Biological and structural characterization of new linear gomesin analogues with improved therapeutic indices
Résumé
Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. The two disulfide bridges Cys2,15 and Cys6,11 facilitate the folding of the molecule in a -hairpin structure, conferring on the peptide a high stability in human plasma. We report herein biological and structural features of new linear Gm analogues, obtained by combining the removal of both disulfide bridges and the incorporation of a D- or L-proline. Regarding their biological properties, two analogues, namely, [D-Thr2,6,11,15, Pro9]-D-Gm and [Thr2,6,11,15, D-Pro9]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. In addition, at 100 M they are approximately threefold less hemolytic than Gm. The best therapeutic indices were found for [D-Thr2,6,11,15, Pro9]-D-Gm and for [(Des-pGlu1, -Thr2, -Arg3), Thr6,11,15, D-Pro9]-Gm with a 32-fold increase of their activity against bacteria, and from 128- to 512-fold against yeast when compared with Gm. Regarding the stability, [D-Thr2,6,11,15, Pro9]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr2,6,11,15, Pro8]-D-Gm and [Thr2,6,11,15, D-Arg4,16, D-Pro9]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display -conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr2,6,11,15, Pro9]-D-Gm was further analyzed by NMR spectroscopy. 1H NMR experiments in SDS micelles demonstrated that [D-Thr2,6,11,15, Pro9]-D-Gm presents a conformation very similar to that of Gm. In our search for Gm analogues with enhanced potential for drug development, we demonstrated that designing cysteine-free analogues can improve the therapeutic index of Gm derivatives.