Lipid small molecules as 3C-like protease targeting agents for antiviral and broad-spectrum anti-inflammatory effects

Abstract

The use of lipid small molecules as 3C-like protease targeting agents in antiviral and broad-spectrum anti-inflammatory effects. Specifically, through the traditional Chinese medicine lipid molecule library, the 3C-like protease of viruses (such as SARS-CoV-2) was screened to obtain drug lead compounds, and virus attack experiments were used to verify that phosphatidylcholine and diglyceride have antiviral and anti-inflammatory effects.

Description

Technical Field

[0001] The present application belongs to the field of medicinal chemistry. It targets 3C-like proteases (3CLpro) of viruses (such as the new coronavirus SARS-CoV-2) and uses lipids derived from traditional Chinese medicine to conduct virtual screening to obtain drug lead compounds, which are then verified by virus attack experiments. Background Art

[0002] 3C-like protease (3CLpro), also known as main protease (Mpro), is a key hydrolase in viral replication and infection. Many viruses (such as porcine epidemic diarrhea virus, coronavirus, foot-and-mouth disease virus, encephalomyocarditis virus) can encode 3CLpro enzymes.

[0003] Novel coronavirus pneumonia (COVID-19) is an acute infectious pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main transmission routes are through air and contact, and there is the possibility of aerosol transmission and fecal-oral transmission. Patients often have respiratory symptoms in the early stage, such as fever, cough, shortness of breath and difficulty breathing. If the disease is not well controlled, in severe cases, the infection can lead to severe acute respiratory syndrome, systemic sepsis and even death.

[0004] There is currently no specific treatment for COVID-19. Therefore, it is of great significance to study targeted drugs against COVID-19. Traditional Chinese medicine plays an indispensable role in the treatment of COVID-19. Taking the decoction after boiling traditional Chinese medicine is the most common way to use traditional Chinese medicine. It has been reported in the literature that traditional Chinese medicine decoction is composed of small chemical molecules, lipids, proteins and sRNA. Among them, lipids have always been an overlooked part of the effective ingredients of traditional Chinese medicine.

[0005] With the development of computer-aided drug design, virtual drug screening has effectively improved the high cost and high risk problems in the traditional drug development process. It has become a complementary technology to high-throughput screening and a traditional Chinese medicine method for discovering lead compounds and optimizing their activity. Establishing a lipid small molecule library derived from traditional Chinese medicine decoctions and screening the main protein targets of the virus plays a vital role in inhibiting viral replication and infection and controlling the spread of the virus. Summary of the invention

[0006] According to some embodiments, a Compound 3C-like protease targeting agent is provided.

[0007] In specific embodiments, the 3C-like protease targeting agent is diglyceride or phosphatidylcholine.

[0008] According to some embodiments, a diglyceride compound is provided, which is represented by Formula III:

[0009]

[0010] According to some embodiments, a phosphatidylcholine compound is provided, which is represented by Formula I:

[0011]

[0012] According to some embodiments, a phosphatidylcholine compound is provided, which is represented by Formula II:

[0013]

[0014] According to some embodiments, a phosphatidylcholine compound is provided, which is represented by formula IV:

[0015]

[0016] According to some embodiments, a phosphatidylcholine compound is provided, which is represented by Formula V:

[0017]

[0018]

[0019] According to some embodiments, there is provided a use of a 3C-like protease targeting agent or a pharmaceutically acceptable salt thereof selected from any one of the following in the preparation of an antiviral drug:

[0020]

[0021] According to some embodiments, there is provided a use of a 3C-like protease targeting agent or a pharmaceutically acceptable salt thereof selected from any one of the following in the preparation of a disinfectant or disinfectant composition:

[0022]

[0023] According to some embodiments, there is provided a use of a 3C-like protease targeting agent or a pharmaceutically acceptable salt thereof selected from any one of the following in the preparation of a viral inhibitor:

[0024]

[0025]

[0026] According to some embodiments, there is provided a use of a 3C-like protease targeting agent or a pharmaceutically acceptable salt thereof selected from any one of the following in the preparation of an anti-inflammatory drug:

[0027]

[0028]

[0029] Wherein, the inflammation is inflammation caused by Gram-negative bacteria or Gram-positive bacteria.

[0030] According to some embodiments, the use of the aforementioned 3C-like protease targeting agent or a pharmaceutically acceptable salt thereof in preparing a drug for treating pulmonary inflammation is provided. Specifically, the pulmonary inflammation is selected from: inflammation caused by viruses, inflammation caused by bacteria, inflammation caused by fungi, or a combination thereof.

[0031] When used in the aforementioned applications, in some embodiments, the compounds of the present application are acidic, such as compounds having carboxyl or hydroxyl groups. These compounds can form pharmaceutically acceptable salts. Examples of these salts can include sodium, potassium, calcium, aluminum. Also included are salts formed with amines (such as ammonia, alkylamines, hydroxyalkylamines, N-methylglucamine, etc.).

[0032] When used in the aforementioned applications, in some embodiments, the compounds of the present application are basic compounds and can also form pharmaceutically acceptable salts, such as acid addition salts. Examples include inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, aminosulfonic acid, etc., and organic acids such as acetic acid, trifluoroacetic acid, trichloroacetic acid, cinnamic acid, citric acid, maleic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, benzenesulfonic acid, glycolic acid, malic acid, lactic acid, malonic acid, oxalic acid, nicotinic acid, succinic acid, salicylic acid, stearic acid, tartaric acid, p-aminobenzenesulfonic acid, trimethylbenzenesulfonic acid, p-toluenesulfonic acid, mandelic acid, pectinic acid, picric acid, propionic acid, etc.

[0033] All of these acid and base salts are pharmaceutically acceptable salts within the scope of the present application.

[0034] The virus suitable for the present application is not limited to a specific virus family, provided that it contains a 3CLpro enzyme. As an example, it can be selected from any one of the following: rhinovirus, coronavirus, adenovirus, influenza virus, parainfluenza virus, respiratory syncytial virus, echovirus, coxsackievirus, porcine epidemic diarrhea virus, foot-and-mouth disease virus, encephalomyocarditis virus.

[0035] When used for the aforementioned applications, in some specific embodiments, the virus is a coronavirus selected from any one of the following: SARS-CoV, SARS-CoV-2, MERS-CoV, or variants thereof.

[0036] When used for the aforementioned applications, in some specific embodiments, the virus is SARS-CoV-2.

[0037] According to some embodiments, an antiviral agent (or antiviral preparation, or viral inhibitor) is provided, comprising a compound selected from any one of the following or a pharmaceutically acceptable salt thereof:

[0038]

[0039]

[0040] According to some embodiments, an anti-inflammatory agent is provided, comprising a compound selected from any one of the following or a pharmaceutically acceptable salt thereof:

[0041]

[0042]

[0043] In some embodiments, any compound of Formula I to Formula V or a pharmaceutically acceptable salt thereof according to the present application is used for therapeutic purposes.

[0044] In other embodiments, the compound of any one of Formula I to Formula V or a pharmaceutically acceptable salt thereof according to the present application is used for non-therapeutic purposes.

[0045] In the present application, an antiviral agent (or virus inhibitor) refers to any compound or composition that can inhibit (or reduce, hinder, interfere with, inactivate, kill) viral activity, vitality, replication, proliferation, growth, infectivity, or virulence in vivo or in vitro. As an example, the virus is selected from any one of the following: rhinovirus, coronavirus (SARS-CoV, SARS-CoV-2, MERS-CoV, or its variants), adenovirus, influenza virus, parainfluenza virus, respiratory syncytial virus, echovirus, coxsackievirus, porcine epidemic diarrhea virus, foot-and-mouth disease virus, encephalomyocarditis virus.

[0046] In the present application, a disinfectant (or disinfectant composition) refers in particular to any compound (or composition) that can inhibit (or reduce, hinder, interfere with, inactivate, kill) viral activity, vitality, replication, proliferation, growth, infectivity, or virulence in vitro (e.g., in the environment, on the surface of an object, etc.).

[0047] The disinfectant (or disinfectant composition) of the present application is used to disinfect environments, areas, objects, samples, surfaces, containers, or foods infected or contaminated by viruses.

[0048] In some embodiments, the present invention provides a disinfection method or antiviral method suitable for treating an environment, area, article, sample, surface, container, or food exposed to or threatened by a pathogen. In an exemplary embodiment, the virus is in contact with an environmental surface, and the method comprises contacting the environmental surface with a compound / composition in an amount sufficient to disinfect the surface. However, disinfection does not necessarily result in the elimination of all pathogens.

[0049] Eliminate viruses from various surfaces (especially hard surfaces where these viruses can remain active for relatively long periods of time), as well as commercial and public environments (e.g. hospitals, clinics, hotels).

[0050] In some embodiments, the compound of any one of Formula I to Formula V or a pharmaceutically acceptable salt thereof according to the present application is used as an additive to prevent harmful or unwanted viral contamination in the environment, area, article, sample, surface, container, or food.

[0051] In a specific embodiment, any compound of Formula I to Formula V of the present application or a pharmaceutically acceptable salt thereof targets 3C-like protease, thereby exerting an inhibitory effect on the virus.

[0052] In a specific embodiment, the drug (or antiviral agent, or disinfectant composition) may also include one or more pharmaceutically acceptable carriers, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants or synergists in the pharmaceutical field.

[0053] The term "carrier" refers to a diluent, adjuvant, excipient or matrix that is administered with the compound. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions (or inhibitors, disinfectants) of the present application include: water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol) or mixtures thereof, oils (such as olive oil, injectable organic esters). Suitable pharmaceutical carriers are described in Remington Pharmaceutical Sciences by EW Martin.

[0054] In a specific embodiment, the drug (or antiviral agent or disinfectant composition) can be prepared into an injection, tablet, pill, capsule, suspension, emulsion, spray, disinfectant, detergent, culture medium for administration. When applicable to human or animal subjects, the route of administration can be oral, percutaneous, intravenous, pulmonary inhalation, nasal drops, or intramuscular injection. When applicable to an environment, region, article, sample, surface, container, or food, it can be prepared into a spray, detergent, lotion.

[0055] According to some embodiments, there is provided a method of treating a viral infection, comprising contacting a subject with a therapeutically effective amount of a compound selected from any one of the following or a pharmaceutically acceptable salt thereof:

[0056]

[0057]

[0058] According to some embodiments, a method of disinfection is provided, comprising contacting an environment, area, object, sample, surface, container, or food with an effective amount of a compound selected from any one of the following or a pharmaceutically acceptable salt thereof:

[0059]

[0060]

[0061] According to some embodiments, there is provided a method for treating inflammation (preferably pulmonary inflammation), comprising contacting a subject with a therapeutically effective amount of a compound selected from any one of the following or a pharmaceutically acceptable salt thereof:

[0062]

[0063]

[0064] The term "subject" as used herein refers to an organism treated with the compounds of the present application. These organisms include those exposed to or suspected of being exposed to viruses. Organisms include, but are not limited to, animals (e.g., humans, domestic animals, wild animals) and plants.

[0065] On the one hand, "effective amount" is the amount required or sufficient to inhibit (or reduce, hinder, interfere, inactivate, kill) the activity, vitality, replication, proliferation, growth, infectivity, or virulence of the virus. In one example, the effective amount of the present application compound is the amount that can produce a statistically significant reduction in viral replication ability in a subject. In another example, the effective amount of the present application compound is the amount that can produce a statistically significant reduction in viral load, viral transmission ability, or viral activity in the environment.

[0066] On the other hand, "effective amount" also refers to an amount required or sufficient to improve the symptoms of inflammation. In one example, an effective amount of the compound of the present application is an amount that can significantly reduce the expression level of inflammatory factors in a subject.

[0067] In specific embodiments, the time for which the compound of the present application is in contact with the subject (or the environment, surface, etc. to be treated) is sufficient to inhibit (or reduce, hinder, interfere with, inactivate, kill) the activity, vitality, replication, proliferation, growth, infectivity, or virulence of the virus.

[0068] The effective amount level selected will depend on a variety of factors, including the activity of the specific compound of the present application used, the route of administration, the time of administration, the metabolic rate of the specific compound used, the duration of treatment, other drugs (compounds and / or materials) used in combination with the specific compound used, the age, sex, weight, condition, general health and medical history of the subject being treated, and other factors well known in the medical field. A physician or veterinarian of ordinary skill in the art can readily determine the required effective amount.

[0069] In general, a suitable daily dose of a compound of the present application will be that amount of the compound effective to produce a therapeutic effect.

[0070] If desired, the daily dose of a compound of the present application may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day; optionally, the sub-doses are doses in unit dosage form.

[0071] The pharmaceutical composition or active ingredient provided in the present application can be administered according to conventional clinical treatment methods. For example, for pneumonia, it can be administered directly by nasal drops, sprays, or infusions into the lungs; the dosage can be used according to the dosage determined by medical practitioners.

[0072] In a specific embodiment, the pharmaceutical composition or active ingredient provided herein is prepared into a dosage form for pulmonary administration.

[0073] As an example, the inhalable powder for pulmonary administration can be produced by conventional technology, such as jet milling, spray drying, solvent precipitation, supercritical fluid concentration, etc. With dry powder inhaler (DPI), such as metered dose inhalers produced by Nektar, Vectura (Gyrohaler), GSK (Discus), or Astra (Turbohaler), the pharmaceutical composition or active ingredient provided by the application is included in a suitable carrier (such as mannitol, sucrose or lactose) and delivered to the end alveolar surface. Ultrasonic sprayer can also be used to deliver the solution preparation with liposome (or without liposome) to the lung.

[0074] While it is possible for a compound of the present application to be administered alone, it is preferable to administer the compound in the form of a pharmaceutical composition (or disinfectant composition).

[0075] As used herein, the articles "a", "an" and "the" are intended to include "at least one" or "one or more" unless otherwise stated. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : Cell viability detection results.

[0077] FIG. 2A to FIG. 2H : Cytotoxicity test (MTT) detection results.

[0078] Figure 3 : Cytopathic effect (CPE) results.

[0079] 4A to 4F : Expression of IL-1β, IL-6 and TNF-α.

[0080] FIG. 5A to FIG. 5F : Expression of IL-1β, IL-6 and TNF-α.

[0081] FIG. 6A to FIG. 6E : Expression of IL-1β, IL-6 and TNF-α.

[0082] Figure 7 A to Figure 7C : SPR results of small lipid molecules binding to 3C-like proteases.

[0083] FIG. 8A to FIG. 8E :IC binding of lipid small molecules to 3C-like proteases 50 . DETAILED DESCRIPTION

[0084] The embodiments of the present application are described in detail below, which are only used to explain the present application and cannot be construed as limiting the present application. Unless otherwise specified, the scientific and technological terms used in the present application have the same meaning as those commonly understood by those skilled in the art to which the present application belongs. Although the technical scheme of the present application is implemented on a specific virus strain, the technician understands that other viruses containing 3C-like proteases also respond to the compounds of the present application.

[0085] Example 1. Establishment of a lipid library of Chinese herbal medicine decoction

[0086] 1. Take 100g of Chinese medicine, add 500mL of water, soak in cold water for 30min, heat to boiling, then change to low heat for 30min, then change to high heat for 10min, open the lid and cool to room temperature to obtain the Chinese medicine soup.

[0087] 2. Add 15 mL of dichloromethane-methanol mixture (dichloromethane: methanol = 1: 2, v / v) to 4 mL of the obtained traditional Chinese medicine decoction to make dichloromethane: methanol: water = 1: 2: 0.8, and vortex mix for 10-15 min.

[0088] 3. Add 5 mL of dichloromethane to the EP tube and vortex mix for 10 minutes.

[0089] 4. Add 5 mL of ddH2O to the EP tube to make the ratio of dichloromethane: methanol: water = 2:2:1.8, and vortex mix for 10 min.

[0090] 5. Remove the upper liquid and the insoluble substances in the middle layer, take the lower dichloromethane layer, blow dry with nitrogen gas to obtain lipid small molecules.

[0091] 6. Sample analysis was performed using ExionLC (SCIEX, USA) and TripleTOF 5600+ (SCIEX, USA) instruments in positive and negative ion modes.

[0092] 7. The raw data were converted into .abf format using ABF CONVERTER, imported into MS-DIAL 4.0.0 for data analysis, and Lipidblast was used to identify the components to obtain a small molecule lipid library of Chinese herbal medicine decoction.

[0093] Example 2. Screening of database

[0094] 1. Target: Mpro (PDB: 6LU7).

[0095] 2. Download the three-dimensional structure of the Mpro protein and inhibitor co-crystal from RCSB PDB (PDB: 6LU7).

[0096] 3. The molecules in the traditional Chinese medicine decoction lipid database established in Example 1 were added into Discovery studio 4.0 through the Build 3D database function module to form a small molecule compound library that can be used for subsequent screening.

[0097] 4. Use the Prepare Protein module to process the protein file of the three-dimensional structure of the Mpro protein and inhibitor co-crystal downloaded in step 1 to remove multiple protein conformations, supplement incomplete amino acid residues, hydrogenate the protein, etc. After processing, the position of the ligand in the crystal structure of the protein ligand is defined as the active pocket.

[0098] 5. Use Discovery Studio's rapid molecular docking tool LibDock / CDOCKER for virtual screening and save the molecular conformations of compounds with high scores. There are a total of 5 lipid molecules with the following structural formulas:

[0099] PC(9:0 / 9:0)CAS No.:27869-45-0:

[0100]

[0101] PC(16:0e / 18:1)CAS No.:26853-31-6:

[0102]

[0103] DG(18:0 / 16:0)CAS No.:17708-08-06:

[0104]

[0105]

[0106] LPC(14:0 / 0:0)CAS No.:20559-16-4:

[0107]

[0108] LPC(16:0)CAS No.:17364-16-8:

[0109]

[0110] 6. Purchase compound molecules for MTT and CPE experimental verification, as well as surface plasmon resonance (SPR) and enzyme half inhibition rate (IC 50 )Test verification.

[0111] Example 3. MTT assay

[0112] 1. Experimental Procedure

[0113] 96-well plates were plated with 5000 Vero E6 cells per well. After 12 hours, 8 groups were set up:

[0114] Untreated group, virus group, blank control, PC (9:0 / 9:0) group, PC (16:0e / 18:1) group, DG (18:0 / 16:0) group, LPC (14:0 / 0:0) group, LPC (16:0) group. Six replicate wells were set up for each group, and the drug treatment concentration was 3 μM.

[0115] 12 hours after drug addition, except for the untreated group, the virus (SARS-COV-2, MOI: 0.1) was challenged, and the drug concentration of each group was kept constant. After 48 hours, observation and photography were carried out, and then 10 μl of MTT dye was added to each well. After 4 hours, the absorbance of each well was measured at a wavelength of 490 nm, and the results were recorded ( Figure 1 , FIG. 2A to FIG. 2H , Figure 3 ).

[0116] 2. Results

[0117] The five compounds obtained in Example 2 were confirmed to have an inhibitory effect on SARS-COV-2 virus infection at the cellular level. Figure 1 In the experiment, the five compounds improved the proliferation of Vero E6 cells after infection. According to the CPE experiment, compared with the model group, the five compounds improved the viability of infected Vero E6 cells.

[0118] Example 4. Alleviation of the inflammation model induced by lipopolysaccharide (LPS) of Gram-negative bacteria in THP-1 cell line

[0119] 1. Cell Culture

[0120] The THP-1 cells used in the experiment were purchased from the Cell Culture Center of Peking Union Medical College. α-MEM medium was purchased from HyClone. The cells were cultured in a 37°C, 5% CO2 incubator. THP-1 cells were cultured with 1640 medium as the basal medium, to which 10% v / v fetal bovine serum and antibiotics (penicillin 100U / mL and streptomycin 100mg / mL) were added. When the cells were cultured to the logarithmic growth phase, they were divided into 12-well plates (1mL culture medium / well), incubated at 37°C overnight, and subsequent experiments were carried out when the cells grew to 80%.

[0121] 2. Experimental grouping of cells and preventive drug administration

[0122] When the cells in the 12-well plate grew to 80%, lipid small molecules were administered preventively 12 hours in advance, with the amount of 0.03 mg per well. Then LPS stimulation was given, with the final concentration of 1 μg / mL per well, and the cell supernatant and cells were collected after 12 hours.

[0123] LPS was purchased from Sigma-Aldrich with a stock concentration of 1 mg / mL. When used, 50 μL of LPS stock solution was added to 450 μL Opti-MEM and 10 μL was added to each well.

[0124] Blank group: refers to the untreated cells, which serves as the blank control group.

[0125] Model group: LPS stimulation was added.

[0126] Lipid small molecule group: lipid small molecules were administered preventively and LPS was given for stimulation.

[0127] 3. Collect the cell supernatant and use ELISA kit to detect the content of IL-1β, IL-6 and TNF-α in the supernatant.

[0128] 4. Collect cells and extract RNA using RNA rapid kit, and use qPCR method to detect IL-1β, IL-6, TNF-α mRNA expression ( 4A to 4F ).

[0129] Example 5. Alleviating effect on polyinosinic-cytidylic acid (Poly I:C)-induced inflammation model in A549 cell line

[0130] 1. Cell Culture

[0131] The A549 cells used in the experiment were purchased from the Cell Culture Center of Peking Union Medical College. F-12 culture medium was purchased from HyClone. The cells were cultured in a 37°C, 5% CO2 incubator. A459 cells were cultured with F-12 culture medium as the basal medium, to which 10v / v% fetal bovine serum and antibiotics (penicillin 100U / mL and streptomycin 100mg / mL) were added. When the cells were cultured to the logarithmic growth phase, they were divided into 12-well plates (1mL culture medium / well), incubated at 37°C overnight, and subsequent experiments were performed after the cells adhered to the wall.

[0132] 2. Experimental grouping of cells and preventive drug administration

[0133] When the cells in the 12-well plate grew to 80%, lipid small molecules were administered preventively 12 hours in advance, with the amount of 0.03 mg per well, and then Poly I:C stimulation was given, with the final concentration of 1 μg / mL per well, and the cell supernatant and cells were collected after 12 hours.

[0134] PolyI: C was purchased from Sigma-Aldrich with a storage concentration of 10 mg / mL. The 12-well plate transfection system was configured as follows: 100 μL opti-MEM + 2 μL lipo2000; 100 μL opti-MEM + 0.1 μL PolyI: C. Let stand for 15 min, 200 μL per well.

[0135] Blank group: refers to the untreated cells, which serves as the blank control group.

[0136] Model group: PolyI:C stimulation was added.

[0137] Lipid small molecule group: preventive administration of lipid small molecules in advance and PolyI:C stimulation were given.

[0138] 3. Collect the cell supernatant and use ELISA kit to detect the content of IL-1β, IL-6 and TNF-α in the supernatant.

[0139] 4. Collect cells and extract RNA using RNA rapid kit, and use qPCR method to detect IL-1β, IL-6, TNF-α mRNA expression ( FIG. 5A to FIG. 5F ).

[0140] Example 6. The alleviating effect of the lipid small molecules of the present application on the inflammatory model induced by lipoteichoic acid (LTA) of Gram-positive bacteria in the U937 cell line

[0141] 1. Cell Culture

[0142] The U937 cells used in the experiment were purchased from the Cell Culture Center of Peking Union Medical College. 1640 culture medium was purchased from HyClone. The cells were cultured in a 37°C, 5% CO2 incubator. The culture of U937 cells was based on 1640 culture medium, to which 10v / v% fetal bovine serum and antibiotics (penicillin 100U / mL and streptomycin 100mg / mL) were added. When the cells were cultured to the logarithmic growth phase, they were divided into 12-well plates (1mL culture medium / well), incubated at 37°C overnight, and the subsequent experiments were carried out when the cells grew to 80%.

[0143] 2. Experimental grouping of cells and preventive drug administration

[0144] When the cells in the 12-well plate grew to 80%, lipid small molecules were administered preventively 12 hours in advance, with the amount of 0.03 mg per well, and then LTA stimulation was given, with the final concentration of 1 μg / mL per well, and the cell supernatant and cells were collected after 12 hours.

[0145] LTA was purchased from Sigma-Aldrich with a storage concentration of 1 mg / mL. When used, 50 μL LTA stock solution was taken into 450 μL Opti-MEM and 10 μL was added to each well.

[0146] Blank group: refers to the untreated cells, which serves as the blank control group.

[0147] Model group: LTA stimulation was added.

[0148] Lipid small molecule group: preventive administration of lipid small molecules in advance and LTA stimulation were given.

[0149] 3. Collect the cell supernatant and use ELISA kit to detect the content of IL-1β, IL-6, and TNF-α in the supernatant.

[0150] 4. Collect cells and extract RNA using RNA rapid kit, and use qPCR method to detect IL-1β, IL-6, TNF-α mRNA expression ( FIG. 6A to FIG. 6E ).

[0151] Example 7. Surface Plasmon Resonance (SPR) Assay Verifies Binding of Small Lipid Molecules to 3C-like Proteases

[0152] 3C-like proteins were immobilized on carboxymethyl-5′-dextran (CM5) sensor chips using amine coupling chemistry. 3C-like proteins were immobilized on a four-channel flow cell, and a three-channel flow cell was used as a reference cell for activation and blocking. 3C-like proteins (20 μg mL-1, 10 mm sodium acetate solution, pH 4.5) were immobilized on the surface by injection for 420 s. The remaining activated groups on the surface were blocked by injection of 1 μM ethanolamine at pH 8.5 for 420 s. During the above steps, the flow rate was maintained at 30 μL min-1, and 1x phosphate-buffered saline (PBS) running buffer was used during fixation. Approximately 13,000 response units (RU) of protein can be routinely captured on a CM5 chip in a four-channel flow cell.

[0153] SPR experiments showed that PC (9:0 / 9:0), LPC (14:0), LPC (16:0) and 3C-like proteins could bind ( FIG. 7A to FIG. 7C ).

[0154] Example 8. Binding half inhibition rate (IC) of lipid molecules to 3C-like proteases 50 )Detection

[0155] 1. Dissolve the compound in DMSO solution and prepare a high concentration stock solution (10 to 100 mM);

[0156] 2. Dissolve the SARS-CoV-2 main protease in the incubation solution (50mM Tris-HCl + 1mM EDTA, pH 7.3) to a final concentration of 30nM;

[0157] 3. Add 80 μL of the main protease solution to the 96-well fluorescence detection plate, and then add the stock solution of the drug to be tested to a final concentration of 0.01 to 100 μM, with a total of 10 concentration gradients;

[0158] 4. After the enzyme and drug mixture is incubated at 30°C for 10 minutes, the substrate is added and the reaction system is incubated in the dark;

[0159] 5. Add 40 μL of fluorescent substrate to a final concentration of 20 μM;

[0160] 6. Set up negative control by adding enzyme, substrate and drug in equal volumes of DMSO solution, in triplicate wells;

[0161] 7. Fluorescence detection (320 nm excitation, 405 nm detection), measured every 30 seconds, a total of 20 times;

[0162] 8. Result statistics and IC 50 calculate( FIG. 8A to FIG. 8E ).

Claims

1. Use of a 3C-like protease targeting agent in the preparation of an antiviral drug, wherein the 3C-like protease targeting agent is diglyceride or phosphatidylcholine, wherein: The virus is a virus comprising a 3C-like protease; The virus is SARS-CoV-2; The diglyceride is a compound shown in formula III: Formula III; The phosphatidylcholine is selected from any one of the following compounds: Formula I; Formula II; Formula IV; Formula V; or a pharmaceutically acceptable salt thereof.

2. Use of a 3C-like protease targeting agent in the preparation of a virus inhibitor, wherein the 3C-like protease targeting agent is diglyceride or phosphatidylcholine, wherein: The virus is a virus comprising a 3C-like protease; The virus is SARS-CoV-2; The diglyceride is a compound shown in formula III: Formula III; The phosphatidylcholine is selected from any one of the following compounds: Formula I; Formula II; Formula IV; Formula V; or a pharmaceutically acceptable salt thereof.

3. Use of a 3C-like protease targeting agent in the preparation of a disinfection composition, wherein the 3C-like protease targeting agent is diglyceride or phosphatidylcholine, wherein: The virus targeted by the disinfection composition is a virus containing a 3C-like protease; The virus is SARS-CoV-2; The diglyceride is a compound shown in formula III: Formula III; The phosphatidylcholine is selected from any one of the following compounds: Formula I; Formula II; Formula IV; Formula V; or a pharmaceutically acceptable salt thereof.

4. The use according to any one of claims 1 to 3, wherein the compound is a compound of formula II, formula III or formula IV or a pharmaceutically acceptable salt thereof.

5. The use according to any one of claims 1 to 3, wherein the compound is a compound of formula III or a pharmaceutically acceptable salt thereof.

6. The use according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium, potassium, calcium, aluminum, sulfate, nitrate, phosphate, hydrochloride, hydrobromide, sulfamate, acetate, trifluoroacetate, trichloroacetate, cinnamate, citrate, maleate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, glycolate, malate, lactate, malonate, oxalate, nicotinate, succinate, salicylate, stearate, tartrate, p-aminobenzenesulfonate, trimethylbenzenesulfonate, p-toluenesulfonate, mandelate, pectinate, picrate, and propionate.

7. A method for inhibiting virus proliferation or growth in an environment or on a surface of an object, comprising contacting the virus with an effective amount of a 3C-like protease targeting agent, wherein the 3C-like protease targeting agent is diglyceride or phosphatidylcholine, wherein: The virus is a virus comprising a 3C-like protease; The virus is SARS-CoV-2; The diglyceride is a compound shown in formula III: Formula III; The phosphatidylcholine is selected from any one of the following compounds: Formula I; Formula II; Formula IV; Formula V; or a pharmaceutically acceptable salt thereof.

8. The method according to claim 7, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium, potassium, calcium, aluminum, sulfate, nitrate, phosphate, hydrochloride, hydrobromide, sulfamate, acetate, trifluoroacetate, trichloroacetate, cinnamate, citrate, maleate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, glycolate, malate, lactate, malonate, oxalate, nicotinate, succinate, salicylate, stearate, tartrate, p-aminobenzenesulfonate, trimethylbenzenesulfonate, p-toluenesulfonate, mandelate, pectinate, picrate, and propionate.

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