In light of the era of microbial drug resistance, the current study aimed to better understand the relationships between sequence, higher-order structure, and mechanism of action for five designed peptides against multidrug-resistant (MDR) pathogens. All peptides studied were 15 residues long, were polycationic, adopted alpha-helical structures within hydrophobic environments (excluding the D-amino acid-substituted peptide MA-d), and contained N-terminal glycine residues, a novel antimicrobial peptide (AMP) design principle. Increasing hydrophobicity enhanced MICs (<500 g/ml to <7.4 g/ml) without significantly increasing hemolytic activity (18% maximum hemolysis at 3,400 g/ml). To the best of our knowledge, this is the first study to have successfully adapted and used a transmission electron microscopy (TEM) immunogold method to investigate the mechanism of action of short (ϳ15 residues long) AMPs within bacteria. We propose a "floodgate" mechanism to possibly explain membrane deformation and the relative absence of membraneassociated peptides 10 h into incubation.

In the current study, indolicidin, a known antimicrobial originally isolated from bovine neutrophils, was modified with respect to hydrophobicity and tryptophan content to maximize bioactivity, and minimize cytotoxicity. Since indolicidin contains five tryptophans (very hydrophobic) of its total 13 amino acids, alanine (mildly hydrophobic) was incrementally substituted in its place to generate five novel derivatives with decreasing hydrophobicity. Antimicrobial testing identified two active derivatives with minimum inhibitory concentrations in the 10(-9) g mL(-1) range against Candida albicans, as well as broad-spectrum activity against various other Gram-positive/negative pathogens in the 10(-3)-10(-6) g mL(-1) range. Cytotoxicity testing yielded minimum hemolytic concentrations of approximately 3 x 10(-3) g mL(-1) for both active derivatives, resulting in hemolytic indices of >1.3 x 10(6) (peptide Delta45) and 3.6 x 10(5) (peptide Delta5) (improvements of >33,000-fold and approximately 10,000-fold, respectively, compared to indolicidin). The potent antimicrobial activity and low cytotoxicity of these derivatives show promise as potential antibiotics.

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We investigated the hydrophobic packing of two previously designed caviteins, LG2 and LG3, which differ by one Gly in the linker regions between the peptide sequence and the cavitand scaffold. We sought to diminish the putative native-like properties of LG2 and LG3, and see if we could diagnose a change in the conformational specificity of the hydrophobic core. We replaced the leucine residues with norleucine residues at the hydrophobic positions in LG2 and LG3, to create NG2 and NG3, respectively. LG2 exhibited more dispersion, but less sharp signals than LG3 in the amide region of its (1)H NMR spectrum. NG3 and NG2 were found to be slightly less helical and significantly less stable toward guanidine hydrochloride compared with their reference caviteins. The (1)H NMR spectrum of NG2 was very similar to that of LG2, whereas there was a noticeable loss in the number and sharpness of the amide signals of NG3 compared with LG3. These data suggest that LG3 is very well packed; a loss in conformational specificity resulted from replacement of the leucine residues with norleucine residues. In contrast, the packing and dynamics of the hydrophobic core in LG2 were similar to those in NG2 (both more modest than LG3), as their (1)H NMR spectra were virtually indistinguishable. Overall, substitution of leucine by norleucine provided an efficient, convenient, and informative probe of the packing and dynamics of our caviteins' hydrophobic cores.

The design, synthesis, and characterization of novel cavitand-based hetero-TASPs, TASPs having different peptide sequences within one bundle, are described. Three families of hetero-TASPs were designed: the LG3/LG2 family (different linker lengths), LG3/AG3 family (altering helix hydrophobicity), and the LG3/LG2C family (anti-parallel caviteins). These first generation hetero-TASPs were found to be alpha-helical, stable towards guanidine hydrochloride, and monomeric in solution. The LG3/LG2 caviteins exhibited primarily native-like properties. The remaining hetero-TASP families were found to exhibit less dispersion and broader signals in the amide regions of their (1)H NMR spectra than their respective reference caviteins. The success in the design of the LG3/LG2 hetero-TASPs suggests that subsequent hetero-TASPs may have potential to manifest superior native-like structure compared with homo-TASPs, and refinement of the linker and peptide sequences may accomplish this goal.