CN115813929B

CN115813929B - Application of S63845 in preparation of anti-influenza virus infection medicines

Info

Publication number: CN115813929B
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: 2023-07-21
Anticipated expiration: 2042-11-15
Also published as: CN115813929A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of S63845 in preparation of anti-influenza virus infection medicines. The invention proves that S63845 can resist influenza virus infection at the cellular level and the animal level, and S63845 inhibits replication of influenza virus in cells in a dose-dependent manner; s63845 inhibits the expression of viral N proteins in cells in addition to inhibiting viral replication in cells; s63846 can inhibit the occurrence of weight loss and mortality in mice caused by influenza virus infection; s63845 also inhibits replication of influenza virus and expression of viral NP proteins in lung tissue. Based on the above, the invention provides a safe and efficient small molecular compound with small toxic and side effects for treating influenza virus clinically.

Description

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

Technical Field

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

Background

Influenza viruses belong to the Orthomyxoviridae (Orthomyxoviridae), the genus influenza virus. Including human influenza virus and animal influenza virus. Influenza viruses are classified into A, B, C types according to the antigenic and genetic properties of viral Nucleoprotein (NP) and matrix protein (M). Influenza a virus whole genome consists of 8 single-stranded negative-strand RNAs of unequal size. Influenza A viruses can be further divided into 17H (H1-H17) and 10N (N1-N10) subtypes, depending on the glycoprotein Hemagglutinin (HA) and Neuraminidase (NA) on the surface of the virion. Human influenza viruses are predominantly of the H1, H2 and H3 subtypes. Highly pathogenic avian influenza viruses are mostly of the H5, H7 and H9 subtypes, with the highest mortality rate of the H5N1 subtype. Influenza B viruses are only found in humans and seals, and generally cause only local epidemics. Influenza C viruses can infect humans and pigs, are mostly in a sporadic form, and primarily infect infants, generally without causing pandemic.

Influenza viruses recognize and bind to sialic acid receptors on the surface of host cells via their surface spike HA, and enter the host cells as endocytoses. Under the acidic pH condition of endocytosis, the conformation of the virus HA protein is changed, so that the virus envelope and the cell membrane are fused. The low pH environment releases the viral particle nucleic acid into the cytoplasm of the infected cell, and then transfers into the nucleus for replication and transcription of the genome, from which transcribed mRNA is transferred into the cytoplasm, translating out viral structural and non-structural proteins. The viral nucleic acids and proteins assemble into new virions, and the newly generated progeny viruses hydrolyze the cell surface glycoproteins by Neuraminidase (NA), causing the virions to be released from the budding site, completing the entire life cycle.

Influenza a viruses are infectious disease pathogens of human and animal co-morbid origin and can be transmitted and mutated continuously among multiple animal hosts. The emergence of new strains may lead to the occurrence of human pandemic diseases. Most alarming is the generation of new variant strains from other species (e.g., the generation of swine influenza H1N1 virus), thereby causing outbreaks of epidemic disease worldwide. Some antiviral compounds have been developed and have been used in clinical influenza prevention and treatment. There are four anti-influenza drugs currently approved by the FDA for sale: amantadine, rimantadine, oseltamivir, zanamivir. The first two belong to M2 ion channel inhibitors, inhibit viral replication by inhibiting release of viral nucleic acids into the cytoplasm; the latter two are NA activity inhibitors, 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 to viral mutants. In addition, when a new epidemic situation is exploded, the identification of a new virus strain and the identification and distribution of a preventive or therapeutic drug are delayed for a long time, so that the development of influenza virus infection drugs is urgent.

S63845 is a novel, selective inhibitor of cell anti-apoptotic protein (MCL-1). S63845 induces death of cancer cell lines by inhibiting MCL-1, and thus has potent antitumor activity. In the mouse experiments, S63845 was well tolerated with no significant weight loss. After looking at the relevant data, no relevant report was found that S63845 could inhibit influenza virus replication.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides an anti-influenza virus drug, which comprises an active ingredient S63845 and a pharmaceutically acceptable carrier, wherein S63845 has an anti-influenza virus effect.

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

Use of S63845 for the manufacture of a medicament for the prevention and/or treatment of a disease caused by influenza virus infection.

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

Further, S63845 is capable of inhibiting influenza virus replication or influenza virus NP protein expression in lung tissue.

Further, S63845 is dose dependent in inhibiting replication of influenza virus in cells.

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

Further, S63846 can be used to increase survival after influenza infection.

In summary, the invention has the advantages and positive effects that:

1. the medicine structure is relatively simple;

2. the medicine can play the role of resisting influenza virus 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 inhibits copy number of influenza Virus in A549 cells

FIG. 1B S63845 inhibits the expression level of NP protein in A549 cells

FIG. 2 is a schematic diagram showing a dose-dependent detection of S63845 for inhibiting replication of influenza virus in A549 cells.

FIG. 3 is a schematic diagram showing the detection of weight change in mice.

FIG. 4 is a graph showing the detection of survival rate of mice.

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

FIG. 5B S63845 inhibits the expression level of NP protein in lung tissue

Detailed Description

The present invention will be described in further detail with reference to the following examples, in which the apparatus and reagents used in the respective examples and test examples are commercially available unless otherwise specified, in order to make the objects, technical schemes and advantages of the present invention more apparent. The specific embodiments described herein are to be considered in an illustrative sense only and are not intended to limit the invention.

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 or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific 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 within the scope of the following claims.

Example 1

S63845 is resistant to influenza infection at the cellular level

A549 cells in this example were purchased from American Type Culture Collection (ATCC) and were kept by the present laboratory. Influenza virus (H1N 1, PR8 strain) was kept by the laboratory.

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

The experimental procedure in this example is as follows:

a549 cells were treated with MCL-1 specific inhibitor S63845 (3 μm) for 4 hours and infected 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 fluorescence absolute quantitative PCR. The template used was the NP gene of influenza virus, the primer sequences were as follows: forward:5'-ATCACTCACTGAGTGACATC-3', reverse 5'-TCGTCCAATTCCACCAATCA-3'.

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

In addition, the applicant treated a549 cells with different concentrations of S63845 (0, 0.3 μm, 3 μm and 30 μm, respectively) for 4 hours, and added influenza virus (moi=0.1) to infect the cells for 48 hours. The copy number of influenza virus in the cell supernatant was detected by a method using fluorescence absolute quantitative PCR. The template used was the NP gene of influenza virus, the primer sequences were as follows: forward:5'-ATCACTCACTGAGTGACATC-3', reverse 5'-TCGTCCAATTCCACCAATCA-3'.

Experimental results the 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

C57BL/6 male mice of 4 to 6 weeks of age in this example were purchased from the medical laboratory animal center in Guangdong province. Influenza virus (H1N 1, PR8 strain) was kept by the laboratory.

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

The experimental procedure of this example is as follows:

a total of 32C 57BL/6 male mice of 4 to 6 weeks of age were randomly divided into 4 groups of 8 mice each.

Group 1 mice were given the designation ctrl+s63845 by intraperitoneal injection of 200 μl S63845 (12.5 mg/kg) and, after 90 minutes, by nasal drip injection of 50 μl pbs;

group 2 mice were given the designation iav+s63845 by intraperitoneal injection of 200 μl S63845 (12.5 mg/kg) and, after 90 minutes, by nasal drip injection of 50 μl influenza virus (1 LD 50);

group 3 mice, designated ctrl+PEG300, were injected with 200 μl of control solvent (100 μl of PEG300 plus 100 μl of PBS) intraperitoneally, and after 90 minutes, with 50 μl of PBS by nasal drip;

group 4 mice, designated iav+peg300, were injected by intraperitoneal injection of 200 μl of control solvent (100 μl PEG300 plus 100 μl PBS) and, after 90 minutes, by nasal drip injection of 50 μl influenza virus (1 LD 50).

After 4 days, groups 1 and 2 mice were re-injected intraperitoneally with 200. Mu.l of S63845 (12.5 mg/kg), groups 3 and 4 mice were re-injected intraperitoneally with 200. Mu.l of control solvent (100. Mu.l of PEG300 plus 100. Mu.l of PBS). The weights of the 4 groups of mice were weighed daily from the start of infection with influenza virus, and the survival rate of the mice was recorded.

The experimental results show that: there was no significant change in body weight of group 1 mice ctrl+s63845 (fig. 3) and no change in survival (fig. 4) compared to group 3 ctrl+peg300 mice. S63845 proved to have no effect on mouse body weight and survival. Group 4 iav+peg300 mice showed a decrease in body weight from day two to day 9 to the lowest (fig. 3) and mice died from day 7, with a final mortality rate of 66.7% for group 4 mice compared to group 3 ctrl+peg300 mice (fig. 4). The above results demonstrate that the influenza virus infected mouse model was successfully constructed. The reduction in body weight was slowed in group 2 iav+s63845 mice compared to group 4 iav+peg300 mice (fig. 3), with 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.

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

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

Use of s63845 in the manufacture of a medicament for the prevention and/or treatment of a disease caused by influenza virus infection;

the molecular formula of S63845 is: c (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, S63845 treats a disease caused by an influenza infection by inhibiting influenza replication or inhibiting viral NP protein expression.
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 replication of influenza virus in a cell in a dose dependent manner.
5. The use of claim 1, wherein S63846 inhibits weight loss caused by influenza virus infection.
6. The use of claim 1, S63846 can be used to increase survival after influenza infection.