Peptide-based Molecules Research Articles

N-Terminal-prolonged vinyl ester-based peptides as selective proteasome β1 subunit inhibitors

The synthesis and biological properties of vinyl ester peptide-based molecules bearing linear N-terminal amino acids are reported. Compounds were tested in vitro for their capacity to inhibit the chymotryptic-, tryptic-like, and post-acidic activities of the proteasome. Some analogues showed selective inhibition of post-acidic (PGPH) activity, which is attributed to the β1 subunit. Interestingly, active compounds demonstrated higher inhibitory activity toward ‘standard’ proteasomes than toward immunoproteasomes. The inhibitory potency was found to be related to the amino acidic sequence and to the length of the N-terminal residues. The new inhibitors demonstrated resistance to plasmatic proteases and a good capacity to permeate the cell membrane.

Novel Genetic and Peptide-Based Strategies Targeting the Bcl-2 Family, an Update

By controlling mitochondrial membrane permeability, the Bcl-2 family of proteins plays a crucial role in the regulation of cell death. Members of this family are functionally categorized as either pro- or anti-apoptotic based on their ability to promote or inhibit mitochondrial-dependent cell death signals. The critical role of Bcl-2 family proteins in human diseases has made them the focus of intense research by the pharmaceutical industry and academia. Anti-apoptotic Bcl-2 homologues have been widely explored as potential therapeutic targets for anticancer therapy through the development of nucleic-acid and peptide-based inhibitory molecules. Recently, strategies for the development of peptides or polypeptides derived from specific Bcl-2 family members have also been devised. This paper provides a brief review of the most recent patents that have been published relating to this area.

Peptide-based cationic molecules for the production of positive charged liposomes and micelles

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8–30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4 ± molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10–17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.

C-terminal constrained phenylalanine as a pharmacophoric unit in peptide-based proteasome inhibitors

Here we report the synthesis and biological properties of peptide-based molecules bearing constrained analogues of phenylalanine at the C-terminal. Compounds were tested as proteasome subunits' inhibitors. Dehydro-peptides showed good inhibition, in particular against trypsin-like (T-L) proteasome activity while some C-terminal Tic-derivatives inhibit only caspase-like activity in enzymatic β1 subunits with a certain degree of efficacy. The best analogues of the series demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.

Synthesis and in Vitro Hydroxyapatite Binding of Peptides Conjugated to Calcium-Binding Moieties

To confer bone-binding properties to proteins and other biological agents that lack specific targeting capacity, model peptide-based molecules were synthesized containing poly(aspartic acid), poly(glutamic acid), or a bisphosphonate (pamidronate). These motifs have well-documented affinities to hydroxyapatite, a property desirable for the targeting of molecules to bone for drug delivery and tissue engineering applications. Model peptides of increasing molecular mass (5-33 amino acids) were directly conjugated to eight aspartic acids (Asp8), eight glutamic acids (Glu8), or pamidronate, purified by high-performance liquid chromatography, and characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The modified peptides were incubated with hydroxyapatite in phosphate-buffered saline at physiological conditions over 24 h. This study revealed a significant amount (>90%) of conjugated peptides adsorbed to the hydroxyapatite as compared to unmodified peptides (<5%). It was found that while there were significant differences between the different hydroxyapatite-binding and control groups for all time points, the size of the peptide had no statistical effect on peptide-hydroxyapatite binding. These results demonstrate that bisphosphonate and oligopeptide conjugates hold great promise for the development of new bioactive molecules for bone-specific applications.

Limited tendency of α‐helical residues to form disulfide bridges: a structural explanation

Disulfide bridges have an enormous impact on the structure of a large number of proteins and polypeptides. Understanding the structural basis that regulates their formation may be important for the design of novel peptide-based molecules with a specific fold and stability. Here we report a statistical analysis of the relationships between secondary structure and disulfide bond formation, carried out using a large database of protein structures. Our analyses confirm the observation sporadically reported in previous investigations that cysteine residues located in alpha-helices display a limited tendency to form disulfide bridges. The very low occurrence of the disulfide bond in all alpha-chains compared to all beta-chains indicates that this property is also evident when proteins with different topologies are investigated. Taking advantage of the large database that endorsed the analysis on relatively rare motifs, we demonstrate that cysteine residues embedded in 3(10) helices present a good tendency to form disulfide bonds. This result is somewhat surprising since 3(10) helices are commonly assimilated into alpha-helices. A plausible structural explanation for the observed data has been derived combining analyses of disulfide bond sequence separation and of the length of the different secondary structure elements.

Domain Subclass Conversion Improved Activity of Anti-Mpl Agonist Antibodies in the Form of Whole IgG.

Since the molecular cloning of thrombopoietin (TPO), two types of recombinant human TPO, full-length recombinant human TPO (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF), have been tested in a variety of clinical trials. The agents showed clinical efficacy in some cases. However, neutralizing antibodies against endogenous TPO were generated in some healthy volunteers who received PEG-rhMGDF, resulting in thrombocytopenia. Recently, several TPO mimetics are being developed, such as peptide-based molecules, nonpeptidyl small compounds and agonist antibodies. Here we report generation of novel fully human anti-human Mpl agonist antibodies. Two lead monoclonal antibodies MA01 and MA02 were cloned from human antibody-producing mice (KM mice™) immunized with human Mpl-expressing cells. Both MA01 and MA02 were of immunoglobulin gamma 1 (IgG1). These IgG1 agonists promoted proliferation of UT7/TPO cells in vitro, but were >100-fold less effective than TPO. We, therefore, genetically modified MA01 and MA02 in order to enhance the activity and to minimize the effector function. First, constant heavy (CH) region of MA01 was converted to IgG4 (MA01G4) to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This conversion affected neither binding affinity nor agonistic activity of MA01. Next, hinge region of MA01G4 was changed to human IgG3 hinge (MA01G4344). MA01G4344 showed no change in binding affinity, but showed 10-fold higher activity than original MA01. In the case of MA02, the CH region was converted from G1 to G3344 to generate MA02G3344, in which CH1-hinge region was of IgG3 and CH2-CH3 region was of IgG4. The conversion drastically improved agonistic activity. Although MA02 (G1) activated Jak2 and Erk1/2 but not STAT5 and Akt, MA02G3344 activated all of them. These data suggest that conversion of CH1-hinge region to IgG3 type is essential for agonistic activity of anti-Mpl antibodies. The effective concentration 50 (EC50) values of MA01G4344 and MA02G3344 in UT7/TPO assay were in the range of 0.01–0.1nM. These antibodies stimulated human cord blood-derived CD34+ cells to give rise to CFU-MK colonies without any other supportive cytokines. A single injection of MA01G4344 increased platelet count of human Mpl transgenic mice for over one month. These domain subclass-converted antibodies are expected to provide a new medical option for treatment of chronic thrombocytopenia without losing good properties of natural antibody such as long serum half-life and low immunogenicity. [Display omitted]

Intracellular Cargo Delivery by an Octaarginine Transporter Adapted to Target Prostate Cancer Cells through Cell Surface Protease Activation

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.

Novel Angiogenic Compounds for Targeted Drug Delivery

ABSTRACTInduction of angiogenesis is necessary for the success of engineered implantable tissues in order to meet oxygen and nutrient requirements of cells during tissue repair. Insufficient vascularization in bone graft reconstruction may impede healing and initiate hypoxic cell death at the interior of the implant. As a result, endogenous growth factors have been studied to enhance angiogenesis during wound repair. However, these peptide-based molecules are highly sensitive to processing that occurs during scaffold biomaterial fabrication and treatment for tissue engineering purposes. We report here the development of new small molecule regulators of angiogenesis that may circumvent the impediments associated with protein-based growth factor delivery. In this study, we report the design and evaluation of SC–3–143 as a regulator of endothelial function. We show that the compound significantly increases the formation of microvascular networks in vitro, and selectively enhances endothelial survivability by reducing endothelial cell death under serum deprived culture conditions.

The development of a catalytic synthesis of münchnones: a simple four-component coupling approach to alpha-amino acid derivatives.

A new palladium-catalyzed route to prepare 1,3-oxazolium-5-oxides (i.e., Münchnones) directly from imine, carbon monoxide, and acid chloride building blocks has been developed. This provides a straightforward catalytic synthesis of Münchnones and is amenable to generating a diverse range of products by simple modification of the imine or acid chloride starting materials. Münchnones are vital synthetic intermediates to a variety of heterocyclic and peptide-based molecules. As such, this methodology has been utilized to design a new catalytic synthesis of alpha-amino acid derivatives via a one-pot coupling of imines, carbon monoxide, and acid chloride followed by alcohol. The latter represents the first reported catalytic synthesis of alpha-amino acids directly from imine and carbon monoxide building blocks.

The future for antibiotics: Bacterial membrane disintegrators

Bacterial resistance to conventional antibiotics is an ever increasing problem in the treatment of infectious disease. The basic problem is that conventional antibiotics, which generally operate by inhibiting some bacterial enzyme(s), can be rendered ineffective by overuse, natural selection and induced bacterial mutations. A novel class of antibiotics that functions by disrupting the superstructure of bacterial membranes rather than by inhibiting any specific enzyme has presented itself as the future for antibiotics. Most of these bactericidal agents are peptides or peptide-based molecules that have been either isolated from natural sources or synthetically designed. Here we review recent research into the development of peptides as bacterial membrane-disintegrating agents.

Sugar amino acids and their uses in designing bioactive molecules.

In search of new molecular entities for discovering new drugs and materials, organic chemists are looking for innovative approaches that try to imitate nature in assembling quickly large number of distinct and diverse molecular structures from 'nature-like' and yet unnatural designer building blocks using combinatorial approach. The main objective in developing such libraries is to mimic the diversities displayed in structures and properties of natural products. The unnatural building blocks used in these assemblies are carefully designed to manifest the structural diversities of the monomeric units used by nature like amino acids, carbohydrates and nucleosides to build its arsenal. Compounds made of such unnatural building blocks are also expected to be more stable toward proteolytic cleavage in physiological systems than their natural counterparts. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to nature's molecular arsenal. Recent advances in the area of combinatorial chemistry give an unprecedented technological support for rapid compilations of sugar amino acid-based libraries exploiting the diversities of carbohydrate molecules and well-developed solid-phase peptide synthesis methods. This review describes the development of sugar amino acids as a novel class of peptidomimetic building blocks and their applications in creating large number of structurally diverse peptide-based molecules many of which display interesting three-dimensional structures as well as useful biological properties.

Factors limiting the oral bioavailability of N-acetylglucosaminyl- N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion

The bioavailability (BA) of radio-labelled N-acetylglucosaminyl- N-acetylmuramyl dipeptide (GMDP) was low when administered by oral gavage as an aqueous solution to conscious male Sprague–Dawley rats (8.3±4.4% (mean±S.D., n=3)). To assess the likely factors contributing to the poor BA of GMDP, the stability of GMDP in the lumen of the gastrointestinal (GI) tract was examined in vitro, using ex vivo GI contents. GMDP was degraded by the contents of the small intestine, caecum and large intestine but was more stable in stomach contents. The permeability coefficient (p app) of GMDP in isolated sections of rabbit ileum was 1.67×10 −6 cm/s in the mucosal to serosal direction and was not significantly different in the serosal to mucosal direction, indicating that GMDP is poorly permeable and passively transported across the intestinal wall. First pass metabolism was considered to be unlikely to be the primary limitation to the oral bioavailability of GMDP and therefore, that the oral bioavailability of GMDP was likely limited by instability in the lumen of the gastrointestinal tract and low intestinal permeability. A water-in-oil (w/o) microemulsion formulation subsequently developed to address these problems was trialed in a preliminary bioavailability study in rats and enhanced the bioavailability of GMDP ten-fold when administered intraduodenally, indicating that w/o microemulsions may represent a viable mechanism for enhancing the bioavailability of poorly GI-stable and poorly permeable peptide-based molecules.

Miniaturized proteins: the backbone cyclic proteinomimetic approach

The field of proteinomimetics utilizes peptide-based molecules to mimic native protein functions. We describe a novel general method for mimicking proteins by small cyclic peptides for the purpose of drug design, and demonstrate its applicability on bovine pancreatic trypsin inhibitor (BPTI). These unique cyclic peptides, which both embody discontinuous residues of proteins in their bio-active conformation and ensure an induced fit, may overcome some of the pharmacological drawbacks attributed to proteins and peptides. This method, which we call the backbone cyclic (BC) proteinomimetic approach, combines backbone cyclization of peptides with a suitable selection method, cycloscan. Following this procedure, we have prepared a bicyclic nonapeptide, which mimics the binding region of BPTI. The X-ray crystal structure of the complex trypsin:mimetic, as well as kinetic studies, show that the BPTI mimetic binds to the specificity pocket of trypsin in a similar manner to BPTI. Inhibition measurements of various constructs revealed that backbone cyclization imposed the conformation crucial to binding.

Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria.

A plethora of novel gene-encoded antimicrobial peptides from animals, plants and bacteria has been described during the last decade. Many of the bacterial peptides possess modified building blocks such as thioethers and thiazoles or unsaturated and stereoinverted amino acids, which are unique among ribosomally made peptides. Genetic and biochemical studies of many of these peptides, mostly the so-called lantibiotics, have revealed the degree to which cells are capable of transforming peptides by posttranslational modification. The biosynthesis follows a general scheme: Precursor peptides are first modified and then proteolytically activated; the latter may occur prior to, concomitantly with or after export from the cell. The genes for the biosynthetic machinery are organized in clusters and include information for the antibiotic prepeptide, the modification enzymes and accessory functions such as dedicated proteases and ABC transporters as well as immunity factors and regulatory proteins. These fundamental aspects are discussed along with the biotechnological potential of the peptides and of the biosynthesis enzymes, which could be used for construction of novel, peptide-based biomedical effector molecules.

Flow cytometry and data analysis: Basic concepts and statistics

Merging concepts from synthetic biology and computational biology may yield antibiotics that are less likely to elicit resistance than existing drugs and that yet can fight drug-resistant infections. Indeed, computer-guided strategies coupled with massively parallel high-throughput experimental methods represent a new paradigm for antibiotic discovery. Infections caused by multidrug-resistant microorganisms are increasingly deadly. In the current post-antibiotic era, many of these infections cannot be treated with our existing antimicrobial arsenal. Furthermore, we may have already exhausted the category of large molecules produced in nature having antimicrobial activity: the antibiotic scaffolds we have discovered so far may represent the majority of those that exist. The rise in drug-resistant bacteria and lack of new antibiotic classes clearly call for out-of-the-box strategies. Recent advances in computational synthetic biology have enabled the development of antimicrobials. New molecular descriptors and genetic and pattern recognition algorithms are powerful tools that bring us a step closer to developing efficient antibiotics. We review several computational tools for drug design and a number of recently generated antibiotic candidates, with an emphasis on peptide-based molecules. Design strategies can generate a diversity of synthetic antimicrobial peptides, which may help to mitigate the spread of resistance and combat multidrug-resistant microorganisms.

Discovery of a peptide-based renin inhibitor with oral bioavailability and efficacy.

Peptidic renin inhibitors have been poorly absorbed across the intestine or rapidly eliminated by the liver and have been reported to have oral bioavailabilities of less than 2%. A peptide-based renin inhibitor, A-72517 (molecular mass of 706 daltons), was devised that has oral bioavailabilities of 8, 24, 32, and 53% in the monkey, rat, ferret, and dog, respectively. Dose-related reductions in blood pressure, plasma renin activity, and plasma angiotensin II in parallel with increased plasma drug concentrations were observed after oral administration of A-72517 to conscious, salt-depleted dogs. Thus, peptide-based molecules of sizable molecular mass can be absorbed intact into the systemic circulation of animals. These findings support the potential of peptide-based drugs for oral administration.