Abstract
The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2045-2058 |
| Number of pages | 14 |
| Journal | Journal of Molecular Biology |
| Volume | 426 |
| Issue number | 10 |
| DOIs | |
| State | Published - May 15 2014 |
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Keywords
- antivirals
- filoviral inhibitors
- in silico drug discovery
- VP35
ASJC Scopus subject areas
- Molecular Biology
Cite this
In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase cofactor activity. / Brown, Craig S.; Lee, Michael S.; Leung, Daisy W.; Wang, Tianjiao; Xu, Wei; Luthra, Priya; Anantpadma, Manu; Shabman, Reed S.; Melito, Lisa M.; Macmillan, Karen S.; Borek, Dominika M.; Otwinowski, Zbyszek; Ramanan, Parameshwaran; Stubbs, Alisha J.; Peterson, Dayna S.; Binning, Jennifer M.; Tonelli, Marco; Olson, Mark A.; Davey, Robert A.; Ready, Joseph M.; Basler, Christopher F.; Amarasinghe, Gaya K.
In: Journal of Molecular Biology, Vol. 426, No. 10, 15.05.2014, p. 2045-2058.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase cofactor activity
AU - Brown, Craig S.
AU - Lee, Michael S.
AU - Leung, Daisy W.
AU - Wang, Tianjiao
AU - Xu, Wei
AU - Luthra, Priya
AU - Anantpadma, Manu
AU - Shabman, Reed S.
AU - Melito, Lisa M.
AU - Macmillan, Karen S.
AU - Borek, Dominika M.
AU - Otwinowski, Zbyszek
AU - Ramanan, Parameshwaran
AU - Stubbs, Alisha J.
AU - Peterson, Dayna S.
AU - Binning, Jennifer M.
AU - Tonelli, Marco
AU - Olson, Mark A.
AU - Davey, Robert A.
AU - Ready, Joseph M.
AU - Basler, Christopher F.
AU - Amarasinghe, Gaya K.
PY - 2014/5/15
Y1 - 2014/5/15
N2 - The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
AB - The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
KW - antivirals
KW - filoviral inhibitors
KW - in silico drug discovery
KW - VP35
UR - http://www.scopus.com/inward/record.url?scp=84899641278&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84899641278&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2014.01.010
DO - 10.1016/j.jmb.2014.01.010
M3 - Article
C2 - 24495995
AN - SCOPUS:84899641278
VL - 426
SP - 2045
EP - 2058
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 10
ER -