Studies On The Regulation Of Membrane Activity Of Cationic Amphiphilic Peptides

Being one of the most well-studied classes of membrane-active peptides,cationic amphipathic peptides(CAPs)show great potential in treatment of diseases.On one hand,CAPs have been considered as promising candidates for new generation of anticancer agents due to their intrinsic capability to overcome drug resistance of cancer cells.However,despite of the growing medical demands,the translation of CAPs to practical applications has been stagnant.One of the main bottlenecks is their hemolytic toxicity and hence a low safety index and narrow therapeutic window for application.A clear understanding of how a peptide’s physicochemical properties determine its mode of action(MOA)will aid the process.On the other hand,biomacromolecules are at the center in cell function regulation and play increasingly important roles in disease treatments.However,the inefficient intracellular delivery of biomacromolecules caused by cell membrane and endosomal membrane barriers hampers their broader applications.CAPs with’penetrative’ or ’lytic’ membrane activities may serve as promising tools for breaking these obstacles.Yet it remains poorly understood how the strength and distribution of hydrophobicity affect CAPs’ cytotoxicity and cargo delivery capacity.Precise regulation of membrane-active peptide activity will facilitate applicational translation of both aspects.Therefore,we have carried out the following studies around this purpose:1.Glutamate scanning strategy-based regulation of membrane activity of CAPsIn this study,anionic glutamate residue-based scanning was applied to the hydrophobic surface of a self-assembling lysine-rich cationic amphipathic peptide KL1.Single-site mutations from leucine to glutamate dramatically changed the MOA of all mutants from membranolytic to nonlytic.An apoptosis-inducing mutant L2E unable to self-assemble under extracellular anions exhibited a different conformational transformation process in the amphiphilic environment than KL1.Further adjustment of the overall positive charge allowed regulation of cytotoxic potency without affecting the MOA determined by the lack of preassembly formation.Compared with KL1,hemolytic toxicities of nonmembranolytic peptides were greatly reduced,with safety indices increased.This work thus provided novel insights into and integrated rationales on the improvement of CAPs for both anticancer activity and safety profile.2.Iterative strategy-based regulation of membrane activity of CAPsIn this study,a structure-function evolution strategy was applied to an imperfect amphipathic model peptide L9E.An added N-terminal hydrophobic domain and the hydrophobic face of the helical domain were subjected to iterative adjustment for hydrophobicity balance.A moderately hydrophobic peptide LP6 with cytosolic delivery ability for biomacromolecules(FITC-dextran,saporin,and human IgG)was obtained.Further research showed that LP6 promotes the entry of biomacromolecules into cells by mediating clathrin-mediated endocytosis.Once in the endosomes,LP6 mediates anion-responsive endosomolysis and release of cargos.Specifically,this action involves the formation of helical conformation in response to negatively charged phospholipids and an aggregation of peptides in response to highly sialylated environment in the endosomal lumen.Our results thus suggest moderate hydrophobicity underlies the environment-sensitive lytic behavior,and provide novel insights into the delicate balance between the toxicity and delivery capacities of CAPs.The studies in these two parts are based on the adjustment of the hydrophobicity of cationic amphiphilic peptides,specifically including the adjustment of the hydrophobicity of the hydrophobic surface and the balance of the overall hydrophobicity.And they constitute a systematic study on the regulation of the membrane activity of CAPs,which may guide the application of CAPs in anticancer and intracellular delivery of biomacromolecules.