New application of emodin as ribonuclease inhibitor

Abstract

The invention discloses a novel application of emodin as an endonuclease inhibitor. The emodin inhibits replication and transmission of viruses by inhibiting the activity of NSP15 ribonuclease, and recovers host interferon reaction so as to enhance the antiviral effect. The application has the advantages that the action target of the emodin is defined, the effectiveness of the emodin in the aspect of preventing and treating coronavirus infection is further revealed, and the broad-spectrum antiviral potential of the emodin is embodied through the link of playing the antiviral effect by inhibiting the virus replication link; the researches provide candidate drugs and scientific basis for research and development of novel broad-spectrum anti-coronavirus drugs, and promote clinical application of the anti-virus traditional Chinese medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a new use of rhein as an endoribonuclease inhibitor. Background Art

[0002] NSP15, or nonstructural protein (NSP) 15, is a uracil-specific endoribonuclease encoded by coronavirus, which is involved in the synthesis of the viral genome and viral immune escape. Specifically, during viral replication, the viral RNA genome is translated and cleaved by host and viral proteases into 1-16 nonstructural proteins that form the replication and transcription complex (RTC). RTC replicates the viral positive-sense RNA to produce negative-sense RNA, which then serves as a template to produce new positive-sense genomic RNA and subgenomic mRNAs (sgmRNAs). This process generates short-chain RNAs with uridine at the 5' end and long double-stranded RNA (dsRNA) intermediates, which can be recognized by pattern recognition receptors in host cells, such as RIG-I-like receptors (RLRs), protein kinase R (PKR), oligoadenylate synthetase (OAS) and melanoma differentiation-associated gene 5 (MDA5), thereby activating type I interferons (IFN-I) and type III interferons (IFN-III) in host cells, promoting innate immunity and antiviral immune response. The C-terminus of NSP15 has an EndoU (uracil-specific endoribonuclease) domain, which enables NSP15 to specifically recognize and cleave uridine residues in short-chain RNAs and dsRNA intermediates produced during viral RNA transcription. This activity prompts it to generate RNA fragments with 2',3'-cyclic phosphate ends. This special fragment is crucial to the virus's immune escape mechanism. NSP15 usually actively participates in the virus replication process in the form of a hexamer. This structure not only stabilizes its spatial configuration, but also significantly enhances the endoribonuclease activity of NSP15. Through the optimization of this structure, NSP15 can more effectively cut uridine in viral RNA, degrade interferon-stimulated messenger RNA, and reduce the production of interferon-mediated antiviral proteins, thereby helping the virus evade immune surveillance in the early stages of infection, allowing it to survive and replicate in host cells, and then effectively spread and infect more cells.

[0003] In 2020, researchers successfully resolved the crystal structure of NSP15 and found that it has a high structural similarity with NSP15 in SARS-CoV and MERS-CoV. Specifically, in structure, all three are two trimers that form an active hexamer, and the positions of the α-helices and β-folds of the three are highly consistent. Secondly, the residues His235, His250, Lys290, Thr341, Tyr343 and Ser294 in the active site are highly conserved among the three viruses, revealing their core roles in the catalytic process. For example, His235 and His250 may be involved in proton transfer, affecting catalytic activity; while Lys290, Thr341 and Tyr343 may play a key role in substrate recognition and binding. In addition, the sequence similarity between SARS-CoV-2 NSP15 and SARS-CoV NSP15 is as high as 95%, and the sequence similarity with MERS-CoV NSP15 is about 65%, which shows that the sequence of NSP15 is highly conserved in the evolution of coronaviruses and emphasizes the importance of NSP15 as a potential broad-spectrum antiviral drug target.

[0004] At present, a variety of clinical drugs have been reported to inhibit the activity of NSP15, including ciclesonide and tipiracil. Ciclesonide is an inhaled corticosteroid used to treat asthma. Studies have found that it can block the replication of SARS-CoV-2 and MERS-CoV by inhibiting the activity of NSP15. However, long-term or high-dose use of glucocorticoids may cause a series of side effects, such as immunosuppression, cortisol suppression, or osteoporosis. Whether the use of higher doses of ciclesonide in the treatment of COVID-19 is safe has not been fully verified. On the other hand, tipiracil, as a compound approved by the US Food and Drug Administration, is mainly used in chemotherapy drugs to prolong the half-life of drugs in the body by inhibiting thymidine phosphate synthase. It is clinically used to treat gastric cancer and rectal cancer. Studies have found that tipiracil can bind to the uridine binding site in NSP15 and inhibit its endonuclease activity. However, given that tipiracil is mainly used to treat cancer, its mechanism of action in normal cells and virus-infected cells may be different, which may lead to cytotoxicity or other side effects. Therefore, the antiviral effect and safety of currently known NSP15 inhibitors still need to be further verified.

[0005] In recent years, research in the field of traditional Chinese medicine has been significantly deepened and expanded, especially in the study of traditional Chinese medicine and its monomer compounds. These traditional Chinese medicines and monomers have shown significant potential and advantages in the prevention and long-term treatment of viral infections due to their excellent tolerability and low side effects. Given the limitations of currently known NSP15 inhibitors, it is particularly important to find natural NSP15 inhibitors to reduce the risk of relying on traditional synthetic drugs. This will not only enrich our understanding of the antiviral mechanism of traditional Chinese medicine, but may also open up new ways to prevent and treat viral infections and contribute new strategies to global public health. 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 emodin as an inhibitor of coronavirus endoribonuclease NSP15, and the purpose is to find that emodin can inhibit the activity of NSP15, thereby inhibiting the replication of SARS-CoV, MERS-CoV, and SARS-CoV-2, and has universality, so that an inhibitor 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 rhein is used as a new use for preparing endoribonuclease inhibitors, and rhein inhibits the replication and spread of viruses by inhibiting the endoribonuclease activity of NSP15.

[0008] Furthermore, the virus is a coronavirus.

[0009] Furthermore, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0010] The present invention also finds that emodin can enhance the host's interferon response by inhibiting the endoribonuclease activity in NSP15, thereby achieving antiviral and enhanced antiviral effects.

[0011] In summary, the beneficial effects of the present invention are that the present invention clarifies the target of action of emodin, and further reveals the effectiveness of emodin in preventing and treating coronavirus infection, and its antiviral effect by inhibiting the viral replication link. It acts on the highly conserved region of NSP15 involved in the replication link, reflecting its broad-spectrum antiviral potential. These studies provide candidate drugs and scientific basis for the research and development of new broad-spectrum anti-coronavirus drugs, and promote the clinical application of antiviral Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1This is the effect of Emodin on the SARS-CoV-2 minireplicon NSP1-16. In the figure: ****P<0.001 compared with the Control group. All figures are mean ± standard error, n=3.

[0013] Figure 2 The effects of emodin on SARS-CoV-2 minireplicons NSP1-4, NSP5-11 and NSP12-16, respectively. In the figure: ****P<0.001 and ns are compared with the Control group. All figures are mean ± standard error, n=3.

[0014] Figure 3 The effect of Emodin on the thermal stability of SARS-CoV-2 NSP12-NSP16 protein.

[0015] Figure 4 The effect of Emodin on the stability of SARS-CoV-2 NSP15 protein.

[0016] Figure 5 The effect of emodin on the activity of SARS-CoV-2 NSP15 enzyme. DMSO was used as the negative control, and Benzopurpurine 4B was used as the positive control. All figures are mean ± standard error, n = 3.

[0017] Figure 6 This is the dose-response curve of emodin on the SARS-CoV-2 NSP15 enzyme activity. All figures are mean ± standard error, n = 3.

[0018] Figure 7 The effect of emodin on interferon response in human embryonic kidney cells HEK-293T, A549 alveolar epithelial cells and human brain glioma cells U118-MG. In the figure: ****P<0.001 compared with untreated cells. Positive control used Poly(I:C). All are mean ± standard error, n = 3.

[0019] Figure 8 The effect of Emodin on the thermal stability of SARS-CoV-2 NSP15 mutant protein.

[0020] Fig. 9 The effect of Emodin on the protein stability of SARS-CoV-2 NSP15 mutant.

[0021] Fig.10The effect of emodin on the enzyme activity of SARS-CoV-2 NSP15 mutant. DMSO was used as the negative control, and Benzopurpurine 4B was used as the positive control. All figures are mean ± standard error, n = 3.

[0022] Fig.11 Comparison of the difference in enzyme activity between wild-type NSP15 and mutant NSP15 of SARS-CoV-2 by emodin. *P<0.05 and ****p<0.001 in the figure compared with the corresponding concentration group of Emodin+NSP15.

[0023] Fig.12 This is the universal characteristic of rhein against highly pathogenic coronaviruses. Comparison results of the NSP15 enzyme active sites of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E and MHV-2.

[0024] Fig.13 The effect of emodin on the thermal stability of SARS-CoV and MERS-CoV NSP15 mutant proteins.

[0025] Fig.14 This is the dose-concentration curve of Emodin on the enzyme activity of MERS-CoV NSP15. DETAILED DESCRIPTION

[0026] 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.

[0027] Embodiment: The present invention found that rhein inhibits the replication and spread of viruses by inhibiting the enzyme activity of NSP15. The specific experimental contents are as follows:

[0028] 1. Experimental results:

[0029] 1. Inhibitory effect of rheumatoid arthritis on viral replication

[0030] In order to explore whether rhamnosine affects viral replication, it is necessary to clarify whether rhamnosine affects the nonstructural protein (NSP) that mediates viral replication, and further clarify the molecular mechanism by which rhamnosine inhibits viral replication.

[0031] 1.1 Rheum officinale inhibits NSP15 to prevent viral replication

[0032] After coronavirus enters host cells, the virus's NSP (NSP1-NSP16) forms the RTC, which plays a key role in the virus's replication and transcription process. By constructing mini-replicons that co-express some or all of the NSP functional modules, we can partially reproduce the key links of virus replication, make up for the deficiency that SARS-CoV-2 Luc-VLPs cannot simulate the virus replication link, and help to clarify the effect of rheumatoid arthritis on different virus replication links.

[0033] like Figure 1 As shown in the figure, compared with the control group, after 24h treatment with 10μM emodin, the luciferase activity of the replicon system decreased by 44.9%±5.57%, while the luciferase activity of the replicon system in the positive control group (Nirmatrelvir) decreased by 37.16%±1.80%. In order to further clarify which region in the replicon system emodin has a specific inhibitory effect on, we detected the interaction between emodin and the NSP1-4, NSP5-11 and NSP12-16 regions. Figure 2 As shown in the figure, the luciferase activity of NSP12-16 in the replicon system decreased by 29.35%±4.23% after treatment with rheumatoid arthritis. The above results show that rheumatoid arthritis can significantly inhibit the replication of SARS-CoV-2 and only exerts an inhibitory effect on the specific region of NSP12-NSP16.

[0034] 1.2 Rheum officinale inhibits NSP15 endoribonuclease that mediates viral replication

[0035] 1.2.1 Interaction between emodin and NSP15

[0036] Based on the results of the replicon system, rheumatoid arthritis has a potential inhibitory effect on the NSP12-NSP16 region in the SARS-CoV-2 replicon system. However, the specific mechanism of which NSP rheumatoid arthritis inhibits is still unclear.

[0037] Through the cellular thermal shift assay (CETSA), we can determine whether rhein interacts with NSP by detecting the effect of rhein on the stability of SARS-CoV-2 NSP ( Figure 3 ), this experiment is to judge whether the small molecule drug interacts with the target protein based on the degree to which it increases the stability of the target protein. Figure 3As shown in the figure, at 52°C, it can be found that the expression of NSP in each group decreases with the increase of temperature. However, the expression level of NSP15 in the rhein-treated group at 52°C is significantly higher than that in the DMSO-treated group, indicating that rhein improves the thermal stability of SARS-CoV-2 NSP15 and interacts with SARS-CoV-2 NSP15. For other NSPs, no obvious expression difference was observed between the rhein-treated group and the control group.

[0038] Therefore, based on the results of the thermal stability experiment of protein interactions, NSP15 may be the target of rheumatoid arthritis to inhibit coronavirus replication.

[0039] 1.2.2 Rhein increases protein stability of NSP15

[0040] Previous experiments have shown that rhein can interact with the virus NSP15. To further confirm the binding effect of rhein and NSP15, we used the nanoDSF detection method, based on the principle that tryptophan and tyrosine in protein samples will be more or less exposed to the medium environment as the temperature rises, and plotted the relationship between temperature and fluorescence intensity by monitoring the fluorescence changes, and used the temperature of protein unfolding to evaluate its stability at different temperatures.

[0041] NanoDSF results show that ( Figure 4 ), compared with the DMSO-treated group, co-incubation of emodin with NSP15 at 37°C for 1 h increased its unfolding temperature by 2.41°C, indicating that emodin can increase the protein stability of NSP15.

[0042] 1.2.3 Rhein inhibits the endoribonuclease activity of NSP15

[0043] During viral replication, the biological function of NSP15 is to recognize and cut the uridine RNA sequence in the viral genome, helping the virus to evade host cell recognition and immune escape. In order to gain a deeper understanding of the effect of rheumatoid arthritis on the enzyme activity of SARS-CoV-2 NSP15, we performed fluorescence resonance energy transfer (FRET).

[0044] The results are as follows Figure 5As shown, compared with the Blank group, the fluorescence intensity increased significantly after the addition of NSP15 (1μM) protein, while the fluorescence intensity decreased significantly after the addition of 1mM of the positive drug Benzopurpurine 4B to treat NSP15, indicating that the experimental model was successfully constructed. The inhibitory effect of Benzopurpurine 4B on NSP15 is due to the fact that the molecular structure of Benzopurpurine 4B allows it to interact with specific amino acid residues of NSP15, compete with the natural substrate (RNA) of NSP15 for the active site, and interfere with the normal catalytic activity of NSP15. After treatment with 40μM rhein, the enzyme activity of NSP15 was significantly reduced compared with the NSP15 group alone, indicating that rhein can inhibit the enzyme activity of NSP15. In addition, the inhibitory effect of rhein on the enzyme activity of NSP15 was concentration-dependent (0.3μM-1000μM), and its IC 50 58.58μM( Figure 6 ).

[0045] 2. Rhein restores NSP15-weakened host interferon response

[0046] Based on the inhibitory effect of emodin on the enzyme activity of NSP15, we will further explore and clarify whether emodin can also affect the biological function of NSP15. In the process of viral synthesis of new genomes, RNA sequences with uridine are produced in large quantities. Pattern recognition receptors in host cells can recognize these uridine sequences and produce interferon β for immune response to play an antiviral role. However, activated NSP15 reduces the host's interferon response by cutting uridine RNA that can be recognized by the host. In order to further clarify whether emodin can restore the host interferon response inhibited by NSP15, we used fluorescent quantitative PCR to detect the level of IFN-β in the host under different conditions to evaluate the effect of emodin on the biological function of NAP15.

[0047] Poly(IC) (a complex of polyinosine and polycytidine) is a dsRNA mimic, which is widely used as a mimic of viral infection. It mainly activates the immune response in the human body by mimicking the structure of viral RNA. Therefore, we used Poly(I:C) as the positive drug group to construct an IFN-β high expression model. Poly(I:C), PUN RNA (uridine RNA recognized and cleaved by NSP15), NSP15 and rhamnosine were transfected separately or in combination into different cell models, including human embryonic kidney cells HEK-293T, A549 alveolar epithelial cells and human brain glioma cells U118-MG. Figure 7As shown in the results, in three different host cells, transfection of Poly(I:C) 4μg and PUN RNA 5μg can cause an increase in the level of IFN-β in host cells, indicating that host cells can recognize and activate immune responses in the presence of exogenous dsRNA. However, the level of IFN-β in host cells co-transfected with PUN RNA and NSP15 was significantly reduced, and on this basis, the immune response that was originally suppressed was reactivated after 48h of intervention with 10μM emodin. The results suggest that emodin can interfere with the biological function of NSP15, help the host restore the recognition and attack of viral components, and then play an antiviral role. At the same time, combined with the inhibition of viral replication and transmission, it can enhance the antiviral effect.

[0048] 3. Rhein targets the active site H235 of NSP15 to exert its inhibitory effect

[0049] In order to further clarify the binding target of emodin and NSP15, we screened 3 clear enzyme activity sites in the EndoU domain of NSP15 for screening. Through the CETSA experiment, 10μM emodin was used to intervene in HEK-293T cells transfected with different mutants of NSP15 to identify the mutants that bind to emodin. The results are as follows Figure 8 As shown, the protein expression level of NSP15-H250A and NAP15-H290A treated with emodin was still higher than that of DMSO treated group at 72℃, indicating that emodin can still bind to NSP15 even if the amino acids at positions 250 and 290 are mutated. However, in NSP15-H235A, within the range of 37℃-77℃, emodin intervention no longer enhances the protein expression of NSP15, that is, it no longer enhances the thermal stability of NSP15, suggesting that the binding site of emodin with NSP15 is the histidine at position 235.

[0050] Similarly, we performed FRET again to detect the effect of rhamnosin on the enzyme activity of the NSP15-H235A mutant. Fig.10The results showed that the enzyme activity of NSP15 itself was quite limited after the mutation of H235, indicating that H235 is a key site affecting the enzyme activity of NSP15. Similarly, compared with the Blank group, the fluorescence intensity increased significantly after the addition of NSP15-H235A (1μM) protein, while the fluorescence intensity decreased significantly after the addition of the positive drug phenylpropionine 4B 1mM to treat NSP15-H235A. After treatment with 40μM rheumatoid arthritis, the enzyme activity of NSP15-H235A was similar to that of NSP15-H235A, with no obvious downward trend, suggesting that after the mutation of H235 of NSP15, rheumatoid arthritis no longer inhibited the enzyme activity of NSP15. In addition, H235A-NSP15 was treated with different concentrations of 0.3 μM-1000 μM rhein. It was found that the highest concentration of rhein had an inhibition rate of only 11.94% ± 3.94% on the enzyme activity of NSP15-H235A, and its effective IC 50 value( Fig.11 ), the above results suggest that rhamnosus can target the H235 site of NSP15 to inhibit the enzymatic activity and biological function of NSP15.

[0051] 4. The NSP15 endoribonuclease active site of highly pathogenic coronavirus is highly conserved, and rhubarb has a wide range of antagonistic effects on NSP15 endoribonuclease

[0052] like Fig.12 As shown, the comparison of the full-length protein sequences and enzyme active sites of SARS-CoV-2, SARS-CoV and MERS-CoV NSP15 proves that the three active sites of NSP15 screened in the early stage are highly conserved and completely consistent in the three viruses. Among them, the yellow background represents similar sequences of SARS-CoV-2, SARS-CoV and MERS-CoV; the red background represents completely similar sequences of SARS-CoV-2, SARS-CoV and MERS-CoV; the black circles mark the six active sites of SARS-CoV-2, SARS-CoV and MERS-CoV. The six active sites of NSP15 are highly conserved. The CETSA experiment was used to detect whether rhubarb interacts with SARS-CoV and MERS-CoV NSP15 ( Fig.13 ), and found that the expression of NSP15 decreased with the increase of temperature, and the expression level of SARS-CoV NSP15 in the rhein group was significantly higher than that in the DMSO group at 72℃, and the expression level of MERS-CoV NSP15 in the rhein group was significantly higher than that in the DMSO group at 62℃ and 67℃, indicating that rhein improved the thermal stability of SARS-CoV and MERS-CoV NSP15 and interacted with NSP15.

[0053] After confirming that rheum officinale can bind to SARS-CoV and MERS-CoV NSP15, FRET experiments were performed again to confirm whether rheum officinale can inhibit the enzyme activity of MERS-CoV NSP15. Using 0.3μM-1000μM rheum officinale to intervene in 1μM MERS-CoV NSP15, it was found that rheum officinale can still inhibit the enzyme activity of MERS-CoV NSP15 in a concentration-dependent manner, and its IC 50 The above results show that rhein can bind to NSP15 and inhibit the enzyme activity and biological function of NSP15 at the highly conserved H235 enzyme active site in coronavirus, indicating that rhein has universal characteristics against coronavirus.

[0054] in conclusion

[0055] In this study, we found that emodin can inhibit the highly conserved endoribonuclease activity in coronavirus NSP15, thereby preventing viral replication and restoring the antiviral immune response of host cells, reducing the risk of host reinfection with the virus, reflecting its potential to become a broad-spectrum anti-coronavirus drug.

[0056] Materials and methods used in the above experiments in this example

[0057] 1. SARS-CoV-2 replicon system based on luciferase reporter system

[0058] 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 NSP15 (0.03μg). M-Luc (0.02μg) plasmid was co-transfected during each transfection. 24 hours after transfection, emodin was added to the cell culture medium for intervention, and DMSO was added to the control group. 20L of lysis buffer was added 24 hours after drug treatment, and the cells were gently shaken for lysis at room temperature for 20 minutes. The 20L lysate was transferred to an opaque white-walled 96-well plate, 100L of Luciferase assay buffer (Promega) was added, and after thorough mixing, the fluorescence value was measured using an enzyme reader (ThermoFisher). The fluorescence value can accurately reflect the expression of each NSP of the virus, thereby indirectly reflecting the replication efficiency of the virus.

[0059] 2. Cell thermal transfer experiment

[0060] Cellular thermal shift assay (CETSA) is a method to detect the binding of small molecule compounds to SARS-CoV-2 S 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 rhamnosine 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; 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 boiled in LDS loading buffer containing β-mercaptoethanol, and the sample was taken for 4-20 % SDS-PAGE gel (GenScript Biotech) was subjected to 120V constant voltage electrophoresis. After electrophoresis, the membrane was transferred to a PVDF membrane (Milipore) at 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 2h. After washing with TBST, ECL chemiluminescent reagent (AoQing) was added for color development, and the ChemiDoc MP imaging system (Bio-Rad) was used to develop and acquire images.

[0061] 4. Fluorescence resonance energy transfer

[0062] Fluorescence resonance energy transfer (FRET) detection of NSP15 activity was carried out in the following buffer: 50 mM Tris-HCl pH 7.5, 5 mM MnCl2, 100 mM KCl, 1 mM DTT; the NSP15 concentration was 1 μM, and the final concentration of the double-stranded RNA substrate (dsRNA, Bioengineering) was 1 μM; emodin (40 μM Emodin) was mixed with NSP15 protein at room temperature and incubated for 1 hour, and then dsRNA was added; finally, the mixture was transferred to a 96-well plate with a black bottom, 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 the fluorescence intensity was measured every 150 seconds during the assay, and 25 cycles of fluorescence intensity were detected.

[0063] 5. Nano-differential scanning fluorescence method

[0064] Nano differential scanning fluorimetry (nanoDSF) was used to absorb about 10 μL of NSP15 protein into a high-sensitivity capillary glass tube. The temperature was raised at a rate of 1°C / min and the temperature range was set at 19°C-90°C. The ratio of absorbance at 350 / 330 nm and the first-order derivative curve were plotted during the heating process of the protein to observe the temperature at which the protein unfolded and to determine whether rhamnosine increased the temperature at which NSP15 unfolded. A negative drug DMSO group, a positive drug phenylpropionine 4B group (1 mM) and a rhamnosine group (40 μM) were set up to detect the changes in the thermal stability of NSP15 caused by rhamnosine and to determine whether the two had a binding effect.

Claims

1. A new use of rhubarb as an endoribonuclease inhibitor, characterized in that: The endoribonuclease is NSP15.

2. The use according to claim 1, characterized in that Rhamnosin inhibits the activity of endoribonuclease NSP15, thereby inhibiting the replication and spread of the virus.

3. The use according to claim 1, characterized in that Rhamnosin inhibits NSP15 enzyme activity and restores the host's interferon response, thereby achieving antiviral and enhanced antiviral effects.

4. The use according to claim 2 or 3, characterized in that The virus is a coronavirus.

5. The method according to claim 4, wherein the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

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