New Uses of Compound CDDO-Im for the Prevention or Treatment of Foot-and-Mouth Disease

Abstract

This invention belongs to the field of foot-and-mouth disease (FMD) treatment technology, specifically relating to a novel use of the compound CDDO-Im for the prevention or treatment of FMD. This invention unexpectedly discovered that the compound CDDO-Im can induce the expression of FMD virus structural proteins VP0, VP3, and VP1, inhibit FMD virus replication, and thus inhibit FMD virus infection. Furthermore, the compound CDDO-Im has no effect on African swine fever virus replication and can specifically inhibit FMD virus, making it suitable for preparing drugs or adjuvants against FMD virus infection to inhibit FMD virus replication.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel use of a compound CDDO-Im for the prevention or treatment of foot-and-mouth disease. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious disease affecting cloven-hoofed animals such as cattle, pigs, sheep, and goats. In my country, it is classified as a Class A animal disease. The pathogen is the foot-and-mouth disease virus (FMDV), belonging to the Picornaviridae family, and is a non-enveloped, single-stranded, positive-sense RNA virus.

[0003] FMDV has seven different serotypes: O, A, C, Asia1, SAT1, SAT2, and SAT3. Each serotype contains numerous subtypes, and there is no cross-protection between different serotypes. Currently, the most effective measure for controlling foot-and-mouth disease is vaccination. However, the foot-and-mouth disease virus has severe immunosuppression properties, allowing it to rapidly penetrate the host's immune system and replicate and spread efficiently. Therefore, while implementing vaccination to control foot-and-mouth disease, it is also essential to develop drugs for treating or preventing the foot-and-mouth disease virus.

[0004] Compound CDDO-Im is an activator of Nrf2 and PPAR. This invention unexpectedly discovered that while compound CDDO-Im cannot inhibit the replication of African swine fever virus, it can reduce the replication level of foot-and-mouth disease virus and inhibit the expression of foot-and-mouth disease virus structural proteins, thus exhibiting an inhibitory effect on foot-and-mouth disease virus infection. Therefore, it can be used as an inhibitor of foot-and-mouth disease virus for the prevention or treatment of foot-and-mouth disease. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a novel use of the compound CDDO-Im for the prevention or treatment of foot-and-mouth disease, specifically including the following:

[0006] In a first aspect, the present invention provides the use of compound CDDO-Im or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing foot-and-mouth disease virus infection, wherein the structural formula of compound CDDO-Im is shown in formula (Ⅰ) below:

[0007]

[0008] Preferably, the compound CDDO-Im or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0009] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, suspensions, and injectable solutions.

[0010] Secondly, the present invention provides the use of compound CDDO-Im or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating foot-and-mouth disease virus infection, wherein the structural formula of compound CDDO-Im is shown in formula (Ⅰ) below:

[0011]

[0012] Preferably, the compound CDDO-Im or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, suspensions, and injectable solutions.

[0014] Thirdly, the present invention provides the application of compound CDDO-Im in the preparation of reagents for inhibiting the expression of foot-and-mouth disease virus structural protein VPO; the structural formula of compound CDDO-Im is shown in formula (Ⅰ) below:

[0015]

[0016] Fourthly, this invention provides the application of compound CDDO-Im in the preparation of reagents that inhibit the expression of the foot-and-mouth disease virus structural protein VP1; the structural formula of the compound CDDO-Im is shown in formula (Ⅰ) below:

[0017]

[0018] Fifthly, this invention provides the application of compound CDDO-Im in the preparation of reagents that inhibit the expression of the foot-and-mouth disease virus structural protein VP3; the structural formula of the compound CDDO-Im is shown in formula (Ⅰ) below:

[0019]

[0020] The beneficial effects of this invention are as follows: This invention unexpectedly discovered that the compound CDDO-Im can reduce the replication level of foot-and-mouth disease virus and inhibit the expression of foot-and-mouth disease virus structural proteins, thus having the effect of inhibiting foot-and-mouth disease virus infection; and the compound CDDO-Im has no effect on the replication of African swine fever virus, can specifically inhibit foot-and-mouth disease virus, and can be used to prepare drugs or adjuvants against foot-and-mouth disease virus infection, for inhibiting the replication of foot-and-mouth disease virus. Attached Figure Description

[0021] Figure 1The graph shows the inhibitory effect of compound CDDO-Im on FMDV.

[0022] Figure 2 Figure showing the experimental results of CDDO-Im inhibiting the FMDV structural protein.

[0023] Figure 3 Figure 1 shows the experimental results of the effect of compound CDDO-Im on the cell viability of PK-15 cells;

[0024] Figure 4 The figure shows the dose-dependent experimental results of the effect of compound CDDO-Im on the levels of FMDV structural proteins;

[0025] Figure 5 The figure shows the effect of compound CDDO-Im on ASFV-eGFP. 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] The experiments described in the following examples obtained biosafety clearance and foot-and-mouth disease laboratory activity permits.

[0028] The structural formula of the compound CDDO-Im described in the following examples is shown in formula (Ⅰ). The compound was purchased from Taoshu Biotechnology (TargetMol, L2510).

[0029]

[0030] Unless otherwise specified, all cells and reagents described in the following examples are commercially available.

[0031] Example 1: Preliminary screening of the effect of compound CDDO-Im on foot-and-mouth disease virus.

[0032] 1. Infection and the interaction of compounds

[0033] PK-15 cells were seeded into 12-well cell culture plates and incubated at 37°C for 12 hours. CDDO-Im (10mM) was diluted to 10μM with DMEM containing 2% serum and added to the cells along with FMDV, 500μL each, for a total volume of 1mL / well. The FMDV infection dose was 1MOI. A 0.1% DMSO group was set up as a control group. The cells were incubated at 37°C and 5% CO2 for 16-20 hours. The cells were harvested when significant cytopathic effects were observed.

[0034] 2. Extract total RNA

[0035] Discard the culture medium and extract total RNA according to the Nucleozol (MACHEREY-NAGEL) procedure. Add 400 μL of RNase-free water, vortex for 15 seconds, incubate at room temperature for 15 minutes, and centrifuge at 12,000 g for 15 minutes at room temperature. Transfer the supernatant to a new centrifuge tube, add isopropanol at a 1:1 ratio, invert to mix, incubate at room temperature for 10 minutes, and centrifuge at 12,000 g for 10 minutes at room temperature. Discard the supernatant, add 500 μL of 75% ethanol to resuspend the precipitate, centrifuge at 8,000 g for 3 minutes at room temperature, discard the ethanol, and dry the precipitate. Dissolve the precipitate in 100-200 μL of RNase-free water.

[0036] 3. Reverse transcription

[0037] An equal amount of extracted RNA was used for reverse transcription, following the ReverTra Ace qPCR RT MasterMix with gDNARemover (Takara) procedure.

[0038] 4. qPCR

[0039] Take equal amounts of reverse transcription product and perform qPCR. qPCR enzyme is... qPCR Mix (Takara) was used with primers 3D-F: 5'-TGGGACCATACAGGAGAAGT-3'; 3D-R: 5'-TTCACCCATCGCAGGTAAAG-3'. The reagent mixture consisted of 10 μL Mix, 0.8 μL 3D-F / R, 2 μL cDNA, and 6.4 μL ddH2O. The qPCR conditions were: 95℃ for 30 s (pre-denaturation), 95℃ for 5 s, and 95℃ for 10 s (40 cycles). The melting curves were: 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. Each sample was run in triplicate. Results were converted to the logarithm of genome copy number according to the standard curve.

[0040] 5. Plot the standard curve:

[0041] The standard plasmid pCDNA3.1-3D containing the FMDV 3D gene was diluted to concentrations of 10... 5 10 6 10 7 10 8 10 9 Using the plasmid as a template for the standard curve, perform the same qPCR as in "4", with three replicates for each concentration. Plot the standard curve with the logarithm of the plasmid concentration on the x-axis and the Ct value on the y-axis.

[0042] The results are as follows Figure 1As shown, compound CDDO-Im significantly inhibits the replication of FMDV in PK-15 cells.

[0043] Example 2: Immunoblotting verification of the effect of compound CDDO-Im on foot-and-mouth disease virus.

[0044] Repeat the experimental procedure in Experiment 1. When significant cytopathic effects appear, collect the samples, discard the supernatant, add 150 μL / well RIPA (containing protease inhibitor) to the cells, lyse on ice for 30 min, add 50 μL 4× protein loading buffer, vortex to mix, centrifuge at 100℃ for 10 min, centrifuge at 8000 rpm for 5 min, perform SDS-PAGE, load 20 μL, incubate at 80V for 30 min, 120V for 60 min; transfer to a membrane, incubate at 230 mA for 2 h, block with 5% skim milk powder for 2 h, incubate with FMDV-O polyclonal antibody (prepared and stored in our laboratory) at 4℃ overnight, incubate with secondary antibody for 1 h, and expose to sunlight.

[0045] The results are as follows Figure 2 As shown, the compound CDDO-Im significantly inhibited the expression levels of FMDV structural proteins VP0, VP3, and VP1.

[0046] Example 3: CCK-8 assay to detect the effect of compound CDDO-Im on the cell viability of PK-15 cells.

[0047] PK-15 cells were prepared at a rate of 1×10⁻⁶. 5 Cells were seeded into 96-well cell culture plates and cultured at 37°C for 12 hours in a 5% CO2 incubator. Cells were then divided into control and experimental groups. Control group cells were treated with 0.1% (v / v) dimethyl sulfoxide (DMSO), while experimental group cells were treated with DMEM (10% FBS) medium containing 0.2, 0.5, 1, 2, 5, 10, and 20 μM of compound CDDO-Im, respectively. A blank control group was also prepared using DMEM (10% FBS) medium containing the same dose of DMSO as the control group. Cells were incubated for 48 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, the plate was gently tapped to mix, and the cells were incubated at 37°C for another 1 hour. After incubation, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula: [(Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance)] × 100% = Cell viability percentage.

[0048] The results are as follows Figure 3 As shown, compound CDDO-Im had no significant effect on cell viability at concentrations of 0.2–10 μM.

[0049] Example 4: Dose-dependent experiment on the inhibition of foot-and-mouth disease virus proliferation in PK-15 cells by compound CDDO-Im

[0050] PK-15 was seeded into 12-well cell plates and cultured at 37°C for 12 hours. CDDO-Im (10 mM) was diluted with DMEM containing 2% serum to 0.4, 1, 2, 4, 10, and 20 μM, and added to the cells along with FMDV, 500 μL each, for a total volume of 1 mL / well. The FMDV infection dose was 1 MOI. A 0.1% DMSO group was set up as a control group. The cells were cultured at 37°C and 5% CO2 for 16-20 hours. When significant cytopathic effects appeared in the cells, samples were collected for Western blot analysis, following the same procedure as in Example 2.

[0051] The results are as follows Figure 4 As shown, the compound CDDO-Im inhibits the expression of FMDV structural proteins VP0, VP3 and VP1 at 1-10 μM.

[0052] Example 5: Specificity experiment of CDDO-Im on the inhibition of foot-and-mouth disease virus proliferation in PK-15 cells.

[0053] Dilute compound CDDO-Im stock solution (10mM) to 10μM with RPMI 1640 (10% FBS, 1% triple antibody) serially, and set aside; dilute ASFV-eGFP fluorescent toxin 1:50 with RPMI 1640 (10% FBS, 1% triple antibody), and set aside; resuscitate porcine primary alveolar macrophages (PAMs) at 1×10 5 Each compound was inoculated into a 96-well plate and incubated at 37°C with 5% CO2 for 6 hours. The culture medium was then discarded, and 100 μL of diluted compound CDDO-Im and 100 μL of diluted virus solution were added. The plate was then incubated at 37°C with 5% CO2 for another 48 hours. Three replicates were prepared for each compound, with a DMSO group serving as a control. Significant fluorescence changes in all three replicates were considered indicative of efficacy.

[0054] The results are as follows Figure 5 As shown, the fluorescence of the CDDO-Im treatment group was not significantly different from that of the DMSO control group. This indicates that the CDDO-Im compound described in this application has no inhibitory effect on African swine fever virus, but can specifically inhibit the replication of foot-and-mouth disease virus.

[0055] In summary, the CDDO-Im of this invention can inhibit FMDV replication in a dose-dependent manner. It can specifically inhibit FMDV replication and can be used as an inhibitor of foot-and-mouth disease virus for the prevention or treatment of foot-and-mouth disease.

Claims

1. The use of compound CDDO-Im or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention of foot-and-mouth disease virus infection, wherein the structural formula of compound CDDO-Im is shown in formula (Ⅰ): Equation (Ⅰ).

2. The application as described in claim 1, characterized in that, The compound CDDO-Im or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier to form any pharmaceutically acceptable dosage form.

3. The application as described in claim 2, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, suspensions, and injectables.

4. The use of compound CDDO-Im or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating foot-and-mouth disease virus infection, wherein the structural formula of compound CDDO-Im is shown in formula (Ⅰ): Equation (Ⅰ).

5. The application as described in claim 4, characterized in that, The compound CDDO-Im or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier to form any pharmaceutically acceptable dosage form.

6. The application as described in claim 5, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, suspensions, and injectables.

7. The application as described in claim 1 or 4, characterized in that, The compound CDDO-Im inhibits the expression of the foot-and-mouth disease virus structural protein VP0.

8. The application as described in claim 1 or 4, characterized in that, The compound CDDO-Im inhibits the expression of the foot-and-mouth disease virus structural protein VP1.

9. The application as described in claim 1 or 4, characterized in that, The compound CDDO-Im inhibits the expression of the foot-and-mouth disease virus structural protein VP3.

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