showed that the average RMSD values ​​of picroside I (A0), B0, and NNM were 0.16±0.02 nm, 0.19±0.03 nm, and 0.16±0.06 nm, respectively, and the corresponding average RMSD values ​​of the receptors were 0.63±0.10 nm, 0.89±0.32 nm, and 0.66±0.08 nm, respectively. This indicates that picroside I (A0) binds tightly to the ligand. The corresponding energy values ​​for the A0-capsid, B0-capsid, and NNM-capsid complexes were -29.0, -23.6, and -10.3 kcal / mol, respectively. This result suggests that the non-covalent binding state of picroside I (A0) to the capsid protein under simulated conditions has a lower binding energy, which is more conducive to binding.

[0077] Example 3

[0078] Microcalorimetry (MST): MST is a commonly used method to detect the dissociation process between biomolecules and obtain interaction patterns and kinetic constants. MST was used to determine the binding force between TMV capsid protein and natural molecules.

[0079] 1. Experimental methods:

[0080] ① First, the purified TMV capsid protein (100 μL) was fluorescently labeled with a fluorescent dye (2 μL) and placed on ice in the dark (30 min);

[0081] ②Then, the labeled TMV capsid protein was separated using a dedicated column and diluted with PBS at pH 7.4 to ensure that the fluorescence absorption value was between 600 and 1000.

[0082] ③Then, different concentrations of molecular solutions or NNM / Ri were diluted with PBS (10 μL) and incubated with labeled TMV capsid protein (0.1 mM, 10 μL) (10 min).

[0083] ④Finally, the mixed solution was absorbed into a standard capillary tube and the MST and dissociation constant (Kd) values ​​of TMV capsid protein were measured under 70% LED power.

[0084] The results showed that the molecules exhibited significantly different binding abilities (ranging from 0.14 μM to 166.89 μM), indicating that the non-sugar ring regions of the compounds also interacted significantly with the capsid protein. Among them, the optimal picroside I (A0) exhibited the strongest binding to the capsid protein (Kd = 0.14 μM), significantly exceeding the antiviral agent NNM (Kd = 3.47 μM) by 24.8-fold. Simultaneously, the isomers 4-methylumbelliferyl-β-D-pyranoside (A3) (Kd = 5.68 μM) and 4-methylumbelliferyl-BD-glucopyranoside (A4) (Kd = 1.43 μM) also exhibited similar binding abilities. 4-methylumbelliferyl-β-D-xyloside (A1) and geniposide (A2) exhibited weaker binding than A3 and A4, with Kd values ​​of 76.92 μM and 26.16 μM, respectively. However, catalpol (A5) (Kd = 166.89 μM) and esculin (sesquihydrate) (A6) (Kd > 200 μM) bound to TMV capsid protein significantly weaker.

[0085] The structural formulas of A0~A6 are as follows:

[0086]

[0087] Example 4

[0088] In vitro biological activity verification.

[0089] 1. Experimental methods:

[0090] ① TMV was inoculated on tobacco (Nicotiana tabacum K326cv) leaves and the inoculated samples were cultured in a greenhouse at 25°C.

[0091] ② Six hours later, samples were collected by punching holes (1 cm in diameter) in K326 leaves containing TMV. The leaf samples were then placed in culture dishes containing different drug concentrations. Blank controls were treated with DMSO at concentrations of 25, 50, 100, and 200 μg / mL. Positive controls were treated with ningnanmycin and ribavirin at concentrations of 25, 50, 100, and 200 μg / mL, respectively. Experimental controls were treated with A0 and A4 at concentrations of 25, 50, 100, and 200 μg / mL. Each sample concentration was replicated three times.

[0092] ③ After 48 hours, the sample genes were extracted using the TransZol Up kit (TransGen Biotech, Beijing, China) for reverse transcription detection.

[0093] ④The iCycleriQ multicolor real-time PCR detection system (Bio-Rad, CA, USA) was used to evaluate the inhibitory effect on the relative expression of TMV capsid protein gene using the 2-ΔΔCt method.

[0094] Results showed that after 48 hours of incubation of infected leaves in the drug solution, picroside I (A0) exhibited the greatest inhibitory effect on the relative expression of tobacco capsid genes (16%), exceeding that of the commercial NNM (32%) by approximately two-fold. Although the compound 4-methylumbelliferyl-BD-glucopyranoside (A4) bound well to the capsid, its anti-capsid gene expression was similar to that of NNM. Compounds were prepared at varying concentrations (25, 50, 100, and 200 μg / mL) to further characterize their inhibitory effects on capsid genes at the transcriptional level. Picroside I (A0) demonstrated a significant ability to reduce capsid gene expression in a dose-dependent manner. Furthermore, within the 50-25 μg / mL range, significant differences were observed between the 4-methylumbelliferyl-BD-glucopyranoside (A4) group and the NNM group. When the concentration of picroside I (A0) increased from 100 μg / mL to 200 μg / mL, the improvement of its anti-capsid gene effect was limited (from 13% to 9%), indicating that high concentration of picroside I (A0) could hardly completely block the replication of capsid genes.

[0095] Example 5

[0096] The application of small molecule inhibitors against pathogenic viruses is used in the test method for anti-tobacco mosaic virus.

[0097] The anti-plant virus activity of the compounds was determined using the half-leaf spot assay. Accurately weigh 3 mg of the test compound into a weighing bottle and add 60 μL of DMSO to fully dissolve it.

[0098] ① Prepare a 500 mg / L solution of compound A0-A6 in double-distilled water containing 1% Tween 20. Separately, take 250 μL of 2% ribavirin aqueous solution, add 60 μL of DMSO solvent and 10 mL of double-distilled water containing 1% Tween 20 to prepare a 500 mg / L ribavirin solution.

[0099] ② The therapeutic activity of the agent against TMV infection. Select the heart-leaf tobacco with the same growth, first dip the virus solution (concentration of 6×10 -3 mg / mL), and inoculate the leaf surface (whole leaf) along its branch vein direction on the leaf sprinkled with corundum by manual friction. The inoculation force of the left and right leaves should be kept consistent as much as possible, and the bottom of the leaf should be supported by a flat wooden board. After the virus liquid dries, rinse the corundum on the leaf with running water. After the leaf is dry, apply the agent to the left half of the leaf, and apply sterile water to the right half of the leaf as a control. Three plants are treated with each agent, each with 3-4 leaves. The plants are then placed in a light incubator for moisturizing culture, controlled at 23°C and 10,000LuX of light. After 2-4 days, the number of dead spots is observed and recorded. Each agent is repeated 3 times according to the above method, and the inhibition rate is calculated.

[0100] ③ The protective activity of the agent against TMV infection. Select the heart-leaf tobacco plants with the same growth. Use a brush to gently apply the agent on the left half of the leaf. Use sterile water to apply on the right half of the leaf as a control. 24 hours later, inoculate the virus. Use a brush to dip the virus juice (concentration of 6×10 -3 mg / mL) and inoculated on the leaf surface (whole leaf) along its veins by manual rubbing onto leaves sprinkled with corundum. Maintain the same inoculation force on both leaves as much as possible, and support the bottom of the leaves with a flat wooden board. After the virus liquid dries, rinse the corundum off the leaves with running water. Three plants were treated with each agent, each with 3-4 leaves. The plants were then placed in a light incubator with moisturizing culture, controlled at 23°C and 10,000 Lux. After 2-4 days, the number of necrotic spots produced was observed and recorded. Three replicates were performed for each agent, and the inhibition rate was calculated.

[0101] The inhibition rate of the compound on tobacco mosaic virus Y (%) = (RL) / R × 100%;

[0102] Wherein, R represents the number of necrotic spots in the control group (right half of the leaf), and L represents the number of necrotic spots in the treatment group (left half of the leaf).

[0103] The present invention also imitated the above method to test the inhibition rate of A0-A6 on plant pathogenic viruses PVY, TSWV and CMV, and the calculation results are shown in Tables 1-4.

[0104] Table 1 Activity test of small molecule inhibitors against plant pathogenic virus tobacco mosaic virus (TMV)

[0105]

[0106] Table 2 Activity test of small molecule inhibitors against plant pathogenic virus Potato virus Y (PVY)

[0107]

[0108]

[0109] Table 3 Activity test of small molecule inhibitors against plant pathogenic virus Tomato spotted wilt virus (TSWV)

[0110]

[0111] Table 4 Activity test of small molecule inhibitors against plant pathogenic virus cucumber mosaic virus (CMV)

[0112]

[0113] As shown in Tables 1-4, the compounds exhibited good antiviral activity against tobacco mosaic virus at a concentration of 500 μg / mL. Regarding therapeutic activity, A0 and A4 exhibited good antiviral activity, with values ​​of 54.4% and 53.3%, respectively, which were superior to the control drug Ningnanmycin (50.9%). Regarding protective activity, compounds A0-A6 were all lower than Ningnanmycin (56.3%). The compounds exhibited good antiviral activity against potato virus Y at a concentration of 500 μg / mL. Regarding therapeutic activity, A0 and A4 exhibited good antiviral activity, with values ​​of 55.3% and 52.3%, respectively, which were superior to the control drug Ningnanmycin (51.0%). Regarding protective activity, compound A0 exhibited an inhibition rate of 54.8%, which was superior to the control drug Ningnanmycin (53.3%). The compounds exhibited good antiviral activity against tomato spotted wilt virus at a concentration of 500 μg / mL. In terms of therapeutic activity, A0 and A4 had good antiviral activity, with values ​​of 60.1% and 54.3%, respectively, which were better than the control drug Ningnanmycin (50.6%). In terms of protective activity, compounds A0-A6 were all lower than Ningnanmycin (52.0%). The compounds had good antiviral activity against cucumber mosaic virus at a concentration of 500 μg / mL. In terms of therapeutic activity, A0 and A4 had good antiviral activity, with values ​​of 58.7% and 58.3%, respectively, which were better than the control drug Ningnanmycin (55.4%). In terms of protective activity, compounds A0-A6 were all lower than Ningnanmycin (62.3%).

[0114] Example 6

[0115] EC 50 Median effective concentration (MEC) is an important indicator for evaluating the effects of compounds on plant pathogens. It is also an important parameter for setting compound concentrations when studying the mechanism of action of target compounds. In the concentration gradient experiment, the two-fold dilution method was used to set 5 appropriate concentrations. Finally, the inhibition rate of the agent on plant pathogenic viruses and the agent concentration were converted into logarithmic values. The toxicity curve was obtained through regression analysis using SPSS software, and the EC was calculated. 50 The half-leaf spot assay was used to determine the anti-plant virus therapeutic activity of the compounds.

[0116] 1. Experimental Methods

[0117] ① Weigh 5 mg of each test compound (A0-A6) into a weighing bottle and add 100 μL of DMSO solvent to fully dissolve it.

[0118] ② Use double distilled water containing 1% Tween 20 to prepare compound solutions of 500, 400, 300, 200, and 100 mg / L respectively.

[0119] ③ Prepare 500 mg / L Ningnanmycin solution as a positive control.

[0120] ④ Select the heart-leaf tobacco with the same growth, first dip the virus solution (concentration of 6×10 -3 The virus solution was inoculated by manual rubbing along the leaf surface (the entire leaf) along the veins onto a leaf sprinkled with corundum. The inoculation force was kept consistent between the two leaves, and the leaves were supported by a flat wooden board. After the virus solution dried, the corundum was rinsed off the leaves with running water.

[0121] ⑤ After the leaves have dried, apply the pesticide to the left half of the leaf and sterile water to the right half as a control. Treat three plants per pesticide, each with 3-4 leaves. Place the plants in a light incubator to maintain moisture, control the temperature at 23°C, and light at 10,000 Lux. After 2-4 days, observe and record the number of necrotic spots. Repeat the above method three times for each pesticide and calculate the inhibition rate.

[0122] The inhibition rate of the compound on tobacco mosaic virus Y (%) = (RL) / R × 100%;

[0123] Wherein, R represents the number of necrotic spots in the control group (right half of the leaf), and L represents the number of necrotic spots in the treatment group (left half of the leaf).

[0124] The calculation results are shown in Table 5.

[0125] Table 5 The therapeutic activity EC of small molecule inhibitors targeting TMV-CP against plant pathogenic virus TMV 50 test

[0126]

[0127] As can be seen from Table 5, the target compounds showed good inhibitory activity against plant pathogenic virus TMV in the in vivo test. The EC values ​​of compounds picroside I (A0) and 4-methylumbelliferyl-BD-glucopyranoside (A4) were 50 The values ​​were 310.9 μg / mL and 371.2 μg / mL, respectively. The activity data tested were superior to the commercial drug ribavirin (588.0 μg / mL), and the activity of the compound was comparable to that of ningnanmycin (343.8 μg / mL). As shown above, the screened molecules have good antiviral activity.

[0128] In summary, the present invention uses the capsid protein of tobacco mosaic virus as an example to screen the small molecule picroside I (A0) containing a sugar ring through virtual screening, molecular dynamics (MD) simulation, molecular mechanics, and MM / GBSA free energy calculations. Based on this, further optimization was performed to obtain six compounds with similar structures to A0: 4-methylumbelliferyl-β-D-xyloside (A1), geniposide (A2), 4-methylumbelliferyl-β-D-pyranoside (A3), 4-methylumbelliferyl-β-D-glucopyranoside (A4), catalpol (A5), and esculin (sesquihydrate) (A6). In vitro and in vivo bioactivity evaluations were performed. The active ingredients screened using the above method showed inhibitory effects on plant viruses such as cucumber mosaic virus, tomato spotted wilt virus, and potato virus Y, and showed particularly good inhibitory effects on tobacco mosaic virus.

[0129] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for screening small molecule inhibitors targeting TMV-CP, characterized in that: The following steps are involved: (1) Pre-processing the TMV capsid protein structure; (2) Download the original virtual natural molecules from the ZINC database and select compounds with a molecular weight of 350-500 as the screening library; introduce the screening library into Discovery Studio for filtering through the intelligent format sugar ring to obtain a natural product ligand screening library containing sugar rings; (3) Virtual screening based on TMV shell protein using Discovery Studio, AutodockVina, Sybyl, and Schrodinger to select the top 100 ligands with the highest affinity scores; (4) Using Venn diagrams for co-ligand presentation, preliminarily screen out lead compounds with strong binding ability to TMV-CP, and perform similarity searches based on lead compounds to obtain molecules with similar structures to the lead compounds and obtain secondary lead compounds; (5) Further screening the lead compound and secondary lead compound to obtain the small molecule inhibitor through molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis.

2. The method according to claim 1, characterized in that: The method for preprocessing the TMV capsid protein structure is: removing all crystal water and non-polar hydrogen atoms in the Autodock tool, determining the TMV capsid protein potential active site coordination in the box (x=0.363, y=-25.381, z=4.010), and setting the grid spacing to 3. The method according to claim 2, characterized in that The grid size is 18×22×20 xyz points.

4. The method according to claim 1, characterized in that: The small molecule inhibitors obtained by further screening the lead compound and the secondary lead compound through molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis include: The docking prediction binding modes of the screened compounds were optimized and each receptor-ligand complex was simulated to further evaluate the binding stability of the ligand-receptor complex; The root mean square fluctuation of each residue was calculated to further evaluate the binding stability of the ligand-receptor complex; The binding free energy of the receptor-ligand complex was calculated by MM / GBSA free energy calculation, and the mmpbsa module in Amber12 was used to perform MM / GBSA free energy decomposition analysis to study the binding interaction between TMV-CP and inhibitors.

5. The small molecule inhibitor obtained by screening according to the method of claims 1-4, characterized in that: The small molecule inhibitor is a glycoside compound, including A0 to A6, and its structural formula is as follows:

6. Use of the small molecule inhibitor as claimed in claim 5 in preventing and controlling plant pathogenic viruses.

7. The use according to claim 6, characterized in that: The plant pathogenic viruses include, but are not limited to, tobacco mosaic virus, cucumber mosaic virus, potato virus Y and tomato spotted wilt virus.

8. A drug for preventing and treating plant pathogenic viruses, characterized in that: Including the small molecule inhibitor according to claim 5.

9. The drug according to claim 8, characterized in that The dosage form of the drug is selected from any one of emulsifiable concentrate, powder, wettable powder, granule, aqueous solution, suspension, ultra-low volume spray, soluble powder, microcapsule, smoke agent, aqueous emulsion or water-dispersible granule.

10. A method for preventing and controlling plant pathogenic viruses, characterized in that: In agricultural production, the small molecule inhibitor according to claim 5 or the drug according to claim 8 is used for prevention and treatment.

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