CN114657149A

CN114657149A - Application of ceratin in preparing medicine for treating coronavirus infectious diseases

Info

Publication number: CN114657149A
Application number: CN202210386551.0A
Authority: CN
Inventors: 童贻刚; 范华昊; 宋立华; 安小平; 王立钦; 刘文丽; 刘振东
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2020-02-16
Filing date: 2021-02-08
Publication date: 2022-06-24
Anticipated expiration: 2041-02-08
Also published as: CN114657149B; CN114908062A; CN114908061B; CN113046327A; CN114908061A; CN114908062B; CN113046327B

Abstract

The invention provides a coronavirus separated from pangolin, named pangolin coronavirus xCoV, which has 92.5 percent of homology with S protein of SARS-COV-2, and receptors of xCoV infected cells are consistent with SARS-COV-2 and are angiotensin converting enzyme 2(ACE 2). However, xCoV does not infect human, so it is very safe for human, and can be used for screening active medicine and vaccine for resisting SARS-COV-2 virus, and also can be used for preparing attenuated vaccine or inactivated vaccine for resisting SARS-COV-2 virus. Based on the pangolin coronavirus xCoV, a plurality of active compounds with anti-coronavirus activity are obtained by screening, and EC is carried out on cepharanthine (cepharanthine), ceratin and mefloquine hydrochloride (mefloquine) in the active compounds₅₀、CC₅₀And evaluation of SI, and research of Qian-Chi- -Chi-by transcriptome sequencing analysisThe mechanism of xCoV inhibition by aurapten.

Description

Application of ceratin in preparing medicine for treating coronavirus infectious diseases

The present application is a divisional application of Chinese patent application 202110172158.7 entitled Pangolin coronavirus xCoV and its application and application of medicine for resisting coronavirus infection, which is filed on No. 2021, No. 02/08.

Technical Field

The invention belongs to the field of medicines, and particularly relates to pangolin coronavirus xCoV and application thereof in screening of anti-SARS-COV-2 virus medicines, a medicine screening model and a medicine screening method comprising the pangolin coronavirus xCoV, and application of an active compound screened based on the xCoV in preparing a medicine for treating SARS-COV-2 virus infectious diseases.

Background

The novel coronavirus (named as SARS-COV-2 by world health organization, and named as 2019 novel coronavirus or 2019nCoV previously) belongs to beta coronavirus, has envelope, and has circular or elliptical particle, usually polymorphism, and diameter of 60-140 nm. The gene characteristics are obviously different from SARS-CoV and MERS-CoV. The present research shows that the homology of the strain and the bat SARS-like coronavirus (bat-SL-CoVZC45) reaches more than 85 percent. The prevention and treatment of SARS-CoV-2 requires an effective vaccine and specific therapy, and the problem of how to rapidly screen drugs capable of inhibiting the replication of the virus is imminent. Meanwhile, because the virus has extremely strong infectivity, how to safely carry out related researches such as drug screening and the like and protect researchers from infection also become a problem to be solved urgently.

Disclosure of Invention

The present inventors isolated and cultured a novel coronavirus xCoV, named Pangolin coronavirus xCoV (also referred to as "Pangolin xCoV" or "xCoV" in the context of the present invention), from killed Pangolin scales located in Customs, and the whole genome sequence analysis result showed that the virus has 92.5% homology with the S protein of SARS-COV-2, and is the virus that has the highest homology with the S protein of SARS-COV-2 and has been successfully isolated and cultured so far. Further experiments showed that the receptors of pangolin xCoV infected cells are consistent with SARS-COV-2 and are angiotensin converting enzyme 2(ACE 2). However, the virus does not infect humans and is therefore very safe for humans.

The invention provides a coronavirus (also called as pangolin coronavirus xCoV "," pangolin xCoV "or xCoV"), wherein the coronavirus strain xCoV is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (the address: Beijing City No. 3 of West Lu 1 of the northward Yangxi, institute of microbiology of China academy of sciences) in 14 days 2 and 2020, and the preservation number is CGMCC No. 19295.

The pangolin coronavirus strain xCoV has a whole genome nucleotide sequence shown as SEQ ID NO.1 in a sequence table.

The nucleic acid sequence of the S gene of the pangolin coronavirus strain xCoV is shown as SEQ ID NO. 2 in a sequence table.

The amino acid sequence of the S protein of the pangolin coronavirus strain xCoV is shown as SEQ ID NO. 3 in a sequence table.

The pangolin coronavirus strain xCoV has 92.5 percent of homology with S protein of SARS-COV-2. Wherein, the nucleic acid sequence of the S gene of SARS-COV-2 is shown as SEQ ID NO. 4 in the sequence table, and the amino acid sequence of the S protein is shown as SEQ ID NO. 5 in the sequence table.

Among them, the results of the sequence identity of xCoV and SARS-COV-2 are shown in FIG. 1.

The invention also provides the application of the pangolin coronavirus strain xCoV, which is used for screening and evaluating active drugs for resisting SARS-COV-2 virus, screening and evaluating vaccines for resisting SARS-COV-2 virus, preparing attenuated vaccines or inactivated vaccines for resisting SARS-COV-2 virus, diagnosing SARS-COV-2 virus infection and preparing therapeutic antibodies. Wherein the vaccine further comprises a pharmaceutically acceptable adjuvant.

The invention also provides a drug screening model for screening and/or evaluating anti-coronavirus active drugs, which comprises the coronavirus with the preservation number of CGMCC No.19295 (also called as pangolin coronavirus xCoV, pangolin xCoV or xCoV).

The drug screening model according to the present invention is a mammalian cell, preferably a Vero E6 cell (Vero green monkey kidney cell), infected with said pangolin coronavirus xCoV.

The drug screening model according to the present invention, wherein the model is preferably used for screening and/or evaluating a drug having an activity against SARS-CoV-2 virus.

The invention also provides a method for screening and/or evaluating the anti-coronavirus active drug, which is carried out by adopting the drug screening model; preferably, the method is used to screen and/or evaluate drugs having anti-SARS-CoV-2 activity.

The method for screening and/or evaluating an anti-coronavirus active drug according to the present invention comprises the step (1): and adding the drug to be tested into the drug screening model and culturing.

The method for screening and/or evaluating an anti-coronavirus active drug according to the present invention, which further optionally comprises the following step (2a) or step (2b) after step (1), or comprises both step (2a) and step (2 b):

step (2 a): observing the cytopathic effect under a microscope;

step (2 b): viral nucleic acid was measured in cells and supernatant.

According to the method for screening and/or evaluating an anti-coronavirus active drug substance of the present invention, the culture time in step (1) may be 12 to 90 hours, such as 24 to 72 hours, 48 to 72 hours, 24 hours, 48 hours, 72 hours, or the like.

In step (2a), when the presence of an intact cell monolayer or no distinct cytopathic effect is observed, it is indicated that the drug to be tested has an activity of inhibiting viral replication.

In addition, the invention also provides application of any one, two or three of the following compounds in preparing a medicament for treating the coronavirus infectious diseases: cepharanthine (cepharanthine), ceratin, mefloquine hydrochloride, and mefloquine.

According to the use of the invention, the coronavirus is SARS-COV-2 virus.

In Addition, the invention also discovers the influence of the cepharanthine, the ceratin and the mefloquine hydrochloride on the life cycle of the xCoV virus through a Time-of-Addition test, and explains the mechanism of the cepharanthine for inhibiting the xCoV virus replication through transcriptomics analysis.

Advantageous effects

The S protein of pangolin coronavirus xCoV and SARS-COV-2 has high homology, and the receptors of xCoV infected cells are consistent with SARS-COV-2 and are angiotensin converting enzyme 2(ACE 2). And the xCoV virus does not infect people, so the xCoV virus is very safe to people and can be used for screening and evaluating the medicine for resisting SARS-COV-2 virus, screening and evaluating vaccines and preparing attenuated and inactivated vaccines. The screening of anti-SARS-COV-2 virus drugs based on the xCoV virus is very safe for developers, and there is no need to worry about infection. Based on the screening model, active drugs of cepharanthine (cepharanthine), cerbrocin and mefloquine hydrochloride (mefloquine) for resisting SARS-CoV-2 are screened out, and the inhibition effect of the cepharanthine, the cerbrocin and the mefloquine hydrochloride after xCoV is introduced into cells is proved, and the mechanism of anti-xCoV of the cepharanthine is explained. Cepharanthine exerts an anti-coronavirus effect by reversing most deregulated genes and pathways in infected cells, mainly by interfering with cellular stress responses, including endoplasmic reticulum stress/unfolded protein response and heat shock factor 1(HSF1) mediated heat shock response.

Drawings

Figure 1 shows the sequence identity results of xCoV with other coronaviruses.

FIG. 2 shows an evolutionary tree analysis of the xCoV genome as a whole and SARS-CoV-2 genome.

FIG. 3 shows a phylogenetic tree analysis of the S gene of xCoV versus the S gene of SARS-CoV-2.

Figure 4 shows ACE2mRNA expression following siRNA knock-down of ACE 2.

Figure 5 shows the effect of siRNA knock-down of ACE2 expression on xCoV virus infection.

Figure 6 shows a flow of drug screening of the present invention.

Figure 7 shows a graph of the morphology of Vero E6 cells 72 hours after xCoV infection with a multiplicity of infection of 0.01, when not dosed.

FIG. 8 shows a morphological map of Vero E6 cells after 72 hours of culture without addition of virus, without addition of drug.

Figure 9 shows a morphogram of Vero E6 cells 72 hours after infection with cepharanthine added to a final concentration of 10 μ M (micromole per liter) and xCoV with a multiplicity of infection of 0.01.

Figure 10 shows a morphogram of Vero E6 cells 72 hours after infection with ceratin added at a final concentration of 10 μ M (micromole per liter) and xCoV with a multiplicity of infection of 0.01.

Figure 11 shows a morphogram of Vero E6 cells 72 hours after infection with mefloquine hydrochloride added to a final concentration of 10 μ M (micromole per liter) and xCoV with a multiplicity of infection of 0.01.

FIG. 12 shows the inhibition of xCoV by three compounds, 10. mu.M cepharanthine inhibits xCoV viral replication by 15393-fold, 10. mu.M ceratin inhibits xCoV viral replication by 5053-fold, and 10. mu.M mefloquine hydrochloride inhibits xCoV viral replication by 31-fold.

FIG. 13 shows the median Effective Concentration (EC) of cepharanthine versus xCoV₅₀) Half the cytotoxic concentration against Vero E6 cells (CC) at 0.9851. mu.M₅₀) 39.32 μ M, and a Selection Index (SI) of 39.91.

FIG. 14 showsSemith Effective Concentration (EC) of ceratin on xCoV₅₀) Half the cytotoxic concentration against Vero E6 cells (CC) at 1.908. mu.M₅₀) 6.227 μ M, and a Selection Index (SI) of 3.290.

FIG. 15 shows the median Effective Concentration (EC) of mefloquine hydrochloride on xCoV₅₀) Half the Cytotoxic Concentration (CC) against Vero E6 cells at 2.728. mu.M₅₀) 10.08 μ M and Selection Index (SI) 3.695.

FIG. 16 shows the results of the Time-of-Addition test of cepharanthine on xCoV.

FIG. 17 shows the results of the Time-of-Addition test of ceratin on xCoV.

FIG. 18 shows the results of the Time-of-Addition test of mefloquine hydrochloride on xCoV.

Figure 19 shows transcriptome analysis results of cepharanthine against xCoV replication.

Detailed Description

The present invention is further described below with reference to examples. It should be noted that the following examples are not intended to limit the scope of the present invention, and any modifications made on the basis of the present invention do not depart from the spirit of the present invention. The raw materials and equipment used in the present invention can be purchased commercially without specific description.

First, experiment method

1. Cell culture and virus culture

The African green monkey kidney cell line Vero E6 was obtained from American type culture Collection (ATCC, No. 1586) at 37 ℃ with 5% CO₂In DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen).

Squama Manis isolate xCoV was propagated in Vero E6 cells and virus titers were determined using the plaque assay. All infection experiments were performed in a biosafety class 2 (BLS2) laboratory.

Marketed drug library (product number L1000, containing 2080 marketed drugs) and antiviral compound library (product number L1700, containing 326 antiviral drugs) were purchased from Shanghai ceramic Biotechnology Ltd. The initial concentration of all drugs was 10mM (millimoles per liter).

Cepharanthine (T0131), ceratin (T0124), and mefloquine hydrochloride (T0860) were purchased from Shanghai ceramic Biotechnology, Inc. The initial concentration of all drugs was 10mM (millimoles per liter).

2. Explore whether ACE2 is an xCoV-infected cell receptor

One day prior to transfection, 12 well cell culture plates were seeded 2X 10 per well⁵Vero E6 cells. The next day, ACE2 gene expression was silenced by trans-transfection with RNAiMax transfection reagent using ACE2 siRNA smart pool (samemar, su) when cells were adherent. Cells were incubated with 2, 10 and 50nM siRNAs transfectants for 48 hours at 37 deg.C. After 48 hours, cells were incubated with xCoV for 2 hours at 37 ℃. Unbound virus was washed off with PBS and incubation was continued for 24 hours with fresh medium. Unbound virus was washed off with PBS, total RNA was extracted, and ACE2mRNA and viral infection were determined using a two-step method qRT-PCR.

3. Screening potential anti-novel pneumovirus medicine from medicine library on the market by utilizing pangolin coronavirus xCoV with high SARS-CoV-2 homology

Seeded 2.5X 10 in 96-well cell plates⁴Vero E6 cells were infected 24 hours later with xCoV with MOI 0.01 to Vero E6 cells, to which various known drugs (2406 marketed drugs and phase III clinical trial drug) were added to a final concentration of 10. mu.M, and cytopathic effect was observed under a microscope on day 3, RNA was extracted from cells and supernatant from culture wells with no significant cytopathic effect, and virus replication was measured in cells and supernatant using qRT-PCR.

4. Viral RNA extraction and real-time quantitative RT-PCR (qRT-PCR)

AxyPrep was used according to manufacturer's instructions^TMHumoral virus DNA/RNA miniprep kit (Axygen, product number AP-MN-BF-VNA-250) and AxyPrep^TMA multipurpose total RNA micro-preparation kit (Axygene, product number AP-MN-MS-RNA-250G) collects cell culture supernatant and Vero E6 cells for RNA extraction. Reverse transcription was carried out using Hifair II 1 chain cDNA Synthesis kit (assist in Shanghai, product No. 11121ES60) with gDNase, and Hieff-qPCR-SYBR-Gre was usedqPCR was performed by en-Master Mix (assist in san Jose Biotechnology Co., Ltd., cat # 11202ES08) or two-step Taqman probe detection qRT-PCR system (Applied-Biosystem), and the sequence information of the primers used is shown in Table 1. After confirmation of sequencing, the PCR product was inserted into a T-vector by bevacizco biotechnology limited, beijing, ruffikco, to generate a standard plasmid. Standard curve is determined by serial dilution of plasmid (10)³-10⁹) The number of copies of (a). qPCR amplification by SYBR-Green method: 95 ℃ for 5min, 40 cycles, 95 ℃ for 10s, 55 ℃ for 20s, 72 ℃ for 31 s.

The Taqman method: the data in FIG. 12 were analyzed using GraphPad-Prism 8 software at 50 ℃ for 2min, 95 ℃ for 10min, 40 cycles, 95 ℃ for 10s, and 60 ℃ for 1 min.

The drug screening procedure of the present invention is shown in FIG. 6.

5.EC₅₀And CC₅₀Detection and Time-of-Addition assay

Experiments were performed using 3 compounds selected in experiment 3, cepharanthine, selamectin, and mefloquine hydrochloride, and Vero E6 cells were infected with xCoV with an MOI of 0.01.

EC₅₀And (3) detection: inoculating Vero E6 cells to a 24-hole cell culture plate, and performing a test when the cell density reaches 60-80%; the drug was diluted to 200. mu.M and then diluted in a two-fold gradient to 0.39. mu.M. After the Vero E6 cells are changed, the drug solution and the virus suspension are diluted 1:1 and then added to the cells. The final concentrations of the test drugs were 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.56. mu.M, 0.78. mu.M, 0.39. mu.M, 0.195. mu.M, and 0. mu.M, respectively. 5% CO at 37 ℃₂Culturing for 60-72h, observing CPE, extracting cell nucleic acid for qPCR detection, and performing data analysis by GraphPad-Prism 8 software to calculate EC₅₀。

CC₅₀And (3) detection: CC Using Cell-Titer-Blue method₅₀Detection of (3). Vero E6 cells were seeded into 96-well cell culture plates and tested at cell densities of 60% -80%. The medicine is diluted twice in proportion, and the diluted medicine is added after the liquid of the Vero E6 cell is changed. The final concentrations of test drugs were 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.56. mu.M, 0.78. mu.M, 0.39. mu.M, 0.195. mu.M, 0. mu.M, respectively. 5% CO at 37 ℃₂Culturing for 48h, adding 20 μ l Cell-Titer-Blue per well, detecting 593nm luminous intensity at 0min, 30min, 60min and 120min, respectively, and performing data analysis with GraphPad-Prism 8 software to calculate CC₅₀。

SI is CC₅₀Divided by EC₅₀And (6) calculating.

Time-of-Addition detection: vero E6 cells were seeded into 24-well cell culture plates and tested at cell densities of 60% -80%. The test drug concentration was selected to be 6.25. mu.M. The whole time course experiment method comprises adding medicine-virus mixed solution, and adding 5% CO at 37 deg.C₂Changing the liquid after culturing for 2h, and adding the medicine-virus mixed liquid; the experimental method of 'before cell entry': adding the mixture of medicine and virus, 5% CO at 37 deg.C₂Changing the culture solution after 2h of culture, and adding a pure culture medium; the experimental method of 'after cell entry': adding pure culture medium, 5% CO at 37 deg.C₂After 2h of culture, the solution is changed and the drug-virus mixed solution is added. 5% CO at 37 ℃₂And (5) continuing to culture for 60-72h, observing CPE, extracting cell nucleic acid, carrying out qPCR detection, and carrying out data analysis by using GraphPad-Prism 8 software.

6. Transcriptomics analysis of cepharanthine against xCoV infection

Cepharanthine (CEP) was tested at a concentration of 6.25 μ M and Vero E6 cells were infected with xCoV with an MOI of 0.01. Four groups were set up for the experiment: vero, Vero + Virus, Vero + CEP, Vero + Virus + CEP. After 72h incubation, cell samples were collected and RNA extraction was performed using TRIzol, rRNA was removed using the QIAseq FastSelect-rRNA HMR Kit (Qiagen, product No. 334387), and NEBNext Ultra was used^TMRNA Library Prep Kit for Illumina (NEB, product No. E7770L) A mRNA sequencing Library was created and RNA sequencing (RNA-seq) was performed using an Illumina Hiseq 2500 sequencing system (Annuodda Biotechnology Co., Ltd.).

FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/FastQC /) tool and FASTX _ trimmers in the FASTX toolkit were used to remove low quality data and linker sequences; mapping the trimmed RNA-seq sequence to a reference green monkey genome ChlSab1.1(GCA _000409795.2) using HISAT2 (v2.1.0); deletion of double-ended data repeats using SAMtools (v 1.5); counting each different gene using HTseq; use of DESeq2 to identify differentially expressed genes between different experimental groups; calculating the False Discovery Rate (FDR) by adjusting the P value by using a Benjamini-Hochberg method; genes with FDR q values <0.05 and | Log2 (fold change) | >1 were considered differentially expressed genes; and (4) drawing a volcanic chart by using a ggplot2 software package of the R language.

Gct format files (including Vero vs. Vero + Virus, Vero + Virus vs. Vero + Virus + CEP) are used as the process files. The gene set includes (1) regulation of heat shock response mediated by heat shock factor 1(HSF1), regulation of cellular pyrogenicity, HSF1 dependent transactivation, HYPOXIA, defense response to viruses, HIF1 targets, adipocyte differentiation and autophagy, available from the MSigDB, KEGG and Reactome databases, (2) up/down regulation genes of viruses, which are differentially expressed genes in the RNA-seq data described above with FDR q values <0.05 and | Log2 (fold change) | > 1. The Normalized Enrichment Score (NES) value and FDR value were obtained from the genome enrichment P values calculated for 1000 permutations using Signal2Noise mode run GSEA4.0.3(https:// www.gsea-msigdb. org/gsea/index. jsp). The visualization heatmap is drawn by the R software package of GENE-E. And a heatmap is drawn from the MSigDB, KEGG, and Reactome databases to display the selected gene set by pathway patterns.

Gene Ontology (GO) analysis was performed on the genes described above that achieved differential expression using the Metascape tool (https:// Metascape. org). Pathways with P values <0.05 were used as significantly enriched pathways, the most significantly enriched pathway being demonstrated using a bubble map created by R package ggplot2, and the Cytoscape in the Metascape website was used to map the interaction network and protein-protein interaction (PPI) network for each important enriched pathway. And each given gene list was analyzed in detail PPI enrichment using BioGrid and OmniPath.

Primer sequences used in the study of Table 1

Second, experimental results

Analysis of the whole genome and the respective virus-encoding genes (nucleotide level and amino acid level) revealed that: xCoV is highly homologous with SARS-CoV-2, has 92.5% homology with S protein of SARS-COV-2, and is the virus with highest homology with S protein of SARS-COV-2 successfully isolated and cultured so far (FIG. 1). Whether at the genome-wide level (FIG. 2) or the critical gene S gene for virus attachment into cells (FIG. 3), the homology of xCoV to SARS-CoV-2 is much higher than that of SARS virus.

By specifically knocking down the expression of ACE2 by adding different concentrations of siRNA, it was found that with the gradual decrease of ACE2mRNA expression level (fig. 4), the ability of xCoV to infect cells was significantly gradually decreased, strongly suggesting that ACE2 is a receptor for xCoV entry into cells (fig. 5).

96-well cell culture wells were filled with one of the various known drugs (2080 marketed drugs and 326 antiviral compounds) and xCoV with an MOI of 0.01 at a final concentration of 10. mu.M, respectively, and treated Vero E6 cells at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator for 72 hours. In the cell culture wells with no added virus compound and most of the cell culture wells with various added compounds, the cells showed significant cytopathic effect (FIG. 7), while the cells did not show any cytopathic effect in the cell culture without added virus and drug (FIG. 8). Then, no obvious cytopathic effect was seen in virus-infected cell culture wells to which were added cepharanthine (fig. 9), ceratin (fig. 10) and mefloquine hydrochloride (fig. 11) at a final concentration of 10 μ M. The cepharanthine, the ceratin and the mefloquine hydrochloride are strongly suggested to be potential strong xCoV infection cell inhibitors.

Further detection by real-time quantitative PCR technique found that 10 micromoles per liter of cepharanthine, ceratin, mefloquine hydrochloride inhibited viral replication 15393-fold, 5053-fold, 31-fold, respectively, 72 hours after xCoV infection of cells at a multiplicity of 0.01, compared to a control with 0.1% DMSO alone (all compounds dissolved in DMSO, thus 0.1% DMSO concentration in each cell culture well after drug addition) (figure 12). The results of this experiment have been repeated 5 times and all can be repeated.

EC₅₀、CC₅₀The results with SI show that the inhibition of xCoV by cepharanthine (fig. 13), ceratin (fig. 14), and mefloquine hydrochloride (fig. 15) appears concentration-dependent. Further, cepharanthine (fig. 16), ceratin (fig. 17), and mefloquine hydrochloride (fig. 18) all exert virus-inhibitory effects after xCoV entered the cell.

Specifically, fig. 16 shows the Time-of-Addition test results of cepharanthine on xCoV, indicating that cepharanthine exerts an inhibitory effect after xCoV enters cells, but cannot inhibit xCoV's invasion. FIG. 17 shows the results of the Time-of-Addition test of ceratin on xCoV, indicating that ceratin exerts an inhibitory effect upon entry of xCoV into cells, but cannot inhibit the entry of xCoV. FIG. 18 shows the results of the Time-of-Addition test of mefloquine hydrochloride on xCoV, which indicates that mefloquine hydrochloride exerts an inhibitory effect after xCoV enters cells, but cannot inhibit the entry of xCoV.

Further transcriptome sequencing analysis suggested that stephanine exerted an anti-coronavirus effect by reversing most deregulated genes and pathways in infected cells, mainly by interfering with cellular stress responses, including endoplasmic reticulum stress/unfolded protein response and HSF 1-mediated heat shock response (fig. 19).

Third, discuss

The study of SARS-CoV-2 virus requires a high level of biological protection facilities, which is in conflict with the urgent need for the study. The pangolin coronavirus xCoV isolated by the inventor has low pathogenicity or no pathogenicity to human bodies, and provides an alternative mode for researching SARS-CoV-2 closely related to the pangolin coronavirus xCoV, and is used by researchers without biosafety level 3 facilities. The inventors believe that this isolate is of low or no pathogenicity to humans because as early as 2017, no suspected infection was found in people in close contact with pangolin scales; the inventors' pangolin coronavirus xCoV isolate was routinely cultured in biosafety secondary facilities.

A coronavirus found in a sample collected from a rhinobat of Yunnan in 2013 was closely related to SARS-CoV-2, and thus it was presumed that the bat may also be a host of SARS-CoV-2. Recently, researchers at university of agriculture in south China declared that squama Manis (Manis japonica) is the intermediate host for SARS-CoV-2. Similarly, in 10 months 2019, a virus genome-wide study of pangolins revealed a SARS-CoV-related sequence, which was re-identified as SARS-CoV-2-related after the appearance of SARS-CoV-2. In addition, the inventor also isolated and cultured a strain of SARS-CoV-2 related coronavirus xCoV from killed and smuggled squama Manis. Through the comparison analysis of the whole genome and various virus coding genes (nucleotide and amino acid), xCoV is highly homologous with SARS-CoV-2, and the S protein homology with SARS-COV-2 reaches 92.5%, so that the virus with the highest S protein homology with SARS-COV-2 is successfully isolated and cultured so far (figure 1). Whether at the genome-wide level (FIG. 2) or the critical gene S gene for virus attachment into cells (FIG. 3), the homology of xCoV to SARS-CoV-2 is much higher than that of SARS virus.

In this study, the inventors have conducted screening of anti-coronavirus active drugs in the SARS-CoV-2-associated coronavirus, pangolin coronavirus xCoV model. Based on the results of preliminary laboratory studies, it was found that xCoV infected mammalian cell Vero E6 (Vero cells) can cause very obvious cytopathic effect. Based on this feature, the inventors used xCoV to infect Vero cells in 96-well cell culture plates at the early stage, and simultaneously added a single drug on the market (2080 multiple drugs on the market and 326 antiviral compounds) to each cell culture well to perform potential active drug screening for inhibiting viral replication. Cytopathic effects were observed under a microscope on day 3, and it was found that there were 3 potential drugs that inhibited virus-infected cells significantly (3 drugs were cepharanthine, ceratin, and mefloquine hydrochloride, respectively). As the xCoV is highly homologous with the current SARS-COV-2, and the receptor of the xCoV infected cell is consistent with the SARS-COV-2, if the medicament has the inhibition effect on the xCoV infected cell, the medicament also has the inhibition effect on the SARS-COV-2 infection.

It is noted that the half inhibitory dose of cepharanthine against SARS-CoV virus causing SARS in 2003 is 8 μ g/ml (13.186 μ M), i.e., 13.186 μ M final concentration of cepharanthine can inhibit 50% of virus infection, as mentioned in the Wangyi et al 2003 patent "application of cepharanthine in preparing anti-SARS virus medicine". The inventors' experimental results showed that the ability to inhibit xCoV virus replication reached 15393-fold using lower concentrations of cepharanthine (10 μ M). Thus, cepharanthine has an inhibitory activity against xCoV virus replication that is at least 30786 times greater than that against SARS-CoV virus replication. Furthermore, the inhibition of SARS-CoV by cepharanthine (which is effectively a low-level inhibition) does not indicate a potent and effective inhibition of xCoV and SARS-CoV-2. In fact, the inventor has tested that the drug library on the market contains many drugs (such as oseltamivir phosphate) which can effectively inhibit the replication of SARS-CoV virus, but the drugs (such as oseltamivir phosphate) have little inhibition effect on the replication of xCoV and SARS-CoV-2. One of the main reasons for this is that, because SARS-CoV virus has a relatively high homology with SARS-CoV-2 virus, and the difference between SARS-CoV virus and SARS-CoV-2 virus in genome and amino acid levels is large, drugs that inhibit SARS-CoV virus probably do not have any effect on xCoV and SARS-CoV-2 (such as oseltamivir phosphate). And xCoV is isolated from dead pangolins obtained from customs, has high homology with SARS-CoV-2, and is the virus with the highest homology with SARS-CoV-2 in coronavirus which can be isolated and cultured at present. Therefore, cepharanthine, ceratin and mefloquine hydrochloride which have strong inhibition effect on xCoV in the invention can also inhibit virus replication of SARS-CoV-2, and are very likely to become specific drugs for novel coronavirus pneumonia, and the drugs are suggested to be used for clinical tests of SARS-CoV-2 patients.

The inventors believe that cepharanthine has a particularly significant pharmaceutical value as a potential drug for the treatment of SARS-CoV-2. The medicine is a double-node javanine alkaloid separated and extracted from Stephania cepharantha and Stephania delavayi of Menispermaceae, and is approved for leucopenia. It has multiple functions, such as inhibiting the efflux transporter ABCC10 of antitumor drugs, inhibiting the entry of HIV-1 by reducing the fluidity of plasma membrane, and binding with the central part of Hsp 90. Importantly, large doses of this drug have low toxicity in animals and no significant side effects in humans. In addition, it has been shown that SARS-CoV-2 can cause the enrichment of cell stress response and autophagy pathway-related genes of peripheral blood mononuclear cells, while stephanin can effectively reverse most deregulated genes and pathways in infected cells, especially endoplasmic reticulum stress/unfolded protein response and HSF 1-mediated heat shock response, thereby exerting an anti-coronavirus infection effect. In view of the strong inhibitory effect observed on viral replication and the established anti-inflammatory effect of this drug, the inventors believe cepharanthine is a promising candidate for the treatment of SARS-CoV-2 infection.

Claims

1. Use of a siramectin for the preparation of a medicament for the treatment of a coronavirus infectious disease.

2. Use according to claim 1, wherein the coronavirus is a SARS-COV-2 virus.