Comparative Properties of Helical and Linear Amphipathicity of Peptides Composed of Arginine, Tryptophan, and Valine.

Background: The persistence of antibiotic resistance has incited a strong interest in the discovery of agents with novel antimicrobial mechanisms. The direct killing of multidrug-resistant bacteria by cationic antimicrobial peptides (AMPs) underscores their importance in the fight against infections associated with antibiotic resistance. Despite a vast body of AMP literature demonstrating a plurality in structural classes, AMP engineering has been largely skewed toward peptides with idealized amphipathic helices (H-amphipathic). In contrast to helical amphipathicity, we designed a series of peptides that display the amphipathic motifs in the primary structure. We previously developed a rational framework for designing AMP libraries of H-amphipathic peptides consisting of Arg, Trp, and Val (H-RWV, with a confirmed helicity up to 88% in the presence of membrane lipids) tested against the most common MDR organisms.

Methods: In this study, we re-engineered one of the series of the H-RWV peptides (8, 10, 12, 14, and 16 residues in length) to display the amphipathicity in the primary structure by side-by-side (linear) alignment of the cationic and hydrophobic residues into the 2 separate linear amphipathic (L-amphipathic) motifs. We compared the 2 series of peptides for antibacterial activity, red blood cell (RBC) lysis, killing and membrane-perturbation properties.

Results: The L-RWV peptides achieved the highest antibacterial activity at a minimum length of 12 residues (L-RWV12, minimum optimal length or MOL) with the lowest mean MIC of 3-4 µM, whereas the MOL for the H-RWV series was reached at 16 residues (H-RWV16). Overall, H-RWV16 displayed the lowest mean MIC at 2 µM but higher levels of RBC lysis (25-30%), while the L-RWV series displayed minor RBC lytic effects at the test concentrations. Interestingly, when the S. aureus strain SA719 was chosen because of its susceptibility to most of the peptides, none of the L-RWV peptides demonstrated a high level of membrane perturbation determined by propidium iodide incorporation measured by flow cytometry, with <50% PI incorporation for the L-RWV peptides. By contrast, most H-RWV peptides displayed almost up to 100% PI incorporation. The results suggest that membrane perturbation is not the primary killing mechanism of the L-amphipathic RWV peptides, in contrast to the H-RWV peptides.

Conclusions: Taken together, the data indicate that both types of amphipathicity may provide different ideal pharmacological properties that deserve further investigation.