Discovering Novel Antimicrobial Peptides using High-Throughput Screening and Rational Variation

Abstract

Antimicrobial peptides (AMPs) have long been attractive drug candidates for the next generation of clinical antibiotics due to their potent antimicrobial activity and low propensity for inducing resistance in pathogens. However, due in part to toxicity concerns and activity loss in vivo, AMPs have yet to have any impact clinically. Our lab hypothesized that the presence of host cells could cause depletion of free peptide available to target bacterial cells and showed this to be true for some AMPs using concentrated human red blood cells (RBCs) as a model eukaryotic cell. To solve this problem, we synthesized a combinatorial peptide library based on the potent AMP, ARVA, and screened the library for activity in the presence of concentrated RBCs. We isolated nine unique, but similar sequences from the screen. We designed a consensus sequence based on the nine peptides and synthesized it using only D-isomer amino acids to form D-NOGCON. D-NOGCON displays excellent antimicrobial activity against multiple human pathogens in the presence and absence of concentrated RBCs, causes very little hemolysis, and is not susceptible to cleavage by cellular or plasma proteases. D-NOGCON also has high activity against bacterial biofilms and does not readily induce leakage. In this work, we created rational variants of D-NOGCON with truncations, insertions and mutations to test various hypotheses about the basis for highly potent antimicrobial activity with low hemolysis and low toxicity against nucleated cells. In the near future, we will use this information to design a next generation combinatorial peptide library based on D-NOGCON that will be screened for i) clinically relevant antimicrobial activity in the presence of serum and concentrated red blood cells, ii) negligible hemolysis, and iii) negligible toxicity against nucleated cells.