Understanding the mechanism of action of cell-penetrating antifungal peptides using the rationally designed hexapeptide PAF26 as a model

Antimicrobial peptides (AMPs) are being actively explored as novel pharmaceuticals, plant protection agents or food preservatives. A decade ago, the cationic peptide PAF26 was identified from a library of hexapeptides using combinatorial chemistry. It was selected as a promising antifungal AMP because of its potency and specificity to inhibit the growth of filamentous fungi. In recent years, different experimental approaches have been undertaken to understand the mechanism of action of PAF26 and the intrinsic determinants of its activity and specificity. These aspects are reviewed here and compared with studies on related antifungal peptides. The small size (six amino acids) of PAF26 has made it simple and easy to design new peptides with different amino acid substitutions, deletions or additions, as well as to label peptides with fluorescent probes. The modes of action of PAF26 and its derivatives have been analyzed in a range of fungi (including Neurospora crassa and Saccharomyces cerevisiae) with the aid of live-cell imaging, inhibitors, mutants and transcriptomic tools. The results obtained have shown that PAF26 has a dynamic antifungal mechanism of action that involves at least three stages: peptide interaction with the fungal cell envelope (cell wall and/or plasma membrane), its internalization, and a series of complex and specific intracellular effects whose relationship with cell death of the target fungus is still unclear. Two functional and separate motifs (cationic and hydrophobic domains) in the peptide amino acid sequence have been identified. As a result of these studies, PAF26 has been proposed as a model peptide for the characterization and study of cationic, cell-penetrating antifungal peptides. Understanding the mechanism of action of PAF26 should help us to design new synthetic peptides and peptidomimetics with improved antifungal activity and stability for use as antifungal drugs.