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We recently described 10 peptides selected from a 16 384-member combinatorial library based on their ability to permeabilize synthetic lipid vesicles in vitro.8 These peptides did not share a common sequence motif, length, or net charge; nonetheless, they shared a mechanism of action that is similar to the natural membrane permeabilizing antimicrobial peptides (AMP). To characterize the selected peptides and to compare the activity of AMPs in vivo and in vitro, we report on the biological activity of the same selected peptides in bacteria, fungi, and mammalian cells. Each of the peptides has sterilizing activity against all classes of microbes tested, at 2-8 μM peptide, with only slight hemolytic or cytotoxicity against mammalian cells. Similar to many natural AMPs, bacteria are killed within a few minutes of peptide addition, and the lethal step in vivo is membrane permeabilization. Single D-amino acid substitutions eliminated or diminished the secondary structure of the peptides, and yet, they retained activity against some microbes. Thus, secondary structure and biological activity are not coupled, consistent with the hypothesis that AMPs do not form pores of well-defined structure in membranes but rather destabilize membranes by partitioning into membrane interfaces and disturbing the organization of the lipids, a property that we have called "interfacial activity". The observation that broad-spectrum activity, but not all antimicrobial activity, is lost by small changes to the peptides suggests that the in vitro screen is specifically selecting for the rare peptides that have broad-spectrum activity. We put forth the hypothesis that methods focusing on screening peptide libraries in vitro for members with the appropriate interfacial activity can enable the design, selection, and discovery of novel, potent, and broad-spectrum membrane-active antibiotics. [ABSTRACT FROM AUTHOR]

Rational design and engineering of membrane-active peptides remains a largely unsatisfied goal. We have hypothesized that this is due, in part, to the fact that some membrane activities, such as permeabilization, are not dependent on specific amino acid sequences or specific three-dimensional peptide structures. Instead they depend on interfacial activity: the ability of a molecule to partition into the membrane—water interface and to alter the packing and organization of lipids. Here we test that idea by taking a nonclassical approach to biomolecular engineering and design of membrane-active peptides. A 16 384-member rational combinatorial peptide library, containing peptides of 9—15 amino acids in length, was screened for soluble members that permeabilize phospholipid membranes. A stringent, two-phase, high-throughput screen was used to identify 10 unique peptides that had potent membrane-permeabilizing activity but were also water soluble. These rare and uniquely active peptides do not share any particular sequence motif, peptide length, or net charge, but instead they share common compositional features, secondary structure, and core hydrophobicity. We show that they function by a common mechanism that depends mostly on interfacial activity and leads to transient pore formation. We demonstrate here that composition—space peptide libraries coupled with function-based high-throughput screens can lead to the discovery of diverse, soluble, and highly potent membrane-permeabilizing peptides. [ABSTRACT FROM AUTHOR]