Barbara Biondi

Therapeutic prospect of Syk inhibitors

Background: The non-receptor spleen tyrosine kinase (Syk; EC 2.7.10.2) is involved in signal transduction in a variety of cell types. In particular, it is a key mediator of immune receptors signaling in host inflammatory cells (B cells, mast cells, macrophages and neutrophils), important for both allergic and antibody-mediated autoimmune diseases. Deregulated Syk kinase activity also allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are able to induce leukemia. Consequently, the development of Syk kinase inhibitors could conceivably treat these disorders and so they have became a major focus in the pharmaceutical and biotech industry. Objective: In this review, we analyze the structure and role of Syk kinase, the use of small molecules, interacting with ATP-binding site, as inhibitors of kinase activity and finally the potential of using inhibitors of Syk kinase expression to attenuate pathological conditions. Conclusion: Syk kinase inhibition is suggested as a powerful tool for the therapy of different pathologies.

Trichogin GA IV: an antibacterial and protease-resistant peptide

The antibacterial and hemolytic activities of the amphiphilic helical, membrane‐active, lipopeptaibol trichogin GA IV and its [Leu11‐OMe] analogue were compared to those of the partially helical or non‐helical 8‐meric or 4‐meric, C‐terminal short sequences, respectively. The study on trichogin GA IV was extended to several methicillin‐resistant Staphylococcus aureus strains. Using a large set of enzymes, we also evaluated the resistance to proteolysis of all of the four peptides. Copyright

Synthesis, preferred conformation, protease stability, and membrane activity of heptaibin, a medium‐length peptaibiotic

The medium‐length peptaibiotics are characterized by a primary structure of 14–16 amino acid residues. Despite the interesting antibiotic and antifungal properties exhibited by these membrane‐active peptides, their exact mechanism of action is still unknown. Here, we present our results on heptaibin, a 14‐amino acid peptaibiotic found to exhibit antimicrobial activity against Staphylococcus aureus. We carried out the very challenging synthesis of heptaibin on solid phase and a detailed conformational analysis in solution. The peptaibiotic is folded in a mixed 310‐/α‐helix conformation which exhibits a remarkable amphiphilic character. We also find that it is highly stable toward degradation by proteolytic enzymes and nonhemolytic. Finally, fluorescence leakage experiments using small unilamellar vesicles of three different compositions revealed that heptaibin, although uncharged, is a selective compound for permeabilization of model membranes mimicking the overall negatively charged surface of Gram‐positive bacteria. This latter finding is in agreement with the originally published antimicrobial activity data. Copyright

Substitution of the arginine/leucine residues in apidaecin Ib with peptoid residues: effect on antimicrobial activity, cellular uptake, and proteolytic degradation.

Several aspects of the mechanism of action of Pro-rich antimicrobial peptides, together with their low toxicity in mammalian cells, make them good candidates for the development of new antibiotic agents. We investigated the effect induced in the insect antimicrobial peptide apidaecin Ib by the replacement of a single arginine/leucine residue with a N-substituted glycine. The resulting peptoid-peptide hybrids are more resistant to proteolysis and devoid of any significant cytotoxic activity, but moving the [NArg]residue from the N- to the C-terminal end of the molecule progressively reduces the antibacterial activity. Cell uptake experiments in E. coli cells suggest that the loss of antibacterial activity of [NArg(17)]apidaecin is a consequence of its inability to translocate into bacterial cells. Conversely, apidaecin and its peptoid-peptide hybrids are able to cross the plasma membrane in eukaryotic cells and to diffuse in the cytosol, although their translocating ability is far less effective than that of other known cell permeant peptides.

Cell-Penetrating Peptides: A Comparative Study on Lipid Affinity and Cargo Delivery Properties.

A growing number of natural and/or synthetic peptides with cell membrane penetrating capability have been identified and described in the past years. These molecules have been considered promising tools for delivering bioactive compounds into various cell types. Although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. In this work we try to connect the capability to interact with model lipid membrane and structural and chemical characteristics of CPPs in order to obtain a biophysical classification that predicts the behavior of CPP-cargo molecules in cell systems. Indeed, the binding with cell membrane is one of the primary step in the interaction of CPPs with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how CPPs may translocate a membrane without destroying it and how this interactions come into play in shuttling CPPs via different routes with different efficiency. We analyzed by CD and fluorescence spectroscopies the binding properties of six different CPPs (kFGF, Nle54-Antp and Tat derived peptides, and oligoarginine peptides containing 6, 8 or 10 residues) in absence or presence of the same cargo peptide (the 392-401pTyr396 fragment of HS1 protein). The phospholipid binding properties were correlated to the conformational and chemical characteristics of peptides, as well as to the cell penetrating properties of the CPP-cargo conjugates. Results show that even if certain physico-chemical properties (conformation, positive charge) govern CPP capability to interact with the model membrane, these cannot fully explain cell-permeability properties.

Opioid peptides: synthesis and biological properties of [(Nγ-glucosyl,Nγ-methoxy)-α,γ-diamino-(S)-butanoyl]4-deltorphin-1-neoglycopeptide and related analogues

The [D-Ala2]deltorphin 1 sequence in which the aspartic acid residue is replaced by the N gamma-OCH3-alpha, gamma-diamino (S) butanoyl residue was synthesized using the Fmoc-chemistry-based solid phase procedure. The resulting deltorphin analogue was chemoselectively glucosylated by reaction with unprotected D-glucose (Glc). The Asn4-, (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-galactopyranosyl)-Asn4- and the (2-acetamido-2-deoxy-D-galactopyranosyl)-Asn4-deltorphin I were also prepared for comparison. The affinity of the new compounds for the delta-opioid receptor was expressed by the inhibition constant (Ki) of the binding of the delta-receptor selective ligand [3H]naltrindole (NTI) to rat brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the mu-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the delta-opioid receptor agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect drastically the Ki and IC50 values for delta-sites, suggesting that an electrostatic interaction does not play an essential role in the binding to delta-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to delta-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the gamma-nitrogen of the Dab residue in comparison with the Asn amide side chain.

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by l-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to l-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.

Synthesis, Preferred Conformation, and Membrane Activity of Medium‐Length Peptaibiotics: Tylopeptin B

The solid‐phase synthesis and full chemical characterization of the medium‐length (14‐amino acid residues) peptaibol with antibiotic properties of tylopeptin B, originally extracted from the fruiting body of the mushroom Tylopilus neofelleus, are described. These data are accompanied by the results on the solution‐phase synthesis via the segment condensation approach of a selected, side‐chain protected, analog. A solution conformational analysis, performed by the combined use of FTIR absorption, circular dichroism, and 2D‐NMR (the latter technique coupled to molecular dynamics calculations), favors the conclusion that the 3D‐structure of tylopeptin B is largely helical with a preference for the α‐ or the 310‐helix type depending upon the nature of the solvent. Helix topology and (partial) amphiphilic character are responsible for the observed membrane‐modifying properties of this peptaibiotic.

Ru(III)-based compounds with sulfur donor ligands: synthesis, characterization, electrochemical behaviour and anticancer activity

In recent years, Ru(iii) complexes have emerged as a new class of effective anticancer agents against tumors that proved to be resistant to all other chemotherapeutic drugs currently in clinical use. To extend our previous studies on metal complexes containing sulfur-donor ligands, we report here on the synthesis and characterization, by means of several spectroscopic and analytical techniques, some [Ru(RSDT)(3)] and [Ru(2)(RSDT)(5)]Cl complexes with dithiocarbamato ligands derived from methyl/ethyl/tert-butyl esters of sarcosine. Their electrochemical behaviour was also studied by cyclic voltammetry. All the complexes were tested for their cytotoxicity on a panel of human tumor cell lines showing highly significant antitumor activity. The chemical and biological properties of the newly synthesized complexes, were compared with those of [Ru(DMDT)(3)] and [Ru(2)(DMDT)(5)]Cl species (DMDT = N,N-dimethyldithiocarbamate) whose chemical (not biological) characterization has been already reported in literature.

Effects of Trehalose on Thermodynamic Properties of Alpha-synuclein Revealed through Synchrotron Radiation Circular Dichroism

Many neurodegenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases, are characterized by protein misfolding and aggregation. The capability of trehalose to interfere with protein misfolding and aggregation has been recently evaluated by several research groups. In the present work, we studied, by means of synchrotron radiation circular dichroism (SRCD) spectroscopy, the dose-effect of trehalose on α-synuclein conformation and/or stability to probe the capability of this osmolyte to interfere with α-synuclein’s aggregation. Our study indicated that a low trehalose concentration stabilized α-synuclein folding much better than at high concentration by blocking in vitro α-synuclein’s polymerisation. These results suggested that trehalose could be associated with other drugs leading to a new approach for treating Parkinson’s and other brain-related diseases.