Use of tylosin derivatives for the preparation of a drug against viruses of the poxviridae family

Abstract

The application discloses application of tylosin derivatives in preparation of anti-poxviridae virus drugs. It is found by the application that tylosin derivative T3 can reduce the replication level of LSDV, the derivative has low toxicity, has no toxic side effect on MDBK cells, can be used as an inhibitor for efficiently resisting bovine tuberculous dermatosis virus, and provides a strong theoretical basis and practical basis for further anti-virus drug research and development, and has wide application prospects in the breeding industry and the biological medicine field.

Description

Technical Field

[0001] This invention relates to the field of biomedicine technology, specifically to the application of tylosin derivative T3 in the preparation of antiviral drugs. Background Technology

[0002] CN117510561A discloses a tylosin derivative with the structure shown in Formula I. Experiments have shown that this tylosin derivative can be used to treat or prevent bacterial infections in animals, but no other uses have been found.

[0003]

[0004] Lumpy skin disease (LSD) in cattle is caused by bovine lumpy skin disease virus (LSDV), a member of the goatpox virus family. The disease is characterized by high fever, swollen lymph nodes, emaciation, oral mucus, reduced milk production, agalactia, nodular lesions, and infertility. LSD poses a serious threat to livestock farming and affects the trade of livestock and their products, but currently there are no effective treatments for LSD.

[0005] Antiviral drugs provide "chemical protection" before vaccines generate neutralizing antibodies, which is particularly important for newborn animals, immunocompromised individuals, and newly emerging variants. They can target the virus itself or host cytokines. Direct antiviral drug targets include polymerases or proteases that inhibit viral attachment, entry, and uncoating. Members of the Poxviridae family follow a highly conserved replication process. With a deeper understanding of the biological structure and replication cycle of orthopoxviruses, some highly effective and low-toxicity small-molecule inhibitors have been discovered, such as cidofovir (CDV) and teviririmat (ST-246), which are currently marketed antipoxvirus drugs. However, these drugs still suffer from low oral bioavailability, easy induction of drug resistance, and strong nephrotoxicity. Therefore, developing a new generation of highly effective, low-toxicity anti-LSDV drugs with a clearly defined mechanism of action is crucial for maintaining animal health, benefiting livestock development, and providing important reference for the control of poxviridae viruses. Summary of the Invention

[0006] Firstly, this invention discovers a novel application of compound T3, a tylosin derivative from CN117510561A, in antiviral drugs targeting poxviral viruses. The structural formula of compound T3 is as follows:

[0007] .

[0008] The poxviruses mentioned include the genera orthopoxvirus and goatpoxvirus.

[0009] The orthopoxvirus genus includes vaccinia virus.

[0010] The goatpoxvirus genus includes bovine nodular dermatovirus.

[0011] In one specific embodiment of the present invention, the viral structural proteins include LSDV028, LSDV050, LSDV047, LSDV033, and LSDV086.

[0012] The concentration of compound T3 in the drug is 25-3200 ppm. Specifically, it is 3200 ppm, 400 ppm, or 25 ppm.

[0013] The drug contains a therapeutically effective amount of compound T3 or its pharmaceutical salt, and contains one or more pharmaceutically acceptable excipients.

[0014] The drug is formulated as a compound drug with compound T3 as the sole active ingredient or in combination with other antiviral active ingredients from the Poxviridae family.

[0015] The drug also includes pharmaceutically acceptable carriers, excipients, or additives; the carriers or excipients are selected from one or more of diluents, binders, adsorbents, fillers, and disintegrants; the additives are selected from one or more of stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants.

[0016] The dosage forms of the drug are tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, and solutions.

[0017] In a second aspect, the present invention also provides a method for treating bovine nodular dermatitis in a subject, comprising administering to the subject an effective amount of compound T3, or a tautomer, cis or trans isomer, meso compound, racemic compound, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof, or the pharmaceutical composition thereof.

[0018] Thirdly, the present invention also provides a pharmaceutical preparation for treating bovine nodular dermatitis, comprising the compound T3.

[0019] The dosage form of the pharmaceutical preparation may be tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, or solutions.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0021] This invention provides the application of tylosin derivative T3 in the preparation of antiviral drugs. Its antiviral effect against LSDV is determined by virtual screening and virus titer determination, and the application safety and low toxicity of compound T3 are determined by cytotoxicity test.

[0022] Specifically, this invention utilizes virtual screening technology in computer-aided drug design to screen small molecule compounds with potential inhibitory effects on target proteins based on the resolved protein crystal structures of 156 LSDV proteins using molecular docking technology. It was found that the binding free energy of tylosin derivative T3 to 122 of the 156 LSDV structural proteins is less than -7.0 kcal / mol, demonstrating good binding to T3 and a strong interaction between the ligand and protein, significantly superior to other tylosin derivatives. Based on molecular docking experiments, it was further discovered that tylosin derivative T3 possesses highly efficient antiviral activity, inhibiting viral replication of LSDV (goatpoxvirus), and exhibits almost no toxicity to MDBK cells. These findings provide a new and reliable technical approach for developing safe and reliable antiviral drugs against poxvirus infections.

[0023] Experimental results showed that when compound T3 was used at concentrations of 25-3200 ppm, it could inhibit the replication of LSDV (Legpoxvirus) in a dose-dependent manner, and T3 exhibited low cytotoxicity and CC (Cellular Activated Toxicology). 50 The value is >3200ppm. Therefore, the compound T3 described in this invention can serve as a highly effective macrolide inhibitor against various viral infections, and has broad application prospects in the fields of aquaculture and biomedicine. Attached Figure Description

[0024] Figure 1 The figure shows the titer results of T3 inhibiting LSDV virus in a dose-dependent manner.

[0025] Figure 2 The graph shows the toxicity of different doses of T3 on MDBK cells. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0029] The experimental methods described below are standard operating procedures in molecular biology, cell biology, or virology, which researchers in the field can easily understand and perform.

[0030] Routine tests in the examples:

[0031] 1. Virtual Filtering

[0032] 1) Protein sequence acquisition: 156 protein sequences were obtained from the literature "Genome of Lumpy Skin Disease". The protein ORF number was found and the sequence (Bos) was searched on NCBI using the ORF number.

[0033] 2) AlphaFold3 One-to-One Fine Modeling Analysis: 156 data points were transferred to AlphaFold3 for conformational prediction analysis and validation, pTM values ​​were calculated, and advanced algorithms were used to refine the protein structures, such as removing water molecules. 3D structural models of 156 LSDV proteins were predicted.

[0034] 3) Virtual Screening and Ligand Docking: Virtual screening was performed using molecular docking. Auto Docking 1.2.5 was used for molecular docking analysis to explore potential binding interactions between selected compounds and 156 proteins with key LSDVs. The docking process aimed to predict the binding mode and affinity of compounds within the target protein's active site. The quality of the predictions was used to screen small molecule compounds, with the highest-scoring compounds selected as candidate compounds for further functional validation.

[0035] 2. Cell preparation

[0036] When passaged MDBK cells, DMEM culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin was used, and DMEM culture medium containing 2.5% fetal bovine serum and 1% penicillin and streptomycin was used for the maintenance medium.

[0037] 3. Virus culture

[0038] LSDV virus culture: Culture approximately 10 flasks of T75 MDBK cells, inoculate with viral stock at 0.01 MOI, scrape off cells 72 hours after infection, transfer to 50 mL centrifuge tubes, centrifuge at 2500 rpm for 10 min, and discard the supernatant. Resuspend in 1.5 mL of 10 mM Tris-Cl per T75 flask and place on ice. Lyse the cell suspension by homogenizing 40 times in a glass homogenizer with a tight pestle, transfer to 50 mL tubes, and centrifuge at 300 g, 4 °C for 5 min, saving the supernatant; sonicate the combined supernatant (lysate) on ice for 90 seconds; separate the sonicated lysate into layers on a 36% sucrose pad in a sterile centrifuge tube, with a maximum tube volume of 38.5 mL, and fill the tube to the top 2-3 mm. Make up the volume with 10 mM Tris-Cl (pH 9.0); centrifuge at 33000g for 80 minutes, aspirate and discard the supernatant, resuspend the virus particles in 1 mL of 1 mM Tris-Cl (pH 9.0), and operate on ice. Store at -80°C.

[0039] 4. Evaluation of the antiviral activity of the test compounds

[0040] 1) Trypsin digestion of fused monolayer MDBK cells;

[0041] 2) In a 24-well plate, lay 1×10⁻⁶ molten metal in each well 5 One MDBK cell was incubated overnight at 37°C in a 5% CO2 incubator.

[0042] 3) Infect the above cells with LSDV-GFP virus at MOI=0.01, and add appropriate concentrations (3200ppm, 400ppm, 25ppm) of the test compound. Replace the maintenance medium containing the test compound after 2 hours.

[0043] 4) Collect virus-containing cells after culturing for 72 hours.

[0044] 5. Fluorescent plaque assay for virus titer

[0045] 1) Trypsin digestion of fused monolayer MDBK cells;

[0046] 2) In a 24-well plate, lay 1×10⁻⁶ molten metal in each well 5 One MDBK cell was incubated overnight at 37°C in a 5% CO2 incubator.

[0047] 3) The collected cells were repeatedly frozen and thawed three times in advance to release the virus. Eight 10-fold serial dilutions of the virus were prepared in DMEM medium (2.5% FBS, 1% PS).

[0048] 4) Remove the culture medium from the 24-well plate with 200 μL of 10 -2 10 -3 10 -4 10 -5 10 -6 Infect cells in wells with the virus dilution (three replicates per dilution), incubate in a 5% CO2 incubator for 2 hours, and then discard the virus inoculum.

[0049] 5) After 2 hours, change the medium to 0.75% w / v CMC medium and incubate the 24-well plate in a 37°C, 5% CO2 incubator for 72 hours to allow the virus to fully infect the cells.

[0050] 6) After 72 hours of incubation, observe the fluorescent patches in the wells under a 4x inverted fluorescence microscope. Choose an appropriate dilution for the number of fluorescent patches (between 20 and 100 patches is recommended) to ensure accurate counting.

[0051] 8) PFU / mL = number of fluorescent patches × virus dilution factor ÷ inoculation volume (mL).

[0052] 6. Detection of cytotoxicity (CCK8 assay)

[0053] 1) Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, and divide them into 96-well plates, 180 μL per well, 3000-10000 cells / well.

[0054] 2) Incubate at 37℃ in a 5% CO2 incubator to allow the cells to adhere to the wall and incubate for 24 hours.

[0055] 3) Add appropriate concentrations (3200ppm, 1600ppm, 800ppm, 400ppm, 100ppm, 25ppm, 6.4ppm, 1.6ppm) of the test compound and continue culturing for 72h.

[0056] 4) Carefully aspirate the supernatant, add 90 μL of fresh culture medium, then add 10 μL of CCK8 solution, and continue culturing for 4 h.

[0057] 5) Measure the absorbance of each well at 450 nm using an ELISA reader.

[0058] 6) Simultaneously set up zeroing wells (culture medium, CCK8 solution) and control wells (cells, drug dissolution medium of the same concentration, culture medium, CCK8 solution), with 8 replicates for each group.

[0059] 7) Use Graphpad Prism software to plot the dose-response curve and calculate the median toxicity concentration (CC). 50 value.

[0060] Example 1: Virtual screening using molecular docking

[0061] Molecular docking was performed between tylosin derivatives in CN117510561A and 156 key proteins of LSDV virus. The results showed that the small molecule compound T3 had a binding energy below -7 kcal / mol with 122 key proteins, making it the compound with the tightest binding among all tylosin derivatives. The top five binding energies for molecular docking are shown in the table below.

[0062] Table 1. Top 5 LSDV key proteins with the highest molecular docking binding energies

[0063]

[0064] Example 2: Effect of tylosin derivative T3 on LSDV-GFP virus replication (evaluation of T3 antiviral activity)

[0065] MDBK cells were infected with LSDV-GFP strain at 0.01 MOI. At the same time as viral infection, different doses of tylosin derivative T3 (3200ppm, 400ppm, 25ppm) (prepared with DMSO, the same below) were added. A DMSO control was also set up. After incubation for 2 hours, the supernatant was discarded, the cells were washed twice with PBS, cell maintenance medium was added, and the cells were cultured in a cell culture incubator for 72 hours. The samples were collected and titrated on MDBK cells, and the titer was determined by fluorescent plaque counting method.

[0066] The results are as follows Figure 1 As shown, compared with the DMSO control group, the tylosin derivative T3 inhibited viral titers in a dose-dependent manner.

[0067] Example 3: Detection of cytotoxicity of tylosin derivative T3 against MDBK cells (CCK8 assay)

[0068] Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, and aliquot 180 μL into 96-well plates (3000-10000 cells / well). Incubate at 37°C in a 5% CO2 incubator until cell adhesion is achieved, and culture for 24 h. Add an appropriate concentration of the test compound T3 and continue culturing for 72 h. Carefully aspirate the supernatant, add 90 μL of fresh culture medium, and then add 10 μL of CCK8 solution, and continue culturing for 4 h. Measure the absorbance of each well at 450 nm using a microplate reader. Simultaneously, set up zero wells (culture medium, CCK8 solution) and control wells (cells, drug solution of the same concentration, culture medium, CCK8 solution), with 8 replicates per group. Plot the dose-response curve using Graphpad Prism software and calculate the half-maximal toxicity concentration (MCC). 50 value.

[0069] The results are as follows Figure 2 As shown, even at the highest tested concentration of 3200 ppm, cell viability remained close to 100%, comparable to the DMSO control group. No significant decrease in cell viability was observed at concentrations ranging from 1.6 ppm to 3200 ppm, indicating that the compound did not exhibit significant toxicity within this concentration range. Therefore, it can be inferred that CC... 50 Value > 3200ppm.

[0070] Furthermore, since the conserved functional gene portions of the orthopoxvirus and goatpoxvirus genera are basically the same and have the same efficacy, it can be inferred that compound T3 can also be used to treat diseases caused by orthopoxvirus.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of compound T3 or its pharmaceutical salt in the preparation of antiviral drugs for poxviruses; The structural formula of compound T3 is as follows: ； The poxvirus in question is a bovine nodular dermatovirus belonging to the genus Goatpoxvirus.

2. The application according to claim 1, characterized in that, The viral structural proteins include LSDV028, LSDV050, LSDV047, LSDV033, and LSDV086.

3. The application according to claim 1, characterized in that, The concentration of compound T3 in the drug is 400-3200 ppm.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients or additives; the excipients are selected from one or more of binders, adsorbents, fillers and disintegrants; the additives are selected from one or more of stabilizers, bactericides, buffers, isotonic agents, chelating agents and surfactants.

5. The application according to claim 1, characterized in that, The dosage forms of the drug are tablets, capsules, oral liquids, lozenges, granules, pills, ointments, elixirs, suspensions, and powders.

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