Application of a compound as an inhibitor of pathogenic fungus Pmk1

Abstract

The present invention discloses the use of a pyrazolopyridine compound as an inhibitor of the pathogenic microorganism Pmk1. The pyrazolopyridine compound is a compound containing a pyrazolopyridine skeleton or a pesticide-acceptable salt thereof, and its structural formula is shown in Formula I. #imgabs0# The Pmk1 is pathogenicity mitogen-activated protein kinase 1 (PMK1) from pathogenic microorganisms. Compound A93-59 provided by the present invention is a Pmk1 inhibitor with strong specificity and high activity, and exhibits excellent ability to inhibit the growth and pathogenicity of pathogenic fungi, particularly rice blast fungus and gray mold lesions.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural disease control, and particularly relates to the application of a pyrazole-pyridine compound as an inhibitor of the pathogenic fungus Pmk1. Background Art

[0002] Crop fungal diseases, such as rice blast and gray mold, pose a serious threat to global crop yields and food security. Chemical control is a crucial component in ensuring safe crop production. However, due to the long-term and excessive use of single-mode fungicides, resistance among plant pathogens is becoming increasingly serious. Discovering new targets is an effective approach to combating resistance.

[0003] Pathogenicity mitogen-activated kinase 1 (Pmk1), a member of the mitogen-activated protein kinase (MAPK) family, transmits signals from the extracellular space to the nucleus through phosphorylation reactions within the MAPK signaling pathway, thereby influencing the growth and pathogenicity of the pathogen. For example, Pmk1 is a key protein in appressorium formation in the rice blast fungus. When Pmk1 deletion mutations occur in rice blast fungus, the conidia adhere and germinate as if they were wild-type, producing normal hyphae and conidia, but they are unable to form appressoria and, therefore, lack the ability to infect and cause disease. Furthermore, Pmk1 regulates hyphal contraction and secreted fungal effector proteins. Previous studies have shown that selective inhibition of Pmk1 by chemical methods can prevent the spread of the fungus within host cells, thereby confining it to a single cell and controlling its spread and expansion. Based on the important influence of Pmk1 on the growth and pathogenicity of plant pathogenic fungi, it can be used as an ideal fungicide target for the development of pesticides for the control of plant pathogenic fungi. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel Pmk1 inhibitor, which can inhibit the activity of Pmk1 and exhibit excellent ability to inhibit the growth and pathogenicity of pathogenic fungi, especially rice blast and gray mold.

[0005] According to one aspect of an embodiment of the present invention, there is provided a use of a pyrazolopyridine compound as an inhibitor of the pathogenic microorganism Pmk1. The pyrazolopyridine compound is a compound containing a pyrazolopyridine skeleton or a pesticide-acceptable salt thereof, and its structural formula is shown in Formula I.

[0006]

[0007] The Pmk1 is pathogenic mitogen-activated protein kinase 1 (PMK1) from pathogenic microorganisms.

[0008] In some embodiments, the pathogenic microorganisms include rice blast, gray mold, and / or other pathogenic fungi having Pmk1 or a homologous protein thereof.

[0009] In some embodiments, the pathogenic microorganism is one or more selected from the group consisting of: rice blast, gray mold, pear black spot Alternaria, cabbage Alternaria, Aspergillus flavus, sesame leaf spot, wheat root rot flat navel helminthes, wheat powdery mildew, ergot, corn leaf spot fungus Helminthosporium, wheat root rot flat navel helminthes (Cochliobolus sativus), gloeosporial anthracnose, cruciferous anthracnose, cucurbit anthracnose, graminearum, oxysporum Fusarium, corn verticillium, wheat leaf blight, oxalic acid Penicillium, wheat stripe fungus, round core cavity fungus barley web spot pathogen, sclerotinia, corn large blight fungus, wheat glumen blight fungus glumen rot shell septoria, corn smut, rice false smut, dahliae, wheat leaf blight fungus.

[0010] According to another aspect of the present invention, a Pmk1 inhibitor is provided, comprising an active ingredient and an adjuvant, wherein the active ingredient is the pyrazole-pyridine skeleton-containing compound described in the first embodiment, or a pesticide-acceptable salt thereof. The inhibitor can inhibit the growth and pathogenicity of pathogenic fungi, particularly rice blast fungus and gray mold fungus.

[0011] In some embodiments, the dosage form of the inhibitor is a pharmacologically acceptable dosage form, which includes at least one of emulsifiable concentrate, wettable powder, suspension, soluble powder, aqueous solution, water-dispersible powder, smoke agent, granule and seed coating agent.

[0012] Compound A93-59 provided in the embodiments of the present invention is an inhibitor of Pmk1, has the characteristics of strong specificity and high activity, and exhibits excellent ability to inhibit the expansion, growth and pathogenicity of pathogenic fungi, especially rice blast and gray mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the results of detecting the interaction between compound A93-59 provided in the examples of the present invention and rice blast fungus Pmk1 and gray mold Pmk1 by surface plasmon resonance.

[0014] Figure 2 This is a schematic diagram of the results of detecting the effect of compound A93-59 provided in an example of the present invention on the pathogenicity of rice blast fungus through in vitro inoculation method.

[0015] Figure 3 This is a schematic diagram of the results of detecting the effect of compound A93-59 provided in the examples of the present invention on the pathogenicity of Botrytis cinerea by in vitro inoculation method. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0017] The present invention is made based on the following knowledge of the inventors:

[0018] Crop fungal diseases pose a serious threat to global crop yields and food safety. For example, the rice blast fungus (Magnaporthe oryzae, asexual form Pyricularia oryzae) can infect a variety of grass crops such as rice and wheat, causing diseases such as rice blast and wheat blast, which seriously threaten the yields of rice and wheat. In addition, gray mold (Botrytis cinerea) has a wide host range and can infect seedlings and fruits of more than 200 crops, potentially causing inestimable losses at all stages of food production. In this regard, chemical control is an important part of ensuring crop yields and food safety. However, due to the long-term and excessive application of fungicides with a single mode of action, the problem of drug resistance in plant pathogens is becoming increasingly serious. Therefore, discovering new targets is an effective way to address drug resistance.

[0019] The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the extracellular space to the nucleus through a step-by-step amplification of phosphorylation reactions. Three MAPK kinases are present in plant pathogenic fungi: Pmk1, Mps1, and Hog1 / Osm1. Among them, pathogenicity mitogen-activated protein kinase 1 (Pmk1), derived from pathogenic microorganisms, plays a crucial role in the growth and pathogenicity of pathogenic microorganisms. Previous studies have shown that selective inhibition of Pmk1 using chemical methods can prevent the spread of rice blast fungi within host cells, thereby confining them to individual cells and controlling their spread and expansion. Given its crucial influence on the growth and pathogenicity of plant pathogens, Pmk1 is an ideal fungicide target for the development of pesticides to control these fungi.

[0020] In some embodiments, the pathogenic microorganisms include rice blast, gray mold, and / or other pathogenic fungi having Pmk1 or a homologous protein thereof.

[0021] It can be understood that the pathogenic microorganisms described in the embodiments of the present disclosure are pathogenic fungi having Pmk1 or its homologous proteins, including but not limited to: Aleraria alernata, Aleraria brassicicola, Aspergillus flavus, Bipolaris oryzae, Bipolaris sorokiniana, Blumeria graminis, Claviceps purpurea, Cochliobolus heterostrophus, Cochliobolus sativus, Colletotrichum gloeosporioides, Colletotrichum higginsianum, Colletotrichum lagenarium, Fusarium graminearum, and the like. graminearum), Fusarium oxysporum, Fusarium verticillioides, Mycosphaerella graminicola, Penicillium oxalicum, Puccinia striiformis, Pyrenophora teres, Sclerotinia sclerotiorum, Setosphaeria turcica, Stagonosporanodorum, Ustilago maydis, Ustilaginoidea virens, Verticillim dahliae, and Zymoseptoria tritici. Fungi in which Pmk1 is a key protein are well known to those skilled in the art.For additional information about Pmk1 in these fungi, please refer to The phosphorylation landscape of infection-related development by the rice blast funus. Cell. 2024 May 9; 187(10): 2557-2573.e18. Cruz-Mireles et al., the contents of which are incorporated herein by reference.

[0022] According to another aspect of the present invention, a Pmk1 inhibitor is provided, comprising an active ingredient and an adjuvant, wherein the active ingredient is the pyrazole-pyridine skeleton-containing compound described in the first embodiment, or a pesticide-acceptable salt thereof. The inhibitor can inhibit the growth and pathogenicity of pathogenic fungi, particularly rice blast fungus and gray mold fungus.

[0023] In some embodiments, the dosage form of the inhibitor is a pharmacologically acceptable dosage form, which includes at least one of emulsifiable concentrate, wettable powder, suspension, soluble powder, aqueous solution, water-dispersible powder, smoke agent, granule and seed coating agent.

[0024] The detection methods used in the following examples are standard methods commonly used in the industry. For example, surface plasmon resonance (SPR) is an internationally accepted method for detecting small molecule-protein interactions, characterized by its high sensitivity and reliability. For example, the in vitro inoculation experiment based on spot-grafting of rice leaves is a standard method for testing the effects of pesticides on the pathogenicity of rice blast fungus (Agricultural Industry Standard of the People's Republic of China NY / T 3257-2018).

[0025] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0026] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0027] Example

[0028] The present invention used the rice blast fungus Pmk1 protein as bait and screened a DNA-encoded compound library (DEL) provided by WuXi AppTec, ultimately yielding compound A93-59 (purchased from WuXi AppTec). The structure of the compound is shown in Formula I. The following examples evaluated compound A93-59's affinity for Pmk1 protein and its effects on the pathogenicity of rice blast fungus and gray mold.

[0029]

[0030] Example 1

[0031] In this example, the interaction between the small molecule compound A93-59 and the Pmk1 protein was detected by surface plasmon resonance. The instrument used in the experiment was Biacore 8k+.

[0032] In the present embodiment, the Pmk1 proteins of rice blast fungus and gray mold were immobilized on the surface of a CM5 chip using amino coupling. A series of concentration gradients (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of the compound of formula I were passed over the chip surface, and the changes in the reaction signals were detected. The signals were then fitted to the steady-state model preset in the software to calculate the equilibrium dissociation constant (K D , the strength of the reaction affinity).

[0033] The results of the interaction between the compound A93-59 provided in the embodiment of the present invention and the rice blast fungus Pmk1 and the gray mold Pmk1 were detected by surface plasmon resonance. Figure 1 This example detects the K of the compound of formula I with Pmk1 of rice blast fungus and gray mold. D The concentrations of the compounds were 57.1 μM and 38.4 μM, respectively, indicating that the compound interacted with both Pmk1s, which was consistent with the DEL screening results.

[0034] Example 2

[0035] This example tested the effect of agent A93-59 on the pathogenicity of rice blast fungus using an in vitro inoculation experiment based on spot-grafting of rice leaves. The specific steps are as follows:

[0036] (1) Cut about 5 cm from the middle of the fourth leaf of a rice seedling at the four-leaf, one-heart stage, scratch it, and place it on a porcelain plate covered with wet absorbent paper. Prepare a suspension of rice blast fungus conidia and adjust its concentration to 5 × 10 4 pieces / mL.

[0037] (2) Take 5 μL of each of the experimental group (a suspension of rice blast fungi mixed with 100 μg / mL of the compound of formula I), the positive control (a suspension of rice blast fungi added with 100 μg / mL of blasticide), and the negative control (a suspension of rice blast fungi without the addition of the drug) and apply them to the scratched areas of rice leaves, evenly applying 3 drops to each leaf.

[0038] (3) Incubate at 26°C in a dark environment for 36 hours.

[0039] (4) Cultivate the leaves in (3) under light conditions for 3-4 days and count the incidence of disease.

[0040] The results are as follows Figure 2As shown in Figure A, the experimental group showed brown resistant spots, which was the same as the positive control, while the negative control showed spindle-shaped spots characteristic of rice blast, indicating that the compound of formula I can inhibit the blast fungus spores from infecting rice leaves, that is, it can inhibit the occurrence of rice blast and has a protective effect on rice plants.

[0041] Example 3

[0042] This example uses an in vitro inoculation experiment based on rice leaves to test the effect of agent A93-59 on the pathogenicity of rice blast fungus at different concentrations and application times. The specific steps are as follows:

[0043] (1) Cut about 5 cm from the middle of the fourth leaf of a rice seedling at the four-leaf, one-heart stage, scratch it, and place it on a porcelain plate covered with wet absorbent paper. Prepare a suspension of rice blast fungus conidia and adjust its concentration to 5 × 10 4 pieces / mL.

[0044] (2) Take 5 μL of the rice blast fungus conidia suspension prepared in (1) and apply it to the scratched part of the rice leaves, with 3 drops evenly applied to each leaf.

[0045] (3) At 12 h and 24 h after inoculation, 5 μL of the control (no drug added) solution, 200 μg / mL aqueous solution of the compound of formula I, and 400 μg / mL aqueous solution of the compound of formula I were taken and applied to the scratched part of the rice leaf.

[0046] (4) Incubate the leaves in (3) at 26°C in a dark environment with moisture retention for 36 hours.

[0047] (5) Cultivate the leaves in (4) under light conditions for 3-4 days and count the incidence of disease.

[0048] The results are as follows Figure 2 As shown in Figure B, compared with the control group, the spindle-shaped lesions characteristic of rice blast in the experimental group were significantly smaller than those in the control group, indicating that the compound of formula I can inhibit the infection of rice blast fungus hyphae in rice leaf tissue, that is, it can inhibit the spread and expansion of rice blast and has a therapeutic effect on rice plants.

[0049] Example 4

[0050] This example tested the effect of agent A93-59 on the pathogenicity of Botrytis cinerea through an in vitro inoculation experiment using strawberries. The specific steps are as follows:

[0051] (1) Place the "Hongyan" strawberry fruits disinfected with alcohol in an inoculation box for use.

[0052] (2) Prepare a conidia suspension of Botrytis cinerea and adjust the conidia concentration of the suspension to 1×10 5 pieces / mL.

[0053] (3) A hole (3 mm in diameter and 10 mm in depth) was punched in the middle of the strawberry fruit, and 6 μL of a spore suspension of Botrytis cinerea mixed with 100 μg / mL or 200 μg / mL of the compound of formula I (experimental group), a positive control (a spore suspension of Botrytis cinerea supplemented with 100 μg / mL boscalid), and a negative control (a spore suspension of Botrytis cinerea without the addition of the drug) were injected into the hole. Five fruits were treated for each treatment.

[0054] (4) The cells were then cultured in a dark environment at 21°C for 12 h and then switched to a 12 h / 12 ​​h alternating light / dark environment.

[0055] (5) Observe the incidence of disease every day and record the incidence of disease 5 days after vaccination.

[0056] The results are as follows Figure 3 As shown, compared with the negative control, the lesion area of ​​the experimental group was significantly reduced, which was the same as the trend of the positive control, indicating that the compound of formula I can inhibit the infection of gray mold on strawberry fruit and has a significant preventive effect on gray mold.

[0057] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0058] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0059] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A pyrazolopyridine compound as an inhibitor of the pathogenic microorganism Pmk1, wherein the pyrazolopyridine compound is a compound containing a pyrazolopyridine skeleton or a pesticide-acceptable salt thereof, and its structural formula is shown in Formula I. The Pmk1 is pathogenicity mitogen-activated kinase 1 from pathogenic microorganisms, and the pathogenic microorganisms are rice blast fungus and gray mold fungus.

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