Application of Remodelin in preparation of medicine for resisting Nipah virus

Abstract

The invention relates to an application of Remodelin in preparation of an anti-Nipah virus drug. The Remodelin can inhibit budding of NiV particles and prevent the virus from being copied in host cells; and the Remodelin can inhibit the duplication of NiV and reduce the virus copy number after NiV infection, and can be used as an NiV inhibitor for preventing or treating NVD.

Description

Technical Field

[0001] This application relates to the field of Nipah virus prevention and control technology, and in particular to the application of Remodelin in the preparation of anti-Nipah virus drugs. Background Technology

[0002] Nipah virus (NiV) is a highly lethal zoonotic single-stranded negative-sense RNA virus, taxonomically belonging to the Paramyxoviridae family (…). Paramyxoviridae Hennipa virus genus ( Henipavirus The Nipah virus was initially discovered in 1998 among pig farmers and slaughterhouse workers in Malaysia and Singapore, and subsequently experienced multiple outbreaks in Southeast and South Asia. Nipah virus has been listed as a priority pathogen, classified as a highly pathogenic pathogen at Biosafety Level 4 (BSL-4). Clinical epidemiological data show that Nipah virus infection has an extremely high mortality rate, typically ranging from 70% to 100% in outbreaks across different regions. The virus has a wide range of transmission routes, primarily through direct transmission to humans via animal hosts such as fruit bats (flying foxes) or pigs, and can also spread horizontally through the ingestion of contaminated food or close person-to-person contact. In terms of pathogenicity, Nipah virus primarily targets the respiratory and central nervous systems of its host. Clinical manifestations present a diverse spectrum of symptoms, ranging from asymptomatic infection and acute respiratory distress syndrome to fatal viral encephalitis. Extensive viral replication within the host and the hijacking of host cellular function lead to severe tissue damage and multiple organ dysfunction. However, despite the serious threat that Nipah virus poses to public health, there are currently no specific antiviral drugs or approved preventative vaccines available clinically. Existing clinical interventions are mainly limited to symptomatic treatment and supportive care, which are insufficient to effectively control disease progression and reduce mortality. Therefore, in-depth exploration of the replication mechanism of Nipah virus and the interaction between the virus and host proteins, and the development of highly effective and low-toxicity antiviral drugs accordingly, are critical scientific issues and clinical needs that urgently need to be addressed in the biomedical field.

[0003] Remodelin (CAS No.: 949912-58-7) is a selective inhibitor of the orally active acetyltransferase NAT10. Remodelin inhibits NAT10 activity in prostate cancer cells, slows DNA replication, and suppresses cell growth. In xenograft models, Remodelin inhibits the growth of prostate cancer and hepatocellular carcinoma and prolongs the healthy lifespan of a mouse model of early aging syndrome. Currently, it is demonstrating broad research and clinical application prospects in various disease models. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Remodelin in the preparation of anti-Nipah virus drugs.

[0005] The first objective of this invention is to provide the use of the compound Remodelin in the preparation of medicaments for the treatment or prevention of Nipah virus.

[0006] A second object of the present invention is to provide the use of a pharmaceutically acceptable salt of the compound Remodelin in the preparation of medicaments for the treatment or prevention of Nipah virus.

[0007] A third objective of this invention is to provide the use of the compound Remodelin in the preparation of medicaments for treating or preventing symptoms caused by Nipah virus.

[0008] A fourth object of the present invention is to provide the use of a pharmaceutically acceptable salt of the compound Remodelin in the preparation of a medicament for treating or preventing symptoms caused by Nipah virus.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention claims protection for the use of the compound Remodelin in the preparation of a medicament for the treatment or prevention of Nipah virus, the compound Remodelin having the following structural formula (Ⅰ): .

[0010] Secondly, the present invention claims the use of a pharmaceutically acceptable salt of the compound Remodelin in the preparation of a medicament for the treatment or prevention of Nipah virus, the compound Remodelin having the following structural formula (Ⅰ): .

[0011] Preferably, the drug inhibits Nipah virus replication.

[0012] Preferably, the drug inhibits budding of Nipah virus.

[0013] Preferably, the drug further contains pharmaceutically acceptable excipients, based on which it can be prepared into different dosage forms.

[0014] Thirdly, the present invention claims protection for the use of the compound Remodelin in the preparation of a medicament for treating or preventing symptoms caused by Nipah virus, the compound Remodelin having the following structural formula (Ⅰ): .

[0015] Fourthly, this invention claims the use of a pharmaceutically acceptable salt of the compound Remodelin in the preparation of a medicament for treating or preventing symptoms caused by Nipah virus, the compound Remodelin having the following structural formula (I): .

[0016] Preferably, the drug inhibits Nipah virus replication.

[0017] Preferably, the drug inhibits budding of Nipah virus.

[0018] Preferably, the drug further contains pharmaceutically acceptable excipients, based on which it can be prepared into different dosage forms.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention unexpectedly discovered that Remodelin can inhibit the budding of NiV particles, preventing further viral infection in host cells; and Remodelin can inhibit NiV replication, and can be used as an inhibitor of NiV for the prevention or treatment of NVD. Attached Figure Description

[0020] Figure 1 Graph showing the cytotoxicity results of the compound Remodelin; Figure 2 The effect of different concentrations of remodelin on Nipah virus replication; Figure 3 The effect of different concentrations of Remodelin on Nipah virus budding; Figure 4 The effect of remodelin on Nipah virus budding was investigated using transmission electron microscopy. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0022] The trVLP-NiV mini genome vector (pNiV-MG-RLuc) and three helper plasmids (pCAGGS-NiV-N, pCAGGS-NiV-P and pCAGGS-NiV-L) are described in patent CN117106735A, "A recombinant viral vector of Nipah virus, pseudovirus and its preparation method and application".

[0023] Example 1: Detection of the cytotoxicity of compound Remodelin I. Experimental Methods Cytotoxicity of the small molecule compound Remodelin was assessed using the CCK-8 assay. HEK293T cell suspension (100 μL / well) was seeded into 96-well plates, with approximately 2 × 10⁶ cells per well for cell proliferation assays. 3 Different concentrations of the compound Remodelin (0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM) were added to the wells. Blank wells (containing only culture medium) and control wells (containing both cells and culture medium) were also included. After incubating the plate for 16 h, 10 μL of CCK-8 solution (manufacturer: Biosharp, catalog number: BS350B) was added to each well, and the plate was incubated for 1–4 h. The plates were gently mixed on a shaker, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0024] II. Experimental Results The results are as follows Figure 1 As shown, the compound Remodelin, at a concentration of less than or equal to 20 μM, exhibits no significant toxicity to cells and demonstrates good safety.

[0025] Example 2: Effects of different concentrations of Remodelin on Nipah virus replication I. Experimental Methods 1. Preparation of a replicable pseudovirus system (NiV virus-like particles) HEK293T cells were used at a rate of 5 × 10⁻⁶ 5 HEK293T cells were seeded at a density of 1 cells / mL in 12-well plates and infected with MAV-T7 vaccinia virus at a dose of 2 MOI. The culture medium was DMEM containing 10% fetal bovine serum and cultured at 37°C with 5% CO2. One h after infection, the trVLP-NiV mini-genome vector (pNiV-MG-Rluc) and three helper vectors (pCAGGS-NiV-N, pCAGGS-NiV-P, and pCAGGS-NiV-L) were co-transfected into the vaccinia virus (MAV-T7 strain) infected HEK293T cells using TransIT®-LT1 (manufacturer: Mirus; catalog number: MIR2300) transfection reagent. Forty-eight h after transfection, the cell culture supernatant was aspirated, centrifuged at 2000×g for 5 min to remove cell debris, and the supernatant was collected, aliquoted, and stored at -80°C to obtain P0 generation trVLP-NiV.

[0026] 2. Remodelin treatment of cells infected with pseudoviruses HEK293T cells were seeded into 12-well plates using DMEM culture medium (containing 10% FBS + 1% antibiotics) and cultured overnight. When the cell density reached 80%, the experimental group was transfected with helper plasmids. After 24 h, each well was inoculated with 300 μL of packaged trVLP-NiV (P0) and then treated with the compound Remodelin (5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM). The control group was transfected with helper plasmids and then treated with DMSO (1%). The medium was changed after 24 h, and the samples were collected after 48 h.

[0027] 3. Luciferase activity detection Cells in a 6-well plate were gently washed twice with pre-chilled PBS, and residual liquid was aspirated. 200 μL of pre-prepared 1× Glo Lysis Buffer was added to each well, and the cells were incubated at room temperature (25°C) for 15 minutes to ensure complete lysis. The expression level of Renaissance luciferase, i.e., the viral replication level, was detected using a Renaissance luciferase assay kit.

[0028] II. Experimental Methods The results are as follows Figure 2 As shown, the compound Remodelin inhibits the replication of Nipah pseudovirus in a dose-dependent manner.

[0029] Example 3: Effect of different concentrations of Remodelin on Nipah virus particle budding I. Experimental Methods Matrix protein (M protein) is a core protein in the NiV life cycle that mediates viral assembly and budding. Studies have shown that ectopic expression of the M protein alone can independently induce the formation and release of virus-like particles (VLPs) without relying on other viral proteins. Exogenous expression of the M protein was used instead of pseudovirus infection to represent the amount of viral budding.

[0030] The pCAGGS-Strep-C vector was digested with Xoh I restriction endonuclease, and the NiV-M gene (Gene ID: 920953) was ligated into the digested vector and named NiV-strep-M.

[0031] HEK293T cells were seeded in 12-well plates using DMEM culture medium (containing 10% FBS and 1% antibiotics) and cultured overnight. When the cell density reached 80%, the experimental group was transfected with the NiV-strep-M plasmid and then treated with the compounds Remodelin (5 μM), Remodelin (10 μM), and Remodelin (20 μM); the control group was transfected with the empty vector and then treated with DMSO (1%).

[0032] After 24 hours, the supernatant was collected and ultracentrifuged (200,000 g for 2 hours). The liquid was discarded, and the cells were resuspended in 40 μL of PBS. The suspension was then transferred to EP tubes, the cells were washed with PBS, centrifuged, and the supernatant was discarded. Total protein was extracted, and M protein expression, i.e., viral budding, was detected by Western blotting.

[0033] The specific method for detecting M protein expression using Western blotting is as follows: Add total protein to NP-40 lysis buffer, lyse on ice for 15 min, centrifuge at 12000 g for 10 min at 4 °C, and collect the supernatant. Mix the supernatant with Loading Buffer at a ratio of 4:1, and heat in a metal bath at 95 °C for 10 min to completely denature the protein; Perform SDS-PAGE electrophoresis, adding 20 μL of protein sample to each well, with the protein marker used as a reference. The initial voltage was 80V, and after bromophenol blue entered the separating gel, it was adjusted to 120V. Electrophoresis continued until the bromophenol blue migrated to the bottom of the gel. For membrane transfer, the NC membrane and filter paper are immersed in transfer solution (containing methanol) to assemble a "sandwich" structure (filter paper-gel-PVDF membrane-filter paper), removing air bubbles. Transfer is performed at a constant pressure of 150 V for 1 h at 4 ℃. Block with 5% skim milk powder (prepared by TBST) at room temperature for 1 h to reduce nonspecific binding. Dilute the M-specific antibody (manufacturer: AntibodySystem; catalog number: YVV18501) and incubate overnight at 4 °C. Wash the membrane for 10 minutes each time, for a total of 3 times. Dilute the HRP-labeled secondary antibody (manufacturer: ABclonal; catalog number AS195, AS079) with TBST buffer and incubate at room temperature for 1 h. Wash the membrane three times for 10 minutes each time, using TBST buffer. Develop using chemiluminescence, cover with ECL reagent, incubate at room temperature for 1 minute, expose using a gel imaging system, and adjust the time to obtain clear bands.

[0034] II. Experimental Methods M protein expression in supernatant as follows Figure 3 As shown, compared with the control group, treatment with the compound Remodelin reduced the budding rate of NiV-M; the reduction in NiV-M budding rate became more pronounced with increasing Remodelin concentration. This indicates that the use of the compound Remodelin can significantly inhibit NiV-M budding, thereby inhibiting Nipah virus replication.

[0035] Example 4: Detection of the effect of Remodelin on Nipah virus budding using transmission electron microscopy I. Experimental Methods HEK293T cells were seeded in 10 cm dishes using DMEM culture medium (containing 10% FBS and 1% antibiotics) and cultured overnight. When the cell density reached 80%, the cells were transfected with the NiV-strep-M plasmid.

[0036] Subsequently, the experimental group was treated with the compound Remodelin (10 μM); the control group was treated with DMSO (1%).

[0037] After 24 h of treatment, discard the supernatant, collect the cells, and fix them overnight in 2.5% glutaraldehyde solution at 4°C. Then, process the samples as follows: Discard the fixative, wash the samples three times with PBS for 15 min each time; fix the samples with 1% osmium tetroxide solution for 1–2 h; discard the fixative, wash the samples again three times with PBS buffer for 15 min each time; dehydrate the samples with a gradient of ethanol solutions (50%, 70%, 80%, 90%, and 95%), treating the samples with each concentration of ethanol solution in ascending order of concentration for 15 min, and finally treating them once with 100% ethanol for 20 min; then treat them with acetone for 20 min; treat the samples with a mixture of embedding agent and acetone (V / V = 1 / 1) for 1 h; then treat the samples with a mixture of embedding agent and acetone (V / V = 3 / 1) for 3 h. h; Finally, treat the sample with pure embedding agent overnight; embed the infiltrated sample and heat at 70℃ overnight to obtain the embedded sample. The sample is sectioned in a Reichert ultramicrotome to obtain sections of 70-90 nm. These sections are stained with lead citrate solution and 50% ethanol saturated solution of uranium acetate for 15 min each, and can then be observed in a transmission electron microscope.

[0038] II. Experimental Methods The results are as follows Figure 4 As shown, the budding rate of VLP cells was significantly reduced after treatment with the compound Remodelin, indicating that the compound Remodelin inhibited the budding level of Nipah virus.

Claims

1. The use of compound Remodelin in the preparation of drugs for the treatment or prevention of Nipah virus, wherein the structural formula of compound Remodelin is shown in formula (Ⅰ) below: 。 2. The use of a pharmaceutically acceptable salt of the compound Remodelin in the preparation of medicaments for the treatment or prevention of Nipah virus, wherein the structural formula of the compound Remodelin is shown in formula (Ⅰ): 。 3. The application according to claim 1 or 2, characterized in that, The drug inhibits Nipah virus replication.

4. The application according to claim 1 or 2, characterized in that, The drug inhibits the budding of Nipah virus.

5. The application according to claim 1 or 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

6. The use of compound Remodelin in the preparation of medicaments for treating or preventing symptoms caused by Nipah virus, wherein the structural formula of compound Remodelin is shown in formula (Ⅰ): 。 7. The use of a pharmaceutically acceptable salt of the compound Remodelin in the preparation of a medicament for the treatment or prevention of symptoms caused by Nipah virus, wherein the structural formula of the compound Remodelin is shown in formula (Ⅰ): 。 8. The application according to claim 6 or 7, characterized in that, The drug inhibits Nipah virus replication.

9. The application according to claim 6 or 7, characterized in that, The drug inhibits the budding of Nipah virus.

10. The application according to claim 6 or 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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