Rational design of novel amphipathic antimicrobial peptides focused on the distribution of cationic amino acid residues

Abstract

Antimicrobial peptides (AMPs) have garnered much attention as novel therapeutic agents against infectious diseases. They exhibit antimicrobial activity through microbial membrane disruption based on their amphipathic properties. In this study, we rationally designed and synthesized a series of novel AMPs Block, Stripe, and Random, and revealed that Stripe exhibits potent antimicrobial activity against Gram-positive and Gram-negative microbes. Moreover, we also demonstrated that Stripe disrupts both Gram-positive and Gram-negative mimetic bacterial membranes. Finally, we investigated the hemolytic activity and cytotoxicity in human blood cells and human cell lines, and found that Stripe exhibited neither. These data indicated that Stripe is a promising antimicrobial reagent that does not display significant cytotoxicity.

Fig. 1. Peptide sequences, structure (side view) and helical wheel (top view) of Block, Stripe, and Random.

Fig. 2. Secondary structures of Mag2, Block, Stripe, and Random in 20 mM phosphate buffer solution with 1% SDS solution, measured using CD spectral analysis. Peptide concentration = 200 μM.

Fig. 3. Fluorescence leakage assay with lipid bilayer mimicking (a) Gram-positive and (b) negative microbial membranes. Composition of the lipid bilayer mimicking Gram-positive, phosphatidylglycerol (PG) : cardiolipin (CL) = 3 : 1; Gram-negative, PG : CL: phosphatidylethanolamine (PE) = 2 : 7 : 1. Fluorescence intensity at 6.25 μM treatment of Triton X-100 is defined as 100%.

Fig. 4. The effect of the antimicrobial peptides Mag2, Block, Stripe, and Random on the cell growth in fibroblasts. TIG-3 cells were incubated with the indicated concentration of the compounds for 72 h and subsequently were subjected to a WST assay. Data in the graphs are means ± standard deviation (n = 4). A mixture of anti-Fas antibodies (100 ng mL^–1), cycloheximide (CHX) (10 μg mL^–1), and LCL-161 (1 μM) was used as an inducer for apoptosis. Although activation of Fas is a well-known inducer of apoptotic cell death, fibroblasts are resistant to Fas-mediated apoptosis., Anti-apoptotic proteins, such as IAP (inhibitor of apoptosis protein) and FLIP (FLICE-like inhibitor protein), are thought to be involved in this resistance. LCL-161 is an inhibitor for IAP. A protein translation inhibitor CHX reduces the levels of FLIP, because FLIP is a short-lived protein. Therefore, we use the treatment with anti-Fas mAb in combination with LCL-161 and CHX as a control.