Method for inhibiting porcine epidemic diarrhea virus infection in vitro by CCG-1423 and application thereof

Abstract

The present application relates to a kind of small molecule inhibitors CCG-1423 inhibit porcine epidemic diarrhea virus infection in vitro method and application, the inhibitor can be used to prepare the antiviral drug of inhibiting porcine epidemic diarrhea virus infection.Prove, in the process of porcine kidney cell LLC-PK1 PEDV infection in vitro, compared with the control group, the protein expression level, RNA content and virus titer of PEDV in the treatment group added with CCG-1423 are significantly reduced, indicating that the inhibitor can be used to inhibit the in vitro infection of porcine epidemic diarrhea virus.

Description

Technical Field

[0001] This invention relates to a method and application of CCG-1423 for inhibiting porcine epidemic diarrhea virus infection in vitro, belonging to the fields of cell biology and virology. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). It can infect pigs of all ages, manifesting as vomiting, loss of appetite, dehydration, and diarrhea. However, newborn piglets are more severely affected, with mortality rates reaching 100%. The primary transmission route is fecal-oral, but it can also be transmitted vertically from sow to newborn piglets through colostrum. PEDV belongs to the genus *Alpha-coronavirus* of the family Coronaviridae. The virus particles are nearly spherical, enveloped, and approximately 130 nm (90-190 nm) in diameter. It is a single-stranded positive-sense RNA virus. In vitro, PEDV primarily infects porcine kidney cells LLC-PK1 and African green monkey kidney epithelial cells (Vero). Currently, there are no specific antiviral drugs against PEDV infection, hindering effective control of PED. Therefore, the development of antiviral drugs for PEDV is urgently needed to reduce the morbidity and mortality of PED.

[0003] The official name of CCG-1423 is N-[2-[4(4-chlorophenyl)amino]-1-methyl-2-oxoethoxy]-3,5-bis(trifluoromethyl)-benzamide, and its structural formula is as follows:

[0004]

[0005] CCG-1423 can directly block serum response factor (SRF). It is a small molecule inhibitor of the RhoA signaling pathway, a member of the Ras homolog gene family A (RhoA) of small G proteins. Studies have shown that the inhibitory effect of CCG-1423 on cancer cell invasion often improves other pathological changes, including insulin resistance, angiogenesis and fibrosis in vascular diseases, demonstrating good clinical application potential. However, there are currently no reports on the application of CCG-1423 in combating porcine epidemic diarrhea virus. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method and application for inhibiting porcine epidemic diarrhea virus infection in vitro using CCG-1423.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Application of a small molecule inhibitor, CCG-1423, in the preparation of drugs that inhibit porcine epidemic diarrhea virus infection.

[0009] The application of the small molecule inhibitor CCG-1423 in the preparation of a drug that inhibits protein expression in porcine epidemic diarrhea virus infection.

[0010] The application of the small molecule inhibitor CCG-1423 in the preparation of drugs that inhibit the RNA content of porcine epidemic diarrhea virus infection.

[0011] The application of the small molecule inhibitor CCG-1423 in the preparation of drugs that inhibit the viral titer of porcine epidemic diarrhea virus infection.

[0012] In the aforementioned application, the concentration of the small molecule inhibitor CCG-1423 is 2 μM.

[0013] A method for inhibiting porcine epidemic diarrhea virus infection in vitro using the small molecule inhibitor CCG-1423, comprising the following steps:

[0014] (1) Porcine kidney cells LLC-PK1 were added at a concentration of 2.5 × 10⁻⁶. 5 Cells / mL were seeded on a 24-well cell culture plate, and 500 μL of DMEM medium was added to each well. The plate was then incubated at 37°C and 5% CO2 for 24 h.

[0015] (2) Replace with new DMEM medium and incubate LLC-PK1 cells at 37°C for 1 h with the prevalent PEDV strains HNXX and DR13 with a multiplicity of infection of 0.5, respectively; discard the culture supernatant and wash the cells three times with PBS buffer to remove virus particles that have not invaded the cells.

[0016] (3) Add 500 μL of DMEM medium containing 2 μM CCG-1423 to the wells of the cell culture plate. For cells infected with the PEDV HNXX epidemic strain, trypsin with a final concentration of 2 μg / mL should be added. Continue to culture at 37℃ for 24 h.

[0017] The DMEM medium contains 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0018] The beneficial effects of this invention are:

[0019] This invention, utilizing cell biology and virology techniques, is the first to discover that CCG-1423 possesses inhibitory activity against porcine epidemic diarrhea virus (PEDV) infection in vitro, providing an application for CCG-1423 in the preparation of anti-PEDV drugs. CCG-1423 exhibits a significant inhibitory effect on PEDV proliferation in host cells, demonstrating good anti-PEDV efficacy.

[0020] In this embodiment of the invention, porcine kidney cells LLC-PK1 were used as a cell model for research. Experimental results showed that CCG-1423 had no significant toxicity to cells within the 10 μM concentration range. During PEDV infection, the addition of 2 μM CCG-1423 significantly reduced PEDV viral infection, protein expression, RNA content, and viral titer compared to the control group without inhibitors. Because CCG-1423 can inhibit viral titer in PEDV in vitro, it is predicted that CCG-1423 can be developed as a safe and effective antiviral drug for porcine epidemic diarrhea virus, showing promising application prospects. Attached Figure Description

[0021] Figure 1 Bar chart showing the toxicity of CCG-1423 to LLC-PK1 cells;

[0022] Figure 2 A graph showing the effect of fluorescence method on the activity of CCG-1423 against porcine epidemic diarrhea virus;

[0023] Figure 3 Graph showing the inhibitory effect of CCG-1423 on the expression of porcine epidemic diarrhea virus N protein as detected by Western blotting;

[0024] Figure 4 The inhibitory effect of CCG-1423 on porcine epidemic diarrhea virus RNA content detected by real-time PCR.

[0025] Figure 5 TCID 50 The experimental results show the inhibitory effect of CCG-1423 on the in vitro proliferation of porcine epidemic diarrhea virus. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, the instruments and equipment used in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the experimental methods involved are all conventional methods.

[0027] The cells, viruses, and main reagents used in this invention are as follows:

[0028] Monoclonal antibodies against porcine kidney cells (LLC-PK1 cells), PEDV DR13 strain, PEDV HNXX strain (a porcine epidemic diarrhea virus strain isolated from clinical samples), and PEDV N protein were all preserved in our laboratory. CCG-1423 (Catalog No.: HY-13991, MedChemExpress), SDS-PAGE Loading Buffer (Catalog No.: P1040, Beijing Solarbio Science & Technology Co., Ltd.), GAPDH Mouse Monoclonal Antibody (Catalog No.: 60004-1-Ig, Proteintech), DyLight 488 Fluorescently Labeled Rabbit Anti-Mouse Antibody (Catalog No.: A23220, Abbkine), HRP-Labeled Goat Anti-Mouse Antibody (Catalog No.: A21010, Abbkine), CCK-8 Cell Proliferation and Toxicity Assay Kit (Catalog No.: CA1210, Beijing Solarbio Science & Technology Co., Ltd.), Trypsin (Catalog No.: T8802, Sigma), RNAiso Plus (Catalog No.: 9109, Takara), PrimeScript RT Master Mix Reverse Transcription Kit (Catalog No.: RR036A, Takara), Premix Ex Taq (Probe qPCR (catalog number: RR390, Takara), all experiments involving live viruses are performed in a biosafety level 2 facility.

[0029] Example 1: Detection of the toxicity of CCG-1423 to LLC-PK1 cells

[0030] LLC-PK1 cells were divided into groups of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well cells. Each well contained 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in DMEM medium. The cells were incubated at 37°C with 5% CO2 for 24 hours. The original cell culture medium was then removed and replaced with DMEM medium containing different concentrations of CCG-1423 (dissolved in DMSO): 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM, with three replicates for each concentration. The cells were then incubated for another 24 hours. Finally, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours. The absorbance at 450 nm was then measured using a multi-sensor microplate reader. Data were analyzed using GraphPad Prism 8.0 software. Statistical analysis was performed using the t-test, where ns indicates no significant difference, and p<0.001 indicates a highly significant difference.

[0031] The results are as follows Figure 1As shown, compared with the DMSO control group (CCG-1423 0 μM) cell viability (cell viability was 100%), the addition of 2 μM, 4 μM, 6 μM, 8 μM and 10 μM CCG-1423 had no significant effect on cell viability (ns), that is, no cytotoxicity, and can be used in subsequent examples.

[0032] Example 2: Detection of the activity of CCG-1423 against porcine epidemic diarrhea virus using fluorescence method.

[0033] LLC-PK1 cells were divided into groups of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL on a 24-well cell culture plate. 500 μL of DMEM medium containing 10% (v / v) 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 then replaced, and LLC-PK1 cells were incubated at 37°C for 1 h with PEDV HNXX and DR13 strains (MOI) of 0.5, respectively. The culture supernatant was discarded, and the cells were washed three times with PBS buffer to remove any virus particles that had not yet invaded the cells. 500 μL of DMEM medium containing 2 μM CCG-1423 (the lowest non-cytotoxic CCG-1423 concentration in Example 1) was added to each well of the cell culture plate. (For cells infected with the PEDV HNXX strain, trypsin was added to a final concentration of 2 μg / mL; the volume added was calculated based on the final concentration.) A DMSO control group (containing CCG-1423) was also included. 0 μM), and continue to incubate at 37℃ for 24 h.

[0034] Next, discard the culture medium, wash three times with PBS, add 250 μL of 4% (m / v) paraformaldehyde to each well, and fix at room temperature for about 20 minutes. Wash three times with PBS, add monoclonal antibody against PEDV N protein and incubate at 37°C for 30 min. After washing three times with PBS, add DyLight 488 fluorescently labeled rabbit anti-mouse antibody and incubate at 37°C for 30 min. After washing three times with PBS, observe under a fluorescence microscope. The intensity of green fluorescence indicates the proliferation of PEDV DR13 or HNXX strains.

[0035] The results are as follows Figure 2 As shown, compared with the DMSO control group (CCG-1423 0 μM), the treatment group with 2 μM CCG-1423 significantly inhibited the proliferation of PEDV HNXX and DR13 strains, indicating that CCG-1423 has good anti-porcine epidemic diarrhea virus activity at this concentration.

[0036] Example 3: Detection of the inhibitory effect of CCG-1423 on the expression of porcine epidemic diarrhea virus N protein by Western blotting.

[0037] To further determine the anti-PEDV activity of CCG-1423, the inhibitory effect of CCG-1423 on the expression of N protein, a major structural protein of PEDV, was detected by Western blotting. The specific method is as follows:

[0038] LLC-PK1 cells were divided into groups of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL on a 24-well cell culture plate. 500 μL of DMEM medium containing 10% (v / v) 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 then replaced, and LLC-PK1 cells were incubated at 37°C for 1 h with the prevalent PEDV strains HNXX and DR13 at an MOI of 0.5. The culture supernatant was discarded, and the cells were washed three times with PBS buffer to remove any virus particles that had not yet invaded the cells. 500 μL of DMEM medium containing 2 μM CCG-1423 was added to the wells of the cell culture plate (for cells infected with the prevalent PEDV HNXX strain, trypsin was added to a final concentration of 2 μg / mL). A DMSO control group (0 μM CCG-1423) was also included. The plates were incubated at 37°C for another 24 h. Cells were washed three times with PBS buffer, and 150 μL of SDS-PAGE Loading Buffer was added to each well to lyse the cells. The cells were boiled for 10 min and centrifuged at 12000 rpm for 3 min. The supernatant was transferred to a new 1.5 mL sterile centrifuge tube to prepare the samples for the DMSO control group and the CCG-1423 treatment group. Then, Western blotting was performed using PEDV N protein antibody (…). Figure 3 (above) and GAPDH antibody ( Figure 3 The primary antibody was HRP-labeled goat anti-mouse antibody, and the secondary antibody was HRP-labeled goat anti-mouse antibody. The expression of PEDV N protein and the intracellular reference protein GAPDH was detected.

[0039] The results are as follows Figure 3 As shown, when LLC-PK1 cells were infected with the prevalent PEDV strains HNXX and DR13, GAPDH was used as an internal reference, and the same GAPDH content represented the same number of cells. In the same number of cells, compared with the DMSO control group (CCG-1423 0 μM), the expression level of PEDV N protein in the treatment group with 2 μM CCG-1423 was significantly reduced, indicating that CCG-1423 can significantly inhibit the expression of PEDV N protein.

[0040] Example 4: Detection of the inhibitory effect of CCG-1423 on porcine epidemic diarrhea virus RNA content by real-time quantitative PCR.

[0041] Quantitative real-time PCR was used to determine the PEDV RNA content to further evaluate the inhibitory effect of CCG-1423 on porcine epidemic diarrhea virus RNA content. The specific method is as follows:

[0042] LLC-PK1 cells were divided into groups of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL on a 24-well cell culture plate. 500 μL of DMEM medium containing 10% (v / v) 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 then replaced, and LLC-PK1 cells were incubated at 37°C for 1 h with the prevalent PEDV strains HNXX and DR13 at an MOI of 0.5. The culture supernatant was discarded, and the cells were washed three times with PBS buffer to remove any virus particles that had not yet invaded the cells. 500 μL of DMEM medium containing 2 μM CCG-1423 was added to the wells of the cell culture plate (for cells infected with the prevalent PEDV HNXX strain, trypsin was added to a final concentration of 2 μg / mL). A DMSO control group (0 μM CCG-1423) was also included. The plates were incubated at 37°C for another 24 h. Cells were collected, cell supernatant was discarded, and cells were washed three times with PBS. Total RNA was extracted from PEDV-infected cells using RNAisoPlus RNA extraction reagent, and complementary DNA (cDNA) was generated using PrimeScript RT Master Mix reverse transcription kit as a template for quantitative real-time PCR. The PEDV N protein RNA content was measured by absolute quantitative real-time PCR to represent the viral RNA content of infected cells. Primers and reaction systems are shown in Tables 1 and 2. Premix Ex Taq (Probe qPCR) was used for probe-based quantitative real-time PCR. The reaction program used was the Fast program built into the ABI quantitative real-time PCR instrument (Applied Biosystems, USA).

[0043] Table 1 Primer sequences for quantitative real-time PCR

[0044]

[0045] Note: FAM and BHQ3 are fluorescent modifying groups.

[0046] Table 2. Real-time PCR reaction system

[0047]

[0048] The experiment was conducted independently three times, with three replicates each time. Experimental data are expressed as group means and standard deviations (SD). Statistical analysis was performed using unpaired, two-tailed Student's t-test in GraphPad Prism 8.0 software. ****p<0.0001 was considered statistically significant.

[0049] The results are as follows Figure 4 As shown, when LLC-PK1 cells were infected with the prevalent PEDV strains HNXX or DR13, the PEDV RNA content in the treatment group with 2 μM CCG-1423 was significantly reduced compared with the DMSO control group (CCG-1423 0 μM), indicating that CCG-1423 can significantly reduce the RNA content of porcine epidemic diarrhea virus in LLC-PK1 cells.

[0050] Example 5 Virus titer test (TCID) 50 ) Detection of the inhibitory effect of CCG-1423 on the in vitro proliferation of porcine epidemic diarrhea virus

[0051] The half-maximal dose of tissue culture infection (TCID) was used. 50 The viral titer was determined to further evaluate the inhibitory effect of CCG-1423 on the production of porcine epidemic diarrhea virus progeny viruses. The specific method is as follows:

[0052] LLC-PK1 cells were divided into groups of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL on a 24-well cell culture plate. 500 μL of DMEM medium containing 10% (v / v) 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 then replaced, and LLC-PK1 cells were incubated at 37°C for 1 h with both the prevalent PEDV strain HNXX and strain DR13 at an MOI of 0.5. The culture supernatant was discarded, and the cells were washed three times with PBS buffer to remove any uninfected viral particles. 500 μL of DMEM medium containing 2 μM CCG-1423 was added to each well of the cell culture plate (for cells infected with the prevalent PEDV HNXX strain, trypsin was added to a final concentration of 2 μg / mL). A DMSO control group (CCG-1423) was also included. The cells were cultured at 37°C for 24 hours with 0 μM of the virus to obtain the supernatant of LLC-PK1 cells infected with strain HNXX or strain DR13.

[0053] Uninfected LLC-PK1 cells were diluted to 2.5 × 10⁻⁶. 596-well cell culture plates were plated with 100 μL of the virus at a concentration of 100 μL / mL, and incubated overnight at 37°C in a 5% CO2 incubator. The supernatant of LLC-PK1 cells infected with HNXX or DR13 strains for 24 hours was serially diluted 10-fold and inoculated into uninfected LLC-PK1 cells plated in 96-well cell culture plates at 100 μL / well. Eight replicates were performed for each dilution. Supernatant of PEDV-infected cells in DMSO control group (CCG-1423 0 μM) was used as a control. The 96-well cell culture plates were incubated at 37°C in a 5% CO2 incubator. Cell growth was observed every 24 hours, and the number of wells showing cytopathic effects was recorded. Observation continued for 5-7 days until the number of wells showing cytopathic effects no longer increased. The TCID of the virus was calculated using the Spearman-Karber method. 50 Experimental data are expressed as group mean and standard deviation (SD). Statistical analysis was performed using unpaired, two-tailed Student's t-test with GraphPadPrism 8.0 software. ***p<0.001 was considered statistically significant.

[0054] The results are as follows Figure 5 As shown, when LLC-PK1 cells were infected with the prevalent PEDV strains HNXX or DR13, the PEDV viral titer was significantly lower in the group treated with 2 μM CCG-1423 compared to the DMSO control group (CCG-1423 0 μM). This indicates that CCG-1423 significantly inhibited the proliferation of PEDV in LLC-PK1 cells, confirming that CCG-1423 can significantly reduce the infection load of PEDV in LLC-PK1 cells.

Claims

1. The application of a small molecule inhibitor CCG-1423 in the preparation of a drug for inhibiting porcine epidemic diarrhea virus infection, characterized in that, The application is in the preparation of drugs that inhibit protein expression, RNA content, or viral titer of porcine epidemic diarrhea virus infection. The structural formula of the small molecule inhibitor CCG-1423 is as follows: 。

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