Use of azilsartan as an inhibitor of insect epicuticular lipid synthesis and for controlling pests

Abstract

The application belongs to the technical field of pesticide preparation and insect prevention and control, and particularly relates to application of Azilsartan as an insect cuticular lipid synthesis inhibitor and in pest control. The application first finds that a small molecule compound Azilsartan is an inhibitor of a target trans-enoyl CoA reductase (TER), and the results of in-vitro enzyme activity inhibition tests and insecticidal tests prove that Azilsartan can significantly inhibit the activity of a key membrane protein TER in an insect cuticular lipid synthesis pathway, and has significant insecticidal activity on Asian corn borer, diamondback moth, yellow mealworm and red flour beetle. Meanwhile, the small molecule compound Azilsartan provides a research basis for development of insecticides, and is conducive to green pesticide research and development.

Description

Technical Field

[0001] This invention belongs to the field of pesticide preparation and insect control technology, specifically involving the application of Azilsartan as an inhibitor of insect epidermal lipid synthesis and in the control of pests. Background Technology

[0002] The insect exoskeleton serves as a protective barrier for its adaptation to the environment. Its main components include chitin, proteins, and lipids, with epidermal lipids being a crucial part. These lipids prevent water evaporation from the insect's body and protect against the invasion of exogenous substances. Insect growth and development are inseparable from the cyclical shedding and formation of the exoskeleton, and the formation of epidermal lipids is essential for maintaining the integrity of the exoskeleton structure. However, plants and mammals do not undergo molting. Therefore, key proteins in the biomass synthesis pathway of insect molting may be potential targets for safe pesticide molecules used in pest control, thus balancing pest control with the safety of plants and humans.

[0003] Epidermal lipid synthesis is a complex, multi-step process that begins with acetyl-CoA and involves a series of enzymes to synthesize hydrocarbons, fatty alcohols, and other substances. In studies known to the inventors, trans-enoyl-CoA reductase (TER) is a key membrane protein in the lipid synthesis pathway, and its deficiency can lead to highly efficient insect mortality. Therefore, screening small molecule compounds that can inhibit trans-enoyl-CoA reductase for use as insecticides holds significant promise. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Azilsartan as an inhibitor of insect epidermal lipid synthesis and in the control of pests. Azilsartan can significantly inhibit the activity of trans-enoyl-CoA reductase, a key membrane protein in the insect epidermal lipid synthesis pathway, and has insecticidal activity against a variety of Lepidoptera and Coleoptera pests.

[0005] This invention provides the application of Azilsartan as an inhibitor of insect epidermal lipid synthesis.

[0006] Preferably, the Azilsartan is a trans-diethyl-CoA reductase inhibitor.

[0007] Preferably, the trans-enoyl-CoA reductase inhibitor includes a trans-enoyl-CoA reductase activity inhibitor.

[0008] Preferably, the trans-enoyl-CoA reductase includes Asian corn borer trans-enoyl-CoA reductase OfTER.

[0009] Preferably, the epidermal lipids include epidermal long-chain fatty acids.

[0010] This invention also provides the application of an insect epidermal lipid synthesis inhibitor with Azilsartan as the active ingredient in the control of pests.

[0011] Preferably, the pest control includes killing pests.

[0012] Preferably, the pests include one or more of the following: corn borer, diamondback moth, mealworm, and red flour beetle.

[0013] Preferably, the concentration of Azilsartan is ≥0.5μM.

[0014] The present invention also provides an insecticide whose active ingredient includes Azilsartan.

[0015] Beneficial effects:

[0016] This invention provides the application of Azilsartan as an inhibitor of insect epidermal lipid synthesis and in pest control. This invention is the first to discover that the small molecule compound Azilsartan is an inhibitor targeting TER (tertiary epidermal protein). In vitro enzyme activity inhibition and insecticidal experiments demonstrate that Azilsartan can significantly inhibit the activity of TER, a key membrane protein in the insect epidermal lipid synthesis pathway, and exhibits significant insecticidal activity against Asian corn borer, diamondback moth, yellow mealworm, and red flour beetle. Furthermore, the small molecule compound Azilsartan of this invention provides a research foundation for the development of insecticides, which is beneficial for the research and development of green pesticides. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a diagram showing the OfTER structure prediction results of the Asian corn borer and its binding with the substrate and Azilsartan in Example 1;

[0019] Figure 2 This is a diagram showing the expression and purification detection of OfTER protein from the Asian corn borer in Example 2;

[0020] Figure 3 This is a graph showing the in vitro inhibitory activity of Azilsartan against OfTER in the Asian corn borer, as described in Example 3.

[0021] Figure 4 The figure shows the effect of Azilsartan on the content of ultra-long chain fatty acids in the epidermis of Asian corn borer and the results of insecticidal activity determination in Example 4. Detailed Implementation

[0022] This invention provides the application of Azilsartan as an inhibitor of insect epidermal lipid synthesis.

[0023] Azilsartan, also known as azithromycin, is a 4-(1-hydroxy-1-methylethyl)-2-propyl-1-imidazol-5-carboxylic acid ester, with CAS number 147403-03-0. In one embodiment, Azilsartan is used as a trans-enoyl-CoA reductase inhibitor; in another embodiment, the trans-enoyl-CoA reductase inhibitor includes an inhibitor of trans-enoyl-CoA reductase activity. In one embodiment, the trans-enoyl-CoA reductase includes the Asian corn borer trans-enoyl-CoA reductase OfTER; the Asian corn borer trans-enoyl-CoA reductase OfTER is disclosed in Chinese patent CN118726413A; the gene encoding the Asian corn borer trans-enoyl-CoA reductase... OfTER

[0024] This invention targets the key membrane protein TER in the insect epidermal lipid synthesis pathway. Based on the predicted three-dimensional structure of TER, small molecule inhibitors were screened, and in vitro activity was assessed to obtain Azilsartan, which exhibits strong inhibitory activity against TER. In vivo activity tests demonstrated that Azilsartan at a concentration of 0.5 μM had significant insecticidal effects against Asian corn borer, diamondback moth, yellow mealworm, and red flour beetle. Specifically, Azilsartan significantly inhibited the activity of insect trans-diethyl-CoA reductase TER. In vivo results showed that it significantly reduced the content of ultra-long chain fatty acids in the epidermis, inhibited epidermal lipid formation, and consequently reduced the insects' waterproofing ability, leading to death.

[0025] Based on the above advantages, this invention also provides the application of an insect epidermal lipid synthesis inhibitor with Azilsartan as the active ingredient in pest control. In one embodiment, the pest control includes killing the pest; in another embodiment, the pest includes lepidopteran pests and / or coleopteran pests; in another embodiment, the lepidopteran pests include the corn borer and / or diamondback moth; the coleopteran pests include the yellow mealworm and / or the red flour beetle; in another embodiment, the corn borer can be the Asian corn borer. In one embodiment, the concentration of Azilsartan is ≥0.5 μM, more preferably 0.5~1 μM.

[0026] This invention also provides an insecticide whose active ingredient includes Azilsartan. This invention does not impose any particular limitations on the formulation and excipients of the insecticide; conventional selections are acceptable as needed.

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] Three-dimensional structure prediction and inhibitor screening of the Asian corn borer by Ofter

[0030] OfTER, the trans-diethyl-CoA reductase of the Asian corn borer, is disclosed in Chinese patent CN118726413A.

[0031] Structure-based virtual screening of small molecules, specifically based on the previously reported Clostridium acetone-butanol (Clostridium perfringens) Clostridium acetobutylicumHomology modeling of the Asian corn borer TER was performed using the three-dimensional structure (PDB: 6X6T) of TER (EC 1.3.1.44) and the AlphaFold3 structure. The three-dimensional protein structure of the Asian corn borer TER was constructed, and its substrate trans-dehydroacyl-CoA binding region was used as a docking pocket for virtual screening. Using AutoDockTools software, a virtual screening was performed on a library of approximately 800 small molecules related to lipid synthesis and metabolism, encompassing both the Lipid Compound Library (catalog number SJ-ML1301, provided by Shandong SikeJ Biotechnology Co., Ltd.) and the Lipid Metabolism Compound Library (catalog number SJ-ML1405, provided by Shandong SikeJ Biotechnology Co., Ltd.). The binding conformations with the highest binding energies were selected (lower binding energies indicate more stable conformations and potentially stronger inhibitory activity against protein receptors). The AutoDockTools output file was opened using the molecular visualization software PyMOL, and the docking pocket was imported to analyze the spatial location of the small molecule ligands within the receptor binding pocket and the type of interaction with the protein. Based on binding energy (absolute value higher than 10 kJ / mol) and the spatial location of the small molecule binding, six small molecule compounds were selected for in vitro and in vivo activity testing, such as... Figure 1 As shown, A is a predicted diagram of the OfTER protein structure and active pocket site; B is a predicted diagram of the binding site of the OfTER protein to its substrate trans-acyl-CoA; and C is a predicted diagram of the binding site of the OfTER protein to the small molecule Azilsartan.

[0032] Depend on Figure 1 It can be concluded that the three-dimensional structure of the OfTER protein consists of 6 β sheets and 11 α helices. Seven amino acid residues participate in the formation of hydrogen bonds when it docks with its substrate, and three amino acid residues participate in the formation of hydrogen bonds when it binds to the small molecule compound Azilsartan.

[0033] Example 2

[0034] The expression and purification of OfTER protein from the Asian corn borer were carried out using the following steps:

[0035] 1. Expression of Ofter protein

[0036] 1) The TER coding sequence of Asian corn borer was obtained based on the transcriptome database of the inventors' team and constructed into the prokaryotic expression vector pET28a.

[0037] 2) Transform the successfully constructed pET28a-OfTER recombinant vector into the BL21(DE3) strain.

[0038] 3) The expression strain containing pET28a-OfTER plasmid was inoculated into 400 μL of Luria-Bertani (LB) liquid medium and cultured for 4-6 h. Then, it was inoculated into 20 mL of LB liquid medium containing 100 μM kanamycin at a volume ratio of 1:500 and cultured overnight at 37°C and 220 rpm on a shaker.

[0039] 4) Expand the 20 mL of overnight culture to 1 L LB liquid medium, add kanamycin to a final concentration of 100 μM, and culture at 37°C and 220 rpm in a shaker.

[0040] 5) Measure the OD of bacterial culture using a cell density meter 600 Value, awaiting 1 / 2 OD 600 If the value is between 0.6 and 0.8, stop the bacterial culture.

[0041] 6) Place the bacterial culture in an ice-water mixture or a four-degree chromatography cabinet to cool it down. After the culture flask and bacterial culture have cooled down completely, add IPTG, the inducer, to a final concentration of 100 μM.

[0042] 7) Transfer the bacterial culture back to a 16℃ shaker and incubate at 220 rpm for 20 h;

[0043] 8) After the expression strains were cultured in stages, they were collected by centrifugation. The bacterial culture was resuspended in a protein buffer with a pH of 8.0 (composed of 50 mM Tris-HCl, 500 mM NaCl and 0.5% Triton X-100) and stored at -20℃.

[0044] 2. Purification of Ofter protein

[0045] 1) High-pressure cell disruption: Thaw the protein expression bacterial culture stored at -20℃ under running water. After the bacterial culture is completely thawed, add an appropriate amount of protein buffer to dilute the bacterial culture until it is no longer thick and clumpy and has suitable fluidity.

[0046] 2) Turn on the high-pressure crusher switch, clean the inside of the high-pressure crushing system, and set the pressure value between 1000 and 1500 psi;

[0047] 3) Add the diluted protein bacterial solution to the sample inlet above the high-pressure crusher, and repeat the crushing of the bacterial solution 3 times.

[0048] 4) After the high-pressure crushed bacterial solution is strictly balanced, it is placed symmetrically in a floor-standing ultra-high speed centrifuge and centrifuged at 15000g and 4℃ for 30min.

[0049] 5) Take out the broken bacterial culture after high-speed centrifugation, transfer the supernatant containing OfTER protein to a clean beaker, and filter out impurities using a 0.45μm filter membrane;

[0050] 6) Mix the filtered supernatant with the equilibrated Ni-NTA packing material evenly, and symmetrically place it on a rotary incubator for 1 hour at 4°C.

[0051] 7) Pour the incubated nickel glue and protein suspension into an empty gravity column, and the incubated protein solution will flow out at a high flow rate to become the flow-through solution.

[0052] 8) Rinse the Ni-NTA packing material with a protein elution buffer (pH 8.0) containing 20 mM imidazole to remove impurities; the elution volume is 200 mL.

[0053] 9) Elute the target protein with a protein elution buffer (pH 8.0) containing 100 mM imidazole, controlling the flow rate to approximately 2-3 s / drop, with an elution volume of 20 mL.

[0054] 3. Obtaining OfTER protein

[0055] Protein purification using molecular sieve was performed with a protein elution buffer of 100 mM imidazole, and the results are as follows: Figure 2 As shown, the protein elution peak and the external water peak are relatively separated, and the target protein OfTER is eluted in large quantities at a volume of 60mL~70mL. The protein elution peak shows a symmetrical peak shape, with the peak tip located at approximately 65mL. The molecular weight corresponding to this position is 34.1kDa, which is consistent with the theoretical molecular weight of OfTER protein, indicating that OfTER protein exists in the solution in monomeric form.

[0056] Example 3

[0057] The in vitro inhibitory activity of Azilsartan against the Asian corn borer (OfTER) was determined using the reaction system shown in Table 1, and the steps were as follows:

[0058] 1) Take 10 μL of Asian corn borer OfTER obtained in Example 2 and 16 μL of NADH and K2HPO4 buffer and pre-incubate in the reaction system for 10 min;

[0059] 2) Add 120 μL of acyl-CoA to initiate the reaction;

[0060] 3) Add 0, 4 μL, 20 μL and 40 μL of small molecule Azilsartan respectively;

[0061] 4) Add K2HPO4 buffer to a final volume of 200 μL, react at 25°C for 20 min;

[0062] 5) Use a spectrophotometer to continuously monitor the change in absorbance (OD value) at 340 nm.

[0063] Table 1 Reaction System

[0064]

[0065] Calculate the relative enzyme activity and inhibition rate. The formula for calculating the relative enzyme activity is: Relative enzyme activity (%) = (OD value of different concentrations of small molecules / ΔOD × 6.22) × 100, where ΔOD is the OD value of the sample plate minus the OD value of the blank plate, and 6.22 is the εNADH value (theoretical molar absorptivity) (mM). -1 cm -1 The formula for calculating the inhibition rate is: Inhibition rate (%) = 100 - relative enzyme activity. The results are as follows: Figure 3 As shown.

[0066] Depend on Figure 3 It can be concluded that as the concentration of small molecule Azilsartan increases, the relative enzyme activity of TER gradually decreases, while the inhibition rate increases significantly, and the inhibition rate reaches its maximum at 50 μM.

[0067] Example 4

[0068] The insecticidal activity of Azilsartan at different concentrations was determined by the following steps:

[0069] Azilsartan, a small molecule compound, was purchased from MCE (MedChemExpress) Biotechnology Company in the United States. Azilsartan was diluted to a 10 μM stock solution using a mixed solution of DMSO and NaCl (DMSO:NaCl ≈ 5:4 volume ratio) as the solvent.

[0070] Azilsartan was mixed with feed at a concentration of 0.1 μM and fed to the first day of the fourth instar of Asian corn borer larvae. Each larva was fed approximately 0.01 μg of Azilsartan once. A control group (ck) was fed the same dose and concentration of a mixed solution of DMSO and NaCl. The results showed that Azilsartan could cause a mortality rate of more than 30% in Asian corn borers.

[0071] Since TER (extrinsic terpenoid ester) is involved in the synthesis of long-chain fatty acids (VLCFAs), the content of VLCFAs in the epidermis of Asian corn borer larvae was detected using an insect VLCFAs detection kit (YJ2000589, Shanghai Yuanju Biotechnology Co., Ltd.) after feeding Azilsartan. The results showed a significant decrease in VLCFA content, such as... Figure 4 As shown in Figure A, Azilsartan can inhibit lipid synthesis in the epidermis of the Asian corn borer.

[0072] To further test the insecticidal activity of Azilsartan, Asian corn borer, diamondback moth, mealworm, and red flour beetle larvae in their final instar were subsequently fed with 0.5 μM (0.05 μg / larvae) and 1 μM (0.1 μg / larvae) concentrations of Azilsartan, respectively, once. Mortality was observed and recorded when they entered the next developmental stage (the control group was fed with the same amount of DMSO:NaCl solution). The results showed that Azilsartan exhibited a concentration-dependent effect; higher concentrations led to higher mortality rates. Figure 4 As shown in Figure B, n represents the number of test insects, the percentage represents the individual survival rate (control group) or the individual mortality rate (feeding small molecular group), and the red diagonal line represents the group with individual insect mortality.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of Azilsartan as an inhibitor of insect epidermal lipid synthesis, wherein Azilsartan is an inhibitor of the activity of trans-diethyl-CoA reductase OfTER in Asian corn borer; The epidermal lipids include epidermal ultra-long chain fatty acids; the amino acid sequence of the Asian corn borer trans-coenzyme A reductase OfTER is shown in SEQ ID NO:

2.

2. Application of insect epidermal lipid synthesis inhibitors with Azilsartan as the active ingredient in pest control, wherein the epidermal lipids include epidermal long-chain fatty acids; and the pests include the Asian corn borer.

3. The application according to claim 2, characterized in that, The concentration of Azilsartan is ≥0.5 μM.

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