[Show abstract][Hide abstract] ABSTRACT: We here report the design, synthesis, and biological activity of a kinked hairpin-loop DNA acting at low nM concentrations as a strong inhibitor of HMGB1 (High-Mobility Group Box-1), a nuclear protein with cytokine activity in a number of inflammatory diseases. Lead compound optimization has been realized by inserting different oligo-ethylene glycol spacers at the 5′-end and loop positions of the natural hairpin DNA, in order to improve its enzymatic stability and structuring capability, as well as its overall pharmacokinetic properties. Thermal stability data as well as activity assays proved that the ODN which contained two hexa-ethylene glycol spacers, one at the 5′-end and the other in the loop, was the best candidate to inhibit HMGB1. Plasma stability assays and hydrodynamic volume measurements afforded further encouraging results in view of future in vivo evaluation of the optimized ligand.
[Show abstract][Hide abstract] ABSTRACT: Hepatoma-derived growth factor related protein 3 (HDGFRP3) belongs to the HDGF polypeptide family. Expression of HDGF is a prognostic factor for NSCLC, pancreatic and gastrointestinal tumors, and silencing of HDGF suppressed cell proliferation, migration and invasion of squamous lung cancer cells. Here we investigated the expression of HDGFRP3 in various tumor cell lines by quantitative PCR and western blot analysis. HDGFRP3 is highly expressed and secreted from neuroblastoma (NB) cell lines. The presence of high levels of HDGFRP3 was confirmed in two animal models of human NB. Since HDGFRP3 shares a conserved Pro-Trp-Trp-Pro motif (PWWP) domain with its family members, we assessed whether it could promote proliferation, migration and angiogenesis in analogy to HDGF. Recombinant HDGFRP3 inhibited migration of endothelial cells in a modified Boyden chamber experiment, while cell survival was not affected. Nevertheless, the protein promoted angiogenesis in vitro, as assessed through vascular sprouting and tubulogenesis assays. Starting from the structural insight of the PWWP domain, an oligonucleotide-based inhibitor was designed and tested to block the pro-angiogenic effect of HDGFRP3. A single stranded DNA inhibited the pro-angiogenic properties of HDGFRP3 in vitro. After modifications to improve its serum stability, the cytotoxicity of the designed oligonucleotide-based molecule (BN210) was tested by an MTT cell viability assay on various NB cell lines. BN210 slightly reduced NB cells proliferation. To determine if BN210 could be used both as single anti-angiogenic agent and in combination with chemotherapy in vivo, orthotopic NB tumor-bearing mice were randomly divided in different groups of treatment: 1 mg/kg of vincristine (VCR), once a week for 4 weeks; 7-21 mg/kg of BN210, either i.p. (5 days/week) or i.v (every 2 days). Other mice were treated with a combination of VCR and BN210. NB-bearing mice treated with BN210 alone showed a partial increase in life span, compared to control mice. However, mice treated with the combination of VCR plus BN210 had statistically significant increased life span, compared to both the control and the single treatment groups, with long term survivors obtained with the combined schedule when BN210 was injected i.v. Thus, the oligonucleotide-based inhibition of the pro-angiogenic activity of HDGFRP3 can be considered as a novel strategy for combined anti-tumor therapy in NB.
Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 07/2011
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In this work, we report thermodynamic, kinetic, and microrheological studies relative to the formation of PNA- and PNA/DNA-based noncovalent polymeric systems, useful tools for biotechnological and bioengineering applications. We realized two kinds of systems: a PNA-based system formed by a self-assembling PNA tridendron, and a PNA/DNA hybrid system formed by a PNA tridendron and a DNA linker. The formation of a three-dimensional polymeric network, by means of specific Watson-Crick base pairing, was investigated by a detailed UV and CD spectroscopic study. Preliminary microrheology experiments were performed on both systems to evaluate their viscoelastic properties which resulted in agreement with the formation of soluble hyperbranched polymers that could be useful for drug/gene delivery, as well as for encapsulating organic pollutants of different shapes and sizes in environmental applications.
[Show abstract][Hide abstract] ABSTRACT: In continuing our studies on new ODN-like molecules suitable for a wide variety of biomedical and bioengineering applications, here we report further studies relative to a chiral nucleopeptide with a diaminobutyric acid (DABA) backbone. In particular, in this work we describe the synthesis of the nucleoaminoacid monomer with the D stereochemistry, performed in analogy to our previous reports on the L-DABA derivative, and the oligomerization using both enantiomers to form an alternate D,L-nucleopeptide. This oligomer was studied for its ability to bind complementary DNA by CD and UV spectroscopies. Furthermore, the new nucleopeptide showed binding evidence with a free nucleobase probably forming a three-dimensional network based on hydrogen bonding. This kind of structures is of particular interest for the development of new nanomaterials with many desirable properties as well as of new ODN-analogues for biotechnological applications.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In this work we explore the ability of a chimeric LNA/DNA bent duplex, in which the kink is induced by 2 unpaired adenines in the middle of one strand, to bind HMGB1, a protein involved in many inflammatory processes. The LNA/DNA duplex was compared with the corresponding full DNA and PNA/DNA chimera duplexes from a thermodynamic and spectroscopic point of view.
[Show abstract][Hide abstract] ABSTRACT: In this work we report a kinetic and thermodynamic study relative to the formation of gel systems based on PNA and PNA/DNA dendrimers, useful for drug delivery or diagnostic applications. We realized two kinds of systems: a PNA-based monomolecular system formed by an autoassembling PNA tridendron (A) and a PNA/DNA bimolecular system based on a PNA tridendron with a mixed sequence and a DNA crosslinker (B). Both systems have the ability to form a three-dimensional network by means of specific W-C base pairing.
[Show abstract][Hide abstract] ABSTRACT: In the present work, we report the synthesis and the characterization of a new chiral nucleoaminoacid, in which a diaminobutyric moiety is connected to the DNA nucleobase by an amidic bond, and its oligomerization to give the corresponding nucleo-gamma-peptide. The ability of this synthetic polymer to bind complementary DNA was studied in order to explore its possible use in antigene/antisense or diagnostic applications. Our interest in the presented DNA analogue was also supported by the importance of gamma-aminoacid-containing compounds in natural products of biological activity and by the known stability of gamma-peptides to enzymatic degradation. Furthermore, our work could contribute to the study of the role of nucleopeptides as prebiotic material in a PNA world that could successively lead to the actual DNA/RNA/protein world, as recently assumed.
[Show abstract][Hide abstract] ABSTRACT: Targeting regulatory RNA regions to interfere with the biosynthesis of a protein is an intriguing alternative to targeting a protein itself. Regulatory regions are often unique in sequence and/or structure and, thus, ideally suited for specific recognition with a low risk of undesired side effects. Targeting regulatory RNA elements, however, is complicated by their complex three-dimensional structure, which poses kinetic and thermodynamic constraints to the recognition by a complementary oligonucleotide. Oligonucleotide mimics, which shift the thermodynamic equilibrium towards complex formation and yield stable complexes with a target RNA, can overcome this problem. Peptide nucleic acids (PNA) represent such a promising class of molecules. PNA are very stable, non-ionic compounds and they are not sensitive to enzymatic degradation. Yet, PNA form specific base pairs with a target sequence. We have designed, synthesised and characterised PNA able to enter infected cells and to bind specifically to a control region of the genomic RNA of coxsackievirus B3 (CVB3), which is an important human pathogen. The results obtained by studying the interaction of such PNA with their RNA target, the entrance into the cell and the viral inhibition are herein presented.