title: Conformation-dependent recognition of HIV gp120 by designed ankyrin repeat proteins provides access to novel HIV entry inhibitors.
abstract: Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.
authors.affiliation: Institute of Medical Virology, University of Zurich, Zurich, Switzerland.
authors.forename: Axel
authors.initials: A
authors.lastname: Mann
authors.forename: Nikolas
authors.initials: N
authors.lastname: Friedrich
authors.forename: Anders
authors.initials: A
authors.lastname: Krarup
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Weber
authors.forename: Emanuel
authors.initials: E
authors.lastname: Stiegeler
authors.forename: Birgit
authors.initials: B
authors.lastname: Dreier
authors.forename: Pavel
authors.initials: P
authors.lastname: Pugach
authors.forename: Melissa
authors.initials: M
authors.lastname: Robbiani
authors.forename: Tina
authors.initials: T
authors.lastname: Riedel
authors.forename: Kerstin
authors.initials: K
authors.lastname: Moehle
authors.forename: John A
authors.initials: JA
authors.lastname: Robinson
authors.forename: Peter
authors.initials: P
authors.lastname: Rusert
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.forename: Alexandra
authors.initials: A
authors.lastname: Trkola
mesh_terms: D017089:Ankyrin Repeat; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015699:HIV Envelope Protein gp120; D023581:HIV Fusion Inhibitors; D011506:Proteins; C514793:gp120 protein, Human immunodeficiency virus 1