Use of celastrol in preparation of anti-hanta virus drugs

Abstract

The application relates to the field of biological medicine, and particularly discloses application of celadixyn in preparation of an anti-Hantaan virus drug, wherein the celadixyn realizes anti-Hantaan virus by inhibiting replication and proliferation of the Hantaan virus. Experiments prove that the celadixyn directly targets HTNV NP to play an anti-virus effect. The chemical structural formula of the celadixyn is as follows:

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of triptolide in the preparation of anti-Hantan virus drugs. Background Technology

[0002] Hantaan virus (HTNV) is a negative-sense RNA virus belonging to the Hantaviridae family. Hantaviridae ), genus Orthohantavirus ( Orthohantavirus The HTNV genome consists of three segments: large (L), medium (M), and small (S), which encode RNA-dependent RNA polymerase (RdRp), a glycoprotein precursor (GPC), and, after protease cleavage, produce Gn, Gc, and nucleocapsid protein (NP). Hantaan virus (HTNV) is a major pathogen causing hemorrhagic fever with renal syndrome (HFRS). A novel approach to combat HTNV is urgently needed. Summary of the Invention

[0003] To develop an anti-HTNV pathway, this invention provides the application of triptolide in the preparation of anti-HTNV drugs. The triptolide exerts its anti-HTNV effect by inhibiting the replication and proliferation of HTNV. Experiments have demonstrated that triptolide exerts its antiviral effect by directly targeting HTNV NPs.

[0004] This invention provides the application of triptolide in the preparation of anti-Hantan virus drugs, and the chemical structural formula of triptolide is as follows: .

[0005] The triptolide described herein exerts its antiviral effect by inhibiting the replication and proliferation of Hantan virus. Experiments have demonstrated that triptolide exerts its antiviral effect by directly targeting HTNV NP.

[0006] Furthermore, the triptolide is used to inhibit the replication of Hantan virus.

[0007] Furthermore, the triptolide is used to inhibit the proliferation of Hantan virus.

[0008] Furthermore, the triptolide directly interacts with the nucleocapsid protein of the Hantan virus.

[0009] Furthermore, the drug is a pharmaceutically permissible dosage form made with triptolide as the active ingredient and any pharmaceutical excipient or pharmaceutical excipient.

[0010] Furthermore, the drug dosage form is a tablet, powder, granule, capsule, or solution.

[0011] Furthermore, the solvent of the solution is dimethyl sulfoxide.

[0012] Furthermore, the concentration of triptolide in the drug is 0.15625 μM to 2.5 μM.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the application of triptolide in the preparation of anti-Hantavirus drugs, wherein triptolide exerts its antiviral effect by inhibiting the replication and proliferation of Hantanvirus. Experiments have demonstrated that triptolide exerts its antiviral effect by directly targeting HTNV NPs. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 To investigate the inhibitory effect and cytotoxicity of triptolide on HTNV infection; In the diagram, A represents the chemical structure of triptolide; B represents the CC50 detection of triptolide in A549; C represents the inhibition rate of different concentrations of triptolide on HTNV (MOI=0.1)-infected A549 cells.

[0016] Figure 2 To investigate the inhibitory effect of triptolide on HTNV-infected A549 cells at different time stages; In the figure, A is a schematic diagram of the administration time of triptolide; B represents the detection of HTNV NP expression by Western blotting; C represents the viral titer detected by FFA. D represents A549 cells infected with HTNV (MOI=0.1) and treated with triptolide for 24 h at different time points. Immunostaining was performed on HTNV NPs (green) and cell nuclei (blue). Images were captured using an IX71 fluorescence microscope system; scale bar = 100 μm.

[0017] Figure 3 Tripterygium wilfordii exerts its anti-HTNV activity in a dose-dependent manner; In the figure, A represents the expression of HTNV NP in A549 cells detected by Western blotting; B represents the expression of viral titer in the supernatant of A549 cells as detected by FFA. C represents the expression of HTNV-S in A549 cells detected by qRT-PCR; D shows the immunostaining pattern of HTNV-infected A549 cells treated with different concentrations of triptolide.

[0018] Figure 4 This verifies the direct interaction between triptolide and HTNV NP; In the figure, A represents the molecular docking results of the triptolide-HTNV NP complex; B represents the molecular docking result of the triptolide-HTNV RdRp complex; C represents the results of surface plasmon resonance (SPR) ligand binding experiments; D represents the results of micro-thermophoresis (MST) detection; E represents the result of the pull-down experiment. Detailed Implementation

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0020] Example 1: Application of triptolide in the preparation of anti-Hantan virus drugs.

[0021] I. Materials and Methods 1. Experimental materials Cells: The human lung adenocarcinoma cell line (A549) and the African green monkey kidney cell line (Vero-E6) used in this invention were both purchased from the China Center for Type Culture Collection (CCTCC, located in Wuhan, Hubei Province).

[0022] Virus: The HTNV virus strain used is the internationally renowned 76-118 strain.

[0023] Celastrol (Cel) was purchased from Maclean's Ltd. The celastrol powder was stored in a dry, light-protected environment, dissolved in dimethyl sulfoxide (DMSO) to prepare a 2.5 mM stock solution, and stored at −80°C for later use.

[0024] 2. Virus preparation and infection Vero-E6 cells were cultured in 10% DMEM medium. When the cells reached approximately 70% confluence, 300 μL of HTNV and 3 mL of serum-free DMEM medium suspension were added. Two hours after infection, 7 mL of DMEM medium containing 2% fetal bovine serum was added. The cells were then cultured in a humidified incubator at 37°C with 5% CO2. After 6 days, the cells and their supernatant were collected and subjected to three freeze-thaw cycles. After the third thaw, the supernatant was transferred to a 50 mL centrifuge tube and centrifuged at 8000×g for 30 min. The supernatant was the amplified HTNV virus stock solution and was frozen at -80°C for later use.

[0025] For use in subsequent infection experiments.

[0026] In the viral infection experiment, cells were seeded in culture plates. When the cell density reached approximately 70%, the cells were washed twice with DPBS, and then HTNV was added with a multiplicity of infection (MOI) of 0.1. After incubation at 37°C for 2 hours, the HTNV was replaced with DMEM containing 2% FBS. After culturing for another 24 hours, subsequent treatments were performed according to different experimental objectives.

[0027] 3. CCK-8 assay for cell viability To determine the safe concentration of triptolide in A549 cells, A549 cells were seeded in 96-well cell culture plates, and three groups were set up according to the experimental design: blank control group, negative control group, and drug treatment group. The blank control group was not seeded with cells, but only 100 µL of DMEM medium containing 10% FBS (containing 10% fetal bovine serum and 1% penicillin-streptomycin-gentamicin solution) was added; the negative control group was seeded with cells and 100 µL of DMEM medium containing 10% FBS was added; after seeding, 100 µL of triptolide solution diluted to different concentrations (2, 4, 6, 8, 10, 12, 14 µM) with 10% DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin-gentamicin solution) was added to the drug treatment groups. Each group was configured with 5 replicates. After incubation at 37 ℃ in a 5% CO2 incubator for 48 h, the cells were then incubated in 10% DMEM medium containing 10% CCK-8 reagent (Targetmol, C0005) in the dark. After incubation at room temperature for 1 h, the absorbance (OD value) at 450 nm was measured using a BioTek Epoch microplate reader to obtain cell viability data after treatment with different concentrations of triptolide. Cell viability (Cv) was calculated using the formula: Cv (%) = [(Ac - Ab) / (As - Ab)] × 100. Where Ac is the OD value of the negative control group, Ab is the OD value of the blank control group, and As is the OD value of the drug-treated group.

[0028] 4. Half-maximal inhibitory concentration of triptolide against HTNV A549 cells were seeded in 12-well cell culture plates and infected with HTNV according to the aforementioned viral infection protocol. Simultaneously, different concentrations of triptolide (0.15625, 0.3125, 0.625, 1.25, and 2.5 μM) were added to the cells. After 2 h of infection, the medium was replaced with the corresponding concentration of the drug. After 24 h of culture, cells were collected and total RNA was extracted. The nucleic acid level of the HTNV S fragment in the cells was detected using quantitative real-time PCR (qRT-PCR). Using the HTNV S fragment expression level in the untreated group (0 μM) as 100%, data analysis was performed using GraphPad Prism 10.1.2 software to calculate the half-maximal inhibitory concentration (IC50) of triptolide on HTNV-S gene expression.

[0029] 5. Time-added experiments 2.5 μM triptolide was administered to six groups according to different routes of administration: Group 1 was the full-course treatment group, with the drug administered throughout the entire course of viral infection (-1 to 24 h); Group 2 was the pretreatment group, with the drug administered only before viral infection (-1 to 0 h); Group 3 was the co-treatment group, with the drug administered only during viral infection (0 to 2 h); and Groups 4, 5, and 6 were the delayed administration groups, with the drug administered during the post-infection period (2 to 24 hours, 6 to 24 hours, and 12 to 24 hours, respectively). Positive control cells were replaced with DMEM containing 2% FBS 2 h after HTNV infection and cultured for 24 h before samples were collected for subsequent experimental detection and analysis.

[0030] 6. Concentration-dependent experiments Tripterygium wilfordii was divided into low, medium, and high dose groups: 0.625, 1.25, and 2.5 μM, respectively. A549 cells infected with HTNV were treated with low, medium, and high doses of triptolide, and cultured at 37 ℃ in a CO2 incubator for 24 h.

[0031] 7. qRT-PCR A549 cells were infected with HTNV (MOI=0.1) and treated with different concentrations of triptolide. Viral RNA levels were relatively quantified by qRT-PCR at 24 h (using 18S as an internal control, calculating 2^(–ΔΔCt)) to assess viral load changes. Total cellular RNA was extracted using the Vazyme Super FastPure Cell RNA Isolation Kit, and 1000 ng of RNA was used for reverse transcription. RNase-free ddH2O and 10 µL of 4×All-in-One Ultra qRTSuperMix enzyme were required for reverse transcription. A reverse transcription program of 50 °C for 10 min and 85 °C for 5 sec was used to obtain cDNA. cDNA was analyzed using 2×qPCR SmArt Mix (SYBR Green) for qRT-PCR to detect the HTNV S gene and the internal reference gene 18S. The upstream and downstream primer sequences for the HTNV S gene were 5'-GAGCCTGGAGACCATCTG-3' and 5'-CGGGACGACAAAGGATGT-3', respectively. The upstream and downstream primer sequences for the internal reference gene 18S were 5'-GTAACCCGTTGAACCCCATT-3' and 5'-CCATCCAATCGGTAGTAGCG-3', respectively.

[0032] 8. Western blotting Cell samples were collected at designated time points and lysed using RIPA lysis buffer (Beyotime, P0013C) containing a protease inhibitor (Targetmol, C0001). Protein concentration was determined using the BCA method (Thermo Fisher Scientific, Waltham, MA, USA). 20 μg of total protein was mixed with 5×SDS-PAGE loading buffer (Beyotime, P0286) and boiled for 5 min. After separation by 10% SDS-PAGE gel electrophoresis (NCM Biotech, P2011), the samples were wet-transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 2.5% skim milk and then incubated with primary antibodies: HTNVmonoclonal antibody 1A8 (prepared by the Department of Microbiology and Pathogenic Biology, Air Force Medical University) and mouse anti-β-actin (Immunoway, PTR2364). Then, it was incubated with infrared dye-conjugated secondary antibody (Li-CorBiosciences, Lincoln, NE, United States). Finally, it was scanned using the Odyssey infrared imaging system (Li-Cor Biosciences).

[0033] 9. Immunofluorescence Cells were fixed with 4% paraformaldehyde at room temperature, then permeabilized with 0.5% Triton X-100 at room temperature. They were subsequently blocked with 3% BSA at room temperature, and incubated overnight at 4 °C with HTNV monoclonal antibody 1A8. The next day, Alexa Fluor 488-labeled goat anti-mouse IgG (Sangon Biotech, D110090) was added and incubated at room temperature in the dark. Nuclei were stained with Hoechst 33258 (MedChemExpress, HY-15558) at room temperature. Finally, images were acquired under a consistent exposure condition using an IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan).

[0034] 10. Enzyme-linked plaque formation assay 100 µL of undiluted and 10-fold diluted cell culture supernatant from each experimental group were seeded in parallel into 96-well plates containing Vero E6 cells, with four replicates per group. Cells were infected at 37 °C for 2 h, then the medium was replaced with DMEM semi-solid maintenance medium containing 1.6% sodium carboxymethyl cellulose and 2% fetal bovine serum. After 6 days of culture, the following treatments were performed: the capping layer was removed, cells were fixed with 4% paraformaldehyde at room temperature, permeabilized with 0.5% Triton X-100, and then non-specific sites were blocked with 0.3% BSA. The culture plates were incubated overnight with 1A8 primary antibody at 4 °C, followed by reaction with secondary antibody HRP-conjugated Goat anti-Mouse IgG (H+L) at room temperature (ABclonal, AS003). Finally, TMB substrate (Biokits, WB003) was added for color development in the dark. The reaction was terminated after viral plaques formed, and the plaque count was performed using scanning imaging. Virus titer (FFU / mL) = number of spots in 96-well plate × virus dilution factor / virus volume inoculated into 96-well plate.

[0035] 11. Molecular docking The NP (PDB ID: 5FSJ) and RdRp (PDB ID: 8C4S) structures related to HTNV were retrieved from the Protein Data Bank (PDB) database, and their corresponding PDB format files were downloaded. After retrieval, PyMOL was used to remove water of crystallization molecules and heterologous ligands, and AutoDock Tools was used to add polar hydrogen atoms to the protein, finally outputting a PDBQT format file. The triptolide (Celastrol, PubChem CID: 122724) 3D SDF file was retrieved from the PubChem chemical database, downloaded, converted to PDB format using Open Babel, and then imported into AutoDock Tools for rotatable bond settings, generating the corresponding PDBQT file. Autodock Vina software was used to perform semi-flexible molecular docking calculations with a rigid protein backbone and rotatable ligands and side chains near the protein active site. The model with the lowest binding energy and highest conformational clustering was selected as the optimal binding posture. The calculated docking conformations were analyzed in depth using PyMOL and LigPlot+ molecular visualization software, and a schematic diagram of the docking mode was generated accordingly.

[0036] 12. Surface plasmon resonance (SPR) technology This invention employs SPR analysis to examine the interaction between triptolide and HTNV NP. HTNV NP (MedChemExpress, HY-P76965) was covalently immobilized on the surface of an esterified CM5 chip (Biacore) via amino coupling to form a stable ligand layer. Triptolide was first dissolved in DMSO to prepare a 100 mM stock solution, followed by serial dilutions with run buffer to obtain a series of sample solutions with concentrations of 100, 50, 25, 12.5, and 6.25 μM. All SPR experiments were performed in a PBS-P buffer system containing 1% DMSO (PBS containing 0.05% Tween-20) to eliminate solvent refractive index interference and reduce nonspecific adsorption. Each concentration of triptolide sample was injected at a constant flow rate for 120 seconds, followed by 300 seconds of dissociation monitoring in run buffer.

[0037] 13. Microscale thermophoresis (MST) The interaction between triptolide and HTNV NP was analyzed using MST. HTNV NP (MedChemExpress, HY-P76965) was labeled with a fluorescent dye (His-Tag Labeling Kit RED-tris-NTA 2nd Generation), and labeled protein samples were obtained after incubation. Triptolide stock solution was serially diluted with buffer to prepare a series of concentration solutions. The labeled protein was mixed with each concentration of triptolide solution and incubated. Fluorescence changes under thermal gradients were detected using an MST instrument (Monolith NT.115). The dissociation constant (KD) of the interaction between triptolide and HTNV NP was calculated by analyzing the changes in molecular migration behavior at different concentrations.

[0038] 14. Protein binding in vitro experiment The triptolide powder was sent to Bio-Tech for biotinylation modification, and the resulting biotin-Celastrol was stored at -20 °C protected from light. A549 cells were seeded in T75 cells, and infected with HTNV when the cell density reached 70%. Cell lysates were collected after 24 h, and the supernatant was collected by centrifugation at 4 °C. The supernatant was divided into two groups: experimental group: Biotin-Celastrol was added to a final concentration of 2.5 μM; control group: an equimolar amount of free biotin was added. After mixing, the mixture was incubated with streptavidin magnetic beads at 4 °C overnight. The beads and liquid were then separated using a magnetic rack. After washing with DPBS, the beads were collected, dissolved in 1× loading buffer, and boiled in a boiling water bath for Western blotting to detect HTNV NP protein expression.

[0039] 15. Statistical Analysis All experimental data were statistically analyzed and graphically visualized using GraphPad Prism 10.1.2 software. Each experiment was independently repeated ≥3 times, and results are expressed as mean ± SD. The dose-response curves were nonlinearly fitted using a four-parameter logistic regression (4PL) model to calculate the IC50. 50 Values. Differences between groups were assessed using one-way ANOVA. NS , P >0.05, significance level set as: * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001.

[0040] II. Experimental Results 1. Effects of Celastrol on A549 cell viability and anti-HTNV activity The chemical structure and detailed information of triptolide are as follows: Figure 1 As shown in Figure A. Cell viability of the A549 cell line was assessed using CCK-8 assay to evaluate the cytotoxic effect of triptolide. The results showed no significant difference in cell survival between A549 cells treated with 2.5 µM triptolide and those without, and the CC50 for A549 cells was 11.97 µM. Therefore, 2.5 µM was subsequently selected as the maximum safe concentration of triptolide. Figure 1 (B). To systematically evaluate the inhibitory efficacy of triptolide against HTNV over a wide concentration gradient, sequential dilutions were set up in the experiment. The results showed that the inhibition of HTNV by triptolide exhibited a typical dose-dependent relationship, and the half-maximal inhibitory concentration (IC50) was determined by fitting a four-parameter logistic model. 50 The value is 0.1095 µM. Figure 1 Based on the above results, triptolide can effectively inhibit HTNV replication at low concentrations, and its therapeutic index (CC50 / IC50) is as high as 47.98, indicating that it has high antiviral activity. This characteristic makes it a potential candidate drug for anti-HTNV, and its mechanism of action deserves further investigation.

[0041] 2. Inhibitory effect of triptolide on HTNV at different stages of infection To further clarify the specific lifecycle of triptolide against HTNV, a time-stacking experiment was conducted on A549 cells. The inhibitory effect of triptolide on HTNV was evaluated by detecting HTNV protein expression levels and viral titers. Six experimental groups were set up, with HTNV infection time and treatment time with 2.5 µM triptolide as follows: Figure 2 As shown in Figure A. A549 cells were infected with HTNV at MOI=0.1 and treated with triptolide at different time points. Cells were collected and total protein was extracted 24 h later. HTNV NP levels were detected by Western blotting to evaluate the inhibitory activity of triptolide at different infection stages. After A549 cells were infected with HTNV at MOI=0.1, triptolide was added at specified time points and cultured for another 24 h. The supernatant was then collected, and viral titers were determined by FFU to assess the inhibitory effect of triptolide on HTNV at different treatment time points. Data are expressed as mean ± standard deviation (mean ± SD, n = 4). Statistical differences were expressed using one-way ANOVA. ns indicated no significant difference. P <0.001, **** P <0.0001.

[0042] The results showed that, compared with the control group, groups 1, 4, 5, and 6 had significantly lower HTNV protein expression and viral titer levels. Figure 2 (B, C, and D) indicates that treatment with triptolide throughout the infection process, at 2 h, 6 h, and 12 h, significantly inhibited HTNV; compared to the control group, the HTNV protein expression and viral titer levels in the three groups were also reduced to varying degrees. Figure 2 (B, C, D). This indicates that triptolide can exert its main antiviral effect to varying degrees in different life cycles of HTNV, namely the replication stage after cell entry, within 24 hours. The inhibitory effect on HTNV is most significant under treatment with triptolide throughout the entire life cycle, at 2 hours, 6 hours, and 12 hours.

[0043] 3. Inhibitory effects of different doses of triptolide on HTNV A549 cells were infected with HTNV at an MOI of 0.1. Two hours later, different concentrations of triptolide were added for treatment, and cells were collected after 24 hours. Total protein was extracted and Western blotting analysis was performed to assess HTNV NP expression levels. Triptolide was administered at concentrations of 0.625, 1.25, and 2.5 µM throughout the infection process. The control group consisted of A549 cells infected with HTNV without triptolide treatment (0 µM). Compared with the control group, HTNV NP protein expression gradually decreased after treatment with 1.25 and 2.5 µM triptolide.Figure 3 A).

[0044] A549 cells were infected with HTNV at an MOI of 0.1, and the supernatant was collected after treatment with different concentrations of triptolide for 24 h. Viral titers were determined using FFU to assess the inhibitory effect of triptolide on HTNV replication. Results are expressed as mean ± SD (n = 4) and were analyzed using one-way ANOVA. P <0.0001. A549 cells were infected with HTNV at an MOI of 0.1 and treated with different concentrations of triptolide for 24 h. Cells were then collected and the relative viral RNA was quantified by qRT-PCR. Results are expressed as mean ± SD (n = 3). One-way ANOVA was used, where **** P <0.0001. HTNV-infected (MOI=0.1) A549 cells were treated with different concentrations of triptolide for 24 h, and HTNV NPs (green) and cell nuclei (blue) were immunostained. Images were captured using an IX71 fluorescence microscope system; scale bar = 100 μm.

[0045] Compared with the control group, treatment with 0.625, 1.25, and 2.5 µM triptolide significantly reduced viral titer, protein expression, and HTNVS fragment expression. Figure 3 (B, C, D). This indicates that different concentrations of triptolide inhibit HTNV, and the higher the concentration, the more significant the inhibitory effect on HTNV, that is, the inhibitory effect of triptolide on HTNV is concentration-dependent.

[0046] 4. Verification of the direct interaction between triptolide and HTNV NP using multiple technologies. Molecular docking data showed that the binding energies of triptolide with HTNV NP and HTNV RdRp were −10.6 and −9.7 kcal / mol, respectively. Triptolide forms one hydrogen bond with HTNV NP at LYS:357. Figure 4 (A). Tripterygium wilfordii forms two hydrogen bonds with HTNVRdRp at THR: 721 and ASP: 1098. Figure 4(B). Tripterygium wilfordii binds to HTNV NP with the lowest binding energy, indicating the highest stability of their complex. HTNV RdRp, primarily responsible for viral gene replication and transcription, is approximately 250 kDa in size. Due to its large molecular weight, it is difficult to express. HTNV NP, as a key protein in viral particle assembly, not only exhibits high stability but also plays a crucial role in viral replication, making it an important target for antiviral drug screening. All conformations were plotted using PyMOL and analyzed in two dimensions using LigPlot+. The left and upper right views of each panel represent the overall three-dimensional structure, with the corresponding HTNV protein presented as a translucent blue surface, tripterygium wilfordii as a green rod, and hydrogen bonds as yellow dashed lines, along with labeling the participating residues. The lower right view shows a two-dimensional interaction diagram, summarizing the interactions between hydrogen bonds (green dashed lines) and hydrophobic contacts (red eyelashes).

[0047] HTNV NPs were immobilized on the surface of a CM5 sensor chip, and kinetic measurements were performed using gradient concentrations of triptolide as the mobile phase. The sensor plots showed typical concentration-dependent binding curves, and the dissociation constant was obtained by fitting a 1:1 binding model. Ligand binding experiments using SPR demonstrated that triptolide exhibits a dose-dependent specific interaction with HTNV NPs, and its dissociation constant (K0) is... D ) is 17μM ( Figure 4 (C).

[0048] Using fluorescently labeled HTNV NP as the target protein and gradient-increased triptolide as the ligand, the assay was performed in a standard buffer. Fluorescence-time loci showed concentration-dependent thermophoretic kinetic signal changes, and the KD value was derived from the fitted sigmoid binding curve. MST detection further confirmed that the migration rate of HTNV NP decreased gradient-decreasing with increasing triptolide concentration, and the sigmoid fitting curve showed an affinity of 3.817 μM, suggesting a specific binding between the two. Figure 4 (D).

[0049] Pull-down experiment. The left figure is a schematic diagram of biotin-labeled triptolide. The right figure shows the incubation of biotin-labeled triptolide (Biotin-Celastrol) or free biotin (control) with streptavidin magnetic beads and HTNV NP for 36 h, respectively. After washing, the complexes were detected by Western blotting. The in vitro pull-down experiment showed that biotin-labeled triptolide (Biotin-Celastrol) significantly enriched HTNV NP, while biotin alone did not have this effect, directly verifying the protein-ligand interaction between triptolide and HTNV NP. Figure 4The above results indicate that triptolide can directly target HTNV NP and form a stable complex. Based on the combined molecular docking and binding kinetics data, the high affinity binding of triptolide to HTNV NP may interfere with NP-mediated viral ribonucleoprotein complex assembly, thereby blocking a key step in viral replication.

[0050] This invention systematically evaluated the inhibitory activity of the pentacyclic triterpenoid compound triptolide against HTNV infection. The results showed that triptolide significantly inhibited HTNV replication in A549 cells, with an IC50 value of [missing information]. 50 0.1095 µM ( Figure 1 To clarify the antiviral action phase, this invention applied drug intervention at different time points after viral infection. The results showed that triptolide exerted an inhibitory effect at all time points after viral adsorption (2, 6, and 12 h), with the most significant effect observed when administered continuously from before viral adsorption to the entire replication process, suggesting that it mainly acts on the replication phase after viral entry into cells. Figure 2 Furthermore, the inhibitory activity of triptolide against HTNV is concentration-dependent; as the drug concentration increases, the viral replication level gradually decreases. Figure 3 ).

[0051] During HTNV infection, viral particles bind to cell surface receptors and enter the host cell via endocytosis. They then release ribonucleoprotein complexes (RNPs) into the cytoplasm, initiating viral genome transcription and replication. As a core component of the viral replication cycle, HTNV NPs can specifically bind to viral RNA to form a helical nucleocapsid, providing a template for RdRp replication. NPs can also promote the release of viral particles from host cells by regulating the translation of host valine-containing proteins, thereby facilitating viral spread between adjacent cells. The multiple functions of NPs in viral assembly and host immune regulation make them an important target for antiviral drug development. RdRp, as a core enzyme in viral genome replication and transcription, is another key target for anti-HTNV drug development, but its large size (approximately 250 kDa) makes it difficult to express. In contrast, NPs have a relatively simple structure and high stability, making them more suitable as targets for small molecule drug intervention.

[0052] To elucidate the molecular mechanism of triptolide's anti-HTNV activity, this invention first performed molecular docking on the NP and RdRp proteins, which play a role in the viral replication phase. The results showed that triptolide possesses binding potential to both HTNV NP and RdRp, with the lowest binding energy observed with NP, suggesting NP as a potential target. Figure 4Subsequently, SPR, MST, and pull-down experiments were used to verify the interaction. The SPR and MST results consistently showed that triptolide specifically bound to HTNV NPs in a dose-dependent manner, with dissociation constants of 17 μM and 3.817 μM, respectively. The pull-down experiment further showed that biotin-triptolide specifically enriched HTNV NPs, while no obvious signal was observed in the blank control. Figure 4 Therefore, this invention systematically elucidates the antiviral activity of triptolide against HTNV at the in vitro level and confirms that it can exert its antiviral effect by directly targeting HTNV NP.

[0053] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0054] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of triptolide in the preparation of anti-Hantan virus drugs, wherein the chemical structural formula of triptolide is as follows: 。 2. The application of triptolide according to claim 1 in the preparation of anti-Hantan virus drugs, characterized in that, The triptolide is used to inhibit the replication of Hantan virus.

3. The application of triptolide according to claim 1 in the preparation of anti-Hantan virus drugs, characterized in that, The triptolide is used to inhibit the proliferation of Hantan virus.

4. The application of triptolide according to claim 1 in the preparation of anti-Hantan virus drugs, characterized in that, The triptolide directly interacts with the nucleocapsid protein of the Hantan virus.

5. The application of triptolide according to claim 1 in the preparation of anti-Hantan virus drugs, characterized in that, The drug is a pharmaceutically permissible dosage form made with triptolide as the active ingredient and any pharmaceutical excipient or pharmaceutical excipient.

6. The application of triptolide according to claim 5 in the preparation of anti-Hantan virus drugs, characterized in that, The drug dosage form is tablet, powder, granule, capsule or solution.

7. The application of triptolide according to claim 6 in the preparation of anti-Hantan virus drugs, characterized in that, The solvent for the solution is dimethyl sulfoxide.

8. The application of triptolide according to claim 7 in the preparation of anti-Hantan virus drugs, characterized in that, The concentration of triptolide in the drug is 0.15625 μM to 2.5 μM.

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