Giovanni N. Roviello

An overview on HMGB1 inhibitors as potential therapeutic agents in HMGB1-related pathologies

HMGB1 (High-Mobility Group Box-1) is a nuclear protein that acts as an architectural chromatin-binding factor involved in the maintenance of nucleosome structure and regulation of gene transcription. It can be released into the extracellular milieu from immune and non-immune cells in response to various stimuli. Extracellular HMGB1 contributes to the pathogenesis of numerous chronic inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, atherosclerosis, chronic kidney disease, systemic lupus erythematosus (SLE), as well as cancer pathogenesis. Interaction of released HMGB1 with the cell-surface receptor for advanced glycation end products (RAGE) is one of the main signaling pathways triggering these diseases. It has been also demonstrated that the inhibition of the HMGB1-RAGE interaction represents a promising approach for the modulation of the inflammatory and tumor-facilitating activity of HMGB1. In this review we describe various approaches recently proposed in the literature to inhibit HMGB1 and the related inflammatory processes, especially focusing on the block of RAGE-HMGB1 signaling. Several strategies are based on molecules which mainly interact with RAGE as competitive antagonists of HMGB1. As an alternative, encouraging results have been obtained with HMGB1-targeting, leading to the identification of compounds that directly bind to HMGB1, ranging from small natural or synthetic molecules, such as glycyrrhizin and gabexate mesilate, to HMGB1-specific antibodies, peptides, proteins as well as bent DNA-based duplexes. Future perspectives are discussed in the light of the overall body of knowledge acquired by a large number of research groups operating in different but related fields.

Nucleobase-containing peptides: an overview of their characteristic features and applications.

Reports on nucleobase-containing chiral peptides (both natural and artificial) and achiral pseudopeptides are reviewed. Their synthesis, structural features, DNA and RNA-binding ability, as well as some other interesting applications which make them promising diagnostic/therapeutic agents of great importance in many areas of biology and therapy are taken into critical consideration.

Synthesis, biological evaluation and supramolecular assembly of novel analogues of peptidyl nucleosides

This work concerns the synthesis, the supramolecular assembly and the evaluation of some biological properties, such as DNA and RNA-binding ability and human serum stability, of novel nucleopeptides. These compounds are of potential interest for the well-known properties that similar compounds, such as natural peptidyl nucleosides, possess in biology and medicine and also for the possibility to realize nucleopeptide-based supramolecular systems useful for drug and gene delivery applications. More particularly, all four nucleobase-containing peptides were synthesized by solid phase synthesis, purified by HPLC and characterized by NMR and ESI-MS. Subsequently, nucleopeptide self-assembly as well as DNA and RNA-binding ability were investigated by CD spectroscopy and further information on the formation of molecular networks, based on the peptidyl nucleoside analogues and nucleic acids, was obtained by Laser Light Scattering. Finally, nucleopeptide enzymatic stability was studied and a half life of about 2 hours was found in the presence of 50% fresh human serum.

RNA-Binding and Viral Reverse Transcriptase Inhibitory Activity of a Novel Cationic Diamino Acid-Based Peptide

A novel cationic peptide based on L-lysine and L-diaminobutyric acid was prepared for the first time by solid phase synthesis. After HPLC purification and ESI MS characterization, we studied by CD and IR spectroscopy the structural features of the novel basic peptide, which is able to form a β-turn-like structure. Furthermore, its interaction with DNA and RNA was investigated by CD and UV spectroscopy, which revealed a preferential RNA-binding ability of the sequential peptide, whereas its inhibitory activity toward HIV and Moloney murine leukemia virus (MMLV) reverse transcriptase action was evaluated by semiquantitative PCR. The cationic sequential peptide was able to inhibit the reverse transcriptase activity in both cases, even if our PCR data suggested a major activity in the case of HIV-RT, probably due to the stronger cationic peptide-protein interaction evidenced by UV spectroscopy.

Faox enzymes inhibited Maillard reaction development during storage both in protein glucose model system and low lactose UHT milk

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (β-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the β-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.

dabPna: Design, Synthesis, And Dna Binding Studies

In continuing our research efforts for developing new oligodeoxynucleotide (ODN)-like drugs and diagnostics, we designed diaminobutyric peptide nucleic acids (dabPNAs), nucleopeptides characterized by a diaminobutyric-based building block that is an isomer of the aminoethylglycyl PNA (aegPNA) unit and the acyclic modification of the aminoprolyl PNA (ampPNA) monomer. In this work we present the solid phase synthesis of a dabPNA oligomer and of two aegPNAs containing a single dabPNA unit. A study relative to their binding ability towards DNA is also reported even in comparison with the well known aegPNAs.

Solid phase synthesis and RNA-binding studies of a serum-resistant nucleo-epsilon-peptide.

In the present work we report the synthesis of a new Fmoc‐protected L‐lysine‐based nucleo‐amino acid suitable for the solid phase assembly and its oligomerisation to the corresponding nucleo‐ε‐peptide that we called ε‐lysPNA. The ability to bind complementary RNA and the stability in serum of this synthetic nucleo‐ε‐peptide were studied to explore its possible use in antisense or diagnostic applications. Our interest to the presented oligonucleotide analogue was also supported by the importance of ε‐peptides and other ε‐amino acid‐containing compounds in natural products with biological activity such as the poly‐ε‐lysines produced by Streptomyces albulus that possess a highly selective antimicrobial activity. Another aspect we intended to evaluate by this work is the possible prebiotic implication of these nucleopeptides, since ε‐peptides, and not α‐peptides, were mainly obtained among the other thermal prebiotic polypeptides in pyrocondensation of lysine, a diamino acid also detected in Mighei meteorite. Besides this intriguing question, all the remarkable properties emerged from the present investigation on ε‐lysPNAs encourage, without doubts, interest in the therapeutic and diagnostic potential of these bioinspired nucleopeptides. Copyright

Synthesis, spectroscopic studies and biological activity of a novel nucleopeptide with Moloney murine leukemia virus reverse transcriptase inhibitory activity.

In this work, we report the synthesis of an alternate nucleo-alpha,epsilon-peptide based on l-lysine moieties, an in vitro study of its biological activity, and spectroscopical binding studies between the novel nucleopeptide and Moloney murine leukemia virus reverse transcriptase as well as RNA. An alternate homothymine hexamer was synthesized by a straightforward solid phase route starting from commercial materials, purified by RP-HPLC and characterized by ESI-MS. The efficiency of the novel nucleo-alpha,epsilon-peptide in interfering with the reverse transcription of eukaryotic mRNA and the noteworthy enzymatic resistance demonstrated by specific assays are in favor of the employment of this nucleopeptide in novel biomedical strategies.

DNA-based strategies for blocking HMGB1 cytokine activity: design, synthesis and preliminary in vitro/in vivo assays of DNA and DNA-like duplexes

In this work we report the design and synthesis of kinked oligonucleotide duplexes as potential inhibitors of HMGB1, a cytokine which triggers a broad range of immunological effects. We found that the designed ligands can interact with HMGB1, as evidenced by circular dichroism spectroscopy, and are able to block some extracellular effects induced by the protein, such as cellular proliferation and migration, as we demonstrated by in vitro biological assays. After selecting the most stable and active kinked duplex, we synthesized the corresponding PNA/DNA chimeric duplex which resulted to be more resistant to enzymatic degradation, and showed a biological activity comparable to that of the natural duplex. Preliminary in vivo assays in a mouse inflammatory model, showed a significant decrease of the mortality after administration of the PNA/DNA kinked duplex to LPS-treated mice.

Synthesis of a l-lysine-based alternate alpha,epsilon-peptide: A novel linear polycation with nucleic acids-binding ability

A novel nucleic acid compaction device based on a positively-charged alpha,epsilon-poly-l-lysine was realized for the first time. The polycationic peptide was obtained by assembling Fmoc and Boc orthogonally protected l-lysine monomers by solid phase synthesis. The route to the novel polycation is very fast and convenient because it allows for the obtainment of the desired product in few synthetic steps exclusively employing Fmoc chemistry. The purification of the poly-amino acid was performed easily by RP-HPLC on C18 stationary phase while ESI-MS analysis confirmed the identity of the novel basic molecule. The structural characteristics of the novel water-soluble peptide, as well as the interaction with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have been investigated by circular dichroism (CD) and ultraviolet (UV) spectroscopy.