Application of glycosides in prevention and treatment of plant pathogenic viruses

Abstract

The application discloses application of glycoside compounds in prevention and treatment of plant pathogenic viruses and belongs to the technical field of pesticide antiviral agents.Taking a tobacco mosaic virus coat protein as an example, small molecules including sugar rings, such as Swertiamarin I (A0) and ZINC85593196 (B0), are screened through virtual screening, molecular dynamics simulation, molecular mechanics and MM / GBSA free energy calculation. Based on this, further optimization is carried out, and six compounds similar to the structure of A0, such as 4-methylumbelliferone-beta-D-xyloside (A1), gardenoside (A2), 4-methylumbelliferone-beta-D-pyranoside (A3), 4-methylumbelliferone-beta-D-pyranoglucoside (A4), catalpol (A5) and esculin (hemihydrate) (A6), are obtained, and in-vivo and in-vitro biological activity evaluation is carried out. The active ingredients screened by the above method have good inhibitory effect on plant viruses.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticide and antiviral, in particular to the application of glycoside compounds in preventing and treating plant pathogenic viruses. BACKGROUND

[0002] Animals, plants and microorganisms such as bacteria and fungi can be infected by corresponding viruses. Viruses seriously threaten human health and 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 appear difficult-to-control viral symptoms in growth, resulting in considerable economic threat to agricultural production. Chemical control as an efficient and labor-saving control means has been widely used in pesticide production. However, some chemical agents have obvious limitations, therefore, it is urgent to develop economically viable and available chemical agents with multiple modes of action to ensure the sustainable development of medical and agricultural systems.

[0003] Traditional drug discovery process has the disadvantages of high risk, long time consumption and high cost. Computer-aided drug design plays a revolutionary role in target identification, virtual screening, molecular docking, dynamics simulation, ADME property prediction, etc., greatly accelerating the speed of drug development, reducing the cost of drug development, improving the efficiency and success rate of research and development, and providing valuable perspectives for studying the potential mechanism of receptor-ligand interaction. Therefore, multiple screening strategies based on cross-validation can effectively improve the hit accuracy. Drug design based on pathogen functional proteins or enzymes is an effective way to develop targeted pesticides. Natural products have the advantages of systematicness, unique function, high efficiency, low toxicity, environmental protection, etc. Natural molecules have been widely used in drug development and disease prevention for a long time due to their abundant resources and excellent performance. Therefore, the development of chemical type of antiviral agents based on glycosyl natural products is a valuable and fruitful way.

[0004] TMV coat protein consists of 158 amino acids (17.5KDa), is a subgenomic RNA translated structural and functional protein, which can self-assemble with TMV RNA to form a complete mature virus. TMV coat protein plays a crucial role in protecting nucleic acids from external factors, assisting TMV long-distance transport, maintaining its infectivity, and participating in nucleic acid replication. These indispensable biological functions make TMV coat protein a promising pesticide target. In order to efficiently and accurately mine potential TMV coat protein inhibitors and their resistance to viral physiological functions, the sugar-modified natural molecule is used as the ligand library, the TMV coat protein is used as the receptor target, and the best picroliv I (A0) structure is selected as the reference. Further screening of 6 compounds similar to picroliv I (A0) structure, 4-methyl umbelliferone-β-D-xyloside (A1), gardenoside (A2), 4-methyl umbelliferone acyl-β-D-pyranoside (A3), 4-methyl umbelliferone acyl-β-D-pyranoglucuronoside (A4), catalpol (A5), esculin (hemihydrate) (A6). The anti-TMV biological activity of these compounds is further confirmed by microscale thermophoresis (MST), quantitative reverse transcription polymerase chain reaction (qRT-PCR) detection, and in vivo verification. SUMMARY

[0005] The purpose of the present application is to provide the application of glycoside compounds in the prevention and treatment of plant pathogenic viruses, in order to solve the problems existing in the prior art. The present application takes tobacco mosaic virus as an example to specifically describe the screening of small molecule inhibitors targeting TMV-CP.

[0006] In order to achieve the above purpose, the present application provides the following scheme:

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

[0008] (1) pretreatment of TMV coat protein structure;

[0009] (2) download the original virtual natural molecule from ZINC database, select the compound with molecular weight of 350-500 as the screening library; introduce the screening library into Discovery Studio through intelligent format sugar ring to obtain natural product ligand screening library containing sugar ring;

[0010] (3) based on TMV coat protein, virtual screening is carried out by Discovery Studio, AutodockVina, Sybyl and Schrodinger, and the top 100 ligands with the highest affinity score are selected;

[0011] (4) using the Venn diagram for co-ligand presentation, screening out the lead compounds with strong TMV-CP binding ability, and performing similarity retrieval based on the lead compounds to obtain molecules with similar structures to the lead compounds, thereby obtaining secondary lead compounds;

[0012] (5) further screening the lead compounds and secondary lead compounds through molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis to obtain the small molecule inhibitors.

[0013] In the second technical scheme of the present application, the small molecule inhibitors screened by the method are glycoside compounds, including A0-A6, and the structural formulae are as follows:

[0014]

[0015] In the third technical scheme of the present application, the small molecule inhibitors are applied to the prevention and treatment of plant pathogenic viruses.

[0016] In the fourth technical scheme of the present application, a medicine for preventing and treating plant pathogenic viruses comprises the small molecule inhibitors.

[0017] In the fifth technical scheme of the present application, a method for preventing and treating plant pathogenic viruses uses the small molecule inhibitors or the medicine for prevention and treatment in agricultural production.

[0018] Based on the above technical scheme, the present application has the following technical effects:

[0019] 1. The number of screened compounds is reduced, the research and development cycle is shortened, and the cost is saved through virtual screening.

[0020] 2. It is found through experiments that the virtual screening hit compounds have obvious TMV-CP activity and can be used for preventing and treating agricultural viruses.

[0021] 3. The virtual screening hit compound skeleton is novel and can be used for further development of new TMV-CP inhibitor type antiviral agents. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical schemes in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0023] Figure 1 TMV-CP inhibitor virtual screening and experimental evaluation process based on molecular docking.

[0024] Figure 2 RMSD plots for A0-capsid, B0-capsid and NNM-capsid complex.

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

[0026] wherein A is the RMSF plot for A0-capsid, B0-capsid and NNM-capsid and B is the MM-GBSA plot for A0-capsid, B0-capsid and NNM-capsid.

[0027] Figure 4 MST experiment plots. Wherein A is the schematic of the microcalorimetric

[0028] Figure 5 Similarity search plot with picroside I (A0) as template. DETAILED DESCRIPTION

[0029] Various illustrative embodiments of the present application are now described in detail. Such description, however, should not be taken to limit the scope of the present application, but rather, should be taken as merely illustrative of certain aspects of the present application. The description herein-including, but not limited to, the background section, the summary section, and the abstract section-is to be taken as illustrative only.

[0030] It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to limit the present application. Additionally, with respect to numerical ranges, it is to be understood that each intervening value, to the upper and lower limits of the ranges stated herein, is also specifically disclosed. Each smaller range between any stated value or intervening value in the stated ranges and any other stated or intervening value in the stated ranges is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited. In case of conflict, the contents of the present specification will control.

[0032] Many modifications and variations of the described implementations of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other implementations will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0033] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0034] Unless otherwise specified, the technical solutions of the present application are conventional solutions in the art, and the reagents or raw materials used are purchased from commercial channels or are already disclosed.

[0035] The embodiment of the present application provides a method for screening a small molecule inhibitor targeting TMV-CP, comprising the following steps:

[0036] (1) pretreating the TMV coat protein structure;

[0037] (2) downloading original virtual natural molecules from the ZINC database, selecting compounds with a molecular weight of 350-500 as a screening library; introducing the screening library into Discovery Studio through intelligent format sugar rings to obtain a natural product ligand screening library containing sugar rings;

[0038] (3) based on the TMV coat protein, performing virtual screening by Discovery Studio, AutodockVina, Sybyl and Schrodinger, and selecting the top 100 ligands with the highest affinity score;

[0039] (4) performing co-ligand presentation using a Venn diagram, preliminarily screening a lead compound with strong binding capacity to TMV-CP, and performing similarity search based on the lead compound to obtain molecules with similar structures to the lead compound, and obtaining secondary lead compounds;

[0040] (5) further screening the lead compound and the secondary lead compound 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 pretreating the TMV coat protein structure is: removing all crystallization water and non-polar hydrogen atoms in the Autodock tool, determining the TMV coat 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 18x22x20 xyz points.

[0043] In some specific embodiments, the smart format sugar 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 compounds and secondary lead compounds by molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis to obtain the small molecule inhibitors includes:

[0045] Optimizing the docking predicted binding mode of the screened compounds, simulating each receptor-ligand complex to further evaluate the binding stability of the ligand-receptor complex;

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

[0047] Calculating the binding free energy of the receptor-ligand complex by MM / GBSA free energy calculation, and performing MM / GBSA free energy decomposition analysis by using the mmpbsa module in Amber12 to study the detailed binding between TMV-CP and the inhibitor.

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

[0049] The embodiments of the present application also provide the small molecule inhibitors screened by the method, and the small molecule inhibitors are glycoside compounds, including A0-A6, and the structural formulae are as follows:

[0050]

[0051] The embodiments of the present application also provide the application of the small molecule inhibitors in preventing and treating 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] The embodiments of the present application also provide a medicine for preventing and treating plant pathogenic viruses, and the medicine comprises the small molecule inhibitors.

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

[0055] The embodiments of the present application also provide a method for preventing and treating plant pathogenic viruses, wherein the small molecule inhibitor or the drug is used for prevention and treatment in agricultural production.

[0056] The design concept of the present application is as follows: first, the compounds in the natural product screening library are filtered by the intelligent format sugar ring to obtain a natural product sugar ring molecular library containing sugar rings, and the TMV-CP is used as the target protein to perform virtual screening by using a molecular docking program, and the molecules with higher scores are selected, considering the drug availability and cost, the secondary lead compounds are quickly obtained by molecular similarity search, and the TMVCP preparation lead compounds with antiviral activity are screened out by biological activity determination and verification, that is, the present application provides a rapid screening method for antiviral lead compounds.

[0057] Embodiment 1

[0058] Virtual screening based on molecular docking: the mining and identification of active natural precursors is a time-consuming and complex engineering. The intelligent screening strategy combined with biological activity evaluation is an intelligent and time-saving method, which can provide clues for discovering treatments for difficult-to-cure plant pathogenic diseases.

[0059] 1. Experimental method:

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

[0061] ② Before molecular docking, pretreat the TMV coat protein, remove all crystal water and non-polar hydrogen atoms in the Autodock tool (1.5.7);

[0062] ③ Then, determine the TMV coat protein potential active site coordination in the box (x = 0.363, y = -25.381, z = 4.010), and the grid spacing is set to (grid size: 18x22x20xyz points);

[0063] ④ Download the original virtual natural molecules from ZINC database (https: / / zinc15.docking.org / substances / subsets / natural-products / ), and there are 80617 kinds of natural products as the screening library;

[0064] V. The natural product ligand screening library containing sugar ring was screened by intelligent format sugar ring ((#8)1-(#6)(-(#6)[#8])—(#6)(-[#8])(#6)(-[#8])(#6)(-[#8])(#6)1-[#8]-[#6] into Discovery Studio (2019) for filtering.

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

[0066] Example 2

[0067] MD simulation and MM / GBSA free energy calculation.

[0068] 1. Experimental method:

[0069] ① MD simulation was performed using GROMACS software (2020), and CHARMM36 force field and TIP3P water model recommended by the tutorial were used 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 coat protein topology;

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

[0072]

[0073] ③ The complex was appropriately placed in a simulation box with a distance of 1 nm from the box edge, and 4 sodium ions were given to the system to neutralize the system charge;

[0074] ④ The system was minimized by the steepest descent method, and was balanced using a combination of NVT and NPT (100 ps, 50,000 steps);

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

[0076] The results show that the average RMSD values of picroside I (A0), B0 and NNM are 0.16 ± 0.02 nm, 0.19 ± 0.03 nm and 0.16 ± 0.06 nm, respectively, and the average RMSD values of the receptors are 0.63 ± 0.10 nm, 0.89 ± 0.32 nm and 0.66 ± 0.08 nm, respectively. It is shown that picroside I (A0) is closely combined with the ligand. The energy values of A0-capsid, B0-capsid and NNM-capsid complexes are -29.0, -23.6 and -10.3 kcal / mol, respectively. This result shows that the non-covalent binding state of picroside I (A0) and the capsid protein has a lower binding energy under the simulated environment, which is more conducive to binding.

[0077] Example 3

[0078] Microscale thermophoresis (MST): MST is a common means for detecting the dissociation process between biomolecules and obtaining the interaction mode and kinetic constant, and is used to determine the binding force between TMV capsid protein and natural molecules.

[0079] 1. Experimental method:

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

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

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

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

[0084] The results showed that the molecules exhibited significantly different binding abilities (from 0.14 μΜ to 166.89 μΜ), indicating that the non-sugar ring region of the compounds also had considerable interaction with the capsid protein. Among them, the optimal picroside I (A0) had the strongest binding ability with the capsid protein (Kd=0.14 μΜ), which was significantly higher than that of the antiviral agent NNM (Kd=3.47 μΜ) by 24.8 times. At the same time, the isomers 4-methyl umbelliferyl-β-D-glucopyranoside (A3) (Kd=5.68 μΜ) and 4-methyl umbelliferyl-β-D-glucopyranoside (A4) (Kd=1.43 μΜ) also exhibited similar binding force, and 4-methyl umbelliferyl-β-D-xyloside (A1) and jatrorrhizine (A2) had weaker binding force than A3 and A4, with Kd values of 76.92 μΜ and 26.16 μΜ, respectively. The binding of catalpol (A5) (Kd=166.89 μΜ) and esculin (A6) (Kd>200 μΜ) with the TMV capsid protein was 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 method:

[0090] ①TMV was inoculated on tobacco (Nicotiana tabacum K326cv) leaves. The inoculated samples were placed in a greenhouse at a temperature of 25°C for culture.

[0091] ②After 6 hours, samples were punched (1 cm in diameter) on K326 leaves containing TMV, and the leaf samples were placed in culture dishes with different drug concentrations. The blank group was DMSO with a concentration of 25, 50, 100, and 200 μg / mL, the positive control group was ningnanmycin and ribavirin with a concentration of 25, 50, 100, and 200 μg / mL, respectively, and the experimental group was A0 and A4 with a concentration of 25, 50, 100, and 200 μg / mL, with each sample concentration repeated three times.

[0092] ③After 48 hours, the sample genes were extracted with 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 inhibition of the relative expression of the TMV capsid protein gene by the 2-ΔΔCt method.

[0094] The results show that the down-regulation of the relative expression of the coat gene of tobacco by picroside I (A0) is the best (16%) after the infected leaves are cultured in the drug solution for 48 hours. It is about 2 times more than that of the commercial NNM (32%). Although compound 4-methylumbelliferyl-B-D-glucuronide (A4) has a good binding force with the coat, its anti-coat gene expression is similar to that of NNM. The compounds are prepared in different concentrations (25, 50, 100, 200 μg / mL) to further identify their inhibitory effects on the coat gene at the transcription level. Picroside I (A0) shows a better ability to reduce the expression of the coat gene in a dose-dependent inhibition manner. Meanwhile, in the range of 50-25 μg / mL, there is a significant difference between the 4-methylumbelliferyl-B-D-glucuronide (A4) group and the NNM group. When the concentration of picroside I (A0) is increased from 100 μg / mL to 200 μg / mL, the improvement of its anti-coat gene effect is limited (from 13% to 9%), indicating that high concentration of picroside I (A0) can hardly completely block the replication of the coat gene.

[0095] Example 5

[0096] Application of small molecule inhibitors against pathogenic viruses, and a method for testing against tobacco mosaic virus.

[0097] The anti-plant virus activity of the compounds is determined by the half-leaf spot method. 3 mg of the test compound is accurately weighed in a weighing bottle, and 60 μL of solvent DMSO is added to dissolve it thoroughly.

[0098] ①A0-A6 are prepared into 500 mg / L compound solutions with double-distilled water containing 1% Tween 20. Another 250 μL of 2% anisomycin aqueous solution is taken, 60 μL of solvent DMSO is added, 10 mL of double-distilled water containing 1% Tween 20 is added, and a 500 mg / L anisomycin solution is prepared.

[0099] ②Therapeutic activity of the agents against TMV infection in vivo. The heart leaves of tobacco with consistent growth are selected, and the virus solution (concentration of 6 x 10 -3 mg / mL) is first taken with a pipette, and is inoculated on the leaves rubbed with diamond sand along the vein direction of the whole leaves. The inoculation strength of the left and right leaves is kept consistent as far as possible, and the leaves are supported with a flat board below. After the virus solution is dried, the diamond sand on the leaves is washed away with running water. After the leaves are dried, the agents are applied on the left half leaves, and sterile water is applied on the right half leaves as a control. Each agent treatment is provided with 3 plants, and each plant has 3-4 leaves. Subsequently, the plants are placed in an illumination incubator for moisture culture, and the temperature is controlled at 23°C, and the illumination is controlled at 10000 LuX. After 2-4 days, the number of dry 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 protection activity of the agent to the living body infected by TMV. The heart leaves of the same growth trend were selected, and the agent was applied to the left half leaf with a brush, and sterilized water was applied to the right half leaf as a control. After 24 hours, the virus was inoculated. Virus juice (concentration of 6 x 10 -3 mg / mL) was taken with a brush, and was inoculated on the leaf surface (whole leaf) along the branch vein direction by artificial rubbing on the leaf blade with emery powder. The inoculation strength of the left and right leaf blades was kept consistent. The emery powder on the leaf blade was washed away with running water after the virus liquid was dried. Three plants were set for each agent treatment, and 3-4 leaves were set for each plant. Then, the plants were placed in a light incubator for moisture culture, and the temperature was controlled at 23℃, and the light intensity was 10000 Lux. After 2-4 days, the number of necrotic spots was observed and recorded. Each agent was repeated for 3 times according to the above method, and the inhibition rate was calculated.

[0101] The inhibition rate Y (%) of the compound to the tobacco mosaic virus is (R-L) / R x 100%;

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

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

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

[0105]

[0106] Table 2: Activity test of small molecule inhibitors to plant pathogenic virus potato virus Y (PVY)

[0107]

[0108]

[0109] Table 3: Activity test of small molecule inhibitors to plant pathogenic virus tomato spotted wilt virus (TSWV)

[0110]

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

[0112]

[0113] From Table 1-4, it can be seen that the compounds have good antiviral activity against tobacco mosaic virus at a concentration of 500 μg / mL. For therapeutic activity, A0, A4 have good antiviral activity, with values of 54.4%, 53.3% respectively, which are better than the control drug ningnanmycin (50.9%). For protective activity, compounds A0-A6 are lower than ningnanmycin (56.3%). The compounds have good antiviral activity against potato Y virus at a concentration of 500 μg / mL. For therapeutic activity, A0, A4 have good antiviral activity, with values of 55.3%, 52.3% respectively, which are better than the control drug ningnanmycin (51.0%). For protective activity, compound A0 has an inhibition rate of 54.8%, which is better than the control drug ningnanmycin (53.3%). The compounds have good antiviral activity against tomato spotted wilt virus at a concentration of 500 μg / mL. For therapeutic activity, A0, A4 have good antiviral activity, with values of 60.1%, 54.3% respectively, which are better than the control drug ningnanmycin (50.6%). For protective activity, compounds A0-A6 are lower than ningnanmycin (52.0%). The compounds have good antiviral activity against cucumber mosaic virus at a concentration of 500 μg / mL. For therapeutic activity, A0, A4 have good antiviral activity, with values of 58.7% and 58.3% respectively, which are better than the control drug ningnanmycin (55.4%). For protective activity, compounds A0-A6 are lower than ningnanmycin (62.3%).

[0114] Example 6

[0115] EC 50 The median effective concentration (EC) is an important indicator for evaluating the effect of a compound on a plant pathogenic disease, and is also an important parameter for setting the concentration of a target compound when studying the mechanism of action of the compound. In the concentration gradient experiment, five appropriate concentrations were set using the double dilution method, and finally the inhibition rate of the agent on the plant pathogenic virus and the concentration of the agent were converted into logarithmic values, and the toxicity curve was obtained by regression analysis using SPSS software, and the EC 50 was calculated. The therapeutic activity of the compound against plant viruses was determined by the half-leaf spot method.

[0116] I. Experimental method

[0117] ①, respectively, 5 mg of test compound (A0-A6) was weighed into a weighing bottle, 100 μL of solvent DMSO was added to dissolve it thoroughly.

[0118] ②, respectively, 500, 400, 300, 200, 100 mg / L of compound solution was prepared with double distilled water containing 1% Tween 20.

[0119] ③Preparation of 500 mg / L ningnanmycin solution as a positive control.

[0120] ④Select heart leaf tobacco with consistent growth, first use the row comparison to dip the virus solution (concentration of 6 x 10 -3 mg / mL), and inoculate on the leaf blade with sandblasting along the branch vein direction of the leaf surface (whole leaf), and the inoculation force of the left and right leaf blades is kept consistent. After the virus solution is dry, wash off the sandblasting on the leaf blade with running water.

[0121] ⑤After the leaf blade is dry, apply the agent on the left half leaf, and apply sterilized water on the right half leaf as a control. Each agent treatment is set with 3 plants, and each plant has 3-4 leaves. Then, place the plants in a light incubator for moisture culture, control the temperature at 23℃, and the light at 10000 LuX. After 2-4 days, observe and record the number of dry spots. According to the above method, repeat each agent 3 times, and calculate the inhibition rate.

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

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

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

[0125] Table 5 EC 50 test

[0126]

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

[0128] In summary, the present application takes tobacco mosaic virus coat protein as an example, through virtual screening, molecular dynamics (MD) simulation, molecular mechanics and MM / GBSA free energy calculation, a small molecule of rhizoma picrorhizae glycoside I (A0) containing sugar ring is screened. Based on this, further optimization is carried out, 6 compounds similar to A0 structure are obtained, 4-methyl umbelliferone-β-D-xyloside (A1), gardenoside (A2), 4-methyl umbelliferone acyl-β-D-pyranoside (A3), 4-methyl umbelliferone acyl-β-D-pyranoglucoside (A4), catalpol (A5), esculin (hemihydrate) (A6), and in vitro and in vivo biological activity evaluation is carried out, the active ingredients screened by the above method have inhibitory effect on plant viruses such as cucumber mosaic virus, tomato spotted wilt virus and potato Y virus, and especially have good inhibitory effect on tobacco mosaic virus.

[0129] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For ordinary skilled users in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method of screening for a small molecule inhibitor targeting TMV-CP, characterized in that, It comprises the following steps: (1) preprocessing the structure of TMV coat protein; (2) downloading original virtual natural molecules from ZINC database, selecting compounds with molecular weight of 350-500 as screening library; introducing the screening library into Discovery Studio for filtering through intelligent format sugar ring to obtain natural product ligand screening library containing sugar ring; (3) based on TMV coat protein, performing virtual screening by Discovery Studio, Autodock Vina, Sybyl and Schrodinger, and selecting the top 100 ligands with the highest affinity score; (4) performing co-ligand presentation by Venn diagram, preliminarily screening out lead compounds with strong binding capacity to TMV-CP, and performing similarity search based on the lead compounds to obtain molecules with similar structure to the lead compounds, and obtaining secondary lead compounds; (5) further screening the lead compounds and secondary lead compounds by molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis to obtain the small molecule inhibitors; The further screening of the lead compounds and secondary lead compounds by molecular dynamics simulation, MM / GBSA free energy calculation and experimental analysis to obtain the small molecule inhibitors comprises: optimizing the docking prediction binding mode of the screened compounds, simulating each receptor-ligand complex to further evaluate the binding stability of the ligand-receptor complex; calculating the root mean square fluctuation of each residue to further evaluate the binding stability of the ligand-receptor complex; calculating the binding free energy of the receptor-ligand complex by MM / GBSA free energy calculation, and performing MM / GBSA free energy decomposition analysis by using the mmpbsa module in Amber 12 to study the binding between TMV-CP and the inhibitors.

2. The method of claim 1, wherein, The method for preprocessing the structure of TMV coat protein is: removing all crystallization water and non-polar hydrogen atoms in Autodock tool, determining the TMV coat protein potential active site coordination in the box (x = 0.363, y = -25.381, z = 4.010), and setting the grid spacing to 0.375 Å.

3. The method of claim 2, wherein, The grid size is 18 × 22 × 20 xyzpoints.

4. The small molecule inhibitor obtained by the method of claims 1-3, wherein, The small molecule inhibitors are glycoside compounds, including A0-A6, and the structural formulae are as follows: 。 5. The application of the small molecule inhibitors in claim 4 in preventing and treating plant pathogenic viruses.

6. Use according to claim 5, characterized in that, The plant pathogenic viruses include but are not limited to tobacco mosaic virus, cucumber mosaic virus, potato Y virus and tomato spotted wilt virus.

7. A medicine for controlling plant pathogenic viruses, characterized by comprising the compound of claim 1 or 2. The small molecule inhibitors in claim 4.

8. The medicament according to claim 7, 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, water emulsion or water dispersible granule.

9. A method for controlling a plant pathogenic virus, characterized by, In agricultural production, the small molecule inhibitors in claim 4 or the drug in claim 7 are used for prevention and treatment.

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