Peptide internalization enabled by folding: triple helical cell‐penetrating peptides

Cell‐penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30‐mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen‐like folding domains. The CPP domains are hexa‐arginine (R₆) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline–hydroxyproline–glycine (POG [proline‐hydroxyproline‐glycine])_n repeats that form a collagen‐like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell‐penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non‐covalent complexation and intracellular delivery

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa‐arginine (R₈) and CHK₆HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep‐3, flanked with CH₃ on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide–plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non‐covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide–plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

In silico predictions of 3D structures of linear and cyclic peptides with natural and non‐proteinogenic residues

We extended the use of Peplook, an in silico procedure for the prediction of three‐dimensional (3D) models of linear peptides to the prediction of 3D models of cyclic peptides and thanks to the ab initio calculation procedure, to the calculation of peptides with non‐proteinogenic amino acids. Indeed, such peptides cannot be predicted by homology or threading. We compare the calculated models with NMR and X‐ray models and for the cyclic peptides, with models predicted by other in silico procedures (Pep‐Fold and I‐Tasser). For cyclic peptides, on a set of 38 peptides, average root mean square deviation of backbone atoms (BB‐RMSD) was 3.8 and 4.1 Å for Peplook and Pep‐Fold, respectively. The best results are obtained with I‐Tasser (2.5 Å) although evaluations were biased by the fact that the resolved Protein Data Bank models could be used as template by the server. Peplook and Pep‐Fold give similar results, better for short (up to 20 residues) than for longer peptides. For peptides with non‐proteinogenic residues, performances of Peplook are sound with an average BB‐RMSD of 3.6 Å for ‘non‐natural peptides’ and 3.4 Å for peptides combining non‐proteinogenic residues and cyclic structure. These results open interesting possibilities for the design of peptidic drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy

Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathepsin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells.     

Targeting of hepatocellular carcinoma with glypican‐3‐targeting peptide ligand

Hepatocellular carcinoma is a common malignancy. The carcinoma cells express glypican‐3 (GPC‐3) on the cell membrane. GPC‐3 is also expressed in melanoma cells. Therefore, GPC‐3 might be a potential target for tumor imaging or therapy. Here, proteomic mass spectrometry was used to identify peptides that target GPC‐3‐expressing tumors. A mammalian expression vector expressing a FLAG‐GPC‐3 fusion protein was cloned for immunoprecipitation. With the use of liposomes, the vector was transfected into HepG2 (HepG2/FLAG‐GPC‐3) and HEK 293 cells, and the transfected cell lines were selected with geneticin. HepG2/FLAG‐GPC‐3 cells were used for immunoprecipitation of FLAG‐GPC‐3 fusion protein. Seven peptide candidates (L1–L7) were selected for GPC‐3‐targeting ligands by mass spectrometric analysis. The L5 peptide with 14 amino acids (Arg‐Leu‐Asn‐Val‐Gly‐Gly‐Thr‐Tyr‐Phe‐Leu‐Thr‐Thr‐Arg‐Gln) showed selective binding to the GPC‐3‐expressing tumor cells, as did a shortened L5 peptide (L5‐2) with seven amino acids (Tyr‐Phe‐Leu‐Thr‐Thr‐Arg‐Gln). These peptide ligands have potential as targeting moieties to GPC‐3‐expressing tumors for diagnostic and/or therapeutic purposes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Enhanced identification of peptides lacking basic residues by LC-ESI-MS/MS analysis of singly charged peptides

Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as "base-less") are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/M Sanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MS schemes. In the present experimental outcome, additional LC-MS/MS analysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MS based identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.     

Dual‐function fluorescent probe for cancer imaging and therapy

To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual‐function quantum dot (QD) fluorescent probes with dual‐targeting action and a therapeutic effect are rare. Here, a dual‐targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin–glycyrrhetinic acid conjugate and arginine‐glycine‐aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as‐prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Cellular internalization of arginine‐rich peptides into tobacco suspension cells: a structure–activity relationship study

Translocation of several fluorescently labeled arginine‐rich peptides into intact plant cells was quantitatively examined in order to investigate the structural factors required for efficient cellular internalization, and thereby, to evaluate the potential of arginine‐rich peptides as intracellular delivery vectors in plants. Cell‐penetrating peptides (CPPs) such as arginine‐rich peptides permit the direct introduction of biologically active macromolecules into plant cytoplasm to manipulate various intracellular processes. While a significant level of adsorption of applied arginine‐rich peptides was observed in the cell walls rich in negative charges, removal of adsorbed peptides by trypsin treatment allowed determination of the amount of internalized peptides in a quantitative manner using spectrofluorometric analysis. The internalization of arginine‐rich peptides depended on the number of arginine residues, and the peptide containing eight arginine residues showed most effective internalization. Besides, the position of small cargoes attached to the arginine‐rich peptides markedly affected the internalization efficiency. The results obtained in this study provide useful information for the development of efficient intracellular delivery tools in plant science. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Design,synthesis and biological activity of cell‐penetrating peptide‐modified octreotide analogs

Four novel octreotide analogs with cell‐penetrating peptides (CPPs) at the N‐terminus or C‐terminus were synthesized by a stepwise Fmoc solid‐phase synthesis strategy. The synthesized peptides were analyzed and characterized using reverse phase HPLC and MALDI‐TOF mass spectrometry. The antiproliferative activity of the analogs was tested in vitro on human gastric (SGC‐7901) and hepatocellular cancer (BEL7402) cell lines using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Interestingly, these analogs showed a higher anticancer activities than the parent octreotide except CMTPT03 analog. The results demonstrate that the designed octreotide analogs enhance their anticancer activity after linking together the CPPs to octreotide at the N‐terminus, and are potential molecules for future use in cancer therapy and drug targeting. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Formation mechanism of cross‐linking Maillard compounds in peptide–xylose systems

The formation mechanism of Maillard peptides was explored in Maillard reaction through diglycine/glutathione(GSH)/(Cys‐Glu‐Lys‐His‐Ile‐Met)–xlyose systems by heating at 120 °C for 30–120 min. Maximum fluorescence intensity of Maillard reaction products (MRPs) with an emission wavelength of 420~430 nm in all systems was observed, and the intensity values were proportional to the heating time. Taken diglycine/GSH–[¹³C₅]xylose systems as a control, it was proposed that the compounds with high m/z values of 379 and 616 have the high molecular weight (HMW) products formed by cross‐linking of peptides and sugar. In (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the m/z value of HMW MRPs was not observed, which might be due to the weak signals of these products. According to the results of gel permeation chromatography, HMW MRPs were formed by Maillard reaction, especially in (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the percentage of Maillard peptides reached 52.90%. It was concluded that Maillard peptides can be prepared through the cross‐linking of sugar and small peptides with a certain MW range. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of cyclic herpes simplex virus peptides containing 281–284 epitope of glycoprotein D‐1 in endo‐ or exo‐position

We have prepared two types of cyclopeptides containing the ²⁸¹DPVG²⁸⁴ sequence from the 276–284 region of glycoprotein gD‐1 of the Herpes simplex virus (HSV). The syntheses were performed by solid phase methodology using MBHA or BHA resin and orthogonal protection schemes. Head‐to‐side‐chain cyclization included the N‐terminal part of the epitope, while side‐chain‐to‐side‐chain lactam bridge formation resulted in a peptide containing a C‐terminal cycle. Peptides elongated by Cys at the N‐terminal of the sequence were also prepared. Boc chemistry using Fmoc and OFm orthogonal protection was applied for on‐resin cyclization. Based on the orthogonality of Bzl and cHex esters under a 1 m TMSOTf‐thioanisole/TFA cleavage condition, a new approach for the cyclization on BHA‐resin has also been developed. Preliminary studies on solution conformation of the cyclic peptides by CD spectroscopy indicated the importance of the location and the size of the cycle within the epitope sequence. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Conformationally constrained peptides for drug delivery

Peptides have shown great potential in acting as template for developing versatile carrier platforms in nanomedicine, aimed at selective delivery of drugs to only pathological tissues saving its normal neighbors. Cell‐penetrating peptides (CPPs) are short oligomeric peptides capable of translocating across the cell membrane while simultaneously employing multiple mechanisms of entry. Most CPPs exist as disordered structures in solution and may adopt a helical conformation on interaction with cell membrane, vital to their penetrative capability. Herein, we report a series of cationic helical amphipathic peptides (CHAPs), which are topologically constrained to be helical. The peptides were tested against cervical and breast cancer cells for their cell penetration and drug delivery potential. The cellular uptake of CHAP peptides is independent of temperature and energy availability. The activity of the peptides is biocompatible in bovine serum. CHAPs delivered functional methotrexate (MTX) inside the cell as CHAP‐MTX conjugates. CHAP‐MTX conjugates were more toxic to cancer cells than MTX alone. However, the CHAP‐MTX conjugates were less toxic to HEK‐293 cells compared with the cancer cells suggesting higher affinity towards cancer cells.     

Identification and characterization of two novel antimicrobial peptides,temporin‐Ra and temporin‐Rb,from skin secretions of the marsh frog (Rana ridibunda)

In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin‐Ra and temporin‐Rb, were identified and purified using RP‐HPLC. Temporin‐Ra and temporin‐Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram‐negative and gram‐positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin‐Rb and 1585.1 Da for temporin‐Ra. Human red blood cells tolerated well exposure to temporin‐Ra and temporin‐Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide‐spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.