Application of anethomycin in preparation of medicine for inhibiting activity of cysteine protease

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of anethomycin in preparation of medicine for inhibiting cysteine protease activity. The invention discovers that anethomycin has the function of inhibiting the activity of virus cysteine protease, especially 3C or 3CL protease, for the first time. Molecular docking proves that anethomycin can effectively bind to the catalytic activity center of Senecavirus (SVA) 3C protease. An in-vitro FRET enzyme activity experiment proves that the inhibition rate of 50 [mu] M of anethomycin on SVA 3C protease reaches up to 88.5%. Cellular level experiments show that the anethomycin can significantly inhibit replication of SVA viruses, shows broad-spectrum antiviral activity on various viruses such as encephalomyocarditis viruses (EMCV), porcine reproductive and respiratory syndrome viruses (PRRSV) and porcine deltacoronaviruses (PDCoV), and is low in cytotoxicity and high in selectivity index (SI).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of anisin in the preparation of drugs that inhibit cysteine ​​protease activity. Background Technology

[0002] Seneca virus A ( Senecavirus A SVA), formerly known as Seneca Valley virus ( Seneca Valley Virus SVA (Senecavirus) belongs to the genus Senecavirus of the family Picornaviridae. The vesicular lesions caused by this virus are similar to those of foot-and-mouth disease (FMD). In 2015, SVA infection was first discovered in pig herds in my country and spread to several provinces and cities, causing certain economic losses to the pig farming industry. Currently, the pathogenic and immune mechanisms of SVA are not fully understood, and effective prevention and control measures are still lacking.

[0003] The SVA genome encodes a cysteine ​​protease 3C, which plays a crucial role in viral replication. Picornaviridae (PVRs) are numerous and can spread through various routes, causing severe economic losses and public health problems. These include poliovirus (PV), which is highly pathogenic to humans, and foot-and-mouth disease virus (FMDV), which causes significant economic losses; both viruses contain 3CLpro. Furthermore, coronaviruses and arteritis viruses also contain 3CLpro, such as porcine deltacoronavirus (PDCOV), encephalomyocarditis virus (EMCV), and porcine reproductive and respiratory syndrome virus (PRRSV). These viruses are widely distributed in human and animal populations in my country, often causing severe economic losses and public health problems. Therefore, the development of novel antiviral drugs with broad-spectrum specificity is both necessary and urgent.

[0004] Anisin is produced by Streptomyces griseus ( Streptomyces griseolus ) or pyrrolidine alkaloids produced by *Streptomyces hygroscopicus*, with the chemical formula C 12 H 19 NO4, with a molecular weight of 265.3. This substance possesses various biological activities and is an effective protein synthesis inhibitor. It exerts its effects by inhibiting the peptidyl transferase activity of eukaryotic ribosomes. It can also activate the JNK (c-Jun N-terminal kinase) and p38 MAPK pathways, promoting the Caspase cascade reaction and thereby inducing tumor cell apoptosis. Anisin has relatively low toxicity; reports indicate that anisin can inhibit CVB3 replication by degrading eEF1A1 via CMA. Antivirus Res (2023). To date, there are no other research reports on the antiviral and molecular mechanisms of anisoxin. Summary of the Invention

[0005] The present application aims at overcoming the deficiencies of the prior art, and discloses, for the first time, that anethol trithione has the function of inhibiting cysteine protease, and provides the application thereof in antiviral drugs.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions. The present application provides the use of anethol trithione in the preparation of a drug for inhibiting the activity of cysteine protease.

[0007] Preferably, the cysteine protease is viral 3C or 3CL protease.

[0008] Preferably, the drug is used for inhibiting the activity of protease in the replication stage of virus.

[0009] The present application also provides the use of anethol trithione in the preparation of an antiviral drug.

[0010] Preferably, the virus is Picornaviridae, Coronaviridae or Arteriviridae.

[0011] The present application also provides the use of anethol trithione in the preparation of an inhibitor for inhibiting the activity of cysteine protease.

[0012] Preferably, the dosage form of the inhibitor is one of a capsule, a tablet or a granule.

[0013] The present application also provides an antiviral drug composition, wherein anethol trithione is used as an active ingredient.

[0014] Preferably, the composition further comprises one or more pharmaceutically acceptable carriers.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) The present application discloses, for the first time, that anethol trithione has the new function of inhibiting the activity of viral cysteine protease (such as 3Cpro), which opens up a new direction for the clinical application thereof.

[0016] (2) The present application confirms that anethol trithione can directly act on the active pocket of SVA 3C protease (3Cpro) to inhibit the enzyme activity thereof through molecular docking and biochemical experiments (FRET).

[0017] (3) Anethol trithione can effectively inhibit SVA replication, and the CC 50 of cytotoxicity is 1473 μM, the IC 50 of inhibition of virus is 0.13 μM.

[0018] (4) Anethol trithione can effectively inhibit the activity of SVA protease, so as to inhibit viral nucleic acid and inhibit viral replication.

[0019] (5) Anethol showed significant inhibitory effect on a variety of 3C / 3CL protease-dependent viruses such as SVA, EMCV, PRRSV and PDCoV, and had broad-spectrum antiviral potential, with a virus inhibition efficiency of 80% to 90%.

[0020] (6) Anethol had no obvious toxicity to host cells at an effective antiviral concentration, had a high selectivity index (SI) and good safety. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0022] Figure 1 A flow chart and a result schematic diagram for high-throughput screening of anethol for anti-SVA virus active compounds in a natural compound library.

[0023] Figure 2 A time point analysis result schematic diagram of anethol in the SVA virus replication stage, wherein from left to right, (A) is the virus adsorption stage, (B) is the virus internalization stage, (C) is the virus replication stage, and (D) is the virus release stage.

[0024] Figure 3 A molecular simulation diagram showing the binding mode of anethol and SVA 3C protease active site.

[0025] Figure 4 A Western blot for verifying the inhibition of anethol on SVA 3C protease activity.

[0026] Figure 5 A Western blot and qPCR for proving the broad-spectrum antiviral effect of anethol on EMCV, PRRSV and PDCoV; wherein from top to bottom, they are EMCV, PRRSV and PDCoV. DETAILED DESCRIPTION

[0027] In order to further illustrate the present application, the technical solutions provided by the present application will be described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0028] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special instructions, and the name and / or abbreviation thereof all belong to the conventional name in the field, which is very clear and explicit in the related application field, and the person skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement it according to the conventional conditions or the conditions recommended by the manufacturer.

[0029] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application have no special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional method well known to the person skilled in the art.

[0030] ST cells, PK-15 cells, HEK-293T cells, Marc-145 cells and BHK-21 cells were preserved by the laboratory; SVACH-SD strain passaged in BHK-21 cells, PDCOV virus passaged in ST cells, PRV passaged in PK-15 cells, PRRSV BB0907 passaged in Marc-145 cells and EMCV NJ08 passaged in BHK-21 cells were all preserved by the laboratory; The natural product drug library containing 160 natural products was purchased from Selleck Chemicals; Anisomycin used for in vitro experiment was purchased from Selleck Chemicals, with a purity of > 99%; Cell viability was detected by enhanced CCK8 kit (Beyotime); The concentration (CC50) that caused 50% cytotoxicity was calculated by GraphPad Prism 6.0 software; Example 1 High-throughput screening of natural compounds against SVA virus 160 natural product libraries were used for screening in BHK-21 cell model. Cells were dissolved in DMSO at a concentration of 10 mM and stored at -80℃ until use. The workflow of high-throughput screening (HTS) is shown in Figure 1 A. BHK-21 cells were seeded in 96-well plates at 5×10 4 When about 90% confluence was reached, 10 μM compounds or DMSO (1 μl) were used for 1 h treatment, followed by infection with SVA at 0.01 MOI for 1 h. Unabsorbed virus particles were washed off with PBS, and then maintenance fluid containing 10 μm compounds or DMSO (1 μl) was added. The virus inhibition rate was calculated by observing cytopathic effect (CPE) and indirect immunofluorescence (IFA).

[0031] The specific steps of indirect immunofluorescence method are as follows: BHK-21 cells are fixed with 4% paraformaldehyde for 20 min, washed with PBS, and 0.1% Trition X-100 is used to permeabilize the cell membrane at 37°C for 20 min. The treated cells are incubated with mouse anti-SVAVP1 protein monoclonal antibody (1:2000 dilution) prepared in the laboratory at 37°C for 2 h. The cells are washed with PBS for 3 times, and FITC-labeled goat anti-mouse IgG (H+L) antibody (1:200 dilution) (Beyotime) is added and incubated at 37°C for 1 h. DAPI staining solution (Beyotime) is used to stain the cell nucleus. After washing with PBS, the cells are observed under a Zeiss inverted fluorescence microscope. The total fluorescence density is determined by using ImageJ software.

[0032] During the preliminary screening, if a compound causes any visible cytotoxicity, or the CPE is reduced by less than 50% compared with the DMSO control group, the compound is excluded. In the second round of screening, the cell survival rate must reach 80% or higher, and the inhibition rate of SVA must reach 80% or higher as determined by IFA. The results are shown in Figure 1 .

[0033] According to the first round of screening results shown in Figure 1 C, a total of 10 compounds were obtained, which had no obvious cytotoxicity and reduced the cytopathic effect (CPE) by more than 80%. Subsequently, the 10 compounds were subjected to the second round of screening; finally, 2 compounds were screened from them: the 2 compounds not only had almost no cytotoxicity, but also had an inhibition effect further verified by immunofluorescence analysis (IFA, as shown in FIG. 1B) and Western blotting (as shown in FIG. 1D), and the results showed that the inhibition rates were higher than 80%. Both of the two compounds exhibited dose-dependent antiviral activity, and then the safety index (SI) of the compound with a safety index (SI) greater than 80 was calculated according to the 50% inhibition concentration (IC 50 ) and the 50% cytotoxicity concentration (CC 50 ) of each candidate compound.

[0034] BHK-21 cells were cultured in DMEM-10% FBS containing different concentrations of anisomycin, and BHK-21 cells were treated with 0.001, 0.01, 0.1, and 1 μmol / L (μM) anisomycin and deguelin, respectively, and incubated at 37°C for 48 h, with DMSO as a negative control.

[0035] As shown in Figure 1As shown in Fig. E, both TCID50 (tissue culture infectious dose 50) and Western blotting results showed that the number of SVA infected cells was significantly reduced after treatment with the two compounds; at the same time, the selection index (SI, reflecting the balance between the antiviral activity and cytotoxicity of the compound) of both was greater than 80, the cytotoxicity CC50 was 1473 μM, and the virus inhibition IC50 was 0.13 μM, as shown in Fig. D. Figure 1 F、 Figure 1 G.

[0036] Considering the comprehensive performance and cost, anisomycin not only has the highest SI value, but also has a lower price, so it is selected as the target compound for further research.

[0037] Example 2 Effect of anisomycin on the proliferation stage of SVA virus To clarify the specific role of anisomycin in the virus replication cycle, different time points were designed for drug administration, and qRT-PCR was used to detect the viral VP1 RNA level to analyze the role of anisomycin in the four stages of virus adsorption, internalization, replication and release.

[0038] 1. Virus adsorption BHK-21 cells were treated with 10 μM anisomycin or DMSO at 37°C for 2 h, and then incubated with SVA virus solution at 4°C for 1 h, as shown in Fig. A. Figure 2 The cells were washed with pre-cooled PBS, and the RNA was extracted for qRT-PCR to detect the SVA VP1 and β-actin mRNA levels in the cells.

[0039] 2. Virus internalization BHK-21 cells were incubated with SVA virus solution at 4°C for 1 h, and then the original culture medium was replaced with culture medium containing anisomycin (10 μM) or DMSO, and incubated at 37°C for 30 min, 1 h and 2 h, as shown in Fig. B. Figure 2 The cells were washed with citric acid buffer to remove non-internalized virus particles, and qRT-PCR was used to detect the SVA VP1 and β-actin mRNA levels in the cells.

[0040] 3. Virus replication BHK-21 cells were incubated with SVA virus solution at 37°C for 1 h, and washed with PBS three times to remove free virus particles. Four hours after infection, the original culture medium was replaced with fresh culture medium containing anisomycin or DMSO. Six hours after continued infection, qRT-PCR was used to detect the SVA VP1 and β-actin mRNA levels in the cells, as shown in Fig. C. Figure 2

[0041] 4. Virus release​ 0.01 MOI SVA infected BHK-21 cells for 1 h, then replaced the culture medium with fresh DMEM. 12 h post-infection, cells were washed with PBS three times, and fresh culture medium containing anisomycin or DMSO was used to replace the original culture medium. The supernatant was harvested after 4 h incubation at 37 °C, and the released virus particles were detected by absolute fluorescent quantitative PCR. Figure 2 D. The VP1 RNA level in the released virus particles was detected by absolute fluorescent quantitative PCR.

[0042] The specific method of qRT-PCR is as follows: total RNA of cells is extracted using a total RNA extraction kit, and reverse transcription is performed using HiScript qRT SuperMix. The above cDNA is used as a template, and amplification is performed on an ABI 7300 real-time PCR instrument using the SYBR Green method.

[0043] The reaction system is as follows: 2x Power SYBR Green PCR Master Mix 10 μL, cDNA 2 μL, and upstream and downstream primers (400 nmol / L each) 0.4 μL each. The reaction program is as follows: 95 °C for 2 min; 95 °C for 15 s, 61 °C for 31 s, for a total of 40 cycles. The primer sequences used for detection are shown in Table 1.

[0044] Table 1 Primers used for detection

[0045] According to the above results, by detecting the SVA VP1 and cell β-actin mRNA levels, anisomycin does not affect the adsorption of the virus, as shown in Figure 2 A; but inhibits the replication of the virus from the internalization stage and continues to act until the release stage of the virus, as shown in Figure 2 B-D.

[0046] Example 3 Molecular docking simulation of the binding of anisomycin to SVA 3C protein To clarify the specific target, Alphafold3 was used to simulate the structure of the non-structural protein of SVA virus, and the three-dimensional structures of SVA 3C, 2B, 2C, 3AB, and 3D were obtained by homology modeling, and anisomycin was docked to the 3C protein active pocket by Autodock, as shown in Figure 3 A.

[0047] The results show that anisomycin (blue stick) forms a hydrogen bond (bond length 2.2 Å) with the key residue 48-HIS of the 3C protease active pocket, and forms additional hydrogen bond interactions with 157-LYS and 47-GLU, thereby closing the catalytic site (magenta area: 48-HIS / 84-ASP / 160-CYS).Figure 3 B, the binding free energy (-5.2 kJ / mol) is significantly lower than other proteins, indicating that anisomycin has the potential to inhibit 3C protease activity.

[0048] Example 4 Inhibition of SVA 3C protease activity in vitro by anisomycin In order to explore the effect of anisomycin on 3C activity, a series of expression plasmids were constructed, and the coding sequence of 3C was amplified from the cDNA of SVA and cloned into pCAGGS-Flag and pGEX to construct pGEX-3C prokaryotic expression plasmid. The plasmid was transformed into BL21 E. coli, which was cultured at 37°C to OD 600 about 0.8, 1 mM IPTG was added and induced at 27°C for 7 h. After collecting the bacterial cells and ultrasonic disruption, GST agarose beads were used for affinity purification, and reduced glutathione was used for elution to obtain high-purity GST-3C fusion protein. The in vitro incubation experiment was used to verify that the GST-3C protein had enzymatic activity. The protein was analyzed and confirmed by SDS-PAGE and Western blot (anti-GST antibody).

[0049] Subsequently, FRET-based enzyme activity experiment was performed: the FRET substrate peptide Dabsyl-LLAKVIQ↓IATSSS-Edans was synthesized. The reaction system contained purified GST-3C protein at a final concentration of 0.5-1.5 μM, 10 μM substrate peptide, and different concentrations (25, 50 μM) of anisomycin (50 μM of Huangbai ketone and DMSO as control). Incubate at 37°C for 20 minutes in a black 96-well plate, monitor the fluorescence value of 485 nm emission under 340 nm excitation light every minute. Relative fluorescence value (RFU) is calculated by subtracting the fluorescence value of the Mock group from each group. Inhibition rate (%) = 100 × [1 - (anisomycin group ΔRFU / negative control group ΔRFU)].

[0050] Western blot general method: cells were lysed with RIPA lysis buffer on ice for 15 min, and proteins were collected for SDS-PAGE electrophoresis, and then transferred to NC membrane. After blocking with 5% skim milk for 2 hours, incubate with primary antibody (1:1000) for 2 hours, then incubate with HRP-labeled secondary antibody (1:1000) for 1 hour, and finally expose with ECL luminescent liquid.

[0051] After transfecting HEK-293T cells with plasmids and treating with anisomycin or DMSO, Western blot analysis showed that anisomycin could inhibit the cleavage of 3C to its substrate DHX30, as Figure 4A. The fluorescence resonance energy transfer experiment is based on the energy transfer of two fluorescent groups, and is used to detect the interaction of two labeled molecules at a very close distance. The specific, single and enzymatically active 3C protein was obtained by prokaryotic expression and GST agarose bead purification, as shown in FIG. 1B. Figure 4 B-E.

[0052] The results show that the prokaryotically expressed GST-3C protein has significant protease activity. The inhibition rates of anisomycin at 50 μM and 25 μM on 3C protease are 88.5% and 33.8%, respectively. At the cellular level, the Western blot results also show that anisomycin can dose-dependently inhibit the cleavage of 3C protease on the substrate DHX30.

[0053] Example 5. Broad-spectrum antiviral activity of anisomycin on multiple viruses It is explored whether anisomycin has broad-spectrum antiviral effect on small RNA viruses containing 3C or 3CL protease.

[0054] The present application selects another small RNA virus EMCV, arterivirus PRRSV, and a coronavirus PDCOV. After the cells are treated with 5 different concentrations (0.001, 0.01, 0.1, 1, 5 μM) of anisomycin, DMSO is used as a negative control, and the corresponding viruses are inoculated, PRRSV, PDCOV, and EMCV are inoculated into Marc-145, ST, and BHK-21 cells, respectively, and cultured for 48, 24, and 18 h, respectively. The proteins and cell supernatants are collected for Western blot and virus titer determination, and the results are shown in FIG. 3. Figure 5

[0055] Western blot and virus titer show that anisomycin can have good antiviral effect on EMCV, PRRSV, and PDCOV at a dose that does not produce cytotoxicity.

[0056] In summary, the results of the examples of the present application first reveal and fully confirm the new function of anisomycin as a viral 3C / 3CL protease inhibitor. By directly and efficiently inhibiting the activity of viral protease, it further blocks the viral replication cycle, and exhibits broad-spectrum antiviral activity on multiple viruses, and has good safety. These findings provide a solid scientific basis and a valuable candidate molecule for the development of new, broad-spectrum antiviral drugs based on anisomycin.

[0057] Although the above examples make a detailed description of the present application, it is only a part of the examples of the present application, but not all the examples, and other examples can be obtained under the premise of no creativity according to the present examples, which all belong to the protection scope of the present application.​

Claims

1. Use of anisomycin in the manufacture of a medicament for inhibiting cysteine protease activity.

2. Use according to claim 1, characterized in that, The cysteine protease is a viral 3C or 3CL protease.

3. Use according to claim 1, characterized in that, The medicament is for inhibiting protease activity at a stage of viral replication.

4. Use of anisomycin in the manufacture of an antiviral medicament.

5. Use according to claim 4, characterized in that, The virus is a Picornaviridae, Coronaviridae, or Arteriviridae.

6. Use of anisomycin in the manufacture of an inhibitor for inhibiting cysteine protease activity.

7. Use according to claim 6, characterized in that, The dosage form of the inhibitor is one of a capsule, a tablet, or a granule.

8. An antiviral pharmaceutical composition, characterized by, The anisomycin is an active ingredient in the composition.

9. The composition of claim 8, wherein, The composition further comprises one or more pharmaceutically acceptable carriers.

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