Use of ngi-1 in the preparation of a medicament for preventing and treating spring viraemia of carp

Abstract

The application discloses application of NGI-1 in preparation of a medicine for preventing and treating spring viremia of carp virus infection, relates to the field of fish medicines, and discloses application of NGI-1 (ML414) with a chemical name of 5-(dimethylsulfamoyl-N-(5-methyl-1,3-thiazol-2-yl)-2-(pyrrolidine-1-yl) benzamide in preparation of a medicine for preventing and treating spring viremia of carp. The NGI-1 can dose-dependently inhibit infection of the spring viremia of carp virus within a safe concentration range. The application has a high biological safety factor, a medicine selection index SI greater than 10, a wide safe medicine window, can efficiently resist viruses without damaging aquatic breeding organisms, and has extremely high industrialization development and practical application values.

Description

Technical Field

[0001] This invention relates to the field of fish medicines, and more particularly to the application of NGI-1 in the preparation of drugs for the prevention and treatment of spring viremia virus infection in carp. Background Technology

[0002] The Food and Agriculture Organization of the United Nations (FAO) recently released its "State of Fisheries and Aquaculture in the World 2026" (SOFIA 2026) report, which indicates that the trade in aquatic animal products has climbed to a record high of US$184 billion, rivaling the value of the terrestrial animal meat trade. In recent years, my country's aquaculture industry has continued to expand, but the general tendency to pursue high density, high yield, and high profits has led to a decline in the disease resistance of aquatic animals and frequent outbreaks of diseases. Statistics show that direct economic losses to farmed aquatic animals caused by diseases have reached tens of billions of yuan, with aquatic viral diseases being the most significant pathogenic factor.

[0003] Carp viremia caused by carp spring viremia virus (SVCV) is characterized by rapid onset, short course, and high mortality. It has already caused outbreaks in multiple regions of Europe, Asia, and North America, and is one of the key viral infectious diseases of fish of international concern. SVCV has a broad host spectrum, infecting not only various carp species (with mortality rates exceeding 70%), but also non-carp aquatic animals such as sunfish, pike, and even shrimp. Currently, this disease has been included in the list of Class II animal diseases by the Ministry of Agriculture and Rural Affairs of my country. Its outbreaks and epidemics not only pose a serious threat to the healthy development and sustainability of carp aquaculture, but also bring potential risks to the breeding of superior broodstock and the export trade of ornamental fish in my country.

[0004] Cyprinid viremia virus (SVCV) is classified under the Rhabdoviridae family. Sprivivirus SVCV is a single-stranded, negative-sense RNA virus. Its genome is approximately 11 kb long and sequentially encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase protein (L). These five proteins assemble together to form the virus's characteristic bullet-shaped morphology. However, currently, there are neither mature vaccines nor effective treatments available for this virus; practical control measures still primarily rely on virus detection combined with the isolation or culling of infected fish. Therefore, the development of drugs that can effectively combat SVCV infection is an urgent practical need.

[0005] NGI-1 (ML414), chemically named 5-(dimethylaminosulfonyl-N-(5-methyl-1,3-thiazolyl)-2-(pyrrolidone-1-yl)benzamide, has a molecular weight of approximately 394.51. It is a white to light beige solid powder, soluble in the organic solvent DMSO, with a solubility of 63.37 mM in DMSO. NGI-1 (ML414) is a potent oligosaccharide syltransferase (OST) inhibitor that directly targets and blocks the function of the OST catalytic subunits STT3A and STT3B. NGI-1 is a cell permeability inhibitor, effectively reducing viral infectivity without affecting cell viability. In mammalian viral systems, NGI-1 has been validated to differentially inhibit different catalytic subtypes of the OST complex: NGI-1 can target and antagonize STT3A-OST or STT3B-OST based on the cellular OST complex subtype expression profile. The catalytic activity of NGI-1 is demonstrated by its dose-dependent inhibition of the proteolytic maturation of Lassa virus envelope glycoproteins through hindering N-glycosylation modification. There are currently no studies on the application of NGI-1 in the prevention and control of aquatic viral diseases, either domestically or internationally.

[0006] Therefore, those skilled in the art are dedicated to developing a method for applying NGI-1 as a drug to prevent and treat carp spring viremia virus infection. Summary of the Invention

[0007] To address the shortcomings of existing antiviral technologies, the technical challenge this invention aims to solve is to provide an application scheme for NGI-1 in the preparation of drugs for the prevention and control of carp viremia virus infection, based on the glycosylation modification mechanism of carp spring viremia virus (SVCV) envelope proteins.

[0008] To achieve the above objectives, this invention provides the application of NGI-1 in the preparation of a drug for preventing and treating spring viremia in carp. The chemical formula of NGI-1 is shown below: .

[0009] Furthermore, NGI-1 is an N-glycosylation inhibitor. The powder is dissolved in the organic solvent DMSO and used as a stock solution for cell experiments. This drug inhibits the glycosylation of the endoplasmic reticulum oligosaccharide transferase (OST) complex.

[0010] Furthermore, the effective dose is generally expressed as the half-maximal inhibitory concentration (IC50) and the half-maximal cytotoxic concentration (IC50). 50 IC50 reflects the efficacy of a drug at its target site. 50 The smaller the value, the stronger the drug's inhibitory activity; the half-maximal cytotoxic concentration (CC) is... 50 CC reflects the toxic side effects of drugs on normal cells. 50The higher the value, the less toxic the drug.

[0011] Furthermore, the antiviral agent is an inhibitor of the aquatic virus carp spring viremia virus.

[0012] Furthermore, the antiviral agent is an inhibitor that suppresses the glycosylation of the envelope protein of carp viremia virus.

[0013] Furthermore, NGI-1 acts on cells.

[0014] Furthermore, the cells were grass carp ovarian cells (CO) and carp epithelial cells (EPC).

[0015] Furthermore, drugs for preventing spring viremia infection in carp are added as additives to fish feed.

[0016] Furthermore, NGI-1's IC50 on CO cells and EPC cells... 50 The values ​​were 0.70 µM and 1.034 µM, respectively.

[0017] Furthermore, NGI-1 showed increased activity in CO cells and EPC cells. 50 All were significantly higher than 30 µM.

[0018] In a preferred embodiment 1 of the present invention, the process for detecting the cytotoxicity of NGI-1 at different concentrations is described in detail. In another preferred embodiment 2 of the present invention, the experimental process of the effect of different concentrations of NGI- on the transcription level of the SVCV N gene in cells is described in detail. In another preferred embodiment 3 of the present invention, the experimental process of the effect of different concentrations of NGI-1 on the expression level of SVCV M protein is described in detail; In another preferred embodiment 4 of the present invention, the experimental process of the effect of different concentrations of NGI-1 on the expression level of G protein of carp spring viremia virus in cells is described in detail.

[0019] The beneficial technical effects of this invention are as follows: NGI-1 is a highly effective cell-permeable OST inhibitor that specifically targets and blocks the biological functions of the OST catalytic subunits STT3A and STT3B, intervening in the virus-dependent OST pathway modification process at the molecular level. This invention breaks through the limitations of NGI-1's original application scenarios by innovatively applying the oligosaccharide transferase inhibitor NGI-1 to the prevention and control of aquatic organism viruses, opening up a new application for its use in the prevention and treatment of spring viremia in carp. This effectively fills the technological gap of NGI-1 in the field of aquatic virus prevention and control, expands the application scope of oligosaccharide transferase inhibitors, and provides a new direction for drug development in the prevention and control of spring viremia in carp.

[0020] NGI-1 possesses excellent cell permeability, and its antiviral mechanism targets viral protein modification pathways without interfering with normal host cell metabolism or adversely affecting host cell viability. Using low-toxicity and highly effective NGI-1 as a control drug allows it to exert antiviral effects within a safe concentration range, balancing antiviral activity with host cell safety, demonstrating significant development potential. It also boasts a high biosafety coefficient (SI greater than 10), a broad safe use window, and can effectively combat viruses without harming aquatic organisms, possessing extremely high value for industrial development and practical application.

[0021] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0022] Figure 1 This is the cytotoxicity test result of different concentrations of NGI-1 on CO cells and EPC cells in a preferred embodiment of the present invention; Figure 2 This is the result of the effect of different concentrations of NGI-1 on the transcription level of the SVCV N gene in CO cells and EPC cells in a preferred embodiment 2 of the present invention; Figure 3 This is the effect of different concentrations of NGI-1 on the expression level of SVCV M protein in CO cells in a preferred embodiment 3 of the present invention; Figure 4 This is the effect of different concentrations of NGI-1 on the expression level of SVCV M protein in EPC cells in a preferred embodiment 3 of the present invention; Figure 5 This is the effect of different concentrations of NGI-1 on the expression level and glycosylation of SVCV G protein in CO cells in a preferred embodiment 4 of the present invention; Figure 6 This is the effect of different concentrations of NGI-1 on the expression level and glycosylation of SVCV G protein in EPC cells in a preferred embodiment 4 of the present invention. Detailed Implementation

[0023] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0024] Example 1: Detection of cytotoxicity of different concentrations of NGI-1

[0025] This experiment used the CCK-8 assay kit to detect the effects of different concentrations of NGI-1 on the viability of CO and EPC cells. The specific steps are as follows: 1. CO cells or EPC cells were cultured in M199 medium containing 10% fetal bovine serum, and passaged by trypsin digestion one day before the experiment.

[0026] 2. Count the cells at a rate of 1×10⁻⁶. 4 The cells were evenly seeded in 96-well plates and cultured at 25°C until the cell confluence reached more than 90%.

[0027] 3. On the day of the experiment, NGI-1 was serially diluted in M199 medium containing 2% fetal bovine serum, starting at 30 µM, and then serially diluted in three-fold increments (10 µM, 3.33 µM, ...) until 0.0137 µM, for a total of 8 concentration gradients.

[0028] 4. Discard the original culture medium in the 96-well plate, add culture medium containing the corresponding concentration of drug, and continue culturing for 24 h. Set up 6 replicates for each concentration, and repeat the experiment at least 3 times.

[0029] 5. After culturing for 24 hours, discard the drug-containing culture medium, add fresh culture medium containing 10% CCK-8, and continue incubation for 2.5 hours.

[0030] 6. The absorbance of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader. Cell viability was calculated at each drug concentration using M199 medium containing 2% fetal bovine serum as a 100% control.

[0031] 7. Using the base-10 logarithm of the diluted compound concentration as the X-axis and cell activity as the Y-axis, a dose-response curve was fitted using GraphPad Prism 8 software, and the CC was calculated. 50 The result is as follows Figure 1 As shown, NGI-1 is involved in the C-cell activity of CO cells and EPC cells. 50 All are greater than 30 µM.

[0032] Example 2: Effects of different concentrations of NGI-1 on the transcriptional level of the SVCV N gene in cells

[0033] 1. Culture CO cells or EPC cells in M199 medium containing 10% fetal bovine serum, and passage them by trypsin digestion one day before the experiment.

[0034] 2. Count the cells at a rate of 1×10⁻⁶. 5 The cells were evenly seeded in 24-well plates and cultured at 25°C until the cell confluence reached more than 90%.

[0035] 3. On the day of the experiment, NGI-1 was serially diluted 3-fold in eight concentration gradients using M199 medium containing 2% FBS. The final concentrations were: 10, 3.33, 1.11, 0.370, 0.123, 0.041, 0.0137, and 0.00457 µM.

[0036] 4. Discard the original culture medium in the 24-well plate, add 240 µL of culture medium containing the corresponding concentration of drug to each well, and incubate with the cells at 25 °C for 1 h.

[0037] 5.1 h later, the SVCV virus was diluted at MOI=0.01, and 10 µL of the virus dilution was added to each cell well and incubated at 20 ℃ for another 1 h.

[0038] 6. After the infection is completed, discard the culture medium containing the drug and virus, replace it with fresh culture medium containing the corresponding concentration of drug, and continue to culture for 23 hours (total drug treatment time is 24 hours).

[0039] 7. Total RNA was extracted from cells using a commercial RNA extraction kit and reverse transcribed into cDNA. The SVCV N gene copy number was detected by RT-qPCR. Virus infection wells without drug treatment were used as a 100% infection rate control, and the virus inhibition rate at each drug concentration was calculated.

[0040] 8. The dilute compound concentration was plotted as the logarithm of base 10 on the X-axis, and the corresponding inhibition rate on the Y-axis. A dose-response curve was fitted using GraphPad Prism 8 software, and the IC50 was calculated. 50 The result is as follows Figure 2 As shown, the IC50 of NGI-1 in CO cells 50 The IC50 concentration was 0.70 µM in EPC cells. 50 The value is 1.031 µM. This experiment should be repeated independently at least 3 times.

[0041] Example 3: Effects of different concentrations of NGI-1 on the expression level of SVCV M protein

[0042] 1. CO cells or EPC cells were cultured in M199 medium containing 10% fetal bovine serum (FBS) at 25°C using standard methods. One day before the experiment, cells were passaged using 0.25% trypsin and further operations were performed when the cell confluence reached over 90%.

[0043] 2. After digestion, the cells were counted at a rate of 1×10⁻⁶. 5 / wells were seeded into 24-well plates, and M199 medium containing 10% FBS was added. The cells were then cultured at 25 °C until the confluence was ≥90%.

[0044] 3. On the day of the experiment, the drug was serially diluted 5-fold using M199 medium containing 2% fetal bovine serum, to 5µM, 1µM, and 0.2µM.

[0045] 4. Discard the original culture medium, add 240 µL of culture medium containing the corresponding concentration of drug to each well, and incubate with the cells at 25°C for 1 h.

[0046] 5. After incubation, dilute the SVCV virus solution with an MOI of 0.01, add 10 µL to each well, and continue infection at 20°C for 1 h.

[0047] 6. Discard the virus-containing culture medium, wash with PBS, and replace with fresh culture medium containing the corresponding concentration of drug. Continue culturing for 23 h (total drug treatment time is 24 h).

[0048] 7. Discard the culture medium and wash twice with PBS. Add 100 µL of cell lysis buffer to each well for complete lysis. Transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 13000 × g for 15 min at 4 °C. Take 80 µL of supernatant, add 20 µL of 5× protein loading buffer, mix well, and boil at 100 °C for 5 min.

[0049] 8. After separation by SDS-PAGE electrophoresis, the expression level of SVCV structural protein M was detected by Western blot to evaluate the inhibitory effect of NGI-1 on viral protein synthesis. The effect of different concentrations of NGI-1 on the expression level of SVCV M protein in CO cells is as follows: Figure 3 As shown, the effect of different concentrations of NGI-1 on the expression level of SVCV M protein in EPC cells is as follows: Figure 4 As shown.

[0050] Example 4: Effects of different concentrations of NGI-1 on the expression level of G protein of carp spring viremia virus in cells

[0051] 1. CO cells and EPC cells were cultured in M199 medium containing 10% fetal bovine serum, respectively. The cells were digested with trypsin one day before the experiment.

[0052] 2. Count the cells and count them at 1×10⁻⁶. 5 / The cells are evenly seeded in a 24-well plate and cultured at 25 ℃ until the cell confluence is above 90%.

[0053] 3. On the day of the experiment, the drug was serially diluted 5-fold using M199 medium containing 2% fetal bovine serum (FBS) to 5 µM, 1 µM, and 0.2 µM.

[0054] 4. Discard the original culture medium in the 24-well plate and replace it with culture medium containing the drug. Incubate for 1 hour, and co-incubate the drug with the cells for 1 hour. The volume of drug-containing culture medium added to each well is 240 µL.

[0055] 5. After one hour, dilute the SVCV virus with an MOI of 0.01. Place 10 μL of the virus dilution into cell wells. Infect the cells with the virus at 20°C for one hour.

[0056] 6. Change the medium in the 24-well plate by aspirating the original culture medium containing the drug and replacing it with M199 medium containing 2% fetal bovine serum. Continue culturing for 23 h.

[0057] 7. Discard the culture medium and wash twice with PBS. Add 100 µL of cell lysis buffer to each well for complete lysis. Transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 13000 × g for 15 min at 4 °C. Take 80 µL of supernatant, add 20 µL of 5× protein loading buffer, mix well, and boil at 100 °C for 5 min.

[0058] 8. After separation by SDS-PAGE electrophoresis, the expression level of SVCV G protein was detected by Western blot to evaluate the inhibitory effect of NGI-1 on viral protein synthesis. The effects of different concentrations of NGI-1 on SVCV G protein expression level and glycosylation in CO cells are as follows: Figure 5 As shown, different concentrations of NGI-1 on SVCV G protein expression levels and glycosylation in EPC cells are as follows: Figure 6 As shown.

[0059] In summary, the results indicate that NGI-1 can inhibit SVCV infection in a dose-dependent manner on different fish cells, and its IC50 value on CO cells and EPC cells is [not specified]. 50 The values ​​were 0.70 µM and 1.034 µM, respectively; meanwhile, NGI-1 showed increased CC in both cell types. 50 All concentrations were significantly higher than 30 µM, and no significant cytotoxicity was observed within the safe concentration range. The drug selectivity index (SI=CC) was [missing value]. 50 / IC 50 A value greater than 10 indicates its potential for further development as an inhibitor of SVCV infection. Based on this, the structure of NGI-1 can be optimized to improve its solubility, lipid-water partition coefficient, and pharmacokinetic and pharmacodynamic properties. Systematic experimental verification of this invention confirms that NGI-1 can effectively inhibit carp spring viremia virus infection at safe concentrations, providing a new and effective means for the prevention and control of carp viral diseases. It can be widely applied to the prevention and treatment of viral diseases in fish, helping to reduce economic losses caused by viral infections in carp farming, and has broad application prospects.

[0060] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of NGI-1 in the preparation of a drug for preventing and treating spring viremia infection in carp, characterized in that, The chemical formula of NGI-1 is shown below: 。 2. The application as described in claim 1, wherein the antiviral agent is an inhibitor of aquatic carp spring viremia virus.

3. The application as described in claim 1, wherein the antiviral agent is an inhibitor that inhibits the glycosylation of the envelope protein of carp viremia virus.

4. The application as described in claim 1, wherein the NGI-1 acts on cells.

5. The application as described in claim 4, wherein the cells are grass carp ovarian cells and carp epithelial cells.

6. In the application as described in claim 4, the half-maximal inhibitory concentrations (IC50) of NGI-1 in grass carp ovarian cells and carp epithelial cells are 0.70 µM and 1.034 µM, respectively.

7. In the application described in claim 4, the half-maximal cytotoxic concentration of NGI-1 in grass carp ovarian cells and carp epithelial cells is significantly higher than 30 µM.

8. The application as described in claim 4, wherein the NGI-1 powder is dissolved in the organic solvent DMSO for use.

9. The application as described in claim 1, wherein the drug for preventing and treating spring viremia infection in carp is added as an additive to fish feed.

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