Application of small molecule aquaporin inhibitors in the fight against pebrine disease in silkworms

Abstract

The present invention discloses the application of a small molecule aquaporin inhibitor in resistance to silkworm pebiosis. The present invention adopts molecular docking technology to screen a small molecule database and performs activity detection on the obtained small molecule inhibitors, thereby obtaining a small molecule aquaporin inhibitor with resistance to silkworm pebiosis. The present invention uses the screened small molecule aquaporin inhibitor as a drug for preventing and treating silkworm pebiosis, thus developing a new application of the inhibitor. When the final concentration of the aquaporin inhibitor is 0.5 μM or more, the germination of silkworm microsporidia in vitro can be inhibited; when the final concentration is 1 μM or more, the proliferation of silkworm microsporidia in silkworm cells can be reduced; and when the final concentration is 100 μM or more when added to feed, the incidence of silkworm pebiosis can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and pathology, and relates to a drug for preventing and treating pebisomesis of the silkworm, in particular to the application of a small molecule inhibitor of aquaporin in preventing and treating pebisomesis of the silkworm. Background Art

[0002] Pebrine disease is a devastating infectious disease of the silkworm caused by oral infection of silkworms by Nosema bombycis. It is also germ-transmissible and often causes huge economic losses to the sericulture industry.

[0003] Research on the chemotherapeutic treatment of pebrine disease in silkworms has been reported for a long time. For example, prior art has found that fumagillin and anisomycin inhibit the proliferation of honeybee pebrine protozoa Nosema apis, and ben-laide significantly inhibits the proliferation of alfalfa weevil pebrine protozoa Nosema sp. However, these drugs are not significantly effective against pebrine disease in silkworms. Currently, the main chemotherapeutic drugs used in production in China are Fangweiling, Yuanzhaojing, and Nongweiling. However, their effectiveness against pebrine disease in silkworms is primarily preventive. Adding these drugs to the diet of silkworms infected with Nosema bombycis has not significantly improved the incidence of the disease or the treatment of pebrine disease. Therefore, research is urgently needed to develop drugs with practical therapeutic effects against pebrine disease in silkworms. Summary of the Invention

[0004] Technical problem to be solved: In order to overcome the shortcomings of the existing technology and obtain drugs that can inhibit the germination of Bombyx mori microsporidia in vitro, reduce the proliferation of Bombyx mori microsporidia in silkworm cells, and reduce the incidence of Bombyx mori pebrine disease, the present invention provides the use of small molecule inhibitors of water channel proteins in the prevention of Bombyx mori pebrine disease.

[0005] Technical solution: Application of a small molecule inhibitor of aquaporin in the prevention of pebrine disease in silkworms. The structural formula of the small molecule inhibitor of aquaporin is:

[0006]

[0007] Targeting the water channel protein of the silkworm microsporidia, a small molecule inhibitor with potential function was obtained based on mainstream virtual modeling and molecular docking technology. The main process is: (1) ligand treatment: using The LigPrep module in was used to prepare ligands for the Enamine Aquaporines compound library. The force field was OPLS_2005, pH 7.0±2.0, and the compounds were converted from 2D structures to 3D structures. (2) Homology modeling: Since the target protein has no protein structure, SwissModel (https: / / swissmodel.expasy.org), Phyre2 (http: / / www.sbg.bio.ic.ac.uk / ~phyre2 / html / page.cgi?id=index), Modeller and I-TASSER (https: / / zhanggroup.org / I-TASSER / ) were used for homology modeling, and SAVES v6.0 (https: / / saves.mbi.ucla.edu / ) was used to evaluate the modeling results. The protein structure with the best result was selected for virtual screening. (3) Receptor processing and grid generation: The protein was processed using the Protein Preparation Wizard to add missing hydrogen atoms and protonate at pH 7.0±2.0. Hydrogen bonds were assigned at pH 7.0 and energy optimization was performed using the force field OPLS_2005. Active sites were predicted using DoGSiteScorer (https: / / proteins.plus / ) and the SiteMap module. The GridGeneration module was used to select the most likely site as the docking active site and generate a grid file. (4) Receptor-ligand docking: After the grid file was generated, all molecules were docked and screened using the Ligand docking in the Glide module. The Standard Precision (SP) mode was used to screen the top 400 compound results. The selected compounds were then screened for one round using the Extra Precision (XP) mode. Finally, a summary analysis was performed and the top 20 compounds were selected based on the two rounds of results for experimental verification. It was finally discovered that the small molecule inhibitor of aquaporin was numbered Z1498802520 in the ZINC database, and its molecular formula was: CCC1=CC(O)=NC(CN2CC(O)CC2C2=CC(F)=CC=C2F)=N1.

[0008] Preferably, the pathogen of the silkworm pebrine disease is Nosema bombycis, or a genetically engineered strain having the same infection characteristics as the natural strain.

[0009] Preferably, the resistance to silkworm pebiosis refers to inhibiting the germination of silkworm Nosema bombycis in vitro, or reducing the proliferation of silkworm Nosema bombycis in silkworm cells, or reducing the incidence of silkworm pebiosis.

[0010] Preferably, the effective concentration of the aquaporin small molecule inhibitor is 0.1 μM to 10 mM. Preferably, the final concentration of the aquaporin small molecule inhibitor for inhibiting the in vitro germination of Nosema bombyx mori is 0.5 μM or more.

[0011] Preferably, the final concentration of the small molecule inhibitor of aquaporin that reduces the proliferation of Nosema bombycis in Bombyx mori cells is 1 μM or more.

[0012] Preferably, the final concentration of the small molecule inhibitor of aquaporin for reducing the incidence of pebivirus disease in silkworms is 100 μM or more when added to the feed.

[0013] The principle of using the small molecule aquaporin inhibitor described in the present invention to combat pebivirus in silkworms is that the microsporidian infection process can be divided into two steps. First, under specific conditions, the spores are activated, the polar filaments inside the spores rapidly evert and pop out, and the sporoplasm is injected into the host cell through the hollow polar filaments to initiate infection. This process, also known as spore germination, is mainly due to the fact that the aquaporins located on the spore wall, under the stimulation of physical and chemical factors such as pH and calcium ions, transfer water outside the spore into the spore, increasing the osmotic pressure inside the spore, causing the polar membrane layer and the posterior polar vesicle to swell, and ultimately triggering the polar filament to pop out. Therefore, by blocking the water transport function of the aquaporins, the germination of the microsporidia can be effectively inhibited, ultimately suppressing the occurrence of the disease.

[0014] Beneficial effects: (1) The present invention adopts molecular docking technology to screen a small molecule database, and performs activity detection on the obtained small molecule inhibitors, thereby obtaining a water channel protein small molecule inhibitor with resistance to silkworm pebbles; (2) The present invention uses the screened water channel protein small molecule inhibitor as a drug for preventing and treating silkworm pebbles, opening up its new use; (3) When the final concentration of the water channel protein small molecule inhibitor is 0.5 μM or more, it can inhibit the germination of silkworm microsporidia in vitro; when the final concentration is 1 μM or more, it can reduce the proliferation of silkworm microsporidia in silkworm cells; when the final concentration is 100 μM or more when added to feed, it can reduce the incidence of silkworm pebbles. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 To investigate the germination rate of Nosema bombycis in vitro after treating pure Nosema bombycis with different concentrations of small molecule inhibitors;

[0016] Figure 2 The titer of Nosema bombycis after cells were treated with different concentrations of small molecule inhibitors;

[0017] Figure 3 To investigate the incidence of silkworms fed with small molecule inhibitors at different concentrations. DETAILED DESCRIPTION

[0018] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0019] Example 1 Inhibition of in vitro germination of Nosema bombyx mori

[0020] Use the following steps:

[0021] (1) Preparation of small molecule Z1498802520 storage solution

[0022] A stock solution of Z1498802520 was prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mM.

[0023] (2) Treatment and in vitro germination of Nosema bombycis

[0024] Purified Nosema bombycis spores were incubated with Z1498802520 inhibitor solutions at final concentrations of 0 μM, 0.1 μM, 0.5 μM, 1 μM, and 10 μM for 30 min, followed by addition of an equal volume of 0.2 M KOH solution and continued incubation at 30°C for 30 min. The germination was observed and counted under a microscope. The germination rate of 100 spores was counted for each sample. Figure 1 As shown: Z1498802520 at a concentration of 0.5 μM and above can significantly inhibit the germination of Nosema bombyx in vitro.

[0025] Example 2: Reducing the proliferation of Nosema bombycis in silkworm cells

[0026] Use the following steps:

[0027] (1) Culture of silkworm BmN cells

[0028] Bombyx mori ovary cell line BmN cells were cultured in TC100 insect cell culture medium supplemented with 10% fetal bovine serum (FBS).

[0029] (2) In vitro germination and cell infection of Nosema bombycis

[0030] Resuspend purified Nosema bombycis spores in 1 ml of 0.1 M KOH solution and incubate at 30°C for 30 minutes. Mix thoroughly with 12 ml of BmN cell suspension. After 5 minutes of simmering, dispense an equal amount of spores into a 12-well cell culture plate. Allow to fully adhere at 28°C for 1 hour. Remove the medium, add fresh medium containing 2 μL of DMSO, and seal the plate for incubation. Replace the medium every 3 days, and lyse the cells after 12 days.

[0031] (3) Z1498802520 processing and result statistics

[0032] When the final concentration of the small molecule is 0μM, 1μM, 5μM and 10μM, the infection of Bombyx mori in cells is prevented and treated: the purified Bombyx mori spores are resuspended with 1ml of 0.1M KOH solution, and the cells are incubated at 30℃ for 30min and then fully mixed with 12ml of cell suspension; after standing for 5min, equal amounts are injected into a 12-well cell culture plate, and the plate is allowed to stand at 28℃ for 1h to allow it to completely adhere to the wall. The culture medium is then removed and fresh culture medium containing small molecule inhibitors at final concentrations of 0μM, 1μM, 5μM and 10μM is added and sealed for culture. Fresh culture medium (containing the corresponding concentration of Z1498802520) is replaced every 3 days. After 12 days, the cells are lysed and the Bombyx mori spores are counted using a hemocytometer. The results are as follows. Figure 2 As shown, Z1498802520 at a concentration of 1 μM and above can effectively inhibit the proliferation of Nosema bombyx in BmN cells.

[0033] Example 3 Reducing the incidence of pebrine disease in silkworms

[0034] Use the following steps:

[0035] When the final concentrations of the small molecule Z1498802520 were 0μM, 1μM, 10μM, 100μM, 1mM and 10mM, the small molecule inhibitor was added to mulberry leaves to prevent and treat Nosema bombycis infection:

[0036] Test silkworm varieties: Qiufeng × Baiyu;

[0037] Age: 3-year-old silkworms;

[0038] Zone setting: Each treatment was repeated in 3 zones, with 100 silkworms in each zone.

[0039] The purified Nosema bombycis was diluted to a suspension of 107 spores / mL using sterile water containing 0μM, 1μM, 10μM, 100μM, 1mM, and 10mM of the small molecule Z1498802520. The suspension was then smeared on the back of a 2cm diameter circular mulberry leaf. Six mulberry leaves were used per test area. After drying, the leaves were fed to the test silkworms. After all the leaves had been eaten, clean mulberry leaves were used instead. After 10 days, the incidence rate was calculated based on the phenotype. The results are shown in the figure below. Figure 3 As shown, the incidence rate of the control group was 100%, while the incidence rate of the group supplemented with Z1498802520 at a concentration of 100 μM and above was significantly reduced.

Claims

1. Application of a small molecule aquaporin inhibitor in the preparation of a drug for treating silkworm pebbles disease, characterized in that: The structural formula of the aquaporin small molecule inhibitor is:

2. The use according to claim 1, characterized in that The aquaporin small molecule inhibitor is numbered Z1498802520 in the ZINC database, and has a molecular formula of: CCC1=CC(O)=NC(CN2CC(O)CC2C2=CC(F)=CC=C2F)=N1.

3. The use according to claim 1, characterized in that The pathogen of the silkworm pebbles disease is Nosema bombycis, or a genetically engineered strain having the same infection characteristics as the natural strain.

4. The use according to claim 1, characterized in that The anti-pearin disease of silkworm refers to inhibiting the in vitro germination of Nosema bombycis, or reducing the proliferation of Nosema bombycis in silkworm cells, or reducing the incidence of Nosema bombycis.

5. The use according to claim 4, characterized in that The effective concentration of the aquaporin small molecule inhibitor is 0.1 μM to 10 mM.

6. The use according to claim 4, characterized in that The final concentration of the small molecule inhibitor of aquaporin that inhibits the germination of Nosema bombyx in vitro is 0.5 μM or above.

7. The use according to claim 4, characterized in that The final concentration of the small molecule inhibitor of aquaporin that reduces the proliferation of Nosema bombycis in Bombyx mori cells is 1 μM or higher.

8. The use according to claim 4, characterized in that The final concentration of the small molecule inhibitor of aquaporin used in the feed for reducing the incidence of pebbles disease in silkworms is 100 μM or more.

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