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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

GC7 disrupts ssRNA association with oligomeric EBOV NP complexes.(A) Measurement of ssRNA binding to oligomeric EBOV NP 36–450 at increasing concentrations by fluorescence anisotropy. (B) Competitive binding between GC7 and fluorophore-labeled ssRNA to oligomeric EBOV NP 36–450. IC50 is derived at the concentration of GC7 that inhibits ssRNA binding to NP oligomers by 50%. Error bars represent SD from independent experiments in triplicate.
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f6: GC7 disrupts ssRNA association with oligomeric EBOV NP complexes.(A) Measurement of ssRNA binding to oligomeric EBOV NP 36–450 at increasing concentrations by fluorescence anisotropy. (B) Competitive binding between GC7 and fluorophore-labeled ssRNA to oligomeric EBOV NP 36–450. IC50 is derived at the concentration of GC7 that inhibits ssRNA binding to NP oligomers by 50%. Error bars represent SD from independent experiments in triplicate.

Mentions: To evaluate the effect of the ligand GC7 in mediating EBOV NP interaction with viral ssRNA, we generated another construct (residue 36–450) in order to purify RNA-free oligomerized EBOV NP. The bacterial RNA co-purified with the protein was removed by collecting the monomer fraction eluted from SEC using high-salt buffer (1.5 M NaCl) (Supplemental Figure S4A). Subsequent dilution to the low-salt condition (200 mM NaCl) triggered spontaneous NP oligomerization confirmed by SEC analysis (Supplemental Figure S4B). The resulting oligomeric RNA-free EBOV NP 36–450 readily binds to an 18 nucleotide ssRNA with low micromolar affinity (~5 μM) measured by fluorescence anisotropy (Fig. 6A). Next a competitive binding assay was conducted by first incubating the monomer of EBOV NP 36–450 with GC7 at various concentrations before diluting the mixture and adding the RNA probe. GC7 incubation with the NP monomer caused protein oligomerization. Importantly, this ligand when bound to the NP oligomer was found to compete with ssRNA for interaction with NP complexes. Increasing GC7 concentration during incubation resulted in a proportional loss of ssRNA from the RNA-NP complex, yielding a half maximal inhibitory concentration (IC50) of ~160 μM (Fig. 6B). These results indicate that the new ligand GC7 disrupts the interaction of EBOV NP with viral ssRNA through either directly occluding the RNA-binding cleft or inducing a conformational change of NP oligomers that would impair ssRNA association.


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)

GC7 disrupts ssRNA association with oligomeric EBOV NP complexes.(A) Measurement of ssRNA binding to oligomeric EBOV NP 36–450 at increasing concentrations by fluorescence anisotropy. (B) Competitive binding between GC7 and fluorophore-labeled ssRNA to oligomeric EBOV NP 36–450. IC50 is derived at the concentration of GC7 that inhibits ssRNA binding to NP oligomers by 50%. Error bars represent SD from independent experiments in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: GC7 disrupts ssRNA association with oligomeric EBOV NP complexes.(A) Measurement of ssRNA binding to oligomeric EBOV NP 36–450 at increasing concentrations by fluorescence anisotropy. (B) Competitive binding between GC7 and fluorophore-labeled ssRNA to oligomeric EBOV NP 36–450. IC50 is derived at the concentration of GC7 that inhibits ssRNA binding to NP oligomers by 50%. Error bars represent SD from independent experiments in triplicate.
Mentions: To evaluate the effect of the ligand GC7 in mediating EBOV NP interaction with viral ssRNA, we generated another construct (residue 36–450) in order to purify RNA-free oligomerized EBOV NP. The bacterial RNA co-purified with the protein was removed by collecting the monomer fraction eluted from SEC using high-salt buffer (1.5 M NaCl) (Supplemental Figure S4A). Subsequent dilution to the low-salt condition (200 mM NaCl) triggered spontaneous NP oligomerization confirmed by SEC analysis (Supplemental Figure S4B). The resulting oligomeric RNA-free EBOV NP 36–450 readily binds to an 18 nucleotide ssRNA with low micromolar affinity (~5 μM) measured by fluorescence anisotropy (Fig. 6A). Next a competitive binding assay was conducted by first incubating the monomer of EBOV NP 36–450 with GC7 at various concentrations before diluting the mixture and adding the RNA probe. GC7 incubation with the NP monomer caused protein oligomerization. Importantly, this ligand when bound to the NP oligomer was found to compete with ssRNA for interaction with NP complexes. Increasing GC7 concentration during incubation resulted in a proportional loss of ssRNA from the RNA-NP complex, yielding a half maximal inhibitory concentration (IC50) of ~160 μM (Fig. 6B). These results indicate that the new ligand GC7 disrupts the interaction of EBOV NP with viral ssRNA through either directly occluding the RNA-binding cleft or inducing a conformational change of NP oligomers that would impair ssRNA association.

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