Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
The increasing number of resistant bacteria has been actively stimulating the search for alternative antibiotics. Antimicrobial peptides poses an interesting alternative as they are actively used by every species as a part of the innate immune system. Gomesin, a 18-residues peptide with two disulfide bonds arranged with ladder connection, is expressed in hemocytes of the Brazilian spider Acanthoscurria gomesiana and was shown to have potent antimicrobial activity towards Gram-negative bacteria and selective anticancer properties against melanoma cells. In a recent study a cyclic analogue of Gomesin was shown to be as active but more stable that its native form. In the current study we rationally designed a series of cyclic Gomesin analogues to improve its antimicrobial activity and investigate its mode-of-action. We successfully improved the activity by ~10-fold against tested Gram negative and Gram positive bacteria and melanoma cells without increasing toxicity. Mode-of-action studies revealed that cyclic Gomesin and its analogues act on bacteria and melanoma cells by targeting and disrupting cell membranes. Interestingly, cyclic Gomesin analogues were found to enter inside other tested human cell lines in a membrane-dependent but non disruptive way suggesting that membrane-disruption vs membrane-permeation is cell-type dependent and very likely determined by cell membrane composition. In summary, we have designed stable cyclic Gomesin analogues with potent antimicrobial activity and efficient cell-penetrating properties that can be used as stable scaffolds to deliver active sequences inside human cells. In addition, we have found that their mode-of-action and potency is cell membrane-dependent.