CN114504573A - Application of MK-0893 in preparing products for inhibiting coronavirus and medicines for resisting coronavirus infection - Google Patents

Abstract

The invention relates to application of MK-0893 in preparation of a product for inhibiting coronavirus and a medicine for resisting coronavirus infection. The invention also provides a coronavirus pawpaw-like protease inhibitor, which is MK-0893 or an optical isomer, a hydrate and a pharmaceutically acceptable salt thereof. By utilizing the strong inhibition effect of MK-0893 on the enzymatic activity of target papain (PLPro) of coronavirus, the invention provides the application of MK-0893 serving as a coronavirus papain (PLPro) inhibitor in preparing products for inhibiting coronavirus and preparing medicaments for resisting coronavirus infection, and provides a new thought for treating and preventing coronavirus infection, particularly resisting new coronavirus infection.

Description

Application of MK-0893 in preparing products for inhibiting coronavirus and medicines for resisting coronavirus infection

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to application of MK-0893 in preparation of a product for inhibiting coronavirus and a coronavirus infection resistant medicine.

Background

Coronavirus is a enveloped virus with a positive-stranded RNA genome, i.e., RNA strand with a methylated "cap" at the 5 'end and a PolyA "tail" structure at the 3' end, and a helically symmetric nucleocapsid protein, with a genome size of 27 to 32kb, which is the largest of the currently known positive-stranded RNA viruses. Under an electron microscope, coronavirus particles are irregular in shape, about 60-220nm in diameter, 100nm in average diameter, spherical or elliptical, and have huge granular protrusions at the edges of the particles to make the shape similar to crown or corona, so that the coronavirus particles are called.

Coronaviruses can be classified into four types according to differences in host, serotype gene sequence and structure: alpha class, beta class, gamma class, and delta class. Among these four classes, the α and β classes only infect mammals, the γ and δ classes mainly infect birds; the alphacoronavirus includes human coronavirus (HCoV-229E, HCoV-NL63), Hepialus rhamnus coronavirus (HKU1, HKU8), canine coronavirus (CCoV), feline coronavirus (FCoV), etc.; the beta genus coronavirus includes human coronavirus (HCoV-OC43, HCoV-HKU1), novel coronavirus (SARS-CoV-2), SARS coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), murine coronavirus, Hepialus coronavirus HKU9, etc.; the gamma-type coronavirus mainly comprises avian coronavirus such as avian Infectious Bronchitis Virus (IBV), Turkey coronavirus (TCoV) and the like; the delta genus coronavirus includes oriole coronavirus (BuCoV HKU11), hydrangeal coronavirus (WECoV), wild duck coronavirus (WiCoV), and black-water chicken coronavirus (CMCoV).

Coronaviruses mainly affect respiratory tract, gastrointestinal tract, nervous system and the like, and often cause respiratory and intestinal diseases, nervous system symptoms and myocarditis. 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.

MK 0893 is a potent selective glucagon receptor antagonist and has been used in studies of type 2 diabetes and therapeutic trials of type 2 diabetes. After examining the relevant data, no report that the MK-0893 compound can inhibit the activity of coronavirus papain (PLPro) is found.

Disclosure of Invention

In order to solve the technical problems, the invention provides the application of MK-0893 in preparing products for inhibiting coronavirus papaya-like protease and medicines for resisting coronavirus infection, so that the activity of coronavirus papaya-like protease can be effectively inhibited, coronavirus is further inhibited, and the aim of treating coronavirus infection is fulfilled.

In order to achieve the purpose, the invention firstly provides the following technical scheme:

application of MK-0893 in preparation of products for inhibiting coronavirus or medicines for resisting coronavirus infection.

The invention further provides a coronavirus papaya-like protease inhibitor, wherein the inhibitor is MK-0893 or an optical isomer, a hydrate and a pharmaceutically acceptable salt thereof.

Preferably, the number of crystal water of the hydrate is any real number from 1 to 16.

Preferably, the pharmaceutically acceptable salt of MK-0893 is a salt of MK-0893 with an acid.

Further preferably, the acid is selected from any one of hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, and malic acid.

In the above scheme, the coronavirus is HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU, SARS-CoV, MERS-CoV, SARS-CoV-2.

The invention also provides a product for inhibiting coronavirus, which contains MK-0893 or an optical isomer, a hydrate and a pharmaceutically acceptable salt thereof. The products for inhibiting coronavirus comprise inhibitors of coronavirus papain-like protease, or disinfection products comprising the inhibitors, such as disinfectant wet tissues, disinfectant liquids, disinfectant sprays, disinfectant hand-washing solutions and the like.

The invention also provides a medicine for resisting coronavirus infection, wherein the active ingredient of the medicine is MK-0893 or an optical isomer, a hydrate and a pharmaceutically acceptable salt thereof.

By utilizing the strong inhibition effect of MK-0893 on the enzymatic activity of target papain (PLPro) of coronavirus, the invention provides the application of MK-0893 serving as a coronavirus papain (PLPro) inhibitor in preparing products for inhibiting coronavirus and preparing medicaments for resisting coronavirus infection, and provides a new thought for treating and preventing coronavirus infection, particularly resisting new coronavirus infection.

Drawings

FIG. 1 is a graph of the fluorescence intensity of a substrate peptide under metabolism of papain (PLPro) with time at various concentrations of MK-0893;

FIG. 2 is a graph showing the inhibition of novel coronavirus papain-like protease (PLPro) by compound MK-0893.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

The invention takes a novel coronavirus (SARS-COV-2) as a model, carries out drug design based on the three-dimensional structure of novel coronavirus pawpaw-like protease (PLPro), theoretically screens thousands of compounds including a natural product library, a clinical compound library and an antiviral drug library, and obtains a compound which has an inhibiting effect on the novel coronavirus pawpaw-like protease (PLPro). Then, enzyme activity test is carried out by adopting a pawpaw-like protease inhibitor screening kit, and inhibition effect of MK-0893 and pharmaceutically acceptable salt thereof on SARS-COV-2 pawpaw-like protease (PLPro) is screened out through experiments.

According to the scheme, the MK-0893 shown as the formula I has an inhibitory effect on novel coronavirus papain (PLPro).

MK-0893, which has inhibitory effects, also includes various optical isomers thereof, hydrates thereof, and pharmaceutically acceptable salts thereof with acids, for example, salts with any one of hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, and malic acid.

The compound can be combined with pharmaceutically acceptable adjuvants to prepare medicines for treating and preventing novel coronavirus infection. The adjuvant comprises diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, and synergist. The medicine can be made into injection, tablet, pill, capsule, suspension or emulsion. The administration route can be oral, percutaneous, intravenous or intramuscular injection.

In the whole experimental process in the examples of the present invention, all the operation methods and operation steps, the reaction conditions of the substrate, and the like are designed and implemented according to methods well known to those skilled in the art.

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 a product invented in the laboratory, has the application patent number of 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 SARS-CoV-2 papain is 0.01-1 mg/mL;

the concentration of the substrate peptide Dabcyl-FTLKGGAPTKVT-E (Edans) is 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.

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 used by the invention needs to be matched with a fluorescence microplate reader for fluorescence detection.

Example 1: the fluorescence intensity of the substrate peptide under the metabolism of papain is changed along with time under the action of MK-0893 at different concentrations

The specific implementation process comprises the following steps:

(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 MK-0893 with different concentrations of 0, 0.05mM, 0.5mM, 1mM and 2.5mM 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 effect of the substrate peptide on the slow increase of fluorescence intensity over time in the presence of MK-0893 under enzymatic metabolism is shown in FIG. 1

The results show that the increase in fluorescence of the substrate peptide produced by the enzyme catalysis can be inhibited by the inhibitor MK-0893, and that this inhibition is concentration-dependent.

Example 2: inhibitory Activity of Compound MK-0893 against novel Coronaviridae 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 MK-0893 with different concentrations of 0, 0.05mM, 0.5mM, 1mM and 2.5mM 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) and counting the fluorescence emission value at 496nm after 342nm excitation before and after incubation of each group. The fluorescence change values before and after incubation of the control group (MK-0893 concentration 0 group) were taken as 100, and the fluorescence change values of the MK-0893 groups at different concentrations were compared to obtain the Residual activity value (Residual activity). 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, which gives the IC50 value for the inhibitor. Table 1 lists the data on the inhibition activity of Compound 1 against SARS-CoV-2 PLpro.

Table 1: compound MK-0893 structure and inhibition of novel coronavirus papain-like protease (PLPro) IC₅₀Value of

As can be seen from FIG. 2 and Table 1, the inhibitory effect on the novel coronavirus papain (PLPro) is very obvious, and the inhibitory rate of the novel coronavirus papain (PLPro) on SARS-CoV-2 papain at the concentration of 25 mu M is over 90 percent, and the IC is₅₀The value was 5.115. mu.M, indicating that MK-0893 is effective in inhibiting the activity of a novel coronavirus papain-like protease. 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.

Claims (10)

1. The application of MK-0893 in preparing products for inhibiting coronavirus or medicines for resisting coronavirus infection is disclosed.
2. 2. A coronavirus papain inhibitor, wherein the inhibitor is MK-0893 or an optical isomer, a hydrate or a pharmaceutically acceptable salt thereof.
3. 3. The coronavirus papain inhibitor according to claim 2, characterized in that: the number of crystal water of the hydrate is any real number from 1 to 16.
4. 4. The coronavirus papain inhibitor according to claim 2, characterized in that: the MK-0893 pharmaceutically acceptable salt is a salt of MK-0893 and an acid.
5. 5. The coronavirus papain inhibitor according to claim 4, characterized in that: the acid is selected from any one of hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid and malic acid.
6. 6. The coronavirus papain inhibitor according to claim 2, characterized in that: the coronavirus is HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU, SARS-CoV, MERS-CoV, SARS-CoV-2.
7. 7. A coronavirus-inhibiting product characterized by: MK-0893 or an optical isomer, hydrate or pharmaceutically acceptable salt thereof is included in the product.
8. 8. The coronavirus-inhibiting product of claim 7, wherein: the product is a disinfection product.
9. 9. A medicament against coronavirus infection, characterized by: a medicament comprising the coronavirus papain inhibitor as defined in any one of claims 2 to 6.
10. 10. A medicament against coronavirus infection, characterized by: MK-0893 or an optical isomer, a hydrate or a pharmaceutically acceptable salt thereof is contained in the drug.

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