Designing and identifying β-hairpin peptide macrocycles with antibiotic potential

Abstract

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.

Development of a Selective and Stable Antimicrobial Peptide

“…DTr18-dab followed a similar trend in propidium uptake as melittin, but it is not as potent in the same time frame. The main target of kanamycin is the ribosome, but it has reported that membrane perturbation can also occur. ,− However, no membrane damage from kanamycin is observed compared to our PBS control under our conditions. ,− …”

“…Loss of peptide sequence abundance can be measured by changes in plasmid number in culture to reveal putative antimicrobial peptide leads. [20][21][22]33,34 From one of these screens, a peptide sequence was discovered that had antimicrobial activity against Escherichia coli W3110. The peptide was designated Tcp18 and, when chemically synthesized and assayed, had a minimum inhibitory concentration (MIC) of 16 μg/mL against both E. coli W3110 and E. coli ATCC 25922 in Muller Hinton broth (MH).…”

“…Displayed peptides with antibacterial activity interact with the cell membranes and kill the cells that produced them, thus reducing their presence in the population. Loss of peptide sequence abundance can be measured by changes in plasmid number in culture to reveal putative antimicrobial peptide leads. − ,, …”

“…Strains used in this paper are described in Table . MICs were performed as previously described. − , Briefly, peptide and bacteria were resuspended in MH broth to the desired concentrations and CFU, respectively, and then mixed for a final bacterial dilution of ∼5 × 10 5 CFU/mL. The plate was incubated for 18–20 h at 37 °C.…”

Adapting antibacterial display to identify serum active macrocyclic peptide antibiotics

“…1C, Table S2). This group had a diverse range of charge (3.88-11.88), length (14)(15)(16)(17)(18) amino acids), and potential cysteine pairs (1-3) (Fig. 1D).…”