Application and screening method of verbascoside in the preparation of drugs for inhibiting the new coronavirus

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the application of verbascoside in the preparation of drugs for inhibiting the new coronavirus and a screening method. The present invention has found through research that verbascoside has a significant inhibitory effect on the main protease of SARS-CoV-2, and for the first time found that verbascoside has a significant inhibitory effect on Omicron BA.1 and BA.2 virus strains, so it can be used for drugs that inhibit the new coronavirus and drugs against Omicron virus. The present invention also provides a screening method for the main protease inhibitor of the new coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of verbascoside in the preparation of drugs for inhibiting the new coronavirus. Background Art

[0002] The new coronavirus (SARS-CoV-2) belongs to the genus β coronavirus, a single-stranded positive-strand RNA, and its genome contains about 30,000 nucleotides, encoding 29 open reading frames (ORFs), 16 non-structural proteins (Nsps) and 4 structural proteins, which are highly contagious. The infection and reproduction rate of the new coronavirus variant strain Omicron in the bronchi is much higher than that of the original coronavirus strain, and it is more contagious. Most of the drugs currently available for the new coronavirus are in the stage of virtual screening of virtual targets. However, after verifying these drugs with the new coronavirus, it was found that the targets disclosed in the existing literature reports of some drugs are not the targets that they can really play a role, resulting in them not being able to really inhibit replication or kill when applied to the real new coronavirus. Drugs that can inhibit replication or kill the new coronavirus may not be effective for the mutant strains of the new coronavirus because the mutant strains may not have the target of the drug. This is also one of the reasons why antiviral drugs need to become ineffective as the virus mutates. Therefore, more effective methods are needed to screen drugs that are effective against the new coronavirus and its mutant strains. Summary of the invention

[0003] In view of the above problems, the present invention provides the use of verbascoside in the preparation of drugs for inhibiting the new coronavirus. Experimental studies have found that verbascoside has an inhibitory effect on SARS-CoV-2M pro (SARS-CoV-2 main protease) has a significant inhibitory effect and has a significant inhibitory effect on Omicron BA.1 and BA.2 virus strains, and can be used as a drug to inhibit the new coronavirus and a drug against Omicron virus.

[0004] In order to achieve the above-mentioned purpose of the invention, the embodiment of the present invention adopts the following technical solutions:

[0005] In a first aspect, an embodiment of the present invention provides the use of verbascoside in the preparation of drugs for inhibiting the new coronavirus.

[0006] The structural formula of verbascoside is shown in Formula I:

[0007]

[0008] The present invention uses SARS-CoV-2M proAs the target protein, SARS-CoV-2M pro The His41 and Cys145 of SARS-CoV-2 were used as docking active pockets, and an in vitro enzyme activity screening system was established. The study found that verbascoside and SARS-CoV-2M pro Active pocket binding; molecular docking showed that verbascoside binds to SARS-CoV-2M through hydrogen bonds and other interactions pro The catalytically active region has a binding capacity comparable to that of the original ligand and forms hydrogen bond interactions with the reported important amino acid residues Phe140 and Cys145. The results of molecular interaction studies showed that verbascoside has an inhibitory effect on SARS-CoV-2M pro The activity has a significant inhibitory effect.

[0009] In addition, in vitro viral inhibitory activity studies have shown that verbascoside has a certain inhibitory effect on Omicron BA.1 and BA.2 virus strains, but no reports have been found on the application of verbascoside to Omicron virus strains.

[0010] In combination with the first aspect, the drug for inhibiting the new coronavirus is SARS-CoV-2M pro Non-covalent inhibitors.

[0011] In a second aspect, an embodiment of the present invention provides the use of verbascoside in the preparation of an in vitro product for inhibiting the new coronavirus.

[0012] In combination with the second aspect, in the product, the concentration of verbascoside is 0.039 μM to 160 μM. The experiment verified that verbascoside has an inhibitory effect on SARS-Cov-2 M in the concentration range of 0.039 μM to 160 μM. pro It has a significant inhibitory effect.

[0013] In a third aspect, an embodiment of the present invention provides the use of verbascoside in the preparation of an anti-Omicron virus drug.

[0014] The present invention found through in vitro pharmacodynamic studies that verbascoside has an EC of 1. 50 The EC value for Omicron BA.2 was 41.29 μM. 50 It is 70.37μM, which can be used as a potential drug against Omicron virus.

[0015] In combination with the third aspect, the strain of Omicron virus includes at least one of the BA.1 and BA.2 strains.

[0016] In a fourth aspect, an embodiment of the present invention further provides a method for screening a novel coronavirus main protease inhibitor, comprising the following steps:

[0017] Step A: SARS-CoV-2M pro The recombinant plasmid is transformed into competent cells of Escherichia coli BL21 (DE3), and positive clones are screened; the positive clones are amplified and cultured, and then the bacterial liquid is collected, the bacteria are broken, and the supernatant is collected;

[0018] Step B: After removing the protein from the supernatant, the supernatant was digested with human rhinovirus 3C protease, and the protein was collected and concentrated to obtain SARS-CoV-2M pro ;

[0019] Step C: the SARS-CoV-2M pro The compounds to be screened are added to the same buffer, and the fluorescence intensity of the buffer is detected after adding the fluorescently labeled substrate, and the main protease inhibitors of the new coronavirus are screened according to the changes in fluorescence intensity.

[0020] In combination with the fourth aspect, the screening method also includes virtual screening verification: including using SARS-CoV-2M pro As the target protein, SARS-CoV-2M pro His41 and Cys145 are docking active pockets, and the obtained compounds are screened in step C for virtual validation and SARS-CoV-2M pro Binding status of active pockets.

[0021] Preferably, the virtual screening validation further comprises validating the compounds obtained by screening in step C and the SARS-CoV-2 M pro The stability of the complex.

[0022] Preferably, the virtual screening validation further comprises validating the compounds obtained by screening in step C and the SARS-CoV-2 M pro The number of hydrogen bonds.

[0023] The beneficial effect of the present invention is that the inventors have found for the first time that verbascoside has an inhibitory effect on SARS-CoV-2 in the concentration range of 0.039 μM to 160 μM through in vitro enzyme inhibition activity screening and virtual screening verification. pro It has a significant inhibitory effect. And the present invention first discovered through virus inhibition research that verbascoside has a significant inhibitory effect on Omicron BA.1 and BA.2 virus strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the IC of verbascoside in Example 1 of the present invention 50 curve;

[0025] Figure 2 The compound of verbascoside and SARS-CoV-2M in Example 1 of the present invention proMolecular docking;

[0026] Figure 3 The effect of verbascoside on SARS-CoV-2M in Example 1 of the present invention pro Molecular dynamics studies of

[0027] Figure 4 The evaluation of the effect of verbascoside on SARS-CoV-2 M by plasma surface resonance and isothermal titration calorimetry in Example 1 of the present invention is as follows: pro The intermolecular interactions;

[0028] Figure 5 Verbascoside-SARS-CoV-2M in Example 1 of the present invention pro Calorimetric titration curve at 298K;

[0029] Figure 6 is the verbascoside in Example 1 of the present invention 1 H NMR spectrum;

[0030] Figure 7 SARS-CoV-2M in Example 1 of the present invention pro of 1 H NMR spectrum;

[0031] Figure 8 The compound of verbascoside and SARS-CoV-2M in Example 1 of the present invention pro Combined 1 H NMR spectrum;

[0032] Fig. 9 The verbascoside and SARS-CoV-2M in Example 1 of the present invention pro STD-NMR spectrum of

[0033] Fig.10 The verbascoside and SARS-CoV-2M in Example 1 of the present invention pro WaterLOGSY-NMR spectrum;

[0034] Fig.11 This is a study on the inhibitory effect of verbascoside on viruses in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] The novel coronavirus is highly contagious, and its mutant strain, Omicron, has a higher infection and reproduction rate in the bronchi and is more contagious. Most of the drugs currently available for the novel coronavirus are based on virtual screening of virtual targets. However, some drugs cannot inhibit replication or kill the real novel coronavirus, and may not be effective against the mutant strains of the novel coronavirus.

[0037] To address this issue, the inventors conducted drug screening and in vitro pharmacodynamics verification using the new coronavirus and its mutant strains in a biosafety level 3 laboratory (P3 laboratory).

[0038] Traditional Chinese medicine has shown good therapeutic effects in treating novel virus infections, but its active ingredients and mechanism of action are still unclear. pro ) is a proteolytic enzyme that can hydrolyze and process the replicase precursor polyprotein and release the polymerase and helicase required for viral RNA replication, participating in the key steps of the viral replication process. pro Structural analysis of SARS-CoV-2 pro The recombinant plasmid was transformed into E. coli and purified to obtain SARS-CoV-2M pro Using an in vitro enzyme activity screening method, we screened hundreds of small molecules of traditional Chinese medicine contained in dozens of commonly used heat-clearing and detoxifying Chinese herbs for enzyme inhibition activity, and found that verbascoside has an inhibitory effect on SARS-CoV-2M pro The present invention establishes a molecular docking model to verify the inhibitory effect of verbascoside on SARS-CoV-2M pro The inhibitory effect of verbascoside on SARS-CoV-2M pro The combination ability and combination method.

[0039] In addition, the inventors used Omicron BA.1 and BA.2 virus strains to infect African green monkey kidney cells Vero E6 (ATCC, CRL-1586) and found for the first time that verbascoside had an inhibitory effect on Omicron strains, and calculated the half-maximal effective concentration (EC) of verbascoside based on the cytopathic effect. 50 ).

[0040] Based on the research results, the embodiments of the present invention provide the use of verbascoside in the preparation of drugs for inhibiting the new coronavirus and drugs against Omicron.

[0041] This application provides a new option for fighting the new coronavirus and its mutant strain Omicron. In clinical applications, verbascoside can be used alone according to specific needs, or combined with its target and mechanism of action for drug use.

[0042] In addition, the present invention also provides a method for screening a novel coronavirus main protease inhibitor, comprising the following steps:

[0043] Step A: SARS-CoV-2M pro The recombinant plasmid is transformed into competent cells of Escherichia coli BL21 (DE3), and positive clones are screened; the positive clones are amplified and cultured, and then the bacterial liquid is collected, the bacteria are broken, and the supernatant is collected;

[0044] Step B: After removing the protein from the supernatant, the supernatant was digested with human rhinovirus 3C protease, and the protein was collected and concentrated to obtain SARS-CoV-2M pro ;

[0045] Step C, the SARS-CoV-2M pro The compound to be screened is added to the same buffer and incubated for a certain period of time. After adding the fluorescently labeled substrate, the fluorescence intensity of the buffer is detected, and the main protease inhibitor of the new coronavirus is screened according to the change in fluorescence intensity.

[0046] The scheme of the present invention is described below through specific embodiments.

[0047] The Chinese medicine small molecule standards used in the following examples were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the HPLC purity was greater than 98%. The SARS-CoV-2Mpro recombinant plasmid was provided by the research group of Academician Rao Zihe of the Institute of Immunochemistry, ShanghaiTech University.

[0048] Example 1

[0049] This example uses the process of screening and discovering verbascoside to specifically illustrate the screening method for the novel coronavirus main protease inhibitor provided by the present invention. The experimental results in this screening method also prove the role of verbascoside in inhibiting the novel coronavirus.

[0050] 1. SARS-CoV-2M pro Expression and purification of

[0051] SARS-CoV-2M pro The recombinant plasmid was transformed into competent cells of Escherichia coli BL21 (DE3), and positive clones were screened using LB plates (containing 100 mg / L ampicillin); SARS-CoV-2M pro The nucleotide sequence of the recombinant plasmid (as shown in SEQ ID NO.1) is as follows:

[0052] SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNID YDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQ

[0053] Pick a positive clone (a single clone grown on an LB plate containing ampicillin) from the LB plate, culture it overnight, and then transfer it to 1L LB medium (containing 100mg / L ampicillin). When OD600 reaches 0.6-0.8, add about 1mM IPTG and culture it at 16℃ for about 16 hours.

[0054] The bacterial solution was collected by centrifugation at 4000 rpm for 15-20 min, and then the bacteria were broken by high-pressure homogenizer. The bacterial solution was centrifuged at 13000 rpm for 40 min and the supernatant was collected;

[0055] The supernatant was added to a nickel affinity chromatography column pre-equilibrated with Tris-HCl, and washed with Tris-HCl (containing 20 mM imidazole) for 5-10 column volumes to remove impurities, and then eluted with 300 mM imidazole. Finally, human rhinovirus 3C protease was added and digested at 4°C for 12-20 hours. After that, the protein was collected and concentrated to obtain SARS-CoV-2M pro .

[0056] 2. SARS-CoV-2M pro Screening of inhibitors

[0057] SARS-CoV-2M pro The activity was determined using the fluorescent substrate MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 (purity ≥ 98%), with the N-terminus of the substrate connected to an MCA fluorescent group (absorption wavelength 320 nm, emission wavelength 405 nm).

[0058] (1) Single point inhibition rate screening:

[0059] SARS-CoV-2 M pro In vitro inhibitory activity study. pro SARS-CoV-2 was added to different buffer solutions (50 mM Tris-HCl (pH 7.3), 1 mM EDTA (with or without DTT) pro (final concentration 0.2μM), add the above-mentioned Chinese medicine small molecule standard to make the final concentration 40μM, incubate at room temperature for 1h, and then immediately add the fluorescent labeled substrate (MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2, final concentration 20μM), the excitation wavelength and emission wavelength are 320nm and 405nm respectively, the temperature is maintained at 298K, and the fluorescence reading is recorded every 10 seconds. The control curve is the measurement curve when only protein and substrate are added without any inhibitor. The lower the curve, the stronger the inhibitory effect. When the inhibition rate of the main protease is greater than 90%, the IC of the compound is further determined. 50 .

[0060] Experiments have shown that verbascoside is effective against SARS-CoV-2M pro The inhibition rate is greater than 90%.

[0061] (2)IC 50 Determination of

[0062] Verbascoside was diluted with 13 serial dilutions of inhibitor and 0.2 μM SARS-CoV-2 M pro Mix under 50mM Tris-HCl pH 7.4, 1mM EDTA or (50mM Tris-HCl pH 7.4, 1mM EDTA, 0.01% Triton X-100), incubate at room temperature for 1h, then add 20μM substrate peptide, react at room temperature, and then immediately place on the microplate reader for detection. All experimental data were analyzed using GraphPad Prism, and IC 50 .like Figure 1 As shown in the figure, verbascoside has an effective inhibitory effect on SARS-CoV-2 in the concentration range of 0.039 μM to 160 μM. pro It has a significant inhibitory effect.

[0063] 3. Based on SARS-CoV-2M pro Structural prediction of activity of verbascoside by virtual screening.

[0064] The experiment used SARS-CoV-2M proThe target protein (PDB ID: 6LU7) was used, and its three-dimensional structure data was derived from the PDB database. Verbascoside was used as the docking ligand. The docking was performed using Discovery Studio 2020 Client and PyMOL 2.3.0 in the ligand-flexible mode to optimize small molecules and proteins, including protein hydrogenation, charge calculation, and addition of atom types. pro The optimized small molecule ligand was docked with the viral protein receptor using His41 and Cys145 as the docking active pockets. Figure 2 As shown, verbascoside and SARS-CoV-2M pro The docking energy of active pocket binding was -68.78 Kcal / mol, which was not statistically different from the docking energy of the original ligand N3 -72.88 Kcal / mol.

[0065] 4. Effect of Verbascum glycoside on SARS-CoV-2M pro Molecular dynamics studies

[0066] Molecular dynamics simulations were performed using GROMACS 2018 software. Protein and ligand topology files were preprocessed by the CHARMM36 force field and ChARMM-GUI server, respectively. The complexes were solvated in a dodecahedral periodic box by the TIP3P water model. Microcanonical ensemble, constant pressure, constant temperature equilibrium simulations of about 50 ns were performed for each system. The results were analyzed using the Gromacs software packages GMX RMS, GMX RMSF, GMX gyration, GMX COVAR, and GMX ANAeig modules.

[0067] (1) Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis

[0068] This study tested the effects of verbascoside and SARS-CoV-2M pro The complexes were subjected to 300 ns molecular dynamics simulations, including RMSD and RMSF analysis, to reveal the stability properties of the complexes:

[0069] RMSD is the root mean square deviation. RMSD analysis is used to determine the degree of conformational difference or trajectory stability. The results are as follows: Figure 3 As shown, verbascoside in the optimal conformation of molecular docking after about 20ns relative to the reference molecule (SARS-CoV-2M pro ) reached equilibrium. At 300ns, the average RMSD of verbascoside was 0.17±0.02nm, and SARS-CoV-2M proThe average RMSD of the results is about 0.15 nm, indicating that the position change (shift) of the verbascoside structure over time is small, indicating that verbascoside is closely related to SARS-CoV-2M pro Has a stable bond.

[0070] RMSF is the root mean square fluctuation. It is used to calculate the fluctuation (variation amplitude) of each atom relative to its average position, measure the freedom of atomic movement, and characterize the flexibility of the molecular structure. Figure 3 As shown, with SARS-CoV-2M pro Compared with the RMSF value of SARS-CoV-2, the RMSF value of verbascoside fluctuated less, indicating that verbascoside has a significant effect on SARS-CoV-2M pro The overall structure of the molecule has little effect and the docking is stable, which is consistent with the RMSD results.

[0071] (2) Hydrogen bond and gyration radius (Rg) analysis

[0072] The radius of gyration (Rg) of a protein is used to describe the distribution characteristics of system atoms along a specific axis, characterize the compactness of molecules, and is an effective indicator for evaluating binding stability. Figure 3 As shown, SARS-CoV-2M pro The Rg of the complex was 2.56±0.02nm. After docking with verbascoside, the Rg of the complex only increased by 0.02-0.05nm, with no statistical difference.

[0073] The affinity of the ligand for the protein was introduced by calculating the hydrogen bonding (H-bond) interactions of the complex and predicted by molecular docking studies. The hydrogen bonding was analyzed within a simulation time of 300 ns and the results are shown in Figure 3 As shown, Verbascoside-SARS-CoV-2M pro The complex has a large number of hydrogen bonds at the beginning of the simulation and tends to be stable.

[0074] The results of gyration radius and hydrogen bond interaction showed that verbascoside is a pro An ideal ligand for stable binding.

[0075] 4. Verbascoside and SARS-CoV-2M pro Molecular interaction studies

[0076] (1) Evaluation of the interaction between verbascoside and SARS-CoV-2M based on surface plasmon resonance (SPR) pro interaction.

[0077] The experiment was performed using Biacore T200, SARS-CoV-2Mpro The glycoside was coupled to the tandem sensor chip CM5 by amino coupling reaction, and the working solution (PBS-P, 5% DMSO) of verbascoside was diluted to 0.977-500mmol / L step by step and injected into the channel at a flow rate of 10μL / min. The coupling time and dissociation time were set at 60-200s. The data were analyzed by Biacore T200 evaluation software, and the affinity curve was fitted to obtain the equilibrium dissociation constant (KD).

[0078] The results are as follows Figure 4 As shown, verbascoside and SARS-CoV-2M pro The KD value of SARS-CoV-2 was 4.67 μM, which was similar to that of SARS-CoV-2 pro Has a strong binding affinity.

[0079] (2) Evaluation of the interaction between verbascoside and SARS-CoV-2M based on isothermal titration calorimetry (ITC) pro interaction.

[0080] ITC determination is an important method for studying biothermodynamics and biokinetics. pro Dissolve the SARS-CoV-2M in the same buffer at appropriate concentrations. pro The sample pool was injected, deionized water was injected into the reference pool, and verbascoside was used as the ligand sample solution and titrated continuously to SARS-CoV-2M within the set time. pro . Collect relevant parameters, such as thermodynamic equilibrium constant (K), free energy change (ΔG), enthalpy (ΔH), entropy change (ΔS), etc.

[0081] The results are as follows Figure 5 As shown, at 298K, verbascoside binds to SARS-CoV-2M pro The thermodynamic equilibrium constant (K) of the glycoside was 0.129 μM, which had a good correlation with the SPR value, indicating that the glycoside had an inhibitory effect on SARS-CoV-2 M pro The activity has a significant affinity effect.

[0082] (3) Evaluation of the interaction between verbascoside and SARS-CoV-2M based on nuclear magnetic resonance spectroscopy (NMR) pro Interaction

[0083] In this study, the NRM method was used to observe the relationship between verbascoside and SARS-CoV-2 M using STD-NMR spectroscopy and WaterLOGSY–NMR spectroscopy.pro Verbascoside as a ligand binds to SARS-CoV-2M pro Dissolved in deuterated reagent (D2O), the concentration of the compound is SARS-CoV-2M pro The concentration of the compound was 100 times that of SARS-CoV-2M. pro The WaterLOGSY sample was prepared with 10 times the concentration of 298K at 600MHz and 256 scans. The data were analyzed using mestrenova software (Version 9.0). Figure 6 to Figure 10 shown.

[0084] STD-NMR spectrum analysis showed that verbascoside was closely related to SARS-CoV-2M pro The signal of the combined hydrogen atom is located at δH = 1.923ppm, which is identified as the 22nd hydrogen atom. The WaterLOGSY-NMR spectrum results show that only the peak signal at δH = 1.923ppm is negative, and the others are positive, that is, verbascoside is detected at δH = 1.923ppm and SARS-CoV-2M pro There is binding, and the binding site may be the 22nd hydrogen atom.

[0085] The above results suggest that verbascoside may serve as a novel SARS-CoV-2 M pro Non-covalent inhibitors play an important role.

[0086] Example 2

[0087] This example provides an in vitro pharmacodynamics experiment and results of verbascoside against the new coronavirus.

[0088] The experiment used African green monkey kidney cell Vero E6 (ATCC, CRL-1586) cell line, and the virus strains used were Omicron BA.1 and BA.2 virus strains. The dose of infected cells was MOI = 0.01, that is, 10 4 100 viruses infect each cell.

[0089] Before administration, 100 μL of Vero E6 cells were added to each well of a 96-well plate to make the number of cells per well 2×10 4 and cultured for 24 h.

[0090] The compound was diluted 3-fold stepwise at the highest concentration of 100 μM, with a total of 8 concentration gradients, and 8 replicates were made for each concentration, i.e., repeated 8 times. Omicron BA.1 or BA.2 was added to each well to infect cells, and the plate was read using a Celigo Image Cytometer 48 hours after infection. The half-maximal effective concentration (EC) was calculated based on the cytopathic effect. 50 ). The result is Fig.11 As shown, the EC value of verbascose for Omicron BA.1 50 The EC value for Omicron BA.2 was 41.29 μM. 50 It is 70.37μM.

[0091] The results show that verbascoside has a significant inhibitory effect on Omicron BA.1 and BA.2 virus strains and can be used to prepare drugs against Omicron virus.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of verbascoside in the preparation of a drug against Omicron virus, characterized in that: The strain of the Omicron virus is selected from any one of the BA.1 and BA.2 virus strains.

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