Drug for resisting coronavirus and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to an anti-coronavirus medicine and application. According to the application disclosed by the embodiment of the invention, experiments prove that the punicalagin and the punicalagin respectively have strong binding force to SARS-CoV-2 main protease Mpro and have a remarkable inhibition effect on SARS-CoV-2 virus replication, so that the punicalagin and the punicalagin can be used as novel SARS-CoV-2 inhibitors. Therefore, punicalagin and / or punicalagin can be used for preparing products for inhibiting novel coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an anti-coronavirus drug and its application. Background Art

[0002] Coronaviruses are a class of enveloped, positive-sense, single-stranded RNA viruses that can infect a wide range of mammals, including humans. SARS-CoV-2 infection causes infectious pneumonia, also known as coronavirus disease 2019 (COVID-19), which is highly pathogenic and contagious.

[0003] Current research has revealed that the SARS-CoV-2 main protease is involved in the hydrolysis of the replicase polyprotein encoded by the coronavirus genome into nonstructural proteins necessary for viral genome replication, thus playing a crucial role in the transcription and replication of the coronavirus. Furthermore, no homologous protein to the main protease exists in the human body, making it a promising anti-coronavirus target. Furthermore, enzymatic experiments have further confirmed that the substrate binding site of the coronavirus main protease is highly conserved. Therefore, inhibiting the hydrolytic activity of the SARS-CoV-2 main protease would effectively protect against SARS-CoV-2 infection in the human body. Therefore, the coronavirus main protease is an ideal target for the design of anti-novel coronavirus drugs. Therefore, there is an urgent need for a drug that inhibits the enzymatic activity of the coronavirus main protease. Summary of the Invention

[0004] The object of the present invention is to provide an anti-coronavirus drug, the active ingredients of which include punicalagin and / or punicalagin, both of which have strong binding activity to the SARS-CoV-2 main protease and have a significant inhibitory effect on SARS-CoV-2 virus replication.

[0005] The first object of the present invention is to provide an anti-coronavirus drug, wherein the active ingredient of the anti-coronavirus drug is punicalagin and / or pomegranate peel tannin.

[0006] Preferably, the structural formula of the punicalagin is as shown in Formula 1:

[0007]

[0008] Preferably, the structural formula of the pomegranate peel tannin is as shown in Formula 2:

[0009]

[0010] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0011] Preferably, the medicine is an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

[0012] The second object of the present invention is to provide an application of punicalagin and / or pomegranate peel tannin in the preparation of anti-coronavirus drugs.

[0013] Preferably, the coronavirus is SARS-CoV-2 or a variant thereof; the variant is Omicron BA.5.

[0014] Preferably, the CC of the punicalagin and / or punicalagin is 50 Greater than 100μM.

[0015] Preferably, the EC of punicalagin to SARS-CoV-2 50 The EC value of the pomegranate peel tannin against SARS-CoV-2 is 2.27 μM. 50 It is 12.37μM.

[0016] Preferably, the EC of punicalagin to Omicron BA.5 50 The EC value of the pomegranate peel tannin for Omicron BA.5 is 3.65 μM. 50 It is 13.30μM.

[0017] The third object of the present invention is to provide a method for preparing a method for inhibiting coronavirus main protease M pro Application of enzyme activity inhibitors.

[0018] Preferably, the punicalagin inhibits M pro Active IC 50 was 2.09±0.20μM; the pomegranate peel tannin inhibited M pro Active IC 50 It is 0.32±0.05μM.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention found that pomegranate peel tannin and punicalagin have the effect on SARS-CoV-2M through in vitro enzyme inhibition activity test and molecular interaction study. pro It has a strong binding affinity and can significantly inhibit its activity. And the present invention found through virus inhibition research that both pomegranate peel tannin and punicalagin have a significant inhibitory effect on the Omicron BA.5 virus strain, with an extremely low half-effective concentration. Therefore, pomegranate peel tannin and / or punicalagin can be made into a product that inhibits the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Schematic diagram of the catalytic pocket of the Mpro active site in Example 1, where green represents the active center.

[0023] Figure 2 Example 1M pro Schematic diagram of the active center, where the circular icon represents the active center.

[0024] Figure 3 is the IC of the positive control PF-07321332 in Example 2 50 Schematic diagram of the curve.

[0025] Figure 4 is the IC of pomegranate peel tannin in Example 2 50 Schematic diagram of the curve.

[0026] Figure 5 is the IC of punicalagin in Example 2 50 Schematic diagram of the curve.

[0027] in, Figure 3-5 The horizontal axis represents the sample concentration, and the vertical axis represents the inhibition rate.

[0028] Figure 6 PF-07321332 and M in Example 3 pro Interaction analysis diagram.

[0029] Figure 7 For example 3 punicalagin and M pro Interaction analysis diagram.

[0030] Figure 8 For example 3 pomegranate peel tannin and M pro Interaction analysis diagram.

[0031] in, Figure 6-8 The vertical axis represents the response units.

[0032] Figure 9 Schematic diagram of the 3D structure of pomegranate peel tannin in Example 4 and its interaction with M pro Schematic diagram of the active site of action.

[0033] Figure 10 Schematic diagram of the 3D structure of punicalagin in Example 4 and its interaction with Mpro Schematic diagram of the active site of action.

[0034] Figure 11 M in Example 4 pro -Schematic diagram of the root mean square deviation of small molecule complexes, where the left figure is a schematic diagram of the RMSD curve of the Mpro-pomegranate peel tannin complex; the right figure is a schematic diagram of the RMSD curve of the Mpro-punicalagin complex.

[0035] Figure 12 Schematic diagram of the root mean square fluctuation of the Mpro-small molecule complex in Example 4, where the left figure is a schematic diagram of the RMSF curve of the Mpro-punicalagin complex; the right figure is a schematic diagram of the RMSF curve of the Mpro-punicalagin complex. The horizontal axis represents the amino acid residues.

[0036] Figure 13 is M in Example 4 pro -Schematic diagram of the radius of gyration of small molecule complexes, where the left figure is M pro -Schematic diagram of the Rg curve of the pomegranate peel tannin complex; the right figure is M pro Schematic diagram of the Rg curve of the punicalagin complex.

[0037] Figure 14 The figure below shows the effect of small molecule compounds on Vero E6 cell viability in Example 5. The upper figure shows the effect of pomegranate peel tannin on Vero E6 cell viability, and the lower figure shows the effect of punicalagin on Vero E6 cell viability. The vertical axis represents cell viability.

[0038] Figure 15 These are analysis diagrams of the inhibitory effects of small molecule compounds on wild-type SARS-CoV-2 in Example 5, wherein the three diagrams are analysis diagrams of the inhibitory effects of S-217622, ​​punicalagin, and punicalagin on wild-type SARS-CoV-2.

[0039] Figure 16 This is an analysis of the inhibitory effect of small molecule compounds on SARS-CoV-2Omicron BA.5 in Example 5; among them, the three figures are analysis of the inhibitory effects of S-217622, ​​punicalagin, and pomegranate peel tannin on SARS-CoV-2Omicron BA.5. DETAILED DESCRIPTION

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0042] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0043] Example 1

[0044] SARS-CoV-2M pro Determination of active sites

[0045] The CDOCKER semi-flexible docking mode was used, and the CDOCKER_INTERACTI ON_ENERGY in the docking results was used as the evaluation criterion. pro The original ligand N3 was used as a positive control and was compared with SARS-Co V-2M pro (SARS-CoV-2 main protease) was docked to obtain the binding free energy. The crystal complex structure formed by Mpro and N3 was obtained from the PDB database. The resolution of the structure is N3 is developed through computer-aided drug design pro Inhibitors that can specifically inhibit M pro Based on the position of N3 in the complex and the position of the original ligand, the active site of Mpro was determined, and its coordinates are as follows: Figure 1 As shown, X = -10.743, Y = 12.460, Z = 68.918, and the radius of the active center is as follows Figure 2 Shown

[0046]

[0047] Example 2 Effects of pomegranate peel tannin and punicalagin on SARS-CoV-2M pro The inhibitory effect

[0048] 1. Determination of Binding Capacity

[0049] Discovery studio 2020 software was used for ligand and protein optimization and molecular docking studies. First, the ligand was processed to minimize the energy of the ligand structure and obtain different conformations of the compound. For the receptor protein, the three-dimensional structure of the target protein SARS-CoV-2Mpro (PDB: 6LU7) was obtained from the PDB database. The protein was pre-processed by deleting excess water molecules, adding hydrogen, and filling in missing amino acid residues and terminal residues. The CDOCKER semi-flexible docking mode was used, and the CDOCKER_INTERACTION_ENERGY in the docking result was used as the evaluation standard. pro The original ligand N3 was used as a positive control to dock with Mpro to obtain the binding free energy, which was used to screen potential active molecules.

[0050] As can be seen from Table 1, M pro The binding energy with the original ligand N3 is -72.88 Kcal / mol. The binding energies of punicalagin and punicalagin with Mpro are both lower than the original ligand, at -80.44 Kcal / mol and -77.89 Kcal / mol, respectively. Lower binding energy indicates more stable binding.

[0051] Table 1 Docking binding energy of punicalagin and punicalagin with Mpro

[0052]

[0053] 2.IC 50 Determination of

[0054] Assay Procedure: Punicalin, punicalagin, and the positive control compound PF-07321332 were precisely weighed. Each compound was dissolved in DMSO to prepare a 10 mM stock solution. Using a gradient dilution method, the stock solution (initial concentration: 320 μM) was serially diluted 10-fold to obtain 10 test samples with varying concentrations.

[0055] 54 μL of a 200 nM Mpro protein solution was added to a light-protected 96-well plate. Subsequently, 1 μL of a 40 μM compound working solution was added, mixed, and incubated at room temperature (25°C) for 1 hour to ensure sufficient binding of the compound to the protein. Following incubation, 5 μL of a 20 μM peptide substrate solution (Mca-AVLQ↓SGFR-Lys(DNP)-Lys-NH2) was added to each well. The 96-well plate was immediately placed in a fluorescence microplate reader for real-time fluorescence detection at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Twenty consecutive measurement cycles were performed, with a 15-second interval between each cycle. During the experiment, fluorescence intensity data was collected in real time using a high-sensitivity detection system, and time-dependent fluorescence intensity curves were constructed using analysis software. To eliminate background interference, a blank control group was included. The slope of each sample curve was calculated using the least-squares method and normalized by subtracting the blank control value.

[0056] Test results: Figure 3 As shown, the IC of the positive control PF-07321332 50 0.05±0.02μM; Figure 4 As shown, pomegranate peel tannin IC 50 The concentration range of SARS-CoV-2 was 0.32±0.05μM. pro Has a significant inhibitory effect; such as Figure 5 As shown, punicalagin IC 50 The concentration range of SARS-CoV-2 was 2.09±0.20μM. pro Has a significant inhibitory effect.

[0057] Example 3: Punicalagin and Punicalagin pro Molecular interaction studies

[0058] Experimental process: The study used the Biacore T200 surface plasmon resonance (SPR) method to conduct molecular interaction research. The specific experimental process is as follows: Accurately measure 52.5mL of 10×PBS P solution and add an appropriate amount of ultrapure water to dilute until the total volume reaches 500mL, and finally obtain a solution with a concentration of 1.05×PBS P. Subsequently, accurately measure 20mL of DMSO and mix it with 380mL of 1.05×PBS P solution to obtain a 1.05×PBS P solution containing 5% DMSO. This solution will be used as the running buffer for subsequent experiments. In addition, according to the preparation schemes detailed in Tables 2 and 3, 1.05×PBS P solutions containing 4.5% and 5.8% DMSO were prepared respectively, and the calibration curve was prepared according to the proportions in Table 3 to ensure the accuracy and reliability of the experimental results.

[0059] Table 2 Preparation of buffer

[0060]

[0061] Table 3 Calibration curve preparation

[0062]

[0063] Experimental results: The results are as follows Figure 6 As shown, PF-07321332 and M pro Interaction K D 6.67×10 -7 M; such as Figure 7 、 8 As shown, punicalagin and punicalagin are related to M pro Both have strong interactions, K D 9.09×10 -7 M, 3.18×10 -7 M.

[0064] Example 4 Molecular dynamics study of pomegranate peel tannin and punicalagin with SARS-CoV-2 Mpro

[0065] Molecular dynamics simulations were performed using GROMACS software. The detailed structures of the ligands were obtained in mol 2 format from databases such as PubChem. Discovery studio 2020 software was used to optimize the ligands and proteins, process the ligand compounds, minimize the energy of the ligand structures, and obtain different conformations of the compounds. Molecular dynamics simulations were used to explore the relationship between active molecules and M pro The binding stability within 500ns is shown in the following table. Figure 9-10 As shown, punicalagin is surrounded by multiple amino acid residues and interacts with multiple amino acid residues, including P140, A142, C145 and A191; punicalagin is surrounded by multiple amino acid residues and interacts with multiple amino acid residues, including T26, L141, C145, M165 and T190.

[0066] (1) Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis

[0067] The present invention is to treat pomegranate peel tannin, punicalagin and SARS-CoV-2M pro The docked complex was subjected to 500ns molecular dynamics simulation, including RMSD and RMSF analysis, to reveal the binding stability of each small molecule to SA RS-CoV-2Mpro:

[0068] RMSD is the root mean square deviation, which is used to determine the overall deviation of the molecular structure relative to the reference conformation during the simulation process. Figure 11 As shown, for the Mpro-pomegranate peel tannin complex, the RMSD values ​​are between 0.4 and Fluctuations indicate good stability and structural fidelity. pro -RMSD values ​​of punicalagin complexes ranged from 0.5 to It shows that it has extremely high stability during the simulation process.

[0069] RMSF is the root mean square fluctuation, which measures the degree of fluctuation of atoms or residues relative to their average position during the entire molecular simulation. Figure 12 As shown in Figure 2, the complexes formed by punicalagin and punicalagin with M pro have similar performance in RMSF curves. This indicates that these two small molecule ligands have a significant effect on M pro The effects of spatial structural fluctuations were relatively similar and did not significantly change the flexibility of the protein.

[0070] (2) Analysis of gyration radius (Rg)

[0071] Rg is an important parameter to measure the compactness of protein structure, which represents the average square distance between the mass center of all atoms in the protein and their geometric center. Figure 13 As shown, by comparing the effects of punicalagin, punicalagin and M pro The average Rg of the complexes was compared and found to be very small, indicating relative stability. This result is consistent with the previously obtained RMSD and RMSF results, further verifying the structural stability of the complex in the bound state.

[0072] Furthermore, the stability of the average Rg indicates that despite ligand binding to the protein's active site, the overall three-dimensional structure of the protein remains consistent. This stability in the bound state suggests that the interaction between the ligand and the protein is highly specific. This stable binding not only enhances the biological activity of the complex but also potentially increases its persistence and effectiveness in biological environments.

[0073] Example 5 In vitro antiviral experiments and results of pomegranate peel tannin and punicalagin

[0074] 1. Effects of Punicalagin and Punicalagin on Vero E6 Cell Viability

[0075] Experimental procedure: Take out the frozen African green monkey kidney cells (Vero E6), quickly put them into a 37℃ water bath, and shake them quickly to melt the cryoprotectant quickly and evenly. Wipe the outer wall of the cryotube with 75% medical alcohol, transfer the cells to a centrifuge tube, and centrifuge at 1000rpm for 5 minutes. After centrifugation, remove the supernatant, add 2mL of complete culture medium, gently pipette the cell pellet to fully disperse and mix, and then inoculate the cells into T 25 cm 2 Add 3 mL of complete culture medium to the culture flask, shake the cells, and culture them in a 37°C, 5% CO2 incubator.

[0076] Observe under a microscope that the cell density reaches 80%-90%, discard the culture medium, wash the cells twice with 2.0 mL of sterile PBS, digest the adherent cells, discard the supernatant after centrifugation, add 5.0 mL of complete culture medium and blow it away to mix thoroughly. Pipette 100.0 μL of complete culture medium containing cells, add sterile PBS to dilute 10 times, count the cells on a cell counter, and calculate the number of cells to 2 × 10 4 The cells were plated at a density of 100 μL per well in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 48 h.

[0077] The stock solutions of pomegranate peel tannin and punicalagin were serially diluted according to the ratios in Table 4 to seven different concentrations: 5μM, 10μM, 20μM, 40μM, 60μM, 80μM, and 100μM. Once the cell density in the 96-well plate reached 70-80% under a microscope, the culture medium was discarded and 100μL of complete culture medium containing the drug at various concentrations was added to each well. Blank controls were set up, with six replicates per group. The cells were cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0078] Table 4 Serial dilution of compound stock solutions

[0079]

[0080] After 48 h of culture, the 96-well plate was removed, the supernatant was discarded, 50 μL of 2.5 mg / mL MTT was added, and the plate was placed in a 37°C incubator for further incubation for 4 h. The supernatant was discarded, 150 μL of DMSO was added, and the absorbance was measured at 490 nm using a microplate reader. The results were visualized and analyzed using GraphPad Prism 9 software.

[0081] Experimental results: The results are as follows Figure 14 As shown in Figure 2, all tested compounds showed no significant inhibitory effect on the viability of Vero E6 cells at the highest concentration of 100 μM. 50 ) values ​​were all above 100 μM, indicating that these compounds had low toxicity to cells at this concentration.

[0082] 2. Study on the inhibition of SARS-CoV-2 wild strain by active ingredients in vitro

[0083] Experimental process: In the antiviral activity study, Ensitrelvir (S-217622) was used as a positive control compound to exclude false positives and exogenous factors. 50 S-217622 is the first oral non-peptide-like, non-covalent SARS-CoV-2 M-like drug candidate to be used as a clinical candidate. pro Inhibitor.

[0084] Experimental results: The results are as follows Figure 15 As shown in Figure 2, the positive control S-217622 has a significant anti-SARS-CoV-2 wild strain effect, and its EC 50 Punicalagin and pomegranate peel tannin both have strong inhibitory effects on SARS-CoV-2 wild strains, EC 50 The values ​​were 2.27 μM and 12.37 μM, respectively, showing dose-dependent antiviral activity.

[0085] 3. Study on the inhibition of SARS-CoV-20micronBA.5 virus strain by active ingredients in vitro

[0086] Experimental procedure: The antiviral activity of the compounds punicalagin and punicalagin, and the positive control S-217622, ​​was tested using a CPE inhibition assay using Vero E6 cells in a biosafety level 3 (BSL-3) laboratory at the Institute of Health and Quarantine, Guangzhou Customs Technology Center.

[0087] Virus strains: SARS-CoV-2 wild type, SARS-CoV-2 Delta, and Omicron BA.5, provided by Guangzhou National Laboratory.

[0088] Vero E6 cells were cultured at 2×10 4The cells were seeded at a density of 100 cells / well in a 96-well plate, 100 μL per well, and placed in a constant temperature and humidity incubator at 37°C for overnight culture. After the cells adhered for 16 hours, the cell culture medium was discarded, and 100 μL of culture medium containing a certain concentration of pomegranate peel tannin, punicalagin and positive control S-217622 was added to each well. The starting concentration of the compound was 100 μM, with 8 gradient points, 6 replicate wells for each concentration, and DMSO treatment and normal cell groups were also set up. Except for the normal cell group, complete virus-containing culture medium (MOI = 0.01) was added to each well and placed in a constant temperature and humidity incubator at 37°C for incubation for 48 hours. 48 hours after infection, the Celigo full-field cell scanner was used to record the cell pathological changes, and the half-effective concentration (EC50) was calculated based on the cell survival rate. 50 ) value, and the EC 50 .

[0089] Experimental results: The results are as follows Figure 16 As shown, the EC value of the positive control S-217622 against SARS-CoV-2Omicron BA.5 50 The value is 0.09μM. Punicalagin and pomegranate peel tannin have strong inhibitory effects on SARS-CoV-2Omicron BA.5 virus strain, EC 50 The values ​​were 3.65 μM and 13.30 μM, respectively, showing dose-dependent antiviral activity.

[0090] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An anti-coronavirus drug, characterized in that The active ingredients of the anti-coronavirus drug are punicalagin and / or punicalagin.

2. The drug according to claim 1, characterized in that The medicine further contains pharmaceutically acceptable excipients.

3. The drug according to claim 1, characterized in that The medicine is in the form of an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

4. Application of punicalagin and / or pomegranate peel tannin in the preparation of anti-coronavirus drugs.

5. The use according to claim 4, characterized in that The coronavirus is SARS-CoV-2 or a variant thereof; the variant is Omicron BA.

5.

6. The use according to claim 4, characterized in that The CC of the punicalagin and / or punicalagin 50 Greater than 100μM.

7. The use according to claim 5, characterized in that The EC of punicalagin against SARS-CoV-2 50 The EC value of the pomegranate peel tannin against SARS-CoV-2 is 2.27 μM. 50 It is 12.37μM.

8. The use according to claim 5, characterized in that The EC of punicalagin on Omicron BA.5 50 The EC value of the pomegranate peel tannin for Omicron BA.5 is 3.65 μM. 50 It is 13.30μM.

9. Punicalagin and / or pomegranate peel tannin in the preparation of a method for inhibiting coronavirus main protease M pro Application of enzyme activity inhibitors.

10. The use according to claim 9, characterized in that Punicalagin inhibits M pro Active IC 50 was 2.09±0.20μM; the pomegranate peel tannin inhibited M pro Active IC 50 It is 0.32±0.05μM.

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