Andrea Calderan

Carnosine and carnosine-related antioxidants: a review.

First isolated and characterized in 1900 by Gulewitsch, carnosine (beta-alanyl-L-hystidine) is a dipeptide commonly present in mammalian tissue, and in particular in skeletal muscle cells; it is responsible for a variety of activities related to the detoxification of the body from free radical species and the by-products of membrane lipids peroxidation, but recent studies have shown that this small molecule also has membrane-protecting activity, proton buffering capacity, formation of complexes with transition metals, and regulation of macrophage function. It has been proposed that carnosine could act as a natural scavenger of dangerous reactive aldehydes from the degradative oxidative pathway of endogenous molecules such as sugars, polyunsaturated fatty acids (PUFAs) and proteins. In particular, it has been recently demonstrated that carnosine is a potent and selective scavenger of alpha,beta-unsaturated aldehydes, typical by-products of membrane lipids peroxidation and considered second messengers of the oxidative stress, and inhibits aldehyde-induced protein-protein and DNA-protein cross-linking in neurodegenerative disorders such as Alzheimers disease, in cardiovascular ischemic damage, in inflammatory diseases. The research for new and more potent scavengers for HNE and other alpha,beta-unsaturated aldehydes has produced a consistent variety of carnosine analogs, and the present review will resume, through the scientific literature and the international patents, the most recent developments in this field.

Neuroprotective actions of a histidine analogue in models of ischemic stroke.

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4‐hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase‐3 protein. These findings suggest a therapeutic potential for HNE‐scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.

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.

Antamanide, a Derivative of Amanita phalloides, Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore

Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors.

Glutathione Transferase (GST)-Activated Prodrugs

Glutathione transferase (formerly GST) catalyzes the inactivation of various electrophile-producing anticancer agents via conjugation to the tripeptide glutathione. Moreover, several data link the overexpression of some GSTs, in particular GSTP1-1, to both natural and acquired resistance to various structurally unrelated anticancer drugs. Tumor overexpression of these proteins has provided a rationale for the search of GST inhibitors and GST activated cytotoxic prodrugs. In the present review we discuss the current structural and pharmacological knowledge of GST-activated cytotoxic compounds.

Synthetic peptides reproducing the EGF-receptor segment homologous to the pp60v-src phosphoacceptor site. Phosphorylation by tyrosine protein kinases

The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.

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.

Recognition of lysine‐rich peptide ligands by murine cortactin SH3 domain: CD, ITC, and NMR studies

Cortactin is a ubiquitous actin‐binding protein that regulates various aspects of cell dynamics and is implicated in the pathogenesis of human neoplasia. The sequence of cortactin contains a number of signaling motifs and an SH3 domain at the C‐terminus, which mediates the interaction of the protein with several partners, including Shank2. A recombinant protein, comprising the murine cortactin SH3 domain fused to GST (GST‐SH3m‐cort), was prepared and used to assess the domain‐binding affinity of potential peptide‐ligands reproducing the proline‐rich regions of human HPK1 and Shank2 proteins. The key residues involved in the SH3m‐cort domain recognition were identified by three different approaches: non‐immobilized ligand interaction assay by circular dichroism, isothermal titration calorimetry, and nuclear magnetic resonance. Our results show that the classical PxxPxK class II binding motif is not sufficient to mediate the interaction with GST‐SH3m‐cort, an event that depends on the presence of additional basic residues located at either the N‐ or the C‐terminus of the PxxPxK motif. Especially effective in promoting the peptide binding is a Lys residue at the ‐5 position, a determinant present in both P2 (HPK1 394‐403) and S1 (Shank2 1168‐1189) peptides. GST‐SH3m‐cort exhibits the highest affinity toward peptide S1, which contains additional Lys residues at the ‐3, ‐5, and ‐7 positions, indicating that the optimal consensus motif may be KPPxPxKxKxK. These results are supported by the in silico models of SH3m‐cort complexed with P2 or S1, which highlight the domain residues that interact with the recognition determinants of the peptide‐ligand and cooperate in binding stabilization.

Phosphorylation of small peptides by spleen TPK‐IIA, a tyrosine protein kinase stimulated by polylysine and by high ionic strength

The specificity of TPK‐IIA, a spleen tyrosine protein kinase whose activity is dramatically enhanced by either polylysine or very high ionic strength, has been inspected with the aid of a variety of very short peptide substrates. Unlike free tyrosine, which is not phosphorylated at all, and the dipeptides YA and AY, whose phosphorylation is hardly detectable, the tripeptides AYA, YYY and YYA, tetrapeptide AYAA and pentapeptide AAYAA are readily phosphorylated, the V max of AYA being 7‐ and 15‐fold lower vs those of AYAA and AAYAA, respectively. In comparison to AYA, the tripeptides YAA, EYA, GYA and VYV are poorer substrates, while EYH is unaffected by TPK‐IIA. The rate of phosphorylation of the pentapeptide EYAA is negligible as compared to AYAA; the hexapeptide EEYAA, however, is a substrate quite comparable to AAYAA, exhibiting a somewhat lower V max but also a 4‐fold lower K m (3.9 vs 18.6 mM). Taken together, the data would indicate that although TPK‐IIA does not exhibit any absolute requirement for a definite consensus sequence it is nevertheless endowed with peptide substrate specificity, mainly resulting from negative determinants that can be variably overcome by additional features of the sequence.

Peptide-receptor ligands and multivalent approach.

The overexpression of peptide receptors in human tumours makes peptide-ligands attractive agents for the development of specific diagnostic imaging and/or therapy of cancers. Solid-phase peptide synthesis, modern phage display technology and combinatorial peptide chemistry have profoundly affected the pool of available targeting peptides for efficient and specific delivery of imaging or therapeutic label molecules. Additionally, the availability of a wide range of bifunctional chelating agents for the radiolabelling of bioactive peptides with radionuclides has produced a wide variety of useful radiopharmaceutical molecules. This review article examines the principal receptors-binding peptides and their overexpression on tumour cells. We discuss the advantage and the challenges in developing multivalent peptide-based ligands summarising their design strategies and applications.