Optimizing Antimicrobial Peptide Activity Through Balancing of Charge and Hydrophobicity

Abstract

Antimicrobial peptides (AMPs) offer themselves as potential drugs for treating bacterial, fungal, and viral infections and thus as a new class of antibiotics. However, AMPs have not yet been able to fulfill early hopes for substituting classical antibiotics in the treatment of infections by multi-resistant bacteria, since greater bacterial membrane selectivity and in vivo stability have to be achieved. Several strategies for optimization of AMPs’ therapeutic potential have been pursued. Previously, we discovered that improved selectivity toward negatively charged liposomes could be achieved by short chain acylation of mastoparan-X (MPX), whereas acylation by a longer chain impaired the selectivity. In the present work, this hypothesis was further explored through an extended library of novel MPX analogues having C1, C4 or C8 carbon chain substitution at three individual positions along the MPX backbone. The peptides were synthesized exchanging selected hydrophobic residues with Ala, Leu or 2-amino-decanoic acid at position 1, 8 and 14 of the native MPX sequence. Isothermal titration calorimetry was employed for gauging the partitioning into liposomes and the pore formation properties of the MPX analogues. Steady-state fluorescence was utilized for addressing the aggregation state of the peptides in aqueous solution. Bacteriocidal and hemolytic properties of the peptides were measured (on E.coli, L.lactis and human red blood cells) and correlated to the biophysical parameters. The results show that both the position of acylation on the peptide backbone and length of the acyl-chain affect the therapeutic window of the peptide analogue. Two MPX analogues with reduced hemolytic activity and retained antimicrobial activity were identified, and the effective charge and hydrophobicity of the peptides were found to be inversely correlated.