Use of the small peptide glp-1 (28-36) amide in the preparation of a medicament for the prevention and treatment of rice blast and methods

Abstract

The application discloses application and a method of a small peptide GLP-1(28-36)amide in preparation of a medicine for preventing and treating rice blast. A scheme discloses application of the small peptide GLP-1(28-36)amide in preparation of a medicine for preventing and treating Pyricularia oryzae, and application of the provided small peptide GLP-1(28-36)amide in inhibiting Pyricularia oryzae and preventing and treating rice blast. The small peptide GLP-1(28-36)amide of the application can effectively inhibit pathogenicity of spores of Pyricularia oryzae in a concentration range of 12.5-100 muM, in particular, the small peptide GLP-1(28-36)amide with a concentration of 100 muM can completely prevent and treat rice blast, and can be used for developing a new type of green pesticide.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control, specifically the application of the small peptide GLP-1(28-36)amide in the preparation of a drug for controlling rice blast fungus. Background Technology

[0002] Rice is one of the world's most important food crops and a major source of carbohydrates for humans. However, pathogenic fungi and bacteria continue to cause severe losses in rice yields worldwide. Of particular concern is rice blast, a fungal disease caused by the rice blast fungus *Magnaporthe oryzae* (syn. *Pyricularia oryzae*), which poses a significant threat to rice production. In recent years, my country has made significant progress in the prevention and control of rice blast, achieving breakthroughs in areas such as variety improvement and resistance breeding, rational application of chemical pesticides, application of biological control technologies, and the construction of monitoring and early warning systems. However, due to the wide host range, strong environmental adaptability, rapid mutation rate, and ease of developing drug resistance of the rice blast fungus, the control and management of rice blast remains extremely challenging and a long road ahead.

[0003] Currently available antifungal agents are still limited in number and insufficient to combat pathogens that rapidly develop resistance. To prevent plant fungal diseases from spiraling out of control globally, we need to improve the management of existing fungicides, accelerate the discovery and development of new fungicides, and utilize emerging technologies to find more reasonable alternative solutions.

[0004] Short peptide pesticides, as emerging biopesticides, have become a research hotspot in agriculture due to their high targeting, environmental friendliness, and multiple biological activities. Short peptides (typically composed of 2-50 amino acids) can achieve plant protection goals by regulating plant immunity, inhibiting pathogenic microorganisms, or interfering with pest physiological functions. They possess high specificity, are easily degraded, and are effective at low doses. Their mechanisms of action are diverse and highly designable, making them considered potential alternatives to traditional chemical pesticides. Currently, the development of short peptide fungal inhibitors is still in its early stages, and the available target proteins are limited.

[0005] Glucagon-like peptide-1 (GLP-1) is a hormone expressed in the gut and brain, acting through a single GLP-1 receptor (GLP-1 receptor, GLP1R) to regulate energy balance and food intake. GLP-1 receptor agonists (GLP1RAs) treat type 2 diabetes (T2D) by increasing glucose-dependent insulin secretion, decreasing glucagon secretion, and slowing gastric emptying. They also suppress food cravings, making them useful for obesity treatment, and in recent years have gained popularity as a weight-loss drug. GLP-1 exists primarily in two forms: GLP-1(7–36)amide and GLP-1(7–37)amide. GLP-1(7–36)amide is rapidly metabolized in the blood by dipeptidyl peptidase-4 (DPP-4) to GLP-1(9–36)amide. In healthy humans, GLP-1(9–36)amide has not shown any regulatory effect on insulin secretion or glucose metabolism. GLP-1(9–36)amide can be further hydrolyzed into shorter fragments, such as GLP-1(28–36)amide. As a metabolite of GLP-1, GLP-1(28–36)amide may possess unique biological activities different from full-length GLP-1. Studies have shown that GLP-1(28–36)amide can enter cells, target mitochondria, and exert insulin-like effects by regulating oxidative phosphorylation. GLP-1(28–36)amide or its analogues may have therapeutic effects on fasting hyperglycemia and metabolic syndrome in patients with type 2 diabetes. However, the physiological and pharmacological significance of GLP-1(28–36)amide is far from clear, and its activity in plant or microbial systems has not been reported. Furthermore, research and applications of GLP-1(28–36)amide in the control of plant diseases have not been reported. Summary of the Invention

[0006] The pathogenicity of rice blast fungus is regulated by the glycerol synthesis metabolic pathway; in rice blast fungus, the key enzyme in the glycerol synthesis metabolic pathway, glycerol-3-phosphate phosphatase (G3PP), is related to the pathogenicity of rice blast fungus; the small peptide GLP-1(28-36)amide can bind to glycerol-3-phosphate phosphatase in vitro; therefore, the applicant speculates that the small peptide GLP-1(28-36)amide may have the potential to inhibit rice blast fungus and control rice blast.

[0007] The purpose of this invention is to provide an application of the small peptide GLP-1(28-36)amide in inhibiting rice blast fungus and preventing rice blast disease. The small peptide GLP-1(28-36)amide has an in vitro control effect of 100 μM against rice blast fungus and has the advantages of significant effect, long duration of action, safety and simple application method in the prevention and control of rice blast disease.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] I. Application of small peptide GLP-1(28-36)amide in the preparation of drugs for the prevention and control of rice blast fungus (Magnaportheoryzae).

[0010] Application of small peptide GLP-1(28-36)amide in the preparation of drugs for inhibiting the penetration and infection of host by appressorium of rice blast fungus.

[0011] The concentration of the small peptide GLP-1(28-36)amide is 12.5~100 μM.

[0012] II. A drug for controlling rice blast fungus, including the small peptide GLP-1(28-36)amide.

[0013] The concentration of the small peptide GLP-1(28-36)amide is 12.5~100 μM.

[0014] III. A method for controlling rice blast fungus, wherein the drug is sprayed onto the leaves of plants.

[0015] The concentration of the small peptide GLP-1(28-36)amide in the drug used to control rice blast fungus is 12.5~100 μM.

[0016] The concentration of the small peptide GLP-1(28-36)amide is 12.5~100 μM. The preferred concentration is 100 μM, and the small peptide GLP-1(28-36)amide at a concentration of 100 μM can completely inhibit the occurrence of rice blast.

[0017] The application of the small peptide GLP-1(28-36)amide provided by this invention in inhibiting rice blast fungus and controlling rice blast disease shows that it can effectively inhibit the pathogenicity of rice blast fungus spores in the concentration range of 12.5~100 μM. In particular, the 100 μM concentration of compound GLP-1(28-36)amide can completely control rice blast disease. The discovery of this lead compound lays the foundation for the development of novel green pesticides.

[0018] The beneficial effects of this invention are:

[0019] The application of the small peptide GLP-1(28-36)amide provided by this invention in inhibiting rice blast fungus and controlling rice blast disease shows that it can significantly inhibit the infection of rice by rice blast fungus in the concentration range of 12.5~100 μM. In particular, the 100 μM concentration of small peptide GLP-1(28-36)amide can completely control rice blast disease. The discovery of this lead compound will lay the foundation for the creation of new green pesticides. Attached Figure Description

[0020] Figure 1 The binding curves of GLP-1(28-36)amide to the target protein glycerol-3-phosphate phosphatase, determined by surface plasmon resonance (SPR) technique, are shown. The test concentrations were 0.39 μM, 0.78 μM, 1.56 μM, 3.13 μM, 12.5 μM, and 25 μM.

[0021] Figure 2 K was obtained by fitting a steady-state model of GLP-1(28-36)amide and its target protein glycerol-3-phosphate phosphatase, as determined by surface plasmon resonance (SPR) technology. D The tested concentrations were 0.39 μM, 0.78 μM, 1.56 μM, 3.13 μM, 12.5 μM, and 25 μM.

[0022] Figure 3 This study investigated the infection status of rice blast fungus in detached barley leaves after 72 h of suspension drop treatment with different concentrations of the small peptide GLP-1(28-36)amide on rice blast fungus spore suspension.

[0023] Figure 4 This study investigated the appressorium infection rate of *Oryza sativa* spore suspension after 72 h of suspension drop treatment with different concentrations of the small peptide GLP-1(28-36)amide in detached barley leaves. At least 100 appressoriums were counted each time to calculate the infection rate. Data are expressed as the mean ± standard deviation (SD) of n=3 independent replicates (P<0.0001; two-tailed t-test).

[0024] Figure 5 The disease incidence of detached barley leaves 72 hours after suspension drop treatment with different concentrations of the small peptide GLP-1(28-36)amide added to rice blast fungus spore liquid.

[0025] Figure 6 The study investigated the disease incidence on rice leaves after 6 days of spraying with different concentrations of the small peptide GLP-1(28-36)amide on rice blast fungus spores.

[0026] Figure 7This study investigated the diseased area of ​​rice leaves after 6 days of spraying with different concentrations of the small peptide GLP-1(28-36)amide from rice blast fungus spore suspension. At least 10 rice leaves were analyzed in each treatment, and data are expressed as the mean ± standard deviation (SD) of n=3 independent replicates (P<0.0001; two-tailed t-test).

[0027] Figure 8 The results show the molecular docking of the small peptide GLP-1(28-36)amide with glycerol-3-phosphate phosphatase predicted by AlphaFold3. The yellow dashed lines represent hydrogen bonding forces, and the purple dashed lines represent salt bridges. Detailed Implementation

[0028] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0029] The experimental results of this invention are as follows:

[0030] The rice blast fungus used in this experiment was the wild-type strain Guy11 (ATCC Strain Center, USA), preserved in the laboratory. The culture medium for culturing the rice blast fungus in the laboratory was complete culture medium (CM). CM culture medium formula: Glucose 10 g, Peptone 140 2 g, Casein amino acids 1 g, Yeast extract 1 g, NaNO3 6 g, KH2PO4 1.52 g, KCl 0.52 g, MgSO4·7H2O 0.52 g, Biotin 0.1 mg, Vitamin B 0.1 mg, Thiamine 0.1 mg, Riboflavin 0.1 mg, Nicotinic acid 0.1 mg, Para-aminobenzoic acid 0.1 mg, Na2MoO4·5H2O 1.5 mg, CuSO4·5H2O 1.6 mg, CoCl2·6H2O 1.7 mg, MnCl2·4H2O 5 mg, FeSO4·7H2O 5 mg, H3BO3 11 mg, ZnSO4·7H2O 22 mg, Na4EDTA·2H2O 50 mg, Agar powder 15 g, adjust pH to 6.5 with NaOH, bring volume to 1 L with H2O, and autoclave at 121 °C for 30 min.

[0031] The small peptide GLP-1(28-36)amide, CAS number 1225021-13-5, is available from MCE (catalog number: HY-P3101), and its structural formula is as follows:

[0032] ;

[0033] Preparation method of GLP-1(28-36)amide stock solution: Add 10 mg of GLP-1(28-36)amide to 918.82 μL of DMSO to a concentration of 10 mM.

[0034] Example 1: In vitro binding of the small peptide GLP-1(28-36)amide to glycerol-3-phosphate phosphatase, a target protein of rice blast fungus.

[0035] Surface plasmon resonance (SPR) technology was used to detect the interaction: After obtaining high-purity glycerol-3-phosphate phosphatase, the protein was concentrated to 1 mg / mL for Biacore analysis. The isoelectric point of the protein to be analyzed was predicted using software (ideally between 3 and 7) to ensure that the protein was suitable for Biacore analysis. The steps are as follows: ① Prepare the compound: Dissolve the compound to be tested in a protein buffer containing 1‰ DMSO and dilute to 10. -5 M and 10 -6 ① Initial analysis at two concentrations (compounds showing positive results in the initial analysis are then diluted with buffer containing 1‰ DMSO to perform kinetic constant analysis at multiple concentration gradients); ② Coupling: immobilize and couple the target protein onto the chip (including determining the optimal coupling pH, optimal concentration, and optimal flow rate through pre-enrichment experiments); ③ On-chip testing: load the target compounds sequentially, ensuring the sample buffer is as consistent as possible with the system carrier buffer; ④ Regeneration: elute the compounds bound to the chip surface for chip reuse; ⑤ Analyze the derived experimental data according to the binding fitting model provided by GE.

[0036] Results Analysis: The interaction between the target protein glycerol-3-phosphate phosphatase of *Oryza sativa* and the small peptide GLP-1(28-36)amide in vitro was detected using surface plasmon resonance (SPR) technology. The results showed that glycerol-3-phosphate phosphatase and GLP-1(28-36)amide have a certain affinity in vitro, exhibiting a significant concentration-dependent relationship within the tested range of 0.39–25 μM. Figure 1 ); dissociation constant K D The value is 86.7 μM ( Figure 2 The results suggest that GLP-1(28-36)amide may be able to target glycerol-3-phosphate phosphatase of rice blast fungus and inhibit its infection.

[0037] Example 2: Determination and results of the antifungal effect of small peptide GLP-1(28-36)amide on rice blast fungus in isolated barley.

[0038] In vitro barley inoculation: Wild-type rice blast fungus strains were inoculated onto CM medium and cultured for 10 days to obtain spores; barley was planted until one week old, and healthy barley leaves were harvested; a compound was prepared, and the compound was mixed with a spore suspension (final concentration after dilution was 1×10⁻⁶). 5 Dilute the compound (cfu / mL) to the target final concentration, with the control group being DMSO of the same concentration; use a pipette to take 20 μL of spore suspension and drop it onto barley leaves, 3 drops per leaf, treat with moisture and light, and incubate under light at 25°C for 3 days, observe the disease condition of the leaves, and take photos for recording.

[0039] Results Analysis: The inhibitory effects of different concentrations of the small peptide GLP-1(28-36)amide on isolated barley and rice blast disease were tested. Figure 5 As shown, treatment with 25-100 μM small peptide GLP-1(28-36)amide has a certain inhibitory effect on isolated barley blast disease; and within this range, the higher the concentration, the stronger the inhibitory effect; the small peptide GLP-1(28-36)amide at a concentration of 100 μM can completely inhibit isolated barley blast disease.

[0040] Example 3: Observation and results of infection of rice blast fungus by small peptide GLP-1(28-36)amide on isolated barley.

[0041] Observation of barley leaf infection: After in vitro barley inoculation, barley leaves were selected 3 days after inoculation for infection observation. Formaldehyde was used to decolorize the barley leaves, and after more than three rinsings, the leaves were decolorized until transparent. The decolorized leaves were then fixed on glass slides and observed using an optical microscope.

[0042] Results Analysis: The inhibitory effects of different concentrations of the small peptide GLP-1(28-36)amide on isolated barley and rice blast disease were tested. Figure 3 As shown, 72 h after barley leaves were infected by *Oryza sativa*, the *Oryza sativa* in the control group (treated with DMSO) had completely invaded the leaf tissue and produced a large number of secondary infective hyphae that invaded adjacent mesophyll cells; in the treatment group (treated with 25–100 μM of the small peptide GLP-1(28–36)amide), *Oryza sativa* spores could all form appressoria normally, but some appressoria could not form infection spikes to penetrate the leaf epidermis. Figure 4 As shown, the application of small peptide GLP-1(28-36)amide significantly reduced the infection rate mediated by appressorium of rice blast fungus; the higher the concentration of small peptide GLP-1(28-36)amide, the lower the infection rate of rice blast fungus; the small peptide GLP-1(28-36)amide at a concentration of 100 μM could almost completely prevent the infection of rice blast fungus appressorium.

[0043] Example 4: Pathogenicity determination and results of small peptide GLP-1(28-36)amide against rice blast fungus in live rice.

[0044] Rice spraying experiment: Rice plants were grown in pots and cultivated to the three-leaf-one-bud stage. Five pots of rice were prepared for each treatment, with 20 rice seedlings in each pot. Spores of the test strains were washed off, centrifuged at 7000 rpm for 20 min, rinsed three times with sterile water, and the spores were collected. An appropriate amount of sterile water was added to dilute the spore suspension to 1×10⁻⁶. 5 CFU / mL and mix with the test drug; dilute the spore suspension with 0.25% gelatin solution to 5 × 10⁻⁶. 4 cfu / mL; use a sprayer to evenly spray rice blast fungus spores onto the surface of rice leaves, spraying 2 mL per pot of rice; first treat in the dark at 25°C with humidity for 24 h, then transfer to a 16 h / 8 h light-dark alternating incubator and incubate at 25°C for 4-6 days, observe the disease incidence of rice, record the data, and take photos.

[0045] Results Analysis: The efficacy of different concentrations of the small peptide GLP-1(28-36)amide in controlling rice blast disease in live rice was tested. Figure 6 As shown, compared with the control group without the application of small peptide GLP-1(28-36)amide, the 12.5~100 μM small peptide GLP-1(28-36)amide treatment groups all effectively inhibited the infection of rice host by rice blast fungus; the statistical results of leaf disease area showed that the 12.5~100 μM small peptide GLP-1(28-36)amide treatment groups all significantly inhibited the infection of rice blast fungus in vivo. Figure 7 In addition, the small peptide GLP-1(28-36)amide had no significant adverse effects on rice growth, indicating that it may be safe for rice and is a lead compound with the potential to control rice blast.

[0046] Example 5: Molecular docking and results of small peptide GLP-1(28-36)amide with a predicted model of glycerol-3-phosphate phosphatase protein from rice blast fungus.

[0047] Protein model prediction: The protein model of glycerol-3-phosphate phosphatase was predicted using the local version of AlphaFold3, and a high-quality protein model was obtained (ipTM=0.76; pTM=0.78).

[0048] Molecular docking: The glycerol-3-phosphate phosphatase protein model was pretreated, optimized, and minimized using the Protein Preparation Wizard module (with constraint minimization using the OPLS3e force field); the small peptide GLP-1(28-36)amide was prepared according to the default settings of the LigPre module; docking boxes were constructed, using the glycerol-3-phosphate phosphatase protein model as the recipient protein to construct 10 Å docking boxes; molecular docking was performed using the Glide module; docking results were exported and analyzed.

[0049] Results analysis: Molecular docking results showed that the small peptide GLP-1(28-36)amide has a certain affinity for glycerol-3-phosphate phosphatase, a protein from rice blast fungus, with a docking score of -9.772. Figure 8 As shown, in the molecular docking simulation, the small peptide GLP-1(28-36)amide can bind to the receptor protein glycerol-3-phosphate phosphatase through multiple intermolecular interactions. The molecular docking results further corroborate the interaction between the small peptide GLP-1(28-36)amide and the glycerol-3-phosphate phosphatase protein of rice blast fungus, and reveal the possible interaction sites.

[0050] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0051] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. The application of the small peptide GLP-1(28-36)amide, characterized in that, Application in the preparation of drugs for the prevention and control of rice blast fungus.

2. The application of the small peptide GLP-1(28-36)amide according to claim 1, characterized in that, Application of small peptide GLP-1(28-36)amide in the preparation of drugs for inhibiting the penetration and infection of host by appressorium of rice blast fungus.

3. The application as described in claim 1 or 2, characterized in that, The concentration of the small peptide GLP-1(28-36)amide is 12.5~100 μM.

4. A method for controlling rice blast fungus, characterized in that, Spray the small peptide GLP-1(28-36)amide onto the leaves of the plant.

5. The method for controlling rice blast fungus as described in claim 4, characterized in that, The concentration of the small peptide GLP-1(28-36)amide is 12.5~100 μM.

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