New use of magnolol as a preparation of coronavirus inhibitors

Abstract

The present invention discloses a new use of magnolol as a coronavirus inhibitor. Magnolia officinalis inhibits S protein-mediated membrane fusion invasion of the host by acting on the highly conserved HR2 in the virus, and / or inhibits the replication and spread of the virus by inhibiting the ExoN enzyme activity in NSP14. The advantage is that the present invention reveals the effectiveness of magnolol in preventing and treating coronavirus infection, and further clarifies that magnolol exerts its antiviral effect by inhibiting the invasion and replication of the virus, reflecting its broad-spectrum antiviral potential. These studies provide candidate drugs and scientific basis for the development of new drugs for broad-spectrum anti-coronavirus, and promote the clinical application of antiviral Chinese medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of virus inhibitors, and specifically relates to a new use of magnolol as a preparation of coronavirus inhibitors. Background Art

[0002] In order to cope with the continuous mutation and rapid spread of coronavirus, it is crucial to develop safe and effective broad-spectrum antiviral drugs.

[0003] SARS-CoV-2, SARS-CoV and MERS-CoV are all classified as betacoronaviruses in the family Coronaviridae of the order Nidovirales, and are linear single-stranded positive-strand RNA viruses with envelope structures. The non-coding regions at both ends of the SARS-CoV-2 viral genome are 5'-UTR and 3'-UTR, respectively. Starting from the 5' end, about 2 / 3 of the genome contains two overlapping open reading frames (ORF) 1a / 1b, which are mainly responsible for encoding enzymes related to viral replication and transcription. After translation, they are cleaved by proteases to produce 16 nonstructural proteins (NSPs), and the sequences are relatively conservative. The remaining 1 / 3 of the genome is responsible for encoding structural proteins and auxiliary proteins including spike (S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein. These structural proteins are the decisive factors for the assembly, stability and invasiveness of the virus.

[0004] Generally speaking, the life cycle of a virus can be divided into: adsorption invasion, viral replication, assembly and release. During the virus invasion of host cells, the invasion mode of SARS-CoV-2 is determined by the distribution of its receptors and host cell proteases that cut the S protein. After the S protein binds to angiotensin converting enzyme 2 (ACE2), it invades host cells mainly through two pathways: viral membrane fusion and endocytosis. When transmembrane serine protease 2 (TMPRSS2) is present on the cell surface, the Furin protease-like cleavage site at the S1 / S2 junction in the middle of the S protein is exposed and recognized and cut by TMPRSS2, causing the S protein to lose the S1 subunit, exposing the S2' cleavage site at the front of the fusion peptide (FP) and being cleaved. Then FP is inserted into the host cell membrane, and the two heptad repeats (HR1 and HR2) in the S2 subunit interact to form a six-helix bundle (6-HB), which shortens the distance between the viral membrane and the cell membrane, initiates membrane fusion, and mediates the virus to enter the host cell. The virus particles are uncoated under the action of host cell enzymes, thereby releasing the viral genome into the cell to complete the invasion process. Afterwards, the SARS-CoV-2 genomic RNA is uncoated and released into the cytoplasm, and ORF1ab begins to encode the translation replication enzyme polyproteins pp1a and pp1ab. Subsequently, under the action of NSP3 and NSP5, 16 NSPs of the virus are cut and assembled into a viral replication-transcription complex (RTC). In the double-membrane vesicles formed by rearrangement of the endoplasmic reticulum, RTC uses positive-sense single-stranded RNA genome (+ssRNA) as a template to replicate and transcribe viral RNA; the generated negative-strand RNA serves as a template for subgenomic mRNAs (sg mRNAs), mediating the encoding of S, M, E and N proteins, and transferring these structural proteins to the endoplasmic reticulum membrane, and transporting them to the endoplasmic reticulum Golgi intermediate compartment through the endoplasmic reticulum. Finally, +ssRNA integrates with other structural units through the structural protein N to form new virus particles, which are released from the host cell by exocytosis or infect new host cells by membrane fusion.

[0005] Among them, NSP12 is an RNA-dependent RNA polymerase (RNΑ-dependent RNA polymerase, RdRP), which is the main factor in guiding the synthesis of progeny virus + ssRNA and sg mRNAs, and NSP7 and NSP8 are auxiliary factors. At the same time, the stable function of RdRp requires the assistance of more molecules, including NSP13 and NSP10 / NSP14 proofreading complex. Among them, NSP13 has helicase activity, which polarizes RNA from the 5' end to the 3' end in a nucleoside triphosphate-dependent manner. Studies have found that once a nucleotide is incorrectly inserted, NSP13 will push RdRp away from the RNA template. At this time, the proofreading function of NSP14 exoribonuclease (ExoN) will identify and correct the incorrect nucleotides on the RNA chain to ensure accurate replication of RNA. As a protein with bifunctional enzyme activity, NSP14 has not only ExoN activity but also guanine-N7-methyltransferase (N7-MTase) function. MTase participates in the synthesis of the 5' end cap structure of viral sg mRNAs. Its RNA cap structure enables coronavirus to mimic the eukaryotic RNA structure, thereby avoiding recognition and degradation by the host immune system. In addition, NSP10 is an auxiliary factor of NSP14 and can significantly improve the enzyme activity of NSP14. From the life cycle of the above-mentioned virus, it can be seen that when looking for virus inhibitors, the above-mentioned targets can be used for design. The existing inhibitors, such as the main protease inhibitors namatevir tablets / ritonavir (Paxlovid) and the RdRp inhibitor remdesivir, have become the hot spots of current antiviral research. However, there are relatively few studies on other enzymes in coronaviruses, which still need further in-depth exploration. Summary of the invention

[0006] In view of the problems of insufficient research on specific enzyme targets and scarcity of broad-spectrum antiviral drugs in existing coronavirus inhibitor research, the present invention provides a new use of magnolol as a preparation of coronavirus inhibitors, and the purpose is to find that magnolol can have an inhibitory effect on different links of SARS-CoV, MERS-CoV, and SARS-CoV-2, and has universality, so that inhibitors with broad-spectrum antiviral properties can be obtained.

[0007] In order to achieve the above-mentioned purpose, the technical solution provided by the present invention is that magnolia officinalis is used as a new use of preparing coronavirus inhibitors, and magnolia officinalis inhibits S protein-mediated membrane fusion invasion of the host by acting on the highly conserved HR2 in the virus, and / or inhibits the replication and spread of the virus by inhibiting the ExoN enzyme activity in NSP14.

[0008] Furthermore, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0009] Furthermore, magnolol enhances the antiviral efficacy of remdesivir by inhibiting the ExoN enzyme activity in NSP14.

[0010] In summary, the beneficial effect of the present invention is that the present invention reveals the effectiveness of magnolol in preventing and treating coronavirus infection, and further clarifies that magnolol exerts its antiviral effect by inhibiting the invasion and replication of the virus, and acts on the highly conserved regions of HR2 of the coronavirus S protein involved in the invasion link and ExoN involved in the replication link, respectively, reflecting its broad-spectrum antiviral potential. These studies provide candidate drugs and scientific basis for the development of new drugs for broad-spectrum anti-coronavirus, and promote the clinical application of antiviral Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The effect of magnolol (Mag) on ​​the invasion of SARS-CoV-2Luc-VLPs into the lungs of humanized ACE2 mice. In the figure: Luciferase activity quantitatively detects the content of SARS-CoV-2Luc-VLPs in the lungs of mice. UDCA represents the positive control drug ursodeoxycholic acid intervention group; ****P<0.001 compared with the Blank group, #P<0.05, ####P<0.001 compared with the Control group.

[0012] Figure 2 The effect of magnolol (Mag) on ​​the invasion of SARS-CoV-2Luc-VLPs into the lungs of humanized ACE2 mice, in which SARS-CoV-2Luc-VLPs are marked in red, ACE2 is marked in green, F-actin is marked in white, and the nucleus is marked in blue. The corresponding magnified area of ​​the dotted box is on the right, and the scale bar is 50μm or 10μm.

[0013] Figure 3 The effect of magnolol (Mag) on ​​the expression of ACE2 and TMPRSS2 in the lungs of humanized ACE2 mice. In the figure: ****P<0.001 compared with the Blank group.

[0014] Figure 4 The effect of magnolol (Mag) on ​​the invasion of SARS-CoV-2Luc-VLPs into human type II alveolar epithelial cells (AECⅡ), in the figure: quantitative detection of intracellular SARS-CoV-2Luc-VLPs levels; ****p<0.001 compared with the Blank group; ####p<0.001 compared with the Control group; S NAb represents the positive control drug S protein neutralizing antibody; all figures are mean ± standard error, n=3.

[0015] Figure 5 This is the effect of magnolol (Mag) on ​​the invasion of SARS-CoV-2Luc-VLPs into AECⅡ. In the figure: Immunofluorescence staining was used to detect the level of intracellular SARS-CoV-2Luc-VLPs, where red represents SARS-CoV-2Luc-VLPs, green represents ACE2, gray represents the cytoskeleton F-actin, and blue represents the cell nucleus; S NAb represents the positive control drug S protein neutralizing antibody; the scale bars in the figures are all 50 μm.

[0016] Figure 6 This is the effect of magnolol (Mag) on ​​membrane fusion mediated by SARS-CoV-2S protein and ACE2. In the figure: green fluorescence represents the formation of multinucleated giant cells through membrane fusion between cells; the fluorescent images represent the membrane fusion levels of the SARS-CoV-2S protein Alpha strain (A), Delta mutant strain (B), OmicronBA.1 mutant strain (C), Omicron BA.2 mutant strain (D), Omicron BQ.1.1 mutant strain (E), Omicron XBB.1 mutant strain (F) control group and magnolol (Mag) intervention group; the left histogram represents the quantitative detection of the corresponding membrane fusion level by luciferase activity. **p<0.01, ***p<0.005, ****p<0.001 compared with the Control group. All figures are mean ± standard error, n≥3. The scale bar is 200μm.

[0017] Figure 7 This is the effect of magnolol (Mag) on ​​membrane fusion mediated by SARS-CoV-1S protein and ACE2. In the figure: green fluorescence represents the formation of multinucleated giant cells through membrane fusion between cells; the fluorescent images represent the membrane fusion levels of the SARS-CoV-1S protein control group and the magnolol (Mag) intervention group; the left histogram represents the quantitative detection of the corresponding membrane fusion level by luciferase activity. ****p<0.001 compared with the Control group. All figures are mean ± standard error, n≥3. The scale bar is 200μm.

[0018] Figure 8 The effect of magnolol (Mag) on ​​the invasion of SARS-CoV-2Luc-VLPs into Vero E6 cells through the endocytosis pathway.

[0019] Fig. 9 The effect of magnolol (Mag) on ​​the thermal stability of SARS-CoV-2S protein S1 and S2 subunits.

[0020] Fig.10The effect of magnolol (Mag) on ​​the secondary structure of SARS-CoV-2 HR1, HR2 and 6-HB, in the figure: Figure A, HR1, HR2 and 6-HB circular dichroism spectra; Figure B, the effect of different concentrations of magnolol (Mag) on ​​the secondary structure of 6-HB; Figure C, the effect of different concentrations of magnolol on the secondary structure of HR1; Figure D, the effect of different concentrations of magnolol on the secondary structure of HR2. The right side is the analysis of the proportion of SARS-CoV-2 HR1, HR2 and 6-HB secondary structures.

[0021] Fig.11 The figure shows the effect of magnolol (Mag) on ​​SARS-CoV-2 HR1, HR2 and 6-HB. In the figure: (A), magnolol of different concentrations was pre-incubated with HR2 and then incubated with HR1 to form 6-HB; (B), the effect of magnolol of different concentrations on the content of HR1; (C), the effect of magnolol of different concentrations on the content of HR2.

[0022] Fig.12 Molecular docking prediction of the binding site between magnolia officinalis (Mag) and HR2. In the figure: the area marked by the black dotted box on the left is enlarged to the black dotted box on the right; the right boxes mark E1188 (the 1188th glutamic acid residue) that forms a hydrogen bond (yellow dotted line) with magnolia officinalis.

[0023] Fig.13 The effect of magnolol (Mag) on ​​E1188A mutant SARS-CoV-2 HR2 and 6-HB. In the figure: (A), 6-HB formation analysis of E1188A mutant HR1 (Mut-HR2) at 37°C; (B), comparison of the effects of different concentrations of magnolol on the content of mutant HR2 (Mut-HR2).

[0024] Fig.14 Effects of magnolol (Mag) on ​​SARS-CoV-2 (A) replicon NSP1-16 and (B) mini-replicons NSP1-4, NSP5-11 and NSP12-16. In the figure: ****P<0.001 compared with the Control group. All figures are mean ± standard error, n=3.

[0025] Fig.15 This is the effect of magnolol (Mag) on ​​the expression of luciferase (M-Luc).

[0026] Fig.16 The effect of magnolia bark ol (Mag) on ​​the thermal stability of SARS-CoV-2 NSP12-NSP16 protein.

[0027] Fig.17 The affinity of magnolol (Mag) to SARS-CoV-2 NSP14.

[0028] Fig.18 This is the effect of magnolia bark phenol (Mag) on ​​the secondary structure of SARS-CoV-2NSP14. In the figure: the left side is the ratio of the secondary structure of SARS-CoV-2NSP14 analyzed.

[0029] Fig.19 The effect of magnolol (Mag) on ​​the thermal stability of SARS-CoV-2 NSP14 ExoN and N7-methyltransferase (N7-MTase) proteins.

[0030] Fig. 20 The effect of magnolol (Mag) on ​​the activity of SARS-CoV-2 ExoN. In the figure: (A), the activity curve of NSP14 of SARS-CoV-2 was measured. RNase A was used as the positive control; (B), the dose-response curve of NSP14 activity under the intervention of magnolol and sofalcone (positive control).

[0031] Fig.21 The effect of magnolia bark phenol (Mag) on ​​the thermal stability of the protein of SARS-CoV-2 ExoN mutant.

[0032] Fig. 22 The universal characteristics of magnolol against highly pathogenic coronaviruses. (A) Comparison of the active sites of SARS-CoV-2, SARS-CoV and MERS-CoV NSP14-ExoN enzymes; (B) Effect of magnolol (Mag) on ​​the thermal stability of SARS-CoV and MERS-CoV NSP14 proteins.

[0033] Fig.23 The effect of magnolol combined with remdesivir on SARS-CoV-2RdRp, in the figure: Sofalcone was used as the positive control; **p<0.01 and ****p<0.001 compared with the control group (M-Luc+RdRp- / - / -) not treated with Sofalcone / Magnolol (Mag); ##p<0.01, ###p<0.005 and ####p<0.001 compared with the control group (M-Luc+RdRp+NSP14- / - / -) not treated with Sofalcone / Mag; Compared with the Sofalcone / Mag-treated control group.

[0034] Fig.24 The effect of magnolia bark ol (Mag) on ​​the inhibition of intracellular protein synthesis and translation by SARS-CoV-2NSP14.

[0035] Fig.25The effect of magnolia bark ol (Mag) on ​​SARS-CoV-2 NSP14 inhibiting IFN-α-induced ISG expression.

[0036] Fig.26 SARS-CoV-2Luc-VLPs invaded 293F-ACE2-TMPRSS2 cells transfected with NSP14 after being treated with IFN and / or magnolol. In the figure: ****p<0.001 compared with the blank control group not treated with SARS-CoV-2Luc-VLPs; ##p<0.01 compared with the control group (SARS-CoV-2Luc-VLPs- / - / -) not treated with IFN / NSP14 / magnolia (Mag); &&p<0.01 compared with the group treated with IFN (SARS-CoV-2Luc-VLPs+ / - / -); Compared with the NSP14 / IFN-treated group (SARS-CoV-2Luc-VLPs+ / + / -). DETAILED DESCRIPTION

[0037] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. Unless otherwise specified, the operation of the present invention adopts the prior art.

[0038] Example 1: The novel use of magnolol provided by the present invention as a preparation of coronavirus inhibitors, magnolol inhibits S protein-mediated membrane fusion invasion of the host by acting on the highly conserved HR2 in the virus. The specific experimental contents are as follows:

[0039] 1. Experimental results:

[0040] 1. Magnolia officinalis inhibits SARS-CoV-2 invasion of hosts in in vivo and in vitro experiments

[0041] To determine the effectiveness of magnolol in inhibiting the invasion of SARS-CoV-2, we used hACE2 transgenic mice and alveolar epithelial cells type II (AECⅡ) derived from human induced pluripotent stem cells (hiPSC) in vitro models as host models. At the same time, in order to avoid the interference of viral replication and focus on studying the process of viral invasion of host cells, we used SARS-CoV-2 virus-like particles (Luciferase virus-likeparticles, Luc-VLPs) labeled with luciferase that do not have the ability to replicate. Luc-VLPs are composed of the main structural proteins of the virus, S, M, N, and E proteins, and have the structure and function of natural viruses, which can be used as a virus replacement model.

[0042] 1.1 Magnolia officinalis inhibits SARS-CoV-2 from invading the lungs of humanized ACE2 mice

[0043] In order to clarify that magnolia officinalis has the effect of preventing viruses from invading the host. In this experiment, we used the intervention measure of preventive oral administration of magnolia officinalis to mice, and then performed tracheal instillation of SARS-CoV-2Luc-VLPs in humanized ACE2 mice to simulate the process of virus invasion of mouse lungs, and finally detected the content of Luc-VLPs in the invaded lungs. We used ursodeoxycholic acid (UDCA), which has a significant effect in preventing SARS-CoV-2 infection, as a positive control drug.

[0044] like Figure 1 As shown, the results of luciferase activity detection showed that compared with the Blank group, Luc-VLPs were detected in the lung tissue of mice 6 hours after instillation of SARS-CoV-2Luc-VLPs, while the Luc-VLPs content in the lung tissue of the magnolol and UDCA groups was significantly lower than that of the Control group, which was statistically significant. Specifically, the inhibition rate of the UDCA group was 79.0%, the inhibition rate of the low-dose magnolol group (100 mg / kg) was 13.3%, the inhibition rate of the medium-dose group (200 mg / kg) was 36.6%, and the inhibition rate of the high-dose group (400 mg / kg) was 55.6%, which was statistically significant ( Figure 1 ). This indicates that through oral administration, magnolia bark phenol intervention can dose-dependently inhibit SARS-CoV-2 from invading the lung tissue of humanized ACE2 mice, and has the effect of preventing SARS-CoV-2 from invading the lungs of mice.

[0045] like Figure 2As shown, immunofluorescence detection further observed that compared with the Control group, the magnolol intervention group had a reduced Luc-VLPs invasion content and showed a dose-dependent relationship, suggesting that magnolol can inhibit the invasion of Luc-VLPs into the lung tissue of hACE2 transgenic mice, while the UDCA group had a higher inhibition rate of Luc-VLPs invasion. The above results are consistent with the results of luciferase activity detection. In addition, the expression of ACE2 in the lungs of mice treated with magnolol did not change, while the expression of ACE2 in the UDCA treatment group was significantly reduced. In summary, these results not only show that oral magnolol can significantly reduce the host's susceptibility to SARS-CoV-2, but also reflect the effect of magnolol in protecting the body from viral invasion without affecting the host's ACE2 expression.

[0046] In order to explore whether magnolol inhibits viral invasion while affecting the host's virus invasion-related receptors, the expression of ACE2 in humanized ACE2 mice and after magnolol intervention was detected by immunoblotting, and semi-quantitative analysis was performed. The experimental results showed that compared with the Control group, the expression levels of ACE2 and TMPRSS2 in the magnolol intervention group did not change; the expression level of TMPRSS2 in the UDCA intervention group did not change, while the expression of ACE2 was significantly downregulated, with statistically significant differences ( Figure 3 ). This indicates that magnolol did not change the expression of ACE2 and TMPRSS2, receptors associated with viral invasion, in the mouse lungs.

[0047] 1.2 Magnolia officinalis inhibits SARS-CoV-2 invasion of human AECⅡ alveolar organoids

[0048] Lung organoids are currently the model that is closest to human lungs, and the lung organoids differentiated from hiPSCs are used as in vitro lung models. Therefore, we used the SARS-CoV-2Luc-VLPs invasion of human AECⅡ alveolar organoids to clarify the inhibitory effect of magnolol on SARS-CoV-2 invasion of the host. Quantitative detection of luciferase activity showed that the level of Luc-VLPs in the model group was significantly increased compared with the blank group, indicating that the difference was statistically significant; compared with the model group, the level of Luc-VLPs in the positive control group was significantly decreased by 54.7%±2.0%, indicating that the model was successfully constructed and the detection method was feasible. The Luc-VLPs level in the 30μM magnolol intervention group decreased by 27.5%±1.2%, which was statistically significant, and magnolol inhibited the invasion of Luc-VLPs in a dose-dependent manner. In summary, these experimental results show that magnolol can effectively inhibit the invasion of SARS-CoV-2Luc-VLPs in AECⅡ cells ( Figure 4 ).

[0049] The results of immunofluorescence staining for SARS-CoV-2Luc-VLPs showed that no Luc-VLPs signal was detected in the blank group; a large number of red fluorescent signals were observed in the cells of the model group, representing SARS-CoV-2Luc-VLPs; there were very few intracellular Luc-VLPs signals in the positive control group (SNAb); the intracellular red fluorescent signals in the magnolol intervention group were significantly reduced, indicating that the level of SARS-CoV-2Luc-VLPs was reduced ( Figure 5 ), which is consistent with the results of quantitative detection of luciferase activity.

[0050] 2 Magnolia officinalis prevents virus invasion by inhibiting membrane fusion between viruses and host cells

[0051] SARS-CoV-2 and other coronaviruses mainly enter the host through two invasion pathways: membrane fusion and endocytosis. Therefore, we studied whether magnolol affects membrane fusion and endocytosis to invade host cells.

[0052] 2.1 Magnolia officinalis inhibits membrane fusion of SARS-CoV-2 mutants and SARS-CoV-1

[0053] SARS-CoV-2 and other coronaviruses mainly invade host cells through S protein-mediated membrane fusion. Therefore, we need to explore the effect of magnolol on S protein-mediated membrane fusion of SARS-CoV-2 and its mutants ( Figure 6 ).

[0054] The fluorescence intensity of green fluorescent protein (GFP) reflects the degree of viral membrane fusion. The analysis results of GFP fluorescence intensity showed that compared with the Control group without any drug treatment, the GFP fluorescence intensity in the magnolia bark phenol intervention group decreased.

[0055] Through quantitative detection of luciferase activity, compared with the Control group without any drug treatment, magnolia bark ol (Mag) can inhibit the membrane fusion mediated by S protein and TMPRSS2 of SARS-CoV-2 and its variants, with the membrane fusion rate of Alpha strain reduced by 37.1%, Delta variant reduced by 29.7%, Omicron BA.1 variant reduced by 24.1%, OmicronBA.2 variant reduced by 24.0%, Omicron BQ 1.1 variant reduced by 19.2%, and Omicron XBB.1 variant reduced by 22.8%. All of the above results were statistically significant.

[0056] The effect of magnolol on SARS-CoV-1S protein-mediated membrane fusion was further tested. The results showed that compared with the control group cells not treated with any drug, magnolol (Mag) could inhibit the membrane fusion mediated by the S protein of SARS-CoV-1, and the fusion rate was reduced by 26.1% ( Figure 7 ).

[0057] In summary, the above data indicate that magnolia officinalis inhibits coronavirus membrane fusion and invasion of the host, which may be related to the S protein of the virus.

[0058] 2.2 Magnolia officinalis has no inhibitory effect on another invasion pathway of SARS-CoV-2 - endocytic invasion

[0059] In addition to invading the host through viral membrane fusion, coronaviruses such as SARS-CoV-2 also invade host cells through the endocytosis pathway. In order to explore whether magnolol has an inhibitory effect on viral endocytosis, we used Vero E6 cells as a host model. This cell does not express TMPRSS2, so the virus mainly invades host cells through the endocytosis pathway. Figure 8 As shown in the figure, compared with the Control group, there was no significant change in the level of SARS-CoV-2Luc-VLPs in the magnolol intervention group, and there was no statistical difference. This indicates that magnolol has no effect on the endocytosis process of SARS-CoV-2Luc-VLPs.

[0060] 3 Magnolia officinalis disrupts HR2 that mediates viral membrane fusion

[0061] In order to explore how magnolia officinalis affects viral S protein-mediated membrane fusion, we need to further clarify the molecular mechanism by which magnolia officinalis inhibits viral membrane fusion.

[0062] 3.1 Interaction between magnolol and S2 subunit of S protein

[0063] According to the previous experimental results ( Figure 6 , Figure 7 ), it is speculated that magnolol may interact with S protein. In order to verify this speculation, the interaction between magnolol and different subunits of S protein was detected based on the cellular thermal shift assay (CETSA). This experiment determines whether a small molecule drug interacts with the target protein based on the degree to which it increases the stability of the target protein. Fig. 9As shown in the results, the expression level of the full-length S protein (about 180kD) gradually decreased with increasing temperature; compared with the Control group, the expression level of the magnolol group increased significantly when the temperature exceeded 46°C. The viral S protein is divided into two subunits, S1 and S2. The results showed that the expression level of the S1 subunit (about 120kD) gradually decreased with increasing temperature; compared with the Control group, the expression of the S1 protein in the magnolol group did not change. The expression level of the S2 subunit (about 90kD) gradually decreased with increasing temperature; compared with the Control group, the expression level of the magnolol group increased significantly when the temperature exceeded 46°C. In summary, these results indicate that magnolol can bind to the SARS-CoV-2S protein and interact with the S2 subunit. Therefore, the S2 subunit may be the target of magnolol in inhibiting membrane fusion.

[0064] 3.2 Magnolia officinalis destroys the secondary structure of HR2 in the S2 subunit and the stability of HR2

[0065] During the process of viral membrane fusion and invasion of host cells, the viral S protein will have a specific interaction with the host cell membrane. Specifically, HR1 and HR2 in the viral S2 subunit will assemble into a hexameric structure, namely 6-HB. The formation of this structure can shorten the distance between the virus and the host cell membrane, thereby promoting the fusion of the virus and the host cell membrane. In order to study the effect of magnolol on HR, we synthesized SARS-CoV-2HR1 and HR2 peptides.

[0066] Circular dichroism spectroscopy was used to detect the secondary structures of HR1, HR2, and 6-HB formed by HR1 and HR2. In the secondary structure of proteins, peptide bonds are highly ordered, and the direction of the arrangement determines the splitting of the peptide bond energy level transition, so the positions and absorption strengths of the CD bands produced by different secondary structures are different. It is generally believed that the characteristic peaks of the α-helix structure appear around 208 and 222nm; the CD spectrum of β-folding is not constant, generally with a larger positive band at 190-200nm and a negative band at 210-225; random curling generally has a negative band at 198nm and a short and wide peak near 220nm.

[0067] The results are as follows Fig.10 As shown in A, the circular dichroism spectrum of 6-HB formed by mixing HR1 and HR2 shows typical double negative peaks at 222nm and 208nm, which is the manifestation of the -helical conformation, which is consistent with the literature reports, indicating that the circular dichroism spectrum detection method and conditions used in the experiment are correct.

[0068] The effect of magnolol on the secondary structure of 6-HB formed by HR1 and HR2 was then tested, and the results showed that ( Fig.10B), the secondary structure of 6-HB in the control group (HR1 / HR2) showed a typical -helix structure, and the -helix ratio accounted for 84.9% of the total conformation. After intervention with different concentrations of magnolol, the -helix structure ratio of 6-HB decreased to 69.1% (150μM magnolol) and 39.0% (300μM magnolol), respectively; while the β-fold ratio accounted for 15.1% of the total conformation. After intervention with different concentrations of magnolol, the β-fold ratio of 6-HB increased to 19.0% (150μM magnolol) and 32.4% (300μM magnolol), respectively, indicating that with the increase of magnolol concentration, the secondary structure of 6-HB gradually changed from α-helix to β-fold, indicating that in the phosphate buffer system, the binding of magnolol to 6-HB will cause the 6-HB peptide chain structure to stretch rapidly, the structure becomes loose, and lead to changes in the secondary spatial conformation.

[0069] The effect of magnolol on the secondary structure of HR1 was further tested. The results showed that ( Fig.10 C), after intervention with different concentrations of magnolol, the proportion of -helix structure decreased from 62.0% to 59.2% (150μM magnolol) and 29.1% (300μM magnolol); and the proportion of β-folded structure decreased from 30.6% to 0%, respectively, indicating that in the phosphate buffer system, with the addition of magnolol, the secondary structure of HR1 becomes loose, resulting in changes in the secondary spatial conformation. In addition, the effect of magnolol on the secondary structure of HR2 was detected, and the results showed that ( Fig.10 D), the -helix ratio accounted for 31.4% of the total conformation. After intervention with different concentrations of magnolol, the -helix structure ratio of HR2 increased to 61.5% (150μM magnolol) and 53.0% (300μM magnolol), respectively; while the β-fold ratio accounted for 34.0% of the total conformation, and the β-fold structure ratio of HR2 decreased to 13.7% (150μM magnolol) and 22.1% (300μM magnolol), respectively, indicating that with the increase of magnolol concentration, the secondary structure of HR2 gradually changed from β-fold to α-helix structure, which indicates that in the phosphate buffer system, magnolol binds to HR2 to cause HR2 to unfold, and the peptide chain structure becomes more compact, resulting in changes in the secondary spatial conformation. The above results suggest that magnolol can affect the secondary structure of HR1 and HR2, thereby inhibiting the formation of 6-HB.

[0070] In order to explore the effect of magnolol on HR stability, HR1, HR2 and 6-HB were quantitatively evaluated using native-PAGE electrophoresis. After incubation of HR1 and HR2 for 1 hour after treatment with different concentrations of magnolol, 6-HB expression (upper band) and HR2 expression (lower band) were detected. Since HR1 has a positive charge (charge: +1), the direction of movement in electrophoresis is opposite to the direction of electron movement and moves away from the gel direction, so no band appears. The results show that with the increase of magnolol concentration, the expression level of 6-HB gradually decreases ( Fig.11 A). The detection of HR1 and HR2 content found that with the increase of magnolol concentration, the band intensity of HR1 did not change ( Fig.11 B), HR2 expression level gradually decreased ( Fig.11 C) These results indicate that, unlike the results of the detection of HR spatial conformation, magnolol only destroyed the stability of HR2, thereby inhibiting the formation of 6-HB, and did not affect HR1.

[0071] 3.3 Binding site of magnolol to HR2 region

[0072] According to previous experimental results, magnolol can destroy the stability of HR2 and cause it to lose its biological function. However, the specific amino acid site where magnolol acts on HR2 is still unclear. Based on this, we used molecular docking technology to predict the binding site of magnolol and HR2. HR2 protein (PDB: 6LVN) is composed of 36 amino acids. In the docking experiment, based on the binding free energy analysis in the molecular docking results, we determined the optimal binding conformation and conducted further interaction analysis on it. The binding conformation of magnolol at the active pocket of HR2 is shown in the figure. Fig.12 As shown, a hydrogen bond was formed between magnolol and E1188 in HR2, and the binding free energy between magnolol and HR2 was -6 kcal / mol. This indicates that magnolol can interact with HR2, and its binding site is located at E1188, which in turn affects the viral membrane fusion process.

[0073] According to the experimental results predicted by molecular docking, we constructed HR2 with glutamic acid mutation (E1188A) by replacing glutamic acid (E) at position 1188 in HR2 with alanine (A), and compared the effects of magnolol on the mutant HR2 to further clarify the effects of magnolol on the structure and function of HR2. The experimental results showed that in the experiment of forming 6-HB with E1188A mutant Mut-HR2, the formation of 6-HB was significantly weakened, indicating that the glutamic acid residues in HR2 play an important role in maintaining the formation of 6-HB, and the E1188A mutant Mut-HR2 inhibited the formation of 6-HB to a certain extent ( Fig.13A); and compared with the Control, the expression of 6-HB and HR2 in the magnolol intervention group did not change significantly ( Fig.13 B), indicating that magnolol acts on the mutated amino acid (E1188A) and binds to HR2 to inhibit the formation of 6-HB. In general, disruption of HR2 is an important target for magnolol's antiviral membrane fusion activity.

[0074] In highly contagious and highly variable coronaviruses such as SARS-CoV-2, the proteins responsible for the fusion of the virus with the host cell membrane are often highly conserved, especially the HR1 and HR2 regions of the viral S2 subunit. These regions are not prone to mutation and can be used as one of the effective targets for the development of broad-spectrum antiviral drugs. At present, most peptidomimetic membrane fusion inhibitors are designed based on these HR sequences, and their mechanism of action is mainly to prevent the fusion of the virus with the host cell through competitive inhibition. However, this study found that the mechanism of action of magnolol in inhibiting membrane fusion is completely different from that of peptidomimetic drugs. It can target the glutamic acid residues in HR2, thereby directly destroying the structure of the S protein HR2 and destroying the integrity of the virus. This mechanism of action that targets the virus and changes its structure can directly affect the activity of the progeny virus.

[0075] in conclusion

[0076] In this study, we found that magnolol can inhibit the membrane fusion between the virus and the host cell mediated by the coronavirus S protein by acting on the highly conserved HR2 region in the coronavirus S protein, thereby preventing the virus from invading the host cell. Therefore, magnolol is expected to become a potential broad-spectrum anti-coronavirus drug, which is of great significance for the possible emergence of SARS-CoV-2 variants and similar coronavirus epidemics in the future.

[0077] 2. Materials and methods required in the above experimental process

[0078] 1. Synthesis of SARS-CoV-2 virus-like particles

[0079] 1) Plasmid construction and molecular cloning

[0080] The gene sequences of human codon-optimized SARS-CoV-2 S (Gene ID: 43740568), M (Gene ID: 43740571), E (Gene ID: 43740570), and N (Gene ID: 43740575) proteins were synthesized, and a Flag tag was added to the carboxyl terminus of each protein, with the polypeptide sequence DYKDDDDK. In addition, we synthesized a vector plasmid containing firefly luciferase, which embedded the RNA packaging sequence PS9 (nt 20080-21171) in its 3' untranslated region (UTR). At the same time, a new pcDNA3.1+PA2 plasmid was designed and synthesized, which can encode both the M and E proteins of SARS-CoV-2. All these plasmids were used to prepare SARS-CoV-2 luciferase-tagged virus-like particles (Luc-VLPs).

[0081] For further study, we constructed chimeric reporter plasmids encoding Renilla luciferase (RL) and green fluorescent protein (GFP), named DSP1-7 and DSP8-11, respectively. The construction of these plasmids was based on the pcDNA3.1 expression vector, and the restriction sites of NheI and NotI restriction endonucleases were introduced at the 5' and 3' ends of the vector, respectively. The target gene sequence was successfully cloned into the expression vector through double restriction digestion and T4 DNA ligase operation.

[0082] For the transformation process of the plasmid, the chemically competent cells DH5α were treated with heat shock. First, the target DNA was added to the cells and then mixed in an ice bath for 30 minutes. Subsequently, the cells were heat shocked in a 42°C water bath for 1 minute and then quickly cooled for 2 minutes. After that, sterile LB medium was added and incubated at 37°C and 200rpm for 45 minutes. Then, the cells were centrifuged for 3 minutes, spread on LB culture plates containing ampicillin, and cultured at 37°C overnight. Finally, the surviving single clones were selected for expansion, and the correctness of the plasmid was confirmed by restriction digestion and DNA sequencing to prepare for subsequent experiments.

[0083] 2) Construction and purification of SARS-CoV-2 luciferase-labeled virus-like particles

[0084] To construct SARS-CoV-2Luc-VLPs, HEK-293T cells were inoculated on cell culture plates, and plasmids expressing SARS-CoV-2 spike (S) protein, membrane (M) protein, envelope (E) protein, nucleocapsid (N) protein and luciferase expression plasmids with Flag tags were transfected into the cells simultaneously using PEI transfection reagent. The luciferase plasmid transfection accounted for 1 / 2 of the total transfected plasmid mass, and the molar ratio of S:M+E:N transfection was 1:1:1. After 48 hours of culture, SARS-CoV-2Luc-VLPs in cell culture supernatant and cell lysate were extracted and purified by sucrose density gradient centrifugation.

[0085] Luc-VLPs were extracted and purified from the cell lysate. The cell suspension was scraped by repeated freezing and thawing of cells in liquid nitrogen, and then centrifuged at 13,000 rpm for 15 minutes at 4°C to obtain the supernatant. 1 mL of 20% sucrose solution was added to a 2 mL EP tube, and the collected supernatant was slowly added to the wall. The tube was centrifuged at 21,690 × g at 4°C for 7 hours, the solution in the tube was discarded, PBS was added for resuspending, the protein concentration was measured using NanoDrop, and the tube was stored at 4°C for later use.

[0086] Extract and purify Luc-VLPs from the cell culture supernatant, centrifuge the collected cell culture supernatant at 1000 rpm for 15 minutes at 4°C; transfer the collected supernatant to a new 50 mL centrifuge tube, centrifuge at 2000×g for 15 minutes at 4°C, and filter using a 0.45 um filter; prepare an ultra-high-speed centrifuge tube, slowly add 20% sucrose solution, then slowly add the collected supernatant, centrifuge at 28000 rpm for 3 hours at 4°C, discard the solution in the tube, add PBS to resuspend; use NanoDrop to measure the protein concentration, and store at 4°C for later use.

[0087] 2. Animal experiments

[0088] 1) Experimental animals

[0089] 8-week-old male SPF hACE2-All CDS-B6J transgenic mice, weighing (21.2±1.06g), were purchased from Saiye (Suzhou) Biotechnology Co., Ltd., license number: SCXK (Suzhou) 2022-0016. Rats and mice were kept in the SPF animal room of the Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, license number: SYX (Beijing) 2021-0017. Mice were caged, 4-6 / cage, a total of 8 cages, temperature 24.0±2℃, relative humidity 40-70%, 12h / 12h light and dark alternation, free drinking water and regular feed were allowed. The experimental protocol was approved by the Experimental Animal Ethics Committee of the Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, approval number: IBTCMCACMS21-2111-01. This experiment was carried out in strict accordance with the requirements of experimental animal ethics review.

[0090] 2) Preparation of drug solution

[0091] First, PBS solution was used to prepare 0.5% carboxymethylcellulose sodium (CMC-Na), and the solution was vortexed to mix.

[0092] Preparation of UDCA suspension: First, UDCA (Sigma) was dissolved in PBS (phosphate buffered saline) to a concentration of 80 mg / mL. Then, the solution was vortexed and stored at 4°C. Before use, the suspension was equilibrated to room temperature, and the drug solution was aspirated and vortexed before oral administration.

[0093] Preparation method of magnolol suspension: Use 0.5% CMC-Na to prepare magnolol (source leaf biological) suspension with a concentration of 10 mg / mL, and vortex mix. The suspension is stored at 4°C, and needs to be equilibrated to room temperature before use, and vortex mix before taking the solution before gavage.

[0094] 3) Animal Experimental Protocol

[0095] The experiment was started after the mice were adaptively fed for 5 days. The mice were divided into 4 groups, namely: blank group, model group (0.5% CMC-Na, dose of 10 mL / kg / d), positive control UDCA group (gavage dose of 0.693 g / kg / d), magnolol group (gavage dose of 100, 200, 400 g / kg / d). Among them, the mice in the model group and UDCA group were gavaged for 7 consecutive days, and the mice in the magnolol group were gavaged for 4 consecutive days. On the 4th day of gavage in the model group and UDCA group, gavage was started on the magnolol group so that all mice ended gavage on the same day. Within 2 hours after the last oral gavage, mice were anesthetized with xylazine hydrochloride (10 mg / kg, Huamu) and Shutai (75 mg / kg, Virbac, France), the throat skin was cut open, blunt separation was performed, and after exposing the trachea, 50 μL of SARS-CoV-2Luc-VLPs at a concentration of 15 mg / mL were injected into the mouse lungs using an insulin syringe. Sutures were then performed. Six hours after the injection, the mice were killed. Lung lavage was performed 2-3 times with PBS to remove residual Luc-VLPs.

[0096] Method for detecting the content of SARS-CoV-2Luc-VLPs in mouse lungs: First, the right lung of the mouse was separated and weighed. Then, 100 mg of lung tissue was added to 500 μL of RIPA lysis buffer (ThermoFisher) containing protease inhibitors (Roche) and PMSF (Solerbo). Next, the sample was ground using a frozen grinder. Centrifuge at 13,000 rpm for 15 minutes at 4°C to collect the supernatant. The protein concentration in the sample was detected using Nanodrop, and the protein concentration of each group of samples was adjusted to be consistent using lysis buffer. Finally, the luciferase level in the lysate was detected using the Luciferase Assay System (promega). Relative to the blank group, by calculating the relative luciferase level, the relative SARS-CoV-2Luc-VLPs content in the lungs can be represented.

[0097] Method for observing the distribution of SARS-CoV-2Luc-VLPs in mouse lungs by immunofluorescence: First, we isolated the lungs of mice and placed them in 10% neutral formalin fixative (Solerbo) and fixed them at 4°C for 48-72 hours. Next, the tissue was pathologically sectioned using the frozen section technique. After the tissue was removed, it was embedded in OCT embedding medium (Sakura), and the sample was slowly frozen to -20°C. Then, a frozen microtome was used to slice at a thickness of 10μm, and the slices were attached to adhesive slides (Shitai). Staining was started after obtaining enough slice samples. First, the slices were soaked in ddH2O for 5 minutes to remove the embedding agent. Then, the tissue was circled using a PAP pen (abcam). Next, 0.2% Triton X-100 (Sigma) was used to break the membrane at room temperature for 15 minutes. Then, 1% BSA (PBS solution) was used to block at room temperature for 1 hour. Subsequently, the sections were incubated overnight at 4°C using a primary antibody solution containing anti-Flag antibody (1:1000, MBL) and anti-hACE antibody (1:1000, proteintech). The next day, the sections were washed three times with PBS for 5 minutes each time. Then, a 1% BSA solution containing fluorescent secondary antibody and Phalloidin-647 (1:1000, abcam) was incubated at room temperature in the dark for 1 hour. The sections were washed again with PBS three times for 5 minutes each time. Next, a PBS solution containing DAPI was incubated at room temperature for 10 minutes. Finally, the sections were sealed with an anti-fluorescence quenching sealant (abcam) and dried overnight at room temperature. We observed and photographed the sections using a laser confocal microscope (Olympus).

[0098] The method for detecting the expression level of protein receptors related to virus invasion in mouse lungs is as follows: First, take a sample of RIPA lysate from mouse lungs and add β-mercaptoethanol (100×, Sigma) and LDS sample buffer (4×, GenScript Biotech) to it. Heat the mixture to 95°C for 10 minutes and then cool it at room temperature. Next, use 4-20% SurePAGE precast gel to electrophoretically separate the sample in Tris-MOPS buffer (GenScript Biotech) at a constant voltage of 180V for 1-1.5 hours. After the electrophoresis, the separated proteins were transferred to a PVDF membrane (Millipore) at a constant current of 300mA for 2 hours. After the transfer, use 5% BSA in TBST solution (Solebo) to block on a shaker at room temperature for 2 hours. Then, the target protein band was cut out and incubated overnight at 4 ° C in a TBST solution containing 3% BSA containing hACE2 (1:1000 dilution), TMPRSS2 (1:1000 dilution, proteintech) and β-actin antibody (1:10000 dilution, proteintech). The next day, the TBST solution was used to wash 3 times, 10 minutes each time. Then, a 3% BSA TBST solution containing the corresponding HRP-labeled secondary antibody was used to incubate on a shaker at room temperature for 2 hours. The TBST solution was used to wash again 3 times, 10 minutes each time. Finally, the gel was developed using an ultrasensitive ECL luminescent solution in a gel imaging system and photographed and recorded.

[0099] 3. Differentiation of human AECⅡ alveolar organoids from human induced pluripotent stem cells

[0100] In this study, we used a specific method to culture and differentiate hiPSCs. First, TeSRTM-E8TM medium (Stemcell) was used to maintain the growth of hiPSCs on Matrigel (Stemcell)-coated culture plates until the cells reached 90% confluence. Then, to induce hiPSCs to differentiate into definitive endoderm (DE), DE differentiation medium was used for 3 days; DE differentiation medium consisted of RPMI 1640 medium (Gibco), Activin A (Novoprotein), CHIR99021 (Selleck Chemicals), NeuroCult SM1neuronal supplement (Stem Cell Technologies), and GlutaMAX (Gibco).

[0101] Subsequently, the cells were differentiated into the intestinal foregut endoderm (AFE); the cells were digested using GentleCell Dissociation Reagent, and then passaged into Matrigel (Corning)-coated culture plates, and then cultured for 1 to 4 days using AFE differentiation medium prepared with complete serum-free differentiation medium (cSFDM). AFE differentiation medium consists of cSFDM, Noggin (Novoprotein), and SB431542 (Selleck Chemicals). cSFDM is a mixture of IMDM medium (Gibco) and Ham's F12 medium (Gibco), with the addition of BSA (Sigma), ascorbic acid (Wako Pure Chemical Industrials), 1-Thioglycerol (Sigma), N2 supplement (Stem Cell Technologies), NeuroCult SM1 neuronal supplement, and 1×GlutaMAX.

[0102] Next, the cells were differentiated into NKX2-1 positive lung progenitor cells and cultured for 8-9 days using lung progenitor differentiation medium. The lung progenitor differentiation medium consists of cSFDM, BMP4 (Selleck Chemicals), CHIR99021 (Selleck Chemicals), and RA (Selleck Chemicals). Finally, the cells were differentiated into alveolar epithelial type II cells (AECⅡ) and formed alveolospheres. The cell suspension was mixed with Matrigel and spread on a culture plate and cultured using CK+DCI medium, i.e. cSFDM containing CHIR99021, KGF (Novoprotein), dexamethasone (SelleckChemicals), 8-Bromo-cAMP (Selleck Chemicals), and 3-Isobutyl-1-methylxanthine (Selleck Chemicals). During the culture process, the cells were passaged according to the size and density of the spheres.

[0103] 4. Firefly luciferase activity detection of intracellular SARS-CoV-2Luc-VLPs content

[0104] The firefly luciferase reporter assay system was used to detect the efficacy of magnolol in inhibiting the invasion of SARS-CoV-2Luc-VLPs into human alveolar organoids. First, human AECⅡ or Vero E6 cells or 293F-ACE2-TMPRSS2 cells were cultured on a cell plate; then SARS-CoV-2Luc-VLPs and drugs were added to human AECⅡ alveolar organoids, Vero E6 cells and 293F-ACE2-TMPRSS2 cells and incubated for 6 hours; then the cells were washed with PBS, digested and collected with Cell recovery solutions, and PLB lysis solution (promega) was added to lyse at room temperature for 20 minutes, and then centrifuged at 13000 rpm for 30 seconds to collect the cell lysate. Then 20 μL of cell lysate was added to each well of a white-walled 96-well plate, followed by 100 μL of Luciferase assay buffer (promega), and the intensity of the bioluminescent signal was detected using a microplate reader (ThermoFisher).

[0105] 5. Dual Split Protein (DSP) Cell-Cell Membrane Fusion Experiment

[0106] The membrane fusion between cells expressing the S protein of SARS-CoV-2 and its variants and cells expressing ACE2 receptor and TMPRSS2 was evaluated by observing the GFP fluorescence intensity and luciferase activity. The S protein plasmid of SARS-CoV-2 and its variants and the DSP 1-7 vector expressing Renilla luciferase or GFP plasmid were transfected into effector cells, i.e., 293T cells; TMPRSS2 and the DSP 8-11 vector expressing Renilla luciferase or GFP plasmid were transfected into target cells, i.e., 293F-ACE2 cells. Finally, the target cells were digested into a cell suspension and mixed with magnolol, and then added to the effector cells for co-culture for 4 hours. The GFP signal was observed using a fluorescence microscope (Zeiss) to evaluate the range of cell-cell fusion. The degree of cell fusion was then quantitatively analyzed by detecting luciferase activity using the firefly luciferase reporter assay system (Promega). The culture medium in the wells was aspirated, and 20 μL of RLB lysis buffer (Promega) was added to each well, and the cells were lysed on a horizontal shaker for 20 minutes. The wells were then transferred to a white-walled 96-well plate, 100 μL of Luciferase assay buffer (Promega) was added, and the intensity of the bioluminescence signal was detected using a microplate reader (ThermoFisher).

[0107] 6. Cell thermal transfer experiment

[0108] Cellular thermal shift assay (CETSA) is a method for detecting the binding of small molecule compounds to SARS-CoV-2S protein. In this experiment, HEK-293T cells were inoculated in a culture dish, and then SARS-CoV-2S protein particles were transfected into the cells and cultured continuously; 48 hours after transfection, DMSO or magnolol was added for incubation for 6 hours, and the cell suspension was collected by cell scraping, heated for 3 minutes in a gradient, cooled for 2 minutes at room temperature, and then placed on ice; the culture medium was aspirated, the cells were washed with PBS, and the cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail and PMSF, placed in liquid nitrogen for repeated freeze-thaw lysis 3 times, and centrifuged at 4°C, 13000rpm for 15 minutes, and the supernatant was extracted and LD containing β-mercaptoethanol was added The samples were boiled with S loading buffer and subjected to 160V constant voltage electrophoresis on 4-20% SDS-PAGE gel (GenScript Biotech). After electrophoresis, the samples were transferred to PVDF membrane (Milipore) by 300mA constant current wet method, and then blocked with 5% BSA. Rabbit anti-Flag-tag antibody (1:1000, MBL) was added and incubated overnight at 4°C. After washing with TBST, HRP-labeled goat anti-rabbit IgG secondary antibody (1:4000, proteintech) was added and incubated at room temperature for 2 hours. After washing with TBST, ECL chemiluminescent reagent (Hanqing) was added for color development, and the ChemiDoc MP imaging system (Bio-Rad) was used to develop and acquire images.

[0109] 7. Molecular docking experiment

[0110] The molecular docking experiment was performed using AutoDock V4.2 software. First, the HR2 protein structure (PDB: 6LVN) was preprocessed, including removing water molecules, adding hydrogen atoms, calculating Gasteiger charges, and repairing missing residues. Then, appropriate Grid Box parameters were set according to the active catalytic center of the enzyme to ensure that it was contained inside the box. Next, docking analysis of 10 binding modes was performed for each ligand, and the binding affinity of the molecule was calculated. Based on the docking conformational results, binding cluster analysis was performed to explore the effects of free energy, hydrogen bonding, hydrophobicity, and van der Waals forces on the conformational stability of the complex, in order to reveal the mechanism of mutual binding between magnolol and HR2.

[0111] 8. Circular dichroism spectroscopy

[0112] Circular dichroism spectroscopy is a fast, simple and relatively accurate method that is widely used to determine protein secondary structure. This experiment aims to detect the secondary structure of 6-HB and HR1 and HR2 using circular dichroism spectroscopy and evaluate the effect of magnolol on them. HR1 and HR2 used in the experiment are peptide samples dissolved in NaH2PO4 / Na2HPO4 / NaF buffer, while magnolol is a drug solution dissolved in isopropyl alcohol (IPA).

[0113] In the experiment to study the effect of magnolol on the formation of 6-HB by HR1 and HR2, magnolol (150 μM, 300 μM) or isopropanol (1%) was first incubated with HR2 at 37°C for 1 hour. Then HR1 was added and incubated at 37°C for 30 minutes to promote the formation of 6-HB by HR1 and HR2. The final concentrations of HR1 and HR2 were both 10 μM.

[0114] When studying the effect of magnolol on the secondary structure of HR1 or HR2, magnolol (150 μM, 300 μM) or isopropanol (1%) was incubated with HR1 or HR2 at 37° C. for 1 hour. The final concentrations of HR1 and HR2 were both 10 μM.

[0115] After incubation, the samples were detected using a JASCO J-815 circular dichroism spectrometer. The scanning parameters were set as follows: the detection temperature was room temperature, the scanning wavelength range was 195nm-260nm, the bandwidth was 1nm, the step was 0.5nm, and the scanning speed was 50nm / min. 300μL of sample was added to a 1mm pathlength cuvette for scanning. The data was processed using Spectra Manager (V2.08.04, JASCO) software and the secondary structure level of the sample was calculated.

[0116] 9. Non-denaturing gel electrophoresis

[0117] Take an appropriate amount of polypeptide and use a distilled water solution containing NaH2PO4 (100mM, sigma), Na2HPO4 (100mM, sigma) and NaF (500mM, sigma) to prepare a polypeptide solution with a concentration of 200μM for HR1 (GenScript Technologies) and 80μM for HR2 (GenScript Technologies).

[0118] In this experiment, native-PAGE (N-PAGE) was used to evaluate the effect of magnolol on the stability of 6-HB. N-PAGE distinguishes different proteins by their charge, conformation, and molecular weight. Therefore, we can easily distinguish the 6-HB hexamer formed by HR1 and HR2 from HR1 / 2 alone by native gel electrophoresis. The specific experimental steps are as follows: First, HR1 or HR2 was incubated with 1% DMSO or different concentrations of magnolol (100μM, 200μM, 300μM) at 37°C for 1 hour. After the incubation, HR2 or HR1 was added and continued to incubate at 37°C for 30 minutes to allow HR1 and HR2 to form 6-HB. In this experiment, the final concentration of HR1 was 100μM and the final concentration of HR2 was 40μM. After the incubation, 4× native loading buffer was added. Electrophoresis was performed using a 10% Bis-Tris protein precast gel (GenScript Biotech) without SDS, at a constant voltage of 125 V, in an ice-water bath for 2 hours. After the electrophoresis, the gel was removed and incubated on a shaker at room temperature for 20-30 minutes using a second blue protein staining solution (AQ Aoqing) until the bands were clearly visible. Finally, the ChemiDoc MP Imaging System developer was used for imaging and photographing. ImageJ (V1.51) was used for quantitative analysis of the bands.

[0119] Secondly, this experiment evaluated the effect of magnolol on the stability of HR1 and HR2. First, HR1 (100 μM) or HR2 (40 μM) was incubated with DMSO (1%) or different concentrations of magnolol (150 μM, 300 μM, 600 μM) at 37 ° C for 1 hour. After the incubation, electrophoresis was performed using a 10% Bis-Tris protein precast gel without SDS. The electrophoresis conditions were 125V constant voltage and performed in an ice water bath for 2 hours. After the electrophoresis is completed, staining can be performed with a staining solution (such as a second blue protein staining solution) and imaging equipment can be used for imaging and recording. For the HR1 sample, since it carries a positive charge, the positive and negative poles of the power supply need to be inverted during electrophoresis. The electrophoresis conditions are 125V constant voltage and performed in an ice water bath for 4 to 5 hours.

[0120] Example 2: The present invention found that magnolol inhibits viral replication and transmission by inhibiting the ExoN enzyme activity in NSP14. The specific experimental content is:

[0121] 1. Experimental results:

[0122] 1. Inhibitory effect of magnolol on viral replication

[0123] In order to explore whether magnolia officinalis affects viral replication, it is necessary to clarify whether magnolia officinalis affects the nonstructural protein (NSP) that mediates viral replication, and further clarify the molecular mechanism by which magnolia officinalis inhibits viral replication.

[0124] 1.1 Magnolia officinalis inhibits NSP to prevent viral replication

[0125] After coronavirus enters the host cell, the virus's NSP (NSP1 to NSP16) forms a replicase-transcriptase complex, which plays a key role in the virus's replication and transcription process. Therefore, by constructing a mini-replicon that co-expresses several or all NSP functional modules, we can partially reproduce the key links of virus replication, make up for the deficiency that Luc-VLPs cannot simulate the virus replication link, and help to clarify the effect of magnolol on different virus replication links.

[0126] like Fig.14 As shown, compared with the Control group, after 24 hours of treatment with 30μM magnolol (Mag), the luciferase activity of the replicon system decreased by 35.2%, which was statistically significant, while the luciferase activity of the replicon system in the positive control group (Nirmatrelvir) decreased by 37.2%; among them, the luciferase activity of NSP12-16 in the replicon system decreased by 33.3% after magnolol treatment, which was statistically significant. The above results show that magnolol can significantly inhibit the replication of SARS-CoV-2, and may inhibit the expression of NSP12, NSP13, NSP14, NSP15 and NSP16.

[0127] In order to exclude the possibility that magnolol may reduce the activity of luciferase in the experiment, thereby causing false positive results, we further tested the effect of magnolol on the expression and activity of luciferase (M-Luc). Fig.15 As shown, compared with the group without drug intervention, there was no significant change in luciferase activity after 24 hours of treatment with Magnolia bark phenol (Mag), Sofalcone (Sofalcone), Remdesivir (Remdesivir), and Nirmatrelvir (Nirmatrelvir). The above results indicate that these drugs did not affect the expression of luciferase itself, but inhibited the formation of SARS-CoV-2 replicons, rather than due to transfection efficiency or other factors.

[0128] 1.2 Magnolia officinalis inhibits NSP14 exoribonuclease that mediates viral replication

[0129] 1.2.1 Interaction between magnolol and NSP14

[0130] Previous replicon system detection results showed that magnolol has a potential inhibitory effect on NSPs (NSP12 to NSP16) in the SARS-CoV-2 replicon system. However, the specific mechanism by which magnolol inhibits which non-structural protein (NSP) is still unclear.

[0131] Through the CETSA experiment, we can determine whether magnolol interacts with NSP by detecting the degree of effect of magnolol on the stability of SARS-CoV-2 NSP ( Fig.16 ). First, the expression of each NSP decreased with the increase of temperature, and the expression level of NSP14 in the magnolol-treated group was significantly higher than that in the control group at 52°C and 55°C, indicating that magnolol increased the thermal stability of SARS-CoV-2 NSP14 and interacted with SARS-CoV-2 NSP14. For other NSPs, no significant expression differences were observed between the magnolol-treated group and the control group.

[0132] SPR detection of the interaction between magnolol and SARS-CoV-2 NSP14 ( Fig.17 ), and found that the equilibrium dissociation constant KD value of magnolol and NSP14 was 51.4 μM. The lower the KD value, the higher the affinity, suggesting that magnolol and NSP14 have a strong binding.

[0133] Therefore, the thermal stability and SPR experimental results based on protein interactions indicate that NSP14 may be the target of magnolia bark phenol's anti-coronavirus action.

[0134] 1.2.2 Magnolia officinalis disrupts the secondary structure of NSP14

[0135] Previous experiments have shown that magnolol can interact with the virus NSP14. In order to explore how magnolol affects the secondary structure of NSP14. We used circular dichroism spectroscopy to detect the changes in the secondary structure of NSP14 under the action of magnolol. We can observe in detail how magnolol affects the structure and function of NSP14. This structural understanding will help us reveal the antiviral mechanism of magnolol and provide an important basis for the development of new antiviral drugs targeting NSP14.

[0136] In order to gain a deeper understanding of how magnolol affects the structure of NSP14, the secondary structure of NSP14 was detected by circular dichroism spectroscopy. The results showed that ( Fig.18), the -helix ratio accounted for 39.4% of the total conformation. After intervention with different concentrations of magnolol, the -helix structure ratio of NSP14 increased to 44.7% (150μM magnolol) and 80.8% (300μM magnolol), respectively; while the β-sheet ratio accounted for 8.2% of the total conformation, and the β-sheet structure ratio decreased to 0%. The above results show that magnolol binds to NSP14 to cause NSP14 to unfold, and the secondary structure of NSP14 changes from β-sheet to α-helix structure. The peptide chain structure of NSP14 becomes more compact, resulting in changes in the secondary spatial conformation.

[0137] 1.2.3 Magnolia officinalis inhibits NSP14 exoribonuclease activity

[0138] During viral replication, NSP14 participates in the proofreading and cap structure synthesis of viral RNA through its bifunctional enzyme activity, namely exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase), thereby ensuring accurate viral replication and immune escape. In order to gain a deeper understanding of the interaction between magnolol and SARS-CoV-2 NSP14, especially which enzyme functional region in NSP14 is affected by magnolol, we constructed a recombinant plasmid expressing NSP14-ExoN and N7-MTase.

[0139] CETSA assay was used to detect whether magnolol interacts with ExoN and N7-MTase proteins of NSP14. Fig.19 As shown in the figure, the expression of NSP14-ExoN protein decreases with the increase of temperature. At 52℃, the expression level of NSP14-ExoN protein in the magnolol group was significantly higher than that in the Control group, indicating that magnolol increased the thermal stability of NSP14-ExoN and interacted with the NSP14-ExoN domain. The sequence and structure of the NSP14-ExoN domain in coronaviruses are significantly different from ExoN in most host cells. In most coronaviruses, the ExoN catalytic site and its surroundings are highly conserved. These characteristics make NSP14 an ideal potential target for broad-spectrum antiviral drugs.

[0140] The expression of NSP14 N7-MTase protein also decreased with the increase of temperature. Compared with the Control group, there was no significant difference in the expression level of NSP14 N7-MTase protein in the magnolol group, indicating that magnolol did not bind to NSP14 N7-MTase.

[0141] Based on previous experimental results, we determined that magnolol acts on NSP14-ExoN. During viral replication, the newly generated viral RNA chain needs to be proofread by ExoN to ensure accurate replication of viral RNA. In order to explore whether magnolol inhibits ExoN activity, we synthesized SARS-CoV-2ExoN. The effect of magnolol on the activity of SARS-CoV-2ExoN was detected by the fluorescence resonance energy transfer (FRET) method.

[0142] like Fig. 20 As shown in A, compared with the Control group, after adding NSP14 (100nM) protein, the fluorescence intensity increased significantly, which was comparable to the fluorescence intensity of the ribonuclease (RNase) group, indicating that the experiment was successfully constructed; and after NSP14 was treated with magnolol, its fluorescence intensity decreased significantly, indicating that magnolol can effectively inhibit the enzyme activity of NSP14-ExoN. In addition, as a member of the DEDD exonuclease superfamily, NSP14-ExoN depends on four conserved acidic residues (aspartic acid D and glutamic acid E) and some metal ions (Mg 2+, ... 2+ , Mn 2+ 、Co 2+ and Zn 2+ Therefore, the experimental results show that ethylenediaminetetraacetic acid (EDTA) (2.5 μM) as Mg 2+ The chelating agent can significantly inhibit the enzyme activity of ExoN, and the fluorescence intensity of the EDTA-treated group decreased significantly.

[0143] According to previous literature reports, sofalcone has been shown to have the ability to inhibit ExoN activity and can be regarded as a positive control drug for inhibiting ExoN. Fig. 20 As shown in B, after adding magnolol to intervene in NSP14, its fluorescence intensity decreased significantly and showed a dose-dependent relationship. 50 was 21.86±11.63μM, and the fluorescence intensity of the positive control sofalcone group also decreased significantly. 50 It is 16.31±7.88μM, which shows that magnolol can significantly inhibit the activity of NSP14 Exon.

[0144] 1.2.4 Binding site of magnolol and NSP14 exoribonuclease

[0145] ExoN is a highly conserved enzyme that can serve as an important target for broad-spectrum anti-coronaviruses. In order to support magnolol as a potential broad-spectrum anti-coronavirus drug, we need to provide more sufficient evidence to clarify the specific action area of ​​magnolol on NSP14-ExoN. Studies have shown that mutations in the active motif and zinc finger domain of the NSP14-ExoN domain can affect the replication accuracy and stability in SARS-CoV-2 and MERS-CoV, thereby reducing the fitness of the virus. Specifically, the key active sites associated with the function of the ExoN enzyme include motif I (D90 and E92), motif II (E191 and D243), and motif III (H268 and D273). These residues are essential for viral replication, and mutations in these key residues will result in the loss or significant reduction of ExoN activity. In this study, alanine (A) was substituted for residues D90, E92, E191, D243, H268, and D273 in the ExoN domain, and the CETSA experiment was used to re-evaluate whether magnolol binds to the SARS-CoV-2 NSP14-ExoN mutant protein.

[0146] like Fig.21 As shown in the figure, the expression of each SARS-CoV-2 NSP14 mutant decreased with the increase of temperature, and the expression of each NSP14 mutant showed the following changes after 6 hours of magnolol treatment: compared with the Control group, the expression level of NSP14-D90A protein in the magnolol group was significantly increased at 55°C; the expression level of NSP14-E92A protein in the magnolol group was significantly increased at 46°C; the expression level of NSP14-E191A protein in the magnolol group was significantly increased at 52°C; the expression level of NSP14-H268A protein in the magnolol group was significantly decreased at 49°C. The expression level of NSP14-D273A protein in the magnolol group was significantly increased at 52°C. The above mutants failed to change the effect of magnolol on the thermal stability of NSP14 protein, indicating that magnolol did not bind to the amino acid residue sites in these mutants to exert its effect. However, there was no significant difference in the protein expression level of NSP14-D243A compared with the Control group, suggesting that after the aspartic acid (D) residue at this site was mutated, magnolol failed to change its thermal stability, indicating that magnolol may bind to the aspartic acid residue site, thereby inhibiting the enzyme activity of NSP14-ExoN.

[0147] 1.2.4 The NSP14 exoribonuclease active site of highly pathogenic coronavirus is highly conserved, and magnolol has a wide range of antagonistic effects on NSP14 exoribonuclease

[0148] like Fig. 22As shown in A, the comparison of the full-length protein sequences and ExoN enzyme active sites of SARS-CoV-2, SARS-CoV and MERS-CoV NSP14 proves that the six active sites of NSP14-ExoN are highly conserved and completely consistent among the three viruses. Among them, the red line marks the range of the nucleic acid exoribonuclease ExoN region of SARS-CoV-2, SARS-CoV and MERS-CoV; the gray background-black bold-red * is the active site of SARS-CoV-2, SARS-CoV and MERS-CoV ExoN enzyme. The six active sites of ExoN are highly conserved. At the biological level, the CETSA experiment was used to detect whether magnolol interacts with SARS-CoV and MERS-CoV NSP14 ( Fig. 22 B), it was found that the expression of NSP14 decreased with the increase of temperature, and the expression level of SARS-CoV NSP14 in the magnolol group was significantly higher than that in the Control group at 46℃ and 49℃, and the expression level of MERS-CoV NSP14 in the magnolol group was significantly higher than that in the Control group at 52℃, indicating that magnolol increased the thermal stability of SARS-CoV and MERS-CoV NSP14, interacted with NSP14, and had universal characteristics against coronaviruses.

[0149] 2 Magnolia officinalis enhances the inhibitory effect of remdesivir on RNA-dependent RNA polymerase

[0150] The RNA-dependent RNA polymerase (RdRp) formed by NSP12 guides the synthesis of progeny viral positive-strand RNA and subgenomic mRNA, and plays an important role in the viral replication process. Remdesivir is an antiviral drug and a nucleotide analog. Its mechanism is that after Remdesivir enters the virus, it will be recognized by the viral RdRp and inserted into the newly synthesized viral RNA chain. This insertion interferes with the normal viral RNA synthesis process, thereby exerting an antiviral effect. However, the NSP14-ExoN of SARS-CoV-2 has the ability to detect and remove erroneous RNA sequences. When Remdesivir is inserted into the viral RNA chain, NSP14-ExoN is able to recognize and remove it, which weakens the effectiveness of Remdesivir in inhibiting SARS-CoV-2 replication, resulting in its clinical efficacy against SARS-CoV-2 being greatly weakened. Therefore, inhibiting NSP14-ExoN activity can prevent RdRp from repairing errors in the viral RNA chain, ultimately enhancing the inhibitory effect of Remdesivir on viral replication. Based on previous experimental results showing that magnolia officinalis can inhibit the enzyme activity of NSP14-ExoN, we further clarified through luciferase activity detection whether magnolia officinalis can enhance the inhibitory effect of remdesivir on SARS-CoV-2RdRp by inhibiting NSP14-ExoN, thereby improving the efficacy of remdesivir against SARS-CoV-2.

[0151] The experimental results show that ( Fig.23 ), in the experiment of transfection of RdRp (NSP12) plasmid, compared with the Control, the fluorescence intensity of the magnolol intervention group and the positive control group (sofalcone intervention group) did not change significantly, while the fluorescence intensity of the remdesivir intervention group decreased, with an inhibition rate of 26.0%, which was statistically significant, suggesting that remdesivir can inhibit the expression of RdRp (NSP12).

[0152] In the experiment of simultaneous transfection of RdRp (NSP12) and NSP14 plasmids, the molar ratio of RdRp (NSP12) and NSP14 plasmids was 1:1. Compared with the Control group (NSP12+NSP14), the fluorescence intensity of the remdesivir intervention group did not change significantly, and there was no statistical difference. However, the fluorescence intensity of the remdesivir combined with magnolol intervention group decreased significantly, and its inhibition rate was 30.9%, which was statistically significant; the fluorescence intensity of the desivir combined with sofalcone intervention group decreased significantly, and its inhibition rate was 36.8%, which was statistically significant. The above results show that magnolol enhances the inhibitory effect of remdesivir on RNA-dependent RNA polymerase (RdRp) by inhibiting ExoN activity, so as to improve the antiviral efficacy of remdesivir, which is also a vivid embodiment of the combination of Chinese and Western medicine and the use of Chinese and Western medicine.

[0153] 3 Magnolia officinalis inhibits NSP14 to protect host cell immune response

[0154] Studies have found that NSP14 has the ability to inhibit host cell translation and interfere with the host cell immune response, thereby promoting viral infection. We aimed to explore whether magnolol can restore the host cell immune response by inhibiting NSP14.

[0155] 3.1 Magnolia officinalis inhibits NSP14 to protect host cell physiological functions

[0156] Studies have shown that during the replication of SARS-CoV-2, the virus inhibits protein translation and synthesis in host cells, thereby promoting the production of viral proteins. In order to explore whether magnolol restores host cell translation by inhibiting NSP14, we used puromycin incorporation experiments to elucidate the effect of magnolol on SARS-CoV-2NSP14 inhibition of intracellular protein synthesis and translation. Puromycin incorporation experiments provide a powerful tool for in-depth study of protein synthesis and translation by interrupting the growth of newly synthesized protein chains, allowing us to effectively label and track proteins being synthesized, thereby in-depth exploration of protein synthesis rate, stability, and its key role in cell biology, providing an important experimental means for analyzing cell regulatory mechanisms, disease research, and drug development.

[0157] like Fig.24 As shown, SARS-CoV-2NSP14 protein can inhibit the protein translation synthesis of host cells, and after the intervention of magnolia ol, the inhibitory effect of its protein translation synthesis was significantly reduced, indicating that magnolia ol may inhibit the expression of SARS-CoV-2NSP14, thereby restoring the protein synthesis translation of virus-infected cells.

[0158] 3.2 Magnolia nobile inhibits NSP14 to restore the host's antiviral immune response

[0159] During the replication of coronavirus, the double-stranded RNA (dsRNA) intermediates produced will trigger the body to produce a type I interferon (IFN-I) response. As the first line of defense against viral invasion, the IFN-I immune response can activate the expression of multiple interferon-stimulated genes (ISG) and exert an antiviral effect. The replication of coronavirus can antagonize the production of IFN-I and the expression of downstream antiviral ISGs. Recent studies have shown that NSP14 is one of the most effective IFN antagonists of SARS-CoV-2. Specifically, during viral replication, NSP14-ExoN can degrade viral dsRNA intermediates, thereby hindering the activation of the host's innate immune response. Secondly, studies have shown that viral RNA that is not modified by the cap structure will be recognized by host pattern recognition receptors (such as ISGs), thereby activating IFN-related antiviral immune responses.

[0160] We used Western blot to evaluate the effect of magnolol on the inhibition of IFN-α-induced endogenous ISG expression by SARS-CoV-2 NSP14. Fig.25 As shown in the figure, in the immune response with the addition of IFN-α, NSP14 can significantly inhibit the protein synthesis of antiviral ISGs (viperin, TRIM21, IFITM1, IFITM2, IFITM3) and ISGs with RNA sensing signaling functions (RIG-I, MDA5), while after the addition of magnolol, except for the expression level of IFITM2, the expression levels of other ISG proteins were significantly upregulated, indicating that magnolol can inhibit the expression of NSP14, thereby restoring the expression level of ISGs in host cells and protecting the antiviral immune ability of host cells. In addition, magnolol did not upregulate the expression level of IFITM2.

[0161] The invasion of SARS-CoV-2Luc-VLPs in 293F-ACE2-TMPRSS2 cells transfected with NSP14 after treatment with IFN and / or magnolol was evaluated by luciferase activity assay to elucidate whether magnolol inhibits viral invasion of host cells by restoring IFN expression antagonized by NSP14.

[0162] The experimental results show that ( Fig.26), the invasion rate of SARS-CoV-2Luc-VLPs in the IFN-I treatment group was significantly reduced, indicating that IFN-α induced the upregulation of antiviral ISG expression, thereby inhibiting the ability of Luc-VLPs to fuse membranes and invade cells. However, in the simulated SARS-CoV-2 reinfection experiment, after transfection with SARS-CoV-2NSP14, the invasion rate of SARS-CoV-2Luc-VLPs still increased despite the immune response induced by IFN-α treatment, indicating that NSP14 can significantly inhibit the expression of IFITM proteins, leading to an increase in the invasion rate of SARS-CoV-2Luc-VLPs, however, after the addition of magnolol, the invasion rate of Luc-VLPs decreased again. In addition, the invasion rate of Luc-VLPs in the magnolol intervention group alone did not change, which further indicates that magnolol does not exert its antiviral effect by acting on the host cells themselves, but by inhibiting the expression of NSP14 to restore the IFN-α immune response, thereby inhibiting the reinvasion of SARS-CoV-2.

[0163] In summary, the overexpression of SARS-CoV-2 NSP14 interferes with the induction of IFN-I, inhibits the expression of ISG in the innate immune response and increases the virus invasion rate. However, the intervention of magnolol can block this inhibitory effect by inhibiting the expression of NSP14, thereby reducing the immune escape of the virus, restoring the natural antiviral immune response in the host cells, and inhibiting the re-invasion of the virus, which shows that magnolol can reduce the risk of SARS-CoV-2 reinfection of the body. This is consistent with the mechanism that magnolol can inhibit virus invasion by directly destroying membrane fusion, that is, magnolol has a dual antiviral invasion mechanism, which can interfere with the process of virus immune escape and inhibit virus invasion at the same time.

[0164] in conclusion

[0165] In this study, we found that magnolol can inhibit the highly conserved exoribonuclease activity in coronavirus NSP14, thereby preventing viral replication and restoring the host cell's antiviral immune response, reducing the risk of the host being re-infected with the virus. It is worth noting that magnolol can also enhance the efficacy of remdesivir against SARS-CoV-2, producing a synergistic antiviral effect, showing its potential to become a broad-spectrum anti-coronavirus drug.

[0166] Materials and methods used in the above experiments in this example

[0167] 1. SARS-CoV-2 replicon system based on luciferase reporter system

[0168] A replicon system consisting of a recombinant plasmid expressing SARS-CoV-2 NSP was constructed as a tool for studying the viral replication process and screening viral replication inhibitors. This system encodes viral genes and fragments required for viral RNA synthesis, including SARS-CoV-2 NSP1 to NSP16 genes. Under the regulation of the M protein transcription regulatory sequence, the luciferase gene was inserted as a reporter gene. Finally, the SARS-CoV-2 replicon system was designed by carrying a CMV promoter. HEK-293T cells were first seeded in 96-well plates. Subsequently, 293T cells were transfected with different combinations of NSP plasmids, including NSP1-4 (0.08μg), NSP5-11 (0.08μg), NSP12-16 (0.08μg), RdRp (0.025μg) and NSP14 (0.03μg). M-Luc (0.02μg) plasmid was co-transfected during each transfection. 24 hours after transfection, magnolol was added to the cell culture medium for intervention, and DMSO was added to the control group. 20 μL of lysis buffer was added 24 hours after drug treatment, and the cells were gently shaken for lysis at room temperature for 20 minutes. 20 μL of lysis product was transferred to an opaque white-walled 96-well plate, 100 μL of Luciferase assay buffer (Promega) was added, and after thorough mixing, the fluorescence value was measured using an enzyme reader (ThermoFisher). This fluorescence value can accurately reflect the expression of each NSP of the virus, thereby indirectly reflecting the replication efficiency of the virus.

[0169] 2. Cell thermal transfer experiment

[0170] Cellular thermal shift assay (CETSA) is a method for detecting the binding of small molecule compounds to SARS-CoV-2S protein and NSP. In this experiment, HEK-293T cells were inoculated in a culture dish, and then the plasmids of SARS-CoV-2 non-structural proteins (NSP12-NSP16) were transfected into the cells and cultured continuously; 48 hours after transfection, DMSO or magnolol was added and incubated for 6 hours, and the cell suspension was collected by cell scraping, heated for 3 minutes, cooled at room temperature for 2 minutes, and then placed on ice; the culture medium was aspirated, the cells were washed with PBS, and the cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail and PMSF, placed in liquid nitrogen for repeated freeze-thaw lysis 3 times and centrifuged at 4°C, 13000rpm for 15 minutes, and the supernatant was extracted and added with β -Mercaptoethanol-containing LDS loading buffer was boiled, and the samples were subjected to 4-20% SDS-PAGE gel (GenScript Biotech) for constant voltage electrophoresis at 160V. After the electrophoresis, they were transferred to PVDF membrane (Milipore) by 300mA constant current wet method, and then blocked with 5% BSA. Rabbit anti-Flag-tag antibody (1:1000, MBL) was added and incubated overnight at 4°C. After washing the membrane with TBST, HRP-labeled goat anti-rabbit IgG secondary antibody (1:4000, proteintech) was added and incubated at room temperature for 2 hours. After washing the membrane with TBST, ECL chemiluminescent reagent (Hanqing) was added for color development, and the ChemiDoc MP imaging system (Bio-Rad) was used to develop and acquire images.

[0171] 3. Surface Plasmon Resonance Technology

[0172] The Biacore T200 system used in this experiment is based on the surface plasmon resonance (SPR) principle to detect the interaction between proteins and small molecule drugs. The protein is used as a ligand and the small molecule drug is used as an analyte. The interaction between the two can be monitored in real time through the sensor, and the generated binding data is displayed in resonance response units (RU). The kinetic analysis software uses a 1:1 interaction model to fit the binding data between the analyte and the ligand, thereby obtaining the association rate constant (Ka) and dissociation rate constant (Kd) between the analyte and the ligand, and calculating the affinity value (KD).

[0173] In this experiment, the concentration range of SARS-CoV-2 NSP14 and the concentration range of the active ingredient of magnolol were first determined. Chip CM5 was selected, 5% DMSO + 1× PBS (pH 7.4) + 0.05% Tween-20 was used as the running buffer; 10mM sodium acetate (pH4.5) was used as the fixed buffer. Ligand pre-enrichment: pH 4.5 was determined as the optimal coupling condition. Ligand coupling: The ligand was diluted to 40μg / mL in a buffer with a pH of 4.5, and the target coupling amount was set to 2000RU. Before injection, the activator was prepared by mixing EDC and NHS, and the carboxyl groups on the chip were activated with the mixture at a flow rate of 10μL / min. Then, ethanolamine was used to inactivate the unbound carboxyl groups on the chip at a flow rate of 10μL / min. Sample detection: magnolol was diluted 1:2 with running buffer for a total of 6 concentrations. Samples were loaded at a flow rate of 30μL / min, cross-linked for 60 seconds, and then dissociated for 90 seconds. After each interaction analysis cycle, the sensor chip surface should be fully regenerated using regeneration buffer to remove the ligand and any bound analyte.

[0174] 4. Circular dichroism spectroscopy

[0175] Circular dichroism spectroscopy is a fast, simple and relatively accurate method that is widely used to determine the secondary structure of proteins. This experiment aims to detect the secondary structure of NSP14 using circular dichroism spectroscopy and evaluate the effect of magnolol on it. The NSP14 used in the experiment is a peptide sample dissolved in ddH2O, while magnolol is a drug solution dissolved in isopropylalcohol (IPA).

[0176] When studying the effect of magnolol on the secondary structure of NSP14, magnolol (150 μM, 300 μM) or isopropanol (1%) was incubated with NSP14 at 37° C. for 1 hour. The final concentration of NSP14 was 1 μM.

[0177] After incubation, the samples were detected using a JASCO J-815 circular dichroism spectrometer. The scanning parameters were set as follows: the detection temperature was room temperature, the scanning wavelength range was 195nm-260nm, the bandwidth was 1nm, the step was 0.5nm, and the scanning speed was 50nm / min. 300μL of sample was added to a 1mm pathlength cuvette for scanning. The data was processed using Spectra Manager (V2.08.04, JASCO) software and the secondary structure level of the sample was calculated.

[0178] 5. Fluorescence resonance energy transfer

[0179] Fluorescence resonance energy transfer (FRET) detection of ExoN activity was performed in the following buffer: 50 mM TRIS-HCI (pH 7.5), 2 mM MgCl2, 2 mM DTT; the NSP14 concentration was 200 nM, and the final concentration of the double-stranded RNA substrate (dsRNA, Ruibo Bio) was 1 μM; magnolol (30 μM Mag) was mixed with NSP14 protein at room temperature and incubated for 1 hour, and then dsRNA was added; finally, the mixture was transferred to a black-bottom 96-well plate, and the fluorescence intensity of each well was measured using a microplate reader (ThermoFisher), with the excitation wavelength set to 480 nm (± 20 nm) / emission wavelength set to 520 nm (± 20 nm), and measurements were taken every 150 seconds during the assay for a total of 60 minutes.

[0180] 6. Puromycin incorporation experiment

[0181] In this experiment, HEK-293T cells were first inoculated in 6-well plates, NSP14 gene was transfected into HEK-293T cells, and magnolol (30 μM Mag intervention; 24 hours after transfection, puromycin labeling was performed, that is, 10 μg / mL puromycin (Sigma) was incubated for 15 minutes, and finally the cells were harvested using RIPA lysis buffer containing Cocktail (Roche) and PMSF (Solabo). After centrifugation to extract the supernatant, LDS loading buffer (GenScript Biotech) containing β-mercaptoethanol (Sigma) was added and boiled, and the samples were subjected to 160V constant voltage electrophoresis on SDS-PAGE gel (GenScript Biotech). After the electrophoresis, they were transferred to PVDF membrane (Millipore) by 300mA constant current wet method, and then blocked with 5% BSA, and anti-puromycin antibody (1:1000, Sigma) was added and incubated overnight at 4°C. After washing with TBST, HRP-labeled secondary antibody (1:4000, proteintech) was added and incubated at room temperature for 2 hours. After washing with TBST, ECL chemiluminescent reagent was added for color development, and the ChemiDoc MP imaging system was used to develop and acquire images. For the positive control, 50 μg / mL actinomycin (CHX, Selleck) was added 15 minutes before the addition of puromycin.

[0182] 7. Western blot detection

[0183] In this experiment, HEK-293T cells were first inoculated in 6-well plates, and the NSP14 gene was transfected into HEK-293T cells for 24 hours; 6 hours after transfection, the cells were treated with 2μg / mL IFN-α for 18 hours, and magnolol (30μM Mag) was added for intervention; finally, the cells were harvested using RIPA lysis buffer containing Cocktail (Roche) and PMSF (Solebo), and the supernatant was extracted by centrifugation and then boiled in LDS loading buffer containing β-mercaptoethanol. The samples were subjected to 160V constant voltage electrophoresis on SDS-PAGE gel. After the electrophoresis, they were transferred to PVDF membrane by 300mA constant current wet method, and then 5% BSA was added for blocking, and anti-viperin (1:1000, CST), anti-RIG-I (1:1000, CST), anti-MDA5 (1:1000, CST), anti-TRIM21 (1:1000, Proteintech), and anti-NSP14 were added. ExoN (1:1000, CST), IFITM1 (1:1000, Proteintech), IFITM2 (1:1000, Proteintech), IFITM3 (1:1000, CST), and β-actin (1:10000) antibodies were incubated at 4°C overnight. After washing with TBST, HRP-labeled secondary antibodies (1:4000) were added and incubated at room temperature for 2 hours. After washing with TBST, ECL chemiluminescent reagent was added for color development, and the ChemiDoc MP imaging system was used to develop and acquire images.

[0184] 8. Firefly luciferase activity detection of intracellular SARS-CoV-2Luc-VLPs content

[0185] First, the NSP14 gene was introduced into HEK-293T cells using PEI transfection reagent for 24 hours; then the cells were treated with 2 μg / mL IFN-α for 24 hours, and magnolol was added for intervention (intervention concentration: 30 μM); finally, the supernatant containing SARS-CoV-2Luc-VLPs was added, incubated for 6 hours and then discarded, then washed with PBS and 20 μL PLB lysis buffer was added, and the cells were gently shaken for lysis at room temperature for 20 minutes; 20 μL of the lysate was transferred to an opaque white-walled 96-well plate, 100 μL Luciferase assay buffer (promega) was added, and after thorough mixing, the content of Luc-VLPs was detected using an enzyme reader (ThermoFisher).

Claims

1. A new use of magnolol combined with remdesivir as a drug for the treatment of coronavirus, characterized in that: Magnolia officinalis enhances the antiviral efficacy of remdesivir by inhibiting the ExoN enzyme activity in NSP14.

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