Study Of Mechanism Of Action Of Alpha-helical Membrane-active Peptides Interacting With Model And Biological Membranes

Bacterial infection is a major concern in clinic treatments.In the traditional treatment,antibiotics play an important role,which has a good inhibitory act on bacterial and fungi.However,the abuse usage of antibiotics is a new problem for human.Studies of the substitutes of antibiotics have become a research hotspot.Cancer is also a common malignant disease,threatening human health and life.Anticancer agents are mostly based on alkylating agents and antimetabolites.However,these drugs have low therapeutic indices and strong side effects.Thus,the development of a novel class of anticancer agents is urgently required.Membrane-active peptides(MAPs),including antibacterial peptides,antifungal peptides,and anticancer peptides,have been widely studied and are promising drug candidates to substitute conventional antibiotics and chemotherapeutics.MAPs have a broad spectrum,rapid action,and low resistance.However,the exact targeting specificity mechanism of MAPs is still unclear,which is important for the design of antibacterial and anticancer peptide as potent drugs.In this thesis,a 26-residue amphipathic MAP of V13 K was used as a framework to design two groups of peptide analogs.The hydrophobicity and charge of peptides was systematically changed and was used to investigate the interactions of peptides with different types of cells(prokaryotic cells,cancer cells and normal eukaryotic cells).In this study,peptide hydrophobicity has been proven as a key parameter for the interaction between MAPs and all tested model and biological membranes,which is the critical process affecting peptide biological activities including antimicrobial activity,anticancer activity,hemolytic activity,cytotoxicity against normal cells,and specificity.Except for the hydrophobicity of MAPs,the net charge of a peptide may also be considered as an important parameter due to the electrostatic interaction between peptides and different membranes.These results indicate that peptides exhibited a strong specificity against bacterial and cancer cell lines in this study.Cell membrane permeabilization of MAPs against the outer and inner bacterial membranes was examined by NPN and ONPG experiments.We further studied the interaction between peptides and cancer cell membranes by flow cytometry.It is clear that the membrane disruption abilities of MAPs against these bacterial membranes and cancer cell membranes correlate with the hydrophobicity and net charge of MAPs,which is consistent with the antimicrobial and anticancer activities of MAPs.In order to investigate the interaction between peptides and difference types of cell membranes,three different large unilamellar vesicles were used to mimic a prokaryotic cell membrane,a normal eukaryotic cell membrane and a cancer cell membrane.Through tryptophan fluorescence and quench experiments,MAPs exhibited strong specificity against negatively charged model membranes.That is,the positively charged peptides exhibited stronger insertion ability against the negatively charged model membrane than in the zwitterionic model membrane;thus,they would display a lower cytotoxicity against normal cells.These results are consistent with the antimicrobial and anticancer activity of MAPs.In this study,we systematically investigated the specificity of MAPs with different types of cell membranes using atomic force microscopy(AFM).To this end,AFM imaging experiment further explores the mechanism of membrane active peptide V13 K interacting with prokaryotic membrane and cancer cell membrane.Peptide interacts with bacterial and tumor cell membranes in a necrotic way.Then we further studied the targeting specificity of MAPs with different types of model and cell membranes using single-molecule force spectroscopy.The unbinding forces of MAPs with three model membranes and four biological membranes were determined.The results show that peptides target specifically against prokaryotic and cancer cell membranes rather than normal eukaryotic cell membranes.The different hydrophobicity and charges of peptides had strong correlations between the interaction probability on model and biological membranes.Peptide specificity against different types of membranes has been proven to be strongly dependent on the lipid composition of membranes.Positively charged MAPs showed strong targeting specificity against negatively charged membranes,such as prokaryotic and cancer cell membranes rather than normal eukaryotic cells,which is consistent with the “membrane discrimination” mechanism.Our results demonstrate that the selectivity of MAPs interacting with different cell membranes to improve targeting may be an applicable approach to design peptide therapeutics against bacterial resistance and cancer in clinical practices.