Antonio Randazzo

Topological Characterization of Nucleic Acid G‐Quadruplexes by UV Absorption and Circular Dichroism

The emergence of nucleic acid four-stranded architectures, denominated G-quadruplexes as a prolific area of research, has led to an interest in the development of inexpensive methods for the rapid assessment of their structural characterization in solution. Research in this area is motivated by their potential impact in regulation of biological mechanisms and technological applications. For many applications, light absorption techniques, such as circular dichroism (CD) and UV, have been sufficient to discriminate the quadruplex fold from other architectures. CD is also useful to discriminate a single quadruplex topology from all other 25 generic folding topologies. Here we demonstrate the use of these techniques for characterizing three different types of G-quadruplex topologies classified through the sequence of glycosidic bond angles (GBA) adopted by guanosines of the G-quadruplex stem.

High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human α-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K+ ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of α-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin–TBA complex formed in the presence of Na+ or K+ and a circular dichroism study of the structural stability of the aptamer both free and complexed with α-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na+ and K+ on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein–aptamer interface. The present data, in combination with those previously obtained on the complex between α-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

Thrombin–aptamer recognition: a revealed ambiguity

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

Tandem Application of Virtual Screening and NMR Experiments in the Discovery of Brand New DNA Quadruplex Groove Binders

In the past decade, DNA G-quadruplexes have come into the limelight thanks to their biological implications and to their potential druggability in anticancer therapy. In particular, it has been found that small molecules that stabilize G-quadruplex structures are effective inhibitors of telomerase which plays a critical role in tumorigenesis. So far, the quadruplex groove recognition, which is expected to give a higher degree of selectivity over the other DNA structures, has been demonstrated for very few compounds. Thus with the aim of detecting new and structurally diverse groove binders, a structure-based virtual screening campaign has been performed using the X-ray structure of the [d(TGGGGT)](4) quadruplex. Remarkable results were achieved, and six brand new different molecular entities have been found to interact with the groove through NMR experiments. The reported results will certainly stimulate further studies aimed at the design and optimization of new quadruplex-specific groove binders to be applied as anticancer agents and for other diseases.

A new modified thrombin binding aptamer containing a 5′–5′ inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(3′GGT5′-5′TGGTGTGGTTGG3′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3′GGT5′-5′TGGTGTGGTTGG3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(3′GGT5′-5′TGGTGTGGTTGG3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.

Targeting DNA quadruplexes with distamycin A and its derivatives: An ITC and NMR study

The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and 1H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)]4 and d[AG3(T2AG3)3] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K+ or Na+ at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.

Axinellins A and B: New Proline‐Containing Antiproliferative Cyclopeptides from the Vanuatu Sponge Axinella carteri

Two new bioactive cyclopeptides, named axinellins A (1) and B (2) have been isolated from the marine sponge Axinella carteri. Their structure elucidation was based on two-dimensional (2D) NMR (500 MHz) as well as HRFABMS and ESMS/MS data. All amino acid residues derived from axinellins A and B were found to possess L configuration at Cα by HPLC analysis of their FDAA derivatives (Marfeys method). The amino acid sequence of 1 and 2 was established on the basis of tandem mass spectrometry data (ESMS/MS) and on 1H-1H through-space connectivities observed in NOESY and ROESY spectra. Axinellins A (1) and B (2) exhibited moderate in vitro antitumor activity against human broncopulmonary non-small-cell-lung-carcinoma lines (NSCLC-N6) with IC50 values of 3.0 and 7.3 μg/ml, respectively.

Selective Binding of Distamycin A Derivative to G-Quadruplex Structure [d(TGGGGT)](4).

Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are found in many regions of human genome and in the telomeres of all eukaryotic organisms. Since small molecules that target G-quadruplexes have been found to be effective telomerase inhibitors, the identification of new specific ligands for G-quadruplexes is emerging as a promising approach to develop new anticancer drugs. Distamycin A is known to bind to AT-rich sequences of duplex DNA, but it has recently been shown to interact also with G-quadruplexes. Here, isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound 1) and the [d(TGGGGT)]4 quadruplex. Additionally, to compare the binding behaviour of netropsin and compound 1 to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)]4 has been performed. Experiments show that netropsin and compound 1 are able to bind to [d(TGGGGT)]4 with good affinity and comparable thermodynamic profiles. In both cases the interactions are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity.

Isolation of callipeltins A–C and of two new open-chain derivatives of callipeltin A from the marine sponge Latrunculia sp. A revision of the stereostructure of callipeltins

Two new callipeltin-related acyclic peptides (2 and 3) have been isolated, together with callipeltins A–C from the marine sponge Latrunculia sp. collected at the Vanuatu Islands. The gross structures of new compounds were elucidated by spectroscopic data. The application of the Marfeys analysis on the new derivatives and on callipeltin A (1), allowed us to revise the configuration of two amino acid units in callipeltin A.

Structural revision of halipeptins: synthesis of the thiazoline unit and isolation of halipeptin C

Abstract The structural revision of the anti-inflammatory marine metabolites halipeptin A ( 1 ) and B ( 2 ) along with the isolation of the new related product halipeptin C ( 3 ) are reported. In particular, the heterocyclic portion of the molecule, incorrectly assigned as an oxazetidine ring, has now been characterised as a thiazoline unit by comparison of the spectral data of the natural products ( 1–3 ) with an appropriate synthetic model ( 10 ). GIAO calculated 13C NMR chemical shifts for oxazetidine and thiazoline model compounds provide additional support to the revised structure.