Structure–Activity Relationship Studies of Gomesin: Importance of the Disulfide Bridges for Conformation, Bioactivities, and Serum Stability Marcos A. Fa ´ zio 1 Vani X. Oliveira, Jr. 1 Philippe Bulet 2 M. Tere ˆ sa M. Miranda 3 Sirlei Daffre 4 Antonio Miranda 1 1 Department of Biophysics, Federal University of Sa ˜o Paulo, Sa ˜ o Paulo, Brazil 2 Atheris Laboratories, Bernex, Geneva, Switzerland 3 Department of Biochemistry, IQ, University of Sa ˜ o Paulo, Sa ˜ o Paulo, Brazil 4 Department of Parasitology, ICB, University of Sa ˜ o Paulo, Sa ˜ o Paulo, Brazil Received 5 May 2005; revised 19 May 2005; accepted 10 October 2005 Published online 18 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20396 Abstract: Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys 2-15 /Cys 6-11 and presents a -hair- pin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure–activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the lin- earized analogues adopted secondary structures according to the environment, while the monocy- clic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lac- tam-bridged analogues tend to assume a -helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged ana- logues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gome- sin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activ- This article includes Supplementary Material available at http:// www.interscience.wiley.com/jpages/0006-3525/suppmat. Correspondence to: A. Miranda; e-mail: miranda@biofis.epm.br Biopolymers (Peptide Science), Vol. 84, 205–218 (2006) # 2005 Wiley Periodicals, Inc. 205