阅读说明：本技术 抑制SARS-CoV-2病毒复制的药物的用途、试剂盒 (Application and kit of medicine for inhibiting SARS-CoV-2 virus replication ) 是由 杜瑞坤 崔清华 荣立军 于 2020-12-26 设计创作，主要内容包括：本发明属于生物制药技术领域,公开了一种抑制SARS-CoV-2病毒复制的药物的用途,诃黎勒酸和安石榴苷以任意比例组合在制备对SARS-CoV-2病毒复制抑制的药物中的用途。本发明通过实验两种广谱抗病毒天然产物诃黎勒酸和安石榴苷对SARS-CoV-2病毒复制的抑制能力表明,诃黎勒酸和安石榴苷能够作为新型SARS-CoV-2抑制剂；在非细胞毒性浓度下,诃黎勒酸和安石榴苷通过靶向病毒3-胰凝乳蛋白酶样半胱氨酸蛋白酶(3CL～(Pro))的酶活性,以可逆的、非竞争性的方式减少病毒诱导的VERO-E6细胞单层空斑的形成,诃黎勒酸和安石榴苷作为新的COVID-19疗法存在潜在用途。(The invention belongs to the technical field of biological pharmacy, and discloses an application of a medicine for inhibiting SARS-CoV-2 virus replication, namely an application of myrobalamin acid and punicalagin in preparing a medicine for inhibiting SARS-CoV-2 virus replication in any proportion. According to the invention, the inhibition capability of two natural broad-spectrum antiviral products of chebulagic acid and punicalagin on SARS-CoV-2 virus replication is tested, so that the chebulagic acid and punicalagin can be used as a novel SARS-CoV-2 inhibitor; at a position other thanTerminalia chebula acid and punicalagin target the viral 3-chymotrypsin-like cysteine protease (3 CL) at cytotoxic concentrations Pro ) Reduces virus-induced VERO-E6 cell monolayer plaque formation in a reversible, non-competitive manner, and the potential use of chebulagic acid and punicalagin as a novel COVID-19 therapy.)

1. Application of chebulagic acid and punicalagin in preparing medicine for inhibiting SARS-CoV-2 virus replication at any ratio is provided.

2. The use of claim 1, wherein the ratio of chebulagic acid to punicalagin is 1-9: 9-1.

3. Use according to claim 1, wherein the chebulagic acid is used in the preparation of a medicament for inhibiting the replication of the SARS-CoV-2 virus in an amount of 100% by mass.

4. Use according to claim 1, wherein the punicalagin is used in the preparation of a medicament for inhibiting the replication of SARS-CoV-2 virus at a mass ratio of 100%.

5. The use according to any one of claims 1 to 4, wherein the chebulagic acid and punicalagin are combined in any ratio for the preparation of novel SARS-CoV-2 inhibitors.

6. A target cell for expressing the novel SARS-CoV-2 inhibitor for use as claimed in claim 5 for inhibiting SARS-CoV-2 replication.

7. A kit for detecting the replication inhibition ability of SARS-CoV-2 virus, which comprises the composition of chebulagic acid and punicalagin in any proportion in the use of any one of claims 1 to 5, and the target cell of claim 6.

8. The use of the combination of chebularianic acid and punicalagin in any ratio in the preparation of 3CL for inhibiting 3-chymotrypsin-like cysteine proteases according to any of claims 1 to 5^ProUse in active medicaments.

9. The use according to claim 8, wherein the preparation inhibits the 3-chymotrypsin-like cysteine protease 3CL^ProThe active drug comprises 3CL, a p-3-chymotrypsin-like cysteine protease^ProScreening of inhibitory Effect of Activity:

SARS-CoV-23CL purified by prokaryotic expression^ProCarrying a natural N end and carrying a His purification tag at the C end; when 3CL is used^proAfter the fluorescent resonance energy transfer base peptide substrate and the fluorescent resonance energy transfer base peptide substrate are mixed in a reaction buffer solution, the substrate is hydrolyzed under the action of enzyme;

after hydrolysis, the fluorophore Edans is no longer affected by the quencher molecule Dabcyl, and the fluorescence signal increases, exciting at 336/20nm and emitting at 490/20 nm.

10. The use of claim 9, wherein the reaction buffer comprises: 50mM Tris-HCl, pH7.3, 1mM EDTA.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to application and a kit of a medicine for inhibiting SARS-CoV-2 virus replication.

Background

Currently, the novel coronavirus pneumonia (COVID-19) is a pneumonia disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, to date, no specific antiviral therapy is available for clinical use. While many potentially effective in vitro antiviral drugs, such as Reidesvir and chloroquine, can inhibit SARS-CoV-2 replication, chloroquine has proven ineffective in clinical trials. Reidesciclovir has been approved for emergency use in many countries, but its therapeutic efficacy is relatively limited. Therefore, there is an urgent need to find effective anti-SARS-CoV-2 virus drugs for use alone or in combination with approved antiviral therapies to cope with new coronary epidemics.

Through the above analysis, the problems and defects of the prior art are as follows: the existing anti-SARS-CoV-2 virus medicine has relatively limited curative effect.

The difficulty in solving the above problems and defects is: there is a need to develop new and more potent antiviral drugs.

The significance of solving the problems and the defects is as follows: can be used for treating new coronavirus infection, controlling new pneumonia epidemic situation and preventing possible related diseases from spreading in the future.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application and a kit of a medicine for inhibiting SARS-CoV-2 virus replication, in particular to a composition with SARS-CoV-2 virus replication inhibition capability and an application thereof.

The invention is realized by combining chebulagic acid (CHLA) and Punicalagin (PUG) in any proportion in the preparation of the medicine for inhibiting SARS-CoV-2 virus replication. The proportion of the invention can be 1-9: 9-1.

Further, the application of the chebulagic acid in the preparation of the medicine for inhibiting the replication of SARS-CoV-2 virus with the content of 100 percent by mass ratio.

Further, the application of the punicalagin in preparing the medicine for inhibiting the replication of SARS-CoV-2 virus by 100 percent of mass ratio content.

Further, the myrobalamin acid and punicalagin are combined in any proportion to prepare the novel SARS-CoV-2 inhibitor.

Another objective of the invention is to provide a target cell for expressing the novel SARS-CoV-2 inhibitor in the use to inhibit the replication of SARS-CoV-2.

The invention also aims to provide a kit for detecting the replication inhibition capability of the SARS-CoV-2 virus, the kit comprises the composition of the chebulagic acid and the punicalagin in any proportion in the application and the target cell.

The invention also aims to provide a method for preparing the medicine for inhibiting the 3-chymotrypsin-like cysteine protease 3CL by combining the chebulagic acid and the punicalagin in any proportion^ProUse in active medicaments. The proportion of the invention can be 1-9: 9-1.

Further, the preparation inhibits 3-chymotrypsin-like cysteine protease 3CL^ProThe active drug comprises 3CL, a p-3-chymotrypsin-like cysteine protease^ProScreening of inhibitory Effect of Activity:

SARS-CoV-23CL purified by prokaryotic expression^ProCarrying a natural N end and carrying a His purification tag at the C end; when 3CL is used^proAfter the fluorescent resonance energy transfer base peptide substrate and the fluorescent resonance energy transfer base peptide substrate are mixed in a reaction buffer solution, the substrate is hydrolyzed under the action of enzyme;

after hydrolysis, the fluorophore Edans is no longer affected by the quencher molecule Dabcyl, and the fluorescence signal increases, exciting at 336/20nm and emitting at 490/20 nm.

Further, the reaction buffer comprises: 50mM Tris-HCl, pH7.3, 1mM EDTA.

By combining all the technical schemes, the invention has the advantages and positive effects that: the composition for inhibiting the SARS-CoV-2 virus replication provided by the invention analyzes the inhibition ability of two broad-spectrum antiviral natural products of chebulagic acid (CHLA) and Punicalagin (PUG) on the SARS-CoV-2 virus replication. Indicating that the chebulagic acid and punicalagin can be used as a novel SARS-CoV-2 inhibitor; CHLA and PUG have potential uses as novel COVID-19 therapies by targeting the enzymatic activity of the viral 3-chymotrypsin-like cysteine protease (3CLPro) to reduce the virus-induced plaque formation of a VERO-E6 cell monolayer in a reversible, non-competitive manner at non-cytotoxic concentrations.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a method for detecting the antiviral activity of CHLA and PUG against SARS-CoV-2 infection as provided in an embodiment of the present invention.

FIG. 2A is a schematic diagram of the experiment for inhibiting the plaque formation of SARS-CoV-2 by CHLA and PUG and Reidesciclovir (3. mu.M) at certain concentrations according to the present invention.

FIG. 2B is a schematic representation of the dose-dependent inhibition of SARS-CoV-2 replication exhibited by CHLA and PUG provided in an embodiment of the invention; wherein, IC₅₀And CC₅₀Shown in the upper left corner of each compound; data represent triplicate measurements ± mean Standard Deviation (SD); IC (integrated circuit)₅₀Values were determined by fitting dose-response curves using four-parameter logistic regression in GraphPad, all data normalized to the control.

FIG. 2C is a CHLA and PUG pair 3CL provided by an embodiment of the present invention^ProDose-dependent inhibition of (a); IC of each compound was determined by Fluorescence Resonance Energy Transfer (FRET) -based lysis₅₀Values are shown in the upper left corner and data represent triplicate measurements ± mean Standard Deviation (SD).

FIG. 2D is a graph showing the relationship between CHLA and PUG at different concentrations for SARS-CoV-23CL^proSchematic representation of the effect of enzyme activity.

FIG. 2E shows the CHLA or PUG pair SARS-CoV-23CL provided by the embodiment of the present invention^proLineweaver-Burk plot of inhibition.

FIG. 2F shows the prediction results of CHLA (i) and PUG (ii) and 3CL according to the embodiment of the present invention^pro(PDB ID:6m2 n).

FIG. 3A is the experimental diagram of SARS-CoV-2 pseudovirus provided by the embodiment of the present invention; among them, 293T cells transiently expressed ACE2 and TMPRSS2 served as target cells.

FIG. 3B is a schematic representation of the anti-pseudoviral activity and cytotoxicity provided by an embodiment of the invention; where, data represent three replicates ± mean Standard Deviation (SD).

FIG. 4A is a diagram of a Fluorescence Resonance Energy Transfer (FRET) -based pair of 3CL, according to an embodiment of the present invention^ProSchematic representation of monitoring of enzymatic activity of (a); wherein, 3CL^ProCleavage results in an increase in the fluorescence signal due to cleavage between the quenching molecule Dabcyl and the fluorophore Edans.

FIG. 4B shows 3CL at a certain concentration of CHLA or PUG according to an embodiment of the present invention^ProInitial fluorescence zymogram curve (250nm) diagram.

FIG. 5A is a graph of CHLA vs PL in vitro assays provided by examples of the invention^proSchematic representation of inhibition of enzyme activity; wherein the fluorescence labeled peptide Z-RLRGG-AMC is used as a substrate for SARS-CoV-2PL^proProkaryotic expression, purification and fluorescence measurement are carried out.

FIG. 5B is a graph showing the in vitro assay of PUG vs PL provided in an embodiment of the present invention^proSchematic representation of the inhibition of enzyme activity.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a composition with SARS-CoV-2 virus replication inhibition ability and the application thereof, and the invention is described in detail with reference to the accompanying drawings. The technical solution of the present invention is further described with reference to the following specific examples.

Example 1

The invention relates to an application of 100% of chebulagic acid in preparing a medicine for inhibiting SARS-CoV-2 virus replication.

The application of the chebulagic acid in preparing a novel SARS-CoV-2 inhibitor is provided.

The invention provides a target cell for expressing the SARS-CoV-2 inhibitor in the application to inhibit the replication of SARS-CoV-2.

The invention provides a kit for detecting the replication inhibition capability of SARS-CoV-2 virus, which comprises the chebulagic acid in the application and the target cell.

The invention provides application of chebulagic acid in preparation of 3CL for inhibiting 3-chymotrypsin-like cysteine protease^ProUse in active medicaments.

The preparation inhibits 3-chymotrypsin-like cysteine protease 3CL^ProThe active drug comprises 3CL, a p-3-chymotrypsin-like cysteine protease^ProScreening of inhibitory Effect of Activity:

SARS-CoV-23CL purified by prokaryotic expression^ProCarrying a natural N end and carrying a His purification tag at the C end; when 3CL is used^proAfter the fluorescent resonance energy transfer base peptide substrate and the fluorescent resonance energy transfer base peptide substrate are mixed in a reaction buffer solution, the substrate is hydrolyzed under the action of enzyme;

after hydrolysis, the fluorophore Edans is no longer affected by the quencher molecule Dabcyl, and the fluorescence signal increases, exciting at 336/20nm and emitting at 490/20 nm.

The reaction buffer comprises: 50mM Tris-HCl, pH7.3, 1mM EDTA.

Example 2

The punicalagin of the invention is used for preparing the medicine for inhibiting the replication of SARS-CoV-2 virus with the mass ratio of 100 percent.

The punicalagin is combined in any proportion and is used for preparing a novel SARS-CoV-2 inhibitor.

The invention provides a target cell for expressing the SARS-CoV-2 inhibitor in the application to inhibit the replication of SARS-CoV-2.

The invention provides a kit for detecting the replication inhibition capability of SARS-CoV-2 virus, which comprises punicalagin in the application and the target cell.

The invention provides application of the punicalagin in preparation of 3CL for inhibiting 3-chymotrypsin-like cysteine protease^ProUse in active medicaments.

The preparation inhibits 3-chymotrypsin-like cysteine protease 3CL^ProThe active drug comprises 3CL, a p-3-chymotrypsin-like cysteine protease^ProScreening of inhibitory Effect of Activity:

SARS-CoV-23CL purified by prokaryotic expression^ProCarrying a natural N end and carrying a His purification tag at the C end; when 3CL is used^proAfter the fluorescent resonance energy transfer base peptide substrate and the fluorescent resonance energy transfer base peptide substrate are mixed in a reaction buffer solution, the substrate is hydrolyzed under the action of enzyme;

after hydrolysis, the fluorophore Edans is no longer affected by the quencher molecule Dabcyl, and the fluorescence signal increases, exciting at 336/20nm and emitting at 490/20 nm.

The reaction buffer comprises: 50mM Tris-HCl, pH7.3, 1mM EDTA.

Example 3

The invention provides an application of myrobalamin acid and punicalagin in any proportion in preparing a medicine for inhibiting SARS-CoV-2 virus replication.

The myrobalamin acid and punicalagin are combined in any proportion to prepare the novel SARS-CoV-2 inhibitor.

The invention provides a target cell for expressing the SARS-CoV-2 inhibitor in the application to inhibit the replication of SARS-CoV-2.

The invention provides a kit for detecting the replication inhibition capability of SARS-CoV-2 virus, which comprises the composition of chebularianic acid and punicalagin in any proportion and the target cell.

The invention provides a method for preparing 3CL for inhibiting 3-chymotrypsin-like cysteine protease by combining chebulagic acid and punicalagin in any proportion in the application^ProUse in active medicaments.

The preparation inhibits 3-chymotrypsin-like cysteine protease 3CL^ProThe active drug comprises 3CL, a p-3-chymotrypsin-like cysteine protease^ProScreening of inhibitory Effect of Activity:

SARS-CoV-23CL purified by prokaryotic expression^ProCarrying a natural N end and carrying a His purification tag at the C end; when 3CL is used^proAfter the fluorescent resonance energy transfer base peptide substrate and the fluorescent resonance energy transfer base peptide substrate are mixed in a reaction buffer solution, the substrate is hydrolyzed under the action of enzyme;

after hydrolysis, the fluorophore Edans is no longer affected by the quencher molecule Dabcyl, and the fluorescence signal increases, exciting at 336/20nm and emitting at 490/20 nm.

The reaction buffer comprises: 50mM Tris-HCl, pH7.3, 1mM EDTA.

The technical solution of the present invention will be further described with reference to specific experiments.

As shown in FIG. 1, the method for detecting the antiviral activity of CHLA and PUG on SARS-CoV-2 infection provided by the embodiment of the invention comprises the following steps:

s101, pretreating VERO-E6 growing into monolayer cells in a 12-well plate for 1h by using CHLA or PUG with different concentrations;

s102, adding SARS-CoV-2 clinical Isolate Isolate USA-WA1/2020 to infect;

s103, after CO-cultivation for 1 hour, the medium was changed to fresh MEM containing 1.25% Avicel, and incubated at 37 ℃ with 5% CO₂Incubating for 48h in the environment;

s104, cells were fixed with 10% formalin, stained with 1% crystal violet and observed.

The present invention will be further described with reference to the following examples.

The emerging SARS-CoV-2 infection is responsible for the COVID-19 pandemic worldwide. To date, treatment regimens for the prevention of this disease are limited. Here, the present invention tested the inhibitory ability of two broad-spectrum antiviral natural products, chebulagic acid (CHLA) and Punicalagin (PUG), against the replication of SARS-CoV-2 virus. In non-cellsCHLA and PUG were targeted to the viral 3-chymotrypsin-like cysteine protease (3 CL) at toxic concentrations^Pro) In a reversible, non-competitive manner, reduces virus-induced formation of VERO-E6 cell monolayer plaques. The experiments of the present invention demonstrate the potential use of CHLA and PUG as novel COVID-19 therapies.

In the emergency experiments for the development of SARS-CoV-2 therapeutics, natural products from medicinal plants have been an important source of novel lead compounds with different mechanisms of action, such as violaxacin, salvianolic acid C, cepharanthine, guava-rine, etc. The invention discloses another two natural products of chebulagic acid (CHLA) and Punicalagin (PUG) separated from traditional Chinese medicinal plants myrobalan as novel SARS-CoV-2 inhibitors.

CHLA and PUG are hydrolyzable polyphenols that have been reported to block the interaction between cell surface glycosaminoglycans (GAG) and viral glycoproteins. Using this mechanism of action, CHLA and PUG are able to inhibit a variety of GAG-dependent viral infections such as herpes simplex virus type 1, human cytomegalovirus and hepatitis C virus into host cells. Furthermore, recent experimental results have found that CHLA and PUG inhibit influenza virus replication by targeting neuraminidase-mediated viral release processes. All of these indicate that CHLA and PUG have the potential for a broad spectrum antiviral drug with low cost and high efficacy.

The invention detects the antiviral activity of CHLA and PUG on SARS-CoV-2 infection in BSL-3 grade laboratory, and uses Reidesvir as positive control. VERO-E6 grown as monolayers in 12-well plates WAs pretreated with varying concentrations of CHLA or PUG for 1h, and then infected with the SARS-CoV-2 clinical isolate IsolateUSA-WA1/2020 (from BEI resource Bank). After CO-cultivation for 1h, the medium was changed to fresh MEM containing 1.25% Avicel and incubated at 37 ℃ with 5% CO₂Incubate for 48h in ambient. Cells were then fixed with 10% formalin, stained with 1% crystal violet and observed. The results showed that both CHLA and PUG inhibited the formation of SARS-CoV-2 plaques in a dose-dependent manner (FIG. 2A), EC₅₀The values were 9.76. + -. 0.42. mu.M and 7.20. + -. 1.08. mu.M, respectively (FIG. 2B). To eliminate the diseaseInhibition of toxic replication is the possibility that results from compound-mediated cytotoxicity, and cell proliferation-based cytotoxicity assays were performed in the present invention. As shown in FIG. 2B, CC for CHLA and PUG₅₀The (50% cytotoxic concentration) values are all around 100 μ M, and the selectivity index (SI [ CC ]₅₀/EC₅₀]) 10 and 13 respectively. These results indicate that CHLA and PUG have the activity of inhibiting the replication of SARS-CoV-2 in vitro.

Two independent experiments have shown that cell surface GAGs, such as heparan sulfate, participate in the entry of SARS-CoV-2 by interacting with viral surface glycoproteins (S).

Thus, the present invention first tests whether CHLA and PUG function by preventing SARS-CoV-2 entry. The present invention prepared a lentivirus pseudovirus (SARS-CoV-2pp) carrying SARS-CoV-2S protein and utilized 293T cells transiently expressing human ACE2 and TMPRSS2 as target cells (FIG. 3A). CHLA or PUG with a final concentration of 100-1.56 μ M was added during the SARS-CoV-2pp vaccination, and after incubation for 48h, luciferase activity was analyzed and virus entry effect was monitored. Neither CHLA nor PUG affected the entry of SARS-coV-2pp without interfering with target cell viability, suggesting that CHLA and PUG inhibit SARS-CoV-2 replication by another mechanism, rather than by inhibiting viral entry mediated by viral glycoprotein S (FIG. 3B).

3-chymotrypsin-like cysteine proteases (3 CL)^Pro) Is one of the most potential drug targets in coronaviruses. And Papain (PL)^Pro) Same, 3CL^ProIs an essential viral protease to treat viral polyprotein, and thus, inhibition of the activity of this enzyme would block viral replication. A simulation experiment result shows that several hydrolyzed polyphenols including CHLA are SARS-CoV-23CL^ProA potential inhibitor of (a). To verify this problem, the present inventors performed SARS-CoV-23CL on CHLA and PUG^ProEnzyme inhibition screening assay. SARS-CoV-23CL purified by prokaryotic expression^ProCarrying a natural N-terminal and carrying a His purification tag at the C-terminal. When 3CL is used^proAnd a Fluorescence Resonance Energy Transfer (FRET) based peptide substrate (Dabcyl-KTSAVLQ/SGFRKME-Edans, 50. mu.M) in reaction buffer (50mM Tris-HCl,ph7.3, 1mM EDTA), the substrate is hydrolyzed by the action of enzymes. After hydrolysis, the fluorophore Edans was no longer affected by the quencher molecule Dabcyl, resulting in an increase in the fluorescence signal, which was excited at 336/20nm and emitted at 490/20nm (FIG. 4A). The results show that CHLA and PUG are able to dose-dependently reduce fluorescence generation, IC₅₀The values were 9.09. + -. 0.87. mu.M and 4.62. + -. 0.27. mu.M, respectively (FIGS. 4B and 2C). Fluorescence interference experiments further showed that CHLA and PUG only affect fluorescence detection at higher concentrations: (>50 μ M, data not shown). In conclusion, the experiments of the invention show that CHLA and PUG can block 3CL^ProThe enzyme activity of (a). Notably, CHLA is on SARS-CoV-2PL^ProHas little effect on the enzyme activity of (A), while PUG weakly inhibits PL^ProIC of₅₀Over 50 μ M (FIG. 5). These data indicate that PUG is a potential dual-target SARS-CoV-2 inhibitor, although it inhibits PL^ProIs less active.

To better understand CHLA and PUG with SARS-CoV-23CL^ProKinetic model of interaction the hydrolytic activity of proteases was determined at a series of concentrations in the presence of different concentrations of inhibitor, the substrate concentration remaining constant at sub-saturation levels. Relative to 3CL with initial reaction rate at each inhibitor concentration^ProWas plotted and fitted to a straight line using linear regression analysis. As shown in fig. 2D, the slope of the line decreased with increasing concentrations of either CHLA or PUG, indicating that both CHLA and PUG are reversible inhibitors. To deduce the binding mode, in 3CL^ProThe enzyme activity was measured at constant concentration (250nm), in the absence or presence of different concentrations of CHLA or PUG, increasing the substrate concentration and plotted against a Lineweaver-Burk plot, respectively. The results show that all lines cross on the x-axis, indicating that both CHLA and PUG exhibit non-competitive inhibition patterns (FIG. 2E).

According to the enzymatic assay of the present invention, both CHLA and PUG are noncompetitive allosteric inhibitors. In order to search potential allosteric binding sites, the invention utilizes AutoDockVina software to perform CHLA and PUG to 3CL^ProVirtual docking of (3). As shown in FIG. 2F, both CHLA and PUG are likely to bind to 3CL^ProHas stable binding freedom at the cleft between domains II and IIICan be used. This binding site is slightly distant from the substrate binding site.

In conclusion, the experimental results of the present invention show that CHLA and PUG are novel against 3CL^ProSARS-CoV-2 inhibitors of proteases and provide evidence that these inhibitors are potential codv-19 therapeutics. At the same time, this indicates the importance of herbal extracts as a means of discovering and developing potential antiviral therapies.

FIG. 2 shows the passage of Chebulagic acid (CHLA) and Punicanagin (PUG) through 3CL blockade^ProThe enzyme activity inhibits SARS-CoV-2 infection. A. CHLA and PUG and Reidesvir (3. mu.M) were tested for plaque formation inhibition of SARS-CoV-2 at certain concentrations. CHLA and PUG exhibit dose-dependent inhibition of SARS-CoV-2 replication. Antiviral activity and cytotoxicity are shown in red and blue, respectively. IC (integrated circuit)₅₀And CC₅₀Shown in the upper left corner of each compound. Data represent triplicate measurements ± mean Standard Deviation (SD). IC (integrated circuit)₅₀Values were determined by fitting dose-response curves to four-parameter logistic regression in GraphPad (version 8.1.2), all data normalized to the control. CHLA and PUG on 3CL^ProDose-dependent inhibition of (a). The assay is performed using a Fluorescence Resonance Energy Transfer (FRET) based lysis method. IC of each compound₅₀The values are shown in the upper left corner. Data represent triplicate measurements ± mean Standard Deviation (SD). D. CHLA and PUG at different concentrations against SARS-CoV-23CL^proInfluence of the enzyme activity. CHLA or PUG on SARS-CoV-23CL^proLineweaver-Burk plot of inhibition. F. Prediction of CHLA (i) and PUG (ii) with 3CL^pro(PDB ID:6m2 n). Red circles indicate catalytic sites. SARS-CoV-23CL^proRepresented by a wheat colored cartoon, while CHLA and PUG appear stick-like. The bond is shown as a dashed line and the bond length is measured between the heavy atoms.

FIG. 3 is a pseudoviral infection assay. SARS-CoV-2 pseudovirus experimental picture, 293T cell used as target cell after transient expression of ACE2 and TMPRSS 2. B. Anti-pseudoviral activity and cytotoxicity are shown in red and blue, respectively. Data represent triplicate measurements ± mean Standard Deviation (SD).

In FIG. 4, A. is based on Fluorescence Resonance Energy Transfer (FRET)To 3CL^ProThe enzyme activity of (a) was monitored. 3CL^ProCleavage results in an increase in the fluorescence signal due to cleavage between the quenching molecule Dabcyl and the fluorophore Edans. B. 3CL at a certain concentration of CHLA or PUG^ProInitial fluorescence zymogram curve (250 nm).

FIG. 5 shows that CHLA and PUG have little effect on PLPro activity.

PL of SARS-CoV-2^proThe gene (957bp) was PCR amplified from pET-32a (+) -pap _ like protease plasmid (the gift from the ancient university of transportation, ceramic doctor), cloned into pET32a (+) expression vector, and transformed into DH5 α competent cells. By sequencing, PL-containing cells were correctly cloned in pET32a (+) vector^proThe gene was plasmid and transformed into BL21(DE3) E.coli for protein expression and purification, and the N-terminal tag was removed by digestion with bovine Enterokinase (EK) prior to activity determination. Protein purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentration was determined by Bradford method using bovine serum albumin as standard.

For PL^ProThe substrate for activity detection is a fluorescent-labeled peptide Z-Arg-Leu-Arg-Gly-AMC (Z-RLRGG-AMC) synthesized by NJpeptide. Hydrolysis of the peptide bond of AMC significantly increases the fluorescence of the AMC molecule, thereby allowing accurate determination of enzyme activity. The total volume of the reaction was 100. mu.L, which contained the following ingredients: 20mM Tris-buffer, pH 8.0, 4mM Dithiothreitol (DTT), 300nM PL^proAnd a gradient concentration of inhibitor (0-100. mu.M). Detection was initiated by the addition of Z-RLRGG-AMC at a final enzyme concentration of 30. mu.M. The reaction progress was monitored continuously on a microplate reader BioTek Synergy LX (λ ex 360 nm; λ em 460 nm; gain 40).

FIG. 5 is an in vitro assay of CHLA (A) and PUG (B) vs PL^proInhibition of enzyme activity. Targeting SARS-CoV-2PL Using Fluorescently labeled peptide Z-RLRGG-AMC as substrate^proProkaryotic expression, purification and fluorescence measurement are carried out.

As shown in FIG. 5, CHLA has little effect on PL activity, while PUG shows weaker inhibitory effect (IC) at higher concentrations₅₀Values above 50 μ M).

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.