CN114617893A - Application of Epiandrosterone in preparation of anti-coronavirus drugs and drugs - Google Patents

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to application of Epiandrosterone in preparation of a coronavirus infection resistant medicament. The invention discovers a new function of Epiandrosterone, the Epiandrosterone has stronger inhibition effect on the enzymatic activity of target spot papain-like protease Plpro of coronavirus, especially novel coronavirus, and has the potential of developing drugs for treating and preventing coronavirus, especially anti-novel coronavirus.

Description

Application of Epiandrosterone in preparation of anti-coronavirus drugs and drugs

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to an application of Epiandrosterone in preparing a medicine for resisting coronavirus inflammation, and more particularly relates to Epiandrosterone or an effective inhibitor of coronavirus papain-like protease PLPro.

Background

Coronaviruses belong to the order of nested viruses, the family of coronaviruses, the genus of coronaviruses, and are a large group of viruses widely existing in nature, and the genome is the largest among currently known positive-strand RNA viruses. Meanwhile, the coronavirus is an RNA virus with a cyst membrane and a genome which is a single-segment positive-strand RNA virus, the genome of the coronavirus is simply composed of one RNA strand, genetic information is carried by RNA, and after the virus enters a host cell, a ribosome of the host can be directly utilized to produce proteins required by virus replication without a transcription process.

Coronaviruses can be classified into four types according to host, serotype gene sequence and structure: alpha, beta, gamma and delta, and no antigen cross reaction exists among different classes; of these four classes, the α and β classes only infect mammals, and the γ and δ classes mainly infect birds. In recent decades, many outbreaks of coronavirus, such as SARS, MERS and COVID-19, caused by coronavirus infection seriously affect human health and even take human life. The repeated outbreaks of coronaviruses suggest that humans are poorly understood about their research and the development of drugs for treating coronaviruses is imminent.

The papain (PLPro) is a hydrolase expressed on a genome nsp3 at the 5' end of the coronavirus, and mainly has the function of cleaving a specific tetrapeptide structure LXGG between nsp1-2, nsp2-3 and nsp3-4 on a polyprotein pp1a which needs to be hydrolyzed into a mature functional protein; in addition, papain has the additional function of detaching ubiquitin from host cells, which protects coronaviruses and thus circumvents the immune response inherent to the host. Therefore, the papain is crucial to the life cycle of the virus and can be used as an ideal target for designing and screening anti-coronavirus medicines. The gene sequence analysis of different coronaviruses shows that the papain of the coronaviruses has high similarity, and the medicine with the inhibiting ability is suitable for the papain of the coronaviruses.

In recent years, "computer-aided drug design" (CADD) has become an important method and tool for modern drug research and development. Therefore, the CADD method has been widely applied to the discovery and optimization of lead compounds. Computer-assisted high-throughput virtualization has become a common computer-assisted drug discovery method. The principle is that a chemical structure capable of being combined with a specific disease target protein is searched in millions of small molecule compound libraries by a molecular docking method through a supercomputer, and efficient and reliable small molecule lead compounds are provided for further molecular, cell, animal and clinical research. In conclusion, the small molecular compound designed and synthesized by using the papain plpro as a target and combining the CADD and biological activity test tests at various levels has important practical significance for developing antiviral drugs.

Disclosure of Invention

The invention aims at the problem that related inflammatory diseases such as lung, respiratory tract and intestinal diseases, nervous system symptoms, myocarditis and the like caused by coronavirus lack of effective prevention and treatment medicines, and points out the application of Epiandrosterone and pharmaceutically acceptable salts thereof in preparing anti-coronavirus inflammatory medicines.

The technology of the invention takes a novel coronavirus (SARS-CoV-2) as a model, carries out drug design based on a three-dimensional structure of novel coronavirus pawpaw-like protease (SARS-CoV-2PLPro), and theoretically screens thousands of compounds including a natural product library, a clinical compound library and an antiviral drug library to obtain the compound which can inhibit the novel coronavirus PLPro. Then, we used the SARS-CoV-2 papain inhibitor screening reagent box constructed in the previous stage to perform experimental screening on these compounds through enzyme activity test.

Through drug screening according to the scheme, the invention discovers that Epiandrosterone shown as the following formula I has an inhibiting effect on novel coronavirus PLPro.

The compound has obvious effect of inhibiting novel coronavirus papain (PLPro), has the inhibition rate of more than 90 percent on SARS-CoV-2 papain at the concentration of 25 mu M, and has IC₅₀The value was 6.010. mu.M, indicating that Epiandrosterone is effective in inhibiting the activity of the novel coronavirus papain. Furthermore, based on the high similarity of coronavirus papain-like proteases shown by sequence analysis, we reasoned that this class of compounds can also effectively inhibit the activity of other coronavirus papain-like proteases, especially against SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU 1.

Preferably, Epiandrosterone having an inhibitory effect also includes various optical isomers, hydrates thereof. The compounds of formula I can be used in combination with pharmaceutically acceptable adjuvants for the preparation of medicaments for the treatment and prophylaxis of novel coronavirus infections. The adjuvant comprises diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, and synergist conventional in pharmaceutical field. The medicine can be made into injection, tablet, pill, capsule, suspension or emulsion. The administration route can be oral, percutaneous, intravenous or intramuscular injection.

Drawings

FIG. 1 is a graph showing the change of fluorescence intensity of a substrate peptide with time under the action of different concentrations of Epiandrosterone under the metabolism of papain in example 1

FIG. 2 is a graph of the inhibition of the novel coronavirus papain-like protease by the compound Epiandrosterone of example 2.

Detailed Description

The following detailed description of specific embodiments of the invention is provided for the purpose of illustrating the invention more clearly and is not to be taken in a limiting sense.

All procedures and procedures, as well as reaction conditions of substrates, etc., are designed and carried out in accordance with methods well known to those of ordinary skill in the art throughout the course of the experiment.

Inhibitor molecules used in the examples described below were all available from mce (medchemexpress) or other common commercial sources.

The inhibitor screening kit used in the invention is invented by the laboratory (application patent number is 202010674650.X), and comprises the following components:

SARS-CoV-2 papain-like protease,

The substrate peptide Dabcyl-FTLKGGAPTKVT-E (Edans),

Boric acid borax buffer solution.

The concentration of the SARS-CoV-2 papain is 0.01-1 mg/mL;

preferably, the substrate peptide Dabcyl-FTLKGGAPTKVT-E (Edans) is present in a concentration of 0.1mM-5 mM; wherein Dabcyl is the quencher 4-dimethylaminoazobenzene 4' -carboxylic acid and Edans is the fluorophore 5- (2-aminoethylamino) -1-naphthalenesulfonic acid;

preferably, the concentration of the borax borate buffer solution is 10-100 millimole/liter; the pH value of the borax borate buffer solution is 7-9.

The SARS-Cov2 papain-like protease used was purchased from CrystalO Biopharma, the substrate Dabcyl-FTLKGGAPTKVT-E (Edans) was custom synthesized from GL biochem, and the borax borate buffer was purchased from Leptoradix organisms. Preferably, SARS-Cov-2 papain (concentration 0.1mg/mL), the substrate peptide Dabcyl-FTLKGGAPTKVT-E (Edans) (concentration 0.1mM-5mM), borax borate buffer (pH 7-9). The kit needs to be subjected to fluorescence detection by a fluorescence microplate reader.

Example 1: the fluorescence intensity of the substrate peptide under the action of Epiandrosterone with different concentrations changes with time under the metabolism of papain

The specific implementation process comprises the following steps:

1) storing the SARS-CoV-2 pawpaw-like protease and the substrate peptide stock solution in a refrigerator at-80 ℃;

2) melting SARS-CoV-2 papain in a freezing plate (-4 to 4 ℃) at room temperature, diluting 1uL into 98uL borax borate buffer solution (PH 7.4), and adding into a detection plate;

3) adding 1uL of inhibitors (Epiandrosterone concentration is 0, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM, 2.5mM) with different concentrations into the solution obtained in the step (2);

4) adding 1uL of substrate peptide (0.5mM) with the same concentration into the solution obtained in the step (3), incubating at 37 ℃ by using a fluorescence microplate reader, monitoring 342nm excitation by using the fluorescence microplate reader, detecting the fluorescence emission value at 496nm while incubating, and taking one point every 1 minute;

5) the slow increase effect of the fluorescence intensity of the substrate peptide under the enzyme metabolism in the presence of the inhibitor is shown in the following FIG. 1, and the results show that the fluorescence intensity of the substrate peptide generated by the enzyme catalysis can be inhibited by the inhibitor Epiandrosterone, and the inhibition phenomenon is concentration-dependent.

Example 2: inhibitory capacity of compound Epiandrosterone on novel coronavirus papain-like protease

The specific implementation process is as follows:

1) storing the stock solutions of SARS-CoV-2 papain and substrate peptide in a refrigerator at-80 deg.C;

2) melting SARS-CoV-2 papain in a freezing plate (-4 to 4 ℃) at room temperature, diluting 1uL into 98uL borax borate buffer solution (PH 7.4), and adding into a detection plate;

3) adding 1uL of inhibitor (Epiandrosterone concentration is 0, 0.05mM, 0.15mM, 0.25mM, 0.5mM, 1mM, 2.5mM) with different concentrations into the solution obtained in the step (2);

4) adding 1uL of substrate peptide (0.5mM) with the same concentration into the solution obtained in the step (3), incubating at 37 ℃ by using a fluorescence microplate reader, monitoring 342nm excitation by using the fluorescence microplate reader, detecting the fluorescence emission value at 496nm while incubating, and incubating for 1 h;

5) the fluorescence emission at 496nm after and before 342nm excitation of each group incubation was counted. The fluorescence change values before and after incubation in the control group (group with inhibitor concentration of 0) were taken as 100, and the Residual activity values (Residual activity) were obtained from the fluorescence change values in the different inhibition groups. The logarithmic value of the inhibitor concentration (logc (inhibitor)) is plotted on the abscissa and the corresponding Residual activity value (Residual activity) is plotted on the ordinate using GraphPad Prism6 software. The results are shown in FIG. 2, giving the IC of the inhibitor₅₀The value is obtained. Table 1 lists the data on the inhibition activity of Compound 1 against SARS-CoV-2 PLpro. IC (integrated circuit)₅₀The formula is calculated as Y is 100/(1+10^ ((X-Logic)₅₀) Y) where Y represents the remaining activity fraction, X represents the common logarithm of the inhibitor compound concentration, a refers to the power algorithm. FIG. 2 is a graph showing the ratio of the concentration of various inhibitors to the activity of the inhibitory enzyme, and the inhibitory activity of the compound can be obtained as expressed by the concentration of the inhibitor at which the activity of the inhibitor is half inhibited.

Table one: compounds Epiandrosterone structure and inhibition of novel coronavirus papain-like protease (PLPro) IC₅₀Value of

As can be seen, the compound Epiandrosterone has obvious inhibition effect on the novel coronavirus papain, and the IC50 value is 6.010 muM, which indicates that Epiandrosterone can effectively inhibit the activity of the novel coronavirus papain. Moreover, the coronavirus pawpaw-like protease is shown to have high similarity based on sequence analysis, so the compound can also effectively inhibit the activity of other coronavirus pawpaw-like proteases, especially SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU 1.

Further, the procedures of example 1-2 (procedures and conditions identical to those of the above examples except that the inhibitors were replaced with the following compounds, respectively) were performed on the following compounds having a sterol structure similar to that of Epiandrosterone, showing that the following compounds do not inhibit or weakly inhibit 3cl and plpro proteases of neocoronavirus.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. The application of Epiandrosterone in preparing anti-coronavirus medicines.
2. 2. The use according to claim 1, wherein said Epiandrosterone has the structure of formula I

   Formula I.
3. 3. The use of claim 1, wherein said Epiandrosterone inhibits corona

   

   Enzymatic activity of the rhabdovirus papain PLPro.
4. 4. Use according to claim 1, wherein the Epiandrosterone is for use in the preparation of a medicament for the treatment of a coronavirus papain-like protease PLPro inhibitor.
5. 5. The use according to claim 1, wherein the coronavirus is HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU, SARS-CoV, MERS-CoV, or a novel coronavirus (SARS-CoV-2).
6. 6. The coronavirus of claim 5, preferably a novel coronavirus (SARS-CoV-2).
7. 7. A compound of formula I according to claim 2, wherein the compounds of formula I include optical isomers thereof, hydrates thereof; the number of crystal water of the hydrate is any real number from 0 to 16.
8. 8. A coronavirus papain inhibitor comprising Epiandrosterone.
9. 9. A medicament for inhibiting coronavirus papain-like protease, comprising Epiandrosterone.
10. 10. The medicament of claim 4 or 9, which is an injection, tablet, pill, capsule, suspension or emulsion of the compound.

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