The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL prot...The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.展开更多
Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly letha...Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly lethal disease caused by porcine epidemic diarrhoea virus(PEDV)and causes substantial losses in the swine industry worldwide.However,currently available commercial drugs have not shown great therapeutic effects.In this study,a fluorescence resonance energy transfer(FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds,3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone,that target the 3C-like protease(3CL^(pro))of PEDV.These compounds are of low molecular weight(MW)and greatly inhibited the activity of this enzyme(IC_(50) values were obtained in this study).Furthermore,these compounds exhibited antiviral capacity against another member of the CoV family,feline infectious peritonitis virus(FIPV).Here,the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified(with EC_(50) values)and cell viability assays were performed.The results of putative molecular docking models indicate that these compounds,labeled compound 1 and compound 2,contact the conserved active sites(Cys144,Glu165,Gln191)of 3CL^(pro) via hydrogen bonds.These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.展开更多
Since December 2019,severe acute respiratory syndrome coronavirus 2 has been found to be the culprit in the coronavirus disease 2019(COVID-19),causing a global pandemic.Despite the existence of many vaccine programs,t...Since December 2019,severe acute respiratory syndrome coronavirus 2 has been found to be the culprit in the coronavirus disease 2019(COVID-19),causing a global pandemic.Despite the existence of many vaccine programs,the number of confirmed cases and fatalities due to COVID-19 is still increasing.Furthermore,a number of variants have been reported.Because of the absence of approved anticoronavirus drugs,the treatment and management of COVID-19 has become a global challenge.Under these circumstances,drug repurposing is an effective method to identify candidate drugs with a shorter cycle of clinical trials.Here,we summarize the current status of the application of drug repurposing in COVID-19,including drug repurposing based on virtual computer screening,network pharmacology,and bioactivity,which may be a beneficial COVID-19 treatment.展开更多
Objective: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot(RDS), was developed based on a classical prescription of traditional Chinese medicine(TCM) and the theoretical understanding of her...Objective: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot(RDS), was developed based on a classical prescription of traditional Chinese medicine(TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019(COVID-19), have been shown. However,the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.Methods: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein–protein interaction(PPI) network, and cluster analysis;further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.Results: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent–target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target–constitu ent–disease network. Twelve disease pathways attributed to the Lung–Large Intestine meridians, with six and five attributed to the Kidney–Urinary Bladder and Stomach–Spleen meridians, respectively. Onehundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-23-chymotrypsin-like protease(3 CLpro), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3 CLproinhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode,the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.Conclusion: RDS acts primarily in the Lung–Large Intestine, Kidney–Urinary Bladder and Stomach–Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS’s dual effects of healthstrengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.展开更多
基金supported by the National Key Plan for Scientific Research and Development of China(2016YFD0500300)National Natural Science Foundation of China(32170651)Hunan Provincial Natural Science Foundation of China(2020JJ3006)。
文摘The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.
基金This work was supported by the National Key R&D Plan of China(grant no.2018YFD0500102)the Natural Science Foundation of Hubei Province of China(grant no.2016CFA069)。
文摘Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly lethal disease caused by porcine epidemic diarrhoea virus(PEDV)and causes substantial losses in the swine industry worldwide.However,currently available commercial drugs have not shown great therapeutic effects.In this study,a fluorescence resonance energy transfer(FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds,3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone,that target the 3C-like protease(3CL^(pro))of PEDV.These compounds are of low molecular weight(MW)and greatly inhibited the activity of this enzyme(IC_(50) values were obtained in this study).Furthermore,these compounds exhibited antiviral capacity against another member of the CoV family,feline infectious peritonitis virus(FIPV).Here,the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified(with EC_(50) values)and cell viability assays were performed.The results of putative molecular docking models indicate that these compounds,labeled compound 1 and compound 2,contact the conserved active sites(Cys144,Glu165,Gln191)of 3CL^(pro) via hydrogen bonds.These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.
基金supported by the Ph D Start-up Fund of Guangdong Medical University(Grant No.:B2019016)Administration of Traditional Chinese Medicine of Guangdong Province(Grant No.:20201180)+4 种基金Science and Technology Special Project of Zhanjiang(Project No.:2019A01009)Natural Science Foundation of Guangdong Province(Grant No.:2016B030309002)Basic and Applied Basic Research Program of Guangdong Province(Grant No.:2019A1515110201)Educational Commission of Guangdong Province(Grant No.:4SG20138G)Fund of Southern Marine Science and Engineering Guangdong Laboratory(Zhanjiang)(Grant No.:ZJW-2019-007)。
文摘Since December 2019,severe acute respiratory syndrome coronavirus 2 has been found to be the culprit in the coronavirus disease 2019(COVID-19),causing a global pandemic.Despite the existence of many vaccine programs,the number of confirmed cases and fatalities due to COVID-19 is still increasing.Furthermore,a number of variants have been reported.Because of the absence of approved anticoronavirus drugs,the treatment and management of COVID-19 has become a global challenge.Under these circumstances,drug repurposing is an effective method to identify candidate drugs with a shorter cycle of clinical trials.Here,we summarize the current status of the application of drug repurposing in COVID-19,including drug repurposing based on virtual computer screening,network pharmacology,and bioactivity,which may be a beneficial COVID-19 treatment.
基金the financial support from National Key Research and Development Program of China (No.2018YFC1707900).
文摘Objective: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot(RDS), was developed based on a classical prescription of traditional Chinese medicine(TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019(COVID-19), have been shown. However,the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.Methods: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein–protein interaction(PPI) network, and cluster analysis;further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.Results: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent–target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target–constitu ent–disease network. Twelve disease pathways attributed to the Lung–Large Intestine meridians, with six and five attributed to the Kidney–Urinary Bladder and Stomach–Spleen meridians, respectively. Onehundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-23-chymotrypsin-like protease(3 CLpro), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3 CLproinhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode,the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.Conclusion: RDS acts primarily in the Lung–Large Intestine, Kidney–Urinary Bladder and Stomach–Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS’s dual effects of healthstrengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.
