title: Biological and structural characterization of new linear gomesin analogues with improved therapeutic indices.
abstract: Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. The two disulfide bridges Cys(2,15) and Cys(6,11) facilitate the folding of the molecule in a beta-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-Thr(2,6,11,15), Pro(9)]-D-Gm and [Thr(2,6,11,15), D-Pro(9)]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. In addition, at 100 microM they are approximately threefold less hemolytic than Gm. The best therapeutic indices were found for [D-Thr(2,6,11,15), Pro(9)]-D-Gm and for [(Des-pGlu(1), -Thr(2), -Arg(3)), Thr(6,11,15), D-Pro(9)]-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-Thr(2,6,11,15), Pro(9)]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr(2,6,11,15), Pro(8)]-D-Gm and [Thr(2,6,11,15), D-Arg(4,16), D-Pro(9)]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display beta-conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr(2,6,11,15), Pro(9)]-D-Gm was further analyzed by NMR spectroscopy. (1)H NMR experiments in SDS micelles demonstrated that [D-Thr(2,6,11,15), Pro(9)]-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.
authors.affiliation: Department of Biophysics, UNIFESP, 04044-020 São Paulo, SP, Brazil.
authors.forename: Marcos A
authors.initials: MA
authors.lastname: Fázio
authors.forename: Laurence
authors.initials: L
authors.lastname: Jouvensal
authors.forename: Françoise
authors.initials: F
authors.lastname: Vovelle
authors.forename: Philippe
authors.initials: P
authors.lastname: Bulet
authors.forename: M Terêsa M
authors.initials: MT
authors.lastname: Miranda
authors.forename: Sirlei
authors.initials: S
authors.lastname: Daffre
authors.forename: Antonio
authors.initials: A
authors.lastname: Miranda
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology* / Q000633:toxicity; D002176:Candida albicans / Q000187:drug effects; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D015195:Drug Design; D004355:Drug Stability; D004926:Escherichia coli / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000187:drug effects; D055672:Static Electricity
chemical_list: D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; D008823:Micelles; C415987:gomesin; D011392:Proline