阅读说明：本技术 雷公藤红素在制备抗冠状病毒产品中的应用 (Application of tripterine in preparation of anti-coronavirus products ) 是由 高伟 赵文华 屠李婵 于 2020-11-09 设计创作，主要内容包括：本发明公开了雷公藤红素在制备抗冠状病毒产品中的应用,属于医药领域的应用。采用分子对接技术证实雷公藤红素对新型冠状病毒COVID-19的S蛋白与ACE2蛋白的抑制作用,并经亲和力实验测定其与S蛋白具有较强的亲和力,雷公藤红素在5μM到40μM范围内在细胞实验中对冠状病毒具有明显抑制作用,具有浓度依赖性。雷公藤红素是冠状病毒的特异性抑制剂具有特定的新颖性。雷公藤红素可用于制备抑制冠状病毒的产品具有广泛的实用性。(The invention discloses an application of tripterine in preparing an anti-coronavirus product, belonging to the application in the field of medicines. The molecular docking technology is adopted to confirm the inhibition effect of the tripterine on the S protein and ACE2 protein of the novel coronavirus COVID-19, and the affinity experiment determines that the tripterine has stronger affinity with the S protein, and the tripterine has obvious inhibition effect on the coronavirus in the cell experiment within the range of 5 mu M to 40 mu M and has concentration dependence. Tripterine is a specific inhibitor of coronavirus and has particular novelty. The tripterine can be used for preparing products for inhibiting coronavirus, and has wide practicability.)

1. The application of the tripterine in preparing an anti-coronavirus product is characterized in that the tripterine is a compound with the following structure:

。

2. a medicament comprising one or more of the celastrol or pharmaceutically acceptable salts thereof of claim 1 in combination with a pharmaceutically acceptable carrier.

3. Use according to claim 2 for the prevention and/or treatment of infections caused by coronaviruses.

4. Use according to claim 2 for the prevention and/or treatment of infections caused by novel coronaviruses.

5. The use of claims 3 and 4, wherein the subject is a human, cat, dog, pig or chicken.

6. The use of claim 3, wherein the medicament is administered orally: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, effervescent tablets, sublingual tablets, capsules, hard capsules, soft capsules, microcapsules, microspheres, granules, pills, dripping pills, powders, oral liquids, suspensions, solutions and sustained or controlled release preparations.

7. The use of claim 4, wherein the medicament is administered orally: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, effervescent tablets, sublingual tablets, capsules, hard capsules, soft capsules, microcapsules, microspheres, granules, pills, dripping pills, powders, oral liquids, suspensions, solutions and sustained or controlled release preparations.

8. The use of claim 3, wherein the medicament is administered by inhalation: aerosols and sustained or controlled release formulations.

9. The use of claim 4, wherein the medicament is administered by inhalation: aerosols and sustained or controlled release formulations.

10. The use of claim 3, wherein the medicament is administered intravenously: injections, injection emulsions, freeze-dried powder injections and sustained-release or controlled-release preparations.

11. The use of claim 4, wherein the medicament is administered intravenously: injections, injection emulsions, freeze-dried powder injections and sustained-release or controlled-release preparations.

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of tripterine in preparation of an anti-coronavirus product.

Background

Coronaviruses belong to the genus coronaviruses of the family coronaviridae, and infection with atypical pneumonia by coronaviruses mainly occurs in winter and early spring, and are known by the shape similar to coronaries formed by spinous processes on the viral envelopes. The most representative of the great harm of the novel coronavirus COVID-19 which is currently popular is the emergent public health event of international attention, and no specific treatment medicine for the novel coronavirus exists at present.

As the research goes forward, scientists have analyzed the main protein S protein of the novel coronavirus COVID-19, ACE2 and RNA-dependent RNA polymerase by X-ray (Science, 2020, 368: 1274-. The novel coronavirus S protein, ACE2, RNA-dependent RNA polymerase and the like are taken as targets to carry out molecular docking to discover an inhibitor, a compound formed by blocking the S protein and ACE2 is further formed to inhibit the invasion of COVID-19 to a human body, affinity parameters are determined by adopting dynamics, and the virus inhibition capacity is detected by adopting a cell experiment, so that the novel coronavirus COVID-19 inhibitor is an important way for discovering the novel coronavirus COVID-19 inhibitor at present.

The novel coronavirus S protein, ACE2, RNA-dependent RNA polymerase and the like are taken as target proteins to carry out molecular docking, affinity parameters are determined by dynamics, the virus inhibition capacity is detected by a cell experiment, and the inhibitor of the coronavirus is found from natural plants. The inventor finds that the compound tripterine has obvious coronavirus inhibiting activity.

Tripterine belongs to triterpenoid, is called Celastrol in English, is mainly present in Tripterygium wilfordii hook F.H.Chen of Celastraceae, and has anti-inflammatory (European Journal of Pharmacology, 2020, 885: 173371; International Immunopharmacology, 2020, 84: 106527), antioxidant (Chemico-Biological Interactions, 2016, 246: 52), antitumor (Archives of Medical Research, 2020, 51: 297; EBiomedicine, 2019, 50: 81) and other effects.

At present, no research report of the tripterine antiviral effect is found.

Disclosure of Invention

The invention takes molecular docking, affinity determination and cell virus inhibition experiments as means for finding the coronavirus inhibitor. The novel coronavirus S protein, ACE2 and RNA-dependent RNA polymerase are adopted as target proteins for molecular docking, affinity parameters are determined by dynamics, virus inhibition capacity is detected by cell experiments, and tripterine is found to have a remarkable inhibition effect on coronaviruses from natural plants.

The invention aims to provide application of tripterine in preparation of coronavirus products.

The tripterine is a compound with a structural formula shown in figure 1.

The tripterine has strong affinity with S protein, ACE2 and novel coronavirus RNA-dependent RNA polymerase, and the novel coronavirus S protein is applied to preparation of a preparation for inhibiting coronavirus through inhibiting the combination of the coronavirus S protein and ACE 2.

The invention adopts molecular docking, kinetic determination and cell antiviral experiments to evaluate the inhibition effect of the tripterine on the coronavirus, and the tripterine can inhibit a novel coronavirus key protein. Cell experiments are adopted to evaluate the antiviral effect of the tripterine, and the tripterine has obvious inhibition effect on coronavirus.

The application of the tripterine in preparing antiviral products has the advantages of definite action mechanism and obvious inhibition effect. Tripterine can be separated from radix Tripterygii Wilfordii.

The invention provides tripterine and a pharmaceutical preparation thereof, which are prepared by the following steps: aerosol, tablet, sugar-coated tablet, injection emulsion, freeze-dried powder injection, film-coated tablet, enteric-coated tablet, effervescent tablet, sublingual tablet, capsule, hard capsule, soft capsule, microcapsule, microsphere, granule, pill, dripping pill, powder, paste, oral liquid, suspension, solution and sustained-release or controlled-release preparation.

Drawings

FIG. 1: the structural formula of the tripterine of the invention

FIG. 2: molecular docking scheme for example 1 of the invention

FIG. 3: molecular docking scheme for example 2 of the invention

FIG. 4: molecular docking scheme for example 3 of the invention

FIG. 5: affinity diagram of example 4 of the invention

FIG. 6: affinity diagram of example 5 of the invention

FIG. 7: the affinity diagram of example 6 of the present invention.

Detailed Description

Example 1 molecular docking with novel coronavirus S protein as target protein

The PDB database downloads a novel coronavirus Spike protein into a PDB format, hydrogen atoms and charges are added to a target protein S protein tertiary structure model, and lost amino acid residues are repaired. The AMBER FF99 force field in the SYBYL X-1.2 package was selected for energy optimization. First, choose AMBER FF99 force field to optimize 1000 times by the steepest gradient method (SD). Then, a conjugate gradient method (CG for short) is adopted for optimization until the convergence gradient is 0.05 kcal/(A mol). The method is characterized in that a tripterine 2D structure is drawn through Chem3D, a tripterine 3D structure is generated by using Chem3D software, energy minimization is carried out, and all small molecule ligands are stored in a mol2 format. Checking a ligand molecular structure, converting a molecular plane structure into a 3D structure, adding polar hydrogen to the molecular structure, adding charges to atoms by adopting a Gasteiger-Huckel method, and performing 1000 times of iterative energy optimization on small molecules by adopting a Tripos force field. The interaction force of the ligand molecules with the protein was calculated as Total score using the Surflex-Dock module in the SYBYL-X1.2 software package. The docking results show that the interaction force of the tripterine and the novel coronavirus Spike protein is classified as 3.112 (figure 2), and the tripterine and the Spike protein are obviously combined.

Example 2 molecular docking with ACE2 as target protein

The PDB database downloads an ACE2 structure into a PDB format, hydrogen atoms and charges are added to a target protein ACE2 tertiary structure model, and lost amino acid residues are repaired. The AMBER FF99 force field in the SYBYL X-1.2 package was selected for energy optimization. After iterative optimization, a conjugate gradient method is adopted to optimize until the convergence gradient is 0.05 kcal/(A mol). And drawing a tripterine structure, performing energy minimization, and storing all the used small molecular ligands in a mol2 format. Then hydrogenation, charging and energy optimization are carried out. The interaction force of the ligand molecule with the protein was calculated as the Total score. Docking results showed that tripterine had a Total score of 5.249 with ACE2 (fig. 3), and that tripterine had significant binding to ACE 2.

Example 3 molecular docking with novel coronavirus RNA-dependent RNA polymerase as target protein

The PDB database downloads a novel coronavirus RNA-dependent RNA polymerase structure into a PDB format, and hydrogen atoms and charges are added to a target protein RNA-dependent RNA polymerase tertiary structure model to repair lost amino acid residues. After energy optimization, iterative optimization was performed, and then CG was used to optimize the convergence gradient to 0.05 kcal/(a mol). And drawing a tripterine structure, performing energy minimization, and storing all the used small molecular ligands in a mol2 format. Checking the molecular structure of the ligand, adding hydrogen to the molecular structure, adding charge and optimizing energy. The interaction force of the ligand molecule with the protein was calculated as the Total score. Docking results showed that the Total score of tripterine and the novel coronavirus RNA-dependent RNA polymerase was 3.663 (FIG. 4), and the tripterine and the RNA-dependent RNA polymerase had significant binding.

Example 4 determination of Tripterine affinity parameters for the RBD Domain of the novel coronavirus S protein Using kinetics

The test chip was CM5 and the running buffer was HEPES-EP +. Firstly, fixing the RBD structure domain of the S protein of the recombined novel coronavirus on a CM5 chip, and after the protein is well fixed on the surface of the chip, detecting the affinity of the tripterine and the RBD structure domain of the S protein of the novel coronavirus by adopting multi-cycle kinetics. Before running the formal samples, the system was allowed to simulate running samples at the start phase by running 3 Startup cycles with running buffer instead of analyte samples to achieve stable baseline and system conditions. The analyte concentration gradient was 625, 1250, 2500, 5000, 10000. mu.M, the flow rate was 30. mu.L/min, the binding time was 180 s, and the dissociation was 320 s. 0.75 mM NaOH was regenerated for 30 s at a flow rate of 20. mu.L/min and stabilized for 60 s after regeneration. The experimental result shows that the tripterine has stronger affinity with the S protein (figure 5), and the KD is 84 mu M.

Example 5 kinetic determination of the affinity parameters of Tripterine to ACE2 protein

The test chip was CM5 and the running buffer was HEPES-EP +. Firstly, the recombinant coronavirus ACE2 is fixed on a CM5 chip, and after the protein is well fixed on the surface of the chip, the affinity of the tripterine and the coronavirus ACE2 protein is detected by adopting multi-cycle kinetics. Before running the formal samples, the system was allowed to simulate running samples at the start phase by running 3 Startup cycles with running buffer instead of analyte samples to achieve stable baseline and system conditions. The analyte concentration gradient was 625, 1250, 2500, 5000, 10000. mu.M, the flow rate was 30. mu.L/min, the binding time was 180 s, and the dissociation was 320 s. 0.75 mM NaOH was regenerated for 30 s at a flow rate of 20. mu.L/min and stabilized for 60 s after regeneration. The experimental result shows that the tripterine has stronger affinity with ACE2 protein (shown in figure 6), and KD is 4.87 mu M.

Example 6 kinetic determination of Tripterine affinity parameters for the novel coronavirus S protein RBD Domain-ACE 2 Complex

The test chip was CM5 and the running buffer was HEPES-EP +. Firstly, an RBD structure domain of a recombinant coronavirus S protein is fixed on a CM5 chip, and after the protein is well fixed on the surface of the chip, the affinity of the protein with a coronavirus ACE2 protein is detected by adopting multi-cycle kinetics when celastrol exists or is deleted. Before running the formal samples, the system was allowed to simulate running samples at the start phase by running 3 Startup cycles with running buffer instead of analyte samples to achieve stable baseline and system conditions. The analyte concentration gradient was 625, 1250, 2500, 5000, 10000. mu.M, the flow rate was 30. mu.L/min, the binding time was 180 s, and the dissociation was 320 s. 0.75 mM NaOH was regenerated for 30 s at a flow rate of 20. mu.L/min and stabilized for 60 s after regeneration. The experimental result shows that the tripterine and the novel coronavirus S protein RBD structure domain-ACE 2 compound have strong affinity (figure 7), and KD is 3.67 nM.

Example 7 detection of Tripterine Virus inhibitory Capacity Using cell assay

50 microliter of 100 TCID50 virus solution was inoculated to each well of 96-well Vero9 cells for 24 hours, adsorbed at 37 ℃ for 90 min, and the virus supernatant was discarded. According to the result of cytotoxicity experiment, in the non-toxic concentration range, adding medicines with different concentrations, and setting normal cell control group and virus control group. The plates were then incubated at 37 ℃ with 5% CO₂Incubate and observe CPE daily. CPE recording method is [1]No CPE; + is CPE in 25% of cells; + is 50% of cells presenting CPE; + + + + 75% of cells present CPE; + + + + + + is 100% of cells presenting CPE. Each well 570 was measured using MTT at approximately the viral controls +++ -. +++OD value under nm wave and calculating the virus inhibition rate of the medicine according to the following formula. The virus inhibition rate = (drug-treated OD value-virus control OD value)/(cell control OD value-virus control OD value) × 100%. The experimental result shows that the tripterine has obvious inhibiting effect on the coronavirus in the range of 5 mu M to 40 mu M.