CN114908062B - Use of mefloquine hydrochloride or mefloquine in preparing medicine for treating coronavirus infectious diseases - Google Patents
Use of mefloquine hydrochloride or mefloquine in preparing medicine for treating coronavirus infectious diseases Download PDFInfo
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Abstract
The application provides a coronavirus which is separated from pangolin, named pangolin coronavirus xCoV, has 92.5 percent of S protein homology with SARS-COV-2, and has the receptor of xCoV infected cells consistent with SARS-COV-2, and is angiotensin converting enzyme 2 (ACE 2). However, xCoV does not infect human, so that the method is very safe for human, can be used for screening active medicaments and vaccines for resisting SARS-COV-2 virus, and can also be used for preparing attenuated vaccines or inactivated vaccines for resisting SARS-COV-2 virus. Screening to obtain multiple active compounds with anti-coronavirus activity based on pangolin coronavirus xCoV, and performing EC on stephanine, siramectin, and mefloquine hydrochloride 50 、CC 50 And SI, and the mechanism of cepharanthine inhibition of xCoV was studied by transcriptome sequencing analysis.
Description
The application is a divisional application of Chinese patent application 202110172158.7, which is filed on the year 2021, month 02 and day 08 and is named as pangolin coronavirus xCoV and application thereof and application of medicines for resisting coronavirus infection.
Technical Field
The application belongs to the field of medicines, and in particular 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 medicines for treating SARS-COV-2 virus infectious diseases.
Background
The novel coronavirus (designated by the world health organization as "SARS-COV-2", previously designated 2019 novel coronavirus or 2019 nCoV) belongs to the genus beta coronavirus, enveloped, particles having a circular or oval shape, often polymorphic, diameter 60-140nm. The gene characteristics are obviously different from SARS-CoV and MERS-CoV. The current research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC 45) is more than 85%. The prevention and treatment of SARS-CoV-2 is an urgent need for effective vaccines and specific therapies, and how to rapidly screen drugs that inhibit replication of the virus is an urgent issue. 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 is also a problem to be solved urgently.
Disclosure of Invention
The present inventors isolated and cultured a novel coronavirus xCoV, called pangolin coronavirus xCoV (also called "pangolin xCoV" or "xCoV" in the context of the present application), from dead pangolin obtained by customs inspection, and found that the whole genome sequence analysis result showed up to 92.5% homology with the S protein of SARS-COV-2, which was the virus having the highest homology with the S protein of SARS-COV-2, which was successfully isolated and cultured so far. Further experiments showed that the receptors for pangolin xCoV infected cells are identical to SARS-COV-2, both being angiotensin converting enzyme 2 (ACE 2). But the virus does not infect humans and is therefore very safe for humans.
The application provides a coronavirus (also called as pangolin coronavirus xCoV, pangolin xCoV or xCoV), wherein the coronavirus strain xCoV is preserved in China general microbiological culture Collection center (address: north Chen West road No.1, 3 of the area of Chachiensis in Beijing, and the institute of microbiology of China academy of sciences) at 14 days of 2 months in 2020, and the preservation number is CGMCC No.19295.
The full genome nucleotide sequence of the pangolin coronavirus strain xCoV is 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.
Pangolin coronavirus strain xCoV according to the application has a homology of 92.5% with the 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 sequence identity results of xCoV and SARS-COV-2 are shown in FIG. 1.
The application also provides application of the pangolin coronavirus strain xCoV, which is used for screening and evaluating active medicaments for resisting SARS-COV-2 viruses, screening and evaluating vaccines for resisting the SARS-COV-2 viruses, preparing attenuated vaccines or inactivated vaccines for resisting the SARS-COV-2 viruses, and preparing diagnostic and therapeutic antibodies for SARS-COV-2 virus infection. Wherein the vaccine further comprises a pharmaceutically acceptable adjuvant.
The application also provides a drug screening model for screening and/or evaluating anti-coronavirus active drugs, which comprises the coronavirus (also called "pangolin coronavirus xCoV", "coronavirus xCoV" or "xCoV") with the preservation number of CGMCC No.19295.
The drug screening model according to the present application is a mammalian cell, preferably Vero E6 cell (african green monkey kidney cell), infected with the pangolin coronavirus xCoV.
The drug screening model according to the present application, wherein the model is preferably used for screening and/or evaluating drugs having anti-SARS-CoV-2 virus activity.
The application also provides a method for screening and/or evaluating anti-coronavirus active drugs, 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 agent according to the present application comprises the steps of (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 agent according to the present application, which further optionally comprises the following step (2 a) or step (2 b) after step (1), or comprises both step (2 a) and step (2 b):
step (2 a): observing the cytopathy under a microscope;
step (2 b): viral nucleic acid in cells and supernatants was assayed.
According to the method of screening and/or evaluating an anti-coronavirus active agent of the present application, the culturing time in step (1) may be 12 to 90 hours, such as 24 to 72 hours, 48 to 72 hours, 24 hours, 48 hours or 72 hours, etc.
In step (2 a), when the presence of an intact cell monolayer or a non-apparent cytopathic effect is observed, the drug to be tested is shown to have an activity in inhibiting viral replication.
In addition, the application also provides the use of any one, two or three of the following compounds for the preparation of a medicament for the treatment of coronavirus infectious diseases: cepharanthine, sirolimus, mefloquine hydrochloride and mefloquine.
According to the use of the application, the coronavirus is SARS-COV-2 virus.
In Addition, the application also discovers the influence of cepharanthine, sirolimus and mefloquine hydrochloride on the life cycle of xCoV viruses through a Time-of-Addition test, and explains the mechanism of inhibiting xCoV virus replication of cepharanthine through transcriptomic analysis.
Advantageous effects
The pangolin coronavirus xCoV has high homology with S protein of SARS-COV-2, and the receptor of xCoV infected cells is consistent with SARS-COV-2, and is angiotensin converting enzyme 2 (ACE 2). The xCoV virus does not infect human, so that the xCoV virus is very safe for human, and can be used for screening and evaluating medicines for resisting SARS-COV-2 virus, screening and evaluating vaccines, preparing attenuated vaccines and inactivating vaccines. Screening for anti-SARS-COV-2 virus based on the xCoV virus is very safe for researchers and does not have to worry about being infected. Active drugs stephanine (stephanine), sirolimus and mefloquine hydrochloride (mefloquine) for resisting SARS-CoV-2 are screened out based on the screening model, and the inhibition effect of stephanine, sirolimus and mefloquine hydrochloride after xCoV enters cells is proved, and the mechanism of stephanine for resisting xCoV is also explained. Cepharanthine exerts an anti-coronavirus effect by reversing most deregulated genes and pathways in infected cells primarily by interfering with cellular stress responses, including endoplasmic reticulum stress/unfolded protein responses and heat shock factor 1 (HSF 1) -mediated heat shock responses.
Drawings
FIG. 1 shows the sequence identity results of xCoV with other coronaviruses.
FIG. 2 shows a phylogenetic tree analysis of xCoV whole genome and SARS-CoV-2 whole genome.
FIG. 3 shows a phylogenetic tree analysis of the S gene of xCoV and the S gene of SARS-CoV-2.
FIG. 4 shows the expression of ACE2mRNA after siRNA knockdown of ACE 2.
Figure 5 shows the effect of siRNA knockdown ACE2 expression on xCoV virus infection.
Figure 6 shows a flow of drug screening of the present application.
FIG. 7 shows the morphology of Vero E6 cells when not dosed 72 hours after xCoV infection at a multiplicity of infection of 0.01.
FIG. 8 shows the morphology of Vero E6 cells without drug after 72 hours incubation without virus.
FIG. 9 shows the morphology of Vero E6 cells after 72 hours of infection with stephanine and xCoV at a multiplicity of infection of 0.01 added at a final concentration of 10. Mu.M (micromole per liter).
FIG. 10 shows a morphology of Vero E6 cells after 72 hours of infection with a final concentration of 10. Mu.M (micromole per liter) of siramectin and a complex number of infection of 0.01 xCoV.
FIG. 11 shows the morphology of Vero E6 cells after 72 hours of infection with mefloquine hydrochloride and xCoV at a multiplicity of infection of 0.01 added at a final concentration of 10. Mu.M (micromole per liter).
FIG. 12 shows the inhibition of xCoV by three compounds, 10. Mu.M of cepharanthine inhibits replication of xCoV virus by 15393 fold, 10. Mu.M of sirolimus inhibits replication of xCoV virus by 5053 fold, and 10. Mu.M of mefloquine hydrochloride inhibits replication of xCoV virus by 31 fold.
FIG. 13 shows the half-maximal Effective Concentration (EC) of cepharanthine on xCoV 50 ) Half-cell toxicity concentration (CC) to Vero E6 cells at 0.9851. Mu.M 50 ) 39.32. Mu.M, and a Selection Index (SI) of 39.91.
FIG. 14 shows the half-Effective Concentration (EC) of siramectin on xCoV 50 ) Half-cell toxicity concentration (CC) to Vero E6 cells at 1.908. Mu.M 50 ) 6.227. Mu.M, and a Selection Index (SI) of 3.290.
FIG. 15 shows the median Effective Concentration (EC) of mefloquine hydrochloride over xCoV 50 ) Half-cell toxicity concentration (CC) to Vero E6 cells at 2.728. Mu.M 50 ) 10.08. Mu.M, and a Selection Index (SI) of 3.695.
FIG. 16 shows the results of the Time-of-Addition test of cepharanthine versus xCoV.
FIG. 17 shows the results of the Time-of-Addition test of sirolimus against xCoV.
FIG. 18 shows the results of the Time-of-Addition test of mefloquine hydrochloride on xCoV.
FIG. 19 shows the results of transcriptome analysis of cepharanthine anti-xCoV replication.
Detailed Description
The application is further described below with reference to examples. It should be noted that the following examples should not be construed as limiting the scope of the application, and any modifications made thereto do not depart from the spirit of the application. The materials and equipment used in the present application are commercially available unless otherwise specified.
1. Experimental method
1. Cell culture and virus culture
Vero E6, an african green monkey kidney cell line, was obtained from the american type culture collection (ATCC, 1586) at 37 ℃, 5% co 2 Is cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; gibco Invitrogen).
Pangolin isolate xCoV was propagated in Vero E6 cells and virus titer was determined using a plaque assay. All infection experiments were performed in biosafety class 2 (BLS 2) laboratories.
The market drug library (product number L1000, containing 2080 drugs already on the market) and the antiviral compound library (product number L1700, containing 326 antiviral drugs) were purchased from Shanghai Tao Su Biochemical technologies Co. The initial concentration of all drugs was 10mM (millimoles per liter).
Cepharanthine (T0131), sirametin (T0124) and mefloquine hydrochloride (T0860) were purchased from Shanghai Tao Su Biotechnology Co. The initial concentration of all drugs was 10mM (millimoles per liter).
2. Investigation of ACE2 as an xCoV infected cell receptor
One day prior to transfection, 2X 10 cells were plated per well in 12-well cell culture plates 5 Vero E6 cells were used. The following day, ACE2 gene expression was silenced by trans-transfection with RNAiMax transfection reagent using ACE2 siRNA smart pool (Ji Ma gene, su) when cells were well adherent. Cells were incubated with 2, 10 and 50nM siRNAs transfection at 37℃for 48 hours, respectively. After 48 hours, cells were incubated with xCoV for 2 hours at 37 ℃. Unbound virus was washed off with PBS and the culture was continued for 24 hours with fresh medium. Washing unbound virus with PBS, extracting total RNA, and performing two-step methodqRT-PCR measures ACE2mRNA and viral infection.
3. Screening potential anti-novel pneumovirus drugs from marketed drug library by utilizing pangolin coronavirus xCoV with high SARS-CoV-2 homology
Planting 2.5X10 in 96-well cell plate 4 Vero E6 cells were infected 24 hours later with xCoV with moi=0.01, while adding thereto various known drugs (2406 drugs on the market and phase III clinical trial drugs) at a final concentration of 10 μm, and on day 3, cytopathic effect was observed by microscopy, RNA was extracted from cells and supernatant from cultured wells without obvious cytopathic effect, and viral replication in cells and supernatant was determined by qRT-PCR.
4. Viral RNA extraction and real-time quantitative RT-PCR (qRT-PCR)
According to the manufacturer's instructions, axyPrep was used TM Body fluid virus DNA/RNA miniprep kit (Axygen, product number AP-MN-BF-VNA-250) and AxyPrep TM A multipurpose Total RNA miniprep kit (Axygene, product number AP-MN-MS-RNA-250G) collects cell culture supernatant and Vero E6 cells for RNA extraction. Reverse transcription was performed using HifairII 1 strand cDNA synthesis kit with gDNase (Shanghai Chemie Biotechnology Co., ltd., product No. 11121ES 60), qPCR was performed using Hieff-qPCR-SYBR-Green-Master Mix (Shanghai Santa Biotechnology Co., ltd., cat., product No. 11202ES 08) or a two-step Taqman probe detection qRT-PCR system (Applied-Biosystem), and sequence information of the primers used is shown in Table 1. After sequencing confirmation, PCR products were inserted into T-vector by beijing, borreliaceae biotechnology limited to generate standard plasmids. The standard curve was obtained by measuring plasmid serial dilutions (10 3 -10 9 ) Is generated by the copy number of the (c). qPCR amplification by SYBR-Green method: 95℃for 5min,40 cycles, 95℃for 10s,55℃for 20s and 72℃for 31s.
Taqman method: the data of FIG. 12 were analyzed with 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 application is shown in FIG. 6.
5.EC 50 And CC 50 Detection and Time-of-Addition testVerification
The 3 compounds screened in experiment 3, stephanine, siramectin, mefloquine hydrochloride, were used to test for infection of Vero E6 cells using xCoV with moi=0.01.
EC 50 And (3) detection: vero E6 cells were inoculated into 24 well cell culture plates and tested when cell densities reached 60% -80%; the drug was diluted to 200. Mu.M and then diluted to 0.39. Mu.M in a two-fold ratio gradient. After the Vero E6 cells were changed, the drug solution was diluted 1:1 with the virus suspension and 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. 37 ℃ 5% CO 2 Culturing for 60-72 hr, observing CPE, extracting cell nucleic acid for qPCR detection, and performing data analysis with GraphPad-Prism 8 software to calculate EC 50 。
CC 50 And (3) detection: CC Using Cell-Titer-Blue method 50 Is detected. Vero E6 cells were inoculated into 96 well cell culture plates and tested when cell densities reached 60% -80%. And (3) diluting the medicine twice by gradient, and adding diluted medicine after changing the liquid of the Vero E6 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. 37 ℃ 5% CO 2 Culturing for 48 hr, adding 20 μl Cell-Titer-Blue into each well, detecting 593nm luminescence intensity at 0min, 30min, 60min, and 120min, respectively, and performing data analysis with GraphPad-Prism 8 software to calculate CC 50 。
SI is CC 50 Divided by EC 50 And (5) calculating to obtain the product.
Time-of-Addition detection: vero E6 cells were inoculated into 24-well cell culture plates and tested when cell densities reached 60% -80%. The test drug concentration was selected to be 6.25 μm. The whole time course experiment method includes adding medicine-virus mixed liquid and 5% CO at 37 deg.c 2 Culturing for 2h, changing liquid, and adding the drug-virus mixed liquid; "Pre-entry" experimental method: adding the drug-virus mixture, and 5% CO at 37deg.C 2 Culturing for 2h, changing the liquid, and adding pure culture medium; the "post-cell" experimental method: adding pure culture medium, and adding 5% CO at 37deg.C 2 Culturing for 2 hr, changing liquid, and adding medicine-virusAnd (3) mixing the liquid. 37 ℃ 5% CO 2 Culturing for 60-72h, observing CPE, extracting cell nucleic acid for qPCR detection, and analyzing data by using GraphPad-Prism 8 software.
6. Transcriptomic analysis of cepharanthine against xCoV infection
Stephanine (CEP) assay was used at a concentration of 6.25 μm to infect Vero E6 cells using xCoV with moi=0.01. Four groupings were set up: vero, vero+virus, vero+cep, vero+virus+cep. After 72h incubation, cell samples were collected and RNA extracted using TRIzol, rRNA was removed using QIAseq FastSelect-rRNA HMR Kit (Qiagen, product number 334387), NEBNEext Ultra was used TM RNA Library Prep Kit for Illumina (NEB, product number E7770L) an mRNA sequencing library was created and RNA sequencing (RNA-seq) was performed using an Illumina Hiseq 2500sequencing system (An Nuo, yoghurt Biotechnology Co., ltd.).
fastQC (http:// www.bioinformatics.babraham.ac.uk/subjects/FastQC /) tools and fastx_trimmers in the FASTX kit are 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; identifying differentially expressed genes between different experimental groups using DESeq 2; the P value was adjusted using the Benjamini-Hochberg method to calculate the False Discovery Rate (FDR); genes with FDR q values <0.05 and |log2 (fold change) | >1 were considered differentially expressed genes; and 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) were used as process files. The gene set includes (1) heat shock factor 1 (HSF 1) -mediated regulation of heat shock response, cell-mediated regulation of heat induction, HSF 1-dependent transactivation, HYPOXIA, defense response against viruses, response to viruses, HIF1 targets, adipocyte differentiation and autophagy, downloadable from MSigDB, KEGG and reactiome databases, (2) up/down-regulating genes of viruses, genes differentially expressed in the above RNA-seq data, with FDR q values <0.05 and |Log2 (fold change) | >1. Genomic enrichment P values were calculated for 1000 permutations using Signal2Noise model run GSEA4.0.3 (https:// www.gsea-msigdb. Org/gsea/index. Jsp), resulting in Normalized Enrichment Score (NES) values and FDR values. The visual heat map is drawn by the R package of GENE-E. And a heat map was drawn from the MSigDB, KEGG, and reactiome databases to display the selected gene set in a pass-through mode.
Gene Ontology (GO) analysis was performed on the genes obtained with differential expression as described above using the Metascape tool (https:// Metascape. Org). The pathway with P <0.05 was used as a significantly enriched pathway, the most significantly enriched pathway was demonstrated using the bubble map created by R-package ggplot2, using the Cytoscape in the Metascape website to map the interaction network and protein-protein interaction (PPI) network for each important enriched pathway. And a detailed PPI enrichment analysis was performed for each given gene list using bio grid and OmniPath.
Table 1 primer sequences used in the study
2. Experimental results
Comparison analysis of the whole genome and the individual virus-encoding genes (nucleotide level and amino acid level) found that: the xCoV has high homology with SARS-CoV-2, and has 92.5% homology with the S protein of SARS-COV-2, and is the virus which has been successfully isolated and cultured so far and has the highest homology with the S protein of SARS-COV-2 (FIG. 1). The homology of xCoV to SARS-CoV-2 is much higher than that of SARS virus, either at the whole genome level (fig. 2) or the key gene S gene (fig. 3) for virus adsorption into cells.
By adding different concentrations of siRNA to specifically knock down ACE2 expression, it was found that as ACE2mRNA expression levels were progressively reduced (fig. 4), xCoV's ability to infect cells was significantly progressively reduced, strongly suggesting that ACE2 is the receptor for xCoV into cells (fig. 5).
In the 96-well cell culture well,each well was added with one of the various known drugs (2080 marketed drugs and 326 antiviral compounds) and xCoV with moi=0.01 at a final concentration of 10 μm, and the treated Vero E6 cells were treated at 37 ℃ with 5% CO 2 The cells were cultured in a cell incubator for 72 hours. At this time, the cells were significantly cytopathic effect in the cells culture wells with no added virus and no added compound and in the vast majority of the cells culture wells with various compounds (FIG. 7), while the cells were not cytopathic in the cell culture without added virus and drug (FIG. 8). Then, no significant cytopathy was seen in the virus-infected cell culture wells to which cepharanthine (FIG. 9), sirolimus (FIG. 10) and mefloquine hydrochloride (FIG. 11) were added at a final concentration of 10. Mu.M. It is strongly suggested that stephanine, siramectin and mefloquine hydrochloride are potential potent inhibitors of xCoV infection.
Further, it was found by real-time quantitative PCR detection that 10. Mu. Mol/L of cepharanthine, sirolimus and mefloquine hydrochloride each had a 15393-fold, 5053-fold and 31-fold inhibition of viral replication after 72 hours of infection of cells with xCoV at a complex number of 0.01, compared to the control group in which only 0.1% DMSO was added (all compounds were dissolved in DMSO, and thus the DMSO concentration in each cell culture well was 0.1%) after the addition of the drug (FIG. 12). The experimental results were repeated 5 times and were all reproducible.
EC 50 、CC 50 The results with SI showed that inhibition of xCoV by stephanine (fig. 13), sirolimus (fig. 14), mefloquine hydrochloride (fig. 15) exhibited concentration-dependent phenomena. In addition, cepharanthine (fig. 16), sirolimus (fig. 17), and mefloquine hydrochloride (fig. 18) all exert viral inhibition after xCoV enters the cell.
Specifically, FIG. 16 shows the results of the Time-of-Addition test of cepharanthine to xCoV, indicating that cepharanthine exerts an inhibitory effect after xCoV enters cells, but cannot inhibit the entry of xCoV. FIG. 17 shows the results of the Time-of-Addition test of sirolimus on xCoV, indicating that sirolimus is inhibited after xCoV enters cells but not xCoV. FIG. 18 shows the results of the Time-of-Addition test of mefloquine hydrochloride to xCoV, indicating that mefloquine hydrochloride acts as an inhibitor after xCoV enters cells, but does not inhibit xCoV entry.
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 responses and HSF1 mediated heat shock responses (fig. 19).
3. Discussion of the application
The study of SARS-CoV-2 virus requires a high level of biological protection, which conflicts with the urgent need for research. The pangolin coronavirus xCoV isolated by the inventor has low pathogenicity or no pathogenicity to human body, and provides an alternative mode for researching SARS-CoV-2 closely related to the pangolin coronavirus xCoV, which 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 the population in intimate contact with pangolin scales; pangolin coronavirus xCoV isolates of the inventors were routinely cultured in biosafety secondary facilities.
One coronavirus found in 2013 from a sample collected from a nasphilic bat in Yunnan was closely related to SARS-CoV-2, and therefore, it was speculated that bat might also be the host for SARS-CoV-2. Recently, researchers at agricultural university of south China announced that pangolin scales (Manis java) were the intermediate host for SARS-CoV-2. Similarly, in month 10 of 2019, a whole genome study of viruses against pangolins found SARS-CoV-associated sequences, which were re-identified as SARS-CoV-2-associated sequences after appearance of SARS-CoV-2. In addition, the inventor also isolated and cultured a strain of SARS-CoV-2 related coronavirus xCoV from dead smuggled pangolin. Through comparative analysis of whole genome and each virus coding gene (nucleotide and amino acid), xCoV is found to be highly homologous with SARS-CoV-2, and the S protein homology with SARS-COV-2 reaches 92.5%, which is the virus with highest S protein homology with SARS-COV-2 which has been successfully isolated and cultured so far (FIG. 1). The homology of xCoV to SARS-CoV-2 is much higher than that of SARS virus, either at the whole genome level (fig. 2) or the key gene S gene (fig. 3) for virus adsorption into cells.
In this study, the inventors performed screening for anti-coronavirus active agents in the SARS-CoV-2 associated coronavirus, i.e., pangolin coronavirus xCoV model. Based on the results of previous laboratory studies, xCoV infection of mammalian cells Vero E6 (Vero cells) resulted in very pronounced cytopathic effects. Based on this feature, the inventors previously used xCoV to infect Vero cells in 96-well cell culture plates while adding a single drug on the market (2080 multiple drugs on the market and 326 antiviral compounds) to each cell culture well, and screened for potential active agents that inhibit viral replication. Cytopathic effect was observed under a microscope at day 3, and as a result, it was found that 3 potential drugs were significantly inhibited on virus-infected cells (3 drugs were cepharanthine, sirolimus and mefloquine hydrochloride, respectively). Since xCoV is highly homologous to current SARS-COV-2 and the receptor of xCoV-infected cells is consistent with SARS-COV-2, if the drug has inhibitory effect on xCoV-infected cells, it also has inhibitory effect on SARS-COV-2 infection.
Notably, the patent "application of cepharanthine in preparing anti-SARS virus medicament" to Wang Yifei et al, mentions that the half-inhibitory dose of cepharanthine against SARS-CoV virus causing SARS in 2003 is 8 μg/ml (13.186 μM), i.e. the final concentration of cepharanthine of 13.186 μM can inhibit 50% of viral infection. The experimental results of the inventors show that the ability to inhibit xCoV virus replication reaches 15393 times with lower concentrations of cepharanthine (10 μm). Thus, stephanine has at least 30786 times greater inhibition of xCoV virus replication than SARS-CoV virus replication. Furthermore, the inhibition of SARS-CoV by stephanine (actually, inefficient inhibition) does not suggest that it can strongly and effectively inhibit xCoV and SARS-CoV-2. In fact, the present inventors have tested that there are many drugs (e.g., oseltamivir phosphate) contained in the marketed drug library that are effective in inhibiting the replication of SARS-CoV virus, but they (e.g., oseltamivir phosphate) have little 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 large homology with SARS-CoV-2 virus, SARS-CoV virus and SARS-CoV-2 virus differ greatly in genome and amino acid level, and it is highly likely that the drugs inhibiting SARS-CoV virus have no effect on xCoV and SARS-CoV-2 (e.g., oseltamivir phosphate). The xCoV is isolated from dead pangolins obtained from customs and has high homology with SARS-CoV-2, and is the virus with the highest homology with SARS-CoV-2 in the coronaviruses which can be isolated and cultured at present. Therefore, the stephanine, the siramectin and the mefloquine hydrochloride which have strong inhibition effect on xCoV can also inhibit the virus replication of SARS-CoV-2, are very likely to become specific medicines for treating novel coronavirus pneumonia, and are suggested to be used for clinical tests of SARS-CoV-2 patients.
The inventor considers that the stephanine has particularly important medicinal value as a potential medicine for treating SARS-CoV-2. The medicine is a double-knot-based Isaline alkaloid separated and extracted from Stephania japonica Diels of Menispermaceae, and is approved for treating leukopenia. It has multiple functions such as inhibiting the efflux transporter ABCC10 of antitumor drugs, inhibiting HIV-1 entry by decreasing plasma membrane fluidity, and binding to the central portion of Hsp 90. Importantly, large doses of this drug have low toxicity in animals and no significant side effects in humans. In addition, studies have shown that SARS-CoV-2 can cause the enrichment of cell stress response and autophagy pathway related genes in peripheral blood mononuclear cells, while stephanine can effectively reverse most deregulated genes and pathways in infected cells, especially endoplasmic reticulum stress/unfolded protein response and HSF1 mediated heat shock response, thereby exerting anti-coronavirus infection effects. In view of the observed strong inhibition of viral replication and the established anti-inflammatory effect of the drug, the inventors believe that cepharanthine is a promising candidate for the treatment of SARS-CoV-2 infection.
Claims (1)
1. Use of mefloquine hydrochloride or mefloquine for the preparation of a medicament for the treatment of SARS-COV-2 virus infectious diseases.
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