No abstract
We have designed beta-amino acid oligomers that are helical, cationic, and amphiphilic with the intention of mimicking the biological activity of amphiphilic, cationic alpha-helical antimicrobial peptides found in nature (e.g., magainins). We have previously identified a 17-residue beta-peptide (called beta-17) with antibiotic activity similar to that of a magainin derivative against four bacterial species, including two clinical isolates that are resistant to common antibiotics. This beta-peptide displays very low hemolytic activity against human red blood cells, which indicates selectivity for bacterial cells over mammalian cells. Here we examine some of the factors important for activity in this class of beta-peptides. An amphiphilic helix is necessary, because a nonamphiphilic isomer proved to be inactive. The ratio of cationic to hydrophobic residues is also important. Active beta-peptides induce the leakage of beta-galactosidase from treated Bacillus subtilis cells, as do alpha-helical antibiotic peptides, and this similarity suggests that the beta-peptide mode of action involves disruption of microbial membranes. This class of beta-peptides is not degraded by proteases, which bodes well for biological applications.
Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.
We have studied the interaction of b-17, a potent synthetic antimicrobial b-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, b-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of membrane interaction relative to magainin II, but both leakage and membrane binding show that b-17, like magainin II, has strong affinity for membranes containing anionic lipids. This is likely to be an important factor contributing to the antimicrobial specificity of the b-peptide.Keywords: b-peptide; antimicrobial; peptide-lipid interactions; membrane leakage; membrane intrinsic curvature.There is currently extensive interest in developing new antimicrobial agents to counter the growing clinical problem of bacterial resistance to traditional antibiotics [1]. A large variety of natural peptides and synthetic derivatives display antimicrobial activity [2], and these peptides have been viewed as potential sources of new therapeutic agents. Nearly all of the synthetic derivatives have been constructed from a-amino acids, as are the natural host-defense peptides. Recently, antimicrobial activity has been reported for a number of b-amino acid oligomers (b-peptides) [3][4][5][6][7][8]. b-Peptides differ from conventional peptides (a-amino acid residues) in two important ways. First, the unnatural backbone of b-peptides confers resistance to degradation by proteolytic enzymes [8][9][10]. Second, b-peptides constructed from appropriately rigidified residues display higher conformational stability, on a per-residue basis, than do conventional peptides [6]. It is therefore of interest to compare the mechanism(s) of b-peptide antibacterial action with the antibacterial mechanisms of analogous conventional peptides.Here we characterize the interactions of one antimicrobial b-peptide, b-17 [7,8], with lipid vesicles, which are simplified models of cell membranes. b-17 was designed to mimic natural host-defense peptides like the magainins, which are thought to exert their antimicrobial effects by disrupting bacterial membranes. The structure of b-17 is shown in Fig. 1. Magainins are cationic peptides that contain 23 a-amino acid residues and adopt an amphiphilic a-helical conformation in the presence of membranes [11]. b-17 contains 17 b-amino acid residues and adopts a 12-helical conformation, which is defined by a network of 12-membered ring C¼O(i) fi NH(i + 3) hydrogen bonds [12][13][14][15]. (The a-helix contains 13-membered ring C¼O(i) fi NH(i + 4) hydrogen bonds.) The b-peptide 12-helix has approximately 2.5 residues per turn and a rise of 5.5 Å per turn; therefore, a 17-residue 12-helix should be similar in length to a 23-residue a-helix formed by a conventional peptide. b-17 contains only two types of b-amino acid residue, hydrophobic trans-2-aminocyclopentanecarboxylic acid (ACPC) and cationic trans-3-aminopyrrolidine-4-carboxylic acid (APC). The repeating APC-ACPC-APC-ACPC-...
Oligomeric backbones with well-defined conformational propensities can serve as scaffolds for displaying sets of functional groups in specific three-dimensional arrangements. beta-Peptides are particularly interesting in this regard because several distinct secondary structures can be induced by appropriate choice of beta-amino acid substitution pattern.3 The beta-peptide 12-helix (defined by 12-membered ring C=O(i)- -H-N(i + 3) hydrogen bonds) is of particular interest because this helix resembles the alpha-helix. To date 12-helices have been observed in beta-peptides comprised exclusively of residues containing a five-membered ring constraint. Here we show that 12-helical propensity is maintained when some cyclic beta-amino acid residues are replaced with more flexible acyclic residues. This result is important because use of acyclic residues greatly facilitates introduction of diverse side chains at specific sites along the 12-helix. We demonstrate the utility of this advance in the context of antibiotic design.
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