Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects.

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA15) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer. Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3'- or 5'-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5'-end and an HPP group at the 3'-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (ΔTm + 14° C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.

Design, Synthesis, and Characterization of an Amphiphilic Lipoic Acid-Based Ru(III) Complex as a Versatile Tool for the Functionalization of Different Nanosystems.

Ru-based chemotherapy is emerging as an effective alternative to the well-established Pt-based one, typically associated with high toxicity. In this context, our recent efforts were devoted to the preparation of nucleolipid-based Ru(III) complexes able to form, under physiological conditions, supramolecular aggregates which can efficiently prevent metal deactivation and convey Ru(III) inside the cells where it exerts its activity. Within an interdisciplinary program for the development of multifunctional nanoparticles for theranostic applications, we here report the design, synthesis, and characterization of a novel functionalized Ru(III) salt, carrying a lipoic acid moiety in the nucleolipid-based scaffold to allow its incorporation onto metal-based nanoparticles.

Exploring the Binding of Natural Compounds to Cancer-Related G-Quadruplex Structures: From 9,10-Dihydrophenanthrenes to Their Dimeric and Glucoside Derivatives.

In-depth studies on the interaction of natural compounds with cancer-related G-quadruplex structures have been undertaken only recently, despite their high potential as anticancer agents, especially due to their well-known and various bioactivities. In this frame, aiming at expanding the repertoire of natural compounds able to selectively recognize G-quadruplexes, and particularly focusing on phenanthrenoids, a mini-library including dimeric (1-3) and glucoside (4-5) analogues of 9,10-dihydrophenanthrenes, a related tetrahydropyrene glucoside (6) along with 9,10-dihydrophenanthrene 7 were investigated here by several biophysical techniques and molecular docking. Compounds 3 and 6 emerged as the most selective G-quadruplex ligands within the investigated series. These compounds proved to mainly target the grooves/flanking residues of the hybrid telomeric and parallel oncogenic G-quadruplex models exploiting hydrophobic, hydrogen bond and π-π interactions, without perturbing the main folds of the G-quadruplex structures. Notably, a binding preference was found for both ligands towards the hybrid telomeric G-quadruplex. Moreover, compounds 3 and 6 proved to be active on different human cancer cells in the low micromolar range. Overall, these compounds emerged as useful ligands able to target G-quadruplex structures, which are of interest as promising starting scaffolds for the design of analogues endowed with high and selective anticancer activity.

Selective Targeting of Cancer-Related G-Quadruplex Structures by the Natural Compound Dicentrine.

Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands. A preliminary screening with the G-quadruplex on the Controlled Pore Glass assay proved that, among the investigated compounds, Dicentrine is the most effective ligand of telomeric and oncogenic G-quadruplexes, also showing good G-quadruplex vs. duplex selectivity. In-depth studies in solution demonstrated the ability of Dicentrine to thermally stabilize telomeric and oncogenic G-quadruplexes without affecting the control duplex. Interestingly, it showed higher affinity for the investigated G-quadruplex structures over the control duplex (Kb~106 vs. 105 M-1), with some preference for the telomeric over the oncogenic G-quadruplex model. Molecular dynamics simulations indicated that Dicentrine preferentially binds the G-quadruplex groove or the outer G-tetrad for the telomeric and oncogenic G-quadruplexes, respectively. Finally, biological assays proved that Dicentrine is highly effective in promoting potent and selective anticancer activity by inducing cell cycle arrest through apoptosis, preferentially targeting G-quadruplex structures localized at telomeres. Taken together, these data validate Dicentrine as a putative anticancer candidate drug selectively targeting cancer-related G-quadruplex structures.

Insights into the Small Molecule Targeting of Biologically Relevant G-Quadruplexes: An Overview of NMR and Crystal Structures.

G-quadruplexes turned out to be important targets for the development of novel targeted anticancer/antiviral therapies. More than 3000 G-quadruplex small-molecule ligands have been described, with most of them exerting anticancer/antiviral activity by inducing telomeric damage and/or altering oncogene or viral gene expression in cancer cells and viruses, respectively. For some ligands, in-depth NMR and/or crystallographic studies were performed, providing detailed knowledge on their interactions with diverse G-quadruplex targets. Here, the PDB-deposited NMR and crystal structures of the complexes between telomeric, oncogenic or viral G-quadruplexes and small-molecule ligands, of both organic and metal-organic nature, have been summarized and described based on the G-quadruplex target, from telomeric DNA and RNA G-quadruplexes to DNA oncogenic G-quadruplexes, and finally to RNA viral G-quadruplexes. An overview of the structural details of these complexes is here provided to guide the design of novel ligands targeting more efficiently and selectively cancer- and virus-related G-quadruplex structures.

Affinity Chromatography-Based Assays for the Screening of Potential Ligands Selective for G-Quadruplex Structures.

DNA G-quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4-targeting synthetic or natural compounds, we developed an affinity chromatography-based assay, named G-quadruplex on Oligo Affinity Support (G4-OAS), by synthesizing G4-forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on-support elongation and deprotection of the G4-forming oligonucleotides can be performed, along with the successive affinity chromatography-based assay, renamed as G-quadruplex on Controlled Pore Glass (G4-CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4-CPG assay are reported.

Natural compounds from Juncus plants interacting with telomeric and oncogene G-quadruplex structures as potential anticancer agents.

Aiming at discovering novel, putative anticancer drugs featuring low-to-null side effects, natural compounds isolated from Juncaceae were studied here for their ability to target G-quadruplex structures originating from cancer-related telomeric and oncogene DNA sequences. Particularly, various dihydrophenanthrene, benzocoumarin and dihydrodibenzoxepin derivatives were firstly screened by the affinity chromatography-based G4-CPG assay, and the compound with the highest affinity and selectivity for G-quadruplexes (named J10) was selected for further studies. Fluorescence spectroscopy and circular dichroism experiments corroborated its capability to selectively recognize and stabilize G-quadruplexes over duplex DNA, also showing a preference for parallel G-quadruplexes. Molecular docking proved that the selective G-quadruplex interactions over duplex interactions could be due to the ability of J10 to bind to the grooves of the telomeric and oncogene G-quadruplex structures. Finally, biological assays demonstrated that J10 induces significant antiproliferative effects on human leukemia cells, with no relevant effects on healthy human fibroblasts. Interestingly, J10 exerts its antiproliferative action on tumor cells by activating the apoptotic pathway.

Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds.

In the quest for selective G-quadruplex (G4)-targeting chemotypes, natural compounds have been thus far poorly explored, though representing appealing candidates due to the high structural diversity of their scaffolds. In this regard, a unique high diversity in-house library composed of ca. one thousand individual natural products was investigated. The combination of molecular docking-based virtual screening and the G4-CPG experimental screening assay proved to be useful to quickly and effectively identify-out of many natural compounds-five hit binders of telomeric and oncogenic G4s, i.e., Bulbocapnine, Chelidonine, Ibogaine, Rotenone and Vomicine. Biophysical studies unambiguously demonstrated the selective interaction of these compounds with G4s compared to duplex DNA. The rationale behind the G4 selective recognition was suggested by molecular dynamics simulations. Indeed, the selected ligands proved to specifically interact with G4 structures due to peculiar interaction patterns, while they were unable to firmly bind to a DNA duplex. From biological assays, Chelidonine and Rotenone emerged as the most active compounds of the series against cancer cells, also showing good selectivity over normal cells. Notably, the anticancer activity correlated well with the ability of the two compounds to target telomeric G4s.

DNA Binding Mode Analysis of a Core-Extended Naphthalene Diimide as a Conformation-Sensitive Fluorescent Probe of G-Quadruplex Structures.

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide (cex-NDI) was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes. Aiming at evaluating how the fluorescence behaviour of this compound is associated with specific binding modes to the different DNA targets, cex-NDI was here studied in its interaction with hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex models by biophysical techniques, molecular docking, and biological assays. cex-NDI showed different binding modes associated with different amounts of stacking interactions with the three DNA targets. The preferential binding sites were the groove, outer quartet, or intercalative site of the hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex, respectively. Interestingly, our data show that the fluorescence intensity of DNA-bound cex-NDI correlates with the amount of stacking interactions formed by the ligand with each DNA target, thus providing the rationale behind the conformation-sensitive properties of cex-NDI and supporting its use as a fluorescent probe of G-quadruplex structures. Notably, biological assays proved that cex-NDI mainly localizes in the G-quadruplex-rich nuclei of cancer cells.

Interference of Polydatin/Resveratrol in the ACE2:Spike Recognition during COVID-19 Infection. A Focus on Their Potential Mechanism of Action through Computational and Biochemical Assays.

In the search for new therapeutic strategies to contrast SARS-CoV-2, we here studied the interaction of polydatin (PD) and resveratrol (RESV)-two natural stilbene polyphenols with manifold, well known biological activities-with Spike, the viral protein essential for virus entry into host cells, and ACE2, the angiotensin-converting enzyme present on the surface of multiple cell types (including respiratory epithelial cells) which is the main host receptor for Spike binding. Molecular Docking simulations evidenced that both compounds can bind Spike, ACE2 and the ACE2:Spike complex with good affinity, although the interaction of PD appears stronger than that of RESV on all the investigated targets. Preliminary biochemical assays revealed a significant inhibitory activity of the ACE2:Spike recognition with a dose-response effect only in the case of PD.

From Prebiotic Chemistry to Supramolecular Biomedical Materials: Exploring the Properties of Self-Assembling Nucleobase-Containing Peptides.

Peptides and their synthetic analogs are a class of molecules with enormous relevance as therapeutics for their ability to interact with biomacromolecules like nucleic acids and proteins, potentially interfering with biological pathways often involved in the onset and progression of pathologies of high social impact. Nucleobase-bearing peptides (nucleopeptides) and pseudopeptides (PNAs) offer further interesting possibilities related to their nucleobase-decorated nature for diagnostic and therapeutic applications, thanks to their reported ability to target complementary DNA and RNA strands. In addition, these chimeric compounds are endowed with intriguing self-assembling properties, which are at the heart of their investigation as self-replicating materials in prebiotic chemistry, as well as their application as constituents of innovative drug delivery systems and, more generally, as novel nanomaterials to be employed in biomedicine. Herein we describe the properties of nucleopeptides, PNAs and related supramolecular systems, and summarize some of the most relevant applications of these systems.

Plant-Derived Stilbenoids as DNA-Binding Agents: From Monomers to Dimers.

Stilbenoids are natural compounds endowed with several biological activities, including cardioprotection and cancer prevention. Among them, (±)-trans-δ-viniferin, deriving from trans-resveratrol dimerization, was investigated in its ability to target DNA duplex and G-quadruplex structures by exploiting NMR spectroscopy, circular dichroism, fluorescence spectroscopy and molecular docking. (±)-trans-δ-Viniferin proved to bind both the minor and major grooves of duplexes, whereas it bound the 3'- and 5'-ends of a G-quadruplex by stacking on the outer quartets, accompanied by rearrangement of flanking residues. Specifically, (±)-trans-δ-viniferin demonstrated higher affinity for the investigated DNA targets than its monomeric counterpart. Additionally, the methoxylated derivatives of (±)-trans-δ-viniferin and trans-resveratrol, i. e. (±)-pterostilbene-trans-dihydrodimer and trans-pterostilbene, respectively, were evaluated, revealing similar binding modes, affinities and stoichiometries with the DNA targets as their parent analogues. All tested compounds were cytotoxic at µM concentration on several cancer cell lines, showing DNA damaging activity consistent with their ability to tightly interact with duplex and G-quadruplex structures.

Disentangling the Structure-Activity Relationships of Naphthalene Diimides as Anticancer G-Quadruplex-Targeting Drugs.

In the context of developing efficient anticancer therapies aimed at eradicating any sort of tumors, G-quadruplexes represent excellent targets. Small molecules able to interact with G-quadruplexes can interfere with cell pathways specific of tumors and common to all cancers. Naphthalene diimides (NDIs) are among the most promising, putative anticancer G-quadruplex-targeting drugs, due to their ability to simultaneously target multiple G-quadruplexes and their strong, selective in vitro and in vivo anticancer activity. Here, all the available biophysical, biological, and structural data concerning NDIs targeting G-quadruplexes were systematically analyzed. Structure-activity correlations were obtained by analyzing biophysical data of their interactions with G-quadruplex targets and control duplex structures, in parallel to biological data concerning the antiproliferative activity of NDIs on cancer and normal cells. In addition, NDI binding modes to G-quadruplexes were discussed in consideration of the structures and properties of NDIs by in-depth analysis of the available structural models of G-quadruplex/NDI complexes.

G-quadruplex-based aptamers targeting human thrombin: Discovery, chemical modifications and antithrombotic effects.

First studies on thrombin-inhibiting DNA aptamers were reported in 1992, and since then a large number of anticoagulant aptamers has been discovered. TBA - also named HD1, a 15-mer G-quadruplex (G4)-forming oligonucleotide - is the best characterized thrombin binding aptamer, able to specifically recognize the protein exosite I, thus inhibiting the conversion of soluble fibrinogen into insoluble fibrin strands. Unmodified nucleic acid-based aptamers, in general, and TBA in particular, exhibit limited pharmacokinetic properties and are rapidly degraded in vivo by nucleases. In order to improve the biological performance of aptamers, a widely investigated strategy is the introduction of chemical modifications in their backbone at the level of the nucleobases, sugar moieties or phosphodiester linkages. Besides TBA, also other thrombin binding aptamers, able to adopt a well-defined G4 structure, e.g. mixed duplex/quadruplex sequences, as well as homo- and hetero-bivalent constructs, have been identified and optimized. Considering the growing need of new efficient anticoagulant agents associated with the strong therapeutic potential of these thrombin inhibitors, the research on thrombin binding aptamers is still a very hot and intriguing field. Herein, we comprehensively described the state-of-the-art knowledge on the DNA-based aptamers targeting thrombin, especially focusing on the optimized analogues obtained by chemically modifying the oligonucleotide backbone, and their biological performances in therapeutic applications.

On the binding of naphthalene diimides to a human telomeric G-quadruplex multimer model.

To selectively target telomeric G-quadruplex (G4) DNA, monomeric and dimeric naphthalene diimides (NDIs) were investigated as binders of multimeric G4 structures able to discriminate duplex DNA. These NDIs were analysed by the affinity chromatography-based screening G4-CPG (G-quadruplex on Controlled Pore Glass), using the sequence d[AGGG(TTAGGG)7] (tel46), folding into two consecutive G4s, as model of the human telomeric G4 multimer. In parallel, a telomeric G4 monomer (tel26) and a duplex structure (ds27) were used as controls. According to G4-CPG screening, NDI-5 proved to be the best ligand in terms of dimeric G4 vs. duplex DNA selectivity and was analysed by circular dichroism (CD), gel electrophoresis, isothermal titration calorimetry (ITC) and fluorescence spectroscopy in its interactions with tel46. NDI-5 strongly binds and stabilizes tel46 G4, favouring a hybrid folding in K+-containing buffer. Under these conditions, the binding process comprises a first event involving three molecules of NDI-5 and a second one in which other six molecules bind to the DNA. In a metal cation-free system, NDI-5 induces tel46 G4 folding, as indicated by CD and PAGE, favouring an antiparallel structuring. Docking simulations showed that NDI-5 can effectively bind to the pocket between two G4 units, representing a promising ligand for multimeric G4s.

Anti-VEGF DNA-based aptamers in cancer therapeutics and diagnostics.

The vascular endothelial growth factor (VEGF) family and its receptors play fundamental roles not only in physiological but also in pathological angiogenesis, characteristic of cancer progression. Aiming at finding putative treatments for several malignancies, various small molecules, antibodies, or protein-based drugs have been evaluated in vitro and in vivo as VEGF inhibitors, providing efficient agents approved for clinical use. Due to the high clinical importance of VEGF, also a great number of anti-VEGF nucleic acid-based aptamers-that is, oligonucleotides able to bind with high affinity and specificity a selected biological target-have been developed as promising agents in anticancer strategies. Notable research efforts have been made in optimization processes of the identified aptamers, searching for increased target affinity and/or bioactivity by exploring structural analogues of the lead compounds. This review is focused on recent studies devoted to the development of DNA-based aptamers designed to target VEGF. Their therapeutic potential as well as their significance in the construction of highly selective biosensors is here discussed.

On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight.

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5'-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5'- and 3'-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range.

Synthesis, Antiproliferative Activity, and DNA Binding Studies of Nucleoamino Acid-Containing Pt(II) Complexes.

We here report our studies on the reaction with the platinum(II) ion of a nucleoamino acid constituted by the l-2,3-diaminopropanoic acid linked to the thymine nucleobase through a methylenecarbonyl linker. The obtained new platinum complexes, characterized by spectroscopic and mass spectrometric techniques, were envisaged to exploit synergistic effects due to the presence of both the platinum center and the nucleoamino acid moiety. The latter can be potentially useful to protect the complexes from early deactivation, as well as to facilitate their cell internalization. The biological activity of the complexes in terms of antiproliferative effects was evaluated in vitro on different cancer cell lines and healthy cells, showing the best results on human cervical adenocarcinoma (HeLa) cells along with good selectivity for cancer over normal cells. In contrast, the metal-free nucleoamino acid did not show any cytotoxicity on both normal and cancer cell lines. Finally, the ability of the novel Pt(II) complexes to bind various DNA model systems was investigated by circular dichroism (CD) spectroscopy and polyacrylamide gel electrophoresis analyses proving that the newly obtained compounds can potentially target DNA, similarly to other well-known anticancer Pt complexes, with a peculiar G-quadruplex vs. duplex selectivity.

Trifunctionalized Naphthalene Diimides and Dimeric Analogues as G-Quadruplex-Targeting Anticancer Agents Selected by Affinity Chromatography.

A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands-according to an affinity chromatography-based screening method named G4-CPG-were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC50 values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays' data.

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs.

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF165 binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.