A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35-NP Interface.
The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.
Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.|Microbiotix Inc. , 1 Innovation Drive, Worcester, Massachusetts 01605, United States.
Gai
G
Liu
Microbiotix Inc. , 1 Innovation Drive, Worcester, Massachusetts 01605, United States.
Peter J
PJ
Nash
Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.
Britney
B
Johnson
Department of Pathology, Department of Microbiology and Immunology, and Galveston National Laboratory, University of Texas Medical Branch at Galveston , Galveston, Texas 77555, United States.
Colette
C
Pietzsch
High Throughput Screening Center, Department of Biochemistry and Molecular Biophysics, and Department of Developmental Biology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.
Ma Xenia G
MX
Ilagan
Department of Pathology, Department of Microbiology and Immunology, and Galveston National Laboratory, University of Texas Medical Branch at Galveston , Galveston, Texas 77555, United States.
Alexander
A
Bukreyev
Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University , Atlanta, Georgia 30303, United States.
Christopher F
CF
Basler
Microbiotix Inc. , 1 Innovation Drive, Worcester, Massachusetts 01605, United States.
Terry L
TL
Bowlin
Microbiotix Inc. , 1 Innovation Drive, Worcester, Massachusetts 01605, United States.
Donald T
DT
Moir
Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.
Daisy W
DW
Leung
Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.
Gaya K
0000-0002-0418-9707
GK
Amarasinghe
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D017124:Conserved Sequence; D004353:Drug Evaluation, Preclinical; D016563:Filoviridae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D005454:Fluorescence Polarization; D057166:High-Throughput Screening Assays; D066298:In Vitro Techniques; D008958:Models, Molecular; D009698:Nucleoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D009698:Nucleoproteins; C080980:VP35 protein, filovirus; D054334:Viral Regulatory and Accessory Proteins
Ebola virus; VP35; fluorescence polarization assay; high-throughput screening; nucleoprotein