CN115804775A

CN115804775A - Application of S63845 in preparation of anti-neocoronavirus infection medicine

Info

Publication number: CN115804775A
Application number: CN202211426549.8A
Authority: CN
Inventors: 潘攀; 申苗苗; 陈俊; 葛威威; 李永奎
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: 2023-03-17
Anticipated expiration: 2042-11-15
Also published as: CN115804775B

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of S63845 in preparation of a medicine for resisting new coronavirus infection. The invention provides an application of S63845 and pharmaceutically acceptable salts thereof in preparing a medicament for preventing and/or treating diseases caused by coronavirus. The inventor finds that S63845 inhibits the replication of new coronavirus and inhibits coronavirus N protein to regulate apoptosis, and S63845 inhibits the interaction of new coronavirus N protein specificity and anti-apoptosis protein MCL-1, so S63845 is a candidate drug for treating diseases caused by coronavirus.

Description

Application of S63845 in preparation of anti-neocoronavirus infection medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to a new application of an S63845 compound.

Background

Coronaviruses (Coronavir) are the largest positive-strand RNA viruses known at present, are important pathogenetic sources of vertebrates, and have a wide infection range. Coronaviruses can infect the respiratory, gastrointestinal, hepatic and central nervous systems of humans, livestock, birds, bats, mice and other wild animals. The sequence alignment analysis shows that SARS-CoV-2 has typical coronavirus genome, belongs to beta coronavirus genus and is similar to SARS-CoV, so that SARS-CoV-2 has the basic features of coronavirus. The genome RNA of the new coronavirus is about 30 kb and encodes about 9860 amino acids. The genome comprises 2 flanking untranslated regions (UTRs) with 5' end cap and 3' end polyA tail structure, and 5' end long open reading frame (ORF 1 a/b) occupies two thirds of the whole genome open reading frame. The replicase complex encoded by it is capable of being hydrolyzed by papain (PLpro) and 3C-like protease (3 CLpro) to produce 16 non-structural proteins including NSP3, NSP5, NSP12 (RNA-dependent RNA polymerase), NSP13 (helicase Hel), exonuclease, and other NSPs that may be involved in viral transcription and replication. The long open reading frame at the 3' end mainly encodes a virus structural protein: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N).

The clinical manifestations of the patients infected by the new coronavirus are fever, dry cough, body ache and hypodynamia, and the patients gradually develop dyspnea and viral pneumonia; further progression can lead to multiple organ damage and failure, and even death, in critically ill patients. Although the FDA has now made urgent approval of drugs for treating new corona, such as gimdesivir (Remdesivir), pfeiri (paxlovid) and Molnupiravir (Molnupiravir), these drugs are expensive and effective, and cannot effectively suppress the spread of new corona virus.

Most of the anti-neocorolla drugs in the prior art are nucleotide drugs, the structures are relatively complex, the requirements on technical difficulty, equipment and fields in production are high, so that the drugs are expensive, the currently approved drugs are authorized for emergency use, the requirements on use conditions are high, and certain drug side effects exist.

At present, no specific medicine for the virus exists in China, and the clinical application mainly aims at symptomatic support treatment. Therefore, the clinical medicine for effectively preventing and treating the novel coronavirus infection is studied at all times.

S63845 is a novel, selective inhibitor of the 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 experiment, S63845 was well tolerated with no significant weight loss. After the relevant data are reviewed, no report that S63845 can inhibit the replication of the new coronavirus is found.

Through the above analysis, the problems and defects of the prior art are as follows: at present, the utilization of S63845 is limited, only the anti-tumor drug can be obtained, and based on the advantages of S63845 as the drug and the limited utilization of S63845, the development of a new application of S63845 is expected.

Based on the defects of the existing medicaments for treating the new coronavirus, the research on the mechanism of S63845 for the new coronavirus is hoped to be carried out, so that a theoretical basis is laid for providing the medicaments with small side effect and better treatment effect for treating the new coronavirus.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of S63845 in preparing a medicine for resisting new coronavirus infection, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention aims to provide a new application of a compound S63845.

The compound S63845 has the structural formula shown as follows:

。

in a first aspect, the new application of the compound S63845 provided by the invention is at least one of the following:

s63845 or its pharmaceutically acceptable salt is used for preparing a medicament for preventing and treating novel coronavirus infection;

s63845 or its pharmaceutically acceptable salt is used for preparing products for inhibiting N protein regulation and control apoptosis;

s63845 or its pharmaceutically acceptable salt can be used for preparing products for inhibiting the replication of new coronavirus or inhibiting the protein expression of new coronavirus;

s63845 or its pharmaceutically acceptable salt is used for preparing a product for inhibiting interaction of new coronavirus structural protein N to MCL-1;

in a second aspect, the invention also claims a product as claimed in the invention, the active ingredient of which is S63845, or a pharmaceutically acceptable salt thereof; the product has any one of the following uses: a preventive and therapeutic agent for a novel coronavirus infection; inhibiting apoptosis of N protein regulation cells; inhibiting the replication of new coronavirus; inhibiting the interaction of structural protein N of the new coronavirus on MCL-1.

In the present invention, the product may be a medicament or a pharmaceutical preparation. The product of the invention can also contain a suitable carrier or excipient besides S63845 or the pharmaceutically acceptable salt thereof. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field. The above medicine can be made into various forms such as injection, tablet, powder, granule, capsule, oral liquid, paste, cream, etc.; the medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The invention detects the fluorescence detection and protein expression level of green fluorescent protein GFP and the quantity of virus secreted into cell culture solution. Shows that S63845 has better inhibiting effect on the expression of new coronavirus protein and the replication of virus nucleic acid when the concentration is 3 mu M. By using molecular biology and cell biology experiments, the results show that S63845 exerts antiviral effects by inhibiting the interaction of the novel coronavirus structural protein N on MCL-1. N inhibits apoptosis, N interacts with MCL-1, S638345 inhibits N-inhibited apoptosis, S63845 is an inhibitor of MCL-1, and MCL-1 inhibits apoptosis, based on which it can be deduced that S63845 promotes apoptosis by inhibiting the relationship between N and MCL-1.

The molecular target of the experimental result is clear, and the result shows that S63845 has wide prospect of being developed into an effective medicament for resisting the new coronavirus for clinical application and further developing as a potential antiviral medicament in clinical application.

In summary, the advantages and positive effects of the invention are: compared with the existing treatment means, the S63845 is used as the medicine for treating the new crown, the advantages are that: 1. the structure of the medicine is relatively simple; 2. the action target of the medicine is clear; 3. the medicine can play a role in resisting new coronavirus under the condition of low concentration (3 mu M); 4. the medicine has low use cost and is simple and convenient to use; 5. the drug has no significant side effects at the cellular level.

Drawings

FIG. 1 is a diagram showing the construction and expression verification of SARS-CoV-2 trVLP infection system.

FIG. 2 is a schematic diagram of the detection of the new coronavirus N protein for inhibiting apoptosis.

FIG. 3 is a schematic diagram of the detection of the interaction between the N protein specificity of a novel coronavirus and the anti-apoptotic protein MCL-1.

FIG. 4 is a schematic diagram of detection of S63845 inhibiting N protein regulation apoptosis.

FIG. 5 is a schematic representation of the detection of S63845 inhibition of replication of new coronavirus.

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.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

EXAMPLE 1 construction of SARS-CoV-2 trVLP infection System

Example the new coronavirus genome with replacement of the N protein by the GFP protein (FIG. 1-a) was from a companion gift. The process and sequence for constructing the genome of a novel coronavirus containing GFP instead of N protein has been described in Ju X, zhu Y, wang Y, li J, zhang J, gong M, et al, (2021) A novel cell culture system modeling the SARS-CoV-2 life cycle PLoS Patholog 17 (3): e1009439.

We transcribed this genome into RNA in vitro, and then transfected the RNA into Caco-2 cells stably expressing N protein by electrotransfer, successfully obtained a novel coronavirus deficient in N protein, which was named SARS-CoV-2 trVLP (FIG. 1-b). The trVLP can complete a complete virus replication cycle in Caco-2 cells stably expressing N protein. Since the progeny virus it produces is deficient in the N protein and therefore not infectious, subsequent experiments can be performed in the secondary biological protection laboratory (P2).

Example 2 the novel N protein of coronavirus can significantly inhibit apoptosis

The A549 cells and THP-1 cells used in this example were purchased from American Type Culture Collection (ATCC) and stored in the laboratory. Plasmid pCDNA3.1-N/M/E/3a carrying the novel structural proteins (N, M, E) of the coronavirus as well as the helper protein (3 a) was prepared by the laboratory of the Applicant.

The structure and sequence of plasmid pCDNA3.1 are given in the following website: https:// www.addgene.org/23252-

The gene sequences of the structural proteins N, M, E of the novel coronavirus and the accessory protein 3a are given in the following website: https:// www.ncbi.nlm.nih.gov/nuccore/MN908947.3

Plasmid pCDNA3.1-N/M/E/3a was prepared as follows:

the N, M, E,3a gene was synthesized by Nanjing Kingsler Biotech Co., ltd, and the target gene was amplified by PCR using the synthesized gene as a template with specific primers and cloned into pCDNA3.1 vector.

The primer sequences are as follows:

N: Forward BamHI 5'-GCGGATCCATGTCTGATAATGGACCCCA-3'

Reverse XbaI 5'-GCTCTAGATTAGGCCTGAGTTGAGTCAG-3'

M: Forward BamHI 5'- GCGGATCCATGGCAGATTCCAACGGTA-3'

Reverse EcoRI 5'- GCGAATTCTTACTGTACAAGCAAAGCA-3'

E: Forward BamHI 5'- GCGGATCCATGTACTCATTCGTTTCGG-3'

Reverse XhoI EcoRI 5'- CGCTCGAGTTAGACCAGAAGATCAGGA-3'

3a: Forward BamHI 5'- GCGGATCCATGGATTTGTTTATGAG-3'

Reverse EcoRI EcoRI 5'- GCGAATTCTTACAAAGGCACGCTAGT-3'

DMEM and RPMI1640 medium used in this example was purchased from GIBCO; lipo2000 was purchased from Invitrogen; flag, GAPDH antibody from sigma; caspase-3 antibodies were purchased from CST; staurosporine was purchased from belleck; apoptosis flow assay kits were purchased from BD.

The experimental method in this example is as follows:

plasmid pCDNA3.1-N/M/E/3a (2. Mu.g) was first transfected into A549 cells and THP-1 cells, respectively, using Lipo2000, 48 hours after transfection, cells were stimulated for 4 hours by addition of the apoptosis activator Staurosporine (5. Mu.M).

The experimental results show that: cleavage of mature body protein clev-Caspase-3, a key protein in the apoptotic pathway Caspase-3, can be significantly inhibited by the new coronavirus N protein (as shown in FIG. 2-a).

Example 3 interaction of the novel coronavirus N protein-specific and anti-apoptotic protein MCL-1

A549 cells in this example were purchased from American Type Culture Collection (ATCC) and stored in the laboratory. Plasmids Pcaggs-HA-MCL-1/BCL-XL/BCL-2/BCL-W with anti-apoptotic family proteins (MCL-1, BCL-XL, BCL-2 and BCL-W) were prepared by the Applicant's laboratory.

For the structure and sequence of the plasmid Pcags see the following websites:

https://www.addgene.org/65974/

the gene sequence of MCL-1 is shown in the following:

https://www.ncbi.nlm.nih.gov/nuccore/NM_021960.5

the gene sequence of BCL-XL (BCL 2L 1) is referred to the following website:

https://www.ncbi.nlm.nih.gov/nuccore/NM_138578.3

the gene sequence of BCL-2 is found at the following website:

https://www.ncbi.nlm.nih.gov/nuccore/XM_047437733.1

the gene sequence of BCL-W is found at the following website:

https://www.ncbi.nlm.nih.gov/nuccore/NM_004050.5

the recombinant plasmid process is as follows:

RNA of A549 cells is extracted, the reverse conversion rate is cDNA, a target gene is amplified by a PCR method by using the cDNA as a template and specific primers of MCL-1, BCL-XL, BCL-2 and BCL-W, and the target gene is cloned to a pCAGGS vector.

DMEM medium in this example was purchased from GIBCO; lipo2000 was purchased from Invitrogen; flag, HA antibody was purchased from sigma.

The experimental method in this example is as follows:

plasmid pCDNA3.1-N (3. Mu.g) and plasmid Pcags-HA-MCL-1/BCL-XL/BCL-2/BCL-W (3. Mu.g) were transfected into A549 cells using Lipo2000, and the interaction between the N protein of the novel coronavirus and the anti-apoptotic family protein was investigated by co-immunoprecipitation experiments 24 hours after transfection.

The experimental results show that: only MCL-1 in the anti-apoptotic family protein can interact with the N protein (FIG. 3-a), and similarly, the N protein interacts only with MCL-1 in the anti-apoptotic family protein (FIG. 3-b).

Example 4S63845 inhibits N protein regulated apoptosis

A549 and THP-1 cells in this example were purchased from American Type Culture Collection (ATCC) and stored in the laboratory.

DMEM and RPMI1640 medium were purchased from GIBCO in this example; caspase-3 and MCL-1 antibodies were purchased from CST; GAPDH antibodies were purchased from sigma; protein N antibodies were purchased from abllone: staurosporine and S63845 were purchased from Selleck; apoptosis flow assay kits were purchased from BD.

The experimental method in this example is as follows:

a549 cells and THP-1 cells stably expressing a new coronavirus N protein are respectively constructed by using a lentivirus infection system, MCL-1 specific inhibition S63845 (3 mu M) is added for treating the cells for 4 hours, and an apoptosis activator Staurosporine (5 mu M) is added for stimulating the cells for 4 hours.

Flow cytometry results showed: compared with the control group of cells, after S63845 treatment, the N protein can not inhibit the apoptosis of late-stage cells (PI) ⁺ And Annexin ⁺ Double positive cells, FIG. 4-a, b), as well as N protein, were unable to inhibit the expression level of clev-caspase-3 (FIG. 4-c, d).

Example 5 S63845 inhibits infection by New coronavirus

Caco-2 cells used in this example were purchased from American Type Culture Collection (ATCC) and stored in the laboratory.

The DMEM medium used in this example was purchased from GIBCO; GAPDH antibodies were purchased from sigma; GFP protein antibodies were purchased from Abclone: s63845 is available from Selleck.

The experimental method in this example is as follows:

the inventor uses a lentivirus infection system to infect Caco-2 cells stably expressing the N protein of the new coronavirus, adds a specific inhibitor S63845 (3 mu M) of MCL-1 to treat the cells for 4 hours, and then adds SARS-CoV-2 trVLP (MOI = 0.5) to infect the cells for 48 hours.

The fluorescence results show that: the level of SARS-CoV-2 replication was significantly reduced after S63845 treatment compared to control treated cells (GFP fluorescence can be used to characterize the viral replication level, FIG. 5-a).

Next, applicants detected the copy number of the virus in the cell supernatant by a method using fluorescent absolute quantitative PCR. The template used is the M gene of the novel coronavirus, and the primer sequences are as follows: forward:5'-GTGCCACTCCATGGCACTAT-3', reverse: 5'-TCCTTGATGTCACAGCGTCC-3'. The experimental results show that: the copy number of SARS-CoV-2 in the cell supernatant was significantly reduced after S63845 treatment compared to control treated cells (FIG. 5-b), and similarly, the western-blot results also demonstrated a significant reduction in the expression level of GFP protein in the cells (FIG. 5-b).

This example was carried out by fluorescence detection and protein expression level detection of green fluorescent protein GFP and the amount of virus secreted into the cell culture broth. The result shows that S63845 (3 mu M) has better inhibiting effect on the expression of new coronavirus protein and the replication of virus nucleic acid.

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

1. Use of compound S63845, said use being at least one of (a) - (c),

(a) S63845 or its pharmaceutically acceptable salt is used for preparing a medicament for preventing and treating novel coronavirus infection;

(b) S63845 or its pharmaceutically acceptable salt is used for preparing products for inhibiting apoptosis of new coronavirus structure N protein regulation cell;

(c) S63845 or its pharmaceutically acceptable salt can be used for preparing products for inhibiting the replication of new coronavirus or expressing new coronavirus protein;

(d) S63845 or its pharmaceutically acceptable salt is used for preparing a product for inhibiting interaction of new coronavirus structural protein N to MCL-1;

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. a product whose active ingredient is S63845 or a pharmaceutically acceptable salt thereof; the product has any one of the following uses: (a) preventive and therapeutic agents for novel coronavirus infections; (b) inhibiting apoptosis of new coronary N protein regulated cells; (c) Inhibiting the replication of new coronavirus or the expression of new coronavirus protein; (d) Inhibiting the interaction of structural protein N of the new coronavirus on MCL-1.

3. The product of claim 2, which is a medicament or pharmaceutical formulation.

4. A product according to claim 2 or 3, which comprises a suitable carrier in addition to S63845, or a pharmaceutically acceptable salt thereof.

5. The product of claim 4, wherein the carrier comprises a pharmaceutically acceptable diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorptive carrier, or lubricant.

6. The product of claim 4, in the form of injection, tablet, powder, granule, capsule, oral liquid, ointment or cream.