Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity

Abstract

To better understand the sequence–structure–function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding–folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the “negative” peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors

“…In reality, close consideration of the interaction between a pHLIP variant and the membrane at pH 8, in conditions more alkaline than physiological conditions where the inserted peptide population should be even less than at physiological conditions, indicates that the most hydrophobic sequences, such as ATRAM and Var3/GLL, and bundled pHLIPs with multiple binding sites within a single construct, exhibit a significant inserted peptide population, as shown by the loss of pH-dependent differences in the translocation of the polar, cell-impermeable cargo amanitin with an increase in construct concentration (i.e., a decrease in potency at higher concentrations). Additionally, as previously shown using the pore-forming peptide melittin, helix formation, membrane binding, and insertion properties are very sensitive to primary structure changes involving glycine and leucine residues (38). Ultimately, due to patient variability, it is highly desirable that potential therapeutic pHLIP constructs are able to discriminate between healthy and tumor tissue over a wide concentration range, meaning that a constant potency is necessary to avoid targeting normal tissue and the resulting significant side effects, suggesting that the properties of these variants may not be well suited for clinical development using agents that require tight targeting.…”

Mechanistic and structural basis of bioengineered bovine Cathelicidin-5 with optimized therapeutic activity

“…The membrane mediated folded and partial folded helical states of BMAP-28 correlates other class of AMPs2636. The leucine to phenylalanine zipper substitution ascertained the differential behavior of the synthetic BMAP-28 analogue where the former facilitates membrane fusion37; and the latter accelerates structural integrity through dimerization27.…”

Interspecies Bombolitins Exhibit Structural Diversity upon Membrane Binding, Leading to Cell Specificity

“…Some experiments have suggested sequential steps of folding, binding, and insertion, whereas other studies have demonstrated more of a cooperative interplay between the two phenomena (9)(10)(11)(12)(13). AMPs are found in all multicellular species and exhibit a wide spectrum of functionality, including antibacterial, antifungal, antiviral, and antitumorigenic effects (6,8,(14)(15)(16)(17). Therefore, there is substantial interest in developing AMPs as novel therapeutics for the treatment of a variety of diseases.…”