Improving oral bioavailability of cyclic peptides by N-methylation

The renaissance of peptides in pharmaceutical industry results from their importance in many biological functions. However, low metabolic stability and the lack of oral availability of most peptides is a certain limitation. Whereas metabolic instability may be often overcome by development of small cyclic peptides containing d-amino acids, the very low oral availability of most peptides is a serious limitation for some medicinal applications. The situation is complicated because a twofold optimization – biological activity and oral availability – is required to overcome this problem. Moreover, most simple “rules” for achieving oral availability are not general and are applicable only to limited cases. Many structural modifications for increasing biological activities and metabolic stabilities of cyclic peptides have been described, of which N-alkylation is probably the most common. This mini-review focuses on the effects of N-methylation of cyclic peptides in strategies to optimize bioavailabilities.     

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.     

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E--F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide–heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide–heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme–peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P₅₀ values. The P₅₀ values ranged from 3.8 mmHg for the heme–peptide B7 complex to 13.7 mmHg for the heme–peptide D13 complex (under the same conditions, P₅₀ values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

Development of a stable and efficient zein based nanopesticides through green and simple way for improving utilization efficiency

Nanopesticides represent one of the effective ways of reducing the use of pesticides and the environmental pollution. Natural zein as a potential carrier for nanoencapsulation of pesticides has the disadvantages of large amount of organic solvents and poor dispersibility. In this study, a water soluble of zein peptides was obtained by hydrolyzed under alkaline conditions with heated and stability of nanopesticides were prepared through crosslinked after self-assemble. As the results, the zein peptides spontaneously assembled into spherical particles in an aqueous solution, the nanopesticides (avermectin, AVM), with uniform size distribution and size can be adjusted by the glutaraldehyde, have a great stability, and redispersed in water after freeze-drying. Moreover, nanoencapsulation protects avermectin from UV light, with 24% of the encapsulated AVM remaining intact after exposure to UV for 60 min, compared to less than 6% for the bare AVM. In addition, the nanoformulation had similar insecticidal activity to commercial EC and the cross-linking of particles slowed down the release rate and the curves were consistent with Higuchi model different from the abnormal diffusion in the uncross linked state. In short, this study provides a simple method to obtain stabilizable nanopesticides based on natural polymer on a green way.     

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.     

Effects of chicken intestinal antimicrobial peptides on humoral immunity of chickens and antibody titres after vaccination with infectious bursal disease virus vaccine in chicken

Abstract

Sixty chickens were randomly divided into two groups (30 chickens in each group) to determine the effect of oral administration of chicken intestinal antimicrobial peptides (CIAMP) on the humoral immune response. Chickens of both groups were fed the same diet. In the treatment group chickens received drinking water supplemented with CIAMP (1 µg/ml) right after hatching. Samples of blood, bursa of Fabricus, spleen and intestine were taken at day 1, 4, 7, 10 and 17 of experiment. CIAMP supplementation enhanced the content of IgG and IgM in serum from day 4 – 10 and day 10 – 17, respectively, (p < 0.05), IgM-forming cells in bursa of Fabricus and spleen at the age of 7 days (p < 0.05) and IgG-forming cells in bursa of Fabricus at the age of 4 days (p < 0.05). In addition, CIAMP enhanced the IgA-forming cells in caecal tonsils diffuse area at day 4 (p < 0.05). Furthermore, CIAMP enhanced the antibody response to infectious bursal disease virus vaccine (IBDV) in chickens 21 days following IBDV vaccine administration (p < 0.05). These results suggested that CIAMP could modulate the humoral immune response of chickens and increased the antibody titres of infectious bursal disease virus vaccine.     

Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus

Aims:  Staphylococcal biofilm-associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system.
Methods and Results:  We tested the activity of the 5-kDa peptide fraction of the cytosol from coelomocytes (5-CC) against a group of Gram-positive, Gram-negative bacteria and fungi. We determined minimal inhibitory concentrations (MICs) ranging from 253·7 to 15·8 mg ml⁻¹. We observed an inhibitory activity and antibiofilm properties of 5-CC against staphylococcal biofilms of reference strains Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. The antimicrobial efficacy of 5-CC against the biofilms of clinical strain Staph. epidermidis 1457 was also tested using live/dead staining in combination with confocal laser scanning microscopy. At a sub-MIC concentration (31·7 mg ml⁻¹) of 5-CC the formation of young (6-h old) and mature (24-h old) staphylococcal biofilms was inhibited.
Conclusions:  The biological activity of 5-CC could be attributed to three peptides belonging to the sequence segment 9–41 of a beta-thymosin of P. lividus .
Significance and Impact of the Study:  The effector cells of P. lividus represent an interesting source of marine invertebrates-derived antimicrobial agents in the development of new strategies to treat staphylococcal biofilms.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.