CN114617880A

CN114617880A - Application of IOWH-032 in preparation of anti-coronavirus medicines and medicines

Info

Publication number: CN114617880A
Application number: CN202011456111.5A
Authority: CN
Inventors: 韩克利; 贾燕; 张洪苓
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2022-06-14
Anticipated expiration: 2040-12-10
Also published as: CN114617880B

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to an application of IOWH-032 in preparation of a coronavirus infection resistant medicine. The IOWH-032 has strong inhibition effect on the enzyme activity of the target main protease 3Clpro of coronavirus, especially novel coronavirus, and can be used for preparing a medicament for resisting coronavirus, especially novel coronavirus infection.

Description

Application of IOWH-032 in preparation of anti-coronavirus medicines and medicines

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to an application of IOWH-032 in preparation of a coronavirus infection resistant medicament

Background

Coronaviruses belong to the single positive strand RNA virus, and the family of coronaviruses mainly includes novel coronaviruses (SARS-CoV-2), SARS coronaviruses (SARS-CoV), middle east respiratory syndrome coronaviruses (MERS-CoV), HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU. Coronaviruses commonly cause respiratory and intestinal diseases, neurological symptoms, and myocarditis. The infection of these diseases seriously affects the health of people. The repeated outbreaks of coronaviruses suggest that human beings are poorly informed about their research and the development of drugs for treating new coronaviruses is imminent.

The main protease of coronavirus, also called 3C-like protease (3CLpro), is a hydrolase expressed in the genome nsp5 at the 5' end of coronavirus and mainly functions to cleave at least 11 sites on the polyproteins pp1a and pp1ab, which need to be processed into mature functional protein after hydrolysis; the 3C-like protease (3CLpro) is a cysteine protease that recognizes sequences with Leu and Glin at positions P2 and P1, respectively. In addition, the substrate binding site of the 3C-like protease (3CLpro) is highly conserved and plays a key role in mediating virus replication and transcription, so the 3CL protease can be used as an ideal target point for designing and screening anti-coronavirus medicines.

IOWH-032 is a potent synthetic inhibitor of the extracellular cystic fibrosis transmembrane conductance regulator (CFTR) and has an IC50 value of 1.01. mu.M [1] in experiments based on T84 and CHO-CFTR cells. The CFTR chloride channel is the major driver of diarrhea caused by enteropathogens and is therefore an attractive drug target. CFTR plays an important role in transepithelial fluid homeostasis by controlling chloride ion entry and exit into cells, ultimately leading to water exchange inside and outside cells [1 ]. Here we demonstrate that IOWH-032 can inhibit the enzymatic activity of the main protease 3Clpro of coronaviruses, especially of the novel coronaviruses. After being examined and reviewed by related data, no report is found about IOWH-032 as a coronavirus main protease 3Clpro inhibitor.

Disclosure of Invention

The invention aims at the problem that relevant inflammatory diseases such as lung, respiratory tract and intestinal tract diseases, nervous system symptoms, myocarditis and the like caused by coronavirus lack of effective prevention and treatment medicines, and points out the application of IOWH-032 in preparing anti-coronavirus inflammation medicines.

The invention aims to provide an IOWH-032 as an inhibitor of coronavirus 3C-like protease (3CLpro) and application thereof in preparing a medicament for preventing and/or treating novel coronavirus infection. The technology of the invention is based on the three-dimensional structure of the novel coronavirus 3CLpro to carry out drug design, and thousands of compounds including a natural product library, a clinical compound library and an antiviral drug library are virtually screened to obtain the compound which can possibly inhibit the novel coronavirus 3 CLpro. We then performed enzyme activity assays using a commercially available 3CLpro fluorescent probe construction method and used for inhibitor screening.

By screening the medicines according to the scheme, the invention discovers that the compounds shown in the following general formula 1 have the inhibition effect on SARS-CoV-23 CL protease:

the compound has obvious effect of inhibiting novel coronavirus 3C-like protease (3CLpro), and IC₅₀6.664uM., indicating that this class of compounds is effective in inhibiting the activity of the novel coronavirus 3C-like protease. Furthermore, given the high similarity of coronavirus main proteases reported in the literature, we theorize that this class of compounds is also effective in inhibiting the activity of other coronavirus main proteases, especially for SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU. Preferably, the IOWH-032 with inhibitory effect also includes the various optical isomers, hydrates thereof and pharmaceutically acceptable salts thereof with acids. 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. 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 time-dependent change in the fluorescence intensity of the substrate peptide under 3C-like protease metabolism at various concentrations of inhibitor IOWH-032 of example 1

FIG. 2 is a plot of the inhibition of the novel coronavirus 3C-like protease by the inhibitor IOWH-032 of example 2.

Detailed Description

In order to illustrate the invention more clearly, the following examples are given for the purpose of illustrating the invention more clearly and are not to be construed as limiting the invention.

In carrying out the experiment, all the procedures and procedures, reaction conditions of the substrate, and the like are designed and carried out according to methods well known to those skilled in the art.

In the examples below, we used inhibitor molecules, available from mce (medchemexpress) or other common commercial sources.

SARS-Cov-23C-like protease used in the present invention is purchased from CrystalO Biopharma, substrate Dabcyl-KTSAVLQ ↓.SGFRKM-E (Edans) -NH2 is purchased from GL biochem, and Borax borate buffer is purchased from Lei root organisms.

Preferably, SARS-Cov-23C-like protease (concentration 0.1mg/mL), substrate peptide Dabcyl-KTSAVLQ ↓ ] SGFRKM-E (Edans) -NH2 (concentration 0.1mM-5mM), borax borate buffer (pH ═ 5-8), the concentration of borax borate buffer is 10-100 millimole/liter.

The invention requires fluorescence detection with a fluorescence microplate reader.

Example 1: determination of inhibitory Activity of IOWH-032 against SARS-Cov-23C-like protease

The specific implementation process comprises the following steps:

1) storing the SARS-Cov-23C-like protease and the substrate peptide stock solution in a refrigerator at-80 ℃;

2) thawing SARS-Cov-23C-like protease (concentration of 0.1mg/mL) in a frozen plate (-4 to 4 ℃) at room temperature, diluting 1uL in 98uL borax borate buffer (pH 7.4), and adding into the detection plate;

3) adding 1uL of inhibitor (IOWH-032, concentration of 0, 0.025mM, 0.125mM, 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 in fluorescence intensity of the substrate peptide in enzyme metabolism with time in the presence of various concentrations of inhibitor is shown in FIG. 1 below, and the results show that the increase in fluorescence intensity of the substrate peptide in enzyme metabolism can be inhibited by the inhibitor IOWH-032, and that the inhibition is increased with the increase in inhibitor concentration.

Example 2: determination of SARS-Cov-23C-like protease inhibitory Capacity of IOWH-032

The specific implementation process is as follows:

1) storing the SARS-Cov-23C-like protease and substrate peptide stock solution in a refrigerator at-80 ℃;

2) thawing SARS-Cov-23C-like protease (concentration 0.1mg/mL) in a frozen plate (-4 to 4 ℃) at room temperature, diluting 1uL in 98uL borax borate buffer (pH 7.4), and adding into the detection plate;

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 (inhibitor concentration 0 group) were taken as 100, and the fluorescence change values before and after incubation of the different inhibition groups were compared to obtain the residual activity value (residual activity). The IC of the inhibitor was obtained as shown in FIG. 2 by plotting the logarithm of the inhibitor concentration (logC (inhibitor)) on the abscissa and the corresponding Residual Activity (Residual Activity) on the ordinate using GraphPad Prism6 software₅₀The value is obtained. Table 1 lists the data on the inhibition activity of Compound 1 against SARS-Cov-23 CLpro. IC (integrated circuit)₅₀The formula is calculated as Y100/(1 +10^ ((X-LogIC50))), where Y represents the residual activity fraction, X represents the common logarithm of the inhibitor compound concentration, and Λ 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: IOWH-032 structureAnd IC for inhibiting novel coronavirus 3C-like protease (3CLpro)₅₀Value of

As can be seen, the compound has obvious inhibiting effect on the novel coronavirus 3C-like protease, and IC₅₀The value was 6.664. mu.M, indicating that IOWH-032 can effectively inhibit the activity of the novel coronavirus 3C-like protease. Moreover, the coronavirus 3C-like protease is shown to have high similarity based on sequence analysis, so the compound can also effectively inhibit the activity of other coronavirus 3C-like proteases, especially SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU 1.

Furthermore, examples 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 structure similar to IOWH-032, and it was found that these compounds did not inhibit the novel coronavirus 3C-like protease.

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

The application of IOWH-032 (compound shown in formula 1) in preparing anti-coronavirus medicines is disclosed.
2. The use according to claim 1, wherein said IOWH-032 comprises a compound having the structure shown in formula 1,
3. the use of a compound according to claim 1 or 2, wherein the IOWH-032 inhibits the enzymatic activity of the coronavirus main protease 3 Clpro.
4. The use of a compound according to claim 1 or 2, wherein said IOWH-032 is used in the preparation of a drug that is an inhibitor of the coronavirus main protease 3 Clpro.
5. Use of a compound according to claim 1 or 2, wherein the coronavirus is HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU, SARS-CoV, MERS-CoV, or a novel coronavirus (SARS-CoV-2).
6. The coronavirus according to claim 5, which is preferably a novel coronavirus (SARS-CoV-2).
7. The compound of formula 1 according to claim 1 or 2, wherein the compound of formula 1 comprises one or more of optical isomers thereof, hydrates thereof; the number of crystal water of the hydrate is any real number in the range of 1-16, and is preferably an integer.
8. A medicament for preventing and/or treating coronavirus infection or inflammation caused thereby, comprising IOWH-032.
9. An inhibitor drug of coronavirus main protease 3Clpro, which comprises IOWH-032.
10. The medicament as claimed in claim 8 or 9, wherein the medicament is an injection, a tablet, a pill, a capsule, a suspension, a granule, a spray or an emulsion.