Discovering Highly Potent Pore-Forming Peptides using Synthetic Molecular Evolution

Abstract

There are many natural and designed peptides that permeabilize membranes, and there are multiple mechanisms by which membrane permeabilization can occur. Yet, peptides that unequivocally self-assemble into equilibrium, membrane-spanning pores at low peptide to lipid ratios (P:L1:1000) are very rare. The design and engineering of such peptide “nanopores” in lipid bilayer membranes is desirable as it could lead to improved biosensor platforms, targeted therapeutics, exogenous ion channels, or drug delivery vehicles. While the few well studied pore-forming peptides have provided a lot of information about the architecture of peptide pores, especially -helical pores, our knowledge of the fundamental molecular principles of pore formation is not detailed enough for rational engineering. This is a roadblock to the design of new pore-forming peptides and to the optimization of known pore-formers for particular applications. In this work we show how novel, highly potent, equilibrium pore-forming peptides can be discovered using synthetic molecular evolution, i.e. iterative cycles of combinatorial library design and high-throughput screening. In the first example, we used two generations of de novo library design and screening to identify highly potent pore-formers that self-assemble into -sheets in membranes. These peptides may be the only known examples of highly potent, pore-forming peptides that have -sheet secondary structure in membranes. In the second example we designed an iterative library that used the helical pore-former melittin as a template. From this library we identified gain-of-function pore-formers that are much more potent than melittin. The results demonstrate the power of synthetic molecular evolution for the discovery and engineering of membrane active peptides.