Dimerization of truncated melittin analogues results in cytolytic peptides.
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ABSTRACT:
SUBMITTER: Rivett DE
PROVIDER: S-EPMC1217380 | biostudies-other | 1996 Jun
REPOSITORIES: biostudies-other
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Project description:Melittin, an antimicrobial peptide, forms pores in biological membranes and triggers cell death. Therefore, it has potential as an anticancer therapy. However, until recently, the therapeutic application of melittin has been impractical because a suitable platform for delivery was not available. Recently, we showed that phospholipid-stabilized perfluorooctyl bromide based nanoemulsion particles (PFOB-NEPs) were resistant to destruction by melittin and enabled specific delivery of melittin to tumor cells, killing them and reducing tumor growth. Earlier, prior work also showed that melittin adsorbed onto the stabilizing phospholipid monolayer of PFOB-NEP but did not disrupt the phospholipid monolayer or produce "cracking" of the PFOB-NEPs. The present work identifies the important structural motifs for melittin binding to PFOB-NEPs through a series of atomistic molecular dynamics simulations. The conformational ensemble of melittin bound to PFOB-NEP lipid monolayer was compared to structure from a control simulation of melittin bound to a lipid bilayer to identify several differences in melittin-lipid interactions between the two systems. First, melittin was deeply buried in the hydrophobic tail region of bilayer, while its depth was attenuated in the PFOB-NEP monolayer. Second, a helical conformation was the major secondary structure in the bilayer, but the fraction of helix was reduced in the PFOB-NEP. Finally, the overall pattern for the direct interaction of melittin with surrounding lipids was similar between liposome and PFOB-NEP, but the level of interaction was slightly decreased in the PFOB-NEP. These results suggest that melittin interacts with the monolayer of PFOB-NEP in a way that is similar way to its interaction with bilayers but that deeper penetration into the hydrophobic interior is inhibited.
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Project description:Traditional transfection agents including cationic lipids and polymers have high efficiency but cause cytotoxicity. While cell penetrating peptide based transfection agents exhibit improved cytotoxicity profiles, they do not have the efficiency of existing lipidic agents due to endosomal trapping. As a consequence, we propose an alternative method to efficient peptide based siRNA transfection by starting with melittin, a known pore-forming peptide. By incorporating modifications to decrease cytotoxicity and improve siRNA binding, we have developed p5RHH, which can complex siRNA to form nanoparticles of 190 nm in diameter. p5RHH exhibits high efficiency with GFP knockdown at concentrations as low as 5 nM, with negligible cytotoxicity. To date, p5RHH has shown the ability to transfect B16 cells, Human Umbilical Vein Endothelial Cells, and RAW264.7 cells with high efficiency. These in vitro models demonstrate that p5RHH mediated transfection can block cancer cell proliferation, angiogenesis, and foam cell formation. Moreover, p5RHH/siRNA nanoparticles maintain their size and transfection efficiency in the presence of serum proteins suggesting the potential for use of p5RHH in vivo. These data suggest that our strategy for development of siRNA transfecting peptides can provide an avenue to safe and effective siRNA therapeutics.
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Project description:The antimicrobial peptide, melittin, is a potential next-generation antibiotic because melittin can spontaneously form pores in bacterial cell membranes and cause cytoplasm leakage. However, the organizations of melittin peptides in cell membranes remain elusive, which impedes the understanding of the poration mechanism. In this work, we use coarse-grained and all-atom molecular dynamics (MD) simulations to investigate the organizations of melittin peptides during and after spontaneous penetration into DPPC/POPG lipid bilayers. We find that the peptides in lipid bilayers adopt either a transmembrane conformation or a U-shaped conformation, which are referred to as T- and U-peptides, respectively. Several U-peptides and/or T-peptides aggregate to form stable pores. We analyze a T-pore consisting of four T-peptides and a U-pore consisting of three U-peptides and one T-peptide. In both pores, peptides are organized in a manner such that polar residues face inward and hydrophobic residues face outward, which stabilizes the pores and produces water channels. Compared with the U-pore, the T-pore has lower energy, larger pore diameter, and higher permeability. However, the T-pore occurs less frequently than the U-pore in our simulations, probably because the formation of the T-pore is kinetically slower than the U-pore. The stability and permeability of both pores are confirmed by 300 ns all-atom MD simulations. The peptide organizations obtained in this work should deepen the understanding of the stability, poration mechanism, and permeability of melittin, and facilitate the optimization of melittin to enhance the antibacterial ability.
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Project description:The selective killing of cancer cells and the avoidance of drug resistance are still difficult challenges in cancer therapy. Here, we report a new strategy that uses enzyme-induced gain of function (EIGF) to regulate the structure and function of phosphorylated melittin analogues (MelAs). Original MelAs have the capacity to disrupt plasma membranes and induce cell death without selectivity. However, phosphorylation of Thr23 on one of the MelAs (MelA2-P) efficiently ameliorated the membrane lysis potency as well as the cytotoxicity for normal mammalian cells. After treatment with alkaline phosphatase (ALP), which is more active in cancer cells than normal cells, MelA2-P restored the pore-forming function around the cancer cells and induced cancer cell death selectively. This mechanism was independent of the receptor proteins and the cell uptake process, which may partially bypass the development of drug resistance in cancer cells.
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Project description:We recently developed an orthogonal, high-throughput assay to identify peptides that self-assemble into potent, equilibrium pores in synthetic lipid bilayers. Here, we use this assay as a high-throughput screen to select highly potent pore-forming peptides from a 7776-member rational combinatorial peptide library based on the sequence of the natural pore-forming peptide toxin melittin. In the library we varied ten critical residues in the melittin sequence, chosen to test specific structural hypotheses about the mechanism of pore formation. Using the new high-throughput assay, we screened the library for gain-of-function sequences at a peptide to lipid ratio of 1:1000 where native melittin is not active. More than 99% of the library sequences were also inactive under these conditions. A small number of library members (0.1%) were highly active. From these we identified 14 potent, gain-of-function, pore-forming sequences. These sequences differed from melittin in only 2-6 amino acids out of 26. Some native residues were highly conserved and others were consistently changed. The two factors that were essential for gain-of-function were the preservation of melittin's proline-dependent break in the middle of the helix and the improvement and extension the amphipathic nature of the α-helix. In particular the highly cationic carboxyl-terminal sequence of melittin, is consistently changed in the gain-of-function variants to a sequence that it is capable of participating in an extended amphipathic α-helix. The most potent variants reside in a membrane-spanning orientation, in contrast to the parent melittin, which is predominantly surface bound. This structural information, taken together with the high-throughput tools developed for this work, enable the identification, refinement and optimization of pore-forming peptides for many potential applications.
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