CN114617880B

CN114617880B - Application of IOWH-032 in preparation of anti-coronavirus drugs and drugs

Info

Publication number: CN114617880B
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: 2023-06-06
Anticipated expiration: 2040-12-10
Also published as: CN114617880A

Abstract

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

Description

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

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to application of IOWH-032 in preparation of medicines for resisting coronavirus infection

Background

Coronaviruses belong to the single positive strand RNA virus, and the family of coronaviruses mainly includes the novel coronavirus (SARS-CoV-2), the SARS coronavirus (SARS-CoV), the middle east respiratory syndrome coronavirus (MERS-CoV), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU. Coronaviruses often cause respiratory and intestinal diseases, neurological symptoms and myocarditis. Infection with these diseases severely affects the health of people. The recurrent attacks of coronaviruses suggest that humans are poorly informed of their studies and that the development of drugs for the treatment of new coronaviruses is urgent.

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

IOWH-032 is a potent inhibitor of synthesis of extracellular cystic fibrosis transmembrane conductance regulator (CFTR), with an IC50 value of 1.01. Mu.M [1] in experiments based on T84 and CHO-CFTR cells. CFTR chloride channel is the primary driving force for diarrhea caused by enteropathogenic bacteria and is therefore an attractive drug target. CFTR plays an important role in transepithelial fluid homeostasis by controlling chloride ions in and out of cells, ultimately leading to intracellular and extracellular water exchange [1]. Here we demonstrate that IOWH-032 can inhibit the enzymatic activity of the main protease 3Clpro of coronaviruses, especially novel coronaviruses. Through looking at the related data, no related report on IOWH-032 as coronavirus main protease 3Clpro inhibitor is seen.

Disclosure of Invention

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

The invention aims to provide IOWH-032 serving as an inhibitor of coronavirus 3C-like protease (3 CLpro) and application thereof in preparing medicaments for preventing and/or treating novel coronavirus infection. The technology of the invention is to carry out drug design based on the three-dimensional structure of novel coronavirus 3CLpro, and carry out virtual screening on thousands of compounds including natural product library, clinical compound library and antiviral drug library, thus obtaining the compounds which can inhibit novel coronavirus 3 CLpro. Then, we used the commercial 3CLpro fluorescent probe construction method to perform enzyme activity test and to perform inhibitor screening.

Through the drug screening according to the scheme, the invention discovers that a class of compounds shown as the following < formula 1) has an inhibition effect on SARS-CoV-2 3CL protease:

the compound has obvious inhibiting effect on novel coronavirus 3C-like protease (3 CLpro), and IC ₅₀ =6.664 μm. The compounds are shown to be effective in inhibiting the activity of novel coronavirus 3C-like proteases. Moreover, 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, it has an inhibitory effectIOWH-032 also includes various optical isomers, hydrates thereof and pharmaceutically acceptable salts thereof with acids. The compounds of formula I can be combined with pharmaceutically acceptable adjuvants to prepare medicaments for the treatment and prophylaxis of novel coronavirus infections. The auxiliary agents comprise diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and synergists which are conventional in the 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 trend of the fluorescence intensity of a substrate peptide with time under the metabolism of a 3C-like protease at various concentrations of the inhibitor IOWH-032 of example 1

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

Detailed Description

In order to more clearly illustrate the present invention, the following examples are set forth only to more clearly illustrate the invention, and are not to be construed as limiting the invention.

All procedures and steps and reaction conditions of the substrate, etc. are designed and carried out according to methods well known to those skilled in the art when the experiment is carried out.

In the examples below, we used inhibitor molecules, purchased from MCE (MedChemExpress) or other common commercial sources.

SARS-Cov-2 3C-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 Raymond organism.

Preferably, SARS-Cov-2 3C-like protease (concentration is 0.1 mg/mL), substrate peptide Dabcyl-KTSAVLQ ∈SGFRKM-E (Edans) -NH2 (concentration is 0.1mM-5 mM), borax borate buffer (pH=5-8) at a concentration of 10-100 mmol/L.

The invention needs to use a fluorescence enzyme-labeled instrument for fluorescence detection.

Example 1: determination of SARS-Cov-2 3C-like protease inhibitory Activity by IOWH-032

The specific implementation process is as follows:

1) Both SARS-Cov-2 3C-like protease and substrate peptide stock solutions were stored in a-80℃refrigerator;

2) Melting SARS-Cov-2 3C-like protease (concentration of 0.1 mg/mL) in a cryoprotectant (-4-4deg.C) at room temperature, diluting 1 μl into 98 μl borax borate buffer (pH=7.4), and adding into detection plate;

3) Adding 1 μl of inhibitor (IOWH-032, concentrations of 0,0.025mM,0.125mM,0.25mM,0.5mM,1mM, and 2.5 mM) to the solution obtained in step (2);

4) Adding 1 mu L of substrate peptide (0.5 mM) with the same concentration into the solution obtained in the step (3), incubating at 37 ℃ by using a fluorescence microplate reader, monitoring fluorescence emission value at 496nm by using the fluorescence microplate reader under 342nm excitation, and detecting at the same time when incubating, wherein each 1 minute is taken;

5) The effect of the slow increase in fluorescence intensity of the substrate peptide over time in the presence of various concentrations of inhibitor in enzyme metabolism is shown in FIG. 1 below, which shows that the increase in fluorescence of the substrate peptide in enzyme metabolism can be inhibited by inhibitor IOWH-032, and that this inhibition increases with increasing inhibitor concentration.

Example 2: determination of the ability of IOWH-032 to inhibit SARS-Cov-2 3C-like protease

The specific implementation process is as follows:

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

5) Fluorescence emission values at 496nm were counted for 342nm excitation before and after incubation of each group. Taking the fluorescence change value before and after incubation of the control group (inhibitor concentration 0 group) as 100, the residual activity value (residual activity) was obtained by comparing the fluorescence change value before and after incubation of the different inhibition groups. The IC of the inhibitor was obtained by plotting the logarithmic value of the inhibitor concentration (logC (Inhibitor)) on the abscissa and the corresponding residual activity value (Residual Activity) on the ordinate using GraphPad Prism6 software, the results being shown in FIG. 2 ₅₀ Values. Table 1 lists the activity data of compound 1 against SARS-Cov-2 3CLpro inhibition. IC (integrated circuit) ₅₀ The formula for the calculation is y=100/(1+10 ((X-LogIC 50))), where Y represents the remaining activity fraction, X represents the usual logarithm of the concentration of inhibitor compound, Λ refers to the power algorithm. FIG. 2 is a graph showing the ratio of various inhibitor concentrations to the inhibition of enzyme activity, and the inhibition capacity of the compound can be obtained, expressed as the concentration of the inhibitor when the inhibition of enzyme activity is half.

Table 1: IOWH-032 structure and IC for inhibiting novel coronavirus 3C-like protease (3 CLpro) ₅₀ Value of

It can be seen that the above compounds have very obvious inhibitory effect on novel coronavirus 3C-like protease, and IC ₅₀ A value of 6.664. Mu.M indicates that IOWH-032 is effective in inhibiting the activity of novel coronavirus 3C-like protease. Furthermore, based on sequence analysis, coronavirus 3C-like proteases have a high similarity, and therefore, such compounds are also capable of effectively inhibiting the activity of other coronavirus 3C-like proteases, especially for SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1.

In addition, examples 1-2 were performed on the following compounds having a structure similar to IOWH-032 (the procedure and conditions were the same as in the above examples, except that the following compounds were used in place of the inhibitors, respectively), and were found to have no inhibition of 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

1. The application of the compound IOWH-032 in preparing the anti-coronavirus medicine is characterized in that the coronavirus is novel coronavirus SARS-CoV-2,

。

2. the use according to claim 1, wherein the IOWH-032 inhibits the enzymatic activity of the coronavirus main protease 3 Clpro.