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The development of radioisotopes of inorganic elements (as 68Ga(III) and 188Re(I)) has gained much interest in recent years for diagnostic and therapeutic application in nuclear medicine1. Small peptides are employed for complexing the radiometal in a biologically stable environment. They have some advantages: easy of synthesis and modification, tumorpenetrating ability and good biocompatibility2. Aim of the present work has been the interaction between Ga3+ ion with a bishistidine oligopeptide, PHIS (Fig.1) in aqueous solutions, by the following methods: Potentiometry, Spectrofluorimetry, FT−IR and 1HNMR Spectroscopy as also by Circular Dichroism. All the measurements have been investigated at 25 °C in 0.1 M NaClO4 media. Information on coordination sites are obtained with model molecule such as histidine (His). By processing the experimental data, it has been possible to obtain the complexation constants. Spectroscopy data are in agreement with the potentiometric measurement. 1H-NMR measurements conducted in H2O as solvent at different metal/ligand ratio indicate that imidazolic rings are involved in coordination with metal ions investigated .

Organometallic tricarbonyl complexes of Rhenium(I) and Technetium(I), fac−[M(H2O)3(CO)3]+ (M = Re, Tc), especially the aqueous complexes, have recently assumed an important role for diagnostic and therapeutic applications. In this regard, fac−[Re(H2O)3(CO)3]+ cation is readily conjugated to biomolecules which contain coordinating N, P, O, S atoms [1], where one or more water molecules can be replaced by ligands to obtain very stable d6 low−spin complexes [2]. Recently study has showed interaction between fac − [Re(H2O)3(CO)3]+ and histidine/imidazole molecule. Aim of this work is the study of the interaction among fac − [Re(H2O)3(CO)3]+ and some aminoacids that have been investigated using different instrumental techniques. Potentiometric titrations conducted with glass electrode in the pH range 2−10 estabilish that a complexation effect is observed. Information on complexes formation can be obtained also by spectrophotometric UV−VIS and fluorimetric measurements in solutions with different metal/ligand ratio.

APEH catalyses the removal of N-terminal acetylated amino acids from proteins destined to be degraded and is now recognized as a new therapeutic target for several diseases. New APEH inhibitors having triazole-based structures have been recently reported. On this basis we have screened a set of click-generated cyclic peptides, previously investigated for peptide conformational stability studies, as possible novel enzyme inhibitors. We have found a clicked peptide, NHB3.3, that inhibits APEH activity and structure–activity studies highlighted that APEH inhibition is mediated by the spatial organization of the triazole ring and by its orientation and distance from the peptide scaffold, whose structural integrity, in turn, also plays a relevant role. In conclusion, our findings confirm that 1,2,3 triazoles are privileged pharmacophores for specific serine protease inhibitors and provide structural insights exploitable for modulating their inhibition activity.

he design of new peptides (QK) targeting two protein systems deeply involved in angiogenesis: integrins and vascular endothelial growth factor receptors is presented. Integrins are cell surface receptors that mediate adhesion of endothelial cells to the extracellular matrix and to the adjacent cells through the interaction with Arg-Gly-Asp (RGD) sequence found within many extracellular matrix protein (i.e. vitronectin). It was shown that integrin avb3 together with avb5 are overexpressed on tumor cells and endothelial cells of the tumor neovasculature, acting as angiogenic markers. It is of great therapeutic and diagnostic interest to develop new compds. able to discriminate between avb3 and avb5. NMR anal. revealed that QK adopts a helical conformation in water, whereas the peptide corresponding to the helix region of VEGF (VEGF15) is unstructured. The conformation of the designed peptide exactly corresponds to the N-terminal helix of VEGF when complexed with Flt-1. Binding studies showed that QK competes with VEGF for a binding site on EC membranes and activates both receptors similarly to VEGF. VEGF15 do not bind to the receptors indicating that the helical structure is necessary for the biol. activity. Cells proliferation expts. and the downstream activation of VEGF-dependent intracellular pathway (ERK1/2) confirmed the agonist behavior of the peptide. Results indicate that QK peptide is able to recapitulate many of the features in signal transduction that are reported for VEGF. The new peptide-based mols. reported may represent the lead compds. for the development of novel angiogenesis modulating drugs for application in diagnostic and targeted therapy.

The solid-phase synthesis of a hemoprotein mimetic, mimochrome III, is reported.

Apolipoprotein A-I (ApoA-I), the major protein component of HDL, plays a key role in reverse cholesterol transport, mainly by stimulating the efflux of cholesterol and activating the enzyme LCAT. LCAT converts cholesterol into cholesteryl esters and addresses them to HDL for transport into the circulation. The binding of haptoglobin (Hpt) to ApoA-I is associated with inhibition of LCAT activity[1]. On the basis of the above information, high levels of Hpt, as occurring during the acute phase of inflammation[2], were suggested to be a major cause of both poor cholesterol removal from peripheral cells and low level of HDL cholesterol in the circulation. Previously, we showed that an ApoA-I mimetic peptide (P2a), with amino acid sequence overlapping the stimulatory site for LCAT, was effective in displacing Hpt from ApoA-I, and able to rescue in vitro the stimulatory function of ApoA-I in the presence of high Hpt levels[3]. In this work we characterized the biological activity of peptide P2a in an experimental model of inflammation. In particular, we verified the ability of peptide P2a to rescue LCAT dependent cholesterol esterification in vivo in presence of high level of Hpt. References 1. Balestrieri, M., Cigliano, L., De Simone, M.L., Dale, B., Abrescia, P. Mol. Reprod. Dev. 59, 186 (2001) 2. Langlois MR & Delanghe JR (1996) Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 42,1589-1600. 3. Spagnuolo, M.S., Cigliano, L., D’Andrea, L.D., Pedone, C. and Abrescia, P. (2005) Assignment of the binding site for Haptoglobin on Apolipoprotein A-I. J. Biol. Chem. 280, 1193-1198.

The invention discloses the use of compds. which bind to the vascular endothelial growth factor (VEGF) receptors and modulate the angiogenic response. The compds., which mimic the VEGF amino acid region 17-25 involved in receptor recognition thereby inhibiting or stimulating the angiogenic process, can be used in the treatment of diseases characterized by excessive or defective angiogenesis VEGF-dependent, such as chronic ischemia, cancer, proliferative retinopathy and rheumatoid arthritis, states or conditions benefiting from the formation or regeneration of new vessels, as well as in the diagnosis of pathologies which present a overexpression of VEGF receptors or as biochem. tools to analyze the cellular pathways dependent on VEGF receptor activation.

The properties of mimochrome II, a hemoprotein model compound, were evaluated in relation to the peptide chain composition and length and its importance in controlling heme properties.

The conformational behavior in solution of a cyclic peptide with sequence cyclo(Pro'-Pro2-Dab3(cHexA)ili[N-'HCO}-Leu4

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.

A one-pot synthesis of homodimeric proteins is described. The synthetic strategy is based on a double expressed protein ligation reaction between thioester peptides and a new bis-cysteinyl linker. The protocol was also applied to the synthesis of heterodimers.

The development of receptor targeting radiolabeled ligands has gained much interest in recent years for diagnostic and therapeutic applications in nuclear medicine. Cholecystokinin (CCK) receptors have been shown to be overexpressed in a subset of neuroendocrine and other tumors. We are evaluating binding and biodistribution properties of a CCK8 peptide derivative labeled with (99m)Tc(I)-tricarbonyl. The CCK8 peptide was modified at its N-terminus by adding to its N-terminus two lysine-histidine modules (KH), where histidine is coupled to the side chain of the lysine ((KH)(2)-CCK8). (99m)Tc(I)-tricarbonyl was generated with the IsoLinktrade mark kit. A431 cells stably transfected with a cDNA encoding for the human CCK2 receptor were utilized to determine binding affinity, internalization, and retention of the labeled peptide, in comparison with wild-type A431 cells. A nude mouse tumor model was obtained by generating A431-CCK2R and A431-control tumors in opposite flanks of the animals. High specific activity labeling with (99m)Tc was achieved. In A431-CCK2R cells, specific saturable binding was observed as well as evident internalization of the radiolabeled peptide after binding. Biodistribution experiments showed rapid, specific localization of (KH)(2)-CCK8 on A431-CCK2R xenografts compared with control tumors, although absolute uptake values were not markedly higher compared with background activity. Clearance of unbound radioactivity was both urinary and hepatobiliary. In imaging experiments, while targeting to CCK2R positive tumors could be appreciated, there was poor contrast between target and nontarget areas. (KH)(2)-CCK8 shows adequate in vitro and in vivo properties for CCK2R targeting although improvement of biodistribution warrant further development.

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.