A composition for use against foot-and-mouth disease virus and its application

Abstract

This invention belongs to the field of biology, specifically relating to a composition and its use against foot-and-mouth disease virus (FMDV). This invention discovered that compounds Pasireotide and Oxyclozanide alone cannot significantly inhibit FMDV replication. Unexpectedly, the combined use of Pasireotide and Oxyclozanide significantly inhibits the expression of FMDV RNA polymerase 3D protein, reducing the half-maximal tissue-cell infection dose (TCID) of FMDV. 50 It can significantly reduce the replication ability of foot-and-mouth disease virus and has the effect of antagonizing foot-and-mouth disease virus infection; and there are currently no clear clinical studies or drug instructions mentioning that there is a direct physical / chemical incompatibility between the two, so it can be used to prepare drugs against foot-and-mouth disease virus and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a composition for combating foot-and-mouth disease virus and its uses. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, highly virulent, and contagious animal disease that primarily infects cloven-hoofed animals such as cattle, pigs, sheep, and goats. In my country, it is classified as a Class A animal disease and falls under the category of key animal infectious diseases requiring prevention and control. The pathogenic agent of this disease is the foot-and-mouth disease virus (FMDV), which belongs to the Picornaviridae family. Its structural characteristics include a lack of envelope and a single-stranded positive-sense RNA genome.

[0003] From a serotype perspective, FMDV has seven different serotypes, including O, A, C, Asia1, and SAT1, SAT2, and SAT3. Notably, each serotype has a large number of subtypes, and there is no cross-protection between different serotypes. This characteristic poses a significant challenge to disease control. Currently, vaccination is the mainstream and effective means of controlling foot-and-mouth disease, but FMDV possesses strong immunosuppressive capabilities, enabling it to rapidly breach the host animal's immune system barrier, thus achieving efficient viral replication and spread.

[0004] Given this, relying solely on vaccines to control foot-and-mouth disease is insufficient to completely address the viral threat. Therefore, simultaneously developing drugs that can be used to combat foot-and-mouth disease virus infection is of paramount practical significance and necessity.

[0005] The compound Pasireotide is a long-acting cyclohexyl peptide somatostatin analog that can enhance the agonist activity of somatostatin receptors and inhibit the secretion of GH, IGF-I and ACTH. It can be used to study acromegaly and Cushing's disease. It also has antisecretory, antiproliferative and pro-apoptotic activities.

[0006] The compound Oxyclozanide is an orally administered salicylaniline anthelmintic with anti-adenovirus, anti-biofilm, antifungal, and antibacterial biological activities. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention has discovered that compounds Pasireotide and Oxyclozanide alone cannot significantly inhibit the replication of foot-and-mouth disease virus. Unexpectedly, the combined use of Pasireotide and Oxyclozanide can significantly inhibit the replication of foot-and-mouth disease virus, and can be used to prepare drugs against foot-and-mouth disease virus. Furthermore, there are currently no clear clinical studies or drug instructions mentioning any direct physical / chemical incompatibilities between the two compounds, indicating promising application prospects.

[0008] Specifically, it includes the following:

[0009] In a first aspect, the present invention provides a composition for inhibiting the replication of foot-and-mouth disease virus, the active ingredient of the composition being composed of the compound Pasireotide or a pharmaceutically acceptable salt thereof and the compound Oxyclozanide or a pharmaceutically acceptable salt thereof; the structural formula of the compound Pasireotide is shown in Formula (I), and the structural formula of the compound Oxyclozanide is shown in Formula (II).

[0010]

[0011] Equation (I);

[0012]

[0013] Formula (II).

[0014] In a second aspect, the present invention provides the use of the composition described in the first aspect in the preparation of an anti-foot-and-mouth disease virus drug.

[0015] Preferably, the composition inhibits the replication of foot-and-mouth disease virus.

[0016] Preferably, the composition inhibits the expression of foot-and-mouth disease virus RNA polymerase 3D protein.

[0017] Preferably, the composition reduces the TCID of foot-and-mouth disease virus. 50 .

[0018] Preferably, the composition is formulated with pharmaceutically acceptable excipients to create a pharmaceutically acceptable dosage form.

[0019] The beneficial effects of this invention are as follows: This invention discovers that compounds Pasireotide and Oxyclozanide alone cannot inhibit the replication of foot-and-mouth disease virus (FMDV). Unexpectedly, the combination of Pasireotide and Oxyclozanide significantly inhibits the expression of FMDV RNA polymerase 3D protein and reduces the TCID of FMDV. 50It can significantly reduce the replication ability of foot-and-mouth disease virus and has the effect of antagonizing foot-and-mouth disease virus infection. It can be used to prepare drugs against foot-and-mouth disease virus. At present, there are no clear clinical studies or drug instructions that mention direct physical / chemical incompatibilities between the two, which shows good application prospects. Attached Figure Description

[0020] Figure 1 The effect of the compound Pasireotide on the activity of BHK21 cells is shown in the figure.

[0021] Figure 2 The effect of compound Oxyclozanide on the activity of BHK21 cells is shown in the figure.

[0022] Figure 3 Figure 1 shows the results of real-time quantitative PCR (RT-qPCR) experiments on the inhibition of foot-and-mouth disease virus replication by the combination of compounds Pasireotide and Oxyclozanide.

[0023] Figure 4 The results of Western blot (WB) experiments showing the inhibition of foot-and-mouth disease virus 3D protein expression by the combination of compounds Pasireotide and Oxyclozanide.

[0024] Figure 5 The combination of the compounds Pasireotide and Oxyclozanide reduces the median cytotoxic dose (TCID) of foot-and-mouth disease virus. 50 The experimental results are shown in the figure.

[0025] Figure 6 Figure 1. Indirect immunofluorescence (IFA) experiment results showing the inhibition of foot-and-mouth disease virus 3D protein expression by the combination of compounds Pasireotide and Oxyclozanide. Detailed Implementation

[0026] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below in conjunction with specific embodiments. However, the scope of protection of this invention is not limited to the embodiments described below.

[0027] DMEM medium, trypsin, fetal bovine serum, goat anti-mouse IgG antibody Alexa Fluor 488, and DAPI nuclear staining solution were purchased from Thermofisher. β-Tubulin monoclonal antibody was purchased from Sigma-Aldrich; RNA extraction TRIzol and reverse transcription kit were purchased from Takara; quantitative real-time PCR kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; BHK21 cells were purchased from ATCC; FMDV strain (FMDV O strain O-BY-CHA-2010) and FMDV-3D protein monoclonal antibody were preserved by Professor Pei Jingjing's research group of the Animal Immunology and Metabolism Team, Research Center for Animal Disease Pathogen Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0028] The structural formulas of the compounds Pasireotide and Oxyclozanide described in the following examples are shown in Formula (I) and Formula (II) respectively. The compounds were purchased from Selleckchem, Houston, TX, USA (P 1127) and Selleckchem, Houston, TX, USA (S 5017).

[0029]

[0030] Equation (I);

[0031]

[0032] Formula (II).

[0033] Unless otherwise specified, all cells and reagents described in the following examples are commercially available.

[0034] This invention uses BHK21 host cells as an example. The study demonstrated that the combination of compounds Pasireotide and Oxyclozanide can inhibit the replication of FMDV in BHK21 host cells, indicating that the combination of compounds Pasireotide and Oxyclozanide described in this invention can inhibit the replication of FMDV and can be used to prepare drugs or adjuvants against FMDV infection.

[0035] Example 1: Effects of compounds Pasireotide and Oxyclozanide on the viability of BHK21 cells

[0036] 1. Preparation of formulations using compounds Pasireotide and Oxyclozanide

[0037] Take 10 μL of 10 mM Pasireotide and Oxyclozanide stock solutions and add them to 190 μL of serum-free DMEM medium for serial dilution to obtain 500 μM Pasireotide and Oxyclozanide working solutions. Store the working solutions in a sealed container at -80℃ for later use.

[0038] BHK21 cell suspension was seeded at 100 μL / well into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h. After cell attachment, the original culture medium in each well was discarded, and 5% DMEM medium containing different concentrations of Pasireotide and Oxyclozanide (final volume 100 μL / well) was added. The corresponding volumes of 500 μM Pasireotide working solution and Oxyclozanide working solution added were 0.5, 1, 2, 4, 6, 8, and 10 μL, respectively, to create a final concentration gradient of 2.5, 5, 10, 20, 30, 40, and 50 μM. Each concentration gradient was set up in triplicate, and the plates were incubated at 37°C with 5% CO2 for another 24 h. After treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 30 min. The absorbance (OD value) of each well was then measured using a microplate reader at 450 nm.

[0039] Cell viability assay results for the compound Pasireotide are as follows: Figure 1 As shown, when the concentration of the compound Pasireotide is ≤10 μM, the viability of BHK21 cells is greater than 90%. This indicates that Pasireotide has no significant toxicity to BHK21 cells at this concentration. Therefore, the compound Pasireotide described in this application has good safety.

[0040] Cell viability assay results for compound Oxyclozanide are as follows: Figure 2 As shown, when the concentration of compound Oxyclozanide is ≤10 μM, the viability of BHK21 cells is greater than 90%. This indicates that Oxyclozanide has no significant toxicity to BHK21 cells at this concentration. Therefore, the compound Oxyclozanide described in this application has good safety.

[0041] Example 2: Inhibitory effect of the combination of compounds Pasireotide and Oxyclozanide on foot-and-mouth disease virus.

[0042] 1. Infection and Compound Treatment

[0043] Cell plating: BHK21 cells were plated at a density of 4 × 10⁶ cells / cm². 5The cells were seeded at a density of cells / well in 12-well cell plates and cultured in a cell culture incubator at 37°C with 5% CO2.

[0044] Inoculation: When cell confluence reaches approximately 80%, inoculate with a 0.1 MOI dose of foot-and-mouth disease virus (FMDV O strain O-BY-CHA-2010). The dosage is calculated using the formula: PFU = number of cells × MOI = 0.7 × TCID. 50 Calculate the required viral volume. Dilute the required viral volume to 980 μL of 2% DMEM medium and seed it into cells in 12-well plates. Incubate at 37°C in a 5% CO2 incubator.

[0045] Single treatment with compounds Pasireotide or Oxyclozanide: 20 μL of working solution of Pasireotide or Oxyclozanide was added to each well of a 12-well plate along with the virus solution, bringing the final concentration of Pasireotide or Oxyclozanide in the wells to 10 μM. The solution was then mixed with the culture medium and incubated at 37°C in a 5% CO2 incubator. The control group was treated with DMSO. Samples were collected for analysis after significant cell lesions were observed.

[0046] Combined treatment with compounds Pasireotide and Oxyclozanide: While adding the virus solution, 10 μL of Pasireotide working solution and 10 μL of Oxyclozanide working solution were added to 12-well cells. The Pasireotide and Oxyclozanide combination was mixed with the culture medium and cultured in a cell culture incubator at 37°C with 5% CO2. The control group was treated with DMSO. When the cells showed significant lesions, the samples were collected for testing.

[0047] 2. Extract total RNA

[0048] Discard the culture medium, wash once with pre-cooled PBS, add 1 mL of RNA extraction reagent (Trizol), mix well by pipetting, transfer to a 1.5 mL EP tube, and incubate at room temperature for 3-5 min. Add 200 μL of chloroform, vortex vigorously for 15 sec, and incubate at room temperature for 5 min. Centrifuge at 12,000 rpm (approximately 14,000 × g) at 4 °C for 10 min. The sample will separate into three layers. Transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, and invert to mix 10-20 times. Centrifuge at 12,000 rpm (approximately 14,000 × g) at 4 °C for 10 min, and discard the supernatant to obtain RNA precipitate. Add 1 mL of 75% ethanol, vortex gently for 15 sec, and wash the RNA precipitate. Centrifuge at 12,000 rpm (approximately 14,000 × g) at 4 °C for 5 min. The RNA precipitate will reach the bottom of the tube; discard the supernatant. Add 100-200 μL of RNA dissolving solution or RNase-free ddH2O to dissolve the RNA.

[0049] 3. Reverse transcription

[0050] Take an equal amount of extracted RNA for reverse transcription, and perform the reverse transcription step according to PrimeScript™ RT Master Mix (Perfect Real Time) (Takara).

[0051] 4. RT-qPCR

[0052] Prepare sterile, RNase-free PCR tubes and place them on an ice box. First, add 2 μL of 5X PrimeScriptRT Master Mix (Perfect Real Time), then add an appropriate amount of Total RNA, and finally add RNase-free ddH2O to bring the total volume of the reaction system to 10 μL.

[0053] Place the PCR tubes containing the prepared reaction solution into a real-time quantitative PCR instrument (or a regular PCR instrument), and set the reaction program: react at 37 ℃ for 15 min, raise the reaction temperature to 85 ℃ and maintain it for 5 sec, and after the reaction is complete, lower the temperature to 4 ℃.

[0054] The results are as follows Figure 3As shown, compared with the solvent control group (DMSO+FMDV group), neither Pasireotide nor Oxyclozanide alone could significantly inhibit the replication of foot-and-mouth disease virus (FMDV). However, the combination of Pasireotide and Oxyclozanide could significantly inhibit the replication of FMDV, indicating that Pasireotide and Oxyclozanide have a synergistic effect and their combined use can significantly inhibit the replication of FMDV.

[0055] Example 3: Immunoblotting verification of the inhibitory effect of the combination of compounds Pasireotide and Oxyclozanide on foot-and-mouth disease virus.

[0056] Repeat the experimental method in Example 2. When significant cytopathic effects appear, collect the cells, discard the supernatant, add 150 μL / well NP 40 (containing protease and phosphatase inhibitors) to each cell, lyse on ice for 30 min, centrifuge at 13000 rpm for 10 min at 4 °C, and discard the precipitate. Add 50 μL of 4× protein loading buffer to the supernatant, vortex to mix, centrifuge at 8000 rpm for 5 min at 100 °C, perform SDS-PAGE (20 μL loading), incubate at 80 V for 30 min, then at 120 V for 60 min; transfer to a membrane, incubate at 230 mA for 2 h, block with 5% skim milk powder for 2 h, incubate overnight at 4 °C with RNA polymerase 3D protein antibody (prepared and stored in our laboratory), wash 3 times for 10 min each time, incubate with secondary antibody for 1 h, wash 3 times for 10 min each time, and expose to sunlight.

[0057] The results are as follows Figure 4 As shown, neither compound Pasireotide nor Oxyclozanide alone could significantly inhibit the expression level of foot-and-mouth disease virus 3D protein (no significant effect). However, the combination of compounds Pasireotide and Oxyclozanide could significantly inhibit the expression level of foot-and-mouth disease virus 3D protein, further indicating that the compounds Pasireotide and Oxyclozanide described in this application have a synergistic effect and can significantly inhibit the replication of foot-and-mouth disease virus when used together.

[0058] Example 4: The effects of compounds Pasireotide and Oxyclozanide on TCID55 foot-and-mouth disease virus in BHK21 cells 50 Impact

[0059] Repeat the experimental method in Example 2. When significant cytopathic effects appeared, collect the supernatant, centrifuge at 3000 rpm for 5 min, and then seed it into a 96-well cell plate. Incubate at 37 ℃ and 5% CO2 until the cell confluence reaches 80%-90%. Then, divide the cells into three groups for treatment, with three biological replicates for each group:

[0060] Pure cell group (blank control group): The original culture medium in the culture plate was removed, and only 100 μL of 5% DMEM culture medium was added. No virus infection or drug treatment was performed.

[0061] Solvent control group (DMSO+FMDV group): Remove the original culture medium from the culture plate, add 100 μL of 5% DMEM culture medium containing DMSO and foot-and-mouth disease virus (MOI=0.1), and incubate at 37℃ and 5% CO2 for 12 h (to ensure that the final concentration of DMSO is consistent with that of the drug treatment group and to eliminate solvent interference).

[0062] Drug treatment groups (combination of Pasireotide and Oxyclozanide + FMDV group, Pasireotide + FMDV group, Oxyclozanide + FMDV group): Remove the original culture medium from the culture plate and add 100 μL of a mixture containing Pasireotide (10 μM) and 5% DMEM medium (MOI=0.1) for foot-and-mouth disease virus, or 100 μL of a mixture containing Oxyclozanide (10 μM) and 5% DMEM medium (MOI=0.1) for foot-and-mouth disease virus, or 100 μL of a mixture containing the combination (both Pasireotide and Oxyclozanide are 5 μM) and 5% DMEM medium (MOI=0.1) for foot-and-mouth disease virus. Gently shake the culture plate to mix the liquid thoroughly and incubate at 37℃ in a 5% CO2 incubator for 12 h. Then collect the samples for detection.

[0063] The results are as follows Figure 5 As shown, neither Pasireotide nor Oxyclozanide, used alone, significantly reduced the TCID of foot-and-mouth disease virus in BHK21 cells. 50 (No significant difference), while the combination of compounds Pasireotide and Oxyclozanide significantly reduced the TCID of foot-and-mouth disease virus in BHK21 cells. 50 This indicates that the combined use of Pasireotide and Oxyclozanide can significantly inhibit the replication of foot-and-mouth disease virus.

[0064] Example 5: Indirect immunofluorescence (IFA) experiment to investigate the effect of the combination of compounds Pasireotide and Oxyclozanide on the expression level of foot-and-mouth disease virus 3D protein.

[0065] BHK21 cells in logarithmic growth phase were adjusted to a suitable concentration using 2% DMEM complete medium and seeded into 96-well cell culture plates at a volume of 100 μL per well (ensuring uniform cell distribution). The 96-well plates were then incubated at 37°C for 12 h.

[0066] Pure cell group (blank control group): The original culture medium in the culture plate was removed, and only 100 μL of 5% DMEM culture medium was added. No virus infection or drug treatment was performed.

[0067] Solvent control group (DMSO+FMDV group): Remove the original culture medium from the culture plate, add 100 μL of 5% DMEM culture medium containing DMSO and foot-and-mouth disease virus (MOI=0.1), and incubate at 37℃ and 5% CO2 for 12 h (to ensure that the final concentration of DMSO is consistent with that of the drug treatment group and to eliminate solvent interference).

[0068] Drug treatment groups (combination of Pasireotide and Oxyclozanide + FMDV group, Pasireotide + FMDV group, Oxyclozanide + FMDV group): Remove the original culture medium from the culture plate and add 100 μL of a mixture containing Pasireotide (10 μM) and 5% DMEM medium (MOI=0.1) for foot-and-mouth disease virus, 100 μL of a mixture containing Oxyclozanide (10 μM) and 5% DMEM medium (MOI=0.1) for foot-and-mouth disease virus, and 100 μL of a mixture containing the combination (both Pasireotide and Oxyclozanide are 5 μM) and 5% DMEM medium (MOI=0.1) for foot-and-mouth disease virus. Gently shake the culture plate to mix the liquid thoroughly and incubate at 37°C in a 5% CO2 incubator for 12 h.

[0069] Discard the culture supernatant from each well of the 96-well plate. Add 100 μL of 4% paraformaldehyde fixative to each well and incubate at room temperature for 30 min. Add 100 μL of 0.2% Triton X-100 permeabilization buffer to each well and incubate at room temperature for 10 min. Wash each well three times for 5 min each time. Discard the PBS wash buffer. Add 100 μL of 5% BSA blocking buffer to each well and incubate at 37°C for 1 h. Discard the blocking buffer. Add 100 μL of primary antibody against foot-and-mouth disease virus RNA polymerase 3D protein diluted 1:1000 with 5% BSA to each well and incubate overnight at 4°C. After incubation, discard the primary antibody solution and wash each well three times with enzyme-free PBS buffer for 10 min each time. Add 100 μL of Alexa Fluor 488-labeled mouse anti-secondary antibody diluted 1:1000 with 5% BSA to each well. Incubate at 37°C for 1 h in the dark. Wash each well three times for 10 min each time. Discard the PBS wash buffer. Add 100 μL of DAPI staining solution diluted 1:5000 with enzyme-free PBS to each well. Incubate at room temperature in the dark for 5 min. Wash each well three times with PBS buffer in the dark for 5 min each time. Then take pictures using an inverted fluorescence microscope.

[0070] The results are as follows Figure 6 As shown, compared with the solvent control group (DMSO+FMDV group), neither Pasireotide nor Oxyclozanide alone could inhibit the expression of foot-and-mouth disease virus 3D protein in BHK21 cells. However, the combination of Pasireotide and Oxyclozanide described in this application can significantly inhibit the expression of foot-and-mouth disease virus 3D protein in BHK21 cells, further demonstrating that the combined use of Pasireotide and Oxyclozanide can significantly inhibit the replication of foot-and-mouth disease virus.

[0071] In summary, the combination of Pasireotide and Oxyclozanide described in this application can significantly inhibit the replication of foot-and-mouth disease virus. This composition can be used as a candidate drug or in combination with other inhibitors for the treatment of foot-and-mouth disease virus.

[0072] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made in accordance with the structure, features and principles of the present invention should be included in the scope of the present invention.

Claims

1. The use of a composition for inhibiting foot-and-mouth disease virus replication in the preparation of an anti-O type foot-and-mouth disease virus drug, characterized in that, The active ingredient of the composition is composed of the compound Pasireotide or a pharmaceutically acceptable salt thereof and the compound Oxyclozanide or a pharmaceutically acceptable salt thereof; the structural formula of the compound Pasireotide is shown in Formula (I) below, and the structural formula of the compound Oxyclozanide is shown in Formula (II) below; Equation (Ⅰ); Equation (II).

2. The application as described in claim 1, characterized in that, The composition inhibits the replication of type O foot-and-mouth disease virus.

3. The application as described in claim 2, characterized in that, The composition inhibits the expression of type O foot-and-mouth disease virus RNA polymerase 3D protein.

4. The application as described in claim 2, characterized in that, The composition reduces the TCID of foot-and-mouth disease virus type O. 50 .

Images