CN111789861B - Application of small molecule compound in resisting African swine fever virus infection - Google Patents

Abstract

The invention discloses an application of a small molecular compound in resisting African swine fever virus infection, belonging to the technical field of medicines. According to the invention, through the crystal structure analysis of the E165R-dUMP compound, the enzyme activity center of the E165R playing a hydrolysis role is determined, and then the 1,2,3,4, 6-O-galloyl glucose which can be matched with the E165R activity center and can obviously inhibit the E165R enzyme activity is obtained by taking the E165R enzyme activity center structure as a target through in vitro screening. Further virus level experiments show that the 1,2,3,4, 6-O-galloyl glucose can obviously inhibit the replication of African swine fever virus in porcine alveolar macrophages, and has high application value in the aspect of clinical treatment or prevention of ASFV infection.

Description

Application of small molecule compound in resisting African swine fever virus infection

Technical Field

The invention relates to application of a small molecular compound in resisting African swine fever virus infection, and belongs to the technical field of medicines.

Background

African Swine Fever (ASF) is a highly fatal disease causing serious harm to the pig industry caused by infection of African Swine Fever Virus (ASFV), and poses serious threat to the world economy and food safety. The ASF epidemic first outbreaks in kenya in 1921 and subsequently epidemic in some countries and regions of africa. In recent decades, ASFV has spread rapidly from Africa to Europe, Asia, and the like, and shows a tendency to spread further. The ASFV is a double-stranded DNA virus with an envelope, has a complex and unique multilayer film structure and sequentially consists of a core, a nucleocapsid, an inner membrane, a capsid and an outer membrane from inside to outside. The diameter of the ASFV virus particle with envelope is about 250nm, the genome total length is 170-194kb, and 150-167 proteins can be coded and respectively participate in the processes of assembling virus particles, replicating genomes and resisting host immunity by viruses. Because ASFV virus particles have complex structures, huge genomes and numerous encoded proteins, the understanding of immunogenic proteins and immune escape mechanisms of the ASFV virus particles is not clear at present. Effective anti-ASFV vaccines and antiviral drugs have not yet been successfully developed, and suicide remains the only means for controlling the spread of ASFV.

dUTPase, an important nucleic acid hydrolase, is capable of hydrolyzing dUTP to produce dUMP and PPi, thereby maintaining the fidelity and integrity of genome replication. An increasing number of pathogens such as trypanosomes, plasmodium falciparum, mycobacterium tuberculosis, and dUTPase from humans, etc. have been targeted for drug development. The dUTPase encoded by the ASFV is called E165R protein, which has important function on the efficient replication of the ASFV in porcine macrophages, and the research shows that the knockout of E165R causes the amplification efficiency of the ASFV in alveolar macrophages to be obviously reduced. Therefore, the ASFV E165R protein can be used as a target of an antiviral drug and is used for developing the anti-ASFV drug.

Disclosure of Invention

The invention takes ASFV E165R as a target spot, screens a small molecular compound with an anti-ASFV effect, and provides a medicament for effectively preventing and treating African swine fever.

The invention has the first aim of providing the application of the small molecular compound in preparing the anti-African swine fever virus medicine; the small molecular compound is 1,2,3,4, 6-O-galloylglucose (Pentagalleylglucose) or a derivative thereof.

In one embodiment, the 1,2,3,4, 6-O-galloylglucose can also be replaced with a compound of formula 1:

wherein R is₁、R₂、R₃、R₄、R₅Can be selected from: h, C1-C3 alkyl, phenyl or galloyl.

In one embodiment, the African swine fever virus includes, but is not limited to, the Pig/HLJ/18 strain, or a DNA virus with more than 99% similarity to this strain.

The second purpose of the invention is to provide a drug for preventing or treating African swine fever, the active ingredient of the drug comprises 1,2,3,4, 6-O-galloylglucose (Pentagalleylglucose) or derivatives thereof.

In one embodiment, the 1,2,3,4, 6-O-galloylglucose can also be replaced with a compound of formula 1:

wherein R is₁、R₂、R₃、R₄、R₅Can be selected from: h, C1-C3 alkyl, phenyl or galloyl.

In one embodiment, the amount of 1,2,3,4, 6-O-galloylglucose in the medicament is not less than 20 μ M/g or 20 μ M/mL.

In one embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.

In one embodiment, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

The invention also claims the application of the compound in the preparation of the African swine fever virus inhibitor.

In one embodiment, the inhibitor is for use in inhibiting african swine fever virus replication.

The invention has the advantages and effects that:

the invention provides a small molecule compound for resisting ASFV infection: 1,2,3,4, 6-O-galloylglucose. The small molecule compound can inhibit the dUTPase activity of E165R. Meanwhile, the small molecular compound can effectively inhibit the replication of ASFV in PAMs, and has high application value in the aspect of clinically treating or preventing ASFV infection.

Drawings

FIG. 1 shows the results of ASFV-E165R protein purification molecular sieve and SDS-PAGE and the results of analytical ultracentrifugation; wherein, A is Superdex^TMProtein sample size was analyzed by 200 Increatase 10/300GL gel filtration chromatography and SDS-PAGE; b is the result of analytical ultracentrifugation of E165R protein.

FIG. 2 is the crystal structure of the E165R-dUMP complex; wherein A is the integral structure of E165R-dUMP compound crystal; b is analysis of interaction force of E165R and dUMP;

FIG. 3 is a graph showing the inhibition of ASFV E165R enzyme activity by 1,2,3,4, 6-O-galloylglucose and the result of ASFV replication in Porcine Alveolar Macrophages (PAMs); wherein, A is the analysis of the hydrolytic activity of the virtually screened small molecule drug inhibiting ASFV E165R; b is 1,2,3,4, 6-O-galloyl glucose which inhibits the activity of ASFV E165R enzyme and is dose-dependent; c is the effect of 1,2,3,4, 6-O-galloylglucose on inhibiting the replication of ASFV in PAMs;

FIG. 4 shows the docking pattern of 1,2,3,4, 6-O-galloylglucose interacting with the active center of ASFV E165R; wherein A is the structure of inhibitor 1,2,3,4, 6-O-galloylglucose; b is the space complementation of inhibitor 1,2,3,4, 6-O-galloyl glucose and ASFV E165R protein; c is the interaction force analysis of 1,2,3,4, 6-O-galloylglucose inhibitor and ASFV E165R protein.

Detailed Description

The preparation method of the enzyme activity experiment related reagent comprises the following steps:

stock solution of pyrophosphoric acid (PPi): an accurately weighed amount of 4.46g of sodium pyrophosphate powder (Na)₄P₂O₇·10H₂O) is added into 100ml deionized water, and is fully dissolved to prepare 100 multiplied storage solution which is stored at 4 ℃ and is diluted to working concentration according to the proportion of 1:100 when in use.

(ii) molybdic acid reagent: A10N sulfuric acid solution is prepared, 27ml of concentrated sulfuric acid is slowly and dropwise added into 73ml of deionized water, and the mixture is uniformly stirred while adding. 2.5g of ammonium molybdate powder is accurately weighed and dissolved in 100ml of prepared sulfuric acid solution, and the solution is preserved at 4 ℃.

③ sulfite reagent: solution A: 0.5g of sodium sulfite powder and 10g of sodium bisulfite powder were accurately weighed and dissolved in 100ml of deionized water, stirred and mixed well, and stored at 4 ℃. Solution B: solution A was diluted with deionized water at a ratio of 1:15 to obtain solution B.

Sulfydryl reagent: sucking 1ml beta mercaptoethanol and 9ml deionized water, and mixing well, preferably using in situ.

Example 1: expression and purification of ASFV-E165R protein

The full-length DNA sequence of the protein of Pig/HLJ/18 strain E165R (GenBank accession CBW46796.1) was ligated into the pET-21a vector by restriction enzyme sites NdeI and XhoI. Wherein the 3' end of the coding region of the ASFV-E165R protein is added with 6 histidine tags (His)₆-tag) and a translation stop codon. After an ASFV-E165R expression vector is constructed, BL21 Escherichia coli competent cells are transformed. Selecting a single clone, inoculating the single clone into 50mL LB culture medium, carrying out shake culture for 12 hours, then transferring 20mL of bacterial liquid into 2L of LB culture medium, and culturing at 37 ℃ to OD₆₀₀When the concentration was 0.6 to 0.8, IPTG was added to a final concentration of 0.5mM, and the culture was continued at 16 ℃ for 16 hours. After expression, the cells were collected, resuspended in protein buffer (20mM Tris, 150mM NaCl, pH8.5), and disrupted at low temperature and high pressure to obtain E165R protein in a soluble form. The E165R protein is subjected to nickel ion affinity chromatography (HisTrap)^TMHP (GE), Resource Q ion exchange chromatography and gel filtration chromatography (Hiload16/60superdex 75pg (GE)) purificationThereafter, the purity of the protein was confirmed by SDS-PAGE. The high-purity E165R protein can be obtained through identification, and the analysis ultracentrifugation result shows that the size of the E165R protein is about 50.5 kDa. The results are shown in FIG. 1.

Example 2: virtual screening of small molecule inhibitor with ASFV-E165R enzyme activity central structure as target spot

The ASFV-E165R protein purified in example 1 is mixed with a substrate dUTP for co-crystallization, and the molecular structure of the high-resolution protein is resolved, so that the crystal structure of a complex of the ASFV-E165R and the product dUMP is obtained, and the complex structure is shown in FIG. 2. The analysis of the crystal structure of the compound confirms the enzyme activity center of the E165R which plays the hydrolytic activity, and then the ASFV-resistant micromolecule medicine is obtained by virtual screening by taking the enzyme activity center structure of E165R as a target. The method comprises the steps of firstly obtaining a drug library containing 41384 small molecules from three companies of TargetMol, Selleck and MCE, and finally obtaining 52 small molecule compounds as candidate drugs through analysis processing of initial processing, drug-like five principle screening, PAINS filtering, molecular docking, docking score analysis, molecular diversity analysis, price control and the like of the drug library, wherein the information of names, targets and the like of the 52 candidate drugs is shown in Table 1.

TABLE 1 virtual screening results for small molecule drugs against ASFV E165R active center

Example 3: virtual screening of drugs to inhibit E165R enzyme activity and ASFV replication

(1) Inhibition of E165R enzyme activity by 1,2,3,4, 6-O-galloylglucose (Pentagalloylglucose)

The 52 small-molecule drugs screened in the example 2 are verified to confirm whether the drugs have dUTPase inhibitory activity, and the production amount of PPi is detected by an in vitro enzyme activity inhibition experiment to determine the capability of the small-molecule drugs in inhibiting the hydrolysis of dUTP by ASFV E165R.

First, a standard curve of pyrophosphate (PPi) was plotted: firstly, 100 times PPi stock solution is diluted into 1 times PPi working solution according to the proportion of 1: 100. ② 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 mul of standard PPi working solution (1mM) are respectively and sequentially added into 10 EP tubes, and deionized water is used for complementing to 800 mul, and the mixture is evenly mixed. ③ sequentially adding 50 mul of molybdic acid solution into each tube, and uniformly mixing; mixing 100 μ l of sulfurous acid A solution; 50 μ l of thiol solution and mix well. Fourthly, standing and developing for 10min at room temperature. Sequentially adding 100 mul of sulfurous acid B solution into each tube, and uniformly mixing; 50 mul of sulfhydryl solution is mixed evenly; 100 μ l of absolute ethanol and mixing. Sixthly, sucking 100 mu l of solution into a 96-well plate per tube, and measuring the light absorption value of the solution at 575 nm. The results were statistically analyzed and a standard curve was drawn.

In the specific experiment process, 52 kinds of micromolecular drugs with the concentration of 100 mu M are respectively mixed and incubated with E165R protein with the concentration of 1 mu M for 30min to combine the protein with the micromolecular drugs, then 100 mu M dUTP is added, each tube is complemented with 50mM Tris-HCl with the pH value of 8.0 to 60 mu l of reaction system, and the mixture is mixed evenly. Then reacting for 10min at 37 ℃, adding deionized water to 800 mu l, and mixing evenly. Finally, drawing according to PPi standard curve ((r)), finally measuring OD value at 575nm, and calculating PPi yield according to standard curve. The result shows that the small molecule drug 1,2,3,4, 6-O-galloylglucose (Pentagalloylglucose) can remarkably inhibit the level of PPi generated by hydrolyzing dUTP by E165R, so that the enzyme activity is reduced to 20% under the condition of a natural state, and the small molecule drug has the capability of inhibiting the hydrolysis activity of E165R (figure 3A). Further, the inhibitory activity against the enzyme activity of E165R was verified in the same manner as described above by adjusting the concentrations of 1,2,3,4, 6-O-galloylglucose to 0. mu.M, 1. mu.M, 10. mu.M, 50. mu.M and 100. mu.M, respectively, and it was found that the enzyme activity of E165R was 50% in the case of 50. mu.M, and the enzyme activity of E165R was only about 20% in the case of 100. mu.M (FIG. 3B).

(2) Inhibition of the ability of 1,2,3,4, 6-O-galloylglucose to replicate ASFV in cells

Infecting PAMs with 0.01MOI ASFV-GFP virus, changing the liquid after 2h, and adding inhibitor 1,2,3,4, 6-O-galloylglucose with different concentrations: 0. mu.M, 4. mu.M, 10. mu.M, 50. mu.M and 100. mu.M, and the fluorescence intensity and cell status of the virus were measured at 24h and 48h, respectively, after addition of the inhibitor. We found that 1,2,3,4, 6-O-galloylglucose was able to significantly inhibit the replication capacity of ASFV in PAMs and was dose-dependent (fig. 3C). At the same time we did not find changes in the cellular morphology of PAMs, suggesting that 1,2,3,4, 6-O-galloylglucose may have lower cytotoxicity (fig. 3C).

Example 4: analysis of interaction between 1,2,3,4, 6-O-galloylglucose inhibitor and ASFV E165R protein

The 1,2,3,4, 6-O-galloylglucose selected in example 3 was subjected to molecular docking analysis with the crystal structure of E165R, and FIG. 4A shows the molecular structure of 1,2,3,4, 6-O-galloylglucose, and the molecular docking results are shown in FIGS. 4B and 4C. The result shows that 1,2,3,4, 6-O-galloyl glucose can be matched with the enzyme activity center of E165R in a spatial structure and is well embedded in the enzyme activity center of E165R, so that dUTP is blocked from entering the enzyme activity center to exert inhibitory activity. The spatial structure between proteins is mainly maintained by non-covalent interactions, including hydrogen bonds, salt bonds, van der waals forces, hydrophobic interactions, electrostatic interactions, and the like. Further analysis of the interaction of 1,2,3,4, 6-O-galloylglucose with the E165R enzyme active center revealed that the R71, S72, S73 and E78 of one subunit and the M99 and K101 amino acid residues of the other subunit of the trimer E165R were able to form intermolecular hydrogen bonds with the 1,2,3,4, 6-O-galloylglucose small molecule to stabilize the complex structure (FIG. 4C). Meanwhile, the R71 residue of one subunit can form electrostatic interaction force with the 1,2,3,4, 6-O-galloylglucose small molecule, and the I48 and Y94 residues of the other subunit can form hydrophobic interaction force with the 1,2,3,4, 6-O-galloylglucose small molecule, so that the complex structure is further stabilized (figure 4C). Whereas in the amino acid residues where E165R forms non-covalent associations with 1,2,3,4, 6-O-galloylglucose small molecules, R71 and S72 of one subunit and Y94 and M99 of the other subunit interact with dUMP, where S72 forms a hydrogen bond with the phosphate group of dUMP and M99 forms a hydrogen bond with deoxyuridine of dUMP (fig. 4B). This indicates that 1,2,3,4, 6-O-galloylglucose can compete with dUMP to bind to the enzyme active center of E165R, thereby blocking the hydrolytic activity of E165R, resulting in the reduction or loss of the enzyme activity.

Example 5: use of 1,2,3,4, 6-O-galloylglucose in preparing medicine

1,2,3,4, 6-O-galloylglucose or a compound shown as a formula 1 is compatible with a pharmaceutically acceptable carrier, or is combined with a plurality of solid or liquid pharmaceutical excipients and/or auxiliary agents to prepare a proper administration form or dosage form which can be used for human;

wherein R1, R2, R3, R4, R5 can be selected from: h, C1-C3 alkyl, phenyl and galloyl.

One or more of the pharmaceutically acceptable filler, wetting agent, disintegrant, binder or lubricant.

The filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (7)

1.1,2,3,4, 6-O-galloylglucose or a salt thereof in the preparation of a medicament for resisting African swine fever virus.

2. The use according to claim 1, wherein the african swine fever virus comprises the Pig/HLJ/18 strain.

3. The use of claim 2, wherein the amount of 1,2,3,4, 6-O-galloylglucose in the medicament is greater than or equal to 20 μ M/g or 20 μ M/mL.

4. The use of claim 3, wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use according to claim 4, wherein the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.

6. Use according to claim 5, wherein the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.

Application of 1,2,3,4, 6-O-galloyl glucose in preparation of African swine fever virus inhibitor is provided.

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