Effects Of Net Charge And Helicity On The Biological Activities Of Membrane Active Peptides

Nowadays, cancer has become a world-wide problem with the incidence of cancerincreasing every year. Both of human health and the safety of life face a huge threatand there are still some greater challenges for present cancer research to go forward.In addition to antibacterial activities of antimicrobial peptides (AMPs), a significantnumber of these bactericidal peptides have shown to have a broad spectrum ofanticancer and antiviral activities. And those cationic antimicrobial peptides withanticancer activities can be called anticancer peptides (ACPs). With low-accessibilityof induction of resistance due to aiming at the membrane of tumor cells, amphipathicα-helical anticancer peptides prove their promising potentials in cancer treatments.Compared with the neutral membrane of human normal cells, anticancer peptides aremore likely to be attracted to the negatively charged membrane of bacterial and cancercells. Thus some of anticancer peptides are more toxic to bacterial and cancer cells,not human normal cells. The plasma membranes of human normal cells carry anoverall neutral charge on their outer surface: primarily due to the fact that the majorlipid components of these membranes are zwitterionic, including:phosphatidylethanolamine, lecithin and sphingomyelin. While the outer membrane ofcancer cells contains only a small amount of phosphatidylserine (PS), being onlyslightly more negatively charged. Although PS is only3%-9%of the totalphospholipides of cancer cell membranes, PS plays an important role in the anticanceractivities of anticancer peptides.We utilized an amphipathic α-helical anticancer peptide A12L/A20L (Peptide P)with significant anticancer activity as the framework, systematically designed a seriesof peptide analogs with different net charges by replacing certain amino acid residues,to study the effects of net charge and the number of positively charged residues on thehydrophilic/polar face of peptide P on biological activity of cationic anticancer peptides. Synthesis of the peptides was carried out by solid-phase synthesis usingFmoc chemistry and purification by reversed-phase high performance liquidchromatography (RP-HPLC) with purification over95%. All the peptides areidentified and analyzed quantitively using ESI-MS and amino acid analysis. Ourresults showed that the ability of anticancer peptides to self-associate decreased as thepositive charge on the polar face of peptides increased and the total hydrophobicitydecreased. CD spectra of the anticancer peptides indicated that high helical structureof all the peptide analogues could be induced in the mimic of the membrane’shydrophobic environment. However, when the net charge of peptide was too high, theelectrostatic repulsions among the intramolecular positive amino acids of peptide willaffect peptide-folding and α-helical stability. We also found that there was an obviousnet charge threshold among the anticancer peptide P analogs. Increases or decreases innet charge beyond the threshold value resulted in a dramatic reduction in bothanticancer activity and therapeutic index. Compared with the neutral membrane ofhuman normal cells, the negatively charged membrane of cancer cells is much moresensitive to net charge changing of the anticancer peptide. As the net charge changes,the hemolytic activity of analogs within the range of concentrations examinedchanged slightly，while the anticancer activity exhibited remarkable differences. Ouramphipathic α-helical anticancer peptide analogs showed good specificity betweencancer cells and human normal cells, which might play a crucial role in makingfurther improvement in target selectivity, and designing anticancer peptide agents withhigher efficacy and lower toxicity.In addition, we altered peptide helicity by replacing L-amino acid using D-aminoacids, to study the effect of helicity on the selectivity of anticancer peptides, and wehave successfully selected two anticancer peptides with high anticancer activity andlow toxicity to the human normal cells. In this paper, we utilized liposomes to mimicprokaryotic membrane and eukaryotic membrane. According to the tryptophanfluorescence emission and the tryptophan quenching data of each peptide, we havefurther identified that rational peptide design by changing helicity can significantly reduce the toxicity of anticancer peptides against liposome mimic eukaryoticmembrane, thus resulting in high selectivity of anticancer peptides, which will make asolid foundation for the research and exploration of anticancer peptides in clinicalpractice.