Optimization of Host Cell-Compatible, Antimicrobial Peptides Effective against Biofilms and Clinical Isolates of Drug-Resistant Bacteria

Here, we describe the continued synthetic molec-ular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called D-amino acid CONsensus with Glycine Absent (D-CONGA), which has excellent antimicro-bial activities in vitro and in vivo. In this newest generation of rational D-CONGA variants, we tested multiple sequence- structure-function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, D-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, D-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of Klebsiella pneumoniae, innate resistance to D-CONGA was surprisingly common despite a lack of inducible resistance in Pseudomonas aeruginosa reported previously. Yet, these same isolates were susceptible to D-CONGA-Q7. D- CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, D-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile.