CN115813929A

CN115813929A - Application of S63845 in preparation of anti-influenza virus infection medicines

Info

Publication number: CN115813929A
Application number: CN202211424357.3A
Authority: CN
Inventors: 潘攀; 李永奎; 刘雨青; 肖珩; 刘金彪; 陈路苗
Original assignee: First Affiliated Hospital of Jinan University
Current assignee: First Affiliated Hospital of Jinan University
Priority date: 2022-11-15
Filing date: 2022-11-15
Publication date: 2023-03-21
Anticipated expiration: 2042-11-15
Also published as: CN115813929B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of S63845 in preparation of an anti-influenza virus infection medicine. The invention proves that S63845 can resist influenza virus infection at a cellular level and an animal level, and S63845 inhibits influenza virus replication in cells in a dose-dependent manner; s63845 can inhibit the expression of virus N protein in cells besides inhibiting the virus replication in cells; s63846 can inhibit weight loss and mortality of mice caused by influenza virus infection; s63845 was also able to inhibit influenza virus replication and expression of viral NP protein in lung tissue. Based on the method, the small molecular compound which is safe, efficient and small in toxic and side effects is provided for clinical treatment of influenza viruses.

Description

Application of S63845 in preparation of anti-influenza virus infection medicines

Technical Field

The invention relates to the technical field of medicines, and particularly relates to application of S63845 in preparation of an anti-influenza virus infection medicine.

Background

Influenza viruses belong to the family Orthomyxoviridae (Orthomyxoviridae), the genus influenza. Including human influenza viruses and animal influenza viruses. Influenza viruses are classified into three types, a, B and C, according to the difference in antigenic and genetic properties between the viral Nucleoprotein (NP) and the matrix protein (M). The influenza A virus whole genome consists of 8 single-stranded negative-strand RNAs of different sizes. Influenza a viruses are further divided into 17H (H1-H17) and 10N (N1-N10) subtypes, depending on the Hemagglutinin (HA) and Neuraminidase (NA) glycoproteins on the surface of the virion. Human influenza viruses are predominantly of the H1, H2 and H3 subtypes. Most of the highly pathogenic avian influenza viruses are H5, H7 and H9 subtypes, and the lethality rate is highest in the H5N1 subtype. Influenza B viruses are found only in humans and seals, and generally cause only local epidemics. Influenza C virus can infect humans and pigs, mostly in a loose form, mainly infect infants, and generally does not cause pandemics.

Influenza viruses, through their surface spikes HA recognize and bind to sialic acid receptors on the surface of host cells, and enter the host cells as endocytoses. Under the acidic pH condition of endocytosis, the conformation of virus HA protein is changed, so that the virus envelope and cell membrane are fused. The low pH environment causes the release of the virion's nucleic acids into the cytoplasm of the infected cell, followed by transfer into the nucleus for genomic replication and transcription, and the resulting transcribed mRNA is transferred from the nucleus to the cytoplasm and translated into the viral structural and non-structural proteins. The virus nucleic acid and protein are assembled into new virus particles, and the newly generated progeny virus hydrolyzes glycoprotein on the cell surface through Neuraminidase (NA), so that the virus particles are released from the budding sites, and the whole life cycle is completed.

Influenza a viruses are zoonotic infectious disease pathogens and can be transmitted and mutated continuously among multiple animal hosts. The emergence of new viral strains may lead to the development of pandemic diseases in humans. The most alarming is the generation of new variant strains derived from other species (e.g. the generation of swine influenza H1N1 virus), thereby causing worldwide outbreaks of epidemics. Several antiviral compounds have been developed and used in clinical influenza prevention and treatment. There are four anti-influenza drugs currently approved by the FDA for marketing: amantadine, rimantadine, oseltamivir, zanamivir. The first two are M2 ion channel inhibitors that inhibit viral replication by inhibiting release of viral nucleic acid into the cytoplasm; the latter two belong to inhibitors of NA activity, which inhibit viral replication by inhibiting the release and spread of viral particles. However, most of these drugs have limited efficacy due to drug toxicity and resistance of viral mutants. Moreover, when a new epidemic outbreak occurs, the identification of a new virus strain and the identification and distribution of preventive or therapeutic drugs are delayed for a long time, and therefore, the development of drugs for influenza virus infection is imminent.

S63845 is a novel, selective inhibitor of the cell anti-apoptotic protein (MCL-1). S63845 induced death of cancer cell lines by inhibiting MCL-1, and thus had potent antitumor activity. In the mouse experiment, S63845 was well tolerated without significant weight loss. Through the examination of related data, no report that S63845 can inhibit the replication of influenza virus is found.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an anti-influenza virus medicament, which comprises an effective component S63845 and a pharmaceutically acceptable carrier, wherein the S63845 has an anti-influenza virus effect.

Further, the anti-influenza virus medicament is a solid preparation, preferably the solid preparation comprises a tablet, a capsule, a pill, an injection preparation, a sustained release preparation or a controlled release preparation.

S63845 application in preparation of medicines for preventing and/or treating diseases caused by influenza virus infection.

Further, S63845 treats diseases caused by influenza virus infection by inhibiting influenza virus replication or inhibiting expression of the viral NP protein.

Further, S63845 was able to inhibit influenza virus replication or influenza virus NP protein expression in lung tissue.

Further, S63845 inhibited influenza virus replication in cells dose-dependently.

Further, S63846 can inhibit weight loss caused by influenza virus infection.

Further, S63846 may be used to improve survival after influenza virus infection.

In summary, the advantages and positive effects of the invention are:

1. the structure of the medicine is relatively simple;

2. the medicine can play a role in resisting new coronavirus under the condition of low concentration of 3 mu M;

3. the medicine has low use cost and is simple and convenient to use;

4. the drug has no obvious side effects at the cellular and animal level.

Drawings

FIG. 1A S63845 inhibition of copy number of influenza viruses in A549 cells

FIG. 1B S63845 inhibition of NP protein expression levels in A549 cells

FIG. 2 is a schematic representation of a dose-dependent assay of S63845 for inhibiting influenza virus replication in A549 cells.

FIG. 3 is a schematic diagram of the detection of the change in body weight of a mouse.

FIG. 4 is a graph showing the survival rate of a mouse.

FIG. 5A S63845 inhibits copy number of influenza viruses in lung tissue

FIG. 5B S63845 inhibition of the expression level of NP protein in Lung tissue

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

Example 1

S63845 against influenza virus infection at cellular level

In this example a549 cells were purchased from american model culture collection (ATCC) and stored by the laboratory. Influenza virus (H1N 1, PR8 strain), was stored in the laboratory.

DMEM medium in this example was purchased from GIBCO; GAPDH antibodies were purchased from sigma; IAV-NP antibodies were purchased from GeneTex; s63845 from Selleck.

The experimental procedure in this example is as follows:

a549 cells were treated with the addition of the specific inhibitor S63845 to MCL-1 (3 μ M) for 4 hours, and then influenza virus (MOI = 0.1) infected cells were added for 48 hours. The copy number of influenza virus in the cell supernatant was detected by a method using fluorescent absolute quantitative PCR. The template used was the NP gene of influenza virus, and the primer sequences were as follows: forward:5 'ATCACTCACTGAGTGACATC-3', reverse 5 'TCGTCCAATTCCACCAACA-3'.

The experimental results show that: the copy number of influenza virus in the cell supernatant was significantly reduced after S63845 treatment compared to the control-treated cells (as shown in fig. 1A), and similarly, the western-blot results also demonstrated a significant reduction in the expression level of NP protein in the cells (as shown in fig. 1B).

In addition, applicants treated a549 cells for 4 hours with different concentrations of S63845 (0, 0.3 μ M, 3 μ M, and 30 μ M, respectively), and then infected cells with influenza virus (MOI = 0.1) for 48 hours. The copy number of influenza virus in the cell supernatant was detected by a method using fluorescent absolute quantitative PCR. The template used was the NP gene of influenza virus, and the primer sequences were as follows: forward:5 'ATCACTCACTGAGTGACATC-3', reverse:5 'TCGTCCAATTCCACAATCA-3'.

The experimental results show that: s63845 inhibited the copy number of influenza virus in cell supernatants in a dose-dependent manner (fig. 2).

Example 2S63845 against influenza Virus infection at animal level

In this example, 4 to 6 week old C57BL/6 male mice were purchased from Guangdong provincial medical laboratory animal center. Influenza virus (H1N 1, PR8 strain), was stored in this laboratory.

GAPDH antibodies in this example were purchased from sigma; IAV-NP antibodies were purchased from GeneTex; s63845 from Selleck.

The experimental procedure for this example is as follows:

a total of 32C 57BL/6 males aged 4 to 6 weeks were randomly assigned to 4 groups of 8 mice each.

Group 1 mice were injected intraperitoneally with 200. Mu.l of S63845 (12.5 mg/kg), and after 90 minutes, with 50. Mu.l PBS by nasal drip, the group was named ctrl + S63845;

group 2 mice were injected intraperitoneally with 200. Mu.l of S63845 (12.5 mg/kg), and 90 minutes later with 50. Mu.l of influenza virus (1 LD 50) by nasal drip, and the group was designated IAV + S63845;

group 3 mice were injected intraperitoneally with 200 μ l of control solvent (100 μ l PEG300 plus 100 μ l PBS) and after 90 minutes, injected nasally with 50 μ l PBS, which was designated ctrl + PEG300;

group 4 mice were injected intraperitoneally with 200 μ l of control solvent (100 μ l of PEG300 plus 100 μ l of PBS) and, after 90 minutes, injected nasally with 50 μ l of influenza virus (1 LD 50), and the group was designated IAV + PEG300.

After 4 days, mice in

groups

1 and 2 were again injected intraperitoneally with 200. Mu.l of S63845 (12.5 mg/kg), and mice in

groups

3 and 4 were again injected intraperitoneally with 200. Mu.l of control solvent (100. Mu.l of PEG300 plus 100. Mu.l of PBS). The weights of the above 4 groups of mice were weighed daily from the time of infection with influenza virus, and the survival rates of the mice were recorded.

The experimental results show that: there was no significant change in ctrl + S63845 body weight (fig. 3) and no change in survival (fig. 4) in group 1 mice compared to group 3 ctrl + PEG300 mice. S63845 was shown to have no effect on mouse body weight and survival. Group 4 IAV + PEG300 mice showed weight loss from day two to day 9 (figure 3) and death of mice beginning on day 7 and finally 66.7% mortality in group 4 mice compared to group 3 ctrl + PEG300 mice (figure 4). The results prove that the influenza virus infected mouse model is successfully constructed. Group 2 IAV + S63845 mice lost less weight compared to group 4 IAV + PEG300 mice (fig. 3), with 1 mouse death occurring only on day 9 with a mortality rate of 16.7% (fig. 4).

The above results demonstrate that S63846 can inhibit the occurrence of weight loss and mortality in mice caused by influenza virus infection.

On day 7 of influenza virus infection, the applicant collected lung tissues of the 4 groups of mice and examined them. The copy number of influenza virus in lung tissue was detected using fluorescence absolute quantitative PCR. The template used was the NP gene of influenza virus, and the primer sequences were as follows: forward:5 'ATCACTCACTGAGTGACATC-3', reverse 5 'TCGTCCAATTCCACCAACA-3'. The experimental results show that: compared with the control group of mice (IAV + PEG 300), the copy number of the influenza virus in the lung tissue is remarkably reduced after the S63845 treatment (as shown in figure 5A), and the western-blot result also proves that the expression level of the NP protein in the cells is also remarkably reduced (as shown in figure 5B).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

The application of S63845 in preparing medicines for preventing and/or treating diseases caused by influenza virus infection;

the molecular formula of S63845 is: c ₃₉ H ₃₇ ClF ₄ N ₆ O ₆ S；

The structural formula of S63845 is as follows:
2. the use of claim 1, wherein S63845 is used for treating diseases caused by influenza virus infection by inhibiting replication of influenza virus or inhibiting expression of viral NP protein.
3. The use of claim 1, wherein S63845 is capable of inhibiting influenza virus replication or influenza virus NP protein expression in lung tissue.
4. The use of claim 1, wherein S63845 inhibits influenza virus replication in cells in a dose-dependent manner.
5. The use of claim 1, wherein S63846 inhibits weight loss due to influenza virus infection.
6. The use of claim 1, wherein S63846 is used to increase survival after infection with influenza virus.
7. An anti-influenza virus medicine comprises an effective component S63845 and a pharmaceutically acceptable carrier, wherein the S63845 has an anti-influenza virus effect.
8. The anti-influenza virus agent of claim 7, which is in the form of a tablet, capsule, pill, injectable formulation, sustained release or controlled release formulation.
9. The anti-influenza virus agent according to claim 7, which can inhibit weight loss caused by influenza virus infection.
10. The anti-influenza virus agent of claim 7, which is useful for increasing survival after influenza virus infection.