Application of 3-denitrified adenosine in preparation of medicine for preventing and / or treating porcine epidemic diarrhea virus
By using the pharmaceutical composition prepared by 3-denitrogenation adenosine, the problem that existing vaccines cannot effectively prevent and control the epidemic diarrhea virus in swine is solved, and the effect of significantly inhibiting virus proliferation is achieved, and a new therapeutic drug regimen is provided.
Patent Information
- Application Number
- CN202411767359.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-04
- Publication Date
- 2025-02-28
AI Technical Summary
The existing swine epidemic diarrhea virus (PEDV) vaccine cannot effectively prevent and control viral infection due to strain drift and new subtypes, resulting in a lack of effective therapeutic drugs.
Using 3-Deazaadenosine as a pharmaceutical ingredient, a pharmaceutical composition containing 3-Deazaadenosine and its pharmaceutically acceptable carrier is used for the prevention and treatment of swine epidemic diarrhea virus.
3-Denitrinosine significantly inhibits the proliferation of swine epidemic diarrhea virus within the concentration range of 5 to 40 μM, and has no effect on host cell activity, providing good anti-swine epidemic diarrhea virus effect.
Smart Images
Figure CN119524003A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicines, and in particular relates to the application of 3-deazaadenosine in the preparation of medicines for preventing and / or treating porcine epidemic diarrhea virus. Background Art
[0002] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive RNA enveloped virus with a single-stranded positive RNA genome (excluding polyA) of approximately 28 kb. The genome consists of a 5' end, a 5' end cap, a 5' UTR, seven open reading frames, a 3' UTR, and a polyadenylation (polyA) tail at the 3' end. The seven open reading frames encode four structural proteins: S protein (spike protein), M protein (membrane protein), E protein (envelope protein), and N protein (nucleocapsid protein), as well as pp1a, pp1ab, and ORF3 proteins.
[0003] PEDV infects the small intestine and intestinal villus cells of pigs, spreads rapidly, and causes a contagious enteric infectious disease characterized by vomiting, diarrhea, and dehydration, resulting in a high mortality rate of newborn piglets (up to 100%), causing huge economic losses to the pig industry. Currently, vaccination is still the main method for preventing PEDV infection. However, over time, due to the occurrence of strain drift and the emergence of new subtypes, new variants make existing vaccines ineffective in preventing and controlling viral infections. Therefore, there is an urgent need to explore potential therapeutic drugs that can inhibit PEDV. Summary of the invention
[0004] Purpose of the invention: The purpose of the present invention is to provide the use of 3-deazaadenosine in the preparation of drugs for preventing and / or treating porcine epidemic diarrhea virus.
[0005] Technical solution: Use of 3-deazaadenosine of the present invention in the preparation of drugs for preventing and / or treating porcine epidemic diarrhea virus.
[0006] The structure of the 3-deazaadenosine is as follows:
[0007]
[0008] The porcine epidemic diarrhea virus is PEDV CV777.
[0009] The prevention is to prevent cells from being infected with porcine epidemic diarrhea virus or to reduce the infection of porcine epidemic diarrhea virus in cells.
[0010] The treatment is to inhibit the growth and replication of porcine epidemic diarrhea virus in cells.
[0011] Preferably, the cells are IPEC-J2 cells.
[0012] Preferably, the non-toxic concentration of 3-deazaadenosine to cells is 5-40 μM.
[0013] Preferably, the concentration of 3-deazaadenosine used to inhibit the proliferation of porcine epidemic diarrhea virus is 5 to 40 μM.
[0014] Further preferably, the concentration of 3-deazaadenosine used to inhibit the proliferation of porcine epidemic diarrhea virus is 5 to 20 μM.
[0015] The pharmaceutical composition for preparing a drug for preventing and / or treating porcine epidemic diarrhea virus comprises 3-deazaadenosine and a pharmaceutically acceptable carrier thereof.
[0016] Preferably, the pharmaceutical composition comprises a stereoisomer of 3-deazaadenosine, a solvate of 3-deazaadenosine, a pharmaceutically acceptable salt of 3-deazaadenosine or a solvate of a pharmaceutically acceptable salt of 3-deazaadenosine as an effective ingredient.
[0017] Preferably, the pharmaceutical composition is at least one of an oral preparation, a spray preparation or an injection preparation.
[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) It is the first time that 3-deazaadenosine is found to have anti-porcine epidemic diarrhea virus activity, and provides an application in the preparation of drugs for preventing and / or treating porcine epidemic diarrhea virus. 3-deazaadenosine exhibits a significant inhibitory effect on the proliferation of porcine epidemic diarrhea virus in host cells, and has a good and stable anti-porcine epidemic diarrhea virus effect; (2) Using porcine intestinal epithelial cells (IPEC-J2) as a cell model, 3-deazaadenosine has no effect on cell activity within a concentration range of 5 to 40 μM, and has a significant inhibitory effect on the proliferation of porcine epidemic diarrhea virus within a concentration range of 5 to 40 μM, and the virus inhibition effect is positively correlated with the concentration of 3-deazaadenosine used, and can be used to prepare drugs for preventing and / or treating porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a graph showing the effect of different concentrations of 3-deazaadenosine on IPEC-J2 cell activity detected by CCK-8 method;
[0020] Figure 2 This is a cell morphology observation diagram of the morphological changes of IPEC-J2 cells at different time points after PEDV infection;
[0021] Figure 3 This is a graph showing the changes in the load of PEDV CV777 at different time points detected by fluorescence quantitative PCR;
[0022] Figure 4The figure shows the change of the load of PEDV CV777 at different time points detected by immunoblotting;
[0023] Figure 5 This is a graph showing the changes in viral load before and after 3-deazaadenosine treatment detected by fluorescent quantitative PCR;
[0024] Figure 6 The figure shows the changes of viral nucleocapsid protein before and after treatment with 3-deazaadenosine detected by immunoblotting;
[0025] Figure 7 This is a diagram showing the distribution of viral nucleocapsid protein before and after 3-deazaadenosine treatment detected by indirect immunofluorescence. DETAILED DESCRIPTION
[0026] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the examples. The cells, viruses and main reagents used in the present invention are as follows:
[0027] Cell line: Porcine intestinal epithelial cells (IPEC-J2 cells) were cultured in DMEM (DMEM, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA).
[0028] Strain: Strain: PEDV CV777 is an existing strain. The strain used in this application is provided by Yangzhou University. The applicant has also used PEDV CV777 before. For details, see the article (Qin W, Qi X, Xie Y, Wang H, Wu S, Sun MA, Bao W. LncRNA446 Regulates Tight Junctions by Inhibiting the UbiquitinatedDegradation of Alix after Porcine Epidemic Diarrhea Virus Infection. J Virol. 2023 Mar 30; 97 (3): e0188422. doi: 10.1128 / jvi.01884-22).
[0029] Main reagents: PEDV-N antibody was purchased from Medgene Labs (Medgene Labs, USA), 3-Deazaadenosine powder was purchased from MCE (MCE, NJ, USA), CCK-8 cell proliferation and toxicity detection kit was purchased from Vazyme (Vazyme, Nanjing, China), and real-time fluorescence quantification kit was purchased from Vazyme (Vazyme, Nanjing, China).
[0030] The reagents not described in detail in the application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.
[0031] Example 1: Detection of IPEC-J2 cell activity by different concentrations of 3-deazaadenosine
[0032] 2000 IPEC-J2 cells were plated in each well of a 96-well cell plate and mixed evenly with DMEM containing 10% FBS (total volume of 100 μL), and cultured overnight in a 37°C, 5% CO2 incubator. When the cell density reached 80%, the 3-deazaadenosine concentrated stock dissolved in ddH2O was diluted with water to 5 μM, 10 μM, 20 μM, and 40 μM and added to the well plate (three replicates were set for each concentration). A control group was also set up, and the treatment time was set to 24 h. The cell viability was then detected using a CCK-8 kit, and 10 μL of CCK-8 solution was added to the well plate per well. The absorbance was detected at 450 nm using a Tecan Infinit200 microplate reader (Sunrise, Tecan, Switzerland). The data were analyzed using GraphPad 9.0 software, and statistical analysis was performed using the Student t test. ns There is no significant difference, the results are as follows Figure 1 shown.
[0033] Depend on Figure 1 It can be seen that compared with the blank group, 3-deazaadenosine had no significant effect on cell activity within the concentration range of 5 to 40 μM.
[0034] Example 2 Cell morphological observation of morphological changes of IPEC-J2 cells at different time points after infection with PEDV CV777
[0035] The cultured IPEC-J2 cells were inoculated into a 12-well cell culture plate. When the cell density reached about 80%, the culture medium was discarded, and the cells were washed three times with PBS. Then, the cells were infected with PEDV virus solution (MOI = 0.1). After 2 hours of infection, the culture medium containing 0.1% trypsin (without FBS) was replaced. After culturing for different time periods (0h, 2h, 12h, 24h, 48h), the cell morphology changes were observed and photographed. The results are shown in Figure 2. Figure 2 shown.
[0036] Depend on Figure 2 It can be seen that as the virus infection time increases, IPEC-J2 cells gradually shrink and deform, and even die and fall off, indicating that PEDV CV777 infection can cause adverse changes in the morphology of IPEC-J2.
[0037] Example 3: Fluorescence quantitative PCR detection of PEDV CV777 proliferation in IPEC-J2 cells in vitro
[0038] In order to detect the in vitro proliferation of PEDV CV777 in IPEC-J2 cells, the virus was added to IPEC-J2 cells and the cells were collected 0 to 48 hours after infection. The purity of viral RNA (Vazyme, Nanjing, China) was determined using the ND-1000 nucleic acid / protein concentration detector according to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit. According to the gene sequence published in the GenBank database, GAPDH was used as the internal reference gene, and qPCR primers were designed using Primer Premier 6.0 software. The upstream primer of PEDV-N was 5'-CACCTCCTGCTTCACGTACA-3', and the downstream primer was 5'-AGCTCCACGACCCTGGTTAT-3'. The primers were synthesized by Sangon Biotech Co., Ltd. (Sangon Biotech, Shanghai, China). qPCR analysis was performed using a real-time fluorescence quantitative kit (Vazyme, Nanjing, China), and 3 independent replicates were set for each analyzed sample. The data were analyzed using GraphPad 9.0 software, and the results are shown in the figure. Figure 3 shown.
[0039] Depend on Figure 3 It can be seen that after PEDV CV777 infected IPEC-J2 cells, the expression of PEDV-N gradually increased from 0h to 24h, reached the maximum at 24h, and decreased at 48h compared with 24h.
[0040] Example 4: Immunoblotting to detect the changes in the loading of PEDV CV777 at different time points
[0041] IPEC-J2 cells were infected with PEDV CV777, and a mock group was set up. Cells were collected at different time points, and the cells were lysed on ice for 30 min using a mixture containing protein lysis buffer (RIPA) (Applygen, Beijing, China), and protease inhibitors (Target MOI, MA, USA) were added. The supernatant was obtained by centrifugation at 14,000 rpm at 4°C for 15 min. The protein concentration was adjusted using a BCA protein assay kit (Beyotime, Shanghai, China), and denatured at 98°C for 10 min for later use.
[0042] The samples were subjected to polyacrylamide gel electrophoresis and the proteins were transferred to PVDF membranes (Millipore, Canada, USA). After blocking with 5% skim milk powder at room temperature for 2 h, PEDV-N and GAPDH primary antibodies were used and incubated overnight at 4°C. The PVDF membrane was rinsed 3 times with 1×TBST and incubated with secondary antibodies (Proteintech, Wuhan, China) at room temperature for 2 h. The samples were washed 3 times with 1×TBST and then soaked in ECL developer for development analysis. The results are shown in Figure 2. Figure 4 shown.
[0043] Depend on Figure 4 It was found that after PEDV CV777 infected IPEC-J2 cells, PEDV-N expression gradually increased from 0 to 24 hours, reached a maximum at 24 hours, and decreased at 48 hours compared with 24 hours.
[0044] Example 5: Fluorescence quantitative PCR detection of the inhibitory effect of 3-deazaadenosine on the proliferation of PEDV CV777 in vitro
[0045] In order to detect the inhibitory effect of 3-deazaadenosine on PEDV CV777, the cells were infected with viruses and cultured for 24 h after adding 0 μM, 5 μM, 10 μM, 20 μM, and 40 μM 3-deazaadenosine. Viral RNA (Vazyme, Nanjing, China) was prepared according to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit, and the purity was determined using an ND-1000 nucleic acid / protein concentration detector. According to the gene sequence published in the GenBank database, GAPDH was used as the internal reference gene, and qPCR primers were designed using Primer Premier 6.0 software. The upstream primer of PEDV-N was 5'-CACCTCCTGCTTCACGTACA-3', and the downstream primer was 5'-AGCTCCACGACCCTGGTTAT-3'. The primers were synthesized by Sangon Biotech, Shanghai, China. qPCR analysis was performed using a real-time fluorescence quantitative kit (Vazyme, Nanjing, China), and three independent replicates were set for each analysis sample. The data were analyzed using GraphPad 9.0 software and Student t test for statistical analysis. *p<0.05 indicates a significant difference, **p<0.01 indicates an extremely significant difference. The results are shown in Figure 5 shown.
[0046] Depend on Figure 5It was found that compared with the toxin-added group, viral proliferation was inhibited after the addition of 3-deazaadenosine, and 10 μM 3-deazaadenosine significantly inhibited the proliferation of PEDV CV777.
[0047] Example 6: Immunoblotting to detect the inhibitory effect of 3-Deazaadenosine on the proliferation of PEDV CV777 in vitro
[0048] PEDV CV777 virus was added to cells with 0μM, 5μM, 10μM, 20μM, and 40μM 3-deazaadenosine, and the cells were collected after 24h of culture. A mixture containing protein lysis buffer (RIPA) (Applygen, Beijing, China) was used, and protease inhibitors (TargetMOI, MA, USA) were added to lyse on ice for 30min, and the supernatant was obtained by centrifugation at 14,000rpm at 4°C for 15min. The protein concentration was adjusted using a BCA protein assay kit (Beyotime, Shanghai, China), and denatured at 98°C for 10min for use.
[0049] The samples were subjected to polyacrylamide gel electrophoresis and the proteins were transferred to PVDF membranes (Millipore, Canada, USA). After blocking with 5% skim milk powder at room temperature for 2 h, PEDV-N and GAPDH primary antibodies were used and incubated overnight at 4°C. The PVDF membrane was rinsed 3 times with 1×TBST and incubated with secondary antibodies (Proteintech, Wuhan, China) at room temperature for 2 h. The samples were washed 3 times with 1×TBST and then soaked in ECL developer for development analysis. The results are shown in Figure 2. Figure 6 shown.
[0050] By Figure 6 It was found that compared with the toxin-added group, viral proliferation was inhibited after the addition of 3-deazaadenosine, and 10 μM 3-deazaadenosine significantly inhibited the proliferation of PEDV CV777.
[0051] Example 7: Indirect immunofluorescence assay to detect the inhibitory effect of 3-deazaadenosine on the proliferation of PEDV CV777 in vitro
[0052] After adding 0μM and 10μM 3-deazaadenosine and culturing for 24h, PEDV CV777 was added to infect the cells for 24 and then the cells were collected. After washing the cells with pre-cooled PBS, the cells were fixed with 4% paraformaldehyde at 4°C for 30min, and then bovine serum albumin (BSA) (Solarbio, Beijing, China) was added for blocking at 37°C for 2h. PEDV-N was used as the primary antibody and incubated overnight at 4°C. The cells were then gently washed three times with PBST, and then a fluorescently labeled secondary antibody (Solarbio, Beijing, China) was added and incubated at room temperature in the dark for 2h. The cells were gently washed three times with PBST, and DAPI was added and incubated at 37°C for 5min for nuclear staining. The fluorescence of the differently treated cells was then observed and photographed using a fluorescent inverted microscope (Leica Microsystems, Wetzlar, Germany). The results are shown in the following figure. Figure 7 shown.
[0053] Depend on Figure 7 It can be seen that compared with the toxin-added group, after the addition of 10 μM 3-deazaadenosine, the distribution of viral N protein decreased, indicating that 10 μM 3-deazaadenosine significantly inhibited the proliferation of PEDV CV777.
[0054] The above results indicate that 3-deazaadenosine has stable anti-porcine epidemic diarrhea virus activity and inhibits the replication of porcine epidemic diarrhea virus.
Claims
1. 3-Use of deazaadenosine in the preparation of drugs for preventing and / or treating porcine epidemic diarrhea virus.
2. The use according to claim 1, characterized in that: The porcine epidemic diarrhea virus is PEDV CV777.
3. The use according to claim 1, characterized in that: The prevention is to prevent cells from being infected with porcine epidemic diarrhea virus or to reduce the infection rate of porcine epidemic diarrhea virus in cells.
4. The use according to claim 1, characterized in that: The treatment is to inhibit the growth and replication of porcine epidemic diarrhea virus in cells.
5. The use according to claim 3 or 4, characterized in that: The cells are IPEC-J2 cells.
6. The use according to claim 5, characterized in that: The 3-deazaadenosine is non-toxic to cells at a concentration of 5 to 40 μM.
7. The use according to claim 1, characterized in that: The concentration of 3-deazaadenosine used to inhibit the proliferation of porcine epidemic diarrhea virus is 5 to 40 μM.
8. The use according to claim 1, characterized in that: The concentration of 3-deazaadenosine used to inhibit the proliferation of porcine epidemic diarrhea virus is 5 to 20 μM.
9. A pharmaceutical composition for preparing a drug for preventing and / or treating porcine epidemic diarrhea virus, characterized in that: The invention comprises 3-deazaadenosine and a pharmaceutically acceptable carrier thereof.
10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition comprises a stereoisomer of 3-deazaadenosine, a solvate of 3-deazaadenosine, a pharmaceutically acceptable salt of 3-deazaadenosine or a solvate of a pharmaceutically acceptable salt of 3-deazaadenosine as an effective ingredient.