Design of perfectly symmetric Trp-rich peptides with potent and broad-spectrum antimicrobial activities

Tritrpticin, a member of the cathelicidin family, is a Trp-rich or Pro/Arg-rich peptide. Since the Trp, Pro and Arg residues are important in membrane disruption and/or cell entry, tritrpticin is a particularly attractive template around which to design novel antimicrobial peptides. Although tritrpticin is effective against a broad spectrum of microorganisms, it also has relatively strong haemolytic activity, which may compromise its therapeutic effects. To identify antimicrobial analogues of tritrpticin that lack cytotoxicity, we have designed and synthesised several molecules based on the amphipathic turn structure of tritrpticin. C-terminal amidation of tritrpticin enhanced its antimicrobial activity, comparable with indolicidin, another Trp-rich peptide. In contrast, the additional insertion of positively-charged amino acids resulted in only small variations in antibiotic activity, suggesting that a total of five positive charges is sufficient for high antimicrobial activity. We found that perfectly symmetric analogues of tritrpticin with C-terminal amidation showed two- to eight-fold improved antimicrobial activity compared with tritrpticin, as well as significantly reduced haemolytic activity. This reduction in cytotoxicity was correlated with decreased permeabilization of the zwitterionic phosphatidylcholine membrane, the major component of the outer leaflet of red blood cells. In addition, we designed a symmetric indolicidin analogue that possessed antimicrobial potency and selectivity. Moreover, we found that these analogues of tritrpticin and indolicidin were effective against several antibiotic-resistant clinical bacterial isolates. Circular dichroism spectroscopy suggested that the structure of these symmetric analogues resembled that of tritrpticin or indolicidin in a membrane mimetic environment. Overall, our findings suggest that these symmetric peptides with an amphipathic turn structure may serve as useful templates for pharmaceutical compounds that may be effective against increasingly antibiotic-resistant microbes.