HighlightsSynthetic peptide display screen identifies a macrocyclic b-hairpin peptide antibiotic Symbah-1 kills through disrupting bacterial membranes, yet is not very hemolytic Symbah-1 loses activity in human serum, likely due to structural instability Structural optimization improves its serum activity by reducing its protease lability

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 β-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 β-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 β-hairpin antimicrobial peptide activity.

Peptide macrocycles are a rapidly emerging new class of therapeutic, yet the design of their structure and activity remains challenging. This is especially true for those with β-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 β-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 β-hairpin antimicrobial peptide activity.