Application of compounds in insecticide and pesticide synergistic enhancement

Abstract

The present invention discloses that compounds such as benzethonium chloride, dichlorophenol, glycyrrhetinic acid, oxycaine, and ciclopirox have effective activating effects on nicotinic acid receptors. The above five compounds can directly or indirectly regulate the activity of nicotinic acid receptors to affect the change of calcium ion concentration in the endoplasmic reticulum, causing calcium ions to be continuously released from the endoplasmic reticulum calcium store, leading to calcium store depletion and finally muscle spasm, thereby achieving an insecticidal effect. The present invention also detects that benzethonium chloride, glycyrrhetinic acid, oxycaine, and ciclopirox have a synergistic effect on diamide insecticides. The above compounds are all small molecules with simple structures and great manufacturability, providing guiding opinions for subsequent in vivo experiments and compound structure modification.

Description

Technical Field

[0001] The invention belongs to the technical field of insecticides, and specifically relates to the application of a compound in insecticide and pesticide synergistic enhancement. Background Art

[0002] Diamide insecticides are a class of highly efficient and selective insecticides that target nicotinic acid receptors. They can directly bind to and activate the insect's RyR, causing uncontrolled release of intracellular calcium stores, ultimately leading to calcium store depletion, muscle spasms and insect death. Since the development and listing of diamide insecticides, they have received great attention and are widely used around the world due to their significant insecticidal effects (especially against lepidopteran pests), low acute mammalian toxicity and environmentally friendly properties. As a result, resistance mutations have been reported in large quantities in many parts of the world. For example: Fall Armyworm (scientific name: Spodoptera frugiperda, sf) is a pest of the Lepidoptera Noctuidae family, which has caused huge agricultural losses in many countries. Due to the large-scale use of diamide, fall armyworm has caused extremely high resistance worldwide, causing a serious resistance crisis. The main countermeasures for the generation of resistance mutations are currently divided into two aspects: one is to structurally modify the mature diamide insecticides that have been on the market to find more efficient analogs; the other is to develop and find new lead compounds (lead compounds: referred to as lead compounds, are compounds with certain biological activity and chemical structure obtained through various channels and means, used for further structural modification and modification). Among them, the structural modification of diamide insecticides is mainly around the o-phenylenediamine mother nucleus. With a large number of modifications and applications, it is found that the space for modification of this mother nucleus is gradually getting smaller, and with the large-dose use of this type of insecticide, its resistance is also rapidly increasing. At present, it is urgent to find another way to find other targeted insecticides. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention aims to provide a compound for use in regulating the calcium ion concentration in the endoplasmic reticulum of insect cells.

[0004] Another object of the present invention is to provide an application of a compound in synergistically enhancing the effect of diamide insecticides.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] The invention discloses an application of a compound in regulating the calcium ion concentration in the endoplasmic reticulum of insect cells. The compound is one of benzethonium chloride, dichlorophenol, glycyrrhetinic acid, oxethacaine and ciclopirox.

[0007] In the above technical solution, the insect is a Lepidoptera insect, a Diptera insect, a Coleoptera insect or a Hemiptera insect.

[0008] In the above technical solution, the compound directly or indirectly regulates the activity of nicotinic acid receptors and thus regulates the calcium ion concentration in the endoplasmic reticulum of insect cells.

[0009] A compound is used in synergistically enhancing the synergy of diamide insecticides. The compound is one of benzethonium chloride, glycyrrhetinic acid, oxethacaine and ciclopirox.

[0010] In the above technical solution, the structural formula of the diamide insecticide is one of the following six:

[0011]

[0012] Ryanodine receptor: The English name is Ryanodine receptor (RyR), which is a calcium release channel present in the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR). It can quickly release Ca 2+ It is released from ER / SR, thus exerting a series of physiological functions. The nicotinic acid receptor plays an important role in maintaining the balance of intracellular calcium. The present invention finds that the compounds benzethonium chloride, dichlorophene, enoxolone, oxethazaine, and ciclopirox have an effective activation effect on the nicotinic acid receptor. The above five compounds can directly or indirectly regulate the activity of the nicotinic acid receptor to affect the change of the endoplasmic reticulum calcium ion concentration, causing the calcium ions to be continuously released from the endoplasmic reticulum calcium store, leading to the depletion of the calcium store, and finally muscle spasm, thereby achieving the insecticidal effect.

[0013] The present invention also detected that benzethonium chloride, enoxolone, oxethazaine and ciclopirox have synergistic effects on diamide insecticides. The above five compounds are all small molecules with simple structures and great manufacturability, providing guidance for subsequent in vivo experiments and compound structure modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the F / F0 of the 2113 small molecules in Example 2;

[0015] Figure 2 is the F / F0 of OXE, BEN, DIC, CIC and ENO at different concentrations in Example 4;

[0016] Figure 3It is the F / F0 of the synergistic effect of OXE, BEN, CIC and ENO on CHL and that of CHL alone in Example 5. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0018] In the following examples, Flp-In T-REx stably expressing the wild-type Spodoptera frugiperda nicotinic acid receptor (WT sfRyR) gene was used. The nucleotide sequence of the wild-type Spodoptera frugiperda nicotinic acid receptor (WT sfRyR) gene is shown in SEQ ID NO1.

[0019] 5% CO2: is the volume percentage, the remaining 95% is air.

[0020] Flp-In T-REx HEK293-R-CEPIA1er cells: First, stably transfect R-CEPIA1er (sequence see DOI:10.1038 / ncomms5153) into HEK293 cells (purchased from Thermo Fisher Scientific) and express it to form the HEK293-R-CEPIA1er cell line. Use the Flp-In T-REx Core Kit (purchased from Life Technologies Corporation) and follow the kit instructions to convert pFRT / lacZeo and pcDNA3 into TM The 6 / TR plasmid was stably transformed into the HEK293-R-CEPIA1er cell line to obtain the Flp-In T-REx HEK293-R-CEPIA1er cell line, so that the Flp-In T-REx HEK293-R-CEPIA1er cell line has a specific recombination site, and the target fragment at this site can be induced to express by doxycycline.

[0021] Example 1

[0022] Construction of the Flp-In T-RExHEK293-R-CEPIA1er cell line stably expressing the wild-type fall armyworm nicotinic acid receptor (WT sfRyR) gene (see patent application number 202110884938.4):

[0023] Add (Dulbecco' Modification of Eagle's Medium) DMEM complete medium to Flp-In T-REx HEK293-R-CEPIA1er cells, culture at 37°C, 5% CO2, and regularly replace DMEM complete medium to revive Flp-In T-REx HEK293-R-CEPIA1er cells. After revival, remove DMEM complete medium, rinse cells with phosphate buffered saline (PBS, pH=7.4), remove PBS, digest cells with EDTA-trypsin digestion solution, and dilute cells to 5.2×10 6 / mL, and obtain cell culture fluid. Add 3 ml of cell culture fluid into a 6 cm cell culture dish and culture it in a 37°C, 5% CO2 incubator for 24 h.

[0024] WT sfRyR (synthesized by GENEWIZ, Suzhou, China) and the pOG44 plasmid in the Flp-In T-REx Core Kit (Flp recombinase expression plasmid, expressing Flp recombinase under the control of the human CMV promoter, which can help the target plasmid to recombine into the genome of the host cell) were transferred into Flp-In T-REx HEK293-R-CEPIA1er cells using the transfection reagent Lipofectamine 3000 at a mass ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0025] The cells were digested with EDTA-trypsin digestion solution and passaged. The cells from a 6 cm culture dish were transferred to two 10 cm culture dishes, each containing 8 ml of DMEM complete medium. The culture dishes were placed in a 37°C, 5% CO2 incubator and cultured. After 4-6 hours, 15 μg / ml of blasticidin, 60 μg / ml of hygromycin and 480 μg / ml of geneticin (G418) were added to screen positive monoclonal clones.

[0026] DMEM complete medium (containing blasticidin at a final concentration of 15 μg / ml, hygromycin at 60 μg / ml, and geneticin (G418) at 480 μg / ml) was replaced every 4-5 days, and positive single clones were screened after 15 days.

[0027] Example 2

[0028] Method for determining calcium ion concentration in the endoplasmic reticulum:

[0029] The cells stably transfected with WT sfRyR gene obtained in Example 1 were digested with EDTA-trypsin digestion solution, and the cells were diluted to 1×10 5 100 μL / well was added to a 96-well cell culture dish and cultured in a 37°C, 5% CO2 incubator for 24 h. DMEM complete medium containing a final concentration of 6 μg / ml doxycycline was added at 50 μl / well to induce the expression of the WT sfRyR gene. The cells were cultured in a 37°C, 5% CO2 incubator. After 48 h of cell induction, the DMEM complete medium was removed, 81 μl of HEPES-buffered Krebs buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM glucose, 5 mM HEPES, pH 7.4) was added to each well, and the cells were placed in a Flexstation III instrument preheated at 37°C.

[0030] The parameters of the Flexstation III instrument were set, and the fluorescence value of R-CEPIA1er was recorded for 10s / time at an excitation wavelength of 560nm and an emission wavelength of 610nm, for a total of 300 seconds. At 100s, 54uL of the compound to be tested was added to each well, so that the volume percentage of DMSO in each well was 0.4%, and the compound in each well was C1, C1 = 100uM, where the compound to be tested was a mixture of the compound, DMSO and HEPES-buffered Krebs buffer. The ratio (F / F0) of the average fluorescence intensity of the first 100s (F0) to the average fluorescence intensity of the last 100s (F) was defined as the fluorescence change caused by the compound to be screened.

[0031] Figure 1 This is the F / F0 graph obtained after screening 2113 small molecules (purchased from Cherry Pick Library at Selleck) as compounds. F / F0 between 0.8-1.1 is defined as basically ineffective. The smaller the value, the better the activation effect on the WTsfRyR gene and the stronger the insecticidal effect. From F / F0, we can see that there are 5 compounds with F / F0 below 0.8 ( Figure 1 The colored dots in the graph (excluding the dots outside the five-pointed star) are the compounds that can effectively activate the WT sfRyR gene. Among them, the black five-pointed star between 0.8-1.1 is the negative control (no compound in the compound to be tested), and the black five-pointed star in the lower right corner is the positive control (the compound in the compound to be tested is replaced with "CHL").

[0032] The information of the 5 compounds with F / F0 below 0.8 obtained from the above screening is as follows:

[0033] 1. Benzethonium Chloride (BEN for short)

[0034] The structural formula of BEN is as follows:

[0035] SMILE:[Cl-].CC(C)(C)CC(C)(C)C1=CC=C(OCCOCC[N+](C)(C)CC2=CC=CC=C2)C=C1

[0036] Known targets: Acetylcholine receptor (AChR)

[0037] Known Uses: Benzethonium Chloride is a quaternary ammonium antibiotic, an effective nAChRs inhibitor, a cationic surfactant with broad-spectrum bactericidal activity, and acts as an antiseptic, disinfectant, antibacterial, antiviral, and antifungal agent. BEC is toxic to aquatic organisms. Benzethonium was screened as a negative regulator of the mitochondrial calcium uniporter (MCU).

[0038] 2. Dichlorophene (DIC)

[0039] The structural formula of DIC is as follows:

[0040] SMILE:OC1=CC=C(Cl)C=C1CC2=CC(=CC=C2O)Cl

[0041] Known targets: Vitamin D receptor

[0042] Known Uses: Dichlorophenol is a nontoxic laxative of the chlorinated phenolic class of compounds. Dichlorophenol is used as a veterinary antiseptic, anthelmintic, and antiprotozoal agent, as well as a component of antimicrobial soaps and shampoos. The agent may work by increasing the clearance of intestinal contents, thereby eliminating tapeworm infections in the intestine.

[0043] 3. Enoxolone glycyrrhetinic acid (abbreviated as: ENO)

[0044] The structural formula of ENO is as follows:

[0045] SMILE:CC1(C)C(O)CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(O)=O

[0046] Known target: Dehydrogenase

[0047] Known uses: Enoxolone is a β-amyrin pentacyclic triterpene derivative obtained by hydrolysis of glycyrrhizic acid extracted from the herb liquorice. Its main applications include anti-inflammatory, antioxidant, anti-tumor, antibacterial, and antiviral effects.

[0048] 4. Name: Oxethazaine (abbreviated as OXE)

[0049] The structural formula of OXE is as follows:

[0050] SMILE:CN(C(=O)CN(CCO)CC(=O)N(C)C(C)(C)CC1=CC=CC=C1)C(C)(C)CC2=CC=CC=C2

[0051] Known targets: Others

[0052] Known Uses: It is a strong local anesthetic that has multiple effects, including numbing the stomach lining and relieving pain in highly acidic conditions. Almost all local anesthetics work by reducing the tendency of voltage-dependent sodium channels to activate. Oxethcaine reduces the conduction of sensory nerve impulses near the site of application, thereby reducing the permeability of the cell membrane to sodium ions.

[0053] 5. Name: Ciclopirox (abbreviation: CIC)

[0054] The structural formula of CIC is as follows:

[0055] SMILE:CC1=CC(=O)N(O)C(=C1)C2CCCC2

[0056] Known targets: ATPase, Anti-infection

[0057] Known Uses: Ciclopirox is a broad-spectrum antifungal agent that acts as an iron chelator.

[0058] Example 3

[0059] Experimental group: Injection experiment was performed with wax moth to verify the reliability of the five compounds screened in Example 2. 0.3-0.4g wax moth was selected (store: Jinmen Daxu Traditional Handmade Bird Food; Trade Name: Free Shipping Cornworms Living Grape Honeyworms Greater Wax Moth Honeyworms Bird Food Fishing Bait Honey Bag Hamster Turtle Pet Food,

[0060] https: / / m.tb.cn / h.UlbcsHw? tk=Ck1UdfwiFRR, last visited on January 6, 2023) to conduct experiments. After resuscitation at room temperature, 20uL of compound solution was injected into the hind legs. The compound solution was a mixture of compound, DMSO and phosphate buffer (pH=7.4). The concentration of the compound in the compound solution was 1mM, and the volume percentage of DMSO in the compound solution was 0.5%. The compound was OXE, BEN, DIC, CIC or ENO. The mortality of G. mellonella was recorded one week after injection.

[0061] Positive control: basically the same as the experimental group, the only difference is that the compound in the experimental group is replaced by "CHL";

[0062] Negative control: basically the same as the experimental group, the only difference is that after resuscitation at room temperature, 20uL of negative control solution was injected into the hind legs. The negative control solution was a mixture of DMSO and phosphate buffer (pH=7.4), and the volume percentage of DMSO in the negative control solution was 0.5%.

[0063] The mortality rates of G. mellonella recorded in the experimental group, positive control and negative control at one week are shown in Table 1.

[0064] Table 1

[0065]

[0066] Example 4

[0067] The method for determining the calcium ion concentration in the endoplasmic reticulum was basically the same as that in Example 2, except that "the compound was one of OXE, BEN, DIC, CIC and ENO, and the C1 of each compound was 0.01, 0.1, 1, 5, 10, 50, 100 and 500 uM, respectively" to conduct a concentration-dependent test. The ratio (F / F0) of the average fluorescence intensity of the first 100 s (F) to the average fluorescence intensity of the last 100 s (F) was as follows: Figure 2 As described above, the EC50 of OXE, BEN, DIC, CIC and ENO are 80.37uM, 85.37uM, 105.5uM, 31.17uM and 49.34uM, respectively.

[0068] Example 5

[0069] In order to explore whether these compounds can assist CHL (Chlorantraniliprole, chlorantraniliprole) to increase the activation effect, synergistic experiments were carried out on cells.

[0070] Example 5-1

[0071] The method for determining the calcium ion concentration in the endoplasmic reticulum is basically the same as that in Example 2, with the only difference being that "54uL of the compound to be detected is added to each well at 100s, so that the volume percentage of DMSO in each well is 0.4%, the concentrations of the compound in each well are C1, CHL and C2, respectively, and C2 is 0.00001, 0.001, 0.005, 0.01, 0.1, 0.5, 1 and 10uM, respectively, wherein the compound to be detected is a mixture of the compound, CHL, DMSO and HEPES-buffered Krebs buffer, and the compounds are OXE, BEN, CIC and ENO, respectively. When the compound is OXE, C1=100uM; when the compound is BEN, C1=100uM; when the compound is ENO, C1=100uM; when the compound is CIC, C1=75uM;".

[0072] Example 5-2

[0073] It is basically the same as Example 5-1, the only difference being that there is no compound in the compound to be detected.

[0074] The ratio (F / F0) of the average value of the fluorescence intensity in the first 100 s (F0) to the average value of the fluorescence intensity in the last 100 s (F) in Example 5-1 and Example 5-2 is obtained, as Figure 3 As shown, by comparing the CHL gradient with added compounds ( Figure 3 Medium colored line, Example 5-1) and CHL gradient effect without adding any compound ( Figure 3 The black line in the middle, Example 5-2) shows that the four compounds OXE, BEN, CIC and ENO can increase the activation effect of CHL, and the synergistic effect is obvious below or above a certain concentration.

[0075] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

[0076] R-CEPIA1er is a fluorescent bioreceptor (excitation wavelength 560nm, emission wavelength 610nm), which is free in the endoplasmic reticulum and can dynamically monitor the concentration of calcium ions in the endoplasmic reticulum. It is composed of a ring-shaped red fluorescent protein, camodulin (CaM), kinase M13, and SEKDEL short peptide. Kinase M13 is connected to the N-terminus and C-terminus of CaM. When calcium ions bind to CaM, the conformation of kinase M13 and CaM changes, which in turn causes changes in the binding interface between CaM and red fluorescent protein and changes in fluorescence. The SEKDEL short peptide at the C-terminus can bind to receptors in the endoplasmic reticulum to ensure that the protein it carries is expressed in the endoplasmic reticulum. When the pesticide acts on the nicotinic acid receptor, the nicotinic acid receptor opens, calcium ions flow out, the calcium ion concentration in the endoplasmic reticulum decreases, and the fluorescence value of R-CEPIA1er decreases.

Claims

1. Use of a compound in regulating calcium ion concentration in the endoplasmic reticulum of insect cells, characterized in that: The compound is one of benzethonium chloride, dichlorophenol, glycyrrhetinic acid, oxethacaine and ciclopirox; The structural formula of the dichlorophenol is as follows:

2. The use according to claim 1, characterized in that: The insects are Lepidoptera, Diptera, Coleoptera or Hemiptera.

3. The use according to claim 1 or 2, characterized in that: The compound directly or indirectly regulates the activity of nicotinic acid receptors and thus regulates the calcium ion concentration in the endoplasmic reticulum of insect cells.

4. Use of a compound in synergistically enhancing the effect of diamide insecticides, characterized in that: The compound is one of benzethonium chloride, glycyrrhetinic acid, oxethacaine and ciclopirox.

5. The use according to claim 4, characterized in that: The structural formula of the diamide insecticide is one of the following six:

Images