Hydrophobicity-tuned anion responsiveness underlies endosomolytic cargo delivery mediated by amphipathic vehicle peptides.

Peptide conformation can change subject to environment cues. This concept also applies to many cationic amphipathic peptides (CAPs) known to have cell membrane lytic or penetrative activities. Well-conditioned CAPs can match the properties of the target membrane to support their intended biological functions, e.g., intracellular cargo delivery; however, the intricacy in such conditioning surpasses our current understanding. Here we focused on hydrophobicity, a key biophysical property that dictates the membrane activity of CAPs, and applied a structure-function strategy to evolve a template peptide for endosomolytic cargo delivery. The template was subjected to iterative adjustment to balance hydrophobicity between its N-terminal linear and C-terminal helical domains. We demonstrate that the obtained peptide, LP6, could dramatically promote cargo cell entry and facilitate cytosolic delivery of biomacromolecules such as FITC-dextran, saporin, and human IgG. Among the evolved peptide series, LP6 has low cytotoxicity and moderate hydrophobicity, exhibits maximum change in helical conformation in response to negatively charged phospholipids, and also shows an apparent aggregational behavior in response to sialic acid enrichment. These attributes of LP6 collectively indicate that its anion-responsive conformational change is a critical underlining of its endosomolytic cargo delivery capability. Our results also suggest that modulation of hydrophobicity serves as a key to the precise tuning of CAP's membrane activity for future biomedical applications.

Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria.

The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2-4 µM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application.

Engineered Cationic Antimicrobial Peptides (eCAPs) to Combat Multidrug-Resistant Bacteria.

The increasing rate of antibiotic resistance constitutes a global health crisis. Antimicrobial peptides (AMPs) have the property to selectively kill bacteria regardless of resistance to traditional antibiotics. However, several challenges (e.g., reduced activity in the presence of serum and lack of efficacy in vivo) to clinical development need to be overcome. In the last two decades, we have addressed many of those challenges by engineering cationic AMPs de novo for optimization under test conditions that typically inhibit the activities of natural AMPs, including systemic efficacy. We reviewed some of the most promising data of the last two decades in the context of the advancement of the field of helical AMPs toward clinical development.

Histidine-Rich Cationic Cell-Penetrating Peptides for Plasmid DNA and siRNA Delivery.

Amphipathic, pH-responsive, membrane-active peptides such as LAH4 and derivatives thereof have the ability to effectively deliver genes and small interfering RNA (siRNA) into mammalian cells. Their ability to bind and protect nucleic acids and then disrupt membranes when activated at low pH enables them to harness the endocytic machinery to deliver cargo efficiently and with low associated toxicity. This chapter describes protocols for the chemical synthesis of transfection peptides of the LAH4 family, complex formation with nucleic acids, and their use for the in vitro delivery of either plasmid DNA or siRNA into mammalian cell lines.

Design, Characterization, and Antimicrobial Activity of a Novel Antimicrobial Peptide Derived from Bovine Lactophoricin.

Lactophoricin (LPcin), which is a part of proteose peptone isolated from bovine milk, is a cationic amphipathic α-helical antimicrobial peptide. Its truncated variants and mutated analogs were designed and their antimicrobial activities were evaluated by using various assays, like broth dilution methods and disk diffusion methods as well as hemolysis assay. Three analogs, LPcin-C8 (LPcin-YK1), LPcin-T2&6W (LPcin-YK2), and LPcin-T2&6W-C8 (LPcin-YK3), which showed better antibiotic activities than LPcin, were selected. Their secondary structures were also characterized by using CD spectropolarimetry. These three analogs of LPcin could be used as an alternative source of powerful antibacterial agents.