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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

Validation of NP ligands in compound mixture screens.(A) LC-HRMS chromatograms of the protein incubation sample and the control from the EBOV NP experiment (upper) and MARV NP experiment (lower). The NP protein was incubated with a mixture of 14 pure compounds known to be licorice constituents and then ligands were identified by affinity MS analysis. (B) S/N ratios of specific compounds from the mixture screen experiment. Note that GC2/GC12 and GC7/GC10 show identical S/N ratios as they are isomeric pairs that cannot be distinguished under the LC condition. GC7/GC10 and GC13 with S/N > 2 are considered ligands to both NPs.
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f3: Validation of NP ligands in compound mixture screens.(A) LC-HRMS chromatograms of the protein incubation sample and the control from the EBOV NP experiment (upper) and MARV NP experiment (lower). The NP protein was incubated with a mixture of 14 pure compounds known to be licorice constituents and then ligands were identified by affinity MS analysis. (B) S/N ratios of specific compounds from the mixture screen experiment. Note that GC2/GC12 and GC7/GC10 show identical S/N ratios as they are isomeric pairs that cannot be distinguished under the LC condition. GC7/GC10 and GC13 with S/N > 2 are considered ligands to both NPs.

Mentions: To verify binding of ligands discovered from the licorice extract and evaluate other known licorice constituents, we incubated the EBOV NP or MARV NP protein with a mixture of fourteen pure standards at an equal amount for affinity MS analysis. Figure 3A shows the LC-HRMS chromatograms for the protein incubation sample and the control in two types of NP experiments. Compounds with an average S/N ratio > 2 (RSD < 30%) indicate significant increase of abundance in the protein complex and thus are considered ligands to the NP target. Both GC7/10 and GC13 from the compound mixture were highly enriched in EBOV NP and MARV NP complexes whereas none of the other licorice constituents showed significant binding (Fig. 3B).


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)

Validation of NP ligands in compound mixture screens.(A) LC-HRMS chromatograms of the protein incubation sample and the control from the EBOV NP experiment (upper) and MARV NP experiment (lower). The NP protein was incubated with a mixture of 14 pure compounds known to be licorice constituents and then ligands were identified by affinity MS analysis. (B) S/N ratios of specific compounds from the mixture screen experiment. Note that GC2/GC12 and GC7/GC10 show identical S/N ratios as they are isomeric pairs that cannot be distinguished under the LC condition. GC7/GC10 and GC13 with S/N > 2 are considered ligands to both NPs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Validation of NP ligands in compound mixture screens.(A) LC-HRMS chromatograms of the protein incubation sample and the control from the EBOV NP experiment (upper) and MARV NP experiment (lower). The NP protein was incubated with a mixture of 14 pure compounds known to be licorice constituents and then ligands were identified by affinity MS analysis. (B) S/N ratios of specific compounds from the mixture screen experiment. Note that GC2/GC12 and GC7/GC10 show identical S/N ratios as they are isomeric pairs that cannot be distinguished under the LC condition. GC7/GC10 and GC13 with S/N > 2 are considered ligands to both NPs.
Mentions: To verify binding of ligands discovered from the licorice extract and evaluate other known licorice constituents, we incubated the EBOV NP or MARV NP protein with a mixture of fourteen pure standards at an equal amount for affinity MS analysis. Figure 3A shows the LC-HRMS chromatograms for the protein incubation sample and the control in two types of NP experiments. Compounds with an average S/N ratio > 2 (RSD < 30%) indicate significant increase of abundance in the protein complex and thus are considered ligands to the NP target. Both GC7/10 and GC13 from the compound mixture were highly enriched in EBOV NP and MARV NP complexes whereas none of the other licorice constituents showed significant binding (Fig. 3B).

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