In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase cofactor activity.
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.
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA; Biochemistry Undergraduate Program, Iowa State University, Ames, IA 50011, USA.
Craig S
CS
Brown
Simulation Sciences Branch, US Army Research Laboratory, Aberdeen, MD 21005, USA; Department of Cell Biology and Biochemistry, USAMRIID, 1425 Porter St., Fort Detrick, MD 21702, USA.
Michael S
MS
Lee
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Daisy W
DW
Leung
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Tianjiao
T
Wang
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Wei
W
Xu
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Priya
P
Luthra
Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
Manu
M
Anantpadma
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Reed S
RS
Shabman
Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Lisa M
LM
Melito
Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Karen S
KS
MacMillan
Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA; Department of Biophysics, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA; Center for Structural Genomics of Infectious Diseases (CSGID), Chicago, IL, USA.
Dominika M
DM
Borek
Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA; Department of Biophysics, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA; Center for Structural Genomics of Infectious Diseases (CSGID), Chicago, IL, USA.
Zbyszek
Z
Otwinowski
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Biochemistry Graduate Program, Iowa State University, Ames, IA 50011, USA.
Parameshwaran
P
Ramanan
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA; Biochemistry Undergraduate Program, Iowa State University, Ames, IA 50011, USA.
Alisha J
AJ
Stubbs
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA; Biochemistry Undergraduate Program, Iowa State University, Ames, IA 50011, USA.
Dayna S
DS
Peterson
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Biochemistry Graduate Program, Iowa State University, Ames, IA 50011, USA.
Jennifer M
JM
Binning
National Magnetic Resonance Facility at Madison, University of Wisconsin, Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Marco
M
Tonelli
Department of Cell Biology and Biochemistry, USAMRIID, 1425 Porter St., Fort Detrick, MD 21702, USA.
Mark A
MA
Olson
Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, TX 78227, USA.
Robert A
RA
Davey
Department of Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Joseph M
JM
Ready
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Christopher F
CF
Basler
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: gamarasinghe@path.wustl.edu.
Gaya K
GK
Amarasinghe
D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology; D003067:Coenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000378:metabolism; D029043:Ebolavirus / Q000187:drug effects* / Q000201:enzymology; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D054334:Viral Regulatory and Accessory Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry
D000998:Antiviral Agents; D003067:Coenzymes; D011758:Pyrroles; D054852:Small Molecule Libraries; C080980:VP35 protein, filovirus; D054334:Viral Regulatory and Accessory Proteins; D012321:DNA-Directed RNA Polymerases
VP35; antivirals; filoviral inhibitors; in silico drug discovery