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Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches.

Fu X, Wang Z, Li L, Dong S, Li Z, Jiang Z, Wang Y, Shui W - Sci Rep (2016)

Bottom Line: Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP.Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources.In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development.

View Article: PubMed Central - PubMed

Affiliation: College of Biotechnology, Tianjin University of Science &Technology, Tianjin 300457, China.

ABSTRACT
The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development.

No MeSH data available.


Related in: MedlinePlus

Identification of chemical ligands bound to Ebola virus and Marburg virus NPs from Chinese licorice.(A) The workflow of combining affinity MS and metabolomics approaches for ligand discovery towards NPs. (B) OPLS-DA score plots of the crude extracts, the NP incubation samples and the control samples show clear separation of three groups in the data sets of EBOV NP (left) and MARV NP (right). (C) VIP and S/N plots of features detected in the protein incubation sample and control from the EBOV NP experiment (left) and MARV NP experiment (right). Each grey symbol represents a feature detected in four independent replicates (RSD of S/N ratios across replicates < 30%). Each feature also matches its accurate mass with the Chinese licorice compound database. Red symbols annotate two putative ligands bound to NPs (GC7 and GC13). Blue symbols annotate the rest of 11 constituents in Chinese licorice that do not bind NPs. All color-coded compounds are identified by HRMS and MSMS analysis according to reference standards.
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f1: Identification of chemical ligands bound to Ebola virus and Marburg virus NPs from Chinese licorice.(A) The workflow of combining affinity MS and metabolomics approaches for ligand discovery towards NPs. (B) OPLS-DA score plots of the crude extracts, the NP incubation samples and the control samples show clear separation of three groups in the data sets of EBOV NP (left) and MARV NP (right). (C) VIP and S/N plots of features detected in the protein incubation sample and control from the EBOV NP experiment (left) and MARV NP experiment (right). Each grey symbol represents a feature detected in four independent replicates (RSD of S/N ratios across replicates < 30%). Each feature also matches its accurate mass with the Chinese licorice compound database. Red symbols annotate two putative ligands bound to NPs (GC7 and GC13). Blue symbols annotate the rest of 11 constituents in Chinese licorice that do not bind NPs. All color-coded compounds are identified by HRMS and MSMS analysis according to reference standards.

Mentions: To comprehensively screen the crude extract of Chinese licorice for virus NP ligand discovery, we devised a workflow combining affinity mass spectrometry and metabolomics approaches (Fig. 1A). The recombinant core domains of Ebola virus NP (EBOV NP, amino acids 36–351) and Marburg virus NP (MARV NP, amino acids 19–370) were purified and separately incubated with Chinese licorice extract before ultrafiltration was performed to isolate ligand-bound NP complexes. The compound mixture dissociated from the NP complexes or the protein-free control was subjected to metabolomic analysis using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) (full datasets in Supplemental Table S1). A multivariate model based on orthogonal partial least-squares discriminant analysis (OPLS-DA) was then created to differentiate the compound profiles from the crude extracts, the protein incubation samples and the control samples. In both types of NP experiments, there was distinct separation of three sample groups, indicating that the composition of compounds associated with the NP complex was substantially deviated from that in the crude extract and the control (Fig. 1B). We then applied multiple criteria to select putative ligands specifically enriched in the EBOV NP or MARV NP complexes which include: 1) variable influence on projection (VIP) score > 1.0, representing compounds most implicated in the difference between the NP complex and control2728; 2) average S/N value > 2.0 and RSD < 30%, indicating significant change of peak intensity of the compound in the protein complex relative to control1920; 3) matching the accurate mass of a putative ligand to the TCM compound database for Chinese licorice (see details in Experimental). Using this set of stringent criteria, we identified 30 and 22 components from the licorice extract to be candidate ligands bound to EBOV NP or MARV NP, respectively (Fig. 1C, Table S1).


Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches.

Fu X, Wang Z, Li L, Dong S, Li Z, Jiang Z, Wang Y, Shui W - Sci Rep (2016)

Identification of chemical ligands bound to Ebola virus and Marburg virus NPs from Chinese licorice.(A) The workflow of combining affinity MS and metabolomics approaches for ligand discovery towards NPs. (B) OPLS-DA score plots of the crude extracts, the NP incubation samples and the control samples show clear separation of three groups in the data sets of EBOV NP (left) and MARV NP (right). (C) VIP and S/N plots of features detected in the protein incubation sample and control from the EBOV NP experiment (left) and MARV NP experiment (right). Each grey symbol represents a feature detected in four independent replicates (RSD of S/N ratios across replicates < 30%). Each feature also matches its accurate mass with the Chinese licorice compound database. Red symbols annotate two putative ligands bound to NPs (GC7 and GC13). Blue symbols annotate the rest of 11 constituents in Chinese licorice that do not bind NPs. All color-coded compounds are identified by HRMS and MSMS analysis according to reference standards.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940736&req=5

f1: Identification of chemical ligands bound to Ebola virus and Marburg virus NPs from Chinese licorice.(A) The workflow of combining affinity MS and metabolomics approaches for ligand discovery towards NPs. (B) OPLS-DA score plots of the crude extracts, the NP incubation samples and the control samples show clear separation of three groups in the data sets of EBOV NP (left) and MARV NP (right). (C) VIP and S/N plots of features detected in the protein incubation sample and control from the EBOV NP experiment (left) and MARV NP experiment (right). Each grey symbol represents a feature detected in four independent replicates (RSD of S/N ratios across replicates < 30%). Each feature also matches its accurate mass with the Chinese licorice compound database. Red symbols annotate two putative ligands bound to NPs (GC7 and GC13). Blue symbols annotate the rest of 11 constituents in Chinese licorice that do not bind NPs. All color-coded compounds are identified by HRMS and MSMS analysis according to reference standards.
Mentions: To comprehensively screen the crude extract of Chinese licorice for virus NP ligand discovery, we devised a workflow combining affinity mass spectrometry and metabolomics approaches (Fig. 1A). The recombinant core domains of Ebola virus NP (EBOV NP, amino acids 36–351) and Marburg virus NP (MARV NP, amino acids 19–370) were purified and separately incubated with Chinese licorice extract before ultrafiltration was performed to isolate ligand-bound NP complexes. The compound mixture dissociated from the NP complexes or the protein-free control was subjected to metabolomic analysis using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) (full datasets in Supplemental Table S1). A multivariate model based on orthogonal partial least-squares discriminant analysis (OPLS-DA) was then created to differentiate the compound profiles from the crude extracts, the protein incubation samples and the control samples. In both types of NP experiments, there was distinct separation of three sample groups, indicating that the composition of compounds associated with the NP complex was substantially deviated from that in the crude extract and the control (Fig. 1B). We then applied multiple criteria to select putative ligands specifically enriched in the EBOV NP or MARV NP complexes which include: 1) variable influence on projection (VIP) score > 1.0, representing compounds most implicated in the difference between the NP complex and control2728; 2) average S/N value > 2.0 and RSD < 30%, indicating significant change of peak intensity of the compound in the protein complex relative to control1920; 3) matching the accurate mass of a putative ligand to the TCM compound database for Chinese licorice (see details in Experimental). Using this set of stringent criteria, we identified 30 and 22 components from the licorice extract to be candidate ligands bound to EBOV NP or MARV NP, respectively (Fig. 1C, Table S1).

Bottom Line: Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP.Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources.In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development.

View Article: PubMed Central - PubMed

Affiliation: College of Biotechnology, Tianjin University of Science &Technology, Tianjin 300457, China.

ABSTRACT
The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development.

No MeSH data available.


Related in: MedlinePlus