Control of Cell-Selective Activity of Membrane-Active Polyleucine-Based Peptides using Database-Guided High-throughput Screening

Abstract

We have developed a new 14-residue antimicrobial peptide template sequence containing four residues: L, D, K, and A. This template, called LDKA, which was designed using atomic detail structural information derived from unbiased long-timescale equilibrium molecular dynamics folding, partitioning, and pore assembly simulations, shows low micro-molar antibacterial activity against several gram-positive and gram-negative bacteria. Furthermore, LDKA was shown to form pores in microbial and eukaryotic membrane models using fluorescent dye leakage assays. A library of 2,916 peptides was designed using this template sequence and high-throughput screened using an orthogonal vesicle leakage assay. Dyes of different sizes were embedded into vesicles with varying lipid composition to simultaneously screen for both pore size and affinity to charged and neutral membranes. From this screen, 9 different LDKA variants were selected, sequenced, synthesized, and further characterized biophysically. Remarkably, even though the selected sequences displayed only minor mutational changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic charged bilayers. Further screening of these peptides for bacterial and haemolytic activity revealed good correlation between charged membrane model leakage and antibacterial activity, as well as neutral membrane leakage and haemolytic activity. Antimicrobial activity is different for each peptide and depends on the bacterial species. Analysis of the sequences reveals no clear motifs or explanation for these strong preferences, but suggests that hydrophobic moment and sequence arrangement are more important for membrane selectivity than net charge. The results reveal that simple sequence-function relationships for peptides that can form a large repertoire of functional structures may remain elusive and that it may be the peptide sequences propensity to aggregate and assemble in a given environment that holds the key to functional prediction.