Refinement of a Quantitative Structure–Activity Relationship Model for Prediction of Cell-Penetrating Peptide Based Transfection Systems

Cell-penetrating peptide (CPP) based transfection systems (PBTS) are a promising class of drug delivery vectors. CPPs are short mainly cationic peptides capable of delivering cell non-permeant cargo to the interior of the cell. Some CPPs have the ability to form non-covalent complexes with oligonucleotides for gene therapy applications. In this study, we use quantitative structure–activity relationships (QSAR) , a statistical method based on regression data analysis. Here, an fragment QSAR (FQSAR) model is developed to predict new peptides based on standard alpha helical conformers and Assisted Model Building with Energy Refinement molecular mechanics simulations of previous peptides. These new peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were capable of achieving plasmid transfection with significant improvement compared to the previous generation of peptides. Our results demonstrate that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity.     

Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides

An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z′ scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.     

The underestimated importance of belowground carbon input for forest soil animal food webs

The present study investigated the relative importance of leaf and root carbon input for soil invertebrates. Experimental plots were established at the Swiss Canopy Crane (SCC) site where the forest canopy was enriched with ¹³C depleted CO₂ at a target CO₂ concentration of c . 540 p.p.m. We exchanged litter between labelled and unlabelled areas resulting in four treatments: (i) leaf litter and roots labelled, (ii) only leaf litter labelled, (iii) only roots labelled and (iv) unlabelled controls. In plots with only ¹³C-labelled roots most of the soil invertebrates studied were significantly depleted in ¹³C, e.g. earthworms, chilopods, gastropods, diplurans, collembolans, mites and isopods, indicating that these taxa predominantly obtain their carbon from belowground input. In plots with only ¹³C-labelled leaf litter only three taxa, including, e.g. juvenile Glomeris spp. (Diplopoda), were significantly depleted in ¹³C suggesting that the majority of soil invertebrates obtain its carbon from roots. This is in stark contrast to the view that decomposer food webs are based on litter input from aboveground.     

In vitro uptake and stability study of pVEC and its all-D analog

A key step in the development of new hydrophilic pharmaceuticals is to get them through biological barriers. Cell-penetrating peptides, CPPs, have been shown previously to enter cells both in vitro and in vivo by a non-endocytotic mechanism and to be able to carry large cargo molecules with them. Recently, we showed that a small peptide, pVEC, from murine vascular endothelial cadherin, has the characteristics to be classified as a protein derived CPP. Here we have further investigated pVEC together with its all-D analog for cellular uptake, intra- and extracellular stability, and their enzymatic degradation. The two peptides, pVEC and all-D pVEC, translocate into aortic endothelial cells and murine fibroblasts by a non-endocytotic mechanism. In phosphate buffer, pVEC remains intact while the C-terminal lysine is quickly removed in human serum and serum-containing media. Both pVEC and pVEC without the C-terminal Lys were detected by mass spectrometry inside the two cell types tested. The pVEC half-life is 10.5 min in phosphate buffer containing 10 units of trypsin and 44.6 min in phosphate buffer containing 4.2 units of carboxypeptidase A and 18 units of carboxypeptidase B. In contrast topVEC, the all-D analog remains intact in serum and resists enzymatic degradation.     

Isolation and characterization of galanin from sheep brain.

Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26.     

Cholesterol prevents interaction of the cell‐penetrating peptide transportan with model lipid membranes

Interaction of the cell‐penetrating peptide (CPP) cysteine‐transportan (Cys‐TP) with model lipid membranes was examined by spin‐label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys‐TP on membrane structure was studied. The influence of Cys‐TP on membrane permeability was monitored by the reduction of a liposome‐trapped water‐soluble spin probe. Cys‐TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys‐TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so‐called liquid‐ordered phase, no effect of Cys‐TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys‐TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP–lipid interaction in laterally heterogeneous membranes, while it implies that the CPP–lipid interaction can be different at different points along the membrane. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Assessing the delivery efficacy and internalization route of cell-penetrating peptides

Developing efficient delivery vectors for bioactive molecules is of great importance within both traditional and novel drug development, such as oligonucleotide (ON)-based therapeutics. To address delivery efficiency using cell-penetrating peptides (CPPs), we here present a protocol based on splice correction utilizing both neutral and anionic antisense ONs, either covalently conjugated via a disulfide bridge or non-covalently complexed, respectively, that generates positive readout in the form of luciferase expression. The decisive advantage of using splice correction for evaluation of CPPs is that the ON induces a biological response in contrast to traditionally used methods, for example, fluorescently labeled peptides. An emerging number of studies emphasize the role of endocytosis in translocation of CPPs, and this protocol is also utilized to determine the relative contribution of different endocytic pathways in the uptake of CPPs, which provides valuable information for future design of novel, more potent CPPs for bioactive cargoes.     

Protein cargo delivery properties of cell-penetrating peptides. A comparative study

Application of cell-penetrating peptides for delivering various hydrophilic macromolecules with biological function into cells has gained much attention in recent years. We compared the protein transduction efficiency of four cell-penetrating peptides: penetratin, Tat peptide, transportan, and pVEC and studied the effects of various medium parameters on the uptake. Depletion of cellular energy and lowering of temperature strongly impaired the internalization of protein complexed with cell-penetrating peptides, confirming the endocytotic mechanism of peptide-mediated protein cellular transduction. Peptide-induced protein association with HeLa cells decreased 3-6-fold in energy-depleted cells. Inhibition of clathrin-dependent endocytosis by the hyperosmolar medium decreased the uptake of peptide-avidin complexes 1.5-3-fold and the removal of cholesterol from the plasma membrane 1.2-2-fold, suggesting that both clathrin-dependent and independent endocytosis were involved in peptide-induced cellular delivery of avidin. However, even under conditions of cellular energy depletion, ceasing of cellular traffic, and partial depolarization of plasma membrane, peptide-protein complexes associated with HeLa cells, as observed by FACS analysis and spectrofluorimetry. Among the studied peptides, pTat and transportan revealed higher protein transduction efficiency than penetratin or pVEC.     

Overexpression of protein-tyrosine phosphatase PTP sigma is linked to impaired glucose-induced insulin secretion in hereditary diabetic Goto-Kakizaki rats

The impaired glucose-induced insulin release in type 2 diabetes mellitus may be accounted for by reduced B-cell ATP/ADP ratio or decreased phosphorylation of proteins regulating exocytosis of insulin. This, in turn, could be due to enhanced phosphatase activity. Using in situ hybridization techniques to assess the expression of 11 different phosphotyrosine phosphatases (PTPases), known to be present in the B-cells, overexpression by approximately 60% of PTP sigma (also known as LAR-PTP2 or PTP NE-3) was demonstrated in pancreatic islets and liver of spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. In agreement with these findings Western blot of islet lysates, using a polyclonal PTP sigma antiserum, showed increased amounts of the protein in GK relative to control rat islets. Exposure of isolated islets for 20 h to 5 muM antisense to PTP sigma, composed of an antisense PNA sequence of 15 bases linked to the cell penetrating peptide transportan, increased glucose-induced insulin secretion from GK rat islets, but not from control islets. In parallel, the amounts of the phosphatase decreased. In conclusion, increased expression of PTP sigma may be of pathogenetic significance for the defective insulin secretion in GK rat islets.     

NickFects,Phosphorylated Derivatives of Transportan 10 for Cellular Delivery of Oligonucleotides

Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for nucleic acid polymers and, consequently, an efficient and non-toxic transfection agent is needed for their delivery into the cell. In this study we present a novel series, NickFects, of chemically modified TP10 peptide-based delivery vectors used for the cellular delivery of single-stranded oligonucleotides. These carriers, obtained by replacement of Ile8 by threonine in stearyl-TP10 and by modifying of tyrosine and/or threonine, respectively, by phosphorylation formed 300–500 nm in size peptide:oligonucleotide nanocomplexes with negative surface charges. The highest splice-correcting effect was obtained when phosphorotiate 2′-O-methyl oligonucleotides were transduced into cells by NickFect1 (NF1) or NickFect2 (NF2). In addition, we also show how a small modification (one or two negative charges) in peptide sequence can affect its ability to deliver ONs into cells and increase their potency in the splicing redirection assay. Our studies demonstrate that NF1 and NF2 have higher transfection efficacy for oligonucleotides as compared to the most commonly used transfection agent Lipofectamine™ 2000 and lead to higher biological response in cells.