Studies on cationic amphipathic peptoids with antimicrobial and mitochondria targeting activities

Abstract

Peptides are involved in various functions in vivo and have inspired the development of therapeutics such as antimicrobial and anticancer agents. The diversity of functional groups of peptides and three-dimensional structures has been actively investigated in various studies from biological applications to materials science. However, the low stability against hydrolytic enzymes in vivo is a hurdle to the high efficacy of peptide-based therapeutics. To circumvent these problems, peptidomimetics, which are resistant to proteolysis, have been widely investigated as possible alternatives. In Chapter I, we describe chemical and compositional diversities of membrane lipids in eukaryotic cells and bacteria. The diversities are based on antimicrobial and anticancer treatments using cationic amphipathic peptides and peptidomimetics. We describe peptoids, oligo-N-substituted glycines which are secondary structure mimetics of peptides. Peptoids are a class of peptidomimetics in which side chains in the peptide backbone are transferred from carbon to nitrogen; they are highly stable in vivo. Submonomer solid-phase synthesis with various functional groups allows peptoids to be actively studied in structural analysis and biological applications. In addition, introducing synthetic approaches to chiral moieties enables the formation and analysis of the helical structure by resolving issues regarding the difficulties in forming the secondary structure of the peptoid because of the absence of hydrogen bonds. In Chapter II, we discuss improvements to the selectivity of antimicrobial peptoids by helicity-modulation. We confirm that the degree of helicity varies depending on the number and position of chiral side chains in the cationic amphipathic peptoid. The helicity-modulated peptoids have potent broad-spectrum antimicrobial activity including multi-drug-resistant pathogens, and the degree of flexibility of the helical structures affects the antimicrobial activity and cytotoxicity. This change in selectivity is likely caused by the difference in the types of lipids that form the membranes of bacteria and mammalian cells and their methods of interaction with the peptoids. We verified this hypothesis by changing the potential of the bacterial membrane in the presence of antimicrobial peptoids and tested it using imaging analysis with confocal microscopy and AFM. The results suggested that helicity-modulated antimicrobial peptoids not only improve selectivity but also can function as novel antimicrobial agents for the treatment of multidrug-resistant bacteria. In Chapter III, we describe the development of cationic amphipathic peptoids that selectively accumulate in the mitochondria. Low-molecular-based mitochondrial transporters have low water solubility, therefore they show limitations in the delivery of hydrophobic therapeutics. Peptide-based transporters also maintain low bio-stability and lack secondary structures, which make them difficult to utilize in vivo. We developed a novel peptoid-based transporter to overcome these issues. In this study, among various hydrophobic side-chains, the transporter showed the most activity with the cyclohexyl functional group. Furthermore, the degree of overlap with the MitotrackerTM dyes was higher when the mitochondria-targeting peptoids formed secondary structures. Therefore, mitochondria-targeting peptoids with high in vivo stability and solubility in water are expected to be efficient in the delivery of various therapeutics. In Chapter IV, we describe the synthesis and anticancer study of a manganese-porphyrin-conjugated mitochondria-targeting peptoid. We synthesized the conjugate to increase the hydrophilicity of the manganese-porphyrin, which mimicked bleomycin extracted from Streptomyces verticillus, as well as to transport the complex to an environment where its anticancer activity was enhanced. We confirmed by UV/vis spectroscopy, that the conjugate formed a manganese (V)-oxo species when H2O2 was added under basic conditions, showing cytotoxic activity selectively against various cancer cells while showing relatively low toxicity against normal cells. In addition, we observed that the conjugate reduced the mitochondrial membrane potential by TMRE staining assay, and damaged DNA by electrophoresis. Through mechanism analysis, we verified that the conjugate formed a manganese (V)-oxo species that inhibited mitochondrial functionality and caused DNA damage, thereby possessing anticancer activity. The conjugate was expected to show anticancer activity using various mechanisms, thereby proving its potential as an anticancer agent to treat solid cancers, with minimal effects.