CN114668751A

CN114668751A - Application of disulfiram in resisting infection of human coronavirus NL63

Info

Publication number: CN114668751A
Application number: CN202210285908.6A
Authority: CN
Inventors: 王泽方; 张晓宇; 肖云杰; 封守琴; 朱倩
Original assignee: TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
Current assignee: TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2022-06-28

Abstract

The invention provides a papain-like protease 2 (PL) aiming at Human Coronavirus NL63(Human Coronavir NL63, HCoV-NL63)^pro2) The compound is disulfiram and has obvious inhibitory activity on papain 2 in human coronavirus NL63, so that the compound provided by the invention can be used for preparing a small molecule inhibitor for the papain 2 of human coronavirus NL63, and is expected to be a potential drug for resisting HCoV-NL63 infection.

Description

Application of disulfiram in resisting infection of human coronavirus NL63

Technical Field

The invention relates to the technical field of pharmacy, in particular to application of disulfiram in resisting infection of human coronavirus NL 63.

Background

Shortly after the SARS outbreak in 2003, a novel coronavirus, HCoV-NL63, was isolated in a hospital in the netherlands and assigned to the alphacoronavirus by researchers. HCoV-NL63 is found in Europe, Asia, North America and oceania mostly, and generally causes upper respiratory diseases such as fever, cough, sore throat and rhinitis, and can cause lower respiratory diseases such as bronchiolitis and bronchiolitis in severe cases, even cause life threatening diseases, and can cause death of patients within 5 days of illness at the fastest speed. Furthermore, in children infected with HCoV-NL63, the incidence of croup was 6.6 times that of uninfected children. Also, studies have shown that HCoV-NL63 may be associated with Kawasaki disease pathogenesis. In the context of gastrointestinal disease, although HCoV-NL63 was detected in stool samples from patients with partial diarrhea, there is no clear evidence that the viral infection is associated with diarrhea, with only minor etiology in childhood acute gastroenteritis. HCoV-NL63 has become one of the important pathogens causing respiratory tract infection of human, but no specific drug aiming at the virus exists at present, so the research on the inhibitor thereof is carried out and the corresponding drug is urgently found.

The viral protease is essential for its pathogenesis and virulence, and the genome of human coronavirus NL63 encodes a cysteine protease, papain-2 (HCoV-NL63 PL)^pro2) The two proprotein pp1a and pp1ab of the replicase complex encoded by coronavirus ORF1 and ORF1b are cleaved, so that the host function is regulated to promote virus infection, and the proprotein plays an important role in the transcription and replication processes of the virus. HCoV-NL63 PL ^pro2 can cleave the non-structural proteins nsp2| nsp3 and nsp3| nsp4 sites, releasing nsp2 and nsp 3. HCoV-NL63 PL ^pro2 also can make the virus evade host immune response, promote virus replication through Deubiquitinating (DUB), deisgylating and antagonistic interferon effects. Therefore, papain 2 is a key drug target against human coronavirus NL63, so inhibitor screening of papain 2 for relevant drug development against human coronavirus NL63 infection is of great significance.

Disulfiram, known as Disulfiam, can irreversibly inhibit acetaldehyde dehydrogenase by in vivo metabolites, so that the concentration of acetaldehyde in the blood of a drinker is increased, and strong discomfort is generated due to the increased acetaldehyde concentration, and the drinker can feel stronger discomfort after drinking again after taking the medicine, so that the Disulfiram is clinically used as an anti-alcohol drug at present. However, disulfiram has not been used to date for the exploration and study of infection with the human coronavirus NL 63.

Disclosure of Invention

In response to the problems in the related art, the present invention provides the use of disulfiram in the treatment of infection with human coronavirus NL 63.

The invention also provides inhibitors against papain 2 in human coronavirus NL 63.

The disulfiram CAS number of the invention is 97-77-8, and is purchased from Shanghai Michelin Biotechnology, Inc. On the molecular level, by setting up a negative control, the disulfiram has good inhibitory activity on papain 2 in human coronavirus NL63, so that the compound is expected to be a potential drug for inhibiting the infection of the human coronavirus NL 63.

The invention provides a medicament for preventing or treating human coronavirus NL63, which comprises disulfiram as an active ingredient and one or more pharmaceutically acceptable carriers. The carrier comprises a diluent, a surfactant, an adsorption carrier, a lubricant and a synergist which are conventional in the pharmaceutical field. The medicine can be made into tablet, pill, capsule, suspension or emulsion. The administration route is oral.

The invention has the advantages and positive effects that:

the present invention is directed to an inhibitor of papain 2 in human coronavirus NL63, which inhibitor is disulfiram. Disulfiram has a significant inhibitory effect on the activity of papain 2 in human coronavirus NL 63.

Drawings

FIG. 1 is a schematic representation of the inhibitory effect of disulfiram on papain 2 in human coronavirus NL 63.

FIG. 2 IC of disulfiram on papain 2 in human coronavirus NL63₅₀Schematic diagram of the measurement of (1).

FIG. 3 is a schematic representation of the type of inhibitor of papain 2 in human disulfiram against human coronavirus NL 63.

The specific implementation mode is as follows:

in order to better illustrate the present invention, specific embodiments thereof will be described in detail below.

1. Expression and purification of papain 2 from human coronavirus NL63

(1) Will contain the code HCoV-NL63 PL^pro2-Gene pET-28a (+) vector was transformed into Escherichia coli BL21(DE3) strain, and positive clones were selected by kanamycin resistance.

(2) Positive clones were picked up on a kanamycin-resistant plate, and after adding 5mL of LB liquid medium containing 50mg/mL of kanamycin, they were cultured at 37 ℃ for 6 hours, then transferred to 0.8L of LB medium, and after further culturing at 37 ℃ for 6 hours, 400. mu.L of 1mM IPTG (isopropyl-. beta. -D-thiogalactopyranoside) was added, and further culturing at 16 ℃ for 16 hours.

(3) Centrifuging at 4000rpm for 30min, collecting bacterial liquid, and breaking the bacterial liquid under high pressure; and centrifuging the bacteria solution after bacteria breaking at 12000rpm for 1h, and collecting supernatant.

(4) The supernatant was applied to a disruption buffer (20mM Tris-HCl,250mM NaCl, pH 8.2) pre-equilibrated Ni-NTA affinity column and the protein was initially purified by Ni-NTA affinity chromatography.

(5) And (3) carrying out heteroprotein washing on the medium containing the target protein by using a washing heteroprotein buffer containing 20mM imidazole, eluting by using an elution buffer containing 250mM imidazole, collecting the target protein, and purifying by using anion exchange chromatography and gel filtration chromatography to obtain the stable and uniform target protein.

2.HCoV-NL63 PL ^pro2 Activity measurement

RLRGG-AMC polypeptide substrate with a purity of greater than 95% (available from King-Share Biotechnology, Inc.); the fluorescence measuring instrument is a VarioSkan Flash full-wavelength microplate reader (ThermoFisher Scientific), 360nm excitation light and 460nm emission light are set, and a fluorescence signal is detected. The protein buffer component was 50mM Tris-HCl, pH 7.5, buffered HCoV-NL63 PL^pro2 (final concentration 1.6. mu.M), disulfiram (final concentration 20. mu.M) was added, the incubation was carried out at room temperature for 2min, and the substrate RLRGG-AMC was added rapidly at a concentration of 160. mu.M. The fluorescence value was measured by shaking at 300rpm for 5 s. All assays were done in triplicate and appropriate positive and negative controls were maintained throughout the experiment.

3. Compound disulfiram IC₅₀Measurement of (2)

HCoV-NL63 PL ^pro2 at a final concentration of 1.6. mu.M, and the substrate RLRGG-AMC at a final concentration of 160. mu.M. We first set 11 inhibitor concentrations based on the screening results, with final concentrations of disulfiram of 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, 3. mu.M, 1. mu.M, 0.75. mu.M, 0.5. mu.M, 0.25. mu.M, 0.125. mu.M, 0.0625. mu.M, respectively. HCoV-NL63 PL ^pro2 and disulfiram are incubated for 2min at room temperature, 10. mu.L of substrate are added, and the time and fluorescence curve is recorded. Analysis of HCoV-NL63 PL Using Graphpad prism 9 software ^pro2, obtaining the relation curve of disulfiram concentration and residual activity at the initial reaction rate, and finally obtaining IC₅₀The value is obtained.

4. Determination of the type of disulfiram inhibitor of the Compound

Disulfiram was formulated at concentrations of 0. mu.M, 0.75. mu.M, 1.5. mu.M, respectively. Preparing HCoV-NL63 PL with different concentrations by using enzyme active buffer ^pro2 at concentrations of 0. mu.M, 0.375. mu.M, 0.75. mu.M, 1.5. mu.M, 2. mu.M and 2.5. mu.M. The time versus fluorescence change was recorded as described for the activity assay. The initial rate of protease light absorption reaction is obtained by Graphpad prism 9 software, and 3 groups of data are parallelly measured to obtain the type of the inhibitor.

The invention relates to the technical field of pharmacy, in particular to application of disulfiram in resisting infection of human coronavirus NL63, wherein the inhibition rate of disulfiram in inhibiting human coronavirus NL63 papain-like protease 2 is more than 80%, the disulfiram has great application potential in preparing a small molecule inhibitor of the human coronavirus NL63 papain-like protease 2, and the disulfiram is expected to become a potential drug for resisting infection of human coronavirus NL 63.

The methods used above are those commonly used in the art unless otherwise specified.

The present invention is not limited to the above embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of disulfiram for the treatment of an infection with human coronavirus NL 63.

2. Disulfiram is a small molecule inhibitor of papain 2 in human coronavirus NL 63.

3. Use according to claim 1 or 2, wherein disulfiram has the formula:

4. the medicament for treating infection by human coronavirus NL63 according to claim 1 or 2, which is prepared by mixing, granulating, drying, granulating, bulk-mixing and tabletting.

5. A medicament for the treatment of infection with human coronavirus NL63, characterized in that it comprises disulfiram according to claim 1 or 2 and one or more pharmaceutically acceptable carriers; the carrier comprises a diluent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and a synergist which are conventional in the pharmaceutical field.

6. The pharmaceutical composition according to claim 1 or 2, characterized in that: the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.

7. A medicament according to claim 1 or 2 for the treatment of infection by human coronavirus NL63, characterized in that the pharmaceutical composition is an oral or injectable formulation; the oral preparation comprises: at least one of tablets, granules, dripping pills, soft capsules, suspensions, solutions, and syrups; the injection comprises: at least one of lyophilized powder, solution injection, suspension injection, and emulsion injection.