High-Throughput Identification and Characterization of LptDE-Binding Bicycle Peptides Using Phage Display and Cryo-EM

The lipopolysaccharide
(LPS) transport (Lpt) system in
Gram-negative
bacteria maintains the integrity of the asymmetric bacterial outer
membrane (OM). LPS biogenesis systems are essential in most Gram-negative
bacteria, with LptDE responsible for the delivery of LPS to the outer
leaflet of the OM. As an externally accessible, essential protein,
LptDE offers a promising target for inhibitor development without
the need for cellular penetration. However, there are no direct inhibitors
of E. coli LptDE, and drug discovery
is made challenging since it is a membrane target without a conventional
active site. Here, the bicycle phage display platform was used in
combination with cryogenic-electron microscopy (cryo-EM) and surface
plasmon resonance to identify and map bicyclic peptide binders to Shigella flexneri LptDE (SfLptDE). Four distinct
epitopes with unique bicycle molecule binding motifs were identified
across the SfLptD β-barrel. This method represents a streamlined
workflow for the identification and prioritization of hit molecules
against LptDE.