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Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial activity towards both Gram-positive and Gram-negative bacteria as well as eukaryotic cell viability. All examined analogs retained the ability to cross eukaryotic membranes giving rise to a distribution within the vacuolar apparatus. Interestingly, a series of shuffled analogs of penetratin with the cationic residues in conserved positions, attain the same α-helical conformation as native penetratin in the presence of cholesterol-containing liposomes, while conformational differences were observed in the presence of highly anionic liposomes. While the antibacterial effect of the two groups of peptides was similar, the eukaryotic cellular uptake of the shuffled analogs was noticeably lower than for native penetratin. Moreover, a point substitution of Met to Leu in native penetratin had no influence on eukaryotic cellular uptake and antimicrobial effect, and only a minor effect on cytotoxicity, in contrast to the fact that the same substitution in the shuffled analog gave rise to reduced eukaryotic cellular uptake while increasing the antibacterial effect and cytotoxicity.

[structure: see text] We describe the synthesis and characterization of the first generation of oligomers consisting of alternating repeats of alpha-amino acids and chiral N-alkyl-beta-alanine (beta-peptoid) residues. These chimeras are stable toward proteolysis, non-hemolytic, and possess antibacterial activity comparable to well-known antimicrobial agents. Moreover, the chimeras exhibit length-dependent, concentration-dependent, solvent-dependent, and ion-strength-dependent ellipticity, indicating the presence of a secondary structure in solution. Thus, alpha-peptide/beta-peptoid oligomers represent a promising novel peptidomimetic backbone construct for biologically active ligands.

Novel remedies in the battle against multidrug-resistant bacterial strains are urgently needed, and one obvious approach involves antimicrobial peptides and mimics hereof. The impact of α- and β-peptoid as well as β(3)-amino acid modifications on the activity profile against β-lactamase-producing Escherichia coli was assessed by testing an array comprising different types of cationic peptidomimetics obtained by a general monomer-based solid-phase synthesis protocol. Most of the peptidomimetics possessed high to moderate activity toward multidrug-resistant E. coli as opposed to the corresponding inactive peptides. Nevertheless, differences in hemolytic activities indicate that a careful choice of backbone design constitutes a significant parameter in the search for effective cationic antimicrobial peptidomimetics targeting specific bacteria.

Multidrug-resistant bacteria pose a serious threat to public health worldwide. Previously, α-peptide/β-peptoid hybrid oligomers were found to display activity against Gram-negative multidrug-resistant bacteria. In the present work, the influence of hydrophobicity, fluorination, and distribution of cationic/hydrophobic residues on antimicrobial, hemolytic, and cytotoxic properties of α-peptide/β-peptoid hybrids were investigated. An array of 22 peptidomimetics was tested. Analogues with enhanced hydrophobicity were found to exhibit increased activity against Gram-positive bacteria. Incorporation of fluorinated residues into the peptidomimetics conferred increased potency against Gram-positive bacteria, while hemolytic properties and activity against Gram-negative bacteria depended on the degree and type of fluorination. Generally, shorter oligomers were less potent than the corresponding longer analogues. However, some short analogues exhibited equal or higher antimicrobial activity. The alternating hydrophobic/cationic design proved superior to other distribution patterns of cationic side chains and hydrophobic moieties.

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The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical.