Radiolabeled peptides and their expanding role in clinical imaging and targeted cancer therapy.

There is an expanding body of evidence showing that synthetic peptides in combination with radioactive isotopes can be utilized for medical purposes. This area is of particular interest in oncology where applications in diagnosis and therapy are at different stages of development. We review the contributions in this area by the group originally founded by Carlo Pedone in Naples many years ago. We highlight the work of this group in the context of other developments in this area, focusing on three biologically relevant receptor systems: somatostatin, gastrin-releasing peptide, and cholecystokinin-2/gastrin receptors. We focus on key milestones, state of the art, and challenges in this area of research as well as the current and future outlook for expanding clinical applications.

Microwave Heating Promotes the S-Alkylation of Aziridine Catalyzed by Molecular Sieves: A Post-Synthetic Approach to Lanthionine-Containing Peptides.

Aziridine derivatives involved in nucleophilic ring-opening reactions have attracted great interest, since they allow the preparation of biologically active molecules. A chemoselective and mild procedure to convert a peptide cysteine residue into lanthionine via S-alkylation on aziridine substrates is presented in this paper. The procedure relies on a post-synthetic protocol promoted by molecular sieves to prepare lanthionine-containing peptides and is assisted by microwave irradiation. In addition, it represents a valuable alternative to the stepwise approach, in which the lanthionine precursor is incorporated into peptides as a building block.

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study.

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions.

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target.

The binding process of A9 peptide toward HER2-DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2-DIVMP, multiple ligand-based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.

Curcumin-Loaded Nanoparticles Based on Amphiphilic Hyaluronan-Conjugate Explored as Targeting Delivery System for Neurodegenerative Disorders.

Identification of molecules able to promote neuroprotective mechanisms can represent a promising therapeutic approach to neurodegenerative disorders including Huntington's disease. Curcumin is an antioxidant and neuroprotective agent, even though its efficacy is limited by its poor absorption, rapid metabolism, systemic elimination, and limited blood-brain barrier (BBB) permeability. Herein, we report on novel biodegradable curcumin-containing nanoparticles to favor the compound delivery and potentially enhance its brain bioavailability. The prepared hyaluronan-based materials able to self-assemble in stable spherical nanoparticles, consist of natural fatty acids chemically conjugated to the natural polysaccharide. The aim of this study is to provide a possible effective delivery system for curcumin with the expectation that, after having released the drug at the specific site, the biopolymer can degrade to nontoxic fragments before renal excretion, since all the starting materials are provided by natural resource. Our findings demonstrate that curcumin-encapsulated nanoparticles enter the cells and reduce their susceptibility to apoptosis in an in vitro model of Huntington's disease.

Lanthionine Peptides by S-Alkylation with Substituted Cyclic Sulfamidates Promoted by Activated Molecular Sieves: Effects of the Sulfamidate Structure on the Yield.

A green and efficient method for preparing lanthionine peptides by a highly chemoselective and stereochemically controlled procedure is presented. It involves an S-alkylation reaction, promoted by activated molecular sieves, on chiral cyclic sulfamidates, both N-protected and unprotected. Of note, the reaction yield was high also for cyclic sulfamidates bearing a free amine group, while other strategies failed to achieve a ring-opening nucleophilic reaction with N-unprotected substrates. To prove the feasibility of the procedure, the synthesis of a thioether ring B mimetic of the natural lantibiotic haloduracin ß was performed.

Design and analysis of EphA2-SAM peptide ligands: A multi-disciplinary screening approach.

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.

Chemoselective Glycosylation of Peptides through S-Alkylation Reaction.

An efficient and rapid procedure for synthesizing S-linked glycopeptides is reported. The approach uses activated molecular sieves as a base to promote the selective S-alkylation of readily prepared cysteine-containing peptides, upon reaction of appropriate glycosyl halides. Considering the very mild conditions employed, the chemoselective linkage of the electrophilic sugar with a peptide sulfhydryl group occurred in satisfactory yield, allowing the incorporation of mono and disaccharide moieties. The sugar-peptide conjugates obtained from α-d-glycosyl derivatives adopt a ß-S-configuration, indicating the high stereoselectivity of the substitution reaction.

The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase.

Microwave heating in peptide side chain modification via cysteine alkylation.

Microwave irradiation has been successfully applied to a selective synthetic procedure for introducing molecular substituents on peptides, providing a noticeable reduction of the reaction time and also an increased crude peptide purity for some compounds.

Air oxidation method employed for the disulfide bond formation of natural and synthetic peptides.

Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.

Endothelial dysfunction: its clinical value and methods of assessment.

Endothelial dysfunction (ED) is a systemic disorder characterized by reduced production of nitric oxide. This pathologic condition, which impairs vascular homeostasis, leads to the loss of protective properties of endothelial cells and is related to the pathogenesis of cardiovascular diseases. ED may affect every vascular bed, accounting for several clinical implications, particularly when the coronary bed is affected. Although the reliability of ED as a cardiovascular disease surrogate is still debated, many methods for its assessment have been proposed. In this review, we underline the clinical value of ED in the cardiovascular field and summarize the principal methods currently available for its assessment.

Lipidated peptides via post-synthetic thioalkylation promoted by molecular sieves.

A thioalkylation procedure, which uses molecular sieves to promote the reaction, was exploited to provide peptides with useful functional groups (lipidic moieties), naturally occurring on proteins as post-translational modifications. The procedure was further implemented to synthesize tailor-made lipidated peptides, interesting tools to investigate biological processes involving their Ras parent proteins. Moreover, the one-pot preparation of multi-alkylated peptides confirms the versatility and flexibility of the employed methodology.

Cysteine co-oxidation process driven by native peptide folding: an example on HER2 receptor model system.

Synthetic models of receptors that have relevant biological roles are valuable tools for studying receptors itself and the corresponding ligands. Their properties can be validated at first by their capacity to fold in solution under native-like conditions and to assume conformations structurally and functionally equivalent to those in the native receptor. In this context, a new strategy to prepare the two-fragments synthetic receptor model HER2-DIVMP, an independent structural and functional motif of HER2, has been developed and the folding properties have been investigated. The strategy is based on a one-step cysteine co-oxidation procedure in slightly alkaline aqueous buffers, whereby the two separate peptide chains are allowed to self-assemble in solution. Under these conditions, the two chains spontaneously form the expected heterodimer with the correct pattern of disulfide bridges. To gain insights on the folding mechanism, we investigated the folding of two scrambled variants of the constituent peptide chains.

Microwave-Assisted, Solid-State Procedure to Covalently Conjugate Hyaluronic Acid to Curcumin: Validation of a Green Synthetic Protocol.

A microwave-assisted esterification reaction to prepare hyaluronan-curcumin derivatives by employing a solvent-free process was developed. In particular, a solid-state strategy to react two molecules characterized by totally different solubility profiles was developed. Hyaluronic acid, a highly hydrosoluble polysaccharide, was reacted with hydrophobic and even water-unstable curcumin. Microwave (MW) irradiation was employed to activate the reaction between the two solid compounds through the direct interaction with them and to preserve the integrity of the sensitive curcumin species. This new protocol can be considered efficient, fast, and also eco-friendly, avoiding the employment of toxic organic bases and solvents. A cytotoxicity test suggested that the developed hyaluronan-curcumin conjugate (HA-CUR) could be considered a candidate for its implementation as a new material. In addition, preliminary studies revealed promising anti-inflammatory activity and open future perspectives of further investigation.

Bioactive Glass-Ceramic Scaffolds Coated with Hyaluronic Acid-Fatty Acid Conjugates: A Feasibility Study.

Promoting bone healing is a key challenge in our society that can be tackled by developing new implantable biomaterials provided with regenerative properties. In this work, the coating of three-dimensional porous glass-derived scaffolds with hyaluronic acid (HA)-fatty acids was investigated for the first time. The starting scaffolds, based on bioactive silicate glass, were produced by foam replication followed by sintering; then, HA-palmitate and HA-oleate conjugate coatings were deposited on the scaffold struts through a dipping procedure. FT-IR analysis confirmed the successful deposition of the coatings on the surface and struts of the scaffolds, the foam-like architecture of which was maintained as assessed by SEM investigations. The in vitro bioactivity of the HA-fatty-acid-coated scaffolds was studied by immersion tests in simulated body fluid and the subsequent evaluation of hydroxyapatite formation. The deposition of the polymeric coating did not inhibit the apatite-forming ability of scaffolds, as revealed by the formation of nanostructured hydroxyapatite agglomerates 48 h from immersion. These promising results motivate further investigation of these novel bioactive systems, which are expected to combine the bone-bonding properties of the glass with the wound-healing promotion carried out by the polymeric conjugates.

Probing membrane topology of the antimicrobial peptide distinctin by solid-state NMR spectroscopy in zwitterionic and charged lipid bilayers.

Distinctin is a 47-residue antimicrobial peptide, which interacts with negatively charged membranes and is active against Gram-positive and Gram-negative bacteria. Its primary sequence comprises two linear chains of 22 (chain 1) and 25 (chain 2) residues, linked by a disulfide bridge between Cys19 of chain 1 and Cys23 of chain 2. Unlike other antimicrobial peptides, distinctin in the absence of the lipid membrane has a well-defined three-dimensional structure, which protects it from protease degradation. Here, we used static solid-state NMR spectroscopy in mechanically aligned lipid bilayers (charged or zwitterionic) to study the topology of distinctin in lipid bilayers. We found that this heterodimeric peptide adopts an ordered conformation absorbed on the surface of the membrane, with the long helix (chain 2), approximately parallel to the lipid bilayer (~5° from the membrane plane) and the short helix (chain 1) forming a ~24° angle with respect to the bilayer plane. Since the peptide does not disrupt the macroscopic alignment of charged or zwitterionic lipid bilayers at lipid-to-protein molar ratio of 50:1, it is possible that higher peptide concentrations might be needed for pore formation, or alternatively, distinctin elicits its cell disruption action by another mechanism.

Solvent-free synthesis of modified pectin compounds promoted by microwave irradiation.

Microwave-assisted solvent-free modification of pectin was successfully accomplished, consisting in the esterification of several fatty acids by pectin alcoholic functions. The reaction was performed by simply mixing the reagents with a catalytic amount of the inorganic base (potassium carbonate) and irradiating the obtained mixture with microwaves for a short time (3-6 min). The replacement of the traditional heating with a microwave source allowed the development of a new synthetic protocol which provided increased yield of the final products, since it eliminates the small amount of degraded polysaccharide produced during traditional oil bath heating. The desired esters were fully characterized by FT-IR spectroscopy and thermogravimetric analysis.

[Adipokines and coronary artery disease]. / Le adipochine e la malattia aterosclerotica coronarica.

Adipose tissue, besides being an important energetic storage, is also a source of cytokines and hormones which act in a paracrine, autocrine and especially endocrine manner, influencing the cardiometabolic axis. Adipokines are a group of mediators with pleiotropic function, that are involved in many physiological processes, so that a disregulation in their secretion can lead to multiple pathological conditions. In this review our aim was to clarify the role of adipokines in the pathogenesis of atherosclerosis, especially in coronary artery disease, and based on current scientific evidence, to analyze the therapeutic and behavioral strategies that are so far available.

Fluorescence Studies: A9 Peptide, Functionalized with a Fluorogenic Probe, Interacts with Its Receptor Model HER2-DIVMP.

A recently developed synthetic protocol allowed for the functionalization of the active peptide A9 with a fluorogenic probe, which is useful for studying biomolecular interactions. Essentially, a nucleophilic attack on a halo-substituted benzofurazan is selectively performed by a cysteine sulfhydryl group. The process is assisted by the basic catalysis of activated zeolites (4 Å molecular sieves) and promoted by microwave irradiation. Fluorescence studies revealed that a donor-acceptor pair within the peptide sequence was introduced, thus allowing a deeper investigation on the interaction process between the peptide ligand and its receptor fragment. The obtained results allowed us to come full circle for all the currently understood structural determinants that were found to be involved in the binding process.