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Distribution of Physicochemical Properties 8.2.2.1 Antimicrobial Peptoids

Antimicrobial amphiphilic peptides such as magainin-II and defensins are a widely distributed component of eukaryotic and prokaryotic host immune systems [152-154]. Although magainin-II forms an a-helix and defensins form b-sheets, both are amphiphilic and display a facial distribution of cationic and hy-drophobic groups on the folded structures [152-154]. While the mechanism of action remains to be fully understood, it is generally presumed that the cationic residues direct peptides to the partially negatively charged outer membrane of bacteria, while the hydrophobic residues interact with the lipid bilayer and disrupt membrane integrity, leading to cell death [152-154]. Amphiphilic peptides exert activity by targeting the bacterial phospholipid membranes, therefore there is minimal concern for the development of drug resistance. However, the most common drawback of amphiphilic peptides is nonspecific recognition binding

and lysis of host cells. Since red blood cells are particularly susceptible, hemolysis assays are used to determine the potential for such undesirable cytotoxic effects. The IC50 values for magainin-II [152] based a-peptides against E. coli have been reported as 1.2 mg mlr1. Varying the helical content of antimicrobial a-peptides affects activity [152, 153]. Increasing helical content by replacing helix-breaking residues with alanine (which is a strong helix former) enhances antimicrobial activity, but at the expense of specificity as hemolysis concomitantly increases [152].

Antimicrobial peptoids have been designed to mimic the distribution of physi-cochemical properties of the magainin-II amide antibacterial peptide (Fig. 8.9) [170]. The peptoids were designed to be amphiphilic in a helical polyproline type I conformation with a cationic face of lysine-like N-(4-aminobutyl)glycine (NLys) side chains and a hydrophobic face of aliphatic or aromatic side chains [170]. Circular dichroism spectroscopy (CD) revealed that some of the peptoids adopted characteristic polyproline type I-like helical structures in aqueous buffer and also in the presence of lipid vesicles [170]. Helical peptoids as short as 12 residues exhibited selective (nonhemolytic) and potent antibacterial activity against both Gram-positive and Gram-negative bacteria with minimal inhibitory concentrations (MICs) in the low micromolar range [170]. This is comparable to previously reported results using a synthetic magainin II analog and other antibacterial a-peptides [152, 153]. Unstructured peptoids were found to be ineffective as antibiotics [170], demonstrating the prerequisite of the helical structure and appropriate distribution of physicochemical properties for antibiotic activity.

Combinatorial libraries of antimicrobial peptoids have also been synthesized and screened [171]. A peptoid library created from a set of multiple statistically unbiased 324 compound library was created. Screening of the library by growth inhibition assays revealed peptoid compounds with antimicrobial activity against a broad range of bacteria, including E. coli and S. aureus, with MICs in the micromolar range. It was deduced from the screens that antimicrobial activity of pep-toids is enhanced (in terms of host range) by the presence of a primary amine and a hydrophobic amine.

8.2.2.2 Antimicrobial b-Peptides

Amphiphilic helix-forming b-peptides with antimicrobial activity comparable to that of a magainin-II analog have been designed based on the 14-helix [34-

a-helix 14-helix 10/12-helix 12-helix

Fig. 8.10 Physicochemical distribution of various helical types for antimicrobial activity (+ = cationic, H = nonpolar).

a-helix 14-helix 10/12-helix 12-helix

Fig. 8.10 Physicochemical distribution of various helical types for antimicrobial activity (+ = cationic, H = nonpolar).

37], 12-helix [46-48], and 10/12-helix (Fig. 8.10) [50]. 14-Helical designs, consisting of repeating hydrophobic-cationic-hydrophobic triad repeats, display a cationic surface constituting approximately one-third of the helix circumference (Fig. 8.10). Conversely, 12-helical structures consisting of cationic-hydrophobic-cationic-hydrophobic-hydrophobic pentads yield a cationic surface on two-fifths of the helix circumference (Fig. 8.10). Antimicrobial 10/12-helical b-peptides can be designed to have two adjacent cationic faces among the four faces along the helical axis (Fig. 8.10).

It has been shown that varying the helical content of antimicrobial a-peptides affects activity [152, 153]. However, increasing the helical content of 14-helical antimicrobial b-peptides by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues had little effect on antimicrobial activity [37]. Increasing helical propensity of the constituent residues did not alter the MIC values against four bacterial species [37], suggesting that there is no relationship between 14-helical stability and antimicrobial potency. However, the identity of C-terminal groups and the ability of the b-peptide to form an amphi-philic helix were crucial for antimicrobial activity [37]. A series of 9-mer b-peptides were shown to have nearly the same efficacy as (Ala8,13,18)-magainin II amide and melittin to permeabilize the membrane of B. subtilis BAU102 [35], as measured by x-gal release. Furthermore, decreasing hydrophobic character by replacing b3-hLeu with b3-hAla side chains increases the selectivity for targeting bacterial cells over red blood cells [35].

12-Helical designs have also demonstrated antimicrobial activity comparable to magainin-II peptides against four species of Gram-positive and Gram-negative bacteria [46, 47]. Designs consisting of hydrophobic ACPC and hydrophilic APC residues yielded facially amphiphilic 12-helices that were structurally rigid compared with b3-peptides because they are composed entirely of cyclic constrained residues [46]. However, 12-helical b-peptides containing noncyclic residues have also demonstrated similar antibacterial activity with comparable specificity to magainin-II peptides [47].

b-Peptides composed of alternating b2- and b3-amino acids have been shown to fold into 10/12-helices. A 10/12-helix forming b-peptide composed of hydrophobic and aromatic residues with two b3-HLys residues presented on one face of the helix was highly active against two species of Gram-positive bacteria and also displayed activity against Gram-negative bacteria [50]. Importantly, these 10/12-helical antimicrobial b-peptides presented low hemolytic activity against human and rat blood cells [50].

8.2.2.3 Antimicrobial Aryl Amides and Aryl Ureas

Aryl amides are smaller than the previously discussed antimicrobial foldamers, and therefore may be advantageous in terms of production costs and bioavailability. Presently, the biological activity of aryl amides is exclusive to those adopting an extended conformation. Aryl amides composed of di-acid and di-amine monomers possess an extended network of hydrogen bonds, which serve to rigidify the backbone and stabilize the extended conformation (Fig. 8.11).

Facially amphiphilic aryl amides have displayed antimicrobial activity comparable to antimicrobial cyclic a-peptides and magainin II derivatives [155, 156]. Chain length studies showed that short aryl amides were the most effective at inhibiting bacterial cell growth, with an optimal length of 8 repeat units [155]. The 8-mer demonstrated bactericidal activity against several species of Gram-positive and Gram-negative bacteria, with MICs below 50 mg ml-1 for each strain. Longer oligomers are suggested to be less active due to reduced solubility, lower molar concentration, or the inability to penetrate the proteoglycan layer [155]. The presence of positively charged aminoethyl groups were also found to be requisite for activity, as acetylation of the most highly active 8-mer resulted in loss of antimicrobial activity. Antimicrobial activity was attributed to disruption of phospho-lipids bilayers, as determined by the ability of aryl amides designs to induce leakage of calcein from unilamellar vesicles composed of mixed phosphatidylserine and phosphatidylcholine lipids [155]. However, these aryl amides were demonstrated to be hemolytic near the MIC. In addition, increasing the hydrophobicity of designed aryl amides has also been demonstrated to increase antimicrobial activity [156]. Aryl amides with increased hydrophobic character display high potency

Fig. 8.11 Antimicrobial aryl amide and aryl urea.

aryl amide

Fig. 8.11 Antimicrobial aryl amide and aryl urea.

against both Gram-negative and Gram-positive bacteria, with MICs of 6-12 mg mL-1 against both E. coli and S. aureus [156]. However, increases in hydropho-bicity were found to be directly proportional to hemolytic activity. Introduction of more polar substituents yielded aryl amides that were significantly less toxic towards erythrocytes [156]. One of these compounds displayed potency similar to a magainin II analog and had significantly greater selectivity. Another analog with similar potency was nonhemolytic at concentrations as high as 800 mM [156].

Compared with aryl amides, antimicrobial aryl ureas exhibit greater structural rigidity [134]. In contrast to aryl amides, aryl urea homo-oligomers are composed of only one type of monomer. Importantly, the number of hydrogen bonds per monomer unit is higher than that for aryl amides, therefore aryl urea backbones can be relatively more rigid (Fig. 8.11). The increased rigidity is due to the inherent presence of internal NH-S bonding. Amphiphilic aryl ureas prepared by one-pot synthesis demonstrated potent antimicrobial activity against E. coli and B. sub-tilis. A trimer displayed the most potency and specificity, with an MIC = 0.7 mg mL-1.

8.2.2.4 Antimicrobial meta-Phenylene Ethynylenes meta-Phenylene ethynylenes (mPEs) with alternating polar/nonpolar functionalities induce leakage of calcein from large phospholipid vesicles [140, 172], most likely due to the amphiphilic nature of the mPE in the extended conformation (Fig. 8.4). The hydrophobicity of mPEs is tunable through the appendage of hydrophobic or hydrophilic functionalities onto the hydrocarbon scaffold. This tun-ability modulates the affinity and selectivity of mPEs for bacterial phosphopilid membranes. A 20-mer mPE with ethylamine functionalities appended to alternating backbone benzenes exhibited antibacterial activity against both Gram-negative and Gram-positive bacteria, with an MIC = 25 mg mL-1 for E. coli [139]. A mPE composed of six repeat units was less potent (MIC = 50 mg mL_1) but was 20 times more selective than the 20-mer in hemolysis assays, which is comparable to a highly active magainin II analog.

8.2.2.5 DNA-binding Peptoids

DNA has a highly-negatively charged ribophosphate backbone that can be readily targeted by positively-charged compounds. The selective binding of a foldamer to DNA can inhibit the transcription of the targeted gene. To enhance the transcription of certain genes, the foldamer would need to bind the targeted DNA and recruit the appropriate transcription machinery. Also, foldamers that can bind DNA and transport DNA across the cell membrane into the cell (also known as trans-fection) may serve as gene delivery systems for gene therapy.

Peptoids have been shown to bind plasmid DNA [71], and thus have potential as gene delivery systems. The distribution of basic side chains on peptoid scaffolds can thus serve to bind DNA, while hydrophobic residues render amphi-philicity and facilitate cell entry. A series of peptoids, with varying lengths, frequency of cationic side chains, hydrophobicity, and side-chain shape, were developed to bind DNA bearing the firefly luciferase gene [71]. Many of these peptoids condensed DNA and protected DNA from nuclease degradation, although only a single type of repeating triplet motif (cationic-hydrophobic-hydrophobic) was able to cross the cell membrane and localize to the nucleus. Direct binding of the peptoid to the target DNA was shown by gel mobility shift assays [71]. A 36-mer peptoid showed the greatest degree of transfection in multiple cell lines in the presence of fetal calf serum [71]. Electron microscopy revealed that the 36-mer peptoid formed highly regular spherical structures (50-100 nm in diameter) when condensed with DNA [71]. Importantly, the efficiency of peptoid-mediated transfection is similar to the standard lipofection method with cationic lipids in serum-free medium.

8.2.2.6 DNA-binding b-Peptides

A 14-helical b-peptide was designed to bind single stranded and duplex DNA [39]. One side of the 14-helix displayed three b3-hAsn residues to potentially form H-bonds to the DNA bases. Positively charged b3-hLys residues were incorporated at the termini to potentially form ionic interactions with the negatively charged backbone of DNA and the introduction of b3-hAla and b3-hPhe residues rendered the amphiphilic character to the helix and facilitated nuclear entry (Fig. 8.12). Circular dichroism and DNA-melting temperature measurements using UV-Vis revealed a structured interaction occurring between the b-peptide and DNA. CD spectra were characteristic of the b-peptide 14-helix, while signal changes were apparent upon addition of DNA, demonstrating interactions between the b-peptide and DNA. In the presence of b-peptide, melted DNA was unable to reform back to a duplex, suggesting that b-peptide binding interferes with the rewinding process.

8.2.2.7 Cholesterol Uptake-inhibiting b-Peptides

Cholesterol and dietary lipid uptake in the lumen of the small intestine is facilitated by integral proteins in the brush-border membrane (BBM), the so-called scavenger receptors of class B (SRB) type I or II proteins [173]. Uptake occurs

Fig. 8.12 DNA-binding b3-peptide.
Fig. 8.13 14-Helical b-peptide inhibitors of cholesterol absorption.

through the binding of carrier particles such as small unilamellar phospholipid vesicles and mixed bile-salt micelles [173]. In liver and steroidogenic tissues, the SRBI receptor protein binds high density lipoproteins (HDL) which is an important pathway of cholesterol homeostasis and metabolism [174]. More specifically, the SRBI receptor protein binds an amphiphilic a-helix motif on HDL [175]. Consequently, amphiphilic helices that bind the SRBI receptor protein have been shown to inhibit cholesterol and lipid uptake at the BBM [173, 175].

Short b-peptides (hexamers, heptamers, and nonamers) were designed to mimic the amphiphilic helices of HDL, bind the SRBI receptor protein, and inhibit cholesterol absorption. Amphiphilicity of the b-peptides was rendered by the presentation of functionalized b3-hLys or b3-hSer side chains along one helical face, and hydrophobic residues (b3-hVal, b3-hPhe, and b3-hAla) on the other helical face two model assays were used to test the bioactivity (Fig. 8.13) [38]. One assay followed the transport of lipids and cholesterol or cholesteroyl esters from unilamellar vesicles into BBM vesicles. The second assay tested uptake into a monolayer of whole CaCO-2 cells. Three of six b-peptide designs were capable of inhibiting cholesterol uptake into BBM vesicles, with IC50 values as low as 590 mg mL_1, compared with Human apolipoprotein A-I. Interestingly, among the b-peptides tested, only those that could form 14-helices in MeOH demonstrated an

Heparin

Heparin-binding aryl amide

Fig. 8.14 Major repeat of heparin and a heparin binding aryl amide. Backbone rigidifying H-bonds are shown as dashed lines.

o2hc

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