biostudies-literatureRandall JRNIAID NIH HHSeade0008https://www.ebi.ac.uk/biostudies/studies/S-EPMC9833666biostudies-literatureUnknown9(2)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.Science advancesDesigning and identifying β-hairpin peptide macrocycles with antibiotic potential.PMC9833666R01 AI148419R21 AI159203R01 AI125337Slater SLRandall JRDavidson GMavridou DAIDavies BWDuPai CDCole TJWilke COGroover KEfalseDesigning and identifying β-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 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.2023-01-01T00:00:00Z2023 Jan2024-12-04T00:10:40.29Z2024-12-04T00:10:40.29ZS-EPMC98336663663051610.1126/sciadv.ade0008