ß-Hairpins as peptidomimetics of human phosphoprotein-binding domains.

Phosphoprotein-binding domains interact with cognate phosphorylated targets ruling several biological processes. The impairment of such interactions is often associated with disease development, namely cancer. The breast cancer susceptibility gene 1 (BRCA1) C-terminal (BRCT) domain is involved in the control of complex signaling networks of the DNA damage response. The capture and identification of BRCT-binding proteins and peptides may be used for the development of new diagnostic tools for diseases with abnormal phosphorylation profiles. Here we show that designed cyclic ß-hairpin structures can be used as peptidomimetics of the BRCT domain, with high selectivity in binding to a target phosphorylated peptide. The amino acid residues and spatial constraints involved in the interaction between a phosphorylated peptide (GK14-P) and the BRCT domain were identified and crafted onto a 14-mer ß-hairpin template in silico. Several cyclic peptides models were designed and their binding towards the target peptide and other phosphorylated peptides evaluated through virtual screening. Selected cyclic peptides were then synthesized, purified and characterized. The high affinity and selectivity of the lead cyclic peptide towards the target phosphopeptide was confirmed, and the possibility to capture it using affinity chromatography demonstrated. This work paves the way for the development of cyclic ß-hairpin peptidomimetics as a novel class of affinity reagents for the highly selective identification and capture of target molecules.

Engineering Short Preorganized Peptide Sequences for Metal Ion Coordination: Copper(II) a Case Study.

Peptides are multidentate chiral ligands capable of coordinating different metal ions. Nowadays, they can be obtained with high yield and purity, thanks to the advances on peptide/protein chemistry as well as in equipment (peptide synthesizers). Based on the identity and length of their amino acid sequences, peptides can present different degrees of flexibility and folding. Although short peptide sequences (<20 amino acids) usually lack structure in solution, different levels of structural preorganization can be induced by introducing conformational constraints, such as ß-turn/loop template sequences and backbone cyclization. For all these reasons, and the fact that one is not restricted to use proteinogenic amino acids, small peptidic scaffolds constitute a simple and versatile platform for the development of inorganic systems with tailor-made properties and functions. Here we outline a general approach to the design of short preorganized peptide sequences (10-16 amino acids) for metal ion coordination. Based on our experience, we present a general scheme for the design, synthesis, and characterization of these peptidic scaffolds and provide protocols for the study of their metal ion coordination properties.

Treatment of post-burns bacterial infections by Fenton reagent, particularly the ubiquitous multiple drug resistant Pseudomonas spp.

Post-burn microbial infections are a major problem in burns, and in cases of third degree burns, the survival of patients can depend not only upon the severity but also upon the extent and the type of infections. If proper measures are not employed, patients may suffer from opportunistic bacterial attacks, which can vary from simple infection, such as those easily treatable by antibiotics, to more complicated types, which may have natural or acquired resistance to drugs. Infection by multiple drug resistant (MDR) bacteria can create further complexity to the treatment. It is proposed that a combination of diluted hydrogen peroxide (H(2)O(2)) and ferrous sulphate (FeSO(4)), which generates hydroxyl radicals (*OH) via Fenton reaction, can effectively be used for the treatment of post-burns bacterial infections. It should be particularly useful for the ubiquitous opportunistic pathogen, Pseudomonas aeruginosa, known to be notoriously resistant to various antibiotics. This reactive oxygen species (ROS)-induced inactivation of the bacterial skin infections may be of particular importance in Third World countries where the incidence of burns and post-burns infections by MDR bacteria (due to the indiscriminate use of antibiotics, lack of stringent safety regulations and proper hygiene) may be more prevalent and where cocktails of antibiotics may be less affordable. Also, since the putative lack of development of bacterial resistance to *OH is not known, it provides an added advantage to the treatment. Finally, although this work addresses the control of bacterial infections in burns cases, it is envisaged that this ROS-induced chemotherapy may also be useful in combating other kinds of skin infections particularly those resisting antibiotic treatment.

Oligonucleotide conjugates of Eu(III) tetraazamacrocycles with pendent alcohol and amide groups promote sequence-specific RNA cleavage.

Eu(III) complexes of two neutral bifunctional tetraaaza macrocyclic ligands ¿1-[1-carboxamido-3-(4-nitrophenyl)propyl]-4,7,10-tris(2-hydroxyethyl)-1 ,4,7,10-tetraazacyclododecane and 2-(4-nitrobenzyl)-1,4,7,10-tetrakis(2-hydroxyethyl)-1,4,7,10-tetraaza cyclododecane¿ are prepared. Eu(III) complexes of the isothiocyanate derivatives of these macrocycles are treated with oligonucleotides containing 2'-O-propyl-amine linkers to form conjugates. Hydrolytic cleavage of an oligoribonucleotide is promoted by Eu(III) macrocyclic oligonucleotide conjugates containing complementary (antisense) sequences. Cleavage is not observed in the presence of Eu(III) conjugates containing scrambled sequences nor by free complex. Despite the fact that one of the free macrocyclic complexes is more reactive than the other, the extent of cleavage observed is similar for conjugates containing either Eu(III) macrocyclic complex.