Selective nematicidal compounds based on pinewood nematode neuropeptide receptors and uses thereof

Abstract

The application relates to a small molecule lead compound for killing Bursaphelenchus xylophilus and application thereof, and the chemical formula of the small molecule lead compound is respectively C 25 H 27 ClN2O3S、C 30 H 32 N2O 6、 C 27 H 30 N6O, and the semi-lethal concentrations of the small molecule lead compound for killing Bursaphelenchus xylophilus are respectively 9.30 mg / L, 8.76 mg / L and 9.19 mg / L. The small molecule lead compound can effectively inhibit the feeding capacity and population quantity of Bursaphelenchus xylophilus, and shows good nematode killing potential. The small molecule lead compound has good application prospect in the prevention and treatment of Bursaphelenchus xylophilus disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural technology, and particularly relates to a selective nematicidal compound based on a pine wood nematode neuropeptide receptor and its use. BACKGROUND

[0002] Pine wood nematode disease is one of the most dangerous and destructive diseases in the global forest ecosystem, with strong spreading and destructive properties. Asia is the most severely affected region of the pine wood nematode disease epidemic. At the present stage, the most common method for preventing and treating pine wood nematodes is chemical control. At present, chemical control mainly relies on abamectin and emamectin benzoate. From 2019 to the first half of 2021, the use amount of abamectin and emamectin benzoate for preventing and treating pine wood nematodes accounted for more than 99.9% of the total use amount. However, from the perspective of reasonable application of pesticides, long-term use of the same or same mechanism of action of pesticides can easily lead to the development of pesticide resistance in pine wood nematodes and medium insects, which is not conducive to the sustainable prevention and control of pine wood nematode disease. Therefore, it is urgent to accelerate the development of new pesticides for pine wood nematode disease prevention and control, so as to continuously improve the overall level of pine wood nematode disease prevention and control.

[0003] In order to cope with the evolution of pest resistance to insecticides and reduce the toxicity to non-target organisms, the discovery and development of new targets have become a research hotspot in recent years. Nematode neuropeptide receptors, as a class of G protein-coupled receptors (GPCR), play a key role in regulating nematode growth, development, reproduction, behavior and stress response, and are a class of potential drug targets. Therefore, the development of selective nematicidal compounds targeting neuropeptide receptors has become a new direction for the prevention and control of pine wood nematode disease. These compounds are expected to become an important breakthrough in the field of pine wood nematode disease prevention and control, and provide strong support for the protection and sustainable development of forest ecosystems. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application aims to provide a selective nematicidal compound based on a pine wood nematode neuropeptide receptor and its application.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0006] A small molecule lead compound for preventing and treating pine wood nematodes, with a chemical formula of C 25 H 27 Cl N2 O3 S, C 30 H 32 N2 O6 or C 27 H 30 N6 O, corresponding to manufacturer numbers E535-6542, 3343-3114 and F646-0173.

[0007] The application of the small molecule lead compound in preventing and treating Bursaphelenchus xylophilus.

[0008] The application of the pharmaceutical composition containing the small molecule lead compound or salt thereof as an effective ingredient in preventing and treating Bursaphelenchus xylophilus.

[0009] The pharmaceutical composition contains the small molecule lead compound, emamectin benzoate or abamectin, and a pharmaceutically acceptable adjuvant.

[0010] The pharmaceutical composition contains a pharmaceutically acceptable dosage form, including dry powder, wettable powder, emulsion, microemulsion, paste, granule, solution or suspension.

[0011] Advantages of the present application:

[0012] (1) The present application uses computer-aided drug design means to efficiently screen the lead compound from the receptor structure. The key neuropeptide receptor protein of Bursaphelenchus xylophilus is selected as the target, and the molecular docking technology based on the receptor strategy is used for virtual screening of the commercial database, so that the selective nematicidal lead compound with a new mechanism of action is efficiently and quickly screened. The present application provides a technical reference for screening new nematicidal lead compounds based on the receptor structure, and also provides a research idea for the future two-way development of pesticides and medicines based on the neuropeptide receptor target.

[0013] (2) The present application uses the selective nematicidal compound based on the neuropeptide receptor of Bursaphelenchus xylophilus to prevent and treat Bursaphelenchus xylophilus. The three compounds with IDs E535-6542, 3343-3114 and F646-0173 obtained by screening and verification have good inhibition effect and insecticidal activity on the growth of nematodes, and can be used as potential drugs for preventing and treating Bursaphelenchus xylophilus, thereby providing a new type of control agent for preventing and treating Bursaphelenchus xylophilus.

[0014] (3) The LC 50 (Half lethal concentration) of the small molecule lead compound of the present application is 9.30 mg / L, 8.76 mg / L and 9.19 mg / L respectively, which can effectively inhibit the feeding ability and population number of Bursaphelenchus xylophilus, and shows good nematicidal potential. The small molecule lead compound can be applied to the preparation of a drug preparation for killing Bursaphelenchus xylophilus, and has good application prospect in controlling the pine wood nematode disease. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Effect of the neuropeptide receptor lead compound of Bursaphelenchus xylophilus on the feeding of Bursaphelenchus xylophilus;

[0016] Figure 2 Effect of the neuropeptide receptor lead compound of Bursaphelenchus xylophilus on the population number of Bursaphelenchus xylophilus;

[0017] Figure 3Bursaphlenchus xylophilus nematode inoculation experiment with lead compound of nematode neuropeptide receptor. DETAILED DESCRIPTION

[0018] The specific embodiments of the present application are further described in detail below with reference to the following examples. Unless otherwise specified, the instruments involved are conventional instruments, the raw materials and reagents involved are commercially available, and the test methods involved are conventional methods.

[0019] The test strain Botrytis cinerea used for the nematode was donated by the laboratory of Zhejiang University, and was then subcultured in the laboratory of the applicant to feed the Bursaphlenchus xylophilus in the experimental environment.

[0020] The Bursaphlenchus xylophilus used for the experiment was provided by the Forest Ecology and Conservation Institute of Chinese Academy of Forestry, which was originally from the diseased Pinus massoniana in Ningbo, Zhejiang Province, and was subcultured after purifying the family in the laboratory.

[0021] Example 1 Preliminary discovery of lead compound of Bursaphlenchus xylophilus nematode neuropeptide receptor

[0022] Based on multi-omics analysis and RNAi functional verification, three important drug target genes Bxy-npr-5 (CAG9123830.1), Bxy-npr-11 (CAD5220757.1), and Bxy-pcdr-1 (CAG9131797.1) were screened out. Using computer-aided drug design methods, the three-dimensional structure of the nematode neuropeptide receptor was predicted using AlphaFold 2, and the original structure was subjected to molecular dynamics simulation using Desmond. The lowest energy and best configuration of the nematode neuropeptide receptor three-dimensional structure was obtained by clustering analysis of the stable receptor conformation. Based on different docking strategies, high-throughput virtual screening was performed.

[0023] First, 13463 compounds were screened using Glide HTVS (high-throughput screening) mode; 1346 compounds were screened out in the second screening using Glide SP (standard precision) mode; then 134 compounds were screened out in the third screening using Glide XP (extra precision) mode, and finally they were rescored by MM-GBSA method to calculate their binding free energy (ΔG Bind ). According to the "drug-like five principles" (molecular weight less than 500, number of hydrogen bond donors less than 5, number of hydrogen bond acceptors less than 10, octanol-water partition coefficient less than 5), the MM-GBSA results were screened, and 21 compounds were screened out which ranked in the top eight in terms of binding free energy score and met the "drug-like five principles" (Table 1). The detailed information of the 21 screened compounds is shown in Table 2. These compounds can be obtained through commercial channels.

[0024] Table 1 Binding free energy values (kcal / mol) of the top 8 compounds in three neuropeptide receptors

[0025]

[0026]

[0027] Example 2 Nematicidal activity assay of lead compounds of pine wood nematode neuropeptide receptors

[0028] 1. Obtaining pine wood nematode juveniles

[0029] The pine wood nematodes were inoculated on a plate of potato dextrose agar medium (PDA) under dark conditions at 25°C and incubated for 3-4 days, at which time most of the female worms entered the egg-laying stage. The worms were separated using a Baermann funnel method, and the mixed-age worms obtained were washed three times with sterile water. The worms were placed in a culture dish under dark conditions at 25°C for 1-4 h to lay eggs. The sterile water containing the worms in the dish was poured out, and then clean sterile water of the appropriate age was carefully and slowly added along the edge of the dish, and repeated multiple times until the worms in the dish were completely washed out, leaving only the embryos tightly adhering to the bottom of the dish. Sterile water was added to cover the bottom of the dish, and synchronized development of the embryos of the pine wood nematode was obtained. The embryos hatched into second instar larvae after 24 h.

[0030] 2. Preliminary screening of insecticidal activity

[0031] The 21 small molecule lead compounds screened were evaluated for insecticidal activity. First, 2 mg of the test compound was accurately weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10,000 mg / L stock solution. Then, the stock solution was diluted to 200 mg / L, and a 96-well cell culture plate was prepared. In each well, 100 μL of a solution containing 200 mg / L of the test compound was added, followed by the addition of 100 μL of a suspension of pine wood nematode second instar larvae (the supernatant obtained by centrifugation of the second instar larvae cultured above), and the final concentration of the test compound was 100 mg / L. Three replicates were set up for each treatment, and an equal amount of DMSO was used as a blank control.

[0032] The 96-well cell culture plate was then placed in a constant temperature incubator at 25°C and incubated in the dark. After 24 h of treatment, the nematodes were transferred to clean water and allowed to stand for 2 h, after which the survival or death of the pine wood nematodes was observed under a microscope, and the corrected mortality rate was calculated.

[0033] The criteria for determining whether the pine wood nematodes were alive or dead were as follows: when moving or the worm body was in the shape of "S", curled, wavy, or spiral, it was determined to be alive; when the worm body was still not moving after being nudged by the outside world, or was in the shape of "J" or "C", the worm body was rigid, and the body wall had no refraction, it was determined to be dead.

[0034] Calculation formula:

[0035]

[0036] The results are shown in Table 3. When the concentration of the small molecule compounds was 100 mg / L, the corrected mortality of the second instar larvae of P. flandersi of five compounds (E535-6542, 3343-3114, T7683, K279-0416 and F646-0173) of the target NPR-11 was more than 72%.

[0037] Table 3 Corrected mortality of small molecule compounds in virtual screening hits on P. flandersi

[0038]

[0039] Note: The final concentration of the application of 21 small molecule compounds was 100 mg / L.

[0040] 2. Re-screening of insecticidal activity of lead compounds of P. flandersi neuropeptide receptor

[0041] The compounds (E535-6542, 3343-3114, T7683, K279-0416 and F646-0173) with corrected mortality more than 50% in the primary screening were further screened.

[0042] The mother liquor of the above small molecule compounds was diluted to 64 mg / L, 32 mg / L, 16 mg / L and 8 mg / L, respectively, and three replicates were set for each concentration; an equal amount of DMSO was used as a blank control, and an existing nematicide Fluopyram was used as a positive control.

[0043] The DPS (v7.05) software and the probability value method were used for toxicity regression analysis of the lead compounds, and the LC 20 , LC 50 , LC 90 values and the corresponding 95% confidence intervals were calculated.

[0044] The results are shown in Table 4. The LC 50 values of compounds E535-6542, 3343-3114 and F646-0173 on the second instar larvae of P. flandersi were 9.30 mg / L, 8.76 mg / L and 9.19 mg / L, respectively, which were close to the positive control Fluopyram (LC 50 = 1.01 mg / L), indicating that the three compounds showed good nematicidal potential and could be used as preferred lead compounds for nematicides.

[0045] Table 4 In vitro toxicity determination of three compounds on P. flandersi

[0046]

[0047] The three compounds E535-6542, 3343-3114 and F646-0173 correspond to the following names respectively:

[0048] 5-{[(3-Chlorophenyl)methanesulfinyl]methyl}-N-[3-(1,2,3,4-tetrahydroisoquinolin-2-YL)pro pyl]furan-2-carboxamide, structural formula as follows I;

[0049] 2-[[1-[3-(2,3-Dihydro-1,4-benzodioxin-3-ylmethoxy)-2-hydroxypropyl]piperidin-4-yl]methyl]benzo[de]isoquinoline-1,3-dione, structural formula as follows II;

[0050] N-[3-(4-Benzylpiperidin-1-YL)propyl]-3-{3H-[1,2,3]triazolo[4,5-B]pyridin-3-YL}benzamide, structural formula as follows III.

[0051]

[0052] Example 3 Effect of lead compounds of neuropeptide receptor on the population reproduction number of pine wood nematode

[0053] In order to study the effect of three lead compounds (E535-6542, 3343-3114 and F646-0173) on the population reproduction number of pine wood nematode, the experiment was divided into control group and treatment group. First, the synchronized reproductive second instar pine wood nematode larvae were collected, and the larvae were soaked in three lead compounds (E535-6542, 3343-3114 and F646-0173) with a concentration of 5 mg / L for 24 h as the treatment group. 100 second instar larvae were randomly selected from the three compound treated nematodes, and 100 second instar larvae were transferred to fresh Botrytis cinerea plates for culture, and the DMSO soaked reproductive second instar larvae for 24 h were used as the control group. When the control group (DMSO) of Botrytis cinerea was eaten up, all other groups of pine wood nematodes were collected by Bellman funnel method, heat-killed, and the number of insect population was counted. The experiment was repeated three times, and the data difference analysis was carried out by t test.

[0054] The experimental results showed that after 7 days, the *Botrytis cinerea* in the control group culture dishes had been completely consumed by *Pinus fibrosum*, but *Botrytis cinerea* could still be seen in the treatment groups treated with the three compounds. Figure 1 The populations of pine wilt nematodes treated with the three compounds were 1091±62, 990±78, and 1059±59, respectively, while the population of the control group was 10533±1103. The populations in each treatment group were significantly different from those in the control group, indicating that the three compounds significantly reduced the population of pine wilt nematodes. Figure 2 ).

[0055] Example 4: Application Test of Neuropeptide Receptor Lead Compound

[0056] Second-instar pine wilt larvae were collected and treated with three lead compounds (E535-6542, 3343-3114 and F646-0173) at a concentration of 5 mg / L for 24 h as treatment groups. Meanwhile, reproductive second-instar larvae treated with DMSO for 24 h served as control groups. Black pine seedlings were then inoculated.

[0057] Using the "T" inoculation method, 1000 treated second-instar larvae were inoculated into the upper and middle parts of the trunk of Japanese black pine seedlings. The inoculated black pines were then cultured under natural conditions, and the disease incidence and mortality rates of the pine trees in different groups were observed and recorded.

[0058] The results are as follows Figure 3 As shown, all black pines in the control group withered after 40 days, with a mortality rate of 100%. All black pines in the three compound treatment groups survived after 40 days, with a mortality rate of 0%, indicating that these three lead compounds have a very good effect in controlling pine wilt nematodes.

[0059] The lead compound or its salts of the present invention can be used alone as a drug for the control of pine wood nematodes, or can be combined with other substances for the control of pine wood nematodes to form a drug composition. The drug composition includes the lead compound of the present invention, and other components include, but are not limited to, substances such as abamectin or emamectin.

[0060] The drug or pharmaceutical composition also contains pharmaceutically acceptable excipients and can be formulated into any pharmaceutically acceptable dosage form, including but not limited to dry powder, wettable powder, emulsion, microemulsion, paste, granules, solution or suspension.

[0061] The lead compound of the present invention or its salts, or a pharmaceutical composition including the lead compound of the present invention, can be directly applied to pine wood nematodes or their habitats to control the damage caused by pine wood nematodes.

[0062] It should be noted that the embodiments of the present application are not limited to the content described in the embodiments, and those skilled in the art can make similar modifications or equivalent replacements based on the concept of the present application, and such modifications or replacements all belong to the protection scope of the present application.

Claims

1. The application of a small molecule lead compound in the control of pine wood nematode, characterized in that: The chemical formula of the compound is C. 27 H 30 N6O, with the corresponding manufacturer number F646-0173, has the following structural formula: 。 2. The application as described in claim 1, characterized in that, The applications include pharmaceutical compositions in which small molecule lead compounds or their salts are active ingredients.

3. The application as described in claim 2, characterized in that, The pharmaceutical composition includes a small molecule lead compound, emamectin benzoate or avermectin, and pharmaceutically acceptable excipients.

4. The application as described in claim 3, characterized in that, The pharmaceutical composition is in a pharmaceutically acceptable dosage form selected from dry powder, wettable powder, emulsifiable concentrate, microemulsion, paste, granules, solution or suspension.

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