Methods and applications of EHop-016 in inhibiting in vitro infection of porcine epidemic diarrhea virus.

Abstract

This invention relates to a method and application of the inhibitor EHop-016 for inhibiting porcine epidemic diarrhea virus (PEDV) infection in vitro. EHop-016 can be used to prepare antiviral drugs for inhibiting PEDV infection. In this invention, during the in vitro infection of African green monkey kidney cells (Vero) with PEDV, the addition of 4 μmol / L EHop-016 significantly reduced the viral load, protein expression level, RNA content, and viral titer of PEDV in the EHop-016-treated group compared to the control group without EHop-016, indicating that this inhibitor can be used to inhibit porcine epidemic diarrhea virus infection in vitro.

Description

Technical Field

[0001] This invention relates to a method and application of EHop-016 to inhibit 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 (PEDvirus, PEDV). It primarily invades the epithelial cells of the small intestinal villi, causing villi atrophy and deformation, leading to acute diarrhea, vomiting, dehydration, and high mortality in newborn piglets, resulting in significant economic losses in the pig industry. The main route of transmission 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 particle is 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 African green monkey kidney cells (Vero cells). Currently, there are no specific antiviral drugs against PEDV infection, hindering effective control of PED. Therefore, there is an urgent need to develop antiviral drugs for PEDV to reduce its morbidity and mortality.

[0003] The official name of EHop-016 is N4-(9-ethyl-9H-carbazole-3-yl)-N2-[3-(4-morpholinyl)propyl]-2,4-pyrimidinediamine, with the chemical formula C. 25 H 30 N6O, with a molecular weight of 430.55, has the following structural formula:

[0004]

[0005] EHop-016 is a selective inhibitor of Rac1 and Rac3 in the Rac-GTPase family, effectively reducing tumor growth and metastasis in mice. Studies have shown that EHop-016 can improve the resistance of various cancer cells to the anticancer drug cisplatin. This in vitro and in vivo combination therapy with chemotherapy drugs provides a promising targeted treatment strategy for cancer in clinical practice. However, there are currently no reports on the application of EHop-016 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 the inhibitor EHop-016.

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

[0008] The application of an inhibitor, EHop-016, in drugs that inhibit porcine epidemic diarrhea virus infection in vitro.

[0009] The application of the inhibitor EHop-016 in the preparation of a drug that inhibits protein expression in porcine epidemic diarrhea virus infection.

[0010] The application of the inhibitor EHop-016 in the preparation of drugs that inhibit the RNA content of porcine epidemic diarrhea virus infection.

[0011] The use of the inhibitor EHop-016 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 inhibitor EHop-016 when treating cells was 4 μmol / L.

[0013] The method for inhibiting porcine epidemic diarrhea virus infection in vitro using the inhibitor EHop-016 includes the following steps:

[0014] (1) The Vero cells were prepared at a concentration of 2.5 × 10⁻⁶. 5 Cells / mL were seeded onto 24-well cell culture plates, and 500 μL of LMEM medium was added to each well. The plates were then incubated at 37°C and 5% CO2 for 24 h.

[0015] (2) Replace with new DMEM medium and incubate Vero cells at 37°C for 1 h with PEDV-HHXX and PEDV-DR13 strains 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 4 μmol / L EHop-016 to the wells of the cell culture plate (for cells infected with PEDV-HHXX strain, trypsin with a final concentration of 2 μg / mL should be added separately), and continue to culture at 37℃ for 24 h.

[0017] The DMEM culture medium in step (1) contains 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin.

[0018] Beneficial effects of this invention:

[0019] This invention, utilizing cell biology and virology techniques, is the first to discover that EHop-016 possesses inhibitory activity against porcine epidemic diarrhea virus (PEDV) infection in vitro, providing an application for EHop-016 in the preparation of anti-PEDV drugs. EHop-016 exhibits a significant inhibitory effect on PEDV proliferation in Vero cells, demonstrating good anti-PEDV efficacy.

[0020] This invention uses Vero kidney cells from African green monkeys as a cell model for research. Experimental results show that EHop-016 has no significant toxicity to cells within a concentration range of 10 μmol / L. During PEDV infection, the addition of 4 μmol / L EHop-016 significantly reduced PEDV viral titer, viral RNA content, and viral N protein expression compared to the control group treated with DMSO. Since EHop-016 can inhibit viral titer in PEDV in vitro, it is predicted that EHop-016 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 of EHop-016 toxicity assay on Vero cells.

[0022] Figure 2 Comparison of EHop-016's inhibition of PEDV strain proliferation as detected by indirect immunofluorescence assay.

[0023] Figure 3 Western blot image of EHop-016 expressing N protein of porcine epidemic diarrhea virus.

[0024] Figure 4 The graph shows the inhibitory effect of EHop-016 on porcine epidemic diarrhea virus RNA content detected by quantitative real-time PCR.

[0025] Figure 5 TCID 50 The experimental results show the inhibitory effect of EHop-016 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] African green monkey kidney cells (Vero cells), PEDV-HNXX strain (GenBank: MK124712.1), PEDV-DR13 strain, and monoclonal antibodies against PEDV N protein were all preserved in our laboratory. EHop-016 (CAS No.: 1380432-32-5, MedChemExpress), SDS-PAGE Loading Buffer (Cat No.: P1040, Beijing Solarbio Science & Technology Co., Ltd.), GAPDH mouse monoclonal antibody (Cat No.: 60004-1-Ig, Proteintech), Alexa Fluor 488 goat anti-mouse secondary antibody (Cat No.: A-11001, Thermo Fisher), HRP-labeled goat anti-mouse antibody (Cat No.: A21010, Abbkine), CCK-8 cell proliferation and cytotoxicity assay kit (Cat No.: CA1210, Beijing Solarbio Science & Technology Co., Ltd.), trypsin (Cat No.: T8802, Sigma), RNAiso Plus (Cat No.: 9109, Takara), PrimeScript RT Master Mix reverse transcription kit (Cat No.: RR036A, Takara), Premix Ex Taq (Probe qPCR) (Cat No.: RR390, Takara), all experiments involving live viruses are performed in a biosafety level 2 facility.

[0029] Example 1: Detection of the toxicity of EHop-016 to Vero cells

[0030] Vero cells were 1×10 5Cells were seeded at a density of 1 / mL on a 96-well cell culture plate. 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin was added to each well. The plate was incubated at 37°C with 5% CO2 for 24 h. The original cell culture medium was then removed and replaced with DMEM medium containing different concentrations of EHop-016 (dissolved in dimethyl sulfoxide DMSO): 0 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, and 10 μmol / L, with three replicates for each concentration. The cell culture plate was incubated for another 24 h. 10 μL of CCK-8 reagent was added to each well, and the plate was incubated for another 2 h. Cell viability was then calculated by measuring absorbance at 450 nm using a multi-mode microplate reader. Data were analyzed using GraphPad Prism 8.0 software, and statistical analysis was performed using the Student t-test. ns indicated no significant difference, and p < 0.001 indicated extremely significant difference.

[0031] The results are as follows Figure 1 As shown, compared with the DMSO control group (EHop-016 0 μmol / L) cell viability (cell viability was 100%), the addition of 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L and 10 μmol / L EHop-016 had no significant effect on cell viability (ns), i.e., no cytotoxicity, and can be used in subsequent examples.

[0032] Example 2: Detection of the activity of EHop-016 against porcine epidemic diarrhea virus by indirect immunofluorescence assay

[0033] Vero cells were divided into groups of 2.5 × 10⁻⁶. 5Vero cells were seeded at 1 / mL in a 24-well cell culture plate. 500 μL of DMEM medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / 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 Vero cells were incubated at 37°C for 1 h with PEDV-HNXX and PEDV-DR13 strains (multiple infection number 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 4 μmol / L EHop-016 was added to the wells of the cell culture plate (for cells infected with the PEDV-HNXX strain, trypsin was added to a final concentration of 2 μg / mL). A DMSO control group (EHop-016 0 μmol / L) was also included. The plates were incubated at 37°C for another 24 h. Next, the culture medium was discarded, and the cells were washed three times with PBS. 250 μL of 4% (m / v) paraformaldehyde fixative was added to each well, and the cells were fixed at room temperature for 20 min, followed by washing three times with PBS. Then, 0.1% Triton X-100 was added for permeation for 5 min, followed by washing three times with PBS. 5% skim milk was added for blocking for 1 h, followed by washing three times with PBS. Then, monoclonal antibody against PEDV N protein was added and incubated at 37°C for 30 min, followed by washing three times with PBS. Finally, DyLight 488 fluorescently labeled goat anti-mouse antibody was added and incubated at 37°C for 30 min, followed by washing three times with PBS. The cells were then observed under a fluorescence microscope, and green fluorescence indicated that PEDV had infected Vero cells and caused lesions.

[0034] The results are as follows Figure 2 As shown, compared with the DMSO control group (EHop-016 0 μmol / L), the treatment group with 4 μmol / L EHop-016 showed a significant reduction in green fluorescence, which significantly inhibited the proliferation of PEDV-HNXX and PEDV-DR13. This indicates that EHop-016 has good anti-porcine epidemic diarrhea virus activity under this concentration.

[0035] Example 3: Detection of the inhibitory effect of EHop-016 on the expression of porcine epidemic diarrhea virus N protein by Western blotting.

[0036] To further determine the anti-PEDV activity of EHop-016, the inhibitory effect of EHop-016 on the expression of N protein, a major structural protein of PEDV, was detected by Western blotting. The specific method is as follows:

[0037] Infected Vero cells were cultured according to Example 2; 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 then boiled for 10 min to prepare samples for the DMSO control group and the EHop-016 treatment group. Western blot was then used to analyze the samples 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.

[0038] The results are as follows Figure 3 As shown, when Vero cells were infected with PEDV-HNXX and PEDV-DR13 strains, 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 (EHop-016 0 μmol / L), the expression level of PEDV N protein in the treatment group with 4 μmol / L EHop-016 was significantly reduced, indicating that 4 μmol / L EHop-016 can significantly inhibit the expression of PEDV N protein.

[0039] Example 4: Detection of the inhibitory effect of EHop-016 on porcine epidemic diarrhea virus RNA content by quantitative real-time PCR.

[0040] The RNA content of PEDV was determined by quantitative real-time PCR to further evaluate the inhibitory effect of EHop-016 on porcine epidemic diarrhea virus RNA. The specific method is as follows:

[0041] Infected Vero cells were cultured according to Example 2; the cell supernatant was discarded, and the cells were washed three times with PBS and collected; total RNA was extracted from PEDV-infected cells using RNAiso Plus RNA extraction reagent, and complementary DNA (cDNA) was generated using the 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).

[0042] Table 1 Primer sequences for real-time PCR

[0043]

[0044] Note: FAM and BHQ3 are fluorescent modifying groups.

[0045] Table 2. Real-time PCR reaction system

[0046]

[0047] 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.

[0048] The results are as follows Figure 4 As shown, when Vero cells were infected with PEDV-HNXX and PEDV-DR13 strains, the PEDV RNA content in the treatment group with 4 μmol / L EHop-016 was significantly reduced compared with the DMSO control group (EHop-016 0 μmol / L), indicating that EHop-016 can significantly reduce the RNA content of PEDV infection in Vero cells.

[0049] Example 5: Virus titer assay (TCID50) to detect the inhibitory effect of EHop-016 on the in vitro proliferation of porcine epidemic diarrhea virus.

[0050] The half-maximal dose of tissue culture infection (TCID) was used. 50 The viral titer was determined to further evaluate the inhibitory effect of EHop-016 on the production of porcine epidemic diarrhea virus progeny viruses. The specific method is as follows:

[0051] Vero cells infected with PEDV-HNXX and PEDV-DR13 strains were cultured according to the method in Example 2. Vero cells were cultured at a density of 2.5 × 10⁶ cells / year. 5Vero cells were seeded at 1 / mL in a 24-well cell culture plate, and 500 μL of DMEM medium containing 10% (v / v) fetal bovine serum was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 h. The DMEM medium was replaced, and Vero cells were incubated at 37°C for 1 h with PEDV-HNXX and PEDV-DR13 strains, respectively, with a multiplicity of infection 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 invaded the cells. 500 μL of DMEM medium containing 4 μmol / L EHop-016 was added to the wells of the cell culture plate (for cells infected with the PEDV-HNXX strain, trypsin was added to a final concentration of 2 μg / mL). A DMSO control group (EHop-016 0 μmol / L) was also included. The plates were incubated at 37°C for another 24 h to obtain Vero cells infected with the PEDV-HNXX and PEDV-DR13 strains.

[0052] Uninfected Vero cells were diluted to 1×10⁻⁶. 5 Vero cells infected with PEDV were seeded at a density of 100 μL / mL in 96-well cells. Each well contained 10% (v / v) fetal bovine serum (FBS) in DMEM medium. The cells were incubated overnight at 37°C with 5% CO2. Vero cells infected for 24 hours were then subjected to three freeze-thaw cycles with their supernatant. The supernatant was centrifuged and serially diluted 10-fold. 100 μL of this supernatant was then seeded into the 96-well cells, with eight replicates per dilution. A control group (DMSO control, EHop-01 60 μmol / L) of PEDV-infected cells was used. The 96-well cells were incubated at 37°C with 5% CO2. Cell growth was observed every 24 hours, and the number of wells showing cytopathic effects was recorded. This observation period lasted 2-3 days until the number of wells showing cytopathic effects stopped increasing. 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 the unpaired, two-tailed Student t-test in GraphPad Prism 8.0 software. ***p<0.001 was considered statistically significant.

[0053] The results are as follows Figure 5 As shown, when Vero cells were infected with PEDV-HNXX and PEDV-DR13 strains, the PEDV viral titer was significantly lower in the group treated with 4 μmol / L EHop-016 compared to the DMSO control group (0 μmol / L). This indicates that EHop-016 significantly inhibited the proliferation of PEDV on Vero cells, further confirming that EHop-016 can significantly reduce the PEDV infection load in Vero cells.

Claims

1. The application of an inhibitor EHop-016 in the preparation of a drug for inhibiting porcine epidemic diarrhea virus, characterized in that, The application is to prepare a drug that inhibits the protein expression, RNA content, and viral titer of porcine epidemic diarrhea virus. The inhibitor EHop-016 has the following structural formula: 。

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