Application of KN-93 in the preparation of drugs for inhibiting porcine delta coronavirus

Abstract

The present invention discloses the use of KN-93 in the preparation of drugs for inhibiting porcine delta coronavirus, and relates to the technical fields of cell biology and virology. The present invention comprehensively utilizes technologies such as cell biology and virology, and finds that the calmodulin-dependent protein kinase II inhibitor KN-93 has the effect of inhibiting porcine delta coronavirus infection in vitro. First, KN-93 is subjected to cytotoxicity testing to determine the maximum safe concentration of the drug, and then fluorescent quantitative PCR (RT-qPCR), TCID 50 The in vitro inhibition of PDCoV infection by 10 μmol / L KN-93 was determined by ELISA and IFA, and it was found to significantly inhibit porcine deltacoronavirus. Therefore, KN-93 can be used to prepare drugs that inhibit porcine deltacoronavirus.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology and virology, and in particular to the application of KN-93 in the preparation of drugs for inhibiting porcine delta coronavirus. Background Art

[0002] Porcine deltacoronavirus (PDCoV) belongs to the Coronaviridae family and a new member of the genus Deltacoronavirus. It is a single-stranded positive-sense RNA virus with an envelope and a diameter of approximately 60-180nm. Under an electron microscope, PDCoV virus particles are spherical with typical crown-like protrusions on the surface of coronaviruses. The genome length is approximately 25.4kb (excluding the Poly A tail), making it the smallest known coronavirus.

[0003] PDCoV is currently prevalent worldwide, severely hampering the development of the pig farming industry. PDCoV infection is widespread in pig populations, with rapid and widespread transmission. It is highly pathogenic, causing clinical symptoms such as vomiting, diarrhea, and dehydration. The mortality rate in suckling piglets can reach 30% to 40%, resulting in significant economic losses for the pig farming industry.

[0004] Unresolved key scientific questions about the virus's infection and pathogenic mechanisms have severely hampered the development of vaccines and antiviral drugs, posing significant challenges to its prevention and control. PDCoV has a broad spectrum of infection, infecting not only pigs but also cattle, mice, chickens, and other animals, posing a significant risk of cross-species transmission and posing a significant challenge to global public health security. Therefore, it is necessary to develop new anti-PDCoV drugs to inhibit PDCoV infection. Summary of the Invention

[0005] The present invention aims to provide the use of KN-93 in the preparation of drugs for inhibiting porcine deltacoronavirus to address the problems of the prior art. The present invention has found that the calmodulin-dependent protein kinase II inhibitor KN-93 has the effect of inhibiting PDCoV infection in vitro and can therefore be used to prepare drugs for inhibiting porcine deltacoronavirus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of KN-93 in preparing a medicament for inhibiting porcine delta coronavirus.

[0008] The present invention also provides a drug for inhibiting porcine delta coronavirus, wherein the active ingredient includes KN-93.

[0009] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the excipients include solubilizers, colorants, binders, disintegrants, fillers, stabilizers and / or preservatives.

[0011] Furthermore, the dosage form of the drug is granules, tablets, powders, capsules, emulsions or injections.

[0012] The present invention also provides the use of KN-93 in the preparation of an environmental disinfectant for inhibiting swine delta coronavirus.

[0013] The present invention also provides an environmental disinfectant for inhibiting swine delta coronavirus, wherein the active ingredient includes KN-93.

[0014] The present invention discloses the following technical effects:

[0015] The present invention uses a combination of cell biology and virology techniques to discover that the calmodulin-dependent protein kinase II inhibitor KN-93 has the effect of inhibiting PDCoV infection in vitro. First, KN-93 is tested for cytotoxicity to determine the highest safe concentration of the drug, and then fluorescent quantitative PCR (RT-qPCR), TCID 50 The in vitro inhibition of PDCoV infection by 10 μmol / L KN-93 was determined by ELISA and IFA, and it was found to significantly inhibit PDCoV. Therefore, KN-93 can be used to prepare drugs to treat PDCoV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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. 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.

[0017] Figure 1 is a bar graph showing the cytotoxicity of KN-93 to LLC-PK1 cells;

[0018] Figure 2 This is a bar graph showing the effect of KN-93 on inhibiting PDCoV infection detected by RT-qPCR. A is the result of 12 hours of cell culture; B is the result of 18 hours of cell culture.

[0019] Figure 3 TCID 50 Methods Histogram showing the effect of KN-93 in inhibiting PDCoV infection;

[0020] Figure 4 This is an immunofluorescence image to detect the effect of KN-93 in inhibiting PDCoV infection. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Terminology Notes:

[0027] KN-93 is a calmodulin-dependent protein kinase II inhibitor with a CAS number of 139298-40-1 and a molecular formula of C 26 H 29 ClN2O4S, the structural formula is as follows:

[0028]

[0029] The MEM culture medium used in the following examples contained 5% (v / v) heat-inactivated fetal bovine serum, 1 wt % penicillin, and 1 wt % streptomycin.

[0030] Example 1

[0031] 1. Materials

[0032] PDCoV HNZK-02 strain (GenBank accession number MH708123) and LLC-PK1 cells were provided by the Animal Molecular Pathogenesis Laboratory of Henan Agricultural University; MEM culture medium and fetal bovine serum were purchased from Gibco, USA; β-actin antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; PDCoV N protein monoclonal antibody has been disclosed in Chinese patent CN202110376120.1 and was prepared and preserved by the Animal Molecular Pathogenesis Laboratory of Henan Agricultural University.

[0033] 2. Methods and Results

[0034] 1. Identification of KN-93 Cytotoxicity to LLC-PK1 Cells

[0035] LLC-PK1 cells were plated into 96-well plates and, after confluence, washed three times with D-Hanks balanced salt solution. Different concentrations of KN-93 were added and incubated at 37°C for 48 hours. Cytotoxicity was assessed according to the CCK8 kit instructions, and statistical analysis was performed using GraphPad software.

[0036] The results are as follows Figure 1 As shown, compared with the cell activity of the untreated group (0 μmo1 / L) (cell activity is 100%), the addition of 20 μmol / L KN-93 had a significant effect on cell activity, while the addition of 5 μmol / L or 10 μmol / L KN-93 had no significant effect on cell activity (ns), that is, no cytotoxicity, so 10 μmol / L KN-93 was used for subsequent experiments.

[0037] 2. RT-qPCR detection of KN-93's inhibitory effect on PDCoV infection

[0038] LLC-PK1 cells were plated into 12-well plates and cultured in a 37°C, 5% CO2 incubator. The cells were washed three times with D-Hanks balanced salt solution and infected with PDCoV HNZK-02 strain (GenBank accession number MH708123) at an MOI of 2 at 37°C for 1 hour. The cells were washed three times with D-Hanks balanced salt solution, and MEM medium containing trypsin and 10 μmol / L KN-93 was added and cultured for 12 and 18 hours. The cells were collected, total RNA was extracted, and reverse transcribed into cDNA. The cDNA was used as a template for RT-qPCR. The primer sequences, reaction system, and reaction procedure are shown in Tables 1, 2, and 3.

[0039] The results are as follows Figure 2 Compared with the untreated group, the PDCoVRNA content in the KN-93-treated group was significantly reduced.

[0040] Table 1 RT-qPCR primer sequences

[0041]

[0042] Table 2 RT-qPCR reaction system

[0043]

[0044] Table 3 RT-qPCR reaction procedure

[0045]

[0046] 3.TCID 50 Methods to detect the effect of KN-93 on inhibiting PDCoV infection

[0047] LLC-PK1 cells were plated into 12-well plates and cultured in a 37°C, 5% CO2 incubator. The cells were washed three times with D-Hanks balanced salt solution, and the PDCoV HNZK-02 strain with an MOI of 2 was used to infect LLC-PK1 cells at 37°C for 1 hour. The cells were washed three times with D-Hanks balanced salt solution, and MEM medium containing trypsin and 10 μmol / L KN-93 was added and cultured for 12 and 18 hours. The cell supernatant was collected and diluted 10-fold, and inoculated into LLC-PK1 cells that had been plated in 96-well cell culture plates. Eight replicates were made for each dilution, and the cells were cultured in a 37°C, 5% CO2 incubator for 3 days. The number of cytopathic wells no longer increased. The TCID of the virus was calculated using the Reed-Muench method. 50 .

[0048] The results are as follows Figure 3 As shown, the PDCoV virus titer in the KN-93-treated group was significantly reduced compared with the untreated group.

[0049] 4. Immunofluorescence detection of KN-93 inhibitory effect on PDCoV infection

[0050] LLC-PK1 cells were plated into 12-well plates and cultured in a 37°C, 5% CO2 incubator. The cells were washed three times with D-Hanks balanced salt solution and infected with the PDCoV HNZK-02 strain at an MOI of 2 for 1 hour at 37°C. The cells were washed three times with D-Hanks balanced salt solution and incubated in MEM medium containing trypsin and 10 μmol / L KN-93 for 18 hours. The cells were fixed with anhydrous ethanol and washed twice with PBS. IFA blocking solution was added to each well and blocked for 2 hours at room temperature. Following blocking, a monoclonal antibody against the PDCoV N protein (1:3000 dilution) was added and incubated overnight at 4°C. The cells were washed three times with PBS and a FITC-conjugated goat anti-mouse secondary antibody (1:3000 dilution) was added and incubated in a 37°C incubator protected from light for 1 hour. The cells were washed three times with PBS and nuclei were stained with DAPI for 15 minutes at room temperature. The cells were placed on a horizontal shaker and washed with PBS at 130 rpm for 5 min, three times, and observed and photographed using a fluorescence microscope.

[0051] The results are as follows Figure 4 As shown, the fluorescence intensity of the KN-93 treated group was significantly decreased compared with the untreated group.

[0052] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of KN-93 in the preparation of drugs for inhibiting porcine delta coronavirus.

2. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that The excipients include solubilizers, colorants, binders, disintegrants, fillers, stabilizers and / or preservatives.

4. The use according to claim 3, characterized in that The dosage form of the medicine is granules, tablets, powders, capsules, emulsions or injections.

5. Application of KN-93 in the preparation of environmental disinfectants for inhibiting swine delta coronavirus.

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