A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction

The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.     

SYNTHESIS OF RGD CONTAINING PEPTIDES. COMPARATIVE STUDY OF THEIR INCORPORATION TO THE SURFACE OF 5-FLUORURIDINE LOADED LIPOSOMES

The synthesis on solid phase of a peptide sequence (GGRGRS) related to an integrin adhesion site as well as the preparation of some hydrophobic derivatives is described.

The incorporation of these peptides to the surface of liposomes was carried out either through the NGPE (N-glutaryl dipalmitoyl phosphatidyl choline) carboxyl-group or mixing hydrophobic peptide derivatives with lipids since the beginning of the process. The influence of these factors on the entrapment yield of 5-FUR (5-fluorouridine) was determined. Best results, calculated as percentage of drug encapsulation, were obtained when the peptide was linked to preformed liposomes via an NGPE-amide bond. On the contrary, the presence of these hydrophobic peptides on the bilayers decreases the overall yield of encapsulation of 5-FUR. Nevertheless, considering drug/lipid relationship and scaling-up requirements it seems that the use of myristoyl peptide derivative should be the procedure of choice.

Physicochemical studies carried out with the peptides indicated that the presence of hydrophobic moieties linked to the parent peptide increases the tendency to self aggregation as detected through fluorescence studies using DPH (1, 6 diphenyl hexatriene) as marker, reducing in this way the efficiency of incorporation of hydrophobic peptides to the surface of liposomes.     

Sortase A‐mediated synthesis of ligand‐grafted cyclized peptides for modulating a model protein‐protein interaction

Specific ligand‐grafted cyclic peptides are promising drug candidates that can modulate protein‐protein interactions (PPIs) with increased proteolytic stability. In this study, we aimed to demonstrate that Sortase A (SrtA)‐mediated peptide transpeptidation can be applied to produce bioactive sequence‐grafted, stable, cyclic peptides. A naturally occurring cyclic peptide, sunflower trypsin inhibitor 1 (SFTI‐1), was selected as the scaffold, and a tetrapeptide motif, Glu‐Ser‐Asp‐Val (ESDV), was grafted into the scaffold as a model ligand. The linear precursor of the grafted peptide with SrtA‐recognition motifs at the N‐ and C‐termini was cyclized in good yield simply by co‐incubation with SrtA. The ESDV‐grafted cyclic SFTI‐1 obtained was confirmed to have high stability against proteolysis by human serum and bound to the target PDZ2 domain of postsynaptic density‐95 protein. An optimized sequence‐grafted cyclic SFTI‐1 could competitively suppress the interaction of PDZ2 with its natural ligand, the C‐terminal peptide of the NR2B subunit of the N‐methyl‐D‐aspartate receptor. These results show that a strategy combining peptide grafting into the SFTI‐1 scaffold with SrtA‐catalyzed cyclization can be a simple and effective method for producing stable peptide drugs.     

A novel delivery platform for therapeutic peptides

Although many peptides have therapeutic effects against diverse disease, their short half-lives in vivo hurdle their application as drug candidates. To extend the short elimination half-lives of therapeutic peptides, we developed a novel delivery platform for therapeutic peptides using an anti-hapten antibody and its corresponding hapten. We selected cotinine because it is non-toxic, has a well-studied metabolism, and is physiologically absent. We conjugated WKYMVm-NH₂, an anti-sepsis therapeutic peptide, to cotinine and showed that the conjugated peptide in complex with an anti-cotinine antibody has a significantly improved in vivo half-life while retaining its therapeutic efficacy. We suggest that this novel delivery platform for therapeutic peptides will be very useful to develop effective peptide therapeutics.     

Engineering a noncarrier to a highly efficient carrier peptide for noncovalently delivering biologically active proteins into human cells

Noncovalent protein delivery into cells via peptide carriers is an emerging concept. Only a handful of such peptides are known. To address various limitations associated with protein delivery for therapeutic purposes, a greater number of different delivery peptides would be required. No general method exists for creating such peptides. By combining a sequence of 16 lysine residues (K16) with the signal peptide (SP) sequence of Kaposi's fibroblast growth factor (K-FGF), we have synthesized a peptide (K16SP) that efficiently and noncovalently delivers functionally intact proteins (immunoglobulin G molecules, beta-galactosidase, and green fluorescent protein) into mammalian cells. The peptides K16 and SP each alone did not show any noncovalent protein-carrying capacity. K16SP appears to be nontoxic to cells and three to four times more efficient than a commercially available peptide reagent. Our approach offers proof-of-concept of a general strategy for creating a diverse array of peptide carriers for eventual therapeutic applications.     

Peptide substrates rapidly modulate expression of dipeptide and oligopeptide permeases in Candida albicans

Previous studies showed that peptide transport activity in Candida albicans was completely repressed by NH4+, and that growth on amino acids as sole nitrogen source stimulated transport to a basal level. Here we show that addition of peptide mixtures to culture media gives a further 5-fold increase in transport of dipeptides and oligopeptides; the effect is specific for peptide transport, amino acid uptake being unaffected. Presence of peptides but not amino acids overrides NH4+ repression of peptide transport. Step-up activation of transport activity, caused by addition of peptides to incubation media, and step-down inhibition that accompanies removal of peptides, occurs rapidly (within 30 min at 28 degrees C). Step-up is independent of de novo protein synthesis. This substrate-induced regulation is compatible with a rapid, reversible activation of plasma membrane-bound peptide permease(s), or a mechanism of endocytosis involving a cycle of insertion and retrieval of preformed permease components. These results are considered in relation to the expression of peptide permeases in vivo, and the development of synthetic anticandidal peptide carrier prodrugs designed to exploit these systems.     

Surface behaviour and peptide-lipid interactions of the antibiotic peptides, Maculatin and Citropin

Surface behaviour of Maculatin 1.1 and Citropin 1.1 antibiotic peptides have been studied using the Langmuir monolayer technique in order to understand the peptide-membrane interaction proposed as critical for cellular lysis. Both peptides have a spontaneous adsorption at the air-water interface, reaching surface potentials similar to those obtained by direct spreading. Collapse pressures (Π_c, stability to lateral compression), molecular areas at maximal packing and surface potentials (ΔV) obtained from compression isotherms of both pure peptide monolayers are characteristic of peptides adopting mainly α-helical structure at the interface. The stability of Maculatin monolayers depended on the subphase and increased when pH was raised. In an alkaline environment, Maculatin exhibits a molecular reorganization showing a reproducible discontinuity in the Π-A compression isotherm. Both peptides in lipid films with the zwitterionic palmitoyl-oleoyl-phosphatidylcholine (POPC) showed an immiscible behaviour at all lipid-peptide proportions studied. By contrast, in films with the anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG), the peptides showed miscible behaviour when the peptides represented less than 50% of total surface area. Additional penetration experiments also demonstrated that both peptides better interact with POPG compared with POPC monolayers. This lipid preference is discussed as a possible explanation of their antibiotic properties.     

Microarrays of peptides elevated on the protein layer for efficient protein kinase assay

Peptide microarrays can be used for the high-throughput analysis of protein-peptide interactions. However, current peptide microarrays are rather costly to make and require cumbersome steps of introducing novel polymeric surfaces and/or chemical derivatization of peptides. Here, we report a novel method for manufacturing peptide microarrays by elevating the peptide on the layer of protein by a fusion protein approach. Using two protein kinases and their peptide substrates as examples, we show that elevating peptides on the layer of protein allows sensitive, specific, and efficient detection of peptide-protein interactions without the need for complicated chemical modification of solid supports and peptides. It was found that kinase activity could be detected with as low as 0.09 fmol of kemptide, which is about 1000-fold more sensitive than the 0.1 pmol obtained with other microarray systems. Furthermore, peptides can be produced as fusion proteins by fermentation of recombinant Escherichia coli and thus the expensive peptide synthesis process can be avoided. Therefore, this new strategy will not only be useful in high-throughput and cost-effective screening of kinase substrate peptides but also be generally applicable in studying various protein-peptide interactions.     

A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditions

Sample preparation prior to analysis by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) usually involves the storage of frozen peptide samples in an acidic environment for variable time periods. Questions arose in our laboratory regarding the stability of peptides in acid under medium- to long-term storage. Thus, a 10-month longitudinal study was designed to assess the effect on storage of tryptic peptides at −20 and −80 °C under acidic conditions. Our conclusion and proposal from this evaluation is that the optimal storage conditions of peptide samples in acid for proteomic experiments is at −80 °C and, ideally, as separate aliquots.     

A high-yield method to extract peptides from rat brain tissue

A process to extract and enrich extracellular peptides and proteins from tissues should have broad utility in the burgeoning proteomics field. To address this need, a novel three-step protocol was developed to extract polypeptides from whole tissue samples and enrich the extracellular components. The initial homogenization of rat brain was carried out at neutral pH to optimize protein and peptide stability and solubility. Subsequent covalent chromatography on an activated thiopropyl resin was employed to debulk the tissue extract by selectively removing a substantial fraction of the intracellular protein component under nondenaturing conditions. Finally, extraction with 0.1% trifluoroacetic acid was used to selectively precipitate large proteins while enhancing the solubility of smaller proteins and peptides. The fractions from each step in the process were compared to a single extract obtained by homogenization in 0.5 M acetic acid. The recovery and yields of endogenous neuropeptides and an exogenously added peptide were evaluated by enzyme immunoassay and Western blotting, respectively. In summary, the three-step protocol was superior to the extraction of tissue with 0.5 M acetic acid in terms of peptide recovery, enrichment, and sample stability. Enrichment of the extracellular protein compartment from tissues should be valuable in proteomics experiments aimed at identifying biomarkers that can partition into serum.     

Dynamic PET and SPECT imaging with radioiodinated,amyloid-reactive peptide p5 in mice: A positive role for peptide dehalogenation

Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72 h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients.     

Peptide chemistry toolbox – Transforming natural peptides into peptide therapeutics

The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market.     

Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion

The peptide GALA undergoes a conformational change to an amphipathic alpha -helix when the pH is reduced, inducing leakage of contents from vesicles. Leakage from neutral or negativelycharged vesicles at pH 5.0 was similar and could be adequately explained by a mathematical model which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of M =10+/-2 peptides. Increasing cholesterol content in the membranes resulted in reduced leakage, and increased reversibility of surface aggregation of the peptide. Employing fluorescently labelled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol containing liposomes. Orientation of the peptide GALA in bilayers was determined by a bodipy-avidin/ biotin binding assay. The peptide was labelled by biotin at the N- or Cterminus and bodipy-avidin molecules were added externally or were preencapsulated in the vesicles. The peptides are arranged in the pore perpendicularly to the membrane, such that 3/4 of the N-termini are on the internal side of the membrane. The pores are stable and persist for at least 10 min. When the peptides form an aggregate of size smaller than M, the orientation of the peptide is mostly parallel to the surface and the biotinylated peptide does not translocate. When a critical size of the aggregate is attained, a rearrangement of the peptide occurs, which amounts to rapid penetration and formation of a pore structure. Induction of fusion by peptides may be antagonistic to pore formation, the outcome being dependent on vesicle aggregation.     

Assessment of Human Immune Response to Mutans Streptococcal Glucosyltransferase Peptides Selected by MHC Class II Binding Probability

Dental decay is a major public health challenge, causing substantial social and economic burdens. In animals, vaccination against mutans streptococci, the causative organism, interferes with dental caries. The mutans streptococcal glucosyltransferase (GTF) has been effectively used as a protein antigen in experimental dental caries vaccines. Compared to whole proteins, peptide subunits can focus immune responses on protective epitopes, and not on potentially harmful cross reactive antigens. In the past we selected peptide subunits of GTF for vaccine discovery based on putative functional significance and conservation of GTF primary structure. To focus on the immunogenicity of peptides, we estimated the probability of MHC class II binding. Twenty 20-mer linear GTF peptides were synthesized on this basis and their immunoreactivity explored. Significant human peripheral blood mononuclear cell (PBMC; n = 12) proliferation was observed in response to amino acids (AA) 502–521 (peptide 7), located in the catalytic domain of GTF. Human serum (n = 36) antibody reactivity was observed to AA 438–457 (peptide 5), AA 502–521 (peptide 7), and AA 1376–1395 (peptide 16). Whole saliva mutans streptococcal levels were used as markers of mutans infection, and dental examinations to determine existing and historic caries (DMFS score) were performed. DMFS scores correlated with mutans streptococcal counts, but not with immune responses. We have identified peptides with projected avid MHC-binding activity that reacted with human PBMC and serum antibody, implying that these peptides are immunogenic and may be of significance in a subunit dental caries vaccine.     

Peptides for disrupting and degrading amyloids

Amyloid proteins can aggregate into insoluble fibrils and form amyloid deposits in the human brain, which is the hallmark of many neurodegenerative diseases. Promising strategies toward pathological amyloid proteins and deposition include investigating inhibitors that can disrupt amyloid aggregation or induce misfolding protein degradation. In this review, recent progress of peptide-based inhibitors, including amyloid sequence–derived inhibitors, designed peptides, and peptide mimics, is highlighted. Based on the increased understanding of peptide design and precise amyloid structures, these peptides exhibit advanced inhibitory activities against fibrous aggregation as well as enhanced druggability.     

蛋白质和多肽药物长效性研究进展

基于分子生物学和重组技术的发展,蛋白质和多肽已经成为一类重要的药物,但是其稳定性差,生物利用率低,半衰期短等问题也日益受到关注。本文重点介绍了一些新的给药途径和给药系统,例如鼻腔、颊等给药途径以及黏膜给药系统、透皮给药系统、缓控释技术等给药系统的进展。综述了对于蛋白质和多肽药物进行定点突变和化学修饰,以达到增加其长效性的一些新方法。     

Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B

Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B.     

Trifluoroacetylated tyrosine-rich D-tetrapeptides have potent antioxidant activity

The term “oxidative stress” indicates a set of chemical reactions unleashed by a disparate number of events inducing DNA damage, lipid peroxidation, protein modification and other effects, which are responsible of altering the physiological status of cells or tissues. Excessive Reactive Oxygen Species (ROS) levels may accelerate ageing of tissues or induce damage of biomolecules thus promoting cell death or proliferation in dependence of cell status and of targeted molecules. In this context, new antioxidants preventing such effects may have a relevant role as modulators of cell homeostasis and as therapeutic agents. Following an approach of peptide libraries synthesis and screening by an ORAC_FL assay, we have isolated potent anti-oxidant compounds with well-defined structures. Most effective peptides are N-terminally trifluoroacetylated (CF₃) and have the sequence tyr-tyr-his-pro or tyr-tyr-pro-his. Slight changes in the sequence or removal of the CF₃ group strongly reduced antioxidant ability, suggesting an active role of both the fluorine atoms and of peptide structure. We have determined the NMR solution structures of the active peptides and found a common structural motif that could underpin the radical scavenging activity. The peptides protect keratinocytes from exogenous oxidation, thereby from potential external damaging cues, suggesting their use as skin ageing protectant and as cell surviving agents.     

CutC is induced late during copper exposure and can modify intracellular copper content in Enterococcus faecalis

The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX_nHX_mHP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.