Synthetic Molecular Evolution to Identify Hemocompatible Antimicrobial Peptides Effective Against Drug-Resistant, Biofilm-Forming Bacteria

Abstract

Previously, we identified antimicrobial peptides (AMPs) that retain activity in the presence of eukaryotic cells by using a synthetic molecular evolution (SME) based approach. We demonstrated that variation in the sequence of antimicrobial peptides selected from primary screening of iterative peptide library can lead to identification of peptides that have superior antimicrobial activity, solubility, and lower cytotoxicity against red blood cells (RBCs) and mammalian cells. D-CONGA, a variant obtained by rational modification of the consensus sequence of peptides selected from stringent screening of iterative, second-generation antimicrobial peptide library is highly effective in vitro against all ESKAPE pathogens in the presence of RBCs and serum. The peptide also has anti-biofilm activity in vitro and shows potency against gram-negative and gram-positive drug-resistant bacteria and their biofilms in relevant infected wound model in mice. We next designed and synthesized 13 variants of D-CONGA and characterized them for their bactericidal activity in the presence and absence of RBCs. Their cytotoxicity against mammalian fibroblast, WI-38, and hemolysis in RBC were also measured. The best D-CONGA variant, D-CONGA-Q7, was then tested against 14 clinical isolates of drug-resistant bacteria. The anti-microbial activity of the new peptide was compared with D-CONGA and eight conventional antibiotics from four different classes and was found to be superior to all. Following this, we designed and synthesized a third-generation peptide library based on D-CONGA-Q7. The library members are being screened against gram-positive and gram-negative bacteria in the presence of RBCs. The selected peptides sterilize broad spectrum bacteria at submicromolar concentrations. Hence, we have shown that using rational design and SME, we can improve AMPs with each generation and discover evolved peptides that have increasingly potent and relevant activity against MDR bacteria, compared to conventional antibiotic or other known AMPs.