The Role and Therapeutic Potential of Host Defense Peptides — The Targets of Cationic Antimicrobial Peptides

The Role and Therapeutic Potential of Host Defense Peptides — The Targets of Cationic Antimicrobial Peptides

Hancock, R. E. W., & Sahl, H.-G. (2006). Cationic host defence peptides: novel antimicrobial strategies against old and new pathogens. Nature Reviews Drug Discovery, *5*(2), 123–130. https://doi.org/10.1038/nrd2201

For many years, cationic antimicrobial peptides (CAMPs) were thought to function solely by increasing membrane permeability at very high concentrations, far above their minimum inhibitory concentration (MIC). It is now established that many peptides are active at much lower effective concentrations and exhibit significant heterogeneity in their interactions with specific membrane systems. Although the mechanisms of peptides that target non-membrane sites remain incompletely understood, they appear to involve multiple targets. This multi-target nature contributes to their high efficacy and the low probability of resistance development against CAMPs.

Membrane Targets

Intrinsic differences exist in the utilization of cationic antimicrobial peptides (CAMPs) against eukaryotic and prokaryotic membranes. A primary distinction is the higher proportion of anionic lipids in the outer monolayer of the bacterial cytoplasmic membrane, a steeper transmembrane electrical potential gradient, and the high cholesterol content in eukaryotic membranes. Bacterial membranes are composed of approximately 30% anionic phosphatidylglycerol and cardiolipin and 70% phosphatidylcholine, presenting more anionic lipids compared to the outer leaflet of higher eukaryotic membranes, which are predominantly composed of uncharged neutral lipids. This enhances the electrostatic interaction between cationic peptides and the bacterial cytoplasmic membrane. Beyond this marked structural difference, the transmembrane potential of bacteria (-150 mV) is significantly higher than that of eukaryotic cells (-15 mV).

The structural disparity is even more pronounced between the outer membranes of Gram-negative bacteria and eukaryotic membranes. The Gram-negative outer membrane is an asymmetric bilayer: its inner phospholipid monolayer resembles the cytoplasmic membrane in composition, while the outer leaflet is composed of lipopolysaccharide (LPS)—an amphipathic polyanionic glycolipid. LPS consists of three main components: lipid A, core oligosaccharide, and O-antigen. The lipid A moiety, primarily responsible for the barrier function of the outer membrane, comprises phosphorylated sugar units at the C1 and C4 positions. These phosphate groups are bridged by divalent calcium and magnesium ions, stabilizing the LPS by preventing charge-charge repulsion. Lipid A is situated in the outer leaflet, anchored via N- and O-linked acyl chains (e.g., hydroxymyristate). The core oligosaccharide is attached to lipid A via a unique sugar (KDO, 3-deoxy-D-manno-oct-2-ulosonic acid) and can be extensively modified with phosphates, pyrophosphates, ethanolamine, and amino acids. Consequently, the core oligosaccharide, O-antigen, and lipid A collectively contribute to the highly negative charge on the surface of the Gram-negative outer membrane. Peptides interact with the binding sites of divalent cations on LPS, leading to localized disruption of the outer membrane and subsequently increased membrane permeability.

Several physical parameters determine a peptide's preference for prokaryotic membranes. Many of these parameters have been leveraged to design synthetic peptides with enhanced activity over natural variants. The first variable is net charge. The overall charge of a given peptide and its initial electrostatic interaction significantly influence its antimicrobial activity. Another key variable is hydrophobicity. Antimicrobial activity generally increases with rising hydrophobicity; however, beyond a certain threshold, selectivity between prokaryotic and eukaryotic membranes is lost, accompanied by increased cytotoxicity.

Peptides considered to be membrane-active should demonstrate significant membrane-disruptive activity at their minimum inhibitory concentration (MIC). Theoretically, this may involve complete membrane lysis, the formation of pores leading to loss of transmembrane electrochemical gradients, or leakage of intracellular contents causing cell death. The precise mechanism of membrane lysis remains debated. This paper posits that peptides first bind to the outer monolayer of the cytoplasmic membrane, inserting at an interface parallel to the plane of the hydrophilic-hydrophobic bilayer. At a critical concentration, the peptide molecules reorient, forming transmembrane pores composed of both lipids and peptides. This leads to the loss of membrane potential and leakage of protons, other ions, and larger molecules.