Application of inhibitor T16Ainh-A01 in preparation of medicine for preventing or treating porcine reproductive and respiratory syndrome virus infection

Abstract

The invention belongs to the technical field of cytobiology and medicine, and particularly relates to application of an inhibitor T16Ainh-A01 in preparation of a medicine for preventing or treating porcine reproductive and respiratory syndrome virus infection. According to the application disclosed by the invention, 20 mu mol / L of T16Ainh-A01 is added in the process that MARC-145 cells or PAMs cells are infected with the PRRSV in vitro, compared with the process that T16Ainh-A01 is not added, the RNA content and the virus titer of the PRRSV in a T16Ainh-A01 treatment group are obviously reduced, the T16Ainh-A01 is proved to be capable of obviously inhibiting the RNA content and the virus titer in the PRRSV in-vitro infection process, and a new strategy is provided for preparing a medicine for preventing or treating PRRSV infection. The invention solves the problems of single drug target, easy drug resistance and the like in the prior art, and can be used as a virus inhibitor in the preparation process of anti-PRRSV vaccines to reduce virus pollution.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and pharmaceutical technology, and specifically relates to the application of T16Ainh-A01, an inhibitor of transmembrane protein 16A. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease" in pigs, is a highly contagious disease of pigs caused by PRRS virus (PRRSV). Discovered in the late 1980s, PRRS subsequently spread widely globally, causing serious harm. Studies have shown that PRRSV is a typical immunosuppressive virus characterized by reproductive disorders, including premature birth, late abortion, stillbirth, weak piglets, and mummified fetuses, as well as respiratory dysfunction in piglets and growing pigs. Furthermore, the immunosuppression caused by PRRSV can further lead to secondary infections of various pathogens in pigs. For over forty years, PRRSV has caused enormous economic losses to the pig industry, but its control remains unsatisfactory.

[0003] PRRSV is an enveloped, single-stranded, positive-sense RNA virus belonging to the order Hellaviridae (Hynovirales). Nidovirales Arteritis Virology Department ( Arteriviridae Arteritis virus genus ( PorartevirusUnlike other members of the arteriovirus genus, which exhibit relatively broad cell tropism, PRRSV infection is highly confined to cells of the mononuclear macrophage lineage, such as porcine alveolar macrophages (PAM) and African green monkey kidney epithelial cells (MARC-145), which are the main targets of PRRSV in vivo. Typical immune features of PRRSV infection include persistent viremia, strong suppression of innate cytokines (IFN-α / β, TNF-α, IL-1β, etc.), NK cell dysfunction, rapid induction of non-neutralizing antibodies, delayed appearance of neutralizing antibodies, delayed CD8 T cell response, and induction of regulatory T cells (Tregs). Currently, there are no specific drugs for PRRSV infection in clinical practice. PRRS prevention and control mainly uses inactivated vaccines and attenuated vaccines for immunization, with attenuated vaccines being more commonly used clinically. However, the prevalence of PRRSV infection in pig herds remains high, and vaccination has had limited effectiveness. Inactivated vaccines have the following drawbacks: (1) they require high-dose vaccination or the use of concentrated antigens, resulting in a short period of immunity and often necessitating booster vaccinations; (2) they cannot induce local immunity, leading to weak cellular immunity; (3) it takes 2-3 weeks to produce complete immunity, which is not conducive to emergency preventive vaccination and reducing vaccine costs; (4) there is a possibility of incomplete inactivation and virus shedding; (5) attenuated vaccines pose risks of virulence reversion, recombinant infections, potential infection, and lack of cross-protection. All of these factors hinder the effective control and spread of PRRS, thus necessitating the development of novel anti-PRRSV drugs.

[0004] In recent years, studies have found that PRRSV infection can be inhibited by regulating host cell pathways. For example, invention publication CN112933066A discloses that the calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 can significantly reduce PRRSV RNA and viral titer at a concentration of 50 μM, but its mechanism of action is unclear, and high concentrations may pose toxicity risks. Invention publication CN117503735A discloses that a natural compound and magnolol can inhibit PRRSV replication by 99% at 10 μM, but problems such as poor water solubility and unclear target sites still exist. Therefore, there is an urgent need to develop novel PRRSV inhibitors with clear mechanisms of action, low toxicity, high efficacy, and broad spectrum. Calcium-activated chloride channels (CaCCs) are a class of cell membrane-expressed chloride channels activated by calcium ions, mediating a series of important physiological functions. Among them, transmembrane protein 16A (TMEM16A / ANO1) inhibitors are representative of CaCCs. Recent studies have shown that CaCCs are significantly upregulated in virus-infected host cells, potentially affecting viral invasion and replication by regulating cell membrane potential and ion homeostasis. Among these, the small molecule inhibitor T16Ainh-A01, an aminophenylthiazole, is a TMEM16A / ANO1 inhibitor that inhibits TMEM16A-mediated chloride ion currents. Currently, it is mainly used in research on diseases such as asthma and tumors. For example, invention publication CN113117088A discloses the application of calcium-activated chloride channel inhibitors in tumor immunotherapy, but no reports have been found on their anti-PRRSV activity. Based on this, this invention proposes for the first time the application of calcium-activated chloride channel inhibitors in anti-PRRSV drugs to overcome the shortcomings of existing technologies. Summary of the Invention

[0005] This invention proposes the application of an inhibitor, T16Ainh-A01, in the preparation of drugs for the prevention or treatment of porcine reproductive and respiratory syndrome virus (PRRSV) infection, providing a new strategy for the development of novel anti-PRRSV drugs and solving problems such as single drug target and easy drug resistance in existing technologies.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention provides an antiviral drug, wherein the active ingredient of the antiviral drug is a transmembrane protein 16A inhibitor T16Ainh-A01, wherein the effective concentration of T16Ainh-A01 is 10-20 μM.

[0007] The virus in question is porcine reproductive and respiratory syndrome virus (PRRSV).

[0008] The drug is intended for use on pigs.

[0009] The antiviral drug also contains a pharmaceutically acceptable carrier, which may be a sugar, such as lactose, glucose and sucrose; a starch, such as corn starch and potato starch; or cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose.

[0010] The drug can be prepared into any one or more of the following dosage forms, suitable for different routes of administration. (a) Injectables: aqueous injections, lyophilized powder injections, and long-acting microsphere injections; (b) Aerosols: metered-dose nasal sprays, lung-targeted dry powder inhalers; (c) Oral preparations: solutions, premixes, coated granules, sustained-release capsules.

[0011] The antiviral drug T16Ainh-A01 can significantly inhibit in vitro infection of porcine reproductive and respiratory syndrome virus.

[0012] This invention provides a method and application for inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection in vitro using the inhibitor T16Ainh-A01. The specific steps are as follows: (1) MARC-145 (or CRL2843-CD163) cells were divided into groups of 2×10⁻⁶. 5 Plant cells at a density of 10 cells / mL in 24-well culture plates, add 500 μL of DMEM (or RPMI 1640) medium to each well, and incubate at 37°C in a 5% CO2 incubator for 12-24 h. (2) Replace with a new culture medium and incubate the cells with PRRSV-2 BJ-4 strain with a multiplicity of 1 at 37°C for 2 hours; discard the cell supernatant and wash the cells three times with PBS buffer to remove virus particles that have not been adsorbed onto the cells. (3) Add 500 μL of cell culture medium containing the drug to each well of the cell culture plate and continue culturing at 37°C for 22 h; (4) Discard the cell supernatant, collect the cells, wash with PBS, and then test the PRRSV RNA content and viral titer of MARC-145 (or CRL2843-CD163) cells.

[0013] The DMEM (or RPMI 1640) medium contains 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0014] The concentration of the inhibitor T16Ainh-A01 in the drug is 20 μM.

[0015] The drug can significantly inhibit in vitro infection of porcine reproductive and respiratory syndrome virus (PRRSV), reducing PRRSV RNA content in MARC-145 cells by 90% and viral titer by more than 100-fold; and reducing PRRSV RNA content in CRL2843-CD163 cells by 95%.

[0016] The present invention also provides the above-mentioned antiviral drugs as drugs for the prevention or treatment of porcine reproductive and respiratory syndrome virus infection, and their application in anti-porcine reproductive and respiratory syndrome virus vaccines.

[0017] The present invention has the following beneficial effects: This invention is the first to discover that the transmembrane protein 16A inhibitor T16Ainh-A01 has an inhibitory effect on PRRSV infection in vitro. Firstly, the cytotoxicity of the commercially available transmembrane protein 16A inhibitor T16Ainh-A01 was detected using quantitative real-time PCR (RT-qPCR) and TCID assay. 50 The researchers identified the inhibitory effect of the non-cytotoxic T16Ainh-A01 on PRRSV in vitro infection and found that it could significantly inhibit RNA content and viral titer during PRRSV in vitro infection.

[0018] The method described in this invention can significantly inhibit PRRSV infection in vitro. When 20 μmol / L T16Ainh-A01 is added during the infection of MARC-145 cells or PAMs cell line CRL2843-CD163 with PRRSV, the PRRSV RNA content and viral titer are significantly reduced in the T16Ainh-A01 treatment group compared with the untreated group.

[0019] The transmembrane protein 16A inhibitor T16Ainh-A01 provided by this invention can also be applied to the research and development of antiviral drugs against PRRSV infection, used as an active ingredient in anti-PRRSV infection drug compositions, and can also be used as a virus scavenger in the PRRSV vaccine preparation process, and has broad market prospects. Attached Figure Description

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The bar chart shows the cytotoxicity of T16Ainh-A01 against MARC-145 cells.

[0022] Figure 2 A bar chart showing the relative content of PRRSV RNA in MARC-145 cells infected with PRRSV.

[0023] Figure 3 A bar chart showing the PRRSV virus titer in MARC-145 cells.

[0024] Figure 4 A bar chart showing the relative content of PRRSV RNA in CRL2843-CD163 cells infected with PRRSV. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0026] This invention is the first to discover that TMEM16A is a key host factor for PRRSV infection, inhibiting PRRSV invasion by targeting calcium-activated chloride channels in host cells. The small molecule inhibitor T16Ainh-A01, a type of aminophenylthiazole, is an inhibitor of TMEM16A, inhibiting TMEM16A-mediated chloride currents. This invention demonstrates that the inhibitor T16Ainh-A01 can significantly inhibit PRRSV infection in vitro. Therefore, this invention provides an antiviral drug with the inhibitor T16Ainh-A01 as the active ingredient.

[0027] The active ingredient of the antiviral drug is the transmembrane protein 16A inhibitor T16Ainh-A01, wherein the effective concentration of T16Ainh-A01 is 10-20 μM.

[0028] The antiviral drug also contains a pharmaceutically acceptable carrier, which may be a sugar, such as lactose, glucose and sucrose; a starch, such as corn starch and potato starch; or cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose.

[0029] The drug can be prepared into any one or more of the following dosage forms, suitable for different routes of administration. (a) Injectables: aqueous injections, lyophilized powder injections, and long-acting microsphere injections; (b) Aerosols: metered-dose nasal sprays, lung-targeted dry powder inhalers; (c) Oral preparations: solutions, premixes, coated granules, sustained-release capsules.

[0030] The antiviral drug T16Ainh-A01 can significantly inhibit in vitro infection of porcine reproductive and respiratory syndrome virus.

[0031] According to the present invention, the above-mentioned antiviral drug is also provided as a drug for the prevention or treatment of porcine reproductive and respiratory syndrome virus infection and its use in anti-porcine reproductive and respiratory syndrome virus vaccines.

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Identification of the cytotoxicity of T16Ainh-A01 against MARC-145 cells MARC-145 cells were divided into 2×10 5 Cells were seeded at a density of 100 μL / mL in 96-well plates. Each well contained 100 μL of DMEM medium with 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The plates were incubated at 37°C for 24 h in a 5% CO2 incubator. The original cell culture medium was then removed and replaced with DMEM medium containing different concentrations of T16Ainh-A01 (CAS No.: 552309-42-9, MCE, catalog number HY-100612; 0, 10, 20 μmol / L). After incubation at 37°C for 24 h, 20 μL of Promega CellTiter 96 AQ was added. ueous One Solution reagent (catalog number G3582) was incubated at 37°C in a 5% CO2 incubator for 2 h; cytotoxicity was identified according to the instructions. Statistical analysis was performed using an unpaired, two-tailed Student t-test in GraphPad software; ns was considered as no significant difference.

[0034] The results are as follows Figure 1 As shown, compared with the untreated group (0 μmol / L) with 10 μmol / L T16Ainh-A01, the addition of 10 and 20 μmol / L T16Ainh-A01 had no significant effect on cell activity, i.e., no cytotoxicity, and can be used in subsequent examples.

[0035] Example 2: Effect of T16Ainh-A01 on PRRSV RNA content in MARC-145 cells infected with PRRSV MARC-145 cells were divided into 2×10 524-well cell culture plates were plated with cells per mL. 500 μL of DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. The DMEM medium was replaced, and MARC-145 cells were infected with PRRSV-2 BJ-4 strain (GenBank ID AF331831) with a multiplicity of infection (MOI) of 1 at 37°C for 2 h. The cell supernatant was discarded, and the cells were washed three times with phosphate-buffered saline (PBS) to remove any remaining uninfected virus particles. 500 μL of DMEM medium containing 20 μmol / L T16Ainh-A01 was added to each well of the cell culture plate, and the plates were incubated at 37°C for another 22 h. Collect cells, discard cell supernatant, and wash cells three times with PBS; extract total RNA from PRRSV-infected cells using TRIzol RNA extraction reagent (Vazyme, catalog number R401-01), and use PrimeScript. TM A reverse transcription kit (Vazyme, catalog number R212-01) was used to generate complementary DNA (cDNA) as a template for RT-qPCR. PRRSV ORF7 RNA content was measured using relative RT-qPCR to represent the viral RNA content of infected cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and viral RNA was normalized using GAPDH mRNA. The results were then analyzed using a 2... -△△CT The method was used for relative quantification; primers and reaction systems are shown in Tables 1, 2 and 3. The reaction program used was the Fast program built into the ABI Real-Time PCR instrument (Applied Biosystems, USA).

[0036] Table 1. Primer sequences for relative quantitative PCR Table 2 Relative Real-Time PCR Reaction System (1) Table 3 Relative Real-Time PCR Reaction System (2) The experiment was conducted independently in triplicate, with three replicates each time. Experimental data are expressed as group means and standard deviations (SD). Statistical analysis was performed using the unpaired, two-tailed Student's t-test in GraphPad software. *** p <0.001 indicates a highly statistically significant difference.

[0037] The results are as follows Figure 2The results showed that, compared with the group without T16Ainh-A01 treatment, the PRRSV RNA content in the group treated with 20 μmol / L T16Ainh-A01 was significantly reduced (by nearly 90%), indicating that T16Ainh-A01 can reduce the PRRSV RNA content in MARC-145 cells.

[0038] Example 3: Effect of T16Ainh-A01 on PRRSV virus titer in MARC-145 cells To further confirm that T16Ainh-A01 inhibits the PRRSV infection rate in MARC-145 cells, MARC-145 cells were cultured as in Example 2. The medium was replaced with fresh DMEM, and MARC-145 cells were infected with the PRRSV-2 BJ-4 strain (MOI 1) at 37°C for 2 h. The cell supernatant was discarded, and the cells were washed three times with phosphate-buffered saline (PBS) to remove unadsorbed virus particles. 500 μL of DMEM containing 20 μmol / L T16Ainh-A01 was added to the wells of the cell culture plate, and the cells were cultured at 37°C for another 22 h for the TCID assay. 50 Uninfected MARC-145 cells were diluted to 2 × 10⁻⁶. 5 MARC-145 cells were seeded at a density of 100 μL / mL in 96-well cell culture plates, and each well was incubated overnight at 37°C with 5% CO2. The supernatant from infected MARC-145 cells (24 h prior) was serially diluted 10-fold and seeded into the 96-well plates with 100 μL of the diluted supernatant at 8 replicates. DMEM was used as a control instead of the infected cell supernatant. The 96-well plates were incubated at 37°C with 5% CO2. Cell growth was observed every 24 h, and the number of wells showing cytopathic effects was recorded. This observation period lasted 5-7 days until the number of wells showing cytopathic effects stopped increasing. The TCID of the virus was calculated using the Reed-Muench method. 50 Experimental data are expressed as group means and standard deviations (SD). Statistical analysis was performed using the unpaired, two-tailed Student's t-test in GraphPad software. p <0.0001 indicates a highly statistically significant difference.

[0039] The results are as follows Figure 3 As shown, compared with the group without T16Ainh-A01 (0 μmol / L), the PRRSV viral titer was significantly reduced in the group treated with 20 μmol / L T16Ainh-A01 (the reduction in viral titer was >2 log). 10 TCID 50 / mL (i.e., greater than 100 times), indicating that T16Ainh-A01 can significantly reduce the PRRSV viral titer in MARC-145 cells, further confirming that T16Ainh-A01 can reduce the PRRSV infection dose in MARC-145 cells.

[0040] Example 4: Effect of T16Ainh-A01 on PRRSV RNA content in PAMs infected with PAMs PAMs are the main target cells for PRRSV infection in pigs. To verify whether T16Ainh-A01 can also reduce the PRRSV infection level in PAM cultures, the PRRSV RNA content after T16Ainh-A01 treatment was measured.

[0041] PAMs cell line CRL2843-CD163 cells were divided into 2×10 5 24-well cell culture plates were plated with cells per mL. 500 μL of RPMI 1640 medium (Beijing Solarbio Science & Technology Co., Ltd., catalog number 31800) containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 12 h. The medium was then replaced with fresh RPMI 1640 medium, and CRL2843-CD163 cells were infected with the PRRSV-2 BJ-4 strain (MOI 1) at 37°C for 2 h. The cell supernatant was discarded, and the cells were washed three times with phosphate-buffered saline (PBS) to remove any virus particles not adsorbed onto the cells. 500 μL of RPMI 1640 medium containing 20 μmol / L T16Ainh-A01 was added to each well of the cell culture plate, and the plates were incubated at 37°C for another 22 h. Collect cells, discard cell supernatant, and wash cells three times with PBS; extract total RNA from PRRSV-infected cells using TRIzol RNA extraction reagent (Vazyme, catalog number R401-01), and use PrimeScript. TM A reverse transcription kit (Vazyme, catalog number R212-01) was used to generate complementary DNA (cDNA) as a template for RT-qPCR. PRRSV ORF7 RNA content was measured using relative RT-qPCR to represent the viral RNA content of infected cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and viral RNA was normalized using GAPDH mRNA. The results were then analyzed using a 2... -△△CT The method was used for relative quantification; primers and reaction systems are shown in Tables 1, 2 and 3. The reaction program used was the Fast program built into the ABI Real-Time PCR instrument (Applied Biosystems, USA).

[0042] The experiment was conducted independently in triplicate, with three replicates each time. Experimental data are expressed as group means and standard deviations (SD). Statistical analysis was performed using the unpaired, two-tailed Student's t-test in GraphPad software. *** p <0.001 indicates a highly statistically significant difference.

[0043] The results are as follows Figure 4 The results showed that, compared with the group without T16Ainh-A01 (0 μmol / L), the group with 20 μmol / L T16Ainh-A01 had a significantly lower PRRSV RNA content (reduced by about 95%), indicating that T16Ainh-A01 can reduce the PRRSV RNA content of PAMs infection.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antiviral drug, characterized in that: The active ingredient of the antiviral drug is the transmembrane protein 16A inhibitor T16Ainh-A01.

2. The antiviral drug according to claim 1, characterized in that: The virus in question is porcine reproductive and respiratory syndrome virus (PRRSV).

3. The antiviral drug according to claim 2, characterized in that: The effective concentration of T16Ainh-A01 in the drug is 10-20 μM.

4. The antiviral drug according to claim 3, characterized in that: The drug is an injection, aerosol, or oral preparation.

5. The antiviral drug according to claim 4, characterized in that: The antiviral drug also contains a pharmaceutically acceptable carrier.

6. The antiviral drug according to claim 5, characterized in that: The carrier is selected from any one of lactose, glucose, sucrose, corn starch, sodium methylcellulose, ethylcellulose, and methylcellulose.

7. The use of the antiviral drug according to any one of claims 1-6 as a drug for the prevention or treatment of porcine reproductive and respiratory syndrome virus infection.

8. The application according to claim 7, characterized in that: The antiviral drug works by inhibiting the RNA content of porcine reproductive and respiratory syndrome virus (PRRSV).

9. The use of the antiviral drug according to any one of claims 1-6 in the preparation of a vaccine against porcine reproductive and respiratory syndrome virus.

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