Design of salt-insensitive glycine-rich antimicrobial peptides with cyclictricystine structures

Cyclic peptide backbone and cystine constraints were used to develop a broa dly active salt insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eigh t Gly residues in an 18-residue sequence. The importance of rigidity and am phipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobi al peptide. The first series, which retained the charge and hydrophobic ami no acids of tachyplesin, but contained zero to four covalent constraints, i ncluded a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] an alogues were prepared in a parallel series with all six bulky hydrophobic a mino acids in their sequences replaced with Gly, Circular dichroism measure ments showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sh eet structures, while the less constrained [Gly(6)] analogues were disorder ed. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad acti vity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large v ariations in activity spectra and significant decreases in potency that rou ghly correlated with the decreases in conformational constraints. Except ag ainst Escherichia coli, the Gly-rich analogues with two or fewer covalent c onstraints were largely inactive under high-salt conditions. Remarkably, th e most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum again st all 10 test microbes in both low- and high-salt assays, Collectively, ou r results show that [Gly(6)]ccTP 1a could serve as a template for further a nalogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.