Structure-activity Relationship And Action Mechanism Of α-helical Membrane-active Peptide Targeting To Different Membranes

Membrane-active peptides present a group of bioactive peptides with the potential to develop into antimicrobial and anticancer medicines. The membrane-active peptides target to the bio-membrane system without the specific receptors. In this study, anα-helical membrane-active peptide V13K was used as a model peptide to explore the antibacterial and anticancer activity together with the action mechanism ofα-helical membrane-active peptides.A series of peptide analogs were designed and synthesized through systematically introducing hydrophilic amino acid into the non-polar face of model peptide with substituting low hydrophobic amino acid with high hydrophobic amino acid to compensate the loss of hydrophobicity due to the introduction of hydrophilic amino acid. The introducing of hydrophilic amino acid on the non-polar face of amphipathicα-helical peptide would optimize its toxicity against eukaryotic cell, but decrease the antibacterial activity at the same time. The therapeutic index would be recovered or even enhanced by using more hydrophobic amino acid to substitute less hydrophobic amino acid, thus increasing the antibacterial specificity. These results are also consistent with the action mechanism of"membrane discrimination mechanism", in which for antimicrobial activity, peptide takes a carpet-like mechanism to cause the lysis of bacterial membrane, in contrast, for hemolytic activity of eukaryotic cells, peptides have to penetrate vertically into the hydrophobic core to form pores or channels, thus causes the lysis of eukaryotic cells. The reason to take different mechanisms for different types of cells is due to the different lipid compositions of cytoplasmic membrane. This part of study showed the different requirement of prokaryotic and eukaryotic membrane to the biophysical parameters of membrane-active peptides and also provided a new approach for the design of antibacterial peptides.Most natural cationic antimicrobial peptides exhibit strong antibacterial activities in vitro at concentrations generally higher than those found in vivo, but their activity may be constrained by body fluids. For future systematic usage, it is important to assess their acitivity under the physiological conditions. Thus, the inhibitory effects and mechanism of physiological conditions on the activity of enantiomeric forms of the modelα-helical membrane-active peptide were exploited. The antibacterial activity of enantiomers displayed a significant reduction in the presence of single element. The inhibitory effects of slats (NaCl and CaCl2) might rely on the interruption of the electrostatic attraction between positively charged peptides and negatively charged membranes. Divalent slat CaCl2 exhibited much greater effect on the decrease of antimicrobial activity of peptides against Gram-negative bacteria by strongly stabilizing the outer-membrane of Gram-negative bacteria. Human serum albumin exhibited the ability to combine enantiomers with equivalent affinities, by which the free concentration of peptide would be decreased resulting in the decreased antibacterial activity. In addition, D-P was highly resistant to enzymatic digestion in contrast to poor L-P. In all, all D-peptide may be a promising candidate for clinical practices.Studies have shown that certain cationic AMPs exhibit anticancer activities and some of them also show strong specificity towards cancer cells. However, the action mechanism of anticancer peptides (ACPs) has not been clarified yet. The lethal effects of most natural and synthetic ACPs to the target cancer cells result from the disruption of membrane system and are distinct from the non-membrane disruptive peptides whose fatal effects account on the induction of apoptosis to the target cells. Our study on the anticancer activity and action mechanism of membrane-active peptides with different biophysical parameters showed that the anticancer activity of the analogs exhibited a strong correlation with the hydrophobicity of peptides. In order to illustrate the action mechanism of anticancer peptides against cancer cells, we utilized atomic force microscopy (AFM) to measure the forces of single peptide interacting with cell membranes in a real-time manner. Based on the facts that AFM with a fine cantilever has high sensitivity and the corresponding force spectroscopy allows the detection of piconewton force, we covalently attached a series of membrane-active peptides, derived from the parent peptide to the AFM tip through PEG linker and challenged the peptides against the native HeLa cell membranes. We found that the single molecule force events (force values and the possibility of force signal) between peptides and native membranes would provide an insight in the action mechanism of membrane-active peptides. Based on the present results, we proposed a hydrophobicity-dependent insertion model for the interaction mechanism between single anticancer peptide and cancer cell membranes.