Influence of charge distribution in the mechanism of action of small-sized cationic peptides acting as antibacterial agents: a theoretical and experimental study.

Among cationic antimicrobial peptides, short sequences are excellent candidates for use as antimicrobial agents because they offer several advantages over more extended segments. However, one of the main limitations of short peptides is that their molecular mechanism of action is not well understood, and there are some doubts about their pharmacophoric profile. We report here a theoretical and experimental study demonstrating the key influence of net positive atomic charges on small peptides with antimicrobial activity. To conduct this study, vesicles composed of 20% POPG and 80% POPE were used to simulate the behavior of these peptides in the bacterial membrane. In parallel, biophysical studies were performed on model liposomes, composed of pure DOPC and DOPC/DOPG (80/20 n/n%) mimicking both mammalian and bacterial membranes. Three small-sized peptides (WQWWWWWQW-NH2 (+0), RQWRRWWQR-NH2 (+4), and RKIRRKFKK-NH2 (+7)) possessing representative structural characteristics of this type of compound were selected for this study. Synthesis, bioassays, and evaluation of different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity, and polarity) were evaluated for these peptides. This study was completed through extensive molecular modelling analysis using various techniques. Coarse-grained calculations were used to simulate the behavior of complete systems. In contrast, QTAIM (quantum theory of atoms in molecules) calculations were employed to evaluate the molecular interactions of the different complexes in detail. Our theoretical and experimental results demonstrated that these small-sized peptides must have an appropriate balance between cationic and hydrophobic amino acids to exert antibacterial effects. In fact, the peptide sequence is also important, as they need to have two distinct faces: one positively charged and the other possessing hydrophobic characteristics, to interact properly with the bacterial membrane. The results reported here indicate that small peptides have a distinct mechanism of action, different from that of large peptides, providing further support for a previously proposed mechanism of action that occurs in three steps. From the medicinal chemistry point of view, the information reported here is of great value for the design of new small-sized antimicrobial peptides possessing these structural characteristics.