Rational Design of Hs02 analogs with improved selectivity as antimicrobial agents

Membrane active peptides (MAPs) have broad biotechnological potential, both as alternatives to conventional antibiotics in combating multidrug-resistant microorganisms (MDR), and as carriers of conjugated drugs. However, their application depends on the selectivity with which these molecules recognize their targets, the prokaryotic membranes, in relation to host membranes. Antimicrobial peptides (AMPs) and intragenic antimicrobial peptides (IAPs), these found within the primary structure of various proteins, are often poorly selective, being both antimicrobial and cytotoxic. The IAP Hs02 exemplifies this, as it shows broad and potent antimicrobial activity, but also exhibits cytotoxicity to eukaryotic typically at a 4-fold higher concentration. Hs02 has 16 amino acid residues, and displays a net positive charge, structuring as an amphipathic α-helix upon adsorption to membranes. Therefore, with the purpose of improving the selectivity of this IAP, two series of Hs02 analogs were synthesized, purified, and characterized. In one of these series, three reduced size analogs were synthesized, and afterwards, three others, with specific residue changes in critical locations. Their secondary structures were evaluated by Circular Dichroism spectroscopy in the presence of large unilamellar vesicles (LUVs) with neutral and negative charges, as well as lipopolysaccharides (LPS). Moreover, their antimicrobial activity was tested for Gram-positive and Gram-negative bacteria, and their cytotoxicity was measured by MTT viability assays using BV-2 microglia as a cell model. The results show a significant increase in the selectivity of all analogs developed in the second series, especially for Hs02_16.3. This peptide showed an almost 5-fold increase in its Therapeutic Index (TI), resulting from the reduction of cytotoxicity and the maintenance of antimicrobial activity compared to the original peptide. These findings indicate that the balance between the positive net charge and minimal hydrophobicity of Hs02 was optimized resulting in an increase in its TI. These optimizations further enhanced the biotechnological potential of Hs02_16.3, enabling its use in the formulation of peptide-drug conjugates.