Application of Vat Orange 9 in preparation of anti-enterovirus medicine

Abstract

The invention discloses application of Vat Orange 9 in preparation of an anti-enterovirus medicine, and belongs to the field of medicine. According to the present invention, the influence of the Vat Orange 9 on the thermal stability of the 2C oligomer and the 2C monomer is researched, and the competition experiment discovers that the Vat Orange 9 and the peptide-18 perform target point competition so as to destroy the combination of the peptide-18 and the 2C monomer; meanwhile, it is found that the high-concentration Vat Orange 9 can be used for remarkably inhibiting the ATPase activity of the 2C-oligomer. The compound has no obvious cytotoxicity, and shows an obvious antiviral effect at the gene level and the protein level.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and particularly relates to application of vat orange 9 in preparation of an anti-enterovirus drug. BACKGROUND

[0002] Enterovirus 71 (EV71) belongs to the Enterovirus genus of the Picornaviridae family, and is a small non-enveloped virus. Infection with EV71 can cause hand-foot-mouth disease, and even cause severe nervous system diseases. Although an EV71 vaccine has been marketed, the incidence of hand-foot-mouth disease in recent years is still ranked first among the class C infectious diseases in China, which has caused a threat to the health and public safety of the people in China.

[0003] EV71 is a single-stranded positive-sense RNA virus, and the full-length of the genome is about 7.4 kb, which encodes a large precursor protein. The precursor protein of the virus needs to be matured and cleaved to have functions, and this process is completed by viral proteases 2A and 3C. The precursor protein is finally enzymatically cleaved to produce 11 mature proteins: VP1, VP2, VP3, VP4, and 2A, 2B, 2C, 3A, 3B, 3C, 3D, etc. The 2C protein is composed of about 330 amino acids, and plays a key role in the life cycle of the virus. The known functions of 2C include: (1) the N-terminal of 2C protein has an amphipathic alpha helix, which is the membrane binding region of 2C. 2C recruits 2B, 3A and 3CD proteins to assemble the RNA genome replication complex, and through the N-terminal membrane binding region, the replication complex is located on the intracellular membrane structure such as vesicles, thereby starting the genome replication; (2) 2C has a conserved ATP hydrolyase domain and RNA helicase domain, and can unwind RNA double strands by using the energy of ATP hydrolysis; (3) the C-terminal cysteine-rich region of 2C protein mediates interaction with viral capsid proteins, participates in viral capsid packaging, and determines the morphology and environmental sensitivity of the virus; (4) 2C participates in the process of virus uncoating and cell innate immune escape, and is related to the pathogenicity of the virus.

[0004] EV712C is one of the most conserved non-structural proteins, which consists of an N-terminal membrane-binding motif, a central ATPase, a zinc finger motif and a C-terminal helical domain. The inventors' team resolved the first three-dimensional structure of an enterovirus 2C protein, EV712C protein, and found that the C-terminal domain of 2C protein folds into a long helix alpha 6 (named as Pocket-Binding Domain, PBD), which binds to a hydrophobic pocket (Pocket) of another molecule, mediating 2C to form oligomers (2C-oligomer). 2C lacking PBD cannot oligomerize, and exists as monomers (2C-monomer). Since the oligomerization of 2C protein is crucial in the life cycle of the virus, the inventors' team proposed an anti-EV71 strategy by interfering the PBD-Pocket interaction to hinder the function of 2C protein. Studies have shown that Peptide-18 consisting of 18 amino acids at the C-terminal of 2C protein can effectively inhibit the oligomerization of 2C protein, and can effectively inhibit the proliferation of the virus in both cell models and animal models. However, peptide inhibitors have the disadvantages of not being orally available and being easily degraded, and therefore, it is of great significance to develop non-peptide small molecule inhibitors based on this target.

[0005] Vat Orange 9 is an orange anthraquinone dye, which is mainly applied to the dyeing of cotton, viscose, silk and wool-cotton, and the printing of cotton cloth. There is no report on its antiviral activity in the published literature. SUMMARY

[0006] The purpose of the present application is to provide a new medical use of Vat Orange 9.

[0007] The new medical use of Vat Orange 9 provided by the present application is the use of Vat Orange 9 in the preparation of the following products:

[0008] 1) an anti-enterovirus drug;

[0009] 2) an enterovirus inhibitor;

[0010] 3) a drug for preventing and / or treating enterovirus infection;

[0011] 4) a drug for preventing and / or treating diseases caused by enterovirus infection.

[0012] Specifically, the enterovirus can be Enterovirus 71 (EV71);

[0013] The diseases caused by enterovirus infection include hand, foot and mouth disease.

[0014] The structural formula of Vat Orange 9 is as follows:

[0015]

[0016] Vat Orange 9 competes with peptide-18 for the target site of 2C-monomer of enterovirus 71, and destroys the interaction between peptide-18 and 2C-monomer;

[0017] Vat Orange 9 significantly inhibits the ATPase activity of 2C-oligomer.

[0018] Based on the crystal structure of EV712C (PDB: 5GQ1) obtained in the early stage, the present patent carries out high-throughput virtual screening on the pocket interacting with PBD. The compound with low binding free energy screened is further screened using the biological membrane interference technology, and the compound Vat Orange 9 with the equilibrium dissociation constant of 2C protein at the nanomolar level is identified. In the present patent, the inventors study the effect of Vat Orange 9 on the thermal stability of 2C-oligomer and 2C-monomer, and find through the competition experiment that Vat Orange 9 competes with peptide-18 for the target site, thereby destroying the binding of peptide-18 and 2C-monomer. At the same time, it is found that high concentration of Vat Orange 9 can significantly inhibit the ATPase activity of 2C-oligomer. The compound has no obvious cytotoxicity, and exhibits significant antiviral effect at the gene level and protein level. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the SDS-PAGE detection result graph of 2C protein after purification in embodiment 1 of the present application.

[0020] Figure 2 It is the particle size characterization of 2C protein after purification in embodiment 1 of the present application.

[0021] Figure 3 It is the dissociation curve of Vat Orange 9 and 2C-monomer in embodiment 2 of the present application.

[0022] Figure 4 It is the effect of Vat Orange 9 on the thermal stability of 2C protein in embodiment 3 of the present application.

[0023] Figure 5 It is the interaction analysis between peptide-18 and 2C-monomer in embodiment 4 of the present application.

[0024] Figure 6To evaluate the effect of Vat Orange 9 on the interaction between peptide-18 and 2C-monomer in Example 4 of the present application.

[0025] Figure 7 To evaluate the effect of Vat Orange 9 on ATPase activity in Example 5 of the present application.

[0026] Figure 8 To evaluate the cytotoxicity and anti-EV71 effect of Vat Orange 9 in Example 6 of the present application.

[0027] Figure 9 To evaluate the anti-EV71 effect of Vat Orange 9 by WB in Example 6 of the present application. DETAILED DESCRIPTION

[0028] The present application will be further described in conjunction with the specific embodiments, and the examples provided below are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0029] The experimental methods in the following examples are all routine methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0030] Example 1, Expression and purification of 2C protein

[0031] 1, pET28a-SUMO-2C 116-319 , pET28a-SUMO-2C 116-329 Plasmid was transformed into 3016 competent cells, and the next day a single colony was inoculated into 10 mL LB medium (containing 50 mg / L kanamycin) and cultured at 37°C, 220 rpm overnight.

[0032] 2, 10 mL of bacterial solution was transferred to 1 L of the above-mentioned LB medium for mass culture, and the culture conditions were 37°C, 180 rpm, and the culture was stopped when the OD was about 0.8 after about 3 hours of culture. The temperature of the shaker was set to 18°C, 180 rpm, and 0.5 mM IPTG was added to the large bottle for induction for 14-16 h.

[0033] 3, The next day, the bacterial solution was centrifuged at 4000 rpm for 15 min, and the bacterial body was collected and resuspended with 50 mL of resuspension buffer (Hepes 20 mM pH 7.5, 100 mM NaCl, 20 mM imidazole, 0.1% β-mercaptoethanol, PMSF 1 mM) and blown evenly.

[0034] 4. Ultrasonic disruption of the cells on ice (power 400W, 5s on, 3s off, 20min), the disrupted cells were centrifuged at 20000rpm for 60min at 4°C, and the supernatant was collected.

[0035] 5. Equilibrate the nickel ion affinity column with 10 column volume of resuspension buffer, then load the collected supernatant to the nickel column, and let it flow through, then wash the column with 10 column volume of resuspension buffer to remove the impurities, and keep 3mL resuspension buffer in the column to resuspend the column matrix, then add 100μL UPL1 enzyme to the column, and incubate overnight to remove the SUMO tag.

[0036] 6. The next day, rinse the nickel column with 10mL resuspension buffer, and the 2C-monomer protein (hereinafter referred to as 2C-monomer) and 2C-oligomer protein (hereinafter referred to as 2C-oligomer) can be obtained. 116-319 116-329

[0037] 7. Further purify the 2C protein using Superdex200 column, and the buffer is 20mM HEPES, 100mM NaCl, 2mM DTT, pH7.5. The purified 2C protein was detected by SDS-PAGE, and the result is shown in Figure 1 The retention volume of 2C-oligomer is 16.6mL, and the retention volume of 2C-monomer is 18.3mL.

[0038] The particle size of 2C-oligomer and 2C-monomer was measured by particle size analyzer, and the result is shown in Figure 2 The particle size of 2C-oligomer is 8.1nm, and the particle size of 2C-monomer is 4.7nm.

[0039] Example 2, primary screening and secondary screening of the compounds interacting with the Pocket of 2C protein

[0040] (1) Primary screening

[0041] Based on the crystal structure of EV712C obtained in the early stage, the inventors carried out high-throughput virtual screening on the Pocket interacting with PBD, and based on the screening results, 11 candidate small molecule inhibitors with low binding free energy and commercialization were determined for further screening and identification, as shown in Table 1.

[0042] (2) Secondary screening

[0043] ​​The 11 small molecule inhibitors obtained by preliminary screening are used as test compounds. The equilibrium dissociation constant of the compound with 2C protein is evaluated by biofilm interference technology (BLI) analysis. Biofilm interference technology is a label-free, real-time monitoring optical detection technology mainly used for comprehensive quantitative analysis of biomolecular interactions. BLI uses optical fiber biosensors to detect changes in sensor optical layer thickness in real time during molecular binding and dissociation. The combination of biomolecule A at the end of the sensor forms a biological membrane, and when molecule A binds to the detected molecule B, it causes changes in the molecular weight at the end of the sensor, resulting in changes in the thickness of the biological membrane. After the light passes through the biological membrane layer of the sensor, transmission and reflection occur to form interference light waves. Changes in the thickness of the biological membrane cause a relative shift in the interference light waves. The interference light waves before and after the combination of biological molecules are detected by a spectrometer to form an interference spectrum, which is displayed in real time by the shift (nm) of the interference spectrum. Finally, the detected molecules are analyzed according to the changes in the spectrum before and after the combination of molecules.

[0044] The specific operation of the experiment is as follows:

[0045] 1. Use the biotin rapid labeling kit (Frdbbio) to biotinylate 2C-monomer.

[0046] 2. The solvent of the compound mother liquor is DMSO, and the concentration is 10 mM. The mother liquor is diluted to 100 μM (containing 1% DMSO) using a molecular sieve buffer. The 100 μM compound is then diluted 2-fold gradient to 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM using a molecular sieve buffer containing 1% DMSO.

[0047] 3. Fix 70 ng / μL biotinylated 2C-monomer on the SSA sensor for 1800 s, and set a column of SSA sensors as background values (without 2C-monomer).

[0048] 4. Combine the 2C-monomer protein-fixed sensor with different concentrations of compounds for 600 s at a shaking speed of 1000, and dissociate in a molecular sieve buffer containing 1% DMSO for 1000 s at a shaking speed of 1000.

[0049] 5. Use the octet's supporting software DataAnalysis11 to analyze the data, and the analysis results are shown in Table 1. The most promising compound is compound Vat Orange 9 (Comp.2), which has a dissociation constant of 134 nM with 2C-monomer, and R 2 The value is 0.9963, and the dissociation curve of the combination of the compound with 2C-monomer is shown in Figure 3 .

[0050] Table 11 Virtual screening scores and results of BLI analysis of 11 compounds

[0051]

[0052] Example 3, Effect of compounds on the thermal stability of 2C protein

[0053] Differential Scanning Fluorimetry (DSF) is a method for evaluating the thermal stability of proteins by detecting the amount of fluorescent dye combined with the structure changed protein during the slow heating of the sample on a fluorescence quantitative PCR instrument. This method has the advantages of small amount of protein, high throughput, wide temperature range, accurate data, etc., and is widely used in the fields of protein stability, protein structure and conformation, protein-ligand interaction, protein stabilizers, inhibitors, etc. SYPRO Orange is an environmentally sensitive hydrophobic dye. As the temperature rises, the protein unfolds and the hydrophobic amino acids are gradually exposed. The dye specifically binds to the hydrophobic amino acids, and the fluorescence is enhanced. DSF can monitor the changes in protein conformation during the heating process by SYPRO Orange dye to calculate the melting temperature Tm.

[0054] The specific operation is as follows

[0055] 1. Protein dilution: 2C-monomer and 2C-oligomer were diluted to 20 μM (2x concentration) using molecular sieve buffer.

[0056] 2. Compound dilution: The compounds were diluted to the tested 2x concentration, i.e. 100 μM, 50 μM, 25 μM, using molecular sieve buffer.

[0057] 3. Protein and compound incubation: The protein and the compound diluted in step 2 were mixed uniformly according to the volume ratio of 1:1. At this time, the final concentration of the protein in the reaction system was 10 μM, the DMSO content was 1%, and the concentration of the compound was 50 μM, 25 μM, and 12.5 μM, respectively. A control group was also set up, which was the same as the experimental group except that it did not contain the compound. The above samples were incubated on ice for 2 h in the dark.

[0058] 4. SYPRO Orange dye was added to the above system to make its concentration 1x working concentration. After the dye was mixed uniformly with the reaction system prepared in step 3, it was transferred to a 96-well plate, and 3 replicate wells were set up for each sample. The program was set as shown in Table 2, and the CFX96 Real-Time PCR Detection System was run. The experimental results are as follows Figure 4As shown, the T m value of 2C-monomer is 43°C, and the T m value of 2C-oligomer is 44°C. After adding Vat Orange 9 at different concentrations, the thermal stability of 2C-monomer is not affected and remains 43°C, but the thermal stability of 2C-oligomer gradually decreases with the increase of the concentration of Vat Orange 9, and when the concentration of Vat Orange 9 is 50 μM, the T m value of 2C-oligomer decreases to 42°C. This result shows that the 2C protein in oligomeric form has a lower T m value and a more stable structure, and after adding Vat Orange 9, its stability will be destroyed, while the 2C protein in monomeric form is not affected, which suggests that this compound can destroy the oligomerization of 2C protein.

[0059] Table 2 DSF procedure

[0060] Procedure Time 4℃ 2 min 1 min ramp 1 °C, up to 95 °C 90 min 4℃ 1 min

[0061] Example 4, Competition experiment

[0062] (1) BLI analysis of the interaction between peptide-18 and 2C-monomer

[0063] The sequence of peptide-18 is REYSNRSAIGNTIEALFQ, which is the 18 amino acids at the C-terminal of EV71 2C protein, and can be tightly combined with the pocket in 2C-monomer. In this experiment, Biotin-peptide-18 was used as the stationary phase, and 2C-monomer was used as the mobile phase, and the K D value between the two was detected by BLI.

[0064] The specific operation of this experiment is as follows:

[0065] 1. 100 μM of biotin-peptide-18 was immobilized on the SA probe for 300 s.

[0066] 2. Protein dilution: 2C-monomer was diluted 2-fold gradient to 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM using the molecular sieve buffer.

[0067] 3. The sensor with immobilized Biotin-peptide-18 was combined with 2C-monomer at different concentrations for 150 s at a shaking speed of 1000, and dissociated in the molecular sieve buffer for 300 s at a shaking speed of 1000.

[0068] 4. Data analysis was performed using the octet's software DataAnalysis 11, and the results are shown in FIG. 4. The experimental data showed that there was interaction between peptide-18 and 2C-monomer, and the Kd value was 2.61 nM. Figure 5 D

[0069] (2) Detection of the effect of the compound on the interaction between peptide-18 and 2C-monomer

[0070] 1. Compound and protein incubation: 10 μM of 2C-monomer and 50 μM, 25 μM, 0 μM of compound (reaction buffer was molecular sieve buffer, and the DMSO content was 1%) were incubated on ice for 2 h.

[0071] 2. 100 μM of biotin-peptide-18 was immobilized on the SA probe for 300 s. In addition, a column of control sensors was set, and the sensors in this column did not need to immobilize biotin-peptide-18.

[0072] 3. The sensors immobilized with Biotin-peptide-18 were combined with 2C-monomer incubated with different concentrations of compound for 600 s, with a shaking speed of 1000, and dissociated in the molecular sieve buffer for 1000 s, with a shaking speed of 1000. The control sensors and different concentrations of compound without 2C-monomer were combined for 600 s, with a shaking speed of 1000, and dissociated in the molecular sieve buffer for 1000 s, with a shaking speed of 1000.

[0073] 4. Data analysis was performed using the octet's software DataAnalysis 11, and the results are shown in FIG. 4. The experimental data showed that there was interaction between peptide-18 and 2C-monomer, and the Kd value was 2.61 nM. Figure 6

[0074] Example 5, Effect of the compound on 2C-oligomer ATPase

[0075] 1. Standard curve: ATPase was determined using QuantiChrom™ ATPase / GTPase Assay Kit (DATG-200), and a linear function curve graph of the concentration of free phosphorus and ΔOD value was drawn according to the instructions, y (ΔOD) = 0.003x (Pi, μM) + 0.0092. 620nm

[0076] ​​​​2、Reaction system configuration: the total reaction system is 40 μL, including Assay Buffer 20 μL, 4 mM ATP 10 μL, 2C-oligomer (2C protein in oligomeric form has ATPase activity) and compound 10 μL, the concentration of 2C-oligomer used in this reaction is 20 μM, the concentration of DMSO is 2%, different compound concentrations (200 μM, 100 μM, 50 μM, 25 μM, 0 μM) are set, 3 repeats are set for each system, and a control system (Assay Buffer 20 μL, 4 mM ATP 10 μL, molecular sieve buffer containing 2% DMSO) is set, the above reaction system is mixed uniformly, and is placed in a 37°C incubator for reaction for 10 min.

[0077] 3、Transfer each reaction system to a black 96-well plate with transparent bottom, add 200 μL Reagent to each reaction system using a row gun, mix uniformly, and incubate at room temperature for 30 min in the dark.

[0078] 4、Use a multifunctional enzyme marker to read the value at 620 nm.

[0079] 5、Calculate the enzyme activity: first calculate ΔOD 620nm (requires greater than 0.5), that is, the value of the experimental group minus the value of the control group, and the ΔOD 620nm value is brought into the standard curve drawn to obtain the concentration of Pi, and the enzyme activity is calculated according to the formula, enzyme activity = [Pi] (μM) x 40 μL ÷ (V μL x 10 min) (U / L), wherein 40 μL represents the total volume of the reaction, V represents the volume of 2C-oligomer added, and 10 min represents the reaction time.

[0080] 6、Calculate the inhibition rate of the compound on 2C-oligomer ATPase,

[0081]

[0082] 7、Use GraphPad Prism to draw an inhibition rate curve, as Figure 7 shown, 200 μM of Vat Orange 9 can significantly inhibit the ATPase activity of 2C-oligomer.

[0083] Example 6, CCK8 experiment for evaluating the cytotoxicity of the compound

[0084] The main component in the CCK8 kit is WST-8, which can be reduced by dehydrogenase produced by mitochondria into a water-soluble formazan product. The generated formazan is orange yellow, and the color depth is proportional to the cell activity. The absorbance value at 450 nm wavelength measured by the enzyme label instrument can represent the number of living cells in each well of the cell plate.

[0085] The specific operation is as follows

[0086] 1. Plating: evenly plate the RD cells in a 96-well plate, 100 μL per well, at a density of 3 x 10 5 After 12-14 h of culture, the cell confluence is about 90%.

[0087] 2. Compound dilution: dilute the compound to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM using 2% FBSDMEM medium.

[0088] 3. Change liquid: discard the original culture medium in the 96-well plate, add 100 μL of the compound diluted in step 2 to each well, add 100 μL of 2% FBSDMEM medium containing 1% DMSO to the negative control group, and place the 96-well plate in the cell culture incubator for continuous culture for 24 h.

[0089] 4. Dilute 10 x CCK8 reagent (DOJINDO, CK04) to 1 x working concentration using 2% FBSDMEM. Discard the medium containing the drug, wash the cells with PBS, and add 100 μL of 1 x CCK8 to it. Continue to culture in the cell culture incubator for 3 h.

[0090] 5. Use a multifunctional enzyme label instrument to measure the absorbance value at 450 nm of each cell well.

[0091] 6. Use GraphPad Prism to draw a cell survival rate curve, and the experimental results are shown in Figure 8 The experimental results show that Vat Orange 9 still has no cytotoxicity at a concentration of 100 μM, and its CC 50 is greater than 100 μM.

[0092] Example 7, evaluation of the antiviral effect of the compound

[0093] (1) q-PCR detects the antiviral effect of the compound at the gene level

[0094] 1. Plating: evenly plate the RD cells in a 12-well plate, 1 mL per well, at a density of 3 x 10 5 After 12-14 h of culture, the cell confluence is about 90%.

[0095] 2, Infection: Use DMEM medium to dilute EV71 of known titer (obtained from infectious clone rescue of Fuyang strain, Chen P, Wojdyla JA, Colasanti O, et al. Biochemical and structural characterization of hepatitis A virus 2C reveals an unusual ribonuclease activity on single-stranded RNA. Nucleic Acids Res. 2022; 50(16): 9470-9489. doi: 10.1093 / nar / gkac671 IF: 14.9Q1) to MOI = 0.1 per well, and place the 12-well plate in the cell incubator for 1 h, gently tap the side of the cell plate every 15 min to distribute the virus particles evenly.

[0096] 3, Compound dilution: Dilute the compound to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM with 2% FBS DMEM medium.

[0097] 4, Change of medium: aspirate the medium containing virus in the 12-well plate, wash the cells twice with PBS, add 1 mL of compound diluted in step 3 to each well, and add 1 mL of 1% DMSO-containing 2% FBS DMEM medium to the negative control group, and continue to incubate the 12-well plate in the cell incubator for 24 h.

[0098] 5, Extraction of total RNA from cells: use EasyPure Fast Cell RNA Kit (full gold, ER111) to follow the operation steps in the instructions, and use Nanodrop to determine the RNA concentration of each sample after RNA elution.

[0099] 6, RNA reverse transcription: use PrimeScript TM RT reagent Kit (TaKaRa, RR047A) and follow the instructions.

[0100] ① Remove the genome from the sample, configure the reaction system according to Table 3, use a pipette to mix the configured system, and react at room temperature for 5 min.

[0101] Table 3 Genome removal system

[0102] Reagent Amount used 5x gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA 1.0 μg RNase Free dH2O upto 10 μL

[0103] 2. According to Table 4, configure the reverse transcription system, and use a pipette to blow and mix the configured system.

[0104] Table 4 Reverse transcription system

[0105]

[0106]

[0107] 3. According to Table 5, set the reverse transcription program.

[0108] Table 5 Reverse transcription program

[0109] Temperature Time 42℃ 15 min 85℃ 5s

[0110] 7. q-PCR detection: use TB Green Premix EX Taq TM II (TaKaRa, RR820A), add the corresponding primers in Table 6, and prepare the reaction system as shown in Table 7

[0111] Table 6 Primer name and sequence

[0112]

[0113] Table 7 qRCR reaction system

[0114] Reagent Amount used TB Green Premix Ex Taq II (2X) 12.5 μL Upstream primer F (10 μM) 1 μL Downstream primer R (10 μM) 1 μL cDNA template 2 μL Sterile water 8.5 μL Total 25 μL

[0115] Set 3 replicate wells for each sample, and set the program shown in Table 8 to run CFX96 Real-Time PCR Detection System.

[0116] Table 8 q-PCR reaction program

[0117]

[0118] 8. Data analysis: assuming that the amplification efficiency of the housekeeping gene and the target gene is 100%, use the delta-deltaCT (ΔΔCT) method to calculate the inhibition rate of different concentrations of compounds on the virus.

[0119] ΔΔCT = (CT value of experimental well VP1 - CT value of experimental well GADPH) - (CT value of negative control empty VP1 - CT value of negative control well GADPH)

[0120] Inhibition rate = (1 - 2 -ΔΔCT ) x 100%

[0121] Use GraphPad Prism to draw the inhibition rate curve, as shown in Figure 8 , EC 50The SI index was greater than 20, and the data showed that Vat Orange 9 could significantly inhibit the proliferation of the virus, and the inhibition rate of the virus could reach more than 90% at a use concentration of 50 μM. The inhibition of EV71 had a significant dose-effect relationship in the concentration range of 1.5625 μM to 100 μM. 50 The SI index was greater than 20, and the data showed that Vat Orange 9 could significantly inhibit the proliferation of the virus, and the inhibition rate of the virus could reach more than 90% at a use concentration of 50 μM. The inhibition of EV71 had a significant dose-effect relationship in the concentration range of 1.5625 μM to 100 μM.

[0122] (2) WB detection of the antiviral effect of the compound at the protein level

[0123] 1. Plating: evenly plate the RD cells in a 12-well plate, 1 mL per well, at a density of 3 x 10 5 cells / mL, and after 12-14 h of culture, the cell confluence is about 90%.

[0124] 2. Infection: dilute the known titer of EV71 using DMEM medium, add 400 μL of the virus diluent to each well to infect the cells at an MOI of 0.1, and place the 12-well plate in a cell culture incubator for 1 h. Every 15 min, gently tap the side of the cell plate to evenly distribute the virus particles.

[0125] 3. Compound dilution: dilute the compound to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM using 2% FBS DMEM medium.

[0126] 4. Medium change: discard the medium containing the virus in the 12-well plate, wash the cells twice with PBS, add 1 mL of the compound diluted in step 3 to each well, and add 1 mL of 2% FBS DMEM medium containing 1% DMSO to the negative control group. Place the 12-well plate in a cell culture incubator for 24 h.

[0127] 5. Wash the cells with PBS, add 200 μL of neutral RIPA lysis buffer to each well, and lyse for 15 min on ice. Use a pipette to thoroughly blow the cells to ensure complete lysis. Transfer to an EP tube and centrifuge at 13000 g for 10 min at 4°C. Take 160 μL of supernatant and add 40 μL of protein loading 5x loading. Mix well and heat in boiling water for 10 min.

[0128] 6. Prepare 10% polyacrylamide gel according to the One-Step PAGE Gel Fast Preparation Kit (Vazyme, E303-01). Use it after it is completely solidified.

[0129] 7. Loading and electrophoresis: After centrifugation, the sample prepared in step 5 was loaded in order, 10 μL of sample was added to each well, and the electrophoresis program was set as 180V for 30 min and 120V for 80 min, and the program was run.

[0130] 8. Transferring: The PVDF membrane was activated in methanol for 2 min, then soaked in the electric transfer liquid (Solarbio, D1060), and the transferring filter paper, sponge, etc. were soaked in the electric transfer liquid. The transferring device was assembled in the order of black transfer clamp, sponge, filter paper, glue, membrane, filter paper, sponge, and white transfer clamp, and transferred on ice at 300 mA for 1 h.

[0131] 9. Blocking: 5% skimmed milk powder was prepared in TBST as blocking solution, and the PVDF membrane was blocked at room temperature for 2 h on a horizontal shaker.

[0132] 10. Incubating primary antibody: The PVDF membrane was washed with TBST to remove the residual skimmed milk powder, and the PVDF membrane was incubated with primary antibody (Enterovirus 71 VP1 Monoclonal Antibody, AB_2815396; β-Actin Mouse mAb, AC004) at 4°C overnight.

[0133] 11. Incubating secondary antibody: The PVDF membrane was washed with TBST for 5 times, 5 min each time. The PVDF membrane was incubated with secondary antibody (HRP Goat Anti-Mouse IgG (H+L), AS003) at room temperature for 2 h on a horizontal shaker.

[0134] 12. Chemiluminescence: The PVDF membrane was washed with TBST for 5 times, 10 min each time. The A and B liquids of SuperSignal West PicoPLUS chemiluminescence substrate (Thermo Fisher, 34577) were mixed at a ratio of 1:1, and evenly applied to the PVDF membrane. The color was developed using a chemiluminescence instrument, and the experimental results are shown in TM Figure 2. The experimental data show that Vat Orange 9 can significantly inhibit the replication of EV71. Figure 9

[0135] The present application has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application.​

Claims

1. Application of Vat Orange 9 in the preparation of drugs against enteroviruses.

2. Application of Vat Orange 9 in the preparation of enterovirus inhibitors.

3. Use of Vat Orange 9 in the preparation of drugs for the prevention and / or treatment of enterovirus infections.

4. Vat Orange 9 is used in the preparation of medicines for the prevention and / or treatment of diseases caused by enterovirus infection.

5. The application according to any one of claims 1-4, characterized in that, The enterovirus mentioned is Enterovirus 71 (EV71).

6. The application according to claim 5, characterized in that, The diseases caused by enterovirus infection include hand, foot, and mouth disease.

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