CN118078827A - Medicine for preventing and treating novel coronavirus and application thereof - Google Patents

Abstract

The invention provides a medicine for preventing and treating novel coronavirus and application thereof, belonging to the technical field of application of traditional Chinese medicines. The compound of the invention can target host factors, is used as an excitant of the host factors, inhibits the replication of novel coronaviruses, has broad-spectrum antiviral activity, and plays an important role in preventing and treating the novel coronaviruses. Compared with the existing novel coronavirus therapeutic drugs, the compound has different antiviral action mechanisms, is a beneficial supplement of the existing antiviral drugs, and provides a new choice for the antiviral therapeutic drugs.

Description

Medicine for preventing and treating novel coronavirus and application thereof

Technical Field

The invention belongs to the technical field of application of traditional Chinese medicines, and in particular relates to a medicine for preventing and treating novel coronaviruses and application thereof.

Background

With the subsequent research and development and application of vaccines and small molecular drugs, the currently reported small molecular drugs for treating the new corona, such as Ruidexivir, VV116, nemactetvir/ritonavir and the like, are designed aiming at viruses, but due to the characteristic of continuous mutation of RNA viruses, the novel coronaviruses are continuously mutated in a short period of three years, from initial wild strains to delta strains, to Omikovin, to recent XBB and EG5, and the effect of the vaccines and the small molecular drugs is more and more limited along with the variation of the viruses, so that the development of broad-spectrum antiviral drugs is currently urgently needed.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a safe, effective and broad-spectrum medicament, and provides application of a compound with the following structure in preparing medicaments for preventing and treating novel coronaviruses:

。

The invention mainly aims at host factors, influences the host factors through the compound (YS-49) with the structure shown above, further inhibits the proliferation of novel coronaviruses, has better broad spectrum and can effectively avoid virus mutation.

Preferably, the novel coronavirus is selected from the novel coronavirus wild strain (WT) or the novel coronavirus ba.5 strain.

Preferably, the medicine for preventing and treating the novel coronavirus comprises a compound or a pharmaceutically acceptable salt of the compound used in the application and a pharmaceutically acceptable auxiliary agent.

Preferably, the pharmaceutically acceptable auxiliary agent comprises one or more of pharmaceutically acceptable carrier, excipient or diluent.

Preferably, the dosage form of the medicine comprises at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral liquids, injections or powders.

Preferably, the medicament further comprises at least one additional anti-novel coronavirus active ingredient.

Preferably, the concentration of the compound YS-49 in the novel coronavirus prevention and treatment drug is more than or equal to 0.01wt%.

The molecular formula of the compound YS-49 is C ₂₀H₁₉NO₂, and the molecular weight is 305.36.

Preferably, the mode of administration of the drug includes at least one of oral, intratumoral, rectal, parenteral injection and topical administration.

The invention breaks away from the conventional micromolecular medicaments designed aiming at viruses, and aims at host factors, thereby achieving the antiviral effect. According to the invention, the host factor actin A4 (ACTN 4) recombinant protein is screened out by using three generations of sequencing, and the ACTN4 is found to have good antiviral effect at the cellular level, and further mechanism research shows that (as shown in figure 1), the ACTN4 is screened out by inhibiting nsp7 and/or nsp8 from combining with nsp12, so that nsp12 combined to a novel coronavirus genome is reduced, further replication of the novel coronavirus is inhibited, and YS-49 is screened out as an agonist of ACTN4 aiming at the ACTN 4. In the process of novel coronavirus replication, nsp7, nsp8 and nsp12 are all minimal replicons, nsp7 and nsp8 bind first, then nsp12 is recruited to form a complex, and then bind to the novel coronavirus RNA to initiate viral replication. However, the novel coronavirus causes reduction of methyltransferase WTAP after infecting cells, resulting in reduced m6A modification on ACTN4 mRNA, which in turn affects RNA degradation and translation, thereby inhibiting ACTN4 expression, so YS-49 acts as an agonist of ACTN4, up-regulates ACTN4 expression, ACTN4 binds to the N-terminus of nsp12, ACTN4 binds to more nsp12 after increasing, and causes ACTN4 to competitively bind to nsp12 with nsp7 and/or nsp8, resulting in reduced nsp12 binding to novel coronarnas, thereby inhibiting viral replication.

Compared with the prior art, the invention has the beneficial effects that:

(1) Aiming at the characteristic that RNA viruses are easy to mutate, the focus of research and development of medicaments is not placed on the viruses, but on host factors, and on the premise that normal vital activities of cells are not influenced, the expression of the host factors is changed to inhibit virus replication, so that the method has better broad spectrum and better avoids the problem of virus mutation.

(2) The invention provides a new idea for drug development and vaccine development in the future.

Drawings

FIG. 1 is a schematic diagram of the mechanism by which ACTN4 regulates SARS-CoV-2 replication.

FIG. 2 is a third generation sequencing flow diagram.

FIG. 3 is a graph depicting that knocking down ACTN4 promotes novel coronavirus replication.

FIG. 4 is a reproduction of novel coronavirus inhibition by overexpressed ACTN 4.

FIG. 5 is a reproduction of YS-49 inhibiting novel coronaviruses.

FIG. 6 is a flow chart of an animal experiment in which YS-49 inhibits novel coronaviruses.

FIG. 7 is a graph of YS-49 inhibiting viral load in lung tissue.

FIG. 8 is a graph of YS-49 inhibiting viral load in brain tissue.

FIG. 9 is a graph showing HE staining results of lung tissue and brain tissue.

Detailed Description

The technical scheme of the invention is further described and illustrated by the following examples. The starting materials used in the examples are all commercially available or prepared by conventional methods.

Unless defined otherwise, 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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The gene sequence of SARS-COV-2 is similar to Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV), which binds to and enters host cells primarily associated with the S protein.

The S protein contains S1 and S2 subunits, wherein the S1 subunit contains a Receptor Binding Domain (RBD) for binding to a cellular receptor, aminopeptidase N (APN); the S2 subunit, which is composed of Fusion Peptide (FP), heptapeptide repeat 1 (HR 1), heptapeptide repeat 2 (HR 2), transmembrane domain (TM) and C-terminal domain (CTD), is responsible for mediating viral fusion and entry.

Related studies have shown that angiotensin converting enzyme ii (ACE 2) is a SARS-COV-2 cell receptor, and that ACE2 is also a receptor for the SARS coronavirus; therefore, the pathway of SARS-COV-2 infection of cells should be similar to that of common coronaviruses.

When a common coronavirus infects cells, a target cell protease cleaves the S protein of the virus, and cleaves the S protein into two subunits S1 and S2, thereby activating the S protein. After the RBD of the S1 subunit binds to the receptor of the host cell, the conformation of the Fusion core formed by the HR1 and HR2 domains in the S2 subunit is changed, and then the Fusion Peptide (FP) of the S2 subunit is exposed and inserted into the target cell membrane, thereby causing membrane Fusion and promoting the virus to enter the cell.

There are three main states in the S protein during membrane fusion, including the pre-fusion natural state, the pre-hairpin intermediate state, and the stable post-fusion hairpin state.

The traditional medicine uses blocking polypeptide (blocking peptide) to prevent cell fusion, but the blocking polypeptide for inhibiting viruses has higher specificity, and different polypeptides are required to be designed aiming at different viruses, if main functional sections are not selected, the polypeptide is overlong, the cost is increased, and the effect is reduced.

In order to solve at least one of the technical problems, the invention provides an application of a compound with the following structure in preparing anti-coronavirus drugs:

。

The compound provided by the invention can excite host factor ACTN4, inhibit the combination of novel coronavirus and nsp12, inhibit the infection activity of coronavirus, has lower cytotoxicity and higher safety, can be applied to the preparation of coronavirus medicaments, reduces coronavirus infection, and has important significance for preventing and treating coronavirus.

Redox is used as a nucleoside analog to stop viral RNA replication by acting on the viral RNA-dependent RNA polymerase (RdRp), and to exert antiviral activity.

The compound of the invention can target host factor ACTN4, activate host factor ACTN4, and the ACTN4 can reduce nsp12 bound to the genome of the novel coronavirus by inhibiting nsp7 from binding to nsp12 and nsp8 from binding to nsp12, thereby inhibiting replication of the novel coronavirus, thus having inhibitory activity on all viruses which complete replication of the novel coronavirus by nsp7 binding to nsp12 and nsp8 binding to nsp12, having good broad spectrum and having important effects on prevention and treatment of coronavirus.

Therefore, the compound has an antiviral action mechanism different from that of the Ruidexivir, and provides a new choice for antiviral therapeutic drugs.

The concentration of WT at 50% of the dose of the compound having the structure as described above for inhibiting cell infection in the present invention is 2.586. Mu.g/mL, and the concentration of BA.5 strain at 50% of the dose of the compound having the structure as described above for inhibiting cell infection in the present invention is 1.170. Mu.g/mL, wherein EC50 means half maximal effect concentration.

The selectivity index SI of the compounds of the formula described above for inhibiting WT infected cells in the present invention is 12.93, where si=tox/AVA, wherein AVA (antiviral activity) denotes EC50, 2.586, TOX (cytocity) denotes CC50 denotes the concentration of the drug at which 50% of the cells undergo a cytotoxic reaction, and is an indicator for measuring the cytotoxicity of the drug, and 33.44.

The selectivity index SI of the compound with the structure shown in the formula above for inhibiting the ba.5 strain from infecting cells is 28.5, wherein si=tox/AVA, AVA (antiviral activity) is EC50, 1.170, TOX (cytoxicity) is the concentration of the drug at which 50% of cells undergo cytotoxicity reaction, and the index for measuring the cytotoxicity of the drug is 33.34.

The invention evaluates the inhibition activity of the compound against novel coronavirus infected cells through the culture of coronaviruses.

In some embodiments, the novel coronavirus is selected from the novel coronavirus wild strain (WT) or the novel coronavirus ba.5 strain.

The invention also provides an anti-novel coronavirus drug, which comprises the compound or the pharmaceutically acceptable salt of the compound used in the application, and pharmaceutically acceptable auxiliary agents.

In particular, "pharmaceutically acceptable" refers to those ligands, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for administration to patients and commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers compatible with pharmaceutical administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) Starches, such as corn starch, potato starch, and substituted or unsubstituted beta-cyclodextrin; (3) Cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The dosage form of the medicament comprises at least one of tablets, capsules, aqueous or oily suspensions, granules, emulsions, oral liquids, injections or powders according to different using modes of the auxiliary agent.

The dosage form and the mode of administration of the compound of the present invention or the pharmaceutical composition thereof are not particularly limited. For different dosage forms of the medicament, the administration can be carried out by selecting a proper administration mode.

Representative modes of administration include, but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances. In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Such as suspensions, may contain suspending agents as, for example, particularly ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants. Is prepared by mixing the active ingredient under aseptic condition with pharmaceutically acceptable carrier and any preservative, buffer or propellant as required.

Embodiments of the present invention will be described in detail below with reference to examples.

Example 1

Three-generation sequencing assay

The flow of the third generation sequencing is shown in FIG. 2: extracting 1 mg total RNA from SARS-CoV-2 infected A549-ACE2 (provided by the Chinese academy of sciences 'Wuhan virus) and Huh7 cells (human hepatoma cells provided by the Chinese academy of sciences' Wuhan virus) and purifying with Oligo (dT) kit; RNA sample libraries were then prepared according to Oxford Nanopore DRS instructions and loaded onto FLO-MIN106D sequencing chips, followed by 48h sequencing run on a MinION device (Oxford Nanopore Technologies). The method comprises the following specific steps:

MESSENGER RNA (mRNA) purification:

(2) Incubating the 1.5. 1.5 mL ribozymal EP tube of step (1) with 100 uL RNA solution in a constant temperature metal bath at 65 ℃ for 2 min to disrupt RNA secondary structure;

(3) 200 mu L of Dynabeads magnetic beads (magnetic beads) resuspended by 1 mg are added into a new 1.5 mL non-ribozyme EP tube, the EP tube is placed on a magnetic rack for standing for 30 s after being uniformly mixed, the supernatant is discarded, and the EP tube is reserved;

(4) Then adding 100 mu L of Binding Buffer into the 1.5 mL non-ribozyme EP tube containing the magnetic beads, uniformly mixing and re-suspending, placing the mixture on a magnetic frame after balancing the magnetic beads, standing for 30: 30 s until the solution is clarified, discarding the supernatant, and reserving the EP tube;

(5) Adding 100 mu L of Binding Buffer re-suspending magnetic beads into the 1.5 mL non-ribozyme EP tube in the step (4), adding 100 mu L of the solution containing 75 mu g of RNA prepared in the step (1), thoroughly mixing, placing the EP tube on a rotary shaking table at room temperature, and incubating for 5 min;

(6) Placing the 1.5 mL non-ribozyme EP tube containing the solution and the magnetic beads in the step (5) on a magnetic rack until the solution is clear, discarding the supernatant, and reserving the EP tube;

(7) Adding 200 mu L Washing Buffer B into the 1.5 mL non-ribozyme EP tube in the step (6), uniformly mixing and re-suspending, placing the mixture on a magnetic rack after balancing magnetic beads, standing for 30 s until the solution is clear, discarding the supernatant, and then repeating the step (7) for one time to reserve the EP tube;

(8) Adding 10 mu L of an addition Buffer into the 1.5 mL non-ribozyme EP tube in the step (7) and incubating for 2 min at 65 ℃ on a constant temperature metal bath, immediately placing the EP tube on a magnetic rack, and standing for 30 s until the solution is clear;

(9) Transferring the supernatant obtained in the step (8) into a brand new 1.5 mL non-ribozyme EP tube for later use.

Library construction:

(1) In a 0.2 mL PCR tube, the following components ： 3.0 μL NEB Next Quick Ligation Reaction Buffer 9.0 μL RNA 0.5 μL RNA CS (RCS), 1.0 μL RT Adapter (RTA),1.5 μL T4 DNA Ligase, were added and incubated at room temperature for 10. 10 min;

(2) Preparing the following components of 9.0 mu L of nucleic-FREE WATER,2.0 mu L of 10 mmol/L dNTPs,8.0 mu L of 5 x first-strand buffer and 4.0 mu L of 0.1M DTT, adding the mixed solution into 0.2 mL PCR tube, and uniformly mixing;

(3) Adding 2 mu L of SuperScript III REVERSE TRANSCRIPTASE, uniformly mixing, incubating at 50 ℃ in a constant temperature metal bath for 5min, and incubating at 70 ℃ in the constant temperature metal bath for 10min to inactivate reverse transcriptase;

(4) Transferring the reverse transcription product into a 1.5 mL EP tube, adding 72 mu L of resuspended RNA Clean XP beads, uniformly mixing, and incubating at room temperature for 5 min;

(5) Adding 150 μl of precooled 70% ethanol, mixing, and discarding supernatant;

(6) After adding 20 μl NF H ₂ O and incubating at room temperature for 5: 5min, the solution was placed on a magnetic rack and transferred to a new EP tube;

(7) In a new 1.5 mL Eppendorf DNA LoBind tube, the following components ：20.0 μL Reverse-transcribed RNA,8.0 μL NEB Next Quick Ligation Reaction Buffer,6.0 μL RNA Adapter,3.0 μL Nuclease-free water,3.0 μL T4 DNA Ligase Mix, were added in sequence and incubated at room temperature for 10 min;

(8) Adding 40 mu L RNACLEAN XP beads, uniformly mixing, and incubating at room temperature for 5 min;

(9) Placing the EP tube on a magnetic rack, standing until the solution is clear, discarding the supernatant, adding 150 mu L Wash Buffer for washing twice, and discarding the supernatant;

(10) Adding 21 mu L of an absorption Buffer to dissolve the precipitate, and incubating for 10min at room temperature;

(11) The EP tube was placed on a magnetic rack and left to settle until the solution was clear and the liquid transferred to a new 1.5 mL Eppendorf DNA LoBind tube.

Sample treatment of the machine: RNA sequencing was performed on the samples prepared as described above using Nanppore DIRECT RNA sequencing (SQK-RNA 002).

By third generation sequencing, it was found that there was one down-regulating host factor ACTN4 in Huh7 cells and a549-ACE2 cells, as shown in fig. 3.

Cell lines for knocking down (as shown in FIG. 3) and over expressing (as shown in FIG. 4) ACTN4 are constructed in Huh7 cells, then WT and BA.5 strains are infected respectively, protein and RNA samples are collected after 48 h, the change condition of viral protein N protein is detected by Western blot experiment, and the change condition of viral protein N protein and S protein is detected by RT-qPCR respectively. As can be seen from fig. 3, protein levels and RNA levels of viral N protein were up-regulated and RNA levels of S protein were up-regulated after ACTN4 knockdown; as can be seen from FIG. 4, both protein and RNA levels of the toxic N protein were down-regulated and RNA levels of the S protein were down-regulated after over-expression of ACTN 4.

As can be seen from FIGS. 3 and 4, ACTN4 is effective in inhibiting replication of the novel coronavirus wild-type strain and BA.5 strain.

Example 2

Cell experiments for YS-49 inhibiting novel coronavirus replication

3 Mug/ml, 6 mug/ml and 18 mug/ml of YS-49 (purchased from Selleck company) solution are respectively prepared by dimethyl sulfoxide (DMSO), after Huh7 cells are respectively treated by YS-49 with different concentrations, WT and BA.5 are infected, protein and RNA samples are collected after 48 hours, the change condition of ACTN4 and virus protein N is detected by Western blot experiment, and the change condition of virus protein N and virus protein S is respectively detected by RT-qPCR.

As can be seen from FIG. 5, after different concentrations of YS-49 were added, the protein level of ACTN4 was up-regulated, the protein level and RNA level of viral protein N were down-regulated, and the RNA level of S protein was also down-regulated, demonstrating that YS-49 effectively inhibits replication of novel coronavirus wild-type strains and BA.5 strains.

Example 3

Animal experiment for YS-49 inhibiting novel coronavirus replication

The experimental procedure is shown in fig. 6: healthy laboratory mice with similar body weights were respectively subjected to gastric lavage administration on the day before and the second day after infection with the novel coronavirus, dissected on the fifth day of infection with the novel coronavirus, and examined for viral loads in lung and brain tissues, respectively (fig. 7 and 8), and observed for tissue lesions with HE staining (fig. 9).

As can be seen from FIGS. 7 and 8, YS-49 has a better effect of inhibiting viral replication than the control group; it can also be seen in FIG. 9 that the extent of lesions in the lung and brain tissues after YS-49 treatment was significantly reduced compared to the control group.

It should be understood that the above examples are only for illustrating the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (8)

1. The application of a compound with the structure shown as the following in preparing a medicament for preventing and treating novel coronavirus:

。

2. the use according to claim 1, wherein the concentration of said compound in the preparation of a medicament for the control of novel coronaviruses is not less than 0.01% by weight.

3. The use according to claim 1, wherein said novel coronavirus comprises a novel coronavirus wild strain (WT) or a novel coronavirus ba.5 strain.

4. The use according to claim 1, wherein the medicament for controlling novel coronaviruses comprises a compound or a pharmaceutically acceptable salt of a compound used in the above application, and a pharmaceutically acceptable adjuvant.

5. The use according to claim 1, wherein the pharmaceutically acceptable adjuvant comprises one or more of a pharmaceutically acceptable carrier, excipient or diluent.

6. The use according to claim 1, wherein the pharmaceutical dosage form comprises at least one of a tablet, a capsule, an aqueous or oily suspension, a granule, an emulsion, an oral liquid, an injection or a powder.

7. The use according to claim 1, wherein the medicament further comprises at least one additional anti-coronavirus active ingredient.

8. The use of claim 1, wherein said medicament is administered by at least one of oral, intratumoral, rectal, parenteral injection and topical administration.