Application of GSK2606414 in preparation of medicine for inhibiting duck tembusu virus infection

Abstract

The invention discloses application of GSK2606414 in preparation of a medicine for inhibiting duck tembusu virus infection, and belongs to the technical field of veterinary biological products. The research finds that the GSK2606414 can effectively inhibit the replication of the duck tembusu virus for the first time, provides a new therapeutic drug for the prevention and treatment of the duck tembusu virus, and is of great significance to the actual production of the poultry breeding industry.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, specifically to the application of GSK2606414 in the preparation of a drug for inhibiting duck Tembusu virus infection. Background Technology

[0002] Duck Tembusu virus disease is an acute, contagious disease caused by Duck Tembusu Virus (DTMUV). Initially, it primarily infects laying and breeding ducks, with infected ducks exhibiting depression, decreased egg production, and stunted growth. Morbidity rates can reach as high as 90% on some farms, and the host spectrum of this disease continues to expand. DTMUV has been reported to infect ducks, chickens, geese, sparrows, and mice. Furthermore, serological studies have shown the presence of DTMUV antibodies in some duck farm workers, with approximately 50% of oral swab samples testing positive for DTMUV. Therefore, this disease not only causes significant economic losses to the poultry industry but also poses a potential public health risk.

[0003] Currently, vaccination remains the core strategy for the prevention and control of DTMUV infection. Approved commercial vaccines mainly include inactivated DTMUV vaccines (such as the HB strain and DF2 strain) and live vaccines (such as the WF100 strain and FX2010-180P strain). These vaccines provide protection to waterfowl by inducing an immune response. Studies have shown that inactivated vaccines primarily stimulate humoral immunity to produce neutralizing antibodies, while live vaccines can simultaneously induce both cellular and humoral immunity. However, existing vaccines still have certain limitations, such as short duration of immunity and large fluctuations in antibody titers with inactivated vaccines, while attenuated live vaccines have the risk of virulence reversion and poor transport stability. Therefore, there is an urgent need to develop new DTMUV treatments. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of GSK2606414 in the preparation of a drug for inhibiting duck Tembusu virus infection.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides the use of GSK2606414 in the following (1) or (2):

[0007] (1) Preparation of drugs to inhibit the replication of duck Tembusu virus;

[0008] (2) Prepare drugs for the prevention and treatment of duck Tembusu virus disease.

[0009] In the above applications, the concentration of GSK2606414 used is 0.1-10 μM.

[0010] Preferably, the concentration of GSK2606414 used is 1 μM.

[0011] In the above applications, in addition to GSK2606414 as an active ingredient, the drug may also contain one or more pharmaceutically acceptable carriers.

[0012] The pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers or gel substances. Substances that can serve as pharmaceutically acceptable carriers include: cellulose and its derivatives, such as sodium carboxymethyl cellulose and sodium ethyl cellulose; solid lubricants, such as calcium stearate and magnesium stearate; vegetable oils, such as soybean oil, sesame oil, peanut oil, and olive oil; polyols, such as propylene glycol, mannitol, and sorbitol; and emulsifiers, wetting agents, colorants, stabilizers, and preservatives.

[0013] Drugs can be formulated as injectable preparations, such as aqueous suspensions, oil suspensions, emulsions, or solutions; or as oral preparations, such as powders, tablets, capsules, or granules.

[0014] In a second aspect, the present invention provides a drug for inhibiting the replication of duck Tembusu virus, wherein the drug uses GSK2606414 as an active ingredient.

[0015] Preferably, the concentration of GSK2606414 in the drug is 1 μM.

[0016] The beneficial effects of this invention are:

[0017] This invention is the first to discover that GSK2606414 can effectively inhibit the replication of duck Tembusu virus, providing a new therapeutic drug for the prevention and control of duck Tembusu virus disease, which is of great significance to the actual production of poultry farming. Attached Figure Description

[0018] Figure 1 The results of the effect of DTMUV infection on endoplasmic reticulum stress: In the figure, A is the endoplasmic reticulum stress induced by DTMUV infection of DEF cells; B is the activation of the PERK and IRE1α pathways in the unfolded protein response by DTMUV infection of DEF cells.

[0019] Figure 2 Western blot results of DTMUV-induced autophagy in cells detected by GSK2606414.

[0020] Figure 3 qPCR detection results of DTMUV replication using GSK2606414. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] There are complex interactions among autophagy, endoplasmic reticulum stress (ERS), and viruses. The interaction between ERS and autophagy triggered by viral infection is an important manifestation of virus-host co-evolution. When cells are infected by a virus, the virus utilizes the host cell's resources for replication, leading to an imbalance in ERS and protein homeostasis. At this time, the cell activates autophagy to clear the virus or viral components as a defense mechanism. However, some viruses have also evolved the ability to counteract autophagy, and even use autophagy to promote their own replication and spread. Therefore, autophagy and ERS play a dual role in antiviral defense; they can be both protective mechanisms for cells and can be hijacked by viruses to facilitate their survival and proliferation.

[0023] Since its onset, DTMUV has not only caused huge economic losses to the duck farming industry, but has also brought significant harm to other poultry farming industries. Currently, the relationship between DTMUV replication, autophagy, and endoplasmic reticulum stress remains unclear.

[0024] Therefore, this invention first investigated the effect of DTMUV infection on endoplasmic reticulum stress. The results showed that DTMUV infection significantly induced endoplasmic reticulum stress and activated the unfolded protein response. Furthermore, this invention investigated the effect of the PERK inhibitor GSK2606414 on DTMUV infection-induced autophagy and DTMUV replication. The results showed that GSK2606414 effectively inhibited the PERK pathway activated by DTMUV, thereby regulating autophagy. More importantly, GSK2606414 significantly reduced the viral copy number of DTMUV, demonstrating that GSK2606414 can inhibit DTMUV replication, thus providing a new therapeutic agent for the prevention and treatment of duck Tembusu virus disease.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0027] The DTMUV strain used in this invention is described in the journal article “Duck Tembusu virus infection induces M1 polarization in avian macrophages” (DOI:10.3969 / j.issn.1007-1733.2023.11.006).

[0028] The CAS number for GSK2606414 is 1337531-36-8.

[0029] β-actin, p-IRE1α, p-eIF2α, eIF2α, and LC3 antibodies were purchased from Cell Signal Technology; GRP78 antibody was purchased from Beyotime; and toxic carotenoid (TG) was purchased from MCE.

[0030] Duck embryonic fibroblasts (DEF) were isolated from 10-year-old duck embryos using trypsin digestion and cultured in a humidified cell culture incubator at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum.

[0031] Example 1: Effects of DTMUV infection on endoplasmic reticulum stress

[0032] 1. Test method:

[0033] Duck embryonic fibroblasts (DEF) were cultured in 96-well cell plates and infected with 100 μL of DTMUV virus solution (MOI=1). DEF cells were treated with ERS activator TG (0.5 μM) as a positive control. Uninfected DTMUV cells and cells not treated with TG were used as blank controls (Mock).

[0034] Samples were collected 12h, 24h and 48h after infection for Western blot analysis, and the expression levels of GRP78, β-actin, p-IRE1α, p-eIF2α, eIF2α and GAPDH proteins were detected by immunoblotting.

[0035] 2. Test Results:

[0036] The results showed that the protein expression level of GRP78 was significantly upregulated in a time-dependent manner after 12 h and 24 h of DTMUV infection of DEF cells; while the expression level of GRP78 in the control group cells did not change significantly within the same culture time. Figure 1 A) indicates that DTMUV infection of DEF cells can significantly induce endoplasmic reticulum stress (ERS).

[0037] The expression of p-IRE1α and p-eIF2α significantly increased 12 h and 24 h after DTMUV infection. Figure 1 B) indicates that DTMUV infection activates the unfolded protein response (UPR).

[0038] Example 2: Effects of GSK2606414 on DTMUV-induced autophagy and viral replication

[0039] 1. Test method:

[0040] The experiment is designed with the following treatment:

[0041] Treatment 1 (Mock): Duck embryo fibroblasts (DEF) were cultured in 96-well cell plates and cultured normally;

[0042] Treatment 2 (DTMUV): Duck embryo fibroblasts (DEF) were cultured in 96-well cell plates and infected with 100 μL DTMUV virus solution;

[0043] Treatment 3 (DTMUV+DMSO): Duck embryo fibroblasts (DEF) were cultured in 96-well cell plates, treated with DMSO for 1 h, and then infected with 100 μL of DTMUV virus solution.

[0044] Treatment 4 (DTMUV+GSK2606414): Duck embryonic fibroblasts (DEF) were cultured in 96-well cell plates. GSK2606414 was prepared into a 1 μM solution with DMSO. DEF cells were treated with the GSK2606414 solution for 1 h, and then infected with 100 μL DTMUV virus solution.

[0045] Thirty-six hours after viral infection, cells were collected for Western blot analysis to detect the expression of p-eIF2α, eIF2α, LC3-I, and LC3-II proteins.

[0046] Following the method described in "Establishment of quantitative RT-PCR method for Tembusu virus [J]. Chinese Agricultural Science, 2012, 45(21):4492-4500," the viral copy number of DTMUV in treatments 2-4 was detected.

[0047] 2. Test Results:

[0048] Western blot results are as follows Figure 2 As shown, 36 hours after viral infection, compared with the DTMUV infection group (treatment 2), the p-eIF2α level in the GSK2606414 treatment group (treatment 4) was significantly decreased, indicating that GSK2606414 can effectively inhibit the PERK pathway activated by DTMUV. Furthermore, GSK2606414 treatment also significantly reduced LC3-II protein expression, suggesting that DTMUV can mediate autophagy through the PERK pathway.

[0049] The virus copy number detection results of DTMUV in processes 2-4 are as follows: Figure 3 As shown, the results revealed that the addition of GSK2606414 (treatment 4) significantly reduced the viral copy number of DTMUV. This demonstrates that GSK2606414 can inhibit the replication of DTMUV and could serve as a novel therapeutic agent for duck Tembusu virus disease.

[0050] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Applications of GSK2606414 in the following (1) or (2): (1) Preparation of drugs to inhibit the replication of duck Tembusu virus; (2) Prepare drugs for the prevention and treatment of duck Tembusu virus disease.

2. The application according to claim 1, characterized in that, The concentration of GSK2606414 is 0.1-10 μM.

3. The application according to claim 2, characterized in that, The concentration of GSK2606414 used is 1 μM.

4. The application according to claim 1 or 2, characterized in that, The drug also contains one or more pharmaceutically acceptable carriers.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: sodium carboxymethyl cellulose, sodium ethyl cellulose, calcium stearate, magnesium stearate, soybean oil, sesame oil, peanut oil, olive oil, propylene glycol, mannitol, sorbitol, emulsifiers, wetting agents, colorants, stabilizers, and preservatives.

6. The application according to claim 5, characterized in that, The dosage form of the drug is an aqueous suspension, an oil suspension, an emulsion, a powder, a tablet, a capsule, or a granule.

7. A drug for inhibiting the replication of duck Tembusu virus, characterized in that, The drug uses GSK2606414 as its active ingredient.

8. The medicament according to claim 7, characterized in that, The concentration of GSK2606414 in the drug is 1 μM.

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