Structure-activity Relationships Of Peptides Against HCV/HIV And Anticancer Mechanism Of Action Of Peptides

Membrane-active peptides present a group of small bioactive molecules withthe potential development to anticancer and antiviral drugs. Menbrane-activepeptides target to biomembrane without specific receptors and possessbroad-spetrum bioactivity. However, the membrane-active peptide have strongtoxicity to normal cells, which is limited its clinical application, and the actionmechanism of peptides have not been a clear conclusion. Based on preliminary study,we studied the structure-activity relationship of peptide against HCV/HIV andanticancer mechanism of action of peptide and the work we reported hereinvestigated the action mechanism of “membrane discrimination” of peptides.We firstly studied the the structure-activity relationship of peptide againstHCV/HIV. An amphipathic peptide C5A derived from the membrane anchor domainof the N-terminus region of NS5A non-structure protein of HCV virus was utilizedas the framework to design a series of peptide analogs to modulate the helicity andhydrophobicity of peptide C5A. The leucine was gradually introduced on thenon-polar face of peptide C5A to substitute for the original isoleucine and valine,and meanwhile the leucine maintained the hydrophobicity. Based on the concept thatthe hydrophobicity of peptides have great effects on antivirus activity and specificity,a series of peptides analogs were designed through systematically introducing aminoacids with different hydrophobicity into central location to modulate peptidehydrophobicity. By modulating peptide helicity and hydrophobicity, we improvedthe specificity of C5A against HCV and HIV by23-fold and69-fold, respectively.peptide hydrophobicity played a crucial role in peptide anti-HCV or anti-HIVactivities; peptide analogs with dimerized structure in an aqueous medium whilemaintaining the ability to be induced into a more helical structure in a hydrophobicenvironment may tend to show comparable or improved antiviral activity andspecificity to C5A. In the prevous study, we have systematically studied the effects of peptidehydrophobicity on the mechanism of action of α-helical cationic anticancer peptidesand demonstrated peptides killed various cancer cells with a fast necrotic mechanismcausing cell membrane lysis and hydrophobicity plays a crucial role during theaction. In this study, the single tryptophan of parent peptide PNW(A12LA20L)wasshifted from the N-terminus to the middle and to the C-terminus of peptidesequence,as a fluorescence probe to study anticancer mechanism of action andspecificity of anticancer peptides.The helicity of peptides, as well as hydrophobicity and self-association had thesame trend of change by shifted the single tryptophan from the N-terminus to themiddle and C-terminus, respectively. It is indicated that tryptophan residue played amore important role on stabilizing helical structure at the N-terminus or at the middleposition of the sequence. The tryptophan fluorescence experiment demonstrated thatpeptide analogs were more selective to LUVs mimicking cancer cell membrane thanLUVs mimicking normal cell membrane. During the interaction with targetmembrane, the N-terminus of anticancer peptide was inserted into hydrophobiccomponent of phospholipid bilayer first. The results are consistent with “membranediscrimination” mechanism of peptides, in which bundles of amphipathic α-helicesform transmembrane channel/pore by penetrating vertically into hydrophobic core ofeukaryotic cell membrane; In contranst, peptide takes a carpet-like mechanism tocause the lysis of prokaryotic cell membrane. The difference of action mechanism ofpeptides is due to the different lipid compositions of cytoplasmic membrane. TheITC assays demonstrated that the two peptides showed more affinity to DMPS thanDMPC due to the negatively charged polar headgroups of DMPS. The interaction ofpeptide with DMPS LUVs is droved by electrostatic interaction and with DMPCLUVs is droved by hydrophobic interaction. In DSC experiment, peptide analogssignificantly modified the phase transition profile of DMPC, demonstrating theirinsertion into the lipid bilayer due to hydrophobic interaction, but not significantlymodified the main phase transition of DMPS due to electrostatic interaction.