Application of compound in preparation of medicine for resisting PEDV (Porcine Epidemic Diarrhea Virus)
The -1 Frameshifting process of PEDV virus was inhibited by compound 54-E8, which solved the problem of difficult control of viral replication and proliferation, and achieved effective viral inhibition and disease prevention and treatment effects.
Patent Information
- Application Number
- CN202510263792.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to effectively inhibit the -1 Frameshifting process in swine epidemic diarrhea virus (PEDV), making it difficult to control viral replication and proliferation.
Compound 54-E8 was used to block the replication and proliferation of the virus by inhibiting the -1 Frameshifting process of the PEDV virus.
Compound 54-E8 significantly inhibits the -1 Frameshifting process of PEDV virus, effectively blocks viral replication and proliferation, and has the potential to treat and prevent epidemic diarrhea in swine.
Smart Images
Figure CN119970739A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular relates to application of a compound in preparing a drug for resisting PEDV virus. Background Art
[0002] Porcine epidemic diarrhea is a viral enteric disease of pigs that affects pigs at different growth stages and is particularly deadly to young pigs and newborn piglets. The disease is an acute infectious disease caused by porcine epidemic diarrhea virus (PEDV), which localizes to the intestinal mucosa of pigs and usually causes severe diarrhea, vomiting, dehydration, weight loss and reduced reproductive capacity in infected pigs.
[0003] PEDV belongs to the alpha genus of the Coronaviridae family. It has an envelope and is roughly spherical or pleomorphic in shape, with a diameter of about 95-190nm. Its genome is a single-stranded positive-sense RNA with a total length of 28,000nt, which contains a 5' untranslated region (UTR), 7 open reading frames, a 3' untranslated region (UTR), and a 3' polyadenylic acid (polyA) tail. These seven open reading frames encode four structural proteins: S protein (spike protein), M protein (membrane protein), E protein (envelope protein), N protein (nucleocapsid protein), and pp1a, pp1ab, and ORF3 proteins. Among them, the two open reading frames located upstream are ORF1a and ORF1b, which occupy two-thirds of the genome and contain at least one ribosomal frameshift site. ORF1a and ORF1b encode two large nonstructural polyproteins: pp1a and pp1ab, of which pp1ab's expression depends on the -1 ribosomal frameshifting element in the ORF1a / ORF1b overlap region.
[0004] The "-1 Frameshifting" phenomenon during ribosome elongation is very common in RNA viruses. PRRSV, HIV-1 and SARS viruses all have this phenomenon. The specific process is as follows: (1) The mRNA pseudoknot structure forces the ribosome to stagnate during the elongation phase, and at this time the anticodon loop of the A-site aminoacyl tRNA and the P-site peptidyl tRNA just binds to the mRNA sliding sequence; (2) The sliding sequence causes tRNAs to shift to the -1 position; (3) The downstream mRNA pseudoknot is opened, and the ribosome continues to move forward, but the reading frame shifts. Previous studies have confirmed that the PEDV virus sliding sequence is "UUUAAAC", and there is a three-necked "pseudoknot" structure sequence after this sequence that mediates the ribosome to retreat. When the ribosome in the translation elongation step moves to the sliding sequence, tRNA falls off the ribosome, and the ribosome slides back one step, causing "-1Frameshifting" of the reading frame. After that, tRNA re-enters the position. At this time, the peptidyl transfer center is not affected, and the new peptide chain does not fall off. The ribosome enters the translation process of ORF1b. The "-1 Frameshifting" phenomenon will make the virus have the characteristics of a streamlined genome, fast replication, and high efficiency in the use of genetic material, which is conducive to the reproduction and growth of the virus. If the -1Frameshifting process of PEDV can be inhibited, then PEDV replication can be blocked, thereby achieving the purpose of inhibiting viral proliferation. Therefore, studying drugs that can effectively inhibit the -1 Frameshifting process of PEDV is of great significance for the development of drugs against PEDV and the treatment of diseases caused by PEDV. Summary of the invention
[0005] The object of the present invention is to provide an application of a compound in the preparation of a drug for resisting PEDV virus.
[0006] The present invention provides an application of a compound in the preparation of a drug for resisting PEDV virus, wherein the compound is 54-E8, and its structural formula is:
[0007] .
[0008] Furthermore, the compound can inhibit the replication and / or proliferation of progeny viruses in PEDV virus.
[0009] Furthermore, the compound has an inhibitory effect on the replication and proliferation of PEDV virus in host cells.
[0010] Furthermore, the compound can inhibit the -1 position ribosome frameshift process of the PEDV virus.
[0011] Furthermore, the safe concentration of the compound capable of inhibiting PEDV virus-1 position ribosomal frameshift is 0.5-5 μM.
[0012] Furthermore, the medicine is a medicine for treating and / or preventing porcine epidemic diarrhea.
[0013] Furthermore, the drug is prepared by using the compound or its chemically acceptable salt as an active ingredient and adding pharmaceutically acceptable excipients.
[0014] Furthermore, the medicine is an injectable preparation.
[0015] Furthermore, the medicine is an oral preparation.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The present application has discovered a new use of compound 54-E8 in inhibiting PEDV virus. Experiments have confirmed that compound 54-E8 has a significant inhibitory and blocking effect on the -1 ribosomal frameshifting process of PEDV virus, and can effectively inhibit the replication of PEDV virus, thereby inhibiting the proliferation of PEDV virus. The compound 54-E8 can be used to treat and prevent porcine epidemic diarrhea caused by PEDV virus infection. The compound 54-E8 can be used to prepare drugs against PEDV virus, as well as drugs for treating and preventing porcine epidemic diarrhea caused by PEDV virus infection, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0019] Figure 1 Schematic diagram (a), schematic diagram of the experimental process (b), and experimental results (c) of compound 54-E8 inhibiting the PEDV virus ribosome frameshift process in the luciferase reporter system.
[0020] Figure 2 The inhibitory effect of compound 54-E8 at different concentrations on PEDV virus ribosome frameshifting.
[0021] Figure 3 The cytotoxicity test results of compound 54-E8 at different concentrations.
[0022] Figure 4Schematic diagram (a), schematic diagram of the experimental process (b), and experimental results (c and d) of compound 54-E8 inhibiting the PEDV virus ribosome frameshifting process in the fluorescent protein reporter system.
[0023] Figure 5 This is the inhibitory effect of compound 54-E8 on the in vitro proliferation of PEDV-GIIc virus. DETAILED DESCRIPTION
[0024] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.
[0025] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the embodiments of the present invention are all commercially available; unless otherwise specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.
[0026] The compound, named 54-E8, CAS number: 2380180-12-9, English name: 6-(2-methoxyphenyl)-2-[1-[oxo(4,5,6,7-tetrahydro-2,1-benzoxazol-3-yl)methyl]-3-azetidinyl]-3-pyridazinone, molecular formula: C 22 H 22 N4O4, molecular weight: 406.4, structural formula as shown in formula (I):
[0027] Formula (I).
[0028] Example 1 Inhibition of PEDV virus-1 Frameshift process by compound 54-E8 (luciferase reporter system)
[0029] 1. Experimental Methods
[0030] In vitro cultured cells were used to study whether the drug (compound 54-E8) had an inhibitory effect on the -1 Frameshift process of PEDV virus. First, a dual-luciferase reporter gene lentiviral vector containing the PEDV virus genome sliding sequence was constructed, and the effects of different drugs were tested in the porcine kidney cell line PK15, with an initial screening concentration of 10 μM.
[0031] 1.1. Vector construction
[0032] (1) Synthesize the PEDV virus-1 Frameshift sliding region gene sequence. Perform double restriction digestion on the lentiviral backbone vector, and select the restriction site: EcoRI+BamHI. Restriction digestion conditions: 37°C, 15 minutes. After the restriction digestion is completed, the restriction digestion product is recovered by nucleic acid electrophoresis. For the synthesized PEDV virus-1 Frameshift sliding region gene sequence, first denature at 95°C for 10 minutes and anneal at 72°C for 30 seconds, then mix the annealing product with the restriction digestion product, the mixing ratio is annealing product: restriction digestion product = 3:1, add 10 μl of T4 ligase to the above mixture, and connect in a constant temperature metal bath at 16°C for 16 hours.
[0033] Plasmid transformation competent cells: Take out the competent cells from the -80℃ refrigerator, melt on ice, add 10 microliters of plasmid (the plasmid is a lentiviral vector carrying the gene sequence of the PEDV virus-1 Frameshift sliding region) at a ratio of 100 microliters of competent cells, add plasmid and mix well, place on ice for 30 minutes, put the mixture of competent cells and plasmid into 42℃ water, and heat shock for 90 seconds. After the heat shock, cool the mixture of competent cells and plasmid on ice for 10 seconds. Transfer the cooled mixture to a solid LB plate, spread the liquid evenly on the surface of the solid LB culture medium with a glass rod, then invert the solid LB culture medium and culture it in a 37℃ incubator for 16 hours. After the culture is completed, pick up the monoclonal plaque with a pipette tip and inoculate it with liquid LB culture medium. After culturing in a 37℃ shaker for 8 hours, send it to the company for sequencing. The monoclonal clone with the correct sequencing result is expanded and the plasmid is extracted.
[0034] (2) Through homologous recombination, Renilla luciferase was constructed upstream of the -1 Frameshift region, and firefly luciferase was constructed downstream of the -1 Frameshift region.
[0035] The specific method is as follows: Amplify the Renilla luciferase gene sequence by PCR, and add homology arms to the upstream and downstream of the sequence. Perform PCR on the vector obtained in step (1) to obtain a linearized vector. Obtain the Renilla luciferase gel recovery product and the linearized vector gel recovery product by nucleic acid electrophoresis and gel recovery. Homologous recombination: Mix the Renilla luciferase gel recovery product and the linearized vector gel recovery product in a mass ratio of 3:1, add 2 μl of homologous recombination enzyme, and react at 37°C for 15 minutes.
[0036] (3) Transform the plasmid into competent cells, select single clones and sequence them, amplify the viral backbone vector with the correct sequence and extract the plasmid. The transformation of competent cells and extraction steps are the same as step (1).
[0037] 1.2. Establishment of screening cell system
[0038] (1) Mix the viral backbone vector containing dual luciferase and -1 Frameshift region, PMD2.G, and plpax2 packaging vector with PEI (cationic polymer) in a volume ratio of 4:2:1. Add 70 μg PEI for every 35 μg DNA and transfect 293T cells.
[0039] (2) The supernatant of 293T cells cultured 48 h and 72 h after transfection with viral plasmids was collected, centrifuged at 12,000 g for 10 min to remove cell impurities, and then the lentiviral particles were collected by cesium chloride gradient centrifugation.
[0040] (3) Add lentiviral particles to PK15 cells, and 7 days after infection, select positive cells using Puro. Digest the cells with trypsin to prepare a single cell suspension, and perform monoclonal sorting using flow cytometry.
[0041] (4) The genotype of the cultured monoclonal cells was identified, and the positive clones were expanded to obtain PK15 cells containing the -1Frameshift region (CMV-Renilla-framshift-Firefly).
[0042] 1.3. Drug screening
[0043] (1) PK15 cells containing the -1 Frameshift region were cultured in a 96-well plate, and different test compounds (DMSO and 54-E8) were added thereto, with the drug concentration being 10 μM.
[0044] (2) After 8 h of culture, the cells were lysed and firefly luciferase substrate was added to the lysate. The luminescence value was detected by an ELISA reader.
[0045] (3) Add Renilla luciferase substrate and measure the luminescence value using an ELISA reader.
[0046] (4) The ratio of firefly luciferase to Renilla luciferase is used as a reference for -1 Frameshift efficiency. A larger ratio indicates a higher -1 Frameshift efficiency, and a lower ratio indicates a lower -1 Frameshift efficiency. That is, a lower ratio indicates that the drug has a higher efficiency in inhibiting the -1 Frameshift process and can better inhibit the -1 Frameshift process.
[0047] 2. Experimental results
[0048] (1) Figure 1 The schematic diagram (a), the schematic diagram of the experimental process (b) and the experimental results (c) of the inhibition of PEDV virus ribosome frameshift by compound 54-E8 in the luciferase reporter system. Figure 1As shown in a, when ribosome frameshifting occurs normally, both reporter genes Renilla and Firefly are expressed. When ribosome frameshifting is blocked, reporter gene Renilla is expressed, but Firefly is not expressed. Figure 1 Figure b shows the workflow of the luciferase reporter system. First, the reporter vector is stably integrated into the host cell (pig kidney cell PK15) through a lentiviral vector, and the positive monoclonal cells are treated with the test compound. The inhibitory effect of the compound on the PEDV ribosome frameshift process is determined by using a dual-luciferase reporter gene detection kit and an ELISA instrument. Figure 1 Figure c shows the inhibitory effect of compound 54-E8 at μM concentration on the -1Frameshift process of PEDV virus in the dual luciferase reporter gene system. Figure 1 As shown in the results in c, compound 54-E8 at a concentration of 10 μM has a very significant inhibitory effect on the -1 Frameshift process of PEDV virus (** indicates P < 0.01).
[0049] (2) To further determine the effect of different concentrations of compound 54-E8 on the -1 Frameshift efficiency of PEDV virus, the -1 Frameshift efficiency screening system and method based on luciferase were used to study the -1 Frameshift efficiency of PEDV virus under different concentrations of compound 54-E8 (0.25 μM, 0.5 μM, 1 μM, 2.5 μM and 5 μM). The relationship between the concentration change of compound 54-E8 and the -1 Frameshift inhibitory effect of PEDV virus was analyzed by firefly luciferase activity.
[0050] Test results such as Figure 2 As shown, compound 54-E8 can significantly inhibit the -1Frameshift process of PEDV virus at a concentration of 0.5 μM (* indicates P < 0.05).
[0051] (3) To further determine the inhibitory effect of compound 54-E8 on PEDV virus-1 Frameshift, cytotoxicity assays were performed in wild-type Vero cells.
[0052] The results are as follows Figure 3 As shown, in Vero cells, compound 54-E8 at concentrations of 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM had no toxicity to cells, and only exhibited cytotoxicity at concentrations above 10 μM.
[0053] The above experimental results show that compound 54-E8 has an inhibitory effect on the -1 Frameshift process of PEDV virus, with the lowest effective concentration being 0.5 μM. At this time, the compound is not toxic to cells and has a large safety window.
[0054] Example 2 Inhibition of PEDV virus-1 Frameshift process by compound 54-E8 (fluorescent protein reporter system)
[0055] 1. Experimental Methods
[0056] To further determine the inhibitory activity of compound 54-E8, a reporter gene system based on short half-life fluorescent proteins was constructed. First, ubiquitin was linked to RFP and GFP to obtain UbRFP (ubiquitin-RFP) and UbGFP. Then, Firefly luciferase and Renilla luciferase in the dual luciferase reporter system were replaced with Ub-RFP and Ub-GFP, respectively. The updated reporter system is: CMV-UbRFP- Frameshift-UbGFP (see Figure 4 As shown in a). For the test, CMV-UbRFP- Frameshift-UbGFP cells were passaged in a 96-well plate 24 h in advance, so that the cell confluence was preferably 80%-90% when the drug (test compound: DMSO and 54-E8) was added. 54-E8 or DMSO (0.5 μM) was added to the culture dish for 1 h, and then 5 μM MG-132 (proteasome inhibitor) was added for further treatment for 3 h. Three replicate wells were set for each group. Finally, the fluorescence ratio information of GFP and RFP was obtained by fluorescence microscopy (see Figure 4 (as shown in b).
[0057] 2. Experimental results
[0058] The detection results of the fluorescent protein reporter gene system are as follows Figure 4 As shown in c and d, compound 54-E8 has a very significant (** indicates P < 0.01) inhibitory effect on the -1 Frameshift process of PEDV virus in the fluorescent protein reporter gene system.
[0059] Example 3 Inhibition of the in vitro proliferation of PEDV-GIIc strain by compound 54-E8
[0060] 1. Experimental Methods
[0061] By culturing Vero cells in vitro, an in vitro culture system of the PEDV-GIIc strain was successfully established. To verify the in vitro inhibitory ability of compound 54-E8 on the PEDV-GIIc strain, PEDV-GIIc virus was first added to the Vero cells. After 6 hours, fresh culture medium was replaced and 0.5 μM compound 54-E8 or DMSO (control group) was added at the same time. After 3 days of culture, the cell pathological changes were observed under a bright field microscope.
[0062] 2. Experimental results
[0063] Bright field microscope observation results (photos) are as follows Figure 5 As shown in the experimental results, no lesions occurred in the compound 54-E8 treatment group, while obvious lesions occurred in the DMSO control group, indicating that compound 54-E8 has a significant inhibitory effect on PEDV-GIIc virus cultured in vitro.
[0064] In summary, the present invention provides an application of compound 54-E8 in the preparation of drugs against PEDV virus. The present invention has discovered a new use of compound 54-E8 in inhibiting PEDV virus. The compound 54-E8 of the present invention has a significant inhibitory and blocking effect on the -1Frameshift process of PEDV virus, and can effectively inhibit the replication and proliferation of PEDV virus; the compound 54-E8 can be used to prepare drugs against PEDV virus and drugs for preventing and treating porcine epidemic diarrhea caused by PEDV virus, and has broad application prospects.
Claims
1. Use of a compound in the preparation of a drug for resisting PEDV virus, characterized in that: The compound is 54-E8, and its structural formula is: 。 2. Use of the compound according to claim 1 in the preparation of a drug for resisting PEDV virus, characterized in that: The compound can inhibit the replication and / or proliferation of progeny viruses in PEDV virus.
3. Use of the compound according to claim 1 in the preparation of a drug for resisting PEDV virus, characterized in that: The compound has an inhibitory effect on the replication and proliferation of PEDV virus in host cells.
4. Use of the compound according to any one of claims 1 to 3 in the preparation of a drug against PEDV virus, characterized in that: The compound can inhibit the -1 position ribosome frameshift process of PEDV virus.
5. Use of the compound according to claim 4 in the preparation of a drug against PEDV virus, characterized in that: The safe concentration of the compound capable of inhibiting PEDV virus-1 position ribosome frameshift is 0.5-5 μM.
6. Use of the compound according to claim 1 in the preparation of a drug for resisting PEDV virus, characterized in that: The medicine is a medicine for treating and / or preventing porcine epidemic diarrhea.
7. Use of the compound according to claim 6 in the preparation of a drug for resisting PEDV virus, characterized in that: The medicine is prepared by taking the compound or its chemically acceptable salt as active ingredient and adding pharmaceutically acceptable auxiliary materials.
8. Use of the compound according to claim 7 in the preparation of a drug for resisting PEDV virus, characterized in that: The medicine is an injection preparation.
9. Use of the compound according to claim 7 in the preparation of a drug against PEDV virus, characterized in that: The medicine is an oral preparation.