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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

K160 is engaged in ligand binding and shift of NP oligomeric state.(A) Binding efficiency of GC7 or GC13 to a specific EBOV NP mutant normalized to ligand binding to wild-type NP (defined as 100%) measured by affinity MS. Error bars represent SD from independent experiments in triplicate. (B) SEC chromatograms of wild-type EBOV NP alone or incubated with a specific ligand. (C) SEC chromatograms of EBOV NP K160A mutant alone or incubated with a specific ligand.
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f5: K160 is engaged in ligand binding and shift of NP oligomeric state.(A) Binding efficiency of GC7 or GC13 to a specific EBOV NP mutant normalized to ligand binding to wild-type NP (defined as 100%) measured by affinity MS. Error bars represent SD from independent experiments in triplicate. (B) SEC chromatograms of wild-type EBOV NP alone or incubated with a specific ligand. (C) SEC chromatograms of EBOV NP K160A mutant alone or incubated with a specific ligand.

Mentions: Based on the apo protein structure, we employed in silico docking analysis to create a potential model for binding of GC7 and GC13 to EBOV NP. The predicted ligand binding site involves residues K160 and R298 that possibly make hydrogen bonds with both ligands (Fig. 4C,D). We prepared EBOV NP mutants harboring alanine substitution at K160 and R298 to examine ligand binding capacity using our affinity MS approach. Compared to the wild-type protein, mutation at K160 resulted in nearly 40% loss of binding of both GC7 and GC13, and mutation at R298 reduced binding efficiency of two ligands to a less extent (Fig. 5A). Importantly, the predicted binding free energy for GC7 (−5.88 kcal/mol) is much lower than that for GC10 (−2.75 kcal/mol), which provides the molecular basis for selective binding of one diostereo-isomer GC7 to the protein target. Furthermore, whereas the binding of GC7 strongly induced oligomerization of the wild-type EBOV NP, less obvious shift of the oligomeric state with the same ligand was observed for mutant K160A (Fig. 5B,C).


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)

K160 is engaged in ligand binding and shift of NP oligomeric state.(A) Binding efficiency of GC7 or GC13 to a specific EBOV NP mutant normalized to ligand binding to wild-type NP (defined as 100%) measured by affinity MS. Error bars represent SD from independent experiments in triplicate. (B) SEC chromatograms of wild-type EBOV NP alone or incubated with a specific ligand. (C) SEC chromatograms of EBOV NP K160A mutant alone or incubated with a specific ligand.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: K160 is engaged in ligand binding and shift of NP oligomeric state.(A) Binding efficiency of GC7 or GC13 to a specific EBOV NP mutant normalized to ligand binding to wild-type NP (defined as 100%) measured by affinity MS. Error bars represent SD from independent experiments in triplicate. (B) SEC chromatograms of wild-type EBOV NP alone or incubated with a specific ligand. (C) SEC chromatograms of EBOV NP K160A mutant alone or incubated with a specific ligand.
Mentions: Based on the apo protein structure, we employed in silico docking analysis to create a potential model for binding of GC7 and GC13 to EBOV NP. The predicted ligand binding site involves residues K160 and R298 that possibly make hydrogen bonds with both ligands (Fig. 4C,D). We prepared EBOV NP mutants harboring alanine substitution at K160 and R298 to examine ligand binding capacity using our affinity MS approach. Compared to the wild-type protein, mutation at K160 resulted in nearly 40% loss of binding of both GC7 and GC13, and mutation at R298 reduced binding efficiency of two ligands to a less extent (Fig. 5A). Importantly, the predicted binding free energy for GC7 (−5.88 kcal/mol) is much lower than that for GC10 (−2.75 kcal/mol), which provides the molecular basis for selective binding of one diostereo-isomer GC7 to the protein target. Furthermore, whereas the binding of GC7 strongly induced oligomerization of the wild-type EBOV NP, less obvious shift of the oligomeric state with the same ligand was observed for mutant K160A (Fig. 5B,C).

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