RU2804307C1 - Phillyrin and its derivatives for suppression of the 3clpro protein and fighting the covid-19 virus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the use of phillyrin and/or its derivatives, and/or phillyrin/philligenin compositions, as well as methods for using the above active ingredients. The use of phillyrin and phillyrin derivatives KD-2-GLU and KD-2-SO₃H is proposed, these compounds are represented by the following formulas:

in the preparation of an agent for inhibiting 3CLpro protein of the COVID-19 virus. Also the methods for inhibiting the 3CLpro protein of the COVID-19 virus are provided, comprising the step of reacting the 3CLpro protein of the COVID-19 virus with phillyrin or any of the above-mentioned phillyrin derivatives KD-2-GLU and KD-2-SO₃H. It is proposed to use a composition of phillyrin/philligenin with a weight ratio of phillyrin to philligenin 2~ 98:2~98 as the sole pharmaceutically active ingredient in the preparation of a medicinal product against the COVID-19 virus or a medicinal product for the treatment of a disease caused by the COVID-19 virus, as well as methods for combating the COVID-19 virus or treating a disease caused by the COVID-19 virus, comprising the step of administering to a patient in need an effective amount of the aforementioned phillyrin/philligenin composition or a medicament containing such phillyrin/philligenin composition as the sole pharmaceutically active ingredient.

EFFECT: phillyrin and the above derivatives of phillyrin have a specific effect inhibiting 3CLpro protein of the COVID-19 virus, the phillyrin/philligenin composition significantly inhibits the COVID-19 virus and overexpression of inflammatory mediators.

11 cl, 8 tbl, 4 ex, 8 dwg

Description

Field of technology

The invention relates to the field of technical medicine and, more specifically, relates to the use of phyllirin and/or its derivatives, and/or a phyllirin/phylligenin composition, which suppresses and inhibits the 3CLpro protein of the COVID-19 virus and can be used for the production of medicines against coronavirus, in particular against the COVID-19 virus, and other similar viruses.

State of the art

COVID-19 is coronavirus disease 2019 (also known as novel coronavirus pneumonia). The causative agent of COVID-19 disease is the COVID-19 virus (also known as the novel coronavirus), which belongs to the coronavirus family, which has high homology to SARS, which broke out in 2003. There are still no specific drugs against the COVID-19 virus. Until now, there are also no specific drugs against the previously outbreak of SARS.

The inventors of the present invention devoted themselves to the study of medicinal extracts of traditional Chinese medicine, and accidentally and unexpectedly discovered that the medicinal composition of phyllirin and a small amount of phylligenin has a synergistic medicinal effect, as well as a pronounced synergistic medicinal effect in terms of antiviral activity. For example, Chinese patent application CN105362283A discloses a phyllirin/phylligenin composition and its use for reducing and/or treating viral diseases, among these viral diseases are diseases caused by influenza virus, parainfluenza virus, Coxsackie virus CoxA16, respiratory syncytial virus RSV, simplex virus herpes zoster HSV-I, herpes simplex virus HSV-II, herpes simplex virus CVB3, adenovirus ADV or enterovirus EV71.

However, there are many antivirals with different mechanisms of action, and it is impossible to predict which existing antivirals will have an inhibitory effect on the emerging COVID-19 virus. In particular, since the coronavirus is largely different from the above viruses that the phyllirin/phylligenin composition targets, and research on the COVID-19 virus and its therapeutic mechanism has not yet been completed, those skilled in the art cannot predict the inhibitory effect of the phyllirin/phylligenin composition for coronavirus (especially the COVID-19 virus).

In addition, the effect of phyllirin, phylligenin, or their derivatives on the 3-chymotrypsin-like cysteine protein (abbreviated 3CL pro protein) of the COVID-19 virus has not been reported.

However, the inventors of the present invention have unexpectedly found that the phyllirin/phylligenin composition exhibits excellent inhibitory effect on the COVID-19 virus, and regarding its high level of safety, the clinical approval of this composition for other applications is nearing completion. Thus, this composition has the potential to become a drug for COVID-19 and can be quickly introduced into clinical practice. In addition, the present inventors have discovered that phyllirin, phylligenin or their derivatives have a specific inhibitory effect on the 3CL pro protein of the COVID-19 virus, and can be used as inhibitors of this protein.

Brief description of the invention

The present invention solves the technical problem of providing a new agent that specifically inhibits the 3CLpro protein of the COVID-19 virus and a drug for combating coronavirus (especially COVID-19 virus) or treating diseases caused by coronavirus (especially COVID-19).

In particular, in a first embodiment, the present invention provides the use of phyllin and/or derivatives thereof in the preparation of an agent for inhibiting the 3CLpro protein of the COVID-19 virus. An agent for inhibiting the 3CLpro protein of the COVID-19 virus includes phyllin and/or derivatives thereof.

The use in the first embodiment of the invention may be a single use of phyllirin or derivatives thereof, or a combined use of phyllirin and derivatives thereof, such as the use of a phyllirin/phylligenin composition. When used in the first embodiment of the present invention, the inhibition may be in vivo or in vitro.

Preferably, when used in the first embodiment of the present invention, the derivative is KD-2-GLU or KD-2-SO ₃ H.

Accordingly, a second embodiment of the present invention provides a method for inhibiting the 3CLpro protein of the COVID-19 virus, comprising the step of reacting the 3CLpro protein of the COVID-19 virus with phyllin and/or derivatives thereof. This method can be in vivo or in vitro.

Preferably, in the method in the second embodiment of the present invention, the derivative is KD-2-GLU or KD-2-SO ₃ H.

In a third embodiment, the present invention provides the use of a phyllirin/phylligenin composition in the preparation of medicinal products to combat coronavirus, and also provides the use of a phyllirin/phylligenin composition in the preparation of a medicinal product for the treatment of diseases caused by coronavirus. The fight against coronavirus can be in vitro, for example, by inhibiting the proliferation of coronavirus growth, but it is preferable to fight the coronavirus in vivo, i.e. diseases caused by coronavirus are being treated.

As used herein, unless otherwise noted, the terms “phyllirin/phylligenin,” “phyllirin and phylligenin,” and “phyllirin and phylligenin composition” may be used interchangeably to refer to a composition consisting of phyllirin and phylligenin, i.e., phyllirin/phylligenin is considered as a whole, non-separable composition. Preferably, in the present invention, the weight ratio of phyllirin to phylligenin is 2~98:2~98, preferably 80~98:2~20, and more preferably 90~98:2~10, such as 90:10 or 98:2.

Preferably, when used in the third embodiment of the present invention, the coronavirus refers to the COVID-19 virus, or the disease caused by the coronavirus being COVID-19. Preferably, the phyllirin/phylligenin composition inhibits the 3CLpro protein of the COVID-19 virus, that is, when used in the third embodiment of the present invention, the phyllirin/phylligenin composition counteracts the COVID-19 virus or treats a disease caused by the COVID-19 virus by inhibiting the 3CLpro protein of the COVID virus -19.

The phyllirin/phylligenin composition may be used in combination with other drug(s) against a coronavirus (eg, the COVID-19 virus) or to treat the disease(s) caused by a coronavirus (eg, COVID-19), or may be used alone. The present inventors have discovered that the mechanism of action of the phyllirin/phylligenin composition against the COVID-19 virus involves inhibition of the 3CLpro protein of the COVID-19 virus.

Preferably, when used in the third embodiment of the present invention, the phyllirin/phylligenin composition is used as the only active pharmaceutical ingredient, that is, the only active ingredient in the drug is the phyllirin/phylligenin composition. Thus, the third embodiment of the present invention preferably provides the use of a phyllirin/phylligenin composition as the sole active pharmaceutical ingredient in the preparation of a medicament for combating coronavirus (eg, COVID-19 virus), or the use of a phyllirin/phylligenin composition as the sole active pharmaceutical ingredient in the production of a medicinal product for the treatment of diseases caused by coronavirus (for example, COVID-19).

The drug may include a pharmaceutically acceptable carrier so that a pharmaceutical composition can be prepared. This action is well known to those skilled in the art. When used according to the third embodiment of the present invention, the drug is preferably present in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasmate, adhesive patch or patch.

Accordingly, the present invention also provides a second medical use of the phyllirin/phylligenin composition and a method of treatment using the same.

A fourth embodiment of the present invention provides the use of a phyllirin/phylligenin composition to combat a coronavirus (eg, the COVID-19 virus). The present invention also provides a phyllirin/phylligenin composition for the treatment of diseases caused by coronavirus (eg, COVID-19); or alternatively, the present invention also provides the use of a phyllirin/phylligenin composition to inhibit the 3CLpro protein of the COVID-19 virus.

In addition, the present invention provides a medicament containing a phyllirin/phylligenin composition that is used to combat coronavirus (eg, COVID-19 virus). The present invention also provides a medicinal product containing a phyllirin/phylligenin composition, which is used for the treatment of diseases caused by coronavirus (eg, COVID-19); alternatively, the present invention also provides a medicament containing a phyllirin/phylligenin composition that is used to inhibit the 3CLpro protein of the COVID-19 virus.

Preferably, in a medicinal product comprising a phyllirin/phylligenin composition, the composition is the only active pharmaceutical ingredient.

Also preferably, the medicament comprising the phyllirin/phylligenin composition is provided in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, creams, cataplasms, adhesive patches or patches.

In a fifth embodiment, the present invention provides a method of combating a coronavirus (eg, the COVID-19 virus) comprising administering an effective amount of a phyllirin/phylligenin composition to a patient in need. Accordingly, the present invention also provides a method of treating diseases caused by coronavirus (eg, COVID-19), which includes administering an effective amount of a phyllirin/phylligenin composition to a patient in need. Preferably, the phyllirin/phylligenin composition counteracts the COVID-19 virus or treats the disease(s) caused by the COVID-19 virus by inhibiting the 3CLpro protein of the COVID-19 virus.

In addition, the present invention provides a method of combating the COVID-19 virus, comprising administering an effective amount of a drug containing a phyllirin/phylligenin composition to a patient in need. Accordingly, the present invention also provides a method of treating diseases caused by coronavirus (eg, COVID-19), which includes administering a medicament containing an effective amount of a phyllirin/phylligenin composition to a patient in need.

Herein, the determination of the dose (effective amount) and route of administration of the drug are generally determined by the physician in accordance with the individual characteristics of the patient (such as age, weight, gender, duration of illness, physical condition and severity of infection, etc.). As the patient's individual parameters change, the dosage of administration will also need to be changed, and the appropriate dose will be determined by the attending physician. The route of administration is determined by the dosage form of the pharmaceutical composition, and suitable routes of administration include oral, parenteral, transmucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal, transdermal, and the like. The route of administration is preferably oral.

Preferably, in this method, the phyllirin/phylligenin composition is the only active pharmaceutical ingredient in the medicinal product.

Also preferably, in this method, the drugs are presented in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, creams, cataplasms, adhesive patches or patches.

The beneficial effect of the present invention is that it provides an agent and method for inhibiting the 3CLpro protein of the COVID-19 virus. The composition of phyllirin/phylligenin can effectively inhibit the COVID-19 virus and other coronaviruses even at low concentrations, and the combination of phyllirin and phylligenin, which has a synergistic effect, may become a promising drug to combat coronavirus such as the COVID-19 virus, and may be quickly introduced into clinical practice.

To facilitate understanding, the present invention is described in detail below with reference to specific embodiments and drawings. It should be noted that the descriptions are illustrative only and do not limit the scope of the present invention. As described herein, various embodiments of the present invention will be apparent to those skilled in the art. In addition, the description of the present invention refers to publicly available literature, which is intended to more clearly describe the present invention. The full text of these published literatures is included herein for your reference as if their full text were duplicated herein.

Brief description of drawings

Fig. 1 shows the structure of phyllin and its derivative.

Fig. Figure 2 shows a simulation of the association of KD-1 with the target protein 3CLpro of the COVID-19 virus.

Fig. Figure 3 shows a simulation of the binding of KD-2 to a target protein of the COVID-19 virus.

Fig. 4 shows a simulation of the binding of KD-2-SO ₃ H to the target protein 3CLpro of the COVID-19 virus.

Fig. 5 shows the results of cloning, expression and purification of the 3CL pro protein.

Fig. 6 shows a plot of UV analysis of 3CLpro and KD-1 protein.

Fig. 7 shows a plot of UV analysis of 3CLpro and KD2-GLU protein.

Fig. 8 shows a plot of UV analysis of 3CLpro and KD2-SO ₃ H protein.

Detailed Description of the Invention

The present invention is further illustrated by the following particular embodiments. Unless otherwise indicated, the methods used in particular embodiments are described in the technical literature of the field to which the present invention belongs and in the regulations of drug regulatory authorities, and the devices, raw materials and reagents were purchased on the open market.

Example 1. Study of the effect of phyllirin/phylligenin composition on the COVID-19 virus in vitro

1. Drug testing: phyllirin/phylligenin composition, in which the weight ratio of phyllirin to phylligenin is 90:10 (Dalian Fusheng Natural Medicine Development Co., Ltd.).

2. Cells: VeroE6 cells (State Key Laboratory of Respiratory Diseases of Guangzhou Institute of Respiratory Health).

3. Virus: SARS-CoV-2 strain of the COVID-19 virus (BSL-3 Laboratory at the Guangzhou Technology Center (laboratory of highly pathogenic microorganisms at the Main State Laboratory of Respiratory Diseases)), TCID50 titer = 10 ^-6 / 100 ml.

4. Experimental design (all experimental procedures were performed in a BSL-3 laboratory):

(1) 100 μl of VeroE6 cells at a concentration of 2 × 10 ⁵ VeroE6 cells/ml were added to the wells of a sterile 96-well culture plate, and cultured in 5% CO ₂ at 37°C for 24 hours;

(2) 100 μl/well of 100 TCID50 virus solution was added to the test and control groups on the culture plate, and incubated at 37°C in 5% CO ₂ for 2 hours to allow virus attachment;

(3) after 2 hours, the cell culture solution was removed from the 96-well plate; the phyllirin/phylligenin composition was diluted to a range of concentrations shown in Table 1. The solution was added at 100 μl/well in triplicate for each concentration;

(4) cell control, blind (solvent control), and viral control (negative control) were performed simultaneously;

(5) cells were incubated in 5% CO ₂ at 37°C for 3-4 days;

(6) Cytopathic effect (CPE) was observed under an optical microscope. CPE levels were recorded using the following 6 ratings: “-” no CPE observed; “±” cytopathic effect is less than 10%; “+” cytopathic effect is about 25%; “+ +” cytopathic effect is about 50%; “+ + +” the cytopathic effect is about 75%: “+ + + +” the cytopathic effect is more than 75%. The half maximum effective concentration (IC50) was calculated using the Reed-Munch method or GraphPad Prism 5.0.

5. Experiment results:

As shown in Table 1-1, the phyllirin/phylligenin composition can effectively inhibit the COVID-19 virus even at low concentrations. It was calculated that the concentration of half-maximal inhibition IC ₅₀ is 63.90 μg/ml (at the same time, studies have shown that for pure phyllirin the concentration of half-maximal inhibition IC ₅₀ is 179.1 μg/ml, and pure phylligenin has an even poorer inhibitory effect). The combination of phyllirin and phylligenin in the composition has a synergistic effect, so this composition is expected to be useful for the treatment of COVID-19.

Table 1-1

Drug concentration (µg/ml) Inhibition (%) 250.00 78.33±10.41 125.00 68.33±7.64 62.50 55.00±13.23 31.25 30.00±10.00 15.63 11.67±2.89

Example 2. Study of the effect of phyllirin/phylligenin composition on the HCoV-229E virus in vitro

Coronavirus HCoV-229E is less pathogenic and usually causes only cold-like respiratory symptoms. This embodiment is mainly based on the process shown in Example 1, with the only difference being that the coronavirus used is the HCoV-229E virus (Guangzhou BSL-3 Technology Center Laboratory (Highly Pathogenic Microorganisms Laboratory of the State Key Laboratory of Respiratory Diseases)) , with a titer of TCID50 = 10 ^-5.5 /100 µl, and the titer of the virus used was 100TCID50; and the phyllirin/phylligenin composition was diluted to a range of concentrations shown in Table 2-1. All experimental procedures were performed in a BSL-3 laboratory.

As shown in Table 2-1, the phyllirin/phylligenin composition can effectively inhibit the HCoV-229E virus even at low concentrations. The half-maximal inhibition concentration IC ₅₀ was calculated to be 64.53 μg/ml (while studies have shown that pure phyllirin and pure phylligenin have a poorer inhibitory effect). The combination of phyllirin and phylligenin in the composition has a synergistic effect, and the composition is expected to be useful for the treatment of diseases caused by the HCoV-229E virus.

Table 2-1

Drug concentration (µg/ml) Inhibition Rate (%) 500.000 88.75±2.50 250.000 83.75±4.79 125.000 75.00±4.08 62.500 47.50±6.45 31.250 28.75±4.79 15.625 11.25±4.79

Example 3. Study of the molecular mechanism of inhibition of the COVID-19 virus by phyllin and its derivatives

I. Initial data of the experiment

The Spike protein (S protein) on the surface of the COVID-19 virus and the COVID-19 virus hydrolase (chymotrypsin-like cysteine protease 3, designated 3CL pro protein) are important links in the life cycle of the COVID-19 virus. The life cycle of COVID-19 is that the S protein on the surface of COVID-19 binds to angiotensin converting enzyme 2 (ACE2), through endocytosis enters the cell to release the RNA genetic material, and with the participation of the 3CL pro protein, the RNA genetic material successfully replicates to form a virus.

For virtual screening, molecular docking technology is mainly used, which simulates the drug screening process on a computer. This screening requires first knowing the molecular structure of the drug target, then calculating the binding capacity of small molecules from a library of compounds to the target using molecular modeling, and predicting the physiological activity of the candidate compounds.

In this experiment, Glide molecular docking technology is used to perform a virtual screen to find the optimal binding effect of phyllin (KD-1) and its derivatives on the target protein 3CL pro. Based on the screening results, the effect of binding of KD-1 and its derivatives to the target protein in vitro was studied, and the intermolecular interaction and binding form and structure-activity relationship of KD-1 and its derivatives were explained. Analysis of target protein activity using KD-1 and its derivatives revealed the molecular mechanism of inhibition of the COVID-19 virus by phyllin (KD-1) and its derivatives.

II. Experimental technique

1. Molecular docking of KD-1 and its derivatives with the target protein

(1) System Preparation: The 2.16Å crystal structure of the main protease of SARS-CoV-2 (3CL pro protein) was obtained from the Protein Database (PDB ID: 6LU7). The enzyme structure was pre-processed using the protein Preparation Wizard at Schrodinger, the water of crystallization was removed and missing hydrogen atoms/side chain atoms were added, and the acidic and basic amino acid residues at pH 7.0 were assigned the appropriate charges and protonation states, and energy minimization of the structure was carried out enzyme using OPLS-2005 force field and finally the active site of the protein was predicted using Sitemap. At the same time, the spatial structure of phyllin and its derivatives was optimized using the LigPrep Schrodinger module.

(2) Molecular Docking: Virtual screening based on molecular docking was performed using Glide Maestro 11.5 technology. The calculation of the addition of phyllin and its derivatives (Fig. 1) was performed using the “ultra-precision” resolution (XP) mode of Glide.

(3) Docking score: The interaction between the small molecule and the target protein is calculated by the value of the Glide score function. This function takes into account the interaction of hydrogen bonding, hydrophobic forces, van der Waals forces, etc. Thus, the larger the absolute value of the function, the more stable the small molecule-target protein docking complex will be, and the better the corresponding binding effect will be.

(4) Target analysis: The docking results of KD-1 and its derivatives with the key target protein of the novel coronavirus and the key amino acids involved in the interaction of KD-1 and its derivatives with the key target protein of the novel coronavirus were analyzed to provide theoretical basis and important will help for the prevention, diagnosis and treatment of new coronavirus infection in clinical practice.

2. Protein expression and purification

(1) Cloning: The full-length gene encoding the 3CL pro protein of SARS-CoV-2 (UniprotKB - P0DTD1, residues 3264-3569) was optimized and synthesized in the E. coli expression system (Wuhan Jingkaizhui Biotech Co., Ltd.).

(2) Expression: The isolated plasmids were transfected into E. coli BL21 (DE3) cells, and then cultured in Luria-Bertani medium containing 100 μg ampicillin/ml at 37°C. When cells had expanded to an OD600 of 0.6-0.8 nm, 0.5 mM isopropylthiogalactoside was added to the cell culture to induce expression at 30°C and 180 rpm. After 10 hours, cells were isolated by centrifugation at 3,000 g.

(3) Purification: The cell pellet was resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM 2-mercaptoethanol), lysed by sonication, and then centrifuged at 13,000 g for 30 minutes. The supernatant was loaded onto a Ni-NTA affinity chromatography column, followed by washing with resuspension buffer containing 20 mM imidazole, and then the histidine tag-bound 3CL pro protein was eluted with lysis buffer containing 300 mM imidazole (50 mM Tris-HCl pH 7.0 , 150 mM NaCl). The 3CL pro protein was further purified by ion exchange chromatography. Finally, 3CL pro protein was obtained as a mixture of monomer and dimer and stored in solution.

3. Spectroscopic experiment

In this experiment, protein structural changes were studied using a JASCO-V560 series spectrophotometer operating in the visible and ultraviolet light regions. The ultraviolet absorption spectrum of the protein has two absorption peaks: the large absorption peak at 210 nm reflects the conformation of the protein framework, which is caused by the n-π* C=O transition in the peptide bond. The appearance of a weak absorption peak at 280 nm is the result of a π-π* transition caused by light absorption by aromatic amino acids (Trp, Tyr and Phe).

In this experiment, visible and ultraviolet light absorption spectra were obtained by scanning in the range of 200–700 nm a protein solution and a mixed solution of phyllin and protein and using a buffer solution as a reference. Gel Buffer 2 ml (50 mM Tris-HCl pH 7.0, 150 mM NaCl) was placed in a quartz cuvette, 10 μl of 3CL pro protein at a concentration of 10 ^-5 M was added, and the absorbance value was measured, and then 10 μl of phyllin and its derivatives at a concentration of 10 ^-4 M, and measured changes in absorbance value.

III. Experiment results

The life cycle of COVID-19 involves the S protein on the surface of COVID-19 binding to angiotensin converting enzyme 2 (ACE2), entering the cell through cellular endocytosis to release the RNA genetic material, and then with the help of the 3CL pro protein, the RNA genetic material is successfully replicated to form virus. This experiment investigated compounds that can block the participation of the 3CL pro protein in the replication of RNA genetic material, and then developed drugs against the COVID-19 virus.

In this experiment, the optimal target site was selected based on the crystal structure of the 3CL pro protein and a virtual screening of compounds KD-1 and its derivatives (KD-2-GLU and KD-2-SO ₃ H) was performed. The binding rates of compounds KD-1, KD-2-GLU and KD-2-SO ₃ H to the 3CL pro protein site were obtained, thereby proving the targeting of the compounds to the 3CL pro protein.

Through calculation and analysis, five potential active sites were discovered in the three-dimensional structure of the 3CLpro protein, of which site 1 is the site where drug molecules can easily bind to the 3CLpro protein. KD-1 docked successfully to 5 potential active sites of the 3CLpro protein, and all 5 active sites docked successfully with the docking scores shown in Table 3-1. The Glide Score of KD-1 in 3CLpro protein active site 1 is significantly higher than that in other 3CLpro protein active sites, and 3CLpro protein active site 1 was selected as the theoretical molecular docking value. The analysis and calculation for compounds KD-2-GLU and KD-2-SO ₃ H were the same as above.

Table 3-1. KD-1 Docking Scores with 5 Potential Active Sites of 3CLpro Protein

Small molecule Protein number 3CLpro Active site Connection points KD-1 6lu7 Site 1 -9.461 KD-1 6lu7 Site 2 -3.954 KD-1 6lu7 Site 3 -4.611 KD-1 6lu7 Site 4 -3.342 KD-1 6lu7 Site 5 -4.141

The optimal active site was selected, in which KD-1, KD-2-GLU and KD-2-SO ₃ H showed optimal activity towards the 3CLpro protein. The Glide Score assessed the ability of small molecules to bind to the active sites of the 3CLpro protein. The higher the absolute value, the more stable the mutual association, the better the correspondence. Table 3-2 shows the docking scores for compounds KD-1, KD-2-GLU and KD-2-SO ₃ H in the active site of the COVID-19 virus target protein.

Also shown in Table 3-2 are the theoretical docking scores for compounds KD-1, KD-2-GLU and KD-2-SO ₃ H relative to the core structure of the COVID-19 pro 3CL protein, which plays an important role in the replication process RNA are significant. KD-1 and KD-2-GLU have high docking scores, indicating that these compounds have different degrees of targeting of the 3CL pro protein.

Table 3-2. Summary table of docking scores for KD-1 and its derivatives relative to 3CLpro COVID-19 target protein sites

No. Connections Protein number 3CLpro Connection points 1 KD-1 6LU7 -9.461 2 KD-2-GLU 6LU7 -8.222 3 KD-2- _SO3H 6LU7 -5.886

Through 3D modeling and plane projection of the molecular docking of KD-1 and the target protein 3CLpro, it was shown that the KD-1 junction can fit into the molecular pocket of the protein (Fig. 2, top). Compound KD-1, when binding to the 3CL pro protein, forms a π-π bond with the amino acid residue His41 of the active site of the 3CL pro protein, forms three hydrogen bonds with the peptide bonds Gly143, Asn142, Glu166, and hydrophobically interacts with the Met165 group (Fig. 2, Bottom part). These results indicate that various amino acid groups, such as His, Gly, Asn, Glu, Met, or the like, may sterically fit the KD-1 structure, thereby determining the conformation of the complex. It is speculated that the KD-1 compound may target the 3CL pro protein and thus prevent the 3CL pro protein from participating in COVID-19 RNA replication when the virus reproduces.

Through 3D modeling and plane projection of the molecular docking of KD-2-GLU and the target protein 3CLpro, it was shown that the KD-2-GLU junction could fit into the molecular pocket of the protein (Figure 3, top). The compound KD-2-GLU binds to the 3CL pro protein by forming three hydrogen bonds with the peptide bonds Gly143, Thr43, Thr25 in the active site of the 3CL pro protein and hydrophobically interacting with Tyr54 (Fig. 3, bottom). These results indicate that various amino acid residues, such as Gly, Thr, Tyr or the like, can sterically fit the KD-2-GLU structure, thereby determining the conformation of the complex. It is suggested that the compound KD-2-GLU can target the 3CL pro protein and prevent the 3CL pro protein from participating in COVID-19 RNA replication to reproduce the virus.

Through 3D modeling and plane projection of the molecular docking of KD-2-SO ₃ H and the target protein 3CLpro, it was shown that the KD-2-GLU junction could fit into the molecular pocket of the protein (Fig. 4, top). The compound KD-2-SO ₃ H interacts with the Gly143 and Asn142 groups by forming hydrogen bonds and hydrophobically interacts with Tyr54, Val42, Cys44, Cys145 (Fig. 4, lower part). These results demonstrate that various amino acid residues such as Gly, Asn, Val and the like can sterically fit the KD-2-SO ₃ H structure, thereby determining the conformation of the complex.

The interactions of compounds KD-1 and its derivatives with the active site of the 3CLpro protein are shown in Table 3-3. These results indicate that the interaction of phyllin and its two derivatives with the active site of the 3CLpro protein is not completely hydrophobic, but also involves hydrogen bonding and bonding of the π–π configuration. Interactions through hydrogen bonds and π-π bonding can help stabilize the complex. The compounds KD-1, KD-2-GLU and KD-2-SO ₃ H were found to interact with the 3CLpro protein via hydrogen bonding only with GLY143 with hydrogen distances of 2.22 Å, 2.22 Å and 2. 06 Å respectively. However, hydrophobic and electrostatic interactions play an important role in the binding of compounds KD-1, KD-2-GLU and KD-2-SO ₃ H to the 3CLpro protein. Different compounds bind to different residues of the 3CLpro protein, which explains the differences in binding results.

Table 3-3. Interaction of KD-1 and its derivatives with the 3CLpro protein

Small molecule Interaction Key amino acids KD-1 Action of hydrogen bond HIS41 Hydrophobic action HIS41, GLY143, ASN142, GLU166 π-π connection; HIS41 KD-2-GLU Action of hydrogen bond THR43, THR25, GLY143 Hydrophobic action TYR54 KD-2- _SO3H Action of hydrogen bond GLY143,ASN142 Hydrophobic action VAL42, CYS44, TYR54, CYS145

The fragmented protein was purified on a nickel column and its molecular weight was found to be 34 kDa (Fig. 5, right). Protein ion exchange chromatography was carried out, and then DNS-PAGE was carried out to thereby detect the protein sample 3CL pro protein concentration of 65 mg/ml (Fig. 5, right).

Based on the results of virtual screening using molecular docking, the binding of compounds KD-1, KD-2-GLU and KD-2-SO ₃ H to the 3CLpro protein was examined in vitro using a UV-vis absorption detection method, which further confirmed that that the compounds can target the 3CLpro protein.

In fig. 6 shows the absorbance for compound KD-1, 3CLpro protein, and a mixture thereof, as detected by ultraviolet-visible spectroscopy. These results indicate that the 3CL pro protein exhibits an absorption peak at 278 nm, and that after the addition of KD-1, the absorbance increases and the peak shifts. KD-1 interacts with the 3CL pro protein and can inhibit the activity of this enzyme. KD-1 can form a new complex when interacting with 3CL pro protein, and KD-1 can bind to 3CL pro protein through hydrogen bonding or hydrophobic interaction.

In fig. 7 shows the absorbance for the KD2-GLU compound, the 3CLpro protein, and a mixture thereof, as detected by ultraviolet-visible spectroscopy. These results indicate that 3CL pro protein exhibits an absorption peak at 278 nm, and that after the addition of KD2-GLU, the absorbance increases and the peak shifts. KD2-GLU interacts with the 3CL pro protein and can inhibit the activity of this enzyme. KD2-GLU can form a new complex when interacting with 3CL pro protein, and they can communicate via hydrogen bonding or hydrophobic interaction.

In fig. 8 shows the absorbance of KD2-SO ₃ H, 3CLpro protein, and a mixture thereof, as detected by ultraviolet-visible spectroscopy. These results indicate that after the addition of KD2-SO ₃ H, there is an increase in the light absorption rate and a shift in the peak of the 3CLpro protein. KD2-SO ₃ H interacts with the 3CL pro protein and can inhibit the activity of this enzyme. KD2-SO ₃ H can bind to 3CL pro protein through hydrogen bonding or hydrophobic interaction, and they can form a new complex.

IV. Conclusions from the experiment

The molecular docking results show that the theoretical docking scores of compounds KD-1, KD-2-GLU and KD-2-SO ₃ H towards 3CL pro protein as a key COVID-19 protein involved in RNA replication were significant and among In them, compounds KD-1 and KD-2-GLU had high docking scores. The analysis showed that the compounds KD-1, KD-2-GLU and KD-2-SO ₃ H were associated with peptide bonds in the active site of the 3CL pro protein through hydrogen bonding and hydrophobic interaction, forming a π-π bond with amino acid residues.

Theoretical data were able to prove that these compounds could change the structure of the 3CL pro protein. In vitro spectroscopic experiments show that compounds KD-1, KD-2-GLU and KD-2-SO ₃ H lead to a shift and increase in the main peak for the 3CL pro protein at a wavelength of 278 nm, and this was most pronounced for compound KD -1. Thus, each of the compounds KD-1, KD-2-GLU and KD-2-SO ₃ H can target the 3CL pro protein, can prevent the participation of the 3CL pro protein of the COVID-19 virus in the process of viral replication, and thus , may be a potential targeted inhibitor of the COVID-19 virus.

Example 4. In vivo study of effectiveness against a new type of coronavirus

I. Materials for the experiment

Mice for experiment: hACE2 mice, age 6-7 weeks, weight 20-40 g, total 120 males, laboratory animal supplier - Jiangsu Jitsui Yuaokang Biotech Co., Ltd., laboratory animal production license: SCXK(SU) 2018-0008 , laboratory animal certificate number: No. 32072720201100243581, feed supplier: Jiangsu Meidisen Biotech Co., Ltd.

Medicine: phyllirin/phylligenin composition, in which the weight ratio of phyllirin to phylligenin is 90:10 (Dalian Fusheng Natural Medicine Development Company Co., Ltd.).

II. Experimental technique

(1) Protective effect of the drug on disease progression in mice infected with COVID-19

hACE2 C57BL/6 transgenic mice were divided into a normal group, a group of mice infected with SARS-CoV-2, which in turn was divided into groups: 80 mg/kg, 40 mg/kg, and a positive control group. (remdesivir 50 mg/kg), with each group containing 8 mice. Only mice in the normal group received PBS instilled into their noses; mice in other groups received SARS-CoV-2 virus instilled into their noses at a concentration of ¹⁰⁵ PFU. Two hours after infection, mice in the infected group were injected with phyllin by intragastric gavage once daily for 5 consecutive days. Weight changes were recorded daily after infection, and mortality among mice was calculated over 5 days.

(2) Study on the effectiveness of the drug against excessive inflammation caused by COVID-19 infection in mice

ACE2 C57BL/6 transgenic mice were divided into normal group, SARS-CoV-2 virus-infected group: 80 mg/kg dosing group, 40 mg/kg dosing group, and positive control (remdesivir 50 mg/kg) group. Only mice in the normal group received PBS instilled into their noses; mice in other groups received SARS-CoV-2 virus instilled into their noses at a concentration of ¹⁰⁵ PFU. Two hours after infection, mice in the infected group were injected with phyllin by intragastric gavage once daily for 5 consecutive days. Animals were necropsied on day 5 postinfection to obtain lungs for tissue homogenization to determine virus titer. Total RNA was extracted from the supernatant of the lung tissue homogenate using the Trizol method, and the mRNA expression of the corresponding inflammatory factors was determined using the RT-qPCR method.

III. Experiment results

(1) Results of studies of a drug against the new coronavirus for protection against death

The death protection results are shown in Table 4-1. These results show that the protective effects of two doses of the drug (80 mg/kg and 40 mg/kg) in mice infected with the novel coronavirus were 87.5% and 42.86%, respectively; in the group of mice infected with the virus, death was 71.43%; the degree of protection against death in the group that was administered 80 mg/kg drugs was comparable to the degree of protection in the group that took remdesivir.

Table 4-1. Results of a study on the protection of mice infected with a new type of coronavirus from death (n = 3)

Group Average number of survival days (days) Survival rate (%) Death rate (%) Normal group 5.00±0.00 100 0.00 Group infected with SARS-CoV-2 4.29±0.49 28.57 71.43 Drug 80 mg/kg 4.88±0.35 87.50 12.50 Drug 40 mg/kg 4.43±0.53 42.86 57.14 Remdesivir 50 mg/kg 4.86±0.38 87.50 12.50

(2) Results of a study of the virus titer in the lung tissue of mice infected with a new type of coronavirus, which were administered drugs

As shown in Table 4-2, when two doses of the drugs (80 mg/kg and 40 mg/kg) were administered, the titer of virus in the lungs of infected mice was significantly reduced, and there was no statistical difference between the titer of virus inhibited in the lungs in the group receiving 80 mg/kg, and titer in the remdesivir group.

Table 4-2. Results of a study of the virus titer in the lung tissue of mice infected with a new type of coronavirus (x̄±s, n=3)

Group Virus titer in the lungs (Log10) Normal group - Group infected
SARS-CoV-2 3.14±0.41 Drug 80 mg/kg 1.99±0.38*** Drug 40 mg/kg 2.54±0.32*** Remdesivir 50 mg/kg 1.68±0.45*

Note: Compared with virus infection group, * P < 0.05, ** P < 0.01, *** P < 0.001

(3) Results of a test to induce excessive inflammation in drug-administered mice infected with the novel coronavirus

The results of the analysis of inflammation indicators in infected mice are shown in Table 4-3. On the fifth day post-infection, the expression of IL-1β, IFN-γ, MCP-1 and IFN-α was significantly increased in the virus-infected group. Administration of two doses of drugs (80 mg/kg and 40 mg/kg) had an inhibitory effect on the inflammatory factors IL-1β, IFN-γ, MCP-1 and IFN-α. The group that received a dose of 80 mg/kg of the drug showed better inhibition of overexpression of inflammatory mediators than remdesivir.

Table 4-3. Effect of drugs on the expression of IL-1β, IFN-γ, MCP-1 and IFN-αmRNA mRNA in the lung homogenate of infected mice (x̄±s, n=3)

Group IL-1β IFN-γ IFN-α MCP-1 Normal group 1.00±0.001*** 1.16±0.29*** 1.03±0.045*** 1.00±0.00*** Group infected with SARS-CoV-2 43.42±10.97 432.60±116.11 246.63±49.28 14.72±4.99 Remdesivir 50 mg/kg 7.41±5.86** 173.79±84.84* 69.99±53.20** 7.31±5.14** Drug 80 mg/kg 6.14±3.71*** 51.56±32.33*** 50.22±16.94** 1.80±0.73*** Drug 40 mg/kg 11.00±3.84*** 228.07±180.55 157.04±42.06** 6.89±2.39**

Note: Compared with virus infection group, * P < 0.05, ** P < 0.01, *** P < 0.001

IV. Conclusion

The above experimental results show that the drug according to the present invention has a significant protective effect against death in mice infected with the novel coronavirus, with a rate of protection against death in infected mice of 87.5%, which is comparable to the effectiveness of remdesivir. The drug of the claimed invention can significantly inhibit the titer of the virus in the lung tissue of mice infected with a new coronavirus infection. It also inhibits the mRNA expression of excessive inflammatory factors IL-1β, IFN-α, MCP-1 and IFN-γ induced by novel coronavirus. Therefore, this drug plays a significant role in the treatment of the new coronavirus infection.

Claims (13)

1. The use of phyllirin in the preparation of a product for inhibiting the 3CLpro protein of the COVID-19 virus. 2. Use of a phyllirin derivative in the preparation of an agent for inhibiting the 3CLpro protein of the COVID-19 virus, where the phyllirin derivative is KD-2-GLU represented by the following formula, or KD-2-SO ₃ H represented by the following formula: 3. A method of inhibiting the 3CLpro protein of the COVID-19 virus, including the stage of interaction of the 3CLpro protein of the COVID-19 virus with phyllin. 4. A method for inhibiting the 3CLpro protein of the COVID-19 virus, including the step of reacting the 3CLpro protein of the COVID-19 virus with a phyllirin derivative, where the phyllirin derivative is KD-2-GLU, represented by the following formula, or KD-2-SO ₃ H, represented by the following formula: 5. Use of a phyllirin/phylligenin composition as the only pharmaceutically active ingredient in the preparation of a drug against the COVID-19 virus or a drug for treating a disease caused by the COVID-19 virus, where the weight ratio of phyllirin to phylligenin in the phyllirin/phylligenin composition is 2~98 :2~98. 6. Use according to claim 5, characterized in that the medicinal product is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, adhesive patch or patch. 7. Use according to claim 5, characterized in that the phyllirin/phylligenin composition inhibits the 3CLpro protein of the COVID-19 virus. 8. A method of combating the COVID-19 virus or treating a disease caused by the COVID-19 virus, comprising the step of administering to a patient in need an effective amount of a phyllirin/phylligenin composition, wherein the weight ratio of phyllirin to phylligenin in the phyllirin/phylligenin composition is 2~98:2~98 , and this phyllirin/phylligenin composition is used as the only pharmaceutically active ingredient. 9. A method of combating the COVID-19 virus or treating a disease caused by the COVID-19 virus, comprising the step of administering to a patient in need an effective amount of a medicinal product containing a phyllirin/phylligenin composition, wherein the weight ratio of phyllirin to phylligenin in the phyllirin/phylligenin composition is 2~98 :2~98, and this phyllirin/phylligenin composition is used as the only pharmaceutically active ingredient. 10. The method according to claim 8 or 9, characterized in that the medicinal product is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, adhesive patch or patch. 11. The method according to claim 8 or 9, characterized in that the phyllirin/phylligenin composition inhibits the 3CLpro protein of the COVID-19 virus.