Abstract

Antimicrobial peptides (AMPs) are key components of the innate immune systems of many organisms. AMPs function by permeabilizing microbial membranes, giving them an important advantage over conventional antibiotics as they may elude the selection of drug-resistance. Therefore, there has been increasing interest in engineering AMPs and improving their bioactivity over the last three decades. Yet the lack of obvious structure-function relationships or molecular design principles has obstructed the development of new AMPs. To circumvent this roadblock, we are developing a high-throughput approach to select AMPs that are optimized in all of the critical factors simultaneously. Previously in our lab, we identified a group of broad-spectrum antimicrobial peptides from a synthetic peptide library. Here, we used one of the broad-spectrum AMPs, ∗ARVA (RRGWALRLVLAY), to study the potency, selectivity and mechanism of action of AMPs in the presence of concentrated human erythrocytes (10⊥9 cells/ml) which mimics the in vivo milieu. ∗ARVA, and other AMPs, lose antimicrobial activity in concentrated erythrocytes. We developed a method to make direct measurements of peptide binding to cells which showed that loss of activity is due to weak host cell binding, coupled with the large mass excess of host cell vs. bacterial cells under physiological conditions. To identify AMPs with clinically-relevant activity, we are developing a novel, orthogonal high-throughput screen in which we select simultaneously for 1) peptide solubility; 2) lack of host cell lysis; 3) sterilization of a Gram positive microbe in the presence of concentrated erythrocytes, and 4) sterilization of a Gram negative microbe in the presence of concentrated erythrocytes. Our results show that rational library design and high-throughput screening is a promising approach to identify AMPs that have the needed balance between antimicrobial potency, solubility and target cell selectivity.

Full Text
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