Alice Sosic

Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase

We have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized.

Development of a Multiplex Sandwich Aptamer Microarray for the Detection of VEGF165 and Thrombin

In this work we have developed a multiplex microarray system capable of detecting VEGF165 and thrombin. We recently described a Sandwich Aptamer Microarray (SAM) for thrombin detection feasible for use in multiplex microarrays; here we describe a new aptasensor for VEGF165 detection employing Vap7 and VEa5, two DNA aptamers recognizing different sites of the protein. The aptamers were modified to be adapted to the solid phase platform of SAM and their capability to simultaneously recognize VEGF165 by forming a ternary complex was analyzed in solution. Having so defined the best tandem arrangement of modified aptamers, we set up the aptasensor for VEGF165, and finally analyzed the multiplex system with the two aptasensors for the simultaneous detection of VEGF165 and thrombin. The results indicate that each sandwich is specific, even when the two proteins are mixed. The system performance is consistent with the behavior evidenced by the biochemical analysis, which proves to be valuable to drive the evaluation and refinement of aptamers prior to or along the development of a detection platform. Since thrombin upregulates VEGF expression, the simultaneous recognition of these two proteins could be useful in the analysis of biomarkers in pathologies characterized by neo-angiogenesis.

Design, synthesis and biological evaluation of TAR and cTAR binders as HIV-1 nucleocapsid inhibitors

We designed, synthesized and tested novel 2,6-disubstituted-anthraquinones able to bind dynamic secondary structures of nucleic acids, such as TAR RNA and its reverse transcript cTAR, leading to inhibition of the chaperone activities of the nucleocapsid NCp7, a highly conserved viral protein implied in crucial steps of HIV-1 replication.

Rational design, synthesis, and DNA binding properties of novel sequence-selective peptidyl congeners of ametantrone.

Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti‐neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10‐anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well‐defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC‐rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4‐bis‐[Gly‐(L‐Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases.

Development and Optimization of a Thrombin Sandwich Aptamer Microarray

A sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method.

Synthesis and in Vitro Screening of New Series of 2,6-Dipeptidyl-anthraquinones: Influence of Side Chain Length on HIV-1 Nucleocapsid Inhibitors.

2,6-Dipeptidyl-anthraquinones are a promising class of nucleic acid-binding compounds that act as NC inhibitors in vitro. We designed, synthesized, and tested new series of 2,6-disubstituted-anthraquinones, which are able to bind viral nucleic acid substrates of NC. We demonstrate here that these novel derivatives interact preferentially with noncanonical structures of TAR and cTAR, stabilize their dynamics, and interfere with NC chaperone activity.

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as alkylating agents.

Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3‐chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis‐3‐chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide‐catalyzed double cyclization of unsaturated bis‐N‐chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen‐bridged bis‐3‐chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues.

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates.

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.

Indenocinnoline derivatives as G-quadruplex binders, topoisomerase IIα inhibitors and antiproliferative agents

DNA intercalating agents are a consolidated therapeutic option in the treatment of tumor diseases. Starting from previous findings in the antiproliferative efficacy of a series of indeno[1,2-c]cinnoline-11-one derivatives, we performed a suitable decoration of this scaffold by means of a simple and straightforward chemistry, aiming to a) enlarge the planar core to a pentacyclic benzo[h]indeno[1,2-c]cinnoline-13-one and b) introduce a basic head tethered through a simple polymethylene chain. In fluorescence melting and fluorescence intercalator displacement assays, these new compounds displayed fair to very good intercalating properties on different nucleic acid strands, with preference for G-quadruplex sequences. Inhibition of human topoisomerase IIα and antiproliferative assays on HeLa and MCF7 tumor cell lines outlined a multitarget antiproliferative profile for tetracyclic 6 and pentacyclic derivative 20, both bearing a N,N-dimethylamine as the protonatable moiety. Particularly, compound 6 displayed a very potent inhibition of tumor cell proliferation, while 20 returned the highest thermal stabilization in melting experiments. In summary, these results outlined a potential of such highly planar scaffolds for nucleic acid binding and antiproliferative effects.