Application of pyridopyrimidine and / or pyrrolopyridine compound as combined inhibitor of pathogenic fungi Mps1
By using pyridopyrimidines and/or pyrrolopyridine compounds as Mps1 inhibitors, the problem of difficult inhibition of the pathogenicity of Mps1 protein in Blastobacteria was solved, and effective inhibition of the infectious structure of Blastobacteria was achieved.
Patent Information
- Application Number
- CN202411663919.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art is difficult to effectively inhibit the Mps1 protein in blastobacteria, making it difficult to control its pathogenic ability to infect plant cell walls.
Pyridopyrimidines and/or pyrrolopyridine compounds are used as inhibitors of Mps1 to inhibit their pathogenicity by binding to specific sites of Mps1.
It significantly inhibits the ability of the infectious structure (attached cells) of blast bacteria to penetrate the cell wall of the plant, and improves the inhibitory effect on pathogenic fungi.
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Figure CN119563639A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of agricultural disease control, and in particular to an application of a pyridopyrimidine and / or pyrrolopyridine compound as an inhibitor of the pathogenic microorganism Mps1. Background Art
[0002] Rice is one of the world's major food crops, and its yield is threatened by the rice blast disease caused by Magnaporthe oryzae. In addition to rice, the rice blast fungus can also infect a variety of grasses such as wheat and barley, causing economic losses. Chemical control is an important strategy for plant disease management, but a single fungicide makes this method threatened by the emergence of pathogen resistance, so new fungicides need to be continuously developed based on their mechanism of action.
[0003] The infection mechanism of rice blast fungus in plants is that its conidia attach to the hydrophobic surface of the plant, germinate and develop into appressorium, and then the appressorium accumulates enough turgor pressure to form an infection spike to penetrate the host cell wall. In rice blast fungus, there are three mitogen-activated protein kinase (MAPK) signaling pathways: cell wall integrity (CWI) pathway, invasive growth (IG) pathway, and high osmotic growth (HOG) pathway. Mitogen-activated protein kinase 1 (Mps1) is a MAPK of the CWI pathway of rice blast fungus. Mps1 is indispensable for the development of appressorium to successfully penetrate the host cell wall and fungal pathogenicity, and has been shown to be a potential molecular target for fungicide research.
[0004] Therefore, there is an urgent need for a fungicide targeting the protein Mps1 that plays an important role in the development of rice blast appressorium and penetration of the host cell wall, and its application as an inhibitor of pathogenic fungus Mps1. Summary of the invention
[0005] The purpose of the present invention is to provide a novel Mps1 inhibitor, which can inhibit the pathogenicity of Mps1 and exhibits excellent ability to inhibit pathogenic fungi, especially the infection structure (appressorium) of pathogenic fungi from penetrating plant cell walls to cause disease.
[0006] According to one aspect of an embodiment of the present invention, there is provided a use of a pyridopyrimidine and / or pyrrolopyridine compound as an inhibitor of the pathogenic microorganism Mps1, wherein the pyridopyrimidine compound is a compound containing a pyridopyrimidine skeleton or a pesticide-acceptable salt thereof, and the structural formula thereof is shown in Formula I; and the pyrrolopyridine compound is a compound containing a pyrrolopyridine skeleton or a pesticide-acceptable salt thereof, and the structural formula thereof is shown in Formula II,
[0007]
[0008] The Mps1 is a mitogen-activated protein kinase 1 from pathogenic microorganisms.
[0009] In some embodiments, the pathogenic microorganisms include Magnaporthe grisea, Fusarium oxysporum and / or other pathogenic fungi having Mps1 or its homologous proteins.
[0010] In some embodiments, the pathogenic microorganism is one or more selected from the group consisting of: rice blast, gray mold, Alternaria alternata, corn smut, Colletotrichum gloeosporioides, Colletotrichum cucurbitae, Fusarium graminearum, Fusarium oxysporum, wheat leaf blight and Sclerotinia sclerotiorum.
[0011] In some embodiments, the pyridopyrimidine compound and the pyrrolopyridine compound are administered in combination.
[0012] According to another aspect of an embodiment of the present invention, an inhibitor of Mps1 is provided, comprising an active ingredient and an adjuvant, wherein the active ingredient is a pyridopyrimidine and / or pyrrolopyridine skeleton-containing compound according to the first aspect of the embodiment of the present invention or a pesticide-acceptable salt thereof.
[0013] 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. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a schematic diagram of the results of detecting the interaction between the compounds PLX-4720 and TAK-733 provided in the embodiments of the present invention and the rice blast fungus Mps1 respectively by surface plasmon resonance method.
[0015] Figure 2 Schematic diagram of the identification results of the interaction sites between compounds PLX-4720, TAK-733 and Mps1 through in vitro experimental methods.
[0016] Figure 3 This is a schematic diagram of the results of detecting the effects of the compounds PLX-4720, TAK-733 and their combined use provided in the embodiments of the present invention on the pathogenicity of rice blast fungus by in vitro inoculation method. DETAILED DESCRIPTION
[0017] 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.
[0018] The present invention is made based on the following knowledge of the inventors:
[0019] Crops are threatened by various fungal pathogens throughout their life cycle. Chemical control is an important strategy for plant disease management, but a single fungicide makes this approach vulnerable to the emerging resistance of pathogens, so new fungicides need to be continuously developed based on their mechanism of action.
[0020] Rice is one of the world's major food crops, and its yield is threatened by the rice blast disease caused by Magnaporthe oryzae. In addition to rice, the fungus also infects a variety of grasses, such as wheat and barley. The infection cycle of rice blast in plants begins with its conidia attaching to the hydrophobic surface of the plant, germinating and developing into appressorium (a specialized cell), which then accumulates sufficient turgor pressure to form an infection spike that penetrates the host cell wall. Therefore, proteins that play an important role in the development of appressorium of rice blast and penetration of the host cell wall may become targets for fungicides.
[0021] Kinases regulate signal transduction in eukaryotic cells by phosphorylating substrates. Kinases have common structural features, including the N-terminal domain (N-lobe), the C-terminal domain (C-lobe), and the active site between the two. The MAPK cascade reaction consists of the sequentially activated MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK. According to the resolved complex structure of MAPKK-MAPK (p38α-MKK6) (PDB number 8A8M), MAPKK interacts with the CD site (common docking site), lipid binding site (lipid binding site) and N-lobe site of MAPK, binds and phosphorylates to activate MAPK. Therefore, molecular docking simulations can be performed on the above sites.
[0022] In Magnaporthe oryzae, there are three MAPK signaling pathways: the cell wall integrity (CWI) pathway, the invasive growth (IG) pathway, and the high osmotic growth (HOG) pathway. Mitogen-activated protein kinase 1 (Mps1) is a MAPK of the CWI pathway of Magnaporthe oryzae. Mps1 is essential for appressorium development to successfully penetrate the host cell wall and fungal pathogenicity, and has been shown to be a potential molecular target for fungicide research.
[0023] Therefore, there is an urgent need for a fungicide targeting the protein Mps1 that plays an important role in the development of rice blast appressorium and penetration of the host cell wall, and its application as an inhibitor of pathogenic fungus Mps1.
[0024] The embodiments of the present invention provide a novel Mps1 inhibitor, which can inhibit the pathogenicity of Mps1 and exhibits excellent ability to inhibit pathogenic fungi, especially the infection structure (appressorium) of pathogenic fungi (Pyricularia oryzae) from penetrating plant cell walls to cause disease.
[0025] According to one aspect of an embodiment of the present invention, there is provided a use of a pyridopyrimidine and / or pyrrolopyridine compound as an inhibitor of the pathogenic microorganism Mps1, wherein the pyridopyrimidine compound is a compound containing a pyridopyrimidine skeleton or a pesticide-acceptable salt thereof, and the structural formula thereof is shown in Formula I; and the pyrrolopyridine compound is a compound containing a pyrrolopyridine skeleton or a pesticide-acceptable salt thereof, and the structural formula thereof is shown in Formula II,
[0026]
[0027] The Mps1 is a mitogen-activated protein kinase 1 from pathogenic microorganisms.
[0028] In some embodiments, the pathogenic microorganisms include Magnaporthe grisea, Fusarium oxysporum, and / or other pathogenic fungi having Mps1 or its homologous proteins.
[0029] In some embodiments, the pathogenic microorganism is one or more selected from the group consisting of: rice blast, gray mold, Alternaria alternata, corn smut, Colletotrichum gloeosporioides, Colletotrichum cucurbitae, Fusarium graminearum, Fusarium oxysporum, wheat leaf blight and Sclerotinia sclerotiorum.
[0030] In some embodiments, the pyridopyrimidine compound and the pyrrolopyridine compound are administered in combination.
[0031] According to another aspect of an embodiment of the present invention, an inhibitor of Mps1 is provided, comprising an active ingredient and an adjuvant, wherein the active ingredient is a pyridopyrimidine and / or pyrrolopyridine skeleton-containing compound according to the first aspect of the embodiment of the present invention or a pesticide-acceptable salt thereof.
[0032] 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.
[0033] The detection methods used in the following examples are standard methods commonly used in the industry. For example, the surface plasmon resonance method has the characteristics of high sensitivity and good reliability, and is an internationally used method for detecting the interaction between small molecules and proteins. For example, the in vitro inoculation experiment based on rice leaf spot grafting is a standard method for detecting the effect of agents on the pathogenicity of rice blast fungus (Agricultural Industry Standard of the People's Republic of China NY / T 3257-2018).
[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0035] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.
[0036] Example
[0037] The present invention uses the rice blast fungus Mps1 protein as bait, and finally obtains the hit compounds TAK-733 and PLX-4720 (purchased from Shelleckchem) by screening the MAPK inhibitor compound library (item number L3400-01) provided by Shelleckchem. The structures of the above compounds are shown in the following formula I and formula II. In the following examples, the affinity of the two compounds to the Mps1 protein, the binding sites on Mps1, and the effects of the two compounds on the pathogenicity of the rice blast fungus when applied alone or in combination are evaluated.
[0038]
[0039] Example 1
[0040] In this example, the interaction between small molecule compounds TAK-733 and PLX-4720 and Mps1 protein was detected by surface plasmon resonance, and the instrument used in the experiment was Biacore 8k+.
[0041] In the present embodiment, the Mps1 protein of rice blast fungus was fixed on the surface of CM5 chip by amino coupling method. A series of concentration gradients (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of compounds of formula I and formula II were passed through the chip surface, the change of reaction signal was detected, and then fitted to the steady-state model preset by the software to calculate the equilibrium dissociation constant (K D , the strength of the reaction affinity).
[0042] Figure 1 The results of the surface plasmon resonance method for detecting the interaction between the compounds PLX-4720 and TAK-733 provided in the embodiments of the present invention and Mps1 of the rice blast fungus are shown in FIG. Figure 1 As shown, this example detected the K of the compounds of formula I and formula II with the rice blast fungus Mps1. D The concentrations of the two compounds were 15.1 μM and 33.5 μM, respectively, indicating that both compounds interacted with Mps1.
[0043] Example 2
[0044] In this example, the interaction sites between the small molecule compounds PLX-4720 and TAK-733 and Mps1 were identified by testing the effects of the above two compound molecules on the Mps1 enzyme activity and affinity testing of the Mps1 point mutants.
[0045] 2.1 Testing the effects of compounds on enzyme activity
[0046] The effects of small molecule compounds PLX-4720 and TAK-733 on Mps1 enzyme activity were tested using the ADP-Glo kinase assay kit (Promega, catalog number V6930). The test substrate used was a peptide of the Mps1 downstream protein Swi6, the sequence of which was EGRYRHLVALAT (SEQ ID NO: 1), synthesized by Solebao. Each test sample contained 40mMTris-HCl, 0.2mg / mL Mps1 protein, 0.4mM substrate, 0.1mM ATP, 20mM MgCl2, different concentrations of compounds (0–100μM), 2% DMSO, and 0.1mg / mL bovine serum albumin. The samples were processed according to the instructions of the kit and finally read using a microplate reader (Tecan, model Infinite F200).
[0047] Figure 2 Schematic diagram of the identification results of the interaction sites between compounds PLX-4720, TAK-733 and Mps1 through in vitro experimental methods. Figure 2 Part A shows the effects of different concentrations of compounds PLX-4720 and TAK-733 on the enzyme activity of Mps1. PLX-4720 has an inhibitory effect on the enzyme activity of Mps1, indicating that the small molecule binds to the active site of Mps1. However, TAK-733 has no inhibitory effect on the enzyme activity of Mps1, indicating that the small molecule does not bind to the active site of Mps1.
[0048] 2.2 Affinity test of compounds with Mps1 point mutants
[0049] TAK-733 was molecularly docked with the CD site, lipid binding site and N-lobe site of Mps1 using AutoDock Vina. Point mutations of Mps1 were designed based on the docking results, and the affinity of TAK-733 with the above mutant proteins was detected to confirm the binding position of TAK-733 on Mps1.
[0050] Figure 2 Schematic diagram of the identification results of the interaction sites between compounds PLX-4720, TAK-733 and Mps1 through in vitro experimental methods. Figure 2Part B shows the results of the affinity test of TAK-733 and the mutant protein. The results show that the affinity of TAK-733 to the mutant protein at the CD site of Mps1 is significantly reduced, from 15.1 μM ( Figure 1 ) decreased to 193μM( Figure 2 The results of the experiments in Figure 2 (part B) indicate that TAK-733 binds to the CD site of Mps1. The affinity of the other mutants for the other two sites did not change significantly. Therefore, TAK-733 and PLX-4720 bind to different sites of Mps1, such as Figure 2 As shown in part C.
[0051] Example 3
[0052] This example uses an in vitro inoculation experiment based on rice leaves to detect the effects of TAK-733, PLX-4720 alone and their combination on the pathogenicity of rice blast fungus. The in vitro inoculation experiment based on rice leaves is a standard method for detecting the effects of agents on the pathogenicity of rice blast fungus (Agricultural Industry Standard of the People's Republic of China NY / T 3257-2018).
[0053] The specific steps are as follows:
[0054] (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 conidia and adjust its concentration to 5 × 10 4 Pieces / mL.
[0055] (2) Take 5 μL of each of the experimental group (a suspension of rice blast fungi mixed with 100 μg / mL of the compound), the positive control (a suspension of rice blast fungi added with 100 μg / mL of oryzae), and the negative control (a suspension of rice blast fungi without the addition of the agent) and apply it to the scratched areas of rice leaves, evenly applying 3 drops to each leaf.
[0056] (3) Incubate at 26°C in a dark environment for 36 hours.
[0057] (4) The leaves in (3) were cultured under light conditions for 3-4 days, and the incidence of disease was counted.
[0058] Figure 3 This is a schematic diagram of the results of detecting the effects of the compounds PLX-4720 and TAK-733 provided in the embodiments of the present invention alone or in combination on the pathogenicity of rice blast fungus by in vitro inoculation method. Figure 3As shown in Part A of the experiment, 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 compounds could inhibit the infection of rice leaves by rice blast fungus spores, that is, compounds PLX-4720 and TAK-733 could inhibit the occurrence of rice blast and have a protective effect on rice plants. More importantly, Figure 3 From the B part, we can see that under the premise of the same total concentration of the agents, the inhibitory effect of the combined use of two agents on rice blast fungus is better than that of using one agent alone.
[0059] 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 as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
[0060] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
[0061] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
Claims
1. A use of a pyridopyrimidine and / or pyrrolopyridine compound as an inhibitor of the pathogenic microorganism Mps1, characterized in that: The pyridopyrimidine compound is a compound containing a pyridopyrimidine skeleton or a pesticide-acceptable salt thereof, and its structural formula is shown in Formula I; and the pyrrolopyridine compound is a compound containing a pyrrolopyridine skeleton or a pesticide-acceptable salt thereof, and its structural formula is shown in Formula II, The Mps1 is a mitogen-activated protein kinase 1 from pathogenic microorganisms.
2. The use according to claim 1, characterized in that: The pathogenic microorganisms include rice blast fungus, Fusarium oxysporum and / or other pathogenic fungi having Mps1 or its homologous proteins.
3. The use according to claim 2, characterized in that: The pathogenic microorganisms are one or more selected from the group consisting of: rice blast, gray mold, Alternaria alternata, corn smut, Colletotrichum gloeosporioides, cucurbit anthracnose, Fusarium graminearum, Fusarium oxysporum, wheat leaf blight and Sclerotinia sclerotiorum.
4. The use according to claim 1, characterized in that: The combined administration of the pyridopyrimidine compound and the pyrrolopyridine compound is included.
5. An inhibitor of Mps1, comprising an active ingredient and an adjuvant, wherein the active ingredient is the pyridopyrimidine and / or pyrrolopyridine skeleton-containing compound according to claim 1 or a pesticide-acceptable salt thereof.
6. The inhibitor according to claim 5, characterized in that The dosage form of the inhibitor is a pharmacologically acceptable dosage form, and the dosage form includes at least one of emulsifiable concentrate, wettable powder, suspension, soluble powder, aqueous solution, water-dispersible powder, smoke agent, granule and seed coating agent.