CN116999444A - Application of tetrahydroprogesterone in preparation of anti-influenza virus drugs - Google Patents
Application of tetrahydroprogesterone in preparation of anti-influenza virus drugs Download PDFInfo
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- AURFZBICLPNKBZ-JMTGGFPBSA-N 1-[(3r,8r,9s,10s,13s,14s,17s)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]ethanone Chemical compound C1CC2C[C@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 AURFZBICLPNKBZ-JMTGGFPBSA-N 0.000 title claims abstract description 61
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pulmonology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to application of tetrahydroprogesterone in preparation of anti-influenza virus drugs. The invention creatively discovers that the tetrahydroprogesterone has obvious inhibitory activity on influenza virus infection, and can prevent influenza virus replication by targeting NP through multiple action modes, thereby effectively reducing the drug resistance of the virus. The invention relocates the medicine function of the tetrahydroprogesterone, further expands the indication, reduces the time and cost spent by pharmacokinetics research and toxicity research, greatly reduces the research and development cost, can be used in the influenza treatment scheme more quickly than other anti-influenza medicines, and provides potential treatment thought and reference basis for clinic.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a novel medicinal application of tetrahydroprogesterone, and in particular relates to an application of tetrahydroprogesterone in preparation of anti-influenza virus medicines.
Background
Influenza viruses are an important class of pathogens that cause acute respiratory infections in humans, resulting in seasonal influenza and large outbreaks of influenza. Currently, antiviral strategies are mainly two aspects of vaccination and antiviral drug therapy. Although vaccination is the primary prophylactic method for preventing influenza infection, vaccine development cycle is long, cost is high, and protection scope and timeliness are limited by high mutation characteristics of viruses, so influenza virus inhibitors are key means for treating influenza.
There are currently three classes of anti-influenza drugs worldwide, mainly including M2 ion channel blockers (amantadine and rimantadine), neuraminidase (NA) inhibitors (oseltamivir, zanamivir, and peramivir), and RNA-dependent RNA polymerase (RdRp) inhibitors (fapila Wei Heba lo Sha Wei). However, almost all influenza strains that are prevalent exhibit resistance to M2 inhibitors. In addition, the increase in NA and RdRp inhibitor resistance also limits the efficacy of the drug, which suggests that the drugs used for influenza infection prevention and treatment are very limited. Thus, there is an urgent need to develop novel influenza virus inhibitors against influenza virus conserved sequences.
Viral Nucleoprotein (NP) is a key protein for many phases of the influenza virus lifecycle. The NP protein may bind to viral RNA to form a viral ribonucleoprotein complex (vRNP). Translocation of NP proteins between the cytoplasm and nucleus plays an important role in viral replication and transcription, NP proteins have a variety of biological functions involved in nuclear import and nuclear export of vRNP and assembly of viral particles. Thus the NP protein, which is more conserved in sequence and has multiple functions, means that it is considered a valuable target for anti-influenza drugs.
Currently available NP inhibitors are mainly inhibiting the relatively single biological function of NP, e.g. Kao, r.y., et al reported that nucleoside analogs nucleozin inhibit their nuclear import function by inducing aberrant aggregation of NP, thereby interfering with viral replication (Kao, r.y.; yang, d.; lau, l.s.; tsui, w.h.; hu, l.; dai, j.; chan, m.p.; chan, c.m.; wang, p.; zheng, b.j.; et al identification of influenza Anucleoprotein as an antiviral target. Nature Biotechnol 2010,28,600-605); the NP protein inhibitor naproxen targets the Y148 residue of the NP protein, antagonizes NP nuclear export mediated by the host export protein CRM1, exerting anti-influenza a virus activity (Zheng, w.n.; fan, w.h.; zhang, s.; jiao, p.t.; shang, y.l.; cui, l.; mahesurushan, m.; li, j.; wang, d.y.; gao, g.f.; et al naproxen Exhibits Broad Anti-influenza Virus Activity in Mice by Impeding Viral Nucleoprotein Nuclear export cell Rep 2019,27). These inhibitors are not effective in reducing viral resistance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel medicinal application of the tetrahydroprogesterone, in particular to an application of the tetrahydroprogesterone in preparing anti-influenza virus medicaments.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides the use of tetrahydroprogesterone for the manufacture of a medicament against influenza virus.
The tetrahydroprogesterone disclosed by the invention is a medicament for treating postpartum depression, which is approved by the FDA and marketed, and is a natural product, and the safety and clinical applicability of the tetrahydroprogesterone are easier to reuse for treating other indications. The chemical structural formula of the compound is shown as follows,
the invention creatively discovers that the tetrahydroprogesterone has obvious inhibitory activity on influenza virus infection, and targets NPs through various action modes to prevent influenza virus replication, and firstly delays the NPs from entering the nucleus in a vRNP form; secondly, preventing NPs from outputting in the form of newly synthesized vRNPs; finally, the higher-order oligomerization of NP proteins is induced, interfering with the formation of new cRNPs and vRNPs, so that it is effective in reducing viral resistance.
The invention relocates the medicine function of the tetrahydroprogesterone, further expands the indication, reduces the time and cost spent by the pharmacokinetics research and the toxicity research, greatly shortens the medicine research and development period, reduces the research and development cost, can be used in the influenza treatment scheme faster than other anti-influenza medicines, and provides potential treatment thought and reference basis for clinic.
Preferably, the influenza virus comprises influenza a virus and/or influenza b virus.
In the above application, the drug delays nuclear import and nuclear export of influenza virus ribonucleoprotein complexes.
In the above application, the drug induces higher order oligomerization of influenza virus nucleoprotein.
In a second aspect, the invention provides the use of tetrahydroprogesterone for the preparation of an influenza virus nucleoprotein targeting formulation.
According to the research result of the invention, the tetrahydroprogesterone has the effect of targeting influenza virus nucleoprotein, so the result shows that the tetrahydroprogesterone can be used as a preparation for scientific research of ex vivo level, such as theoretical research of influenza virus life cycle and the like.
In a third aspect, the invention provides the use of tetrahydroprogesterone for the preparation of an influenza a virus inhibitor or an influenza b virus inhibitor.
According to the research result of the invention, the tetrahydroprogesterone has the effect of inhibiting the replication of influenza A virus or influenza B virus, so the result shows that the tetrahydroprogesterone can be used as a preparation for scientific research of ex vivo level, such as theoretical research of life cycle of influenza A virus or influenza B virus, and the like.
Preferably, the medicament also contains pharmaceutically acceptable auxiliary materials.
Preferably, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifying agents, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
Preferably, the dosage form of the medicament is any pharmaceutically acceptable dosage form, such as spray, inhalant, tablet, powder, suspension, granule, capsule, solution and the like. The medicaments of the various formulations can be prepared according to the conventional method in the pharmaceutical field.
Preferably, the medicament also contains other anti-influenza virus medicaments.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively discovers that the tetrahydroprogesterone has obvious inhibitory activity on influenza virus infection, and targets NPs through various action modes to prevent influenza virus replication, and firstly delays the NPs from entering the nucleus in a vRNP form; secondly, preventing NPs from outputting in the form of newly synthesized vRNPs; finally, the higher-order oligomerization of NP proteins is induced, interfering with the formation of new cRNPs and vRNPs, so that it is effective in reducing viral resistance. The invention relocates the medicine function of the tetrahydroprogesterone, further expands the indication, reduces the time and cost spent by the pharmacokinetics research and the toxicity research, greatly shortens the medicine research and development period, reduces the research and development cost, can be used in the influenza treatment scheme faster than other anti-influenza medicines, and provides potential treatment thought and reference basis for clinic.
Drawings
FIG. 1 is a graph of the results of a quantitative analysis test of the anti-influenza virus of tetrahydroprogesterone;
FIG. 2 is a statistical graph of the results of the inhibitory effect of tetrahydroprogesterone on different strains;
fig. 3 is a graph of the results of experimental analysis of the addition of tetrahydroprogesterone;
FIG. 4 is a graph of RdRp analysis results of tetrahydroprogesterone;
FIG. 5 is a graph of subcellular localization of Nucleoprotein (NP) during influenza infection by tetrahydroprogesterone;
fig. 6 is a graph of thermal drift analysis results of tetrahydroprogesterone;
FIG. 7 is a graph showing the results of molecular docking analysis of the binding pattern of tetrahydroprogesterone to NPs.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited. The experimental methods in each example, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions recommended by the manufacturer.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control.
Recombinant influenza A reporter viruses PR8-PB2-Gluc, influenza strain A/Puerto Rico/8/1934 (A/PR 8,79H1N 1) according to the examples below were maintained by the laboratory; A/Brisbane/10/2007 (A/Brisbane, H3N 2) is provided by the national academy of medical science; influenza virus strains of type B-Yamagata and type B-Victoria are supplied by the disease prevention control center of Shandong province (Jinan, china); A/PR8/Mount Sinai recombinant NP protein was purchased from Sino Biological Co., under the model 11675-V08B-B.
The drug of the following examples was tetrahydroprogesterone, which was obtained from MCE company, USA and was designated as model 516-54-1, and the concentrations of the following tetrahydroprogesterone were calculated as the actual concentrations of the tetrahydroprogesterone contained in the drug.
Example 1
Viral inhibition and cytotoxicity evaluation of tetrahydroprogesterone:
evaluation of inhibition ratio of virus: white flat bottom 96 well plates are spread 10 per well 5 Individual canine kidney epithelial cells (MDCK cells), 37 ℃, 5% CO 2 After incubation in The incubator for 24h, PR8-PB2-Gluc virus was infected at 0.01MOI, incubated at 37℃for 2h, and each well was added with OPti-MEM medium (containing 1.5. Mu.g/mL TPCK-trypsin, sigma-Aldrich, st.Louis, MO, USA) starting at 100. Mu.M, 3-fold diluted tetrahydroprogesterone, incubated at 37℃for 36h, and then incubated with Pierce Gaussia luciferase luminescence assay kit (Thermo Scientific, usa) measured Gaussia luciferase activity and calculated the inhibition of the virus by the compound.
Cytotoxicity evaluation: in a transparent flat bottom 96-well plate, 10 wells are plated in each 5 Canine kidney epithelial cells (MDCK cells). The absorbance was measured at 450nM after incubation for 36h at 37℃with addition of 10. Mu. L Cell Counting Kit-8 per well starting at 100. Mu.M with Opti-MEM medium (containing 1.5. Mu.g/mL TPCK-trypsin, sigma-Aldrich, st.Louis, MO, USA) and 3-fold dilution of tetrahydroprogesterone.
As shown in FIG. 1, it is evident from the graph that tetrahydroprogesterone inhibits replication of PR8-PB2-Gluc virus in a dose-dependent manner, and IC thereof 50 ＝6.27μM，CC 50 >100μM，SI>15.9。
Example 2
Virus titer reduction assay:
canine kidney epithelial cells (MDCK cells) were inoculated in 24 well plates and infected with a/PR8, a/Brisbane, B/Yamagata and B/Victoria strains, respectively, at an MOI of 0.01. After incubation at 37℃for 2h, different concentrations of tetrahydroprogesterone diluted with Opti-MEM medium (containing 1.5. Mu.g/mL TPCK-trypsin, sigma-Aldrich, st.Louis, MO, USA) were added to each well, the plates were incubated at 37℃for 36h, the culture supernatants were collected, and the virus titer in the culture supernatants (TCID 50 /mL)。
As shown in FIG. 2, it is evident from the graph that tetrahydroprogesterone exhibits a dose-dependent inhibition of influenza A virus A/PR8 and A/Brisbane, has a lower sensitivity to B/Yamagata and B/Victoria viruses, and a higher concentration of tetrahydroprogesterone inhibits replication of influenza B virus.
Example 3
Time-adding test:
lay-in 10 per well in 24 well plate 5 Individual canine kidney epithelial cells (MDCK cells) were adsorbed at 0.1MOI a/PR8 virus at 4 ℃ for 1h and then transferred to 37 ℃ for 1h. After 1h, unbound viral particles were washed off with PBS and incubated at 37 ℃. Respectively within a specified time period (-2-12 h, -2-0 h, 0-12 h, 2-12 h, 4-12 h, 6-12 h and 8-12 h), wherein the time for adding virus is-2 hPoint, PBS washes unbound virus for 0h time point) cells were treated with 20 μm tetrahydroprogesterone and DMSO. After incubation for 12h at 37 ℃, the supernatants were collected and assayed for viral titer.
The results are shown in FIG. 3, which shows that the intervention of tetrahydroprogesterone completely inhibited viral replication at 0h up to 4h and that the inhibition was reduced at 6h and subsequent time points. These results indicate that tetrahydroprogesterone acts on the later stages of the life cycle of influenza virus.
Example 4
Microreplication assay:
lay-in of each well in a 6-well plate 10 8 Human embryonic kidney cell lines (293T cells), rdRp plasmids pCAG-VN04-NP, pCAG-VN04-PA, pCAG-VN04-PB1, pCAG-VN04-PB2, and internal reference plasmids pPolI-NS-Fluc and pRL-TK were transfected with Lipofectamine 2000. 5h after transfection, cells were transfected at 10 5 The density of individual cells/wells was seeded into white flat bottom 96-well plates and DMSO or different concentrations of tetrahydroprogesterone were added. After 24h, the effect of Allopregnanolone on viral genome replication was assessed by polar chemiluminescence detection using a multifunctional microplate reader according to the Dual-luciferase reporter assay system kit instructions.
As shown in FIG. 4, it was shown that tetrahydroprogesterone significantly inhibited viral RdRp activity in a dose-dependent manner, further indicating that tetrahydroprogesterone may target viral ribonucleoprotein (vRNP).
Example 5
Immunofluorescence assay:
canine kidney epithelial cells (MDCK cells) were seeded in 24-well plates, 10 per well 5 And (3) cells. a/PR8 virus infected cells at moi=1 while 40 μm tetrahydroprogesterone was added, adsorbed for 1h at 4 ℃ and invaded for 1h at 37 ℃. This was counted as the 0h time point, and then washed 3 times with PBS, and replaced with fresh medium containing 40. Mu.M tetrahydroprogesterone, and at 8h, the medium was discarded, washed 1 time with PBS, and each well was fixed at 20℃for 30min with 500. Mu.L of 4% formaldehyde. And permeabilized with 0.5% Triton-X100 for 15min, followed by blocking with 5% BSA for 30min. Subsequently, the cells were incubated for 1h with a primary antibody to NP protein of 1% BSA (dilution 1:500), followed by incubation with a secondary antibody to 1% BSA (dilution 1:500)And 1h. Finally, the cells were washed and counterstained with 4', 6-diamidino-2-phenylindole Dihydrochloride (DAPI) for nuclear localization and image analysis by confocal microscopy.
As shown in FIG. 5, it is clear from the graph that the intervention of-2-0 h of tetrahydroprogesterone does not affect the virus entry. The nuclear import of 2-4h vRNP is delayed under the intervention of tetrahydroprogesterone, and NP is mainly retained in the nucleus 8h after infection, and the result shows that NP protein can be delayed to enter the nucleus under the intervention of tetrahydroprogesterone, but then import into cytoplasm in the form of vRNP is significantly inhibited, and immunofluorescence analysis finds that NP protein is dispersed around the nucleus in the form of oligomer, which affects abnormal oligomerization of NP.
Example 6
Thermal drift analysis:
the solution system is 30 mu L, and comprises 10 mu L of 10 mu MA/PR8/Mount Sinai recombinant NP protein solution; 5 XSYPRO Orange dye 10. Mu.L and 10. Mu.L tetrahydroprogesterone (1.6 mM) or 10. Mu.L DMSO were mixed and the mixture was tested using CFX Real-Time PCR detection system. The temperature was raised from 25℃to 90℃at a rate of 1℃per minute and the change in fluorescence per minute was monitored.
The results are shown in FIG. 6, which shows that the Tm of NP protein shifted to the left of-4.54.+ -. 0.13 ℃ under the intervention of tetrahydroprogesterone, indicating that tetrahydroprogesterone is able to bind to NP protein and destabilize the protein.
Example 7
Molecular docking assay of tetrahydroprogesterone with NP protein:
AutoDock was used for molecular docking simulation of the tetrahydroprogesterone-NP protein interactions and predicts binding affinity and site of NPs. The crystal structure of A/H1N1/PR8 NP was downloaded from the RCSB protein database (PDB ID:2 IQH). The three-dimensional structure of tetrahydroprogesterone is generated by Chem 3D. The NP protein structure is pre-treated, i.e., water molecules are removed and hydrogen atoms are added prior to docking simulation. The interaction between tetrahydroprogesterone and NP was visualized using molecular visualization tools PyMOL.
As shown in FIG. 7, it is clear that tetrahydroprogesterone is able to bind to the pocket near residue L266 of the NP protein, residue L266 representing the C-terminus of nuclear export signal 3 (NES 3, amino acids 256-266), while residue L418 of the adjacent NP monomer plays an important role in tetrahydroprogesterone-NP protein binding by forming hydrogen bonds.
The applicant states that the use of the inventive tetrahydroprogesterone in the manufacture of anti-influenza virus drugs is illustrated by the above examples, but the invention is not limited to, i.e. it is not meant that the invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. The application of tetrahydroprogesterone in preparing anti-influenza virus medicines.
2. The use according to claim 1, wherein the influenza virus comprises influenza a virus and/or influenza b virus.
3. The use of claim 1, wherein the medicament delays nuclear import and nuclear export of influenza virus ribonucleoprotein complexes.
4. The use according to claim 1, wherein the medicament induces a higher order oligomerization of influenza virus nucleoprotein.
5. Application of tetrahydroprogesterone in preparing influenza virus nucleoprotein targeting preparation.
6. The application of tetrahydroprogesterone in preparing influenza A virus inhibitor or influenza B virus inhibitor.
7. The use according to any one of claims 1 to 4, wherein the medicament further comprises pharmaceutically acceptable excipients.
8. The use according to claim 7, wherein the pharmaceutically acceptable excipients comprise any one or a combination of at least two of carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilisers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.
9. The use according to any one of claims 1 to 4, wherein the pharmaceutical dosage form is any one of pharmaceutically acceptable dosage forms.
10. The use according to any one of claims 1 to 4, wherein the medicament further comprises an additional anti-influenza virus medicament.
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