title: Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets.
abstract: Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry.
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, 84112-5650.
authors.forename: Tracy R
authors.initials: TR
authors.lastname: Clinton
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Weinstock
authors.forename: Michael T
authors.initials: MT
authors.lastname: Jacobsen
authors.forename: Nicolas
authors.initials: N
authors.lastname: Szabo-Fresnais
authors.forename: Maya J
authors.initials: MJ
authors.lastname: Pandya
authors.forename: Frank G
authors.initials: FG
authors.lastname: Whitby
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Herbert
authors.forename: Laura I
authors.initials: LI
authors.lastname: Prugar
authors.forename: Rena
authors.initials: R
authors.lastname: McKinnon
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Hill
authors.forename: Brett D
authors.initials: BD
authors.lastname: Welch
authors.forename: John M
authors.initials: JM
authors.lastname: Dye
authors.forename: Debra M
authors.initials: DM
authors.lastname: Eckert
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D016503:Drug Delivery Systems; D029043:Ebolavirus / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus
keywords: N-trimer; designed coiled coil; ebolavirus; ebolavirus GP2; filovirus entry; mirror-image phage display; phage display; prehairpin intermediate