WO2019015223A1

WO2019015223A1 - Agent against plant virus

Info

Publication number: WO2019015223A1
Application number: PCT/CN2017/114842
Authority: WO
Inventors: 朱水芳; 胡帆; 邓宇芳; 雷荣; 李桂芬; 李新实
Original assignee: 中国检验检疫科学研究院
Priority date: 2017-07-20
Filing date: 2017-12-06
Publication date: 2019-01-24
Also published as: CN109275662A

Abstract

The present invention relates to the field of agricultural chemicals. In particular, the present invention provides an agent against a plant virus, the agent comprising a small molecule agent shown in formula (I) or a pharmaceutically acceptable salt containing the chemical structure shown in formula (I), wherein R1, R2, and R3 are independently selected from one of hydrogen, nitro, nitroso, a sulfo group, carboxyl, amino or azo, a halogen atom, C1-10 alkyl, C2-10 linear alkenyl, and C2-10 linear alkynyl. The small molecule agent of the present invention shows good inhibitory effect on viral silencing suppressors. As proved by pharmacodynamic experiments, the small molecule agent of the present invention has highly effective antiviral effect.

Description

An anti-plant virus agent

Technical field

The present invention relates to the field of pesticides, and in particular to a pesticide against plant viruses.

Background technique

Plant virus disease is one of the important diseases of crops. It is called “plant cancer” and causes billions of dollars in damage to global agricultural production every year. In recent years, especially for cash crops, the risk of plant virus diseases has become increasingly serious. Such as tobacco virus disease, once the quality of the diseased tobacco leaves at least 2-3 grades, even in the case of serious loss of economic value, and because of the extremely difficult to control, the economic losses caused by it have far exceeded the tobacco fungal disease, becoming a threat to tobacco production. The biggest class of diseases.

Since the plant virus infects the plant and relies on the metabolism of the host to proliferate, it is difficult to specifically eliminate the virus without affecting the normal metabolic function of the host, so it is extremely difficult to control the viral disease. Antiviral drugs such as Ningnanmycin, S-methylbenzo[1,2,3]thiadiazole-7-thiocarboxylate (BTH, syn.acibenzolar-S-methyl), etc. It mainly works by inducing plant acquired resistance, and the effect is poor and only preventive. In fact, there are currently very few drugs that have a therapeutic effect on plant viruses.

The RNA silencing mechanism discovered in recent years is now considered to be the most important and important defense mechanism for plants against viruses. Simply put, after the virus is infected, its own nucleic acid causes the plant to produce specific degradation to the nucleic acid, thereby achieving the purpose of eliminating the virus. But similar to arms competition, the virus has also evolved a Viral RNA silencing suppressor to counteract plant RNA silencing. For example, potato Y virus, Hac-Pro encoded by the poxvirus, 2b encoded by the Cucumber mosaic virus gene, and P19 encoded by the tomato bush dwarf virus. Its mechanism of action is mainly to interfere with the entire silencing mechanism by binding to small RNAs that play an important role in RNA silencing. Therefore, if you find an agent that can effectively inhibit the virus silencing suppressor, it can help plants win the battle for arms competition and achieve the purpose of effectively treating viral diseases.

As people's concept of environmental protection becomes more and more clear, the problem of high-toxicity and high residue of traditional chemical pesticides has become more and more important. Many of the natural products have high efficiency and low toxicity, good environmental compatibility, and low toxicity to humans and the environment. Therefore, it is of great significance and value to select a highly efficient new type of antiviral pesticide with accurate molecular targets from the natural product library.

Summary of the invention

In order to solve the problems in the prior art, the object of the present invention is to provide a highly efficient novel antiviral agent having a precise molecular target according to the latest viral interaction molecular mechanism.

In order to achieve the object of the present invention, the technical solution of the present invention is as follows:

The present invention targets a virus silencing suppressor, and selects a small molecule agent (ZINC compound database, ID: ZINC72332803) which has a good inhibitory effect on the target protein from a natural product library and proves that the small molecule is confirmed by pharmacodynamic experiments. The agent has an effective antiviral effect.

On the basis of this, the present invention provides an anti-plant virus agent comprising the small molecule agent represented by the formula (I) or a pharmaceutically acceptable salt comprising the chemical structure represented by the formula (I) .

The small molecule medicament has the structure shown in formula (I):

Wherein R ₁ , R ₂ and R ₃ are each independently selected from the group consisting of hydrogen, nitro, nitroso, sulfonate, carboxyl, amino or azo, halogen atom, C1-10 alkyl, C 2-10 alkenyl or C 2 One of the -10 alkynyl groups.

In a specific embodiment of the invention, the medicament comprises a small molecule medicament of formula (II):

It is a case where R ₁ is hydrogen, R ₂ is a methyl group, and R ₃ is a methyl group based on the small molecule drug represented by the formula (I), and is named 2-(((6aR, 6bR, 12aR)). -2,2,6a,6b,9,9,12a-heptamethyl-15-oxocosahedron-1H-1,4a-(epoxymethylalkyl)methyl-10-yl)oxy)carbonyl )benzoic acid. According to the present invention, the small molecule agent represented by the formula (II) exhibits a significant inhibitory effect on the virus silencing suppressor.

Further, the present invention provides the use of a small molecule agent of the formula (I) / formula (II) or a pharmaceutically acceptable salt thereof for inhibiting a Viral RNA silencing suppressor. The viral silencing suppressor includes, but is not limited to, P19, 2b, Hc-Pro, and the like.

Since most viruses have a silencing suppressor, the target virus to which the anti-plant virus agent is directed is not particularly limited.

Optionally and preferably, such as potato virus Y containing Hc-Pro inhibitor, plum pox virus, etc.; tomato infertility virus containing 2b inhibitor, cucumber mosaic virus, etc.; carnation Italian ring spot containing P19 inhibitor Virus, tomato bush dwarf virus, etc.

The agent is also understood to be a pharmaceutical composition comprising an effective amount of a compound of formula (I) / formula (II) or a pharmaceutically acceptable salt thereof. The same applies to the prevention and treatment of diseases caused by plant viruses.

The beneficial effects of the invention are:

The present invention provides a novel use of a compound represented by the formula (I) / formula (II), and finds its novel application in inhibiting a virus silencing suppressor, and based on this, provides an anti-plant virus agent.

DRAWINGS

1 is a EMSA test for detecting the activity of a small molecule agent for inhibiting the silencing suppressor P19 in Example 1 of the present invention.

2 is a EMSA test for detecting the activity of a small molecule agent for inhibiting silencing suppressor 2b in Example 2 of the present invention.

Detailed ways

Preferred embodiments of the present invention will be described in detail below with reference to the embodiments. It is to be understood that the following examples are presented for illustrative purposes only and are not intended to limit the scope of the invention. Various modifications and alterations of the present invention can be made by those skilled in the art without departing from the spirit and scope of the invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

The small molecule agent is of the following formula:

The small molecule drug was applied to an activity detection gel electrophoresis mobility shift assay (EMSA) for inhibiting the silencing suppressor P19, and the results are shown in FIG. 1 . P19 was mixed with the small molecule agent in a binding buffer, and fully reacted at room temperature for 25 minutes. Wherein the concentration of P19 is 2 μM, and the concentration of the small molecule agent is 0.5-10 ppm. Subsequently, siRNA was added to a final concentration of 50 nM, and after thorough mixing, the reaction was continued at room temperature for 25 minutes. Then electrophoresis after film transfer Color observation results. Of the color development results, only the siRNA showed a band and was in the lower part of the figure (shown by the Unbound siRNA arrow). When the silencing suppressor protein binds to the siRNA, the siRNA electrophoresis rate is slowed down and the electrophoresis distance is shortened, so it is in the upper position of the figure (P19-siRNA complex arrow). When the compound is added and the protein is inhibited from binding to the siRNA, the siRNA is restored to the original electrophoresis rate, so the band signal enhancement is shown below the figure.

In Figure 1, lane 1 is a negative control and contains only siRNA. Lane 2 is a positive control containing siRNA and P19. Lanes 3-9 are experimental groups, both containing siRNA and P19, and containing the small molecule agent, the concentration gradient is 0.5, 1, 2, 4, 6, 8, 10 ppm. It can be seen that the negative control bands appear in the unbound siRNA position at the bottom of the figure. After the positive control was added to the repressor protein, some of the siRNA bound to the protein and was shown in the P19-siRNA complex band at the top of the figure. When the concentration of the small molecule drug was added, the band at the top of the figure gradually weakened, indicating that the small molecule agent can effectively prevent the binding reaction between the silence inhibitor protein and the siRNA. As shown in the figure, the 10 ppm concentration of the small molecule agent can well inhibit the binding reaction of P19 and siRNA at a concentration of 2 μM.

Example 2

The small molecule agent is of the following formula:

The small molecule drug was applied to the activity detection EMSA test for inhibiting the silencing suppressor 2b, and the results are shown in Fig. 2 . 2b was mixed with the small molecule drug in a binding buffer, and fully reacted at room temperature for 25 minutes. Wherein the concentration of 2b is 2 μM and the concentration of the small molecule agent is 0.5-20 ppm. Subsequently, siRNA was added to a final concentration of 50 nM, and after thorough mixing, the reaction was continued at room temperature for 25 minutes. Then, after electrophoresis, the color observation results were observed. Among the color development results, only siRNA can display bands. It is in the lower part of the figure (shown by the Unbound siRNA arrow). When the silencing suppressor protein binds to the siRNA, the siRNA electrophoresis rate is slowed down and the electrophoresis distance is shortened, so it is in the upper position of the figure (P19-siRNA complex arrow). When the compound is added and the protein is inhibited from binding to the siRNA, the siRNA is restored to the original electrophoresis rate, so the band signal enhancement is shown below the figure.

In Figure 2, lane 1 is a negative control and contains only siRNA. Lane 2 is a positive control containing siRNA and 2b. Lanes 3-9 were experimental groups, both containing siRNA and 2b, and containing the small molecule agent with a concentration gradient of 0.5, 2, 3, 4, 5, 10, 20 ppm. It can be seen that the negative control bands appear in the unbound siRNA position at the bottom of the figure. After the positive control was added to the repressor protein, some of the siRNA bound to the protein and was shown in the upper 2b-siRNA complex band of the figure. When the concentration of the small molecule drug was added, the band at the top of the figure gradually weakened, indicating that the small molecule agent can effectively prevent the binding reaction between the silence inhibitor protein and the siRNA. As shown in the figure, the small molecule agent at a concentration of 4 ppm can well inhibit the binding reaction of 2b to siRNA at a concentration of 2 μM.

Example 3

The control effect of the compound of the formula (II) of the invention on the tomato bush dwarf virus.

experimental method:

In the 4-5 leaf stage, the N. benthamiana with similar growth was fully ground with 0.3 mL of fresh diseased leaves with 30 mL of double distilled water, and 1% of the virus inoculum was inoculated by diatomaceous earth, and each plant was inoculated with 2 leaves. Two hours, one, and three days after the inoculation, the drug was sprayed with an aqueous solution of 150 ppm of the drug. There are 20 strains per treatment group.

The disease index was calculated on the 8th, 11th and 14th day after inoculation. The results are shown in Table 1.

a. Disease classification criteria:

Level 0 - asymptomatic.

Level 1 - mild symptoms in the inoculated leaves.

Level 2—One to two system blades with veins and deformation.

Grade 3 - Most of the upper leaves are deformed or the main lateral veins are necrotic, and the diseased plants are dwarfed.

Grade 4 - The whole plant is severely deformed or necrotic.

b. Disease index

Disease index = [∑ (number of leaves at each level × relative value) / (total number of leaves investigated × 9)] × 100%

c. Control effect

Control effect (%) = [(Control disease index - treatment of disease index) / control disease index] × 100%

Table 1

Example 4

The control effect of the compound of the formula (II) of the invention on cucumber mosaic virus.

In the 4-5 leaf stage, the N. benthamiana with similar growth was fully ground with 0.3 mL of fresh diseased leaves with 30 mL of double distilled water, and 1% of the virus inoculum was inoculated by diatomaceous earth, and each plant was inoculated with 2 leaves. Two hours, one, and three days after the inoculation, the drug was sprayed with an aqueous solution of 150 ppm of the drug. There are 20 strains per treatment group. The disease index was calculated at 9, 9, and 20 after inoculation. The results are shown in Table 2.

a. Disease classification criteria:

Level 0 - asymptomatic.

Level 1 - mild symptoms in the inoculated leaves.

Level 2—One to two system blades with veins and deformation.

Level 3 - Most upper leaves are sessile, sallow or deformed.

Grade 4—The leaves of the whole plant are full of leaves, severe deformation or necrosis, and the diseased plants are dwarfed.

b. Disease index

c. Control effect

Table 2

Example 5

The control effect of the compound of the formula (II) of the present invention on the phthalocyanine mosaic virus.

The virus is inoculated as a turnip mosaic virus carrying a GFP fluorescent protein, and the virus can be quantified by directly observing fluorescence. Select 4-5 leaf stage, the similarity of N. benthamiana, fully grind 0.4g fresh diseased leaves with 20mL double distilled water, 2% virus inoculum was inoculated by diatomaceous earth friction, and then rinse with water after the leaves are dry. The drug was administered 2 hours, 1 day and 3 days after the inoculation, and sprayed with an aqueous solution of 150 ppm of the drug. The number of viruses with fluorescent signals in the control and drug-treated groups was recorded on

days

3, 4, and 5 after inoculation.

14 plants per treatment group, 2-3 leaves per plant. Different leaves were taken at each recording time point and repeated 3 times. The inhibition rate of the virus was calculated by the following formula.

Y=(C-A)/C*100%

Where Y is the inhibition rate of the compound against the virus, C is the number of viruses in the control group, and A is the number of viruses in the compound treatment group. The results are shown in Table 3.

table 3

Example 6

The control effect of the compound of the formula (II) of the present invention on the poxvirus.

Choose 4-5 leaf stage, the similarity of Ninth's smoke, fully ground with 30mL double distilled water. 0.3 g of fresh diseased leaves, 1% of the virus inoculum was inoculated by diatomaceous earth rubbing, and each plant was inoculated with 2 leaves. Two hours, one, and three days after the inoculation, the drug was sprayed with an aqueous solution of 150 ppm of the drug. Fifteen strains were set for each treatment group, and the disease index was calculated on the 8th, 10th, and 12th day after inoculation.

The results are shown in Table 4.

a. Disease classification criteria:

Level 0 - asymptomatic.

Level 1 - mild symptoms in the inoculated leaves.

Level 2—One to two system blades are mottled and deformed.

Level 3 - Most of the upper leaves are chlorotic and deformed, and the diseased plants are dwarfed.

Grade 4—The leaves of the whole plant are chlorotic, severely deformed or necrotic, and the diseased plants are dwarfed.

b. Disease index

c. Control effect

Table 4

The application of the small molecule pharmaceutical compound of the present invention as an antiviral agent has been described by specific examples, and those skilled in the art can refer to the content of the present invention, appropriately change the raw materials, process conditions and the like to achieve the corresponding other purposes, and the related changes. It will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Industrial applicability

The present invention provides an anti-plant virus agent. The medicament comprises a small molecule agent of the formula (I) or a pharmaceutically acceptable salt comprising a chemical structure represented by the formula (I), wherein R ₁ , R ₂ and R ₃ are each selected from the group consisting of hydrogen and nitrate One of a nitro group, a nitroso group, a sulfonic acid group, a carboxyl group, an amino group or an azo group, a halogen atom, a C1-10 alkyl group, a C2-10 alkenyl group or a C2-10 alkynyl group. The small molecule medicament provided by the invention exhibits a good inhibitory effect on the virus silencing suppressor, has a high antiviral effect, and has good economic value and application prospect.

Claims

An agent against plant viruses, which comprises a small molecule agent represented by formula (I) or a pharmaceutically acceptable salt comprising a chemical structure represented by formula (I):

Wherein R 1 , R 2 and R 3 are each independently selected from the group consisting of hydrogen, nitro, nitroso, sulfonate, carboxyl, amino or azo, halogen atom, C1-10 alkyl, C 2-10 alkenyl or C 2 One of the -10 alkynyl groups.
The anti-plant virus agent according to claim 1, wherein the agent comprises a small molecule agent represented by formula (II):
Use of a small molecule agent of the formula (I) or a pharmaceutically acceptable salt thereof for inhibiting a viral silencing suppressor.
The use according to claim 3, wherein the viral silencing suppressor comprises, but is not limited to, P19, 2b or Hc-Pro.
The use of the small molecule agent represented by the formula (II) or a pharmaceutically acceptable salt thereof for inhibiting a virus silencing suppressor.
The use according to claim 5, wherein the viral silencing suppressor comprises, but is not limited to, P19, 2b or Hc-Pro.