Use of cyclooxygenase in preparation of medicine for resisting Singapore grouper iridovirus

Abstract

The application discloses application of an epoxy enzyme in preparation of a medicine for resisting Singapore grouper iridovirus. The experimental results of the application show that the epoxy enzyme has low cytotoxicity and high biological safety in a concentration range of 0.02-50 muM, can significantly inhibit a cytopathic effect caused by Singapore grouper iridovirus (SGIV) infection, and effectively inhibit transcription of virus genes MCP and VP19 and synthesis of virus proteins, and significantly reduce the number of MCP positive cells and the fluorescence signal intensity. Further quantitative analysis shows that the epoxy enzyme can significantly inhibit the copy number of the SGIV MCP gene at the DNA and mRNA levels. That is, the application shows that the epoxy enzyme can effectively block the replication of SGIV in GS cells by inhibiting the expression of SGIV related genes and interfering with the virus replication process, thereby providing a new medicine candidate scheme for preventing and treating Singapore grouper iridovirus infection.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture disease prevention and control and marine biotechnology, specifically relating to the application of cyclooxygenase in the preparation of drugs against Singapore grouper iridovirus. Background Technology

[0002] In recent years, with increasing aquaculture density and accumulated environmental pressures, frequent disease outbreaks have become a major factor restricting the sustainable development of the grouper industry. Among these, Singapore grouper iridovirus (SGIV) is a significant pathogen causing losses in grouper farming, with mortality rates exceeding 90% in infected groupers. Infected fish typically exhibit symptoms such as lethargy, decreased appetite, darkening of body color, and spleen enlargement. SGIV belongs to the genus Ranavirus in the family Iridoviridae and is a large cytoplasmic DNA virus. The virus particle has an icosahedral structure, with a genome length of approximately 140 kb. Current research has established a virus-sensitive grouper spleen cell infection model, and systematic studies have been conducted on viral morphology, replication kinetics, and ultrastructure.

[0003] Epoxomicin, a natural product containing an epoxetine structure, has been reported as a highly efficient and selective irreversible proteasome inhibitor, capable of inhibiting the replication and late-stage gene expression of DNA viruses such as vaccinia virus. The article "Viral entry and the ubiquitin-proteasome system" points out that the invasion process of many viruses relies on the ubiquitin-proteasome system (UPS) to mediate their release from the endocytic pathway. Proteasome inhibitors, represented by MG132, epoxomicin, lactocytin, and bortezomib, can block the invading virus at multiple stages of the viral endocytic pathway. Z-axis tomography analysis showed that Kaposi's sarcoma-associated herpesvirus (KSHV) accumulates in the cytoplasm of infected cells after treatment with MG132 or epoxomicin. However, current techniques do not demonstrate the inhibitory effect of epoxomicin on grouper iridovirus.

[0004] It is worth noting that although MG132, lactocytin, and bortezomib all belong to the proteasome inhibitor class, they differ fundamentally from cyclooxygenase in terms of pharmacophore structure, binding reversibility, subunit selectivity, and off-target effects. Specifically, MG132 is a reversible peptidaldehyde inhibitor that, in addition to inhibiting the proteasome, also inhibits calpain, serine proteases, and the NF-κB signaling pathway, exhibiting significant off-target activity; bortezomib is a reversible borate inhibitor that can lead to abnormally high intracellular peptide levels; while lactocytin, although an irreversible inhibitor, acts via a β-lactone intermediate, and its subunit selectivity spectrum differs from that of cyclooxygenase. Cyclooxygenase's unique α',β'-epoxyketone pharmacophore allows it to bind irreversibly and selectively to the N-terminal threonine residue of the proteasome β5 subunit, with almost no action on targets other than the proteasome. The aforementioned mechanistic differences indicate that the antiviral activity and safety profiles of different proteasome inhibitors cannot be inferred from each other. The anti-SGIV effects of MG132, lactosin, and bortezomib in the existing technology cannot reasonably be expected to be that cyclooxygenase also has anti-SGIV activity. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the use of cyclooxygenase or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating grouper iridovirus.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention utilizes the grouper spleen cell line (GS cells) to investigate the inhibitory effect of epoxomicin on the SGIV replication cycle and screen compounds with in vitro anti-SGIV potential, providing experimental evidence for the prevention and control of grouper iridovirus disease. Experiments show that epoxomicin exhibits significant antiviral activity in the concentration range of 0.02-50 μM, effectively inhibiting SGIV replication and infection in grouper cells while demonstrating good host cell safety. Immunofluorescence assays confirmed that the number of SGIV major capsid protein (MCP) positive cells was significantly reduced after epoxomicin treatment, indicating that viral invasion or replication was inhibited. Further experiments revealed that epoxomicin treatment significantly downregulated the transcription levels of SGIV structural protein genes MCP and VP19, and effectively reduced viral DNA genome copy number and MCP mRNA expression abundance, indicating that this compound can inhibit viral gene expression at both the transcriptional and replication levels. Western blot analysis further confirmed that cyclooxygenase significantly inhibited the translation and synthesis of the SGIV capsid protein MCP, validating its molecular mechanism of antiviral action by blocking viral gene expression at the protein level. In summary, the cyclooxygenase provided by this invention significantly inhibits SGIV infection at low concentrations and remains effective for 36 hours after infection, exhibiting low toxicity to host cells and high safety. This drug, by inhibiting proteasome activity and blocking viral gene expression, provides a novel drug option for the prevention and control of grouper iridovirus disease, expands the application of proteasome inhibitors in aquaculture, and has significant practical value and market potential.

[0007] This invention provides the use of cyclooxygenase or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating grouper iridovirus.

[0008] Furthermore, the anti-grouper iridovirus is for the prevention or treatment of grouper iridovirus disease caused by grouper iridovirus infection in fish.

[0009] Furthermore, the grouper iridovirus is the Singapore grouper iridovirus.

[0010] Furthermore, the fish in question is a grouper.

[0011] Furthermore, the drug achieves treatment by inhibiting proteasome activity and blocking the expression of viral genes MCP and VP19.

[0012] Furthermore, the concentration of cyclooxygenase in the drug is 0.02–50 μM. Cyclooxygenase selectively binds irreversibly to the β5 subunit of the proteasome via its α',β'-epoxyketone pharmacophore group, thereby inhibiting proteasome activity.

[0013] Furthermore, the concentration of cyclooxygenase in the drug is 1 μM.

[0014] Furthermore, the drug also contains other pharmaceutically acceptable excipients.

[0015] Furthermore, the excipients are at least one of preservatives, colorants, flavoring agents, stabilizers, and isotonic agents.

[0016] Furthermore, the dosage form of the drug is injection, powder, capsule, or granule.

[0017] The present invention also provides a method for preventing and treating grouper iridovirus, wherein the method involves culturing grouper with 0.02-50 μM cyclooxygenase for 2-48 hours to achieve the purpose of preventing and treating grouper iridovirus.

[0018] This invention reveals a new pharmaceutical application of cyclooxygenase in the fight against SGIV virus, aiming to address the current technical bottleneck of the lack of highly effective and specific anti-SGIV drugs in aquaculture and provide a new chemical treatment strategy for the prevention and control of viral diseases in aquatic animals.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses the application of cyclooxygenase in the preparation of a drug against Singapore grouper iridovirus. Experimental results of this invention show that: (1) In this invention, cyclooxygenase begins to show antiviral activity at 0.02 μM, and 1 μM can significantly inhibit the cytopathic effect of SGIV and the transcription and protein synthesis of viral genes MCP and VP19; (2) Cyclooxygenase exhibits low cytotoxicity and high safety at effective antiviral concentrations. After treatment with 0.02-50 μM for 24 h, cell viability remained above 80% in all groups. Its high selectivity stems from cyclooxygenase's unique α',β'-epoxyketone pharmacophore, a structure that prevents it from acting on targets other than the proteasome, unlike MG132 which simultaneously inhibits off-target activities such as calpain and NF-κB, thus mitigating cytotoxic risks. (3) Cyclooxygenase can effectively block the replication of SGIV in GS cells by inhibiting the expression of SGIV-related genes and interfering with the viral replication process; This invention provides a novel drug candidate for the prevention and treatment of iridovirus infection in grouper. Attached Figure Description

[0020] Figure 1 The figure shows the effect of different concentrations of cyclooxygenase on the viability of GS cells.

[0021] Figure 2Figure 1 shows the effect of different concentrations of cyclooxygenase treatment on the GS cell pathogenesis effect induced by SGIV infection.

[0022] Figure 3 The figure shows the results of detecting the transcription and protein expression levels of viral genes in GS cells by different concentrations of cyclooxygenase.

[0023] Figure 4 The image shows the immunofluorescence results of cyclooxygenase treatment on the expression of SGIV MCP protein.

[0024] Figure 5 The figure shows the effects of cyclooxygenase treatment on viral gene transcription and protein expression at different time points after SGIV infection.

[0025] Figure 6 The figure shows the results of detecting the effect of cyclooxygenase treatment on the copy number of the SGIV MCP gene. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0027] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0028] (1) Epoxomicin (CAS No.: 134381-21-8, analytical purity ≥99.60%) was purchased from MedChemExpress. The molecular formula of epoxomicin is C 28 H 50 N4O7 has a molecular weight of 554.72 and its structural formula is as follows: .

[0029] (2) The real-time quantitative PCR primers were synthesized by Qingke Biotechnology Co., Ltd., and the specific sequences are as follows: SGIV Major Capsid Protein (MCP) gene: Forward primer (qMCP-F): 5′-GCACGCTTCTCTCACCTTCA-3′, Reverse primer (qMCP-R): 5′-AACGGCAACGGGAGCACTA-3′; SGIV envelope protein VP19 gene: Forward primer (qVP19-F): 5′-TCCAAGGGAGAAACTGTAAG-3′, Reverse primer (qVP19-R): 5′-GGGGTAAGCGTGAAGACT-3′; Internal reference gene β-actin: Forward primer (β-actin-F): 5′-TACGAGCTGCCTGACGGACA-3′, Reverse primer (β-actin-R): 5′-GGCTGTGATCTCCTTCTGCA-3′.

[0030] (3) Statistical analysis: All quantitative experiments were independently repeated three times, and the results are expressed as mean ± standard error (Mean ± SEM). SPSS 20.0 software was used for statistical analysis. One-way ANOVA was used for comparisons among multiple groups, combined with Bonferroni correction for post-hoc multiple comparisons; unpaired two-tailed t-test was used for comparisons between two groups. The significance level was set as follows: *P<0.05 was considered statistically significant, and **P<0.01 was considered extremely significant.

[0031] Example 1 Cytotoxicity test of cyclooxygenase I. Experimental Methods Grouper Spleen Cell (GS) cells were injected at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in 96-well plates and cultured at 28°C for 18 h. Cyclooxygenase was serially diluted to a series of final concentrations (0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 μM) with complete culture medium. Experimental groups were added to culture medium containing the corresponding drug concentration and incubated at 28°C for 24 h or 48 h; the control group received drug-free culture medium containing 1‰ DMSO (solvent control). After incubation, the supernatant was discarded, and cells were washed with fresh culture medium, followed by the addition of 100 μL / well of CCK-8 working solution diluted 1:10 with fresh culture medium, and incubated at room temperature in the dark for 1–4 h. The absorbance (OD value) at 450 nm was measured using a Thermo Fisher Scientific microplate reader. Cell viability was calculated using the following formula: Relative cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. The experiment was repeated three times.

[0032] II. Experimental Results The results are as follows Figure 1As shown, within the concentration range of 0.02-50 μM, the cell viability of all groups remained above 80% after 24 h of cyclooxygenase treatment; however, after 48 h of treatment, the cell viability of all groups decreased significantly, and the viability remained at around 50%. Therefore, subsequent experimental treatment times were all controlled within 48 h, and the drug concentration range was 0.02-50 μM.

[0033] Example 2: Detection of antiviral activity of cyclooxygenase I. Experimental Methods GS cells were seeded in 24-well plates and cultured at 28°C for 18 h until monolayer confluence. The experimental group was pretreated with cyclooxygenase at concentration gradients of 0.02-50 μM for 2 h before being inoculated with Singapore grouper iridovirus (SGIV) (SGIV infection experiments were all performed with a multiplicity of infection (MOI) of 2); the control group was treated with an equal volume of solvent. Cytopathic effect (CPE) was observed under a microscope 24 h after infection, and cell samples were collected.

[0034] 1. RNA Extraction and Quantitative PCR Analysis. Total RNA was extracted using the Cell Total RNA Isolation Kit, and cDNA was synthesized according to the ReverTraAce® qPCR RT Kit instructions. The reverse transcription system (10 μL) consisted of: 2 μL 5×RT buffer, 0.5 μL RT Enzyme Mix, 0.5 μL RT Primer Mix, 1 μg template RNA, and nuclease-free water to a final volume of 10 μL. Reaction conditions: 37℃ for 15 min, 98℃ for 5 min. Using β-actin as an internal reference gene, RT-qPCR was performed using 2×SYBR Green Real-time PCR Mix to analyze the expression levels of SGIV MCP and VP19 genes. The reaction was performed on a QuantStudio 5 real-time quantitative PCR system. -ΔΔCt The relative expression level was calculated using a method with 3 parallel wells for each sample, and the experiment was independently repeated 3 times.

[0035] 2. Western blot analysis. Cell proteins were collected, separated by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane. Separating gel preparation (10 mL): 5.0 mL of 30% acrylamide / bisacrylamide mixture, 2.6 mL of 1.5 mol / L Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 0.01 mL of TEMED. Stacking gel preparation (4 mL): 0.6 mL of 30% acrylamide / bisacrylamide mixture, 0.5 mL of 1 mol / L Tris-HCl (pH 6.8), 0.04 mL of 10% SDS, 0.04 mL of 10% ammonium persulfate, and 0.01 mL of TEMED. Electrophoresis conditions: 70 V constant voltage for 30 min, then adjusted to 110 V constant voltage for 60 min. Wet transfer method (100 mA, 60 min). Block with 5% skim milk at room temperature for 2-3 hours or overnight at 4°C. Incubate with primary antibody at room temperature for 2-3 hours or overnight at 4°C, then wash twice with PBST (5 min / wash). Incubate with HRP-labeled secondary antibody at room temperature for 45 min, then wash three times with PBST (10 min / wash). Develop color using an HRP-DAB colorimetric kit, acquire images using a chemiluminescence imaging system, and analyze grayscale values ​​using ImageJ software. Standardize using β-Tubulin as an internal control.

[0036] II. Experimental Results Microscopic observation results as follows Figure 2 As shown, the untreated control group cells exhibited obvious CPE (cytopathic effect) characteristics such as shrinkage, rounding, and intercellular voids, while the CPE in the cyclooxygenase-treated group was significantly reduced, indicating that cyclooxygenase can effectively inhibit cytopathic effects induced by SGIV infection.

[0037] RT-qPCR results are as follows Figure 3 As shown in Figure A, compared with the control group, the relative expression levels of SGIV MCP and VP19 genes were significantly reduced in the experimental group. Western blot analysis results are as follows... Figure 3 As shown in Figure B, cyclooxygenase treatment also significantly downregulated the expression level of SGIV MCP protein. These results showed a dose-dependent relationship: with increasing cyclooxygenase concentration, both viral gene transcription levels and protein expression gradually decreased. In conclusion, cyclooxygenase has a significant inhibitory effect on SGIV replication in GS cells.

[0038] Example 3: Fluorescence detection analysis of cyclooxygenase's antiviral activity I. Experimental Methods 1. Immunofluorescence analysis. GS cells were seeded in 24-well plates and cultured at 28°C for 18 h until a monolayer formed. The experimental group was pretreated for 2 h with L-15 medium containing 1 μM cyclooxygenase (dissolved in 0.1% DMSO), while a control group was prepared using 0.1% DMSO solvent. SGIV virus was inoculated at MOI=2 and cultured at 28°C for another 24 h. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed three times with PBS. The cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min, blocked with 0.2% bovine serum albumin (BSA, Sigma) for 30 min, and washed three times with PBS. SGIV MCP mouse monoclonal primary antibody (1:500) was added and incubated at room temperature for 2 h, followed by three washes with PBS. Alexa Fluor 555-labeled anti-mouse IgG secondary antibody (1:200) was added and incubated in the dark for 1 h, followed by three washes with PBS. The nuclei were stained with 4′,6-diamidinyl-2-phenylindole (DAPI) for 5 min. Fluorescence images are acquired using a laser scanning confocal microscope (CLSM).

[0039] II. Experimental Results CLSM observation results as follows Figure 4 As shown, compared with the DMSO control group, the number of MCP protein-positive cells was significantly reduced and the fluorescence signal intensity was significantly weakened in the 1 μM cyclooxygenase treatment group. The results indicate that cyclooxygenase can effectively inhibit the expression of the SGIV structural protein MCP.

[0040] Example 4: Analysis of Cyclooxygenase Antiviral Activity at Different Time Points I. Experimental Methods 1. Cell Processing and Sample Collection: GS cells were pretreated with 1 μM cyclooxygenase for 2 h and then inoculated with SGIV virus. Samples were collected at 12, 24, and 36 h post-infection. The following analyses were performed on samples at each time point: Total RNA was extracted using the Cell Total RNA Isolation Kit, and the transcriptional levels of viral MCP and VP19 genes were detected by RT-qPCR; DNA was extracted using the TIANampGenomic DNA Kit, and viral genome replication was analyzed by RT-qPCR; Total protein was extracted using Pierce IP lysis buffer, and the expression of viral structural proteins was detected by Western blot.

[0041] DNA extraction method: After resuspending the cell pellet in buffer GA, proteinase K was added for digestion, followed by buffer GB and incubation at 70°C for 10 min until the solution became clear. Then, anhydrous ethanol was added and mixed. The lysis buffer was transferred to an adsorption column CB3, centrifuged at 12,000 rpm for 30 s, and washed sequentially with buffer GD and wash buffer PW (twice). The empty column was centrifuged at 12,000 rpm for 2 min and air-dried at room temperature. Finally, 50-200 μL of TE buffer was added for elution, and DNA was collected by centrifugation at 12,000 rpm for 2 min and stored at -20°C for later use.

[0042] II. Experimental Results The results are as follows Figure 5 As shown, the 1 μM cyclooxygenase treatment group exhibited significant antiviral activity at all time points. RT-qPCR results showed that the mRNA levels of SGIV MCP and VP19 in the treated group were significantly lower than those in the control group. Western blot analysis further confirmed that the expression level of SGIV MCP protein was significantly downregulated. Viral nucleic acid analysis results are shown below. Figure 6 As shown, cyclooxygenase significantly reduced viral DNA copy number and mRNA accumulation at all time points. These results indicate that cyclooxygenase effectively inhibited SGIV gene transcription, genome replication, and structural protein synthesis at 12, 24, and 36 hours post-infection, exhibiting a sustained antiviral effect.

Claims

1. The use of cyclooxygenase or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating grouper iridovirus.

2. The application according to claim 1, characterized in that, The anti-grouper iridovirus is for the prevention or treatment of grouper iridovirus disease caused by grouper iridovirus infection in fish.

3. The application according to claim 2, characterized in that, The grouper iridovirus mentioned is the Singapore grouper iridovirus.

4. The application according to claim 2, characterized in that, The fish in question is a grouper.

5. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by inhibiting proteasome activity and blocking the expression of viral genes MCP and VP19.

6. The application according to claim 1, characterized in that, The concentration of cyclooxygenase in the drug is 0.02–50 μM.

7. The application according to claim 6, characterized in that, The concentration of cyclooxygenase in the drug is 1 μM.

8. The application according to claim 1, characterized in that, The drug also contains other pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients are at least one of the following: preservatives, colorants, flavoring agents, stabilizers, and isotonic agents.

10. The application according to claim 1, characterized in that, The dosage forms of the drug are injections, powders, capsules, and granules.

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