Designing and identifying -hairpin peptide macrocycles with antibiotic potential

Peptide macrocycles are a rapidly emerging class of therapeutic, yet the design of their structure and activity remains challenging. This is especially true for those with -hairpin structure due to weak folding properties and a propensity for aggregation. Here, we use proteomic analysis and common antimicrobial features to design a large peptide library with macrocyclic -hairpin structure. Using an activity-driven high-throughput screen, we identify dozens of peptides killing bacteria through selective membrane disruption and analyze their biochemical features via machine learning. Active peptides contain a unique constrained structure and are highly enriched for cationic charge with arginine in their turn region. Our results provide a synthetic strategy for structured macrocyclic peptide design and discovery while also elucidating characteristics important for -hairpin antimicrobial peptide activity.Synthetic identification of membrane selective beta-hairpin antimicrobial peptides enriched for arginine in their loop region.