Application of active agent for inhibiting catalytic activity of JHAMT enzyme of tribolium cavaleriei in medicine for preventing and treating tribolium cavaleriei and method

Abstract

The invention discloses application of an active agent for inhibiting JHAMT enzyme catalytic activity of tribolium castaneum in a medicine for preventing and treating tribolium castaneum and a method. According to the present invention, the active agent capable of inhibiting the enzymatic activity of Tribolium cassiae JHAMT is screened by using Tribolium cassiae JHAMT as the target, and the results show that the larva ecdysis failure, the pupation ecdysis failure or the pupa blackening and the expression quantity of the juvenile hormone downstream response gene are affected in the experiment process, such that the new insecticide can be used for preventing and treating Tribolium cassiae.

Description

Technical Field

[0001] The present invention relates to the field of insect control, in particular to the use of an active agent for inhibiting the catalytic activity of JHAMT enzyme of red flour beetle in a drug for controlling red flour beetle, and also to a method for controlling red flour beetle. Background Art

[0002] Food is the fundamental material foundation for human survival. Seasonal production necessitates post-harvest storage of grain. During this storage process, pests account for the largest portion of grain losses. As a representative coleopteran pest in storage ecosystems, the red flour beetle occupies a special research position in the field of global food security. Due to its outstanding environmental adaptability, pronounced omnivorous nature, unique gregarious behavior, and long life cycle (averaging 1-2 years), this species has become a dominant pest in the post-harvest storage of grass crops.

[0003] Juvenile hormone (JH) is a key hormone in insect growth, development, and reproduction. Fluctuations in its levels influence metamorphosis. Within the insect endocrine system, JH is synthesized and secreted by the Corpora Allata (CA), forming a cascade with the prothoracic steroid hormone 20-hydroxyecdysone (20E). These two hormones synergistically mediate insect development through temporal titer oscillations: JH reaches a characteristic peak at the beginning of each larval instar, maintaining larval morphogenesis; 20E increases significantly toward the end of each instar, triggering the larval-to-pupae transition. During the final larval instar, JH levels plummet, accompanied by a surge in 20E concentration (reaching the metamorphosis threshold), jointly initiating larval-pupae developmental remodeling. Furthermore, the JH signaling pathway is involved in regulating insect reproductive physiology and aging.

[0004] As the terminal rate-regulating enzyme in the juvenile hormone biosynthesis pathway, JHAMT continues to attract research attention due to its critical role in metabolic control. In 2003, Shinoda's team first identified the coding sequence of juvenile hormone acid methyltransferase (JHAMT) in the silkworm Bombyx mori. This landmark discovery provided a key molecular target for the study of insect developmental endocrine regulation. The cloning of the JHAMT gene not only revealed the final enzymatic step in JH biosynthesis, but also highlighted its evolutionary functional importance within its highly conserved catalytic domain. Because JHAMT exhibits strict substrate specificity in insects and lacks homology to vertebrate methyltransferases, inhibitors targeting its active site can achieve selective toxicity, providing a theoretical basis for the design of novel insect growth regulators (IGRs). In the red castaneum beetle, RNAi-mediated knockout of target genes is highly efficient and easy to perform. In 2008, Minakuchi et al. reported three methyltransferase genes from the red castanea beetle. Studies of developmental and spatial expression patterns, as well as the enzymatic properties of the recombinant proteins, revealed that only TcMT3 encodes a functional JHAMT. TcMT3 RNAi resulted in premature pupation in larvae, with most larvae emerging at the end of the sixth instar. These pupae and adults injected with dsTcMT3 appeared normal in appearance but were significantly smaller than normal individuals. These results indicate that the JHAMT gene is essential for JH biosynthesis and maintenance of the larval state.

[0005] Therefore, blocking the JH biosynthesis pathway is an effective approach for developing new pesticides. As the key rate-limiting enzyme in the JH biosynthesis pathway, JHAMT is an important target for designing new pesticides. Furthermore, since humans, livestock, and most aquatic animals do not undergo juvenile hormone-related physiological processes, new pesticide molecules designed with JHAMT as a target are harmless to humans and animals and are more environmentally friendly. Summary of the Invention

[0006] In view of this, one object of the present invention is to provide an active agent for inhibiting the catalytic activity of the red flour beetle JHAMT enzyme for use in drugs for preventing and controlling red flour beetles; a second object of the present invention is to provide a method for preventing and controlling red flour beetles.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: Application of an active agent for inhibiting the catalytic activity of JHAMT enzyme of red castaneum in medicine for preventing and controlling red castaneum.

[0008] Preferably, the active agent of the present invention is (S)-1-(2-oxoindolin-3-yl)-3-(2-(quinolin-8-yl)ethyl) urea.

[0009] Preferably, the control of red flour beetle is through larval molting failure, pupation molting failure or pupal melanization.

[0010] Preferably, the control of red castanea is to reduce the expression of JHAMT gene, JH receptor Met gene, Kr-h1 gene, juvenile hormone binding protein JHBP; and increase the expression of ecdysone early response gene broad complex Br gene.

[0011] A method for controlling red flour beetles comprises mixing an active agent (S)-1-(2-oxoindolin-3-yl)-3-(2-(quinolin-8-yl)ethyl) urea with stored grains or flour, and then feeding the mixture to red flour beetle larvae.

[0012] The beneficial effects of the present invention lie in its ability to effectively develop new insecticides by blocking the JH synthesis pathway, while designing JHAMT, a key rate-limiting enzyme in the JH synthesis pathway, as a key target site for new pesticides. Furthermore, since humans, livestock, and most aquatic animals do not have physiological processes associated with juvenile hormone, new insecticide molecules designed with JHAMT as a target are harmless to humans and animals and more environmentally friendly. This study targeted JHAMT in the red flour beetle (Tribolium castaneum) to screen for agents that inhibit the catalytic activity of the JHAMT enzyme. Experimental findings revealed that larval molting failure, pupation failure, or melanization of pupae affected the expression of genes downstream of juvenile hormone, suggesting potential applications for controlling the red flour beetle. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration: Figure 1 Screening for small molecule inhibitors; Figure 2 IC50 values ​​of small molecule inhibitors (A: structural model of JI3 binding to TcJHAMT; B: IC50 value of inhibitor JI3 against TcJHAMT); Figure 3 To investigate the effect of feeding JI3 on ​​the growth and development of Tribolium castaneum; Figure 4 Death phenotype after adding JI3 and expression levels of JH downstream response genes. DETAILED DESCRIPTION

[0014] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0015] Example 1 Small molecule virtual screening: Based on the reported three-dimensional structure of the juvenile hormone acid methyltransferase of Tribolium castaneum (PDB: 00009un4), the JHA binding region was used as the docking pocket for virtual screening. Schrödinger Maestro 11.4 software was used to virtually screen the Enamine small molecule compound library containing approximately 2 million molecules. Based on the scoring function and manual selection, 38 small molecules were selected for in vitro activity testing. The specific molecules are shown in Table 1.

[0016] Table 1. Candidate small molecule compounds N ID_number docking_score Drug_Names Mw 1 Z224215260 -13.504 N-(5-(3-chlorobenzyl)thiazol-2-yl)-2-(4-(4-hydroxyphenyl)piperazin-1-yl)acetamide 442.96 2 Z1590977493 -13.447 (S)-1-(4-(morpholine-4-carbonyl)phenyl)-3-(1-(4-phenyl-1H-imidazol-2-yl)propyl)urea 433.50 3 Z485131088 -13.191 (S)-1-(2-oxoindolin-3-yl)-3-(2-(quinolin-8-yl)ethyl)urea 346.38 4 Z242812356 -12.967 (S)-1-((4-(benzyloxy)phenyl)amino)-1-oxopropan-2-yl 3-((2,5-dioxoimidazolidin-1-yl)methyl)benzoate 487.50 5 Z1567480713 -12.696 (S)-N-(5-(3-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-2-(3-phenyl-1H-1,2,4-triazol-5-yl)pyrrolidine-1-carboxamide 449.50 6 Z729482416 -12.524 (R)-2-(2-acetyl-1,2-dihydroisoquinolin-1-yl)-N-(2-(2-fluorobenzyl)-1H-benzo[d]imidazol-5-yl)acetamide 454.50 7 Z235433512 -12.351 3-(2-oxo-3-propyl-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-N-(3-(3-phenylureido)phenyl)propanamide 457.52 8 Z1476770574 -12.279 N-(4,5-diphenyloxazol-2-yl)-2-((3-methyl-1,2,4-oxadiazol-5-yl)methoxy)acetamide 390.39 9 Z406436052 -12.211 1-(3-((benzyloxy)methyl)benzyl)-3-cyclopropylthiourea 326.46 10 Z1420248082 -12.185 N-((1H-imidazol-2-yl)methyl)-3-acetamido-5-(trifluoromethyl)benzamide 326.27 11 Z806233880 -12.061 (S)-1-(2-(1H-pyrrol-2-yl)pyrrolidin-1-yl)-3-(2,5-difluorophenyl)propan-1-one 304.33 12 Z2865591287 -12.052 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-methyl-1H-pyrazole-3-carboxylic acid 363.37 13 Z183079270 -12.048 3-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)-N-(3,3-diphenylpropyl)propanamide 427.50 14 Z1833746849 -11.973 (S)-1-(2-ethoxy-5-(trifluoromethyl)phenyl)-3-(5-oxopyrrolidin-3-yl)urea 331.29 15 Z278073194 -11.885 (S)-N-(5-(3,5-difluorobenzyl)thiazol-2-yl)-2-(1,3-dioxoisoindolin-2-yl)-4-(methylthio)butanamide 487.54 16 Z1429162780 -11.866 2-(dibenzylamino)-N-(3-methoxy-4-ureidophenyl)acetamide 418.49 17 Z18766510 -11.830 2-((2-chloro-5-(trifluoromethyl)phenyl)amino)-2-oxoethyl 2-benzylbenzoate 447.83 18 Z414876960 -11.821 N-(3-((3-oxo-[1,2,4]triazolo[4,3-a]pyridin-2(3H)-yl)methyl)phenyl)-3,3-diphenylpropanamide 448.52 19 Z85929273 -11.757 (E)-N'-((3-bromo-2-hydroxy-6,7,8,9-tetrahydrodibenzo[b,d]furan-1-yl)methylene)-2-oxo-1,2-dihydroquinoline-4-carbohydrazide 480.31 20 Z48964194 -11.744 (S,Z)-2-(2-((2-methoxybenzyl)imino)-4-oxothiazolidin-5-yl)-N-(naphthalen-1-yl)acetamide 419.50 21 Z1090047148 -11.724 (S)-4-(((4,5-dihydro-1H-imidazol-2-yl)amino)methyl)-N-((tetrahydrofuran-2-yl)methyl)benzenesulfonamide 338.43 22 Z59756648 -11.712 N-(3,3-diphenylpropyl)-1H-benzo[d][1,2,3]triazole-5-carboxamide 356.42 23 Z56976914 -11.670 2-((3-cyano-4,5-diphenylfuran-2-yl)amino)-2-oxoethyl 2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 456.41 24 Z1655373272 -11.650 (S)-N-((R)-1-(1H-benzo[d]imidazol-2-yl)-2-(4-hydroxyphenyl)ethyl)-2-(3,5-dimethyl-1H-pyrazol-4-yl)propanamide 403.48 25 Z744820942 -11.639 (S)-1-(2-(1H-pyrrol-2-yl)pyrrolidin-1-yl)-2-(3-(trifluoromethyl)phenyl)ethanone 322.32 26 Z217185964 -11.629 2-([1,1'-biphenyl]-2-ylamino)-N-(6-amino-1-benzyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-isobutylacetamide 497.59 27 Z443937456 -11.570 5-(2-(3-(cyclohex-1-en-1-yl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamido)-2-fluorobenzamide 408.43 28 Z739677182 -11.543 2-((4-fluoro-3-(2-phenylacetamido)phenyl)amino)-N-(2-methylbenzyl)acetamide 405.46 29 Z651881592 -11.527 N-(1-(2-amino-2-oxoethyl)-1H-pyrazol-4-yl)-3,3-diphenylpropanamide 348.40 30 Z1631823889 -11.520 N-(5,8-dichloroquinolin-2-yl)-2-oxo-1,2-dihydropyridine-4-carboxamide 334.16 31 Z997775990 -11.496 4-(N-(4-(cyanomethyl)phenyl)sulfamoyl)-1H-pyrrole-2-carboxylic acid 305.31 32 Z51932439 -11.487 2-(benzhydryloxy)-N-(4-(pyridin-2-yl)thiazol-2-yl)acetamide 401.48 33 Z335395056 -11.480 2-((S)-4-cyclopropyl-4-methyl-2,5-dioxoimidazolidin-1-yl)-N-((3aR,4R,5R,7R,7aR)-octahydro-1H-4,7-methanoinden-5-yl)acetamide 345.44 34 Z1741964537 -11.473 4-((((9H-芴-9-基)甲氧基)羰基)氨基)-1-甲基-1H-吡咯-2-羧酸 362.38 35 Z33448130 -11.464 N-(2,3-二氢-1H-茚-5-基)-3-(N-(2-甲氧基苯基)氨磺酰基)苯甲酰胺 422.50 36 Z51853606 -11.431 N-(3-乙酰氨基苯基)-2-(二苯甲氧基)乙酰胺 374.43 37 Z2863938052 -11.426 ((1R,2S)-2-环己基环丙基)(3-羟基-3-(1H-1,2,3-三唑-5-基)氮杂环丁烷-1-基)甲酮 290.36 38 Z220035498 -11.582 3-((2-((4-氯-3-(三氟甲基)苯基)氨基)-2-氧代乙基)(甲基)氨基)-N-(2-氟苯基)丙酰胺 431.81 Initial in vitro screening of candidate inhibitors: Based on the crystal structure analysis and catalytic mechanism analysis of insect JHAMT, computer-assisted screening was used to identify small molecules with optimal binding phases. These selected small molecules were then tested for their inhibitory efficacy in vitro. High-performance liquid chromatography was used as the method for determining the inhibitory efficacy of small molecules.

[0017] The specific detection methods are as follows: (1) Pipette 25 μL of 2 mg / mL JHAMT protein solution and inhibitor small molecule compound (final concentration 300 μM) and place in the reaction chamber at room temperature in the dark for 30 min; (2) Add the methyl donor small molecule SAM with a final concentration of 400 μM and the substrate small molecule FA with a final concentration of 80 μM to the reaction solution in sequence; (3) Add 50 mM HEPEs buffer (pH 7.5) to the reaction system to make up to 500 μL; (4) Gently invert the mixture 3-5 times and react at room temperature in the dark for 30 min. (5) Add an equal volume of acetonitrile solution to terminate the reaction and vortex for 5 min; (6) Centrifuge at 15,000 g for 6 min at room temperature and carefully aspirate the upper product extract; (7) Pipette 150 μL of the upper layer solution into a liquid chromatography glass liner tube and use the liquid chromatography machine to detect the yield of the enzyme-catalyzed reaction product MF. Each group of samples was repeated three times. (8) Turn on the high performance liquid chromatograph and replace the mobile phase with ultrasonically degassed acetonitrile solution and ultrapure water solution.

[0018] The liquid chromatograph program settings are as follows: Mobile phase: 80% acetonitrile in water Flow rate 1.0 mL / min Detection temperature 30℃ Detection wavelength 217nm Test duration: 15 minutes In vitro activity assays such as Figure 1 The results show that small molecule compound No. 3 (JI3, (S)-1-(2-oxoindolin-3-yl)-3-(2-(quinolin-8-yl)ethyl) urea) exhibited high inhibitory efficiency against JHAMT in Tribolium castaneum, reaching 80%. This suggests that a certain conformation or group within JI3 is highly specific for insect JHAMT. Small molecules or groups with this conformation are more affinity for insect JHAMT, thereby blocking the methylation of the substrate FA and affecting MF production.

[0019] Small molecule compound No. 3 was purchased from MCE Company and has the following structure: Example 2 IC of inhibitor 50 The detection process of the inhibitor parameter IC was the same as that of the initial screening of small molecule inhibitor compounds, with the addition amount of the small molecule compound as the only variable. The addition concentrations of the inhibitor small molecule compound in the test were set as: 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 150 μM, 250 μM, 350 μM, and 500 μM. The analysis software Graphpad Prism 8.0 was used to analyze the inhibitor parameter IC of the liquid phase test data. 50 The fitting value.

[0020] IC of inhibitor 50 It is one of the criteria for judging whether a drug is highly effective in drug screening analysis. The JI3 with high in vitro enzyme activity inhibition efficiency screened above is used as the inhibitor IC 50 The detection data were analyzed and processed by Graphpad Prism 9.0 software. IC of each inhibitor small molecule 50 The fitting diagram is as follows Figure 2 The results show that the curve trend of the inhibitor and the inhibitor parameter IC 50 The numerical comparison shows that: JI3 IC 50 The trend is good and IC 50 The value is relatively low, which makes it an ideal inhibitor small molecule.

[0021] Example 3 Use a 60-mesh sieve to screen the eggs laid by adult red flour beetles within 24 hours. Collect the eggs and place them in an incubator until the larvae hatch. Collect the larvae that hatch within 12 hours and place the 12-hour larvae in fresh flour for 14 days. At this time, the growth status and age of the larvae are basically the same. The feeding experiment is carried out with 30-40 larvae per group.

[0022] Screening for appropriate concentration of the agent: Set 5 concentration gradients of the agent (20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm), acetone solution as the control group, take 6 disposable cups, accurately weigh 2 g of flour and put it in the cup, take 1 mL of each concentration of the agent and put it into a clean atomizing spray bottle, spray it evenly on the flour in small amounts several times, stir it evenly with a clean brush, dry it in a 40℃ oven, put the prepared 14-day-old larvae of Tribolium castaneum in each group of 30-40 in disposable cups, and write the group on the wall of the cup. After 48 hours, feed the larvae with normal flour, count the larval mortality rate every 48 hours, take pictures and record the phenotype of the dead individuals in time, and the mortality rate of the control group shall not exceed 15%. The results showed that the survival rate of the larvae decreased to varying degrees after the addition of JI3, and most of the larvae could not successfully pupate and metamorphose into adults. The dead individuals mainly appeared in the larval and pupal stages ( Figure 3 ). In addition, the red flour beetle individuals showed death phenotypes such as larval molting failure, pupation molting failure, and pupal melanization ( Figure 4 , A).

[0023] To further determine whether this mortality was due to blocked JH synthesis, a 100 ppm concentration was selected, resulting in approximately 50% larval mortality. The experiment was repeated using acetone as a control. Flour treated as described above was then added to 30 larvae, with three replicates per treatment. Larvae were sampled at 0, 4, and 6 days after feeding to examine the expression of downstream genes responsive to juvenile hormone in Tribolium castaneum. Quantitative PCR was used to examine the expression levels of JH downstream genes (Krüppel homologue 1, Kr-h1), the methoprene-tolerant JH receptor (Met), the JH binding protein (JHBP), and the ecdysone early response gene broad complex (Br). Results showed that after feeding JI3, JHAMT gene expression significantly decreased after the addition of the inhibitor, JH receptor (Met) gene expression significantly decreased on the 6th day, JH downstream response gene (Kr-h1) expression was extremely significantly downregulated, juvenile hormone binding protein (JHBP) expression significantly decreased on the 4th day, and ecdysone early response gene broad complex (Br) was significantly upregulated. This suggests that the addition of JI3 not only blocked JH signaling, but also caused ecdysone to function prematurely, leading to molting failure and death in red castaneum individuals, resulting in reduced individual survival.

[0024] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Application of an active agent that inhibits the catalytic activity of the JHAMT enzyme of red flour beetle in drugs for preventing and controlling red flour beetle.

2. The use according to claim 1, characterized in that: The active agent is (S)-1-(2-oxoindolin-3-yl)-3-(2-(quinolin-8-yl)ethyl)urea.

3. The use according to claim 1, characterized in that: The method of preventing and controlling the red flour beetle is to prevent and control the red flour beetle by larval molting failure, pupation molting failure or pupal melanization.

4. The use according to claim 1, characterized in that: The method for preventing and controlling red castaneum beetle is to reduce the expression of JHAMT gene, JH receptor Met gene, Kr-h1 gene and juvenile hormone binding protein JHBP; and to increase the expression of Br gene, a broad complex of ecdysone early response genes.

5. A method for preventing and controlling red flour beetle, characterized in that: The active agent, (S)-1-(2-oxoindolin-3-yl)-3-(2-(quinolin-8-yl)ethyl) urea, was mixed with stored grain or flour and then fed to red flour beetle larvae.

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