Alessandra Romanelli

Transcription Factor Decoy Molecules Based on a Peptide Nucleic Acid (PNA)-DNA Chimera Mimicking Sp1 Binding Sites

Peptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDP-based decoy molecules are more resistant than DNA/DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure.

Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Peptides from Royal Jelly: studies on the antimicrobial activity of jelleins, jelleins analogs and synergy with temporins

Peptides isolated from natural fonts are the object of several studies aimed at finding new molecules possessing antibacterial activity. We focused our studies on peptides originally isolated from the Royal Jelly, the jelleins and on some analogs having a UV reporter at the N‐ or C‐terminus. We found that jelleins are mainly active against gram‐positive bacteria; interestingly, they act in synergy with peptides belonging to the family of temporins such as temporin A and temporin B against Staphylococcus aureus A170 and Listeria monocytogenes. Copyright

Cationic liposomes as delivery systems for double-stranded PNA–DNA chimeras exhibiting decoy activity against NF-κB transcription factors

Peptide nucleic acids (PNAs) have been recently proposed as useful molecules in pharmacogenetic therapy, especially due to the fact that they show a very high stability with respect to DNA and RNA. However, PNAs are not efficient decoy molecules, are characterized by negligible cell internalization and low solubility and are not suitable to be delivered by liposomes. With respect to the biological activity of PNA-based molecules, PDP deserve great consideration, due to the fact that they exhibit high levels of solubility, and are expected to be resistant to proteinases and exonucleases. In this manuscript we determined whether double-stranded molecules based on PNA-DNA chimeras containing NF-kappaB binding sites, exhibit decoy activity against NF-kappaB transcription factors. In addition, we determined whether they can be complexed by cationic liposomes. The results obtained demonstrated that hybrids based on PNA-DNA chimeras are powerful decoy molecules against NF-kappaB p52 transcription factor. In addition, we found that cationic liposomes can be proposed for in vitro delivery to target cells of these decoy molecules. The results presented in this paper are thus of practical importance, since the simplicity and the versatility of the cationic liposome technology have made cationic liposomes useful nonviral gene delivery systems for human gene therapy.

Structural Determinants of the Unusual Helix Stability of a De Novo Engineered Vascular Endothelial Growth Factor (VEGF) Mimicking Peptide

Understanding how an amino acid sequence folds into a well organized three-dimensional structure remains a challenge. The interest in protein folding comes from the possibility to predict the protein structure from genome-derived sequence, design proteins with new fold and understand protein misfolding. Peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. Many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of nonnatural amino acids or introducing constraints. In this paper, we report on the stability characterization, using CD, NMR and MD studies, of a designed, a-helical, 15-mer peptide (named QK), composed only of natural amino acids (sequence AcKLTWQELYQLKYKGI-NH2), which activates the VEGFdependent angiogenic response. The QK peptide shows an unusual thermal stability, whose structural determinants have been determined. These results could have implication in the field of protein folding and in the design of helical structured scaffolds for the realization of peptides for applications in chemical biology. As recently described, the NMR structure of QK in pure water presents a central helical sequence (residues 4–12), which corresponds to the VEGF N-terminal helix (residues 17–25), flanked by Nand C-capping regions. The helical conformation of QK represents an important prerequisite for its biological activity, since the isolated peptide, corresponding to the helix region of VEGF, does not assume a helical conformation and does not have significant biological activity. Interestingly, QK represents one of the very few examples of bioactive helical designed peptides, composed of only natural amino acids. To gain an insight into the molecular determinants of QK helical propensity, we examined the effect of the temperature on the QK structure through NMR and CD analyses. Primarily, the aggregation state of the peptide under conditions identical to those used in the NMR structure determination was confirmed by NMR DOSY experiments (see Supporting Information). The DOSY-derived diffusion coefficient value of 1.98@10 10 ms 1 is consistent with a QK monomer state. QK structure variations upon temperature increase were followed by TOCSY experiments. In the 298– 343 K range only small changes of the backbone chemical shifts were observed (Table 1 Supporting Information). The temperature dependences of Ha chemical shift deviations from the random coil values (DdHa) are reported in Figure 1a. Unusually, the chemical shift index (CSI) analysis indicates that at 343 K the peptide retains at least the 80% of the helix conformation at 298 K and the slight reduction occurs uniformly in 4–12 region (Figure 1a). The thermal behavior was also analyzed by CD spectroscopy which allowed [a] D. Diana, Prof. Dr. R. Fattorusso Dipartimento di Scienze Ambientali, Seconda UniversitC di Napoli via Vivaldi 43, 81100 Caserta (Italy) Fax: (+39)0823-274605 E-mail : roberto.fattorusso@unina2.it [b] B. Ziaco, Prof. Dr. C. Pedone, Dr. L. D. DIAndrea Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16 80134 Napoli (Italy) Fax: (+39)081-2534574 E-mail : ldandrea@unina.it [c] Dr. G. Colombo, Dr. G. Scarabelli Istituto di Chimica del Riconoscimento Molecolare, CNR via Bianco, 9, 20131 Milano (Italy) [d] Dr. A. Romanelli Dipartimento delle Scienze Biologiche UniversitC di Napoli “Federico II” via Mezzocannone 16, 80134 Napoli (Italy) Supporting information for this article is available on the WWW under http://www.chemistry.org or from the author: Peptide synthesis, circular dichroism, nmr spectroscopy and molecular dynamic simulations.

Design, structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

BACKGROUND Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. METHODS Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. RESULTS TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. CONCLUSION Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. GENERAL SIGNIFICANCE The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.

Synergistic antibacterial and anti-inflammatory activity of temporin A and modified temporin B in vivo.

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10–14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3–6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.

A new ferrocenemethyl-thymidine nucleoside: Synthesis, incorporation into oligonucleotides and optical spectroscopic studies on the resulting single strand, duplex and triplex structures

Abstract A new thymidine analogue, bearing a ferrocenemethyl residue at the N-3 position of the base, was synthesized in high yields via Mitsunobu reaction of ferrocenemethanol with sugar protected thymidine, converted into the corresponding 3′-phosphoramidite and incorporated into oligonucleotides. Duplex and triplex formation experiments, evaluated by UV and CD spectroscopy, showed a dramatic decrease of the affinity towards complementary single strands, while for triplexes, the introduction of a ferrocene residue in the third strand resulted in higher melting temperatures, associated with a reduced content of triplex structure.

Peptides Targeting Angiogenesis Related Growth Factor Receptors

Growth factors (GFs) are extracellular signaling polypeptides regulating cell proliferation, differentiation and survival. They exert a wide spectrum of biological activities selectively binding to and activating specific membrane receptors which then transfer the message to cell interior inducing specific biochemical pathways. GFs are especially involved in the regulation of angiogenesis, a physiological process underlining several pathologies. Molecules able to modulate angiogenesis, interfering with the molecular recognition between a GF and its receptor, have a big pharmacologic interest. Either GF and the receptor are potential drug target. Peptides are useful molecules to develop new lead compounds disrupting protein-protein interface for pharmacological applications. In this review we describe peptides targeting the receptors of the pro-angiogenic growth factors FGF, PDGF and VEGF. The biological function and the structure of each growth factor/receptor system are discussed, as well as the molecular interaction between peptides and the receptors. Finally, we highlight the pharmacological and diagnostic applications of these peptides in angiogenesis related diseases.

Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3-1 cells: recruitment of NF-kappaB to the IL-8 gene promoter and transcription of the IL-8 gene.

One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3–1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.