Design of salt-insensitive glycine-rich antimicrobial peptides with cyclic tricystine structures.
Cyclic peptide backbone and cystine constraints were used to develop a broadly active salt-insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eight Gly residues in an 18-residue sequence. The importance of rigidity and amphipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobial peptide. The first series, which retained the charge and hydrophobic amino acids of tachyplesin, but contained zero to four covalent constraints, included a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] analogues were prepared in a parallel series with all six bulky hydrophobic amino acids in their sequences replaced with Gly. Circular dichroism measurements showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sheet structures, while the less constrained [Gly(6)] analogues were disordered. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad activity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large variations in activity spectra and significant decreases in potency that roughly correlated with the decreases in conformational constraints. Except against Escherichia coli, the Gly-rich analogues with two or fewer covalent constraints were largely inactive under high-salt conditions. Remarkably, the most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum against all 10 test microbes in both low- and high-salt assays. Collectively, our results show that [Gly(6)]ccTP 1acould serve as a template for further analogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.
Tam
J P
JP
Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232-2363, USA. tamjp@ctrvax.vanderbilt.edu
Lu
Y A
YA
Yang
J L
JL
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000737:chemistry* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003553:Cystine / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D015195:Drug Design; D004926:Escherichia coli / Q000187:drug effects; D005658:Fungi / Q000187:drug effects; D005998:Glycine / Q000737:chemistry; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D012492:Salts
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D004268:DNA-Binding Proteins; D004220:Disulfides; D010456:Peptides, Cyclic; D012492:Salts; C057636:tachyplesin peptide, Tachypleus tridentatus; D003553:Cystine; D005998:Glycine