Application of licochalcone in inhibition of coronavirus 3CL enzyme

Abstract

The invention belongs to the technical field of natural product separation and medicine, and provides application of licochalcone in inhibition of coronavirus 3CL enzyme. The application of licochalcone C in preparation of antiviral products has the advantages of clear action mechanism, remarkable inhibition effect, high safety and convenience in administration. The licochalcone C can be prepared into oral preparations and the like, is used in antiviral products, and has good market prospects and application values.

Description

Technical Field

[0001] The invention belongs to the field of natural product separation and pharmaceutical technology, and specifically relates to an application of licorice chalcone in inhibiting coronavirus 3CL enzyme. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] COVID-19 (Coronavirus Disease 2019) is pneumonia caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccines are the main strategy for preventing and treating viruses, but the rapid mutation of the virus causes the vaccine to fail. Therefore, drug intervention remains the main means of treating SARS-CoV-2.

[0004] SARS-CoV-2 3-chymotrypsin-like protease (3CL pro ), also known as the main protease of the new coronavirus (Mpro), is a non-structural protein in SARS-CoV-2. It plays an irreplaceable role in the life cycle of the new coronavirus, assisting in the replication and transcription of the virus and interfering with the host's antiviral innate immune response. pro The structure and function of 3CL are highly conserved among all known coronaviruses. pro Drugs that can significantly reduce mutation-mediated drug resistance. pro It has become a major target for the development of new anti-SARS-CoV-2 drugs. Targeting 3CL pro Inhibitors of this virus would block viral replication and are a promising strategy for SARS-CoV-2 drug design.

[0005] Licorice is a plant of the Leguminosae family. Glycyrrhiza uralensis Fisch.), Glycyrrhiza inflata ( Glycyrrhiza inflata Bat.) or Glycyrrhiza glabra ( Glycyrrhiza glabra L.) dried roots and rhizomes. Licorice has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, and relieving pain. In addition, in the clinical literature reports on the treatment of COVID-19 with traditional Chinese medicine, licorice is used most frequently. At the same time, the research team conducted a preliminary study on 100 traditional Chinese medicines for the treatment of 3CL pro Activity studies have found that licorice has an effect on 3CL prohas a strong inhibitory effect and shows inhibitory activity against SARS-CoV-2 virus replication at the cellular level.

[0006] Licochalcone compounds are an important class of active ingredients in licorice, with a unique α, β-unsaturated ketone nucleus. Among them, common chalcone compounds in licorice include isoliquiritigenin, licochalcone A, B, C, D, E, and isoliquiritin. Literature studies have shown that isoliquiritigenin has antiviral activity and can inhibit the replication of human immunodeficiency virus in T cells, and it can inhibit the activity of HCoV-229E. Licochalcone A is a multi-target natural drug molecule with great potential in the fields of anti-infection, anti-tumor, and skin care. Some studies have used computer simulation to calculate its binding energy with SARS-CoV-2 PL pro , but there is no experimental verification, nor has its inhibitory activity against 3CL pro been explored. Some studies have reported that licochalcone B can bind to the S protein in SARS-CoV-2, but its half-maximal effective concentration for coronaviruses is 15 μM, with a relatively high effective concentration and low efficacy. Licochalcone C has antioxidant, anti-inflammatory, antibacterial, and anti-tumor effects. Currently, studies have reported that its toxicity is relatively low, but its anti-coronavirus effect has not been reported.

[0007] In summary, there is no research report on the anti-3CL enzyme activity of licochalcone C. Summary of the Invention

[0008] The object of the present invention is to provide a drug that can inhibit SARS-CoV-2: licochalcone C.

[0009] In view of the problems in the prior art, the present invention provides an application of licochalcone in inhibiting coronavirus 3CL enzyme.

[0010] To achieve the above object, the present invention adopts the following technical solutions.

[0011] A coronavirus 3CL inhibitor, comprising licochalcone A and / or licochalcone C.

[0012] The structural formula of the licochalcone A is: .

[0013] The structural formula of the licochalcone C is: .

[0014] The coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or SARS-CoV-2.

[0015] A drug containing the above-mentioned coronavirus 3CL inhibitor.

[0016] The drug also includes other inert excipients.

[0017] The present invention has the following advantages: The present invention uses affinity ultrafiltration, enzyme activity assay, and cell inhibition assay as means to discover SARS-CoV-2 3CL pro inhibitors. Glycyrrhetin A and Glycyrrhetin C were screened from licorice by affinity ultrafiltration, and their inhibitory activity against 3CL pro was determined by enzyme activity experiments, and their anti-coronavirus replication ability was detected by cell experiments. Glycyrrhetin A and C found from natural products have significant inhibitory activity against coronaviruses. The present invention provides the application of Glycyrrhetin C in anti-coronavirus, providing a small molecule compound with good development prospects for anti-COVID-19. The advantages of the application of Glycyrrhetin C of the present invention in the preparation of antiviral products are clear action mechanism, significant inhibitory effect, high safety, and convenient administration. Glycyrrhetin C can be prepared into oral preparations, etc., for use in antiviral products, and has good market prospects and application value. Description of the Drawings

[0018] Figure 1 is the HPLC of the methanol extract of licorice and its ultrafiltration results. Among them, A is the HPLC of the methanol extract of licorice, and B is the HPLC of the ultrafiltrate; Figure 2 is the dose-effect curve for identifying the inhibitory effect of the compound on 3CL pro Among them, A is liquiritigenin, B is isoliquiritigenin, C is Glycyrrhetin A, and D is Glycyrrhetin C; Figure 3 is the 3CL pro hydrolysis activity at different concentrations of Glycyrrhetin. Among them, A is Glycyrrhetin A, and B is Glycyrrhetin C; Figure 4 is the Lineweaver-Burk curve of the inhibition of 3CL pro by Glycyrrhetin. Among them, A is Glycyrrhetin A, and B is Glycyrrhetin C; Figure 5 is the inhibitory activity of Glycyrrhetin against HCoV-229E. Among them, A is Glycyrrhetin A, and B is Glycyrrhetin C; Figure 6 is the inhibitory activity of Glycyrrhetin C against SARS-CoV-2. Detailed Embodiments

[0019] The present invention will be further described below in conjunction with the embodiments and the drawings, but the present invention is not limited by the following embodiments.

[0020] Example 1 Screening of 3CL protease inhibitor components in Glycyrrhiza uralensis Fisch 1. Affinity ultrafiltration screening of 3CL protease active components in Glycyrrhiza uralensis Fisch Take an appropriate amount of the crude powder of dried Glycyrrhiza uralensis Fisch, and extract it by ultrasonic extraction with methanol (10 times the amount of methanol, 70 W, 60 min each time, extract 4 times). Combine the extracts, filter, and dry under reduced pressure using a rotary evaporator. Take an appropriate amount of the extract, dissolve it in methanol to obtain the methanol extract of Glycyrrhiza uralensis Fisch

[0021] Take 400 μL of 3CL pro solution (2.0 mg / mL), and add 30 μL of the methanol extract of Glycyrrhiza uralensis Fisch. After incubating at 30 °C for 20 min, transfer the mixture to an ultrafiltration tube and centrifuge at 15000 x g for 10 min. Discard the lower filtrate, add 200 μL of methanol to the ultrafiltration tube, let it stand for 30 min to release the bound ligand, and collect the ultrafiltrate for HPLC and UHPLC-MS / MS analysis. Take an equal amount of the methanol extract of Glycyrrhiza uralensis Fisch in a 1.5 mL EP tube, add a buffer solution with the same volume as 3CL pro as a control group, and perform the remaining operations in the same way as the experimental group

[0022] 2. Identification of compounds in the ultrafiltrate Under appropriate incubation conditions, potential small molecule inhibitors in the crude extract can enter the active pocket cavity of 3CL pro and form a 3CL pro -inhibitor complex through intermolecular forces. The volume of the complex is larger than the pore size of the ultrafiltration membrane. During high-speed centrifugation, 3CL pro and the 3CL pro -inhibitor complex are retained in the tube, while small molecule compounds not bound to the protein can pass through the ultrafiltration membrane. Subsequently, an organic solvent is used to denature the protein to release the bound compounds, and the ultrafiltrate is collected by high-speed centrifugation. The ultrafiltrate is analyzed by UHPLC-MS / MS to obtain small molecule compounds bound to 3CL pro

[0023] Table 1 UHPLC-MS / MS analysis results of the ultrafiltrate The HPLC results of the ultrafiltrate are as Figure 1 shown. By comparing the HPLC results of the experimental group and the control group, it can be found that the peak area in the experimental group is significantly higher than that in the control group, indicating that there are small molecule compounds bound to 3CL pro in the ultrafiltrate of the experimental group. The ultrafiltrate of the experimental group was analyzed by UHPLC-MS / MS. According to the mass spectrometry characteristic fragments, fragmentation rules and reference documents, we identified 4 potential 3CL​pro Inhibitors (Table 1). They are liquiritigenin, isoliquiritigenin, licochalcone A, and licochalcone C, respectively.

[0024] 3. Identification of the inhibitory activity of the compounds against 3CL enzyme It was divided into an experimental group and a control group, with Ebselen as the positive drug. Experimental group: 40 μL of 250 nM 3CL pro solution and 10 μL of the sample solution were mixed in a 96-well plate and incubated at room temperature for 30 min. Control group: 10 μL of DMSO was used instead of 10 μL of the sample solution. After incubation, 50 μL of 25 μM 3CL pro fluorescent substrate peptide was added, and the fluorescence intensity value was detected by an enzyme-linked immunosorbent assay (excitation light: 336 nm, emission light: 490 nm, continuously monitored for 1 h). Each well was repeated 3 times, the inhibition rate was calculated, and the half-maximal inhibitory concentration (IC 50 ) was fitted.

[0025] Identification of the inhibition rate curve of the compounds against 3CL pro is shown as follows. The IC Figure 2 of the positive control drug Ebselen was 0.894 ± 0.0009 μM, and the IC 50 values of the identified compounds were: liquiritigenin (271.600 ± 6.324 μM), isoliquiritigenin (165.400 ± 4.504 μM), licochalcone A (37.617 ± 1.129 μM), licochalcone C (43.940 ± 10.208 μM). Among the 4 compounds, licochalcone A and licochalcone C had stronger inhibitory activities against 3CL 50 and were natural small molecule inhibitors of 3CL pro . pro

[0026] Example 2 Inhibitory type of licochalcone against 3CL pro To understand the kinetic mode of the interaction between licochalcone A and licochalcone C and 3CL pro , increasing concentrations of 3CL pro (175 nM, 150 nM, 100 nM, 75 nM, 50 nM) were incubated with different concentrations of licochalcone A and licochalcone C at room temperature for 30 min, and then the fluorescence signal was detected after adding 25 μM of the polypeptide substrate. A graph of the initial reaction rate V 0 versus the protein concentration was plotted and linearly fitted. Then, different concentrations of licochalcone A and licochalcone C were incubated with 100 nM 3CL proIncubate at room temperature for 30 min, add fluorescent substrates at different concentrations to initiate the reaction, detect the fluorescence signal, plot the Lineweaver-Burk curve, and determine the type of inhibition according to the curve.

[0027] The types of enzyme inhibition can be divided into reversible inhibitors and irreversible inhibitors. Irreversible inhibitors refer to compounds that form covalent bonds with enzymes, which are relatively stable and not easily broken once formed. Reversible inhibitors include competitive inhibitors, non-competitive inhibitors, and uncompetitive inhibitors. As Figure 3 shown, as the concentrations of liquiritigenin A and liquiritigenin C increase, the slope of the straight line gradually decreases, indicating that both are reversible inhibitors of 3CL pro . Therefore, further explore their inhibitory behavior on 3CL pro .

[0028] Then, keep the protein unchanged, change the substrate concentration and compound concentration, plot the reciprocal of the initial reaction rate against the reciprocal of the compound concentration to obtain the Lineweaver-Burk curve. If the straight lines intersect at the ordinate, it indicates that the compound is a competitive inhibitor; if the straight lines intersect at the abscissa, it indicates that the compound is a non-competitive inhibitor; if the straight lines intersect in the second quadrant, it means that the compound is a mixed (including both competitive and non-competitive) competitive inhibitor. As Figure 4 shown, as the concentrations of liquiritigenin A and liquiritigenin C increase, the maximum initial rate (Vmax) of the Lineweaver-Burk plot decreases, and the Michaelis constant (Km) increases. The straight lines intersect in the second quadrant, indicating that both belong to the mixed competitive inhibition mode. In summary, liquiritigenin A and liquiritigenin C bind to 3CL pro in a reversible mixed manner.

[0029] Example 3 Inhibitory Activity of Liquiritigenin on HCoV-229E 1. Cytotoxicity of Liquiritigenin on Huh-7 Cells Huh-7 cells (1.86 × 10 4 cells / well) were seeded in 96-well plates and cultured at 37 °C with 5% CO 2 for 24 h. 100 μL of the compound was added to each well and incubated at 37 °C for 48 h. The supernatant was discarded, 100 μL of DMEM and 10 μL of CCK-8 solution were added to each well, and the cells were cultured in an incubator at 37 °C for 1 - 1.5 h. The absorbance at 450 nm was measured using a microplate reader. Five replicates were set for each concentration, and a cell control group and a blank solvent group were also set. The maximum non-toxic concentration was defined as a cell survival rate > 90%, and the half cytotoxic concentration (Cellular Cytotoxicity Concentration, CC 50 ) was fitted.

[0030] 2. Inhibitory Activity of Licorice Chalcones against HCoV-229E To further verify the anti-coronavirus effects of licorice chalcone A and licorice chalcone C, an HCoV-229E-infected Huh-7 cell model was constructed: Huh-7 cells (1.60×10 4 cells / well) were seeded in 24-well plates and cultured at 37 °C for 24 h, and then the culture medium was discarded. 500 μL of sample solution was added to each well, and after culturing for 4 h, the culture medium was discarded. Then, 500 μL of HCoV-229E virus dilution (MOI = 0.1) was added to each well. After incubating at 34 °C for 1 h, the supernatant was discarded, 500 μL of sample solution was added, and the supernatant was collected after incubating at 34 °C for 48 h. The TCID 50 of the collected supernatant was measured using the Reed-Muench method, and the inhibition rates of licorice chalcone A and licorice chalcone C against coronavirus 229E were calculated, and the median effective concentration (EC 50 ) and selectivity index (SI value) were fitted.

[0031] The results are as Figure 5 shown. The CC 50 values of licorice chalcone A and licorice chalcone C against Huh-7 cells were 21.18 ± 0.80 μM and 18.53 ± 0.20 μM, respectively. The EC 50 values of the two compounds against HCoV-229E were 5.19 ± 0.30 μM and 2.28 ± 0.44 μM. Among them, the SI value of licorice chalcone A was 4.10, and the SI value of licorice chalcone C was 8.13. Considering effectiveness and safety, licorice chalcone C was selected for subsequent in vitro anti-SARS-CoV-2 replication experiments.

[0032] Example 4 Inhibitory Activity of Licorice Chalcone C against SARS-CoV-2 1. Cytotoxicity of Licorice Chalcone C against Vero-E6 Cells Vero-E6 cells cultured in 96-well plates were added with 100 μL of licorice chalcone C per well and incubated at 37 °C for 48 h. After discarding the supernatant, the proliferation toxicity of the active ingredient against Huh-7 cells was detected using CCK-8. Five replicates were set for each concentration, and a cell control group and a blank solvent group were also set.

[0033] 2. Inhibitory Activity of Licorice Chalcone C against SARS-CoV-2 Vero-E6 cells cultured in 96-well plates were inoculated with 100 TCID at 37 °C50 SARS-CoV-2 for 2 h. The inoculum was removed, and the cells were then treated with different concentrations of liquiritigenin C (10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM). After incubation for 72 h, the infected cells showed 100% CPE under the microscope. The percentage of CPE in the cells treated with the active ingredient was recorded, and the 50% inhibitory concentration (EC 50 ) was calculated.

[0034] The results are as Figure 6 shown. The CC 50 of liquiritigenin C against Vero-E6 was 41.83 ± 2.18 μM. Liquiritigenin C inhibited SARS-CoV-2 in a dose-dependent manner, with an EC 50 value of 3.711 μM and an SI value of 11.27. Therefore, the cell-based antiviral experiment showed that liquiritigenin C could inhibit the replication of the novel coronavirus in a dose-dependent manner.

[0035] The above are only examples of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A coronavirus 3CL inhibitor, characterized in that Including licochalcone A and / or licochalcone C.

2. The coronavirus 3CL inhibitor according to claim 1, characterized in that The coronavirus is selected from HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or SARS-CoV-2.

3. A medicine containing the coronavirus 3CL inhibitor as claimed in claim 1.

4. The drug according to claim 3, characterized in that Other inert excipients are also included.

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