Cyclization of the Antimicrobial Peptide Gomesin with Native Chemical Ligation: Influences on Stability and Bioactivity

Abstract

Gomesin is an 18-residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti-Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.

Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials

“…Using the central active α-helix folding portion (residues 13-31) of LL-37 (designated as IG-19), Shin and coworkers have also found that Trp substitution at the hydrophobichydrophilic interface of the amphipathic helical wheel projection of IG-19 confers a 2.8 fold enhancement in selectivity index with the retention of LPS neutralizing properties compared to the parent LL-37 peptide [19]. Cyclization of naturally derived AMP fragments has also been performed through an intramolecular head-to-tail backbone amide linkage [20][21][22], disulfide bridging [21,[23][24][25][26][27] or native chemical ligation [28] in efforts to increase the stability of AMPs in biological milieus. The conformational constraints introduced with the cyclization of AMPs are believed to enhance protease stability due to the reduced accessibility of the mobile ends of the peptide chains to protease binding and cleavage.…”

In Vivo Efficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

“…Although cyclization was shown previously to enhance stability and bioactivity (28,60), in this case an improvement in stability was observed but bioactivity was reduced. Structural or charge distribution changes upon cyclization might be responsible for the decreased antimicrobial activity.…”

“…Fmoc-protected amino acids (CS Bio Co.) were coupled with HCTU [O-(6-chlorobenzotriazol-1-yl)-N,N,N=,N=-tetramethyluronium hexafluorophosphate] (Peptide International) and DIPEA (N,N-diisopropylethylamine) (Auspep Pty. Ltd.) in DMF (RCI Labscan Ltd.) (28). The cyclic peptides were synthesized by t-butyloxycarbonyl (Boc)-based solid-phase peptide synthesis using standard protocols (29,30).…”

“…Cyclization has been used as one of the strategies in drug design to improve stability; for example, cyclization of the antimicrobial peptide pyrrhocoricin improved its in vivo pharmaceutical properties (27). The in vitro stability and antimalarial activity of the antimicrobial peptide gomesin, from the Brazilian spider Acanthoscurria gomesiana, were also improved after backbone cyclization (28).…”

Vipericidins, Snake Venom Cathelicidin-Related Peptides, in the Milieu of Reptilian Antimicrobial Polypeptides

“…However, the Rhesus y-defensin (RTD-1) represents a particular example of a mini-cyclotide from mammals with antiviral activity . However, artificial cyclization has been applied to create a head-to-tail cyclic peptide aiming to improve the peptide stability to proteases of several noncyclic peptides, preserving the respective antimicrobial activity (Rink et al 2010;Molhoek et al 2011;Chan et al 2013;Arias et al 2014;Sikorska and Kamysz 2014). Although the great majority of AMPs possess a net positive charge, being cationic compounds with hydrophobic patches, thus featuring amphipathic structures, a small but not less important number of anionic AMPs (AAMPs) play a role in the innate immunity of higher vertebrates, invertebrates, and plants (Harris et al 2009).…”