Cationic Alpha-helical Peptides Research Articles

The synthetic peptide LyeTxI-b derived from Lycosa erythrognatha spider venom is cytotoxic to U-87 MG glioblastoma cells.

Antimicrobial peptides present a broad spectrum of therapeutic applications, including their use as anticancer peptides. These peptides have as target microbial, normal, and cancerous cells. The oncological properties of these peptides may occur by membranolytic mechanisms or non-membranolytics. In this work, we demonstrate for the first time the cytotoxic effects of the cationic alpha-helical antimicrobial peptide LyeTx I-b on glioblastoma lineage U87-MG. The anticancer property of this peptide was associated with a membranolytic mechanism. Loss of membrane integrity occurred after incubation with the peptide for 15min, as shown by trypan blue uptake, reduction of calcein-AM conversion, and LDH release. Morphological studies using scanning electron microscopy demonstrated disruption of the plasma membrane from cells treated with LyeTx I-b, including the formation of holes or pores. Transmission electron microscopy analyses showed swollen nuclei with mild DNA condensation, cell volume increase with an electron-lucent cytoplasm and organelle vacuolization, but without the rupture of nuclear or plasmatic membranes. Morphometric analyses revealed a high percentage of cells in necroptosis stages, followed by necrosis and apoptosis at lower levels. Necrostatin-1, a known inhibitor of necroptosis, partially protected the cells from the toxicity of the peptide in a concentration-dependent manner. Imaging flow cytometry confirmed that 59% of the cells underwent necroptosis after 3-h incubation with the peptide. It is noteworthy that LyeTx I-b showed only mild cytotoxicity against normal fibroblasts of human and monkey cell lines and low hemolytic activity in human erythrocytes. All data together point out the anticancer potential of this peptide.

Abstract 3679: Nanotechnology delivery and mechanism of action of telomere oligonucleotides in melanoma

Abstract In nearly 90% of cancer cell types, telomere ends are stabilized by inappropriate activation of telomerase, thereby preventing telomeres from sequential shortening and establishing conditions which promote malignant transformation and immortality. As a result, telomeres and telomerase have recently become attractive targets in the development of novel anticancer therapeutics. Administration of an 11-mer oligonucleotide homologous to the 3′ overhang of the telomere, T-oligo, is presumed to mimic or signal for damaged telomeric DNA, thereby inducing inherent telomere-based DNA damage responses. Exposure to T-oligo induces cell cycle arrest, senescence, apoptosis, and differentiation in a cancer cell-type specific manner. Intravenous administration of T-oligo drastically reduces tumor burden in mice. Remarkably, T-oligo has minimal side-effects on normal cells, and has shown no detectable toxicity in in vivo mouse studies. However, as an oligonucleotide, T-oligo has limited stability due to serum and intracellular nucleases. We have previously improved the delivery of T-oligo by complexing it with a novel cationic alpha helical peptide, PVBLG-8, which improved its efficacy both in vitro and in vivo. Moreover, the T-oligo-PVBLG (TOP) nanocomplex demonstrated increased charge stability and diameter in comparison to T-oligo. In this study we have further optimized the TOP¬¬¬ nanocomplex, thereby improving the physiochemical properties of T-oligo for delivery and bringing T-oligo closer to clinical fruition as an innovative, anticancer therapeutic. The binding efficiency of T-oligo and PVBLG was optimized by measuring the amount of naked DNA under a range of nitrogen/phosphorus (N/P) ratios. We then compared the biodistribution of both T-oligo and TOP in vivo at various N/P ratios. T-oligo was conjugated with FITC and intravenously administered to the tail vein of mice in the presence or absence of PVBLG and monitored using molecular imaging. We found that T-oligo distribution was isolated to the kidneys, indicating that its small size makes it susceptible to renal filtration. In contrast, the TOP complex, which has a larger diameter, was able to bypass renal filtration and enter the liver and/or lungs. We also found that PVBLG was able to protect T-oligo from nucleolytic degradation in vitro. ¬¬¬¬In addition, to further elucidate T-oligo's mechanism of action, we investigated its ability to form G-quadruplexes, since increasing G-quadruplex stability is known to induce apoptosis. G-quadruplex formation by T-oligo was confirmed by NMR and native-PAGE. Furthermore, since increasing G-quadruplex stability is known to activate the SAPK/JNK signaling pathway, we also investigated T-oligo's effect on the SAP/JNK pathway using immunoblotting. Collectively, these studies will significantly advance the therapeutic utility of T-oligo against melanoma. Citation Format: Luke Wojdyla, Neelu Puri. Nanotechnology delivery and mechanism of action of telomere oligonucleotides in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2015-3679

Abstract 4577: Telomere oligonucleotides induce DNA damage responses in cancer cells: Mechanism of action and novel nanocomplex delivery system

Abstract Telomeres, the ribonucleoprotein complexes found at the ends of eukaryotic chromosomes, protect genomic DNA from end-to-end fusions and degradation by nucleases. During each round of cell division, the ends of telomeres are incompletely replicated, resulting in a net loss of telomeric DNA. When critically shortened, telomeres lose their ability to protect chromosomes from sustaining damage, thereby triggering cellular senescence or apoptosis. In numerous cancer types, administration of an oligonucleotide homologous to 3′-telomere overhang (T-oligo) induces potent DNA damage responses, presumably by mimicking or inducing telomere DNA damage, resulting in cell cycle arrest, senescence, or apoptosis. Remarkably, T-oligo induces minimal side-effects in normal cells, and thus, is actively being investigated as a novel anticancer therapeutic. However, as an oligonucleotide, T-oligo's intrinsic instability makes it susceptible to degradation by intracellular and serum nucleases, which severely limits its clinical use. To develop T-oligo as an innovative, effective therapeutic drug, it was complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG-8). PVBLG has been shown to be highly effective in delivering plasmid DNA and siRNA to a variety of cell lines, however, it has not been investigated using oligonucleotides. Here, our studies focus on its mechanism of action, stabilization and improved delivery. We show that PVBLG effectively stabilizes T-oligo by forming a nanocomplex (TOP complex), and enhances its anticancer properties both in vitro and in vivo. In addition, we identify new proteins involved in T-oligo's mechanism of action. The uptake of T-oligo by MM-AN melanoma cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the TOP complex increased uptake by 15-fold. In MM-AN cells, T-oligo complexed with the PVBLG (0.025-.25mg/ml) inhibited growth in vitro in a dose dependent manner by 4.5-8.3-fold, compared to 3-fold by T-oligo alone. Similar results were seen in H358 lung cancer cells. Preformed MM-AN tumors in immunodeficient mice treated with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. In addition, T-oligo treatment demonstrated downregulation of telomere repeat factor 1, a negative regulator of telomere length, in MU melanoma cells, indicating a role for TRF1 in T-oligo-induced DNA damage responses. In summary, our investigation further defines T-oligo's mechanism of action, and demonstrates the potential of a novel nanoparticle, the TOP complex, as an effective anticancer therapeutic. Citation Format: Luke M. Wojdyla, Srijayaprakash B. Uppada, Neelu Puri. Telomere oligonucleotides induce DNA damage responses in cancer cells: Mechanism of action and novel nanocomplex delivery system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4577. doi:10.1158/1538-7445.AM2014-4577

Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

Oligonucleotides homologous to 3′-telomere overhang (T-oligos) trigger inherent telomere-based DNA damage responses mediated by p53 and/or ATM and induce senescence or apoptosis in various cancerous cells. However, T-oligo has limited stability in vivo due to serum and intracellular nucleases. To develop T-oligo as an innovative, effective therapeutic drug and to understand its mechanism of action, we investigated the antitumor effects of T-oligo or T-oligo complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG), in a p53 null melanoma cell line both in vitro and in vivo. The uptake of T-oligo by MM-AN cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the T-oligo-PVBLG nanocomplex increased uptake by 15-fold. In vitro results showed a 3-fold increase in MM-AN cell growth inhibition by the T-oligo-PVBLG nanocomplex compared with T-oligo alone. Treatment of preformed tumors in immunodeficient mice with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. Moreover, T-oligo treatment downregulated procaspase-3 and procaspase-7 and increased catalytic activity of caspase-3 by 4-fold in MM-AN cells. Furthermore, T-oligo induced a 10-fold increase of senescence and upregulated the melanoma tumor-associated antigens MART-1, tyrosinase, and thrombospondin-1 in MM-AN cells, which are currently being targeted for melanoma immunotherapy. Interestingly, siRNA-mediated knockdown of p73 (4–10-fold) abolished this upregulation of tumor-associated antigens. In summary, we suggest a key role of p73 in mediating the anticancer effects of T-oligo and introduce a novel nanoparticle, the T-oligo-PVBLG nanocomplex, as an effective anticancer therapeutic.

The helical propensity of KLA amphipathic peptides enhances their binding to gel-state lipid membranes

The role and importance of the conformation of antimicrobial peptides for their binding and incorporation into lipid membranes as well as for their bioactivity are still not well understood. In this paper, we studied the interaction between four cationic alpha-helical KLA peptides, which differ primarily in their helical propensity, and the anionic gel-state lipid DPPG (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol). Of particular interest was the influence of the peptide conformation and membrane surface properties on the electrostatic binding process. Dynamic light scattering (DSL) showed that generally the KLA peptides possess high aggregation power but modest solubilization power. Circular dichroism spectroscopy (CD) spectra revealed that the KLA peptides with the low helical propensity tend to form beta-structures at low lipid/peptide ratios. Differential scanning calorimetry (DSC) thermograms showed that the helical KLA peptides stabilize the DPPG bilayer, whereas the beta-structured peptides induce pronounced membrane perturbations. Isothermal titration calorimetry (ITC) isotherms showed that the helical KLA peptides bind more efficiently to DPPG vesicles than the beta-structured KLA peptides, and that the binding affinity of the peptides is proportional to the peptide helical propensity and membrane negative surface charge. The stoichiometry values (N) deduced from the ITC isotherms suggest that the helical KLA peptides have a higher capacity to translocate the DPPG lipid bilayer. The new data presented in this study demonstrate the flexibility of KLA peptides in adopting various conformations in response to the surrounding and also how the peptide structuring controls the mode of peptide–membrane interaction.

Dynamics and orientation of a cationic antimicrobial peptide in two membrane-mimetic systems

In order to investigate the functional and structural properties of cationic α-helical peptides in two different membranes, we studied the 20-residue peptide maximin H6 in two membrane-mimetic systems by NMR spectroscopy using partially 15N-labeled peptide and paramagnetic relaxation enhancements. Maximin H6, which is found in skin secretions of frogs of the Bombinae family, attacks gram-negative bacteria and acts haemolytically. While the peptide spontaneously folds into similar structures in both neutral dodecylphosphocholine (DPC) and negatively charged sodium dodecyl sulphate (SDS) micelles, its structure is more flexible in SDS as shown by 15N relaxation measurements. In addition, it is bound closer to the surface of the micelle and rotated by ∼70° around its helix axis in the negatively charged membrane surrogate compared to the structure in DPC. This might form the basis for peptide–peptide interactions through a GxxxG motif, which could finally lead to membrane disruption and, thus, preferential attack of negatively charged microbial cell walls.

Dermaseptin S9, an α-Helical Antimicrobial Peptide with a Hydrophobic Core and Cationic Termini

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.

Processing of lysozyme at distinct loops by pepsin: A novel action for generating multiple antimicrobial peptide motifs in the newborn stomach

C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix-loop-helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the alpha-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic alpha-helical peptides generated from a helix-loop-helix domain (residues 1-38 of cLZ) by nicking at leucine17. A third peptide consisting of an alpha-helix (residues 18-38) and a two-stranded beta-sheet (residues 39-56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylglycerol bilayers.

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of anionic n-saturated diacyl phosphatidylglycerols (PGs). Analogues of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (L(24)DAP), or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (WL(22)W), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the host PG bilayers is altered by these peptides in a hydrophobic mismatch-dependent manner, as previously found with zwitterionic phosphatidylcholine (PC) bilayers. However, all three peptides reduce the phase transition temperature and enthalpy to a greater extent in anionic PG bilayers than in zwitterionic PC bilayers, with WL(22)W having the largest effect. All three peptides form very stable alpha-helices in PG bilayers, but small conformational changes are induced in response to a mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. Moreover, electrostatic and hydrogen-bonding interactions occur between the terminal lysine residues of L(24) and L(24)DAP and the polar headgroups of PG bilayers. However, such interactions were not observed in PG/WL(22)W bilayers, suggesting that the cation-pi interactions between the tryptophan and lysine residues predominate. These results indicate that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer, but also by the tryptophan-modulated electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the negatively charged polar headgroups of the PG bilayers.

Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides.

We have designed beta-amino acid oligomers that are helical, cationic, and amphiphilic with the intention of mimicking the biological activity of amphiphilic, cationic alpha-helical antimicrobial peptides found in nature (e.g., magainins). We have previously identified a 17-residue beta-peptide (called beta-17) with antibiotic activity similar to that of a magainin derivative against four bacterial species, including two clinical isolates that are resistant to common antibiotics. This beta-peptide displays very low hemolytic activity against human red blood cells, which indicates selectivity for bacterial cells over mammalian cells. Here we examine some of the factors important for activity in this class of beta-peptides. An amphiphilic helix is necessary, because a nonamphiphilic isomer proved to be inactive. The ratio of cationic to hydrophobic residues is also important. Active beta-peptides induce the leakage of beta-galactosidase from treated Bacillus subtilis cells, as do alpha-helical antibiotic peptides, and this similarity suggests that the beta-peptide mode of action involves disruption of microbial membranes. This class of beta-peptides is not degraded by proteases, which bodes well for biological applications.

Mitochondrion as a novel target of anticancer chemotherapy.

Mitochondrial membrane permeabilization is a critical event in the process leading to physiologic or chemotherapy-induced apoptosis (programmed cell death). This permeabilization event is, at least in part, under the control of the permeability transition pore complex (PTPC). Oncoproteins from the Bcl-2 family and tumor suppressor proteins from the Bax family interact with PTPC to inhibit or facilitate membrane permeabilization, respectively. Conventional chemotherapeutic agents elicit mitochondrial permeabilization in an indirect fashion by induction of endogenous effectors that are involved in the physiologic control of apoptosis. However, an increasing number of experimental anticancer drugs, including lonidamine, arsenite, betulinic acid, CD437, and several amphipathic cationic alpha-helical peptides, act directly on mitochondrial membranes and/or on the PTPC. Such agents may induce apoptosis in circumstances in which conventional drugs fail to act because endogenous apoptosis induction pathways, such as those involving p53, death receptors, or apical caspase activation, are disrupted. However, stabilization of the mitochondrial membrane by antiapoptotic Bcl-2-like proteins reduces the cytotoxic potential of most of these drugs. Targeting of specific PTPC components may overcome this Bcl-2-mediated apoptosis inhibition. One strategy involves cross-linking of critical redox-sensitive thiol groups within the PTPC; another involves the use of ligands to the mitochondrial benzodiazepine receptor. Thus, the design of mitochondrion-targeted cytotoxic drugs may constitute a novel strategy for overcoming apoptosis resistance.