Investigating the Ruthenium Metalation of Proteins: X‑ray Structure and Raman Microspectroscopy of the Complex between RNase A and AziRu

A Raman-assisted crystallographic study on the adduct between AziRu, a Ru­(III) complex with high antiproliferative activity, and RNase A is presented. The protein structure is not perturbed significantly by the Ru label. The metal coordinates to ND atoms of His105 or of His119 imidazole rings, losing all of its original ligands but retaining octahedral, although distorted, coordination geometry. The AziRu binding inactivates the enzyme, suggesting that its antitumor action can be exerted by a mechanism of competitive inhibition

Interaction of Anticancer Ruthenium Compounds with Proteins: High-Resolution X‑ray Structures and Raman Microscopy Studies of the Adduct between Hen Egg White Lysozyme and AziRu

The binding properties of AziRu, a ruthenium­(III) complex with high antiproliferative activity, toward a hen egg white lysozyme have been investigated by X-ray crystallography and Raman microscopy. The data provide clear evidence on the mechanism of AziRu–protein adduct formation and of ligand exchange in the crystal state

G‑Quadruplex on Oligo Affinity Support (G4-OAS): An Easy Affinity Chromatography-Based Assay for the Screening of G‑Quadruplex Ligands

A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known G-quadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility

Cholesterol-Based Nucleolipid-Ruthenium Complex Stabilized by Lipid Aggregates for Antineoplastic Therapy

A novel ruthenium complex, linked to a cholesterol-containing nucleolipid (named ToThyCholRu), stabilized by lipid aggregates for antineoplastic therapy is presented. In order to retard the degradation kinetics typically observed for several ruthenium-based antineoplastic agents, ToThyCholRu is incorporated into a liposome bilayer formed by POPC. The resulting nanoaggregates contain up to 15% in moles of the ruthenium complex, and are shown to be stable for several weeks. The liposomes host the ruthenium–nucleolipid complex with the metal ion surrounded by POPC lipid headgroups and the steroid moiety inserted in the more external acyl chain region. These ruthenium-containing liposomes are more effective in inhibiting the growth of cancer cells than a model NAMI-A-like ruthenium complex, prepared for a direct evaluation of their anti-proliferative activity. These results introduce new perspectives in the design of innovative transition-metal-based supramolecular systems for anticancer drug vectorization

Anticancer Cationic Ruthenium Nanovectors: From Rational Molecular Design to Cellular Uptake and Bioactivity

An efficient drug delivery strategy is presented for novel anticancer amphiphilic ruthenium anionic complexes, based on the formation of stable nanoparticles with the cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP). This strategy is aimed at ensuring high ruthenium content within the formulation, long half-life in physiological media, and enhanced cell uptake. An in-depth microstructural characterization of the aggregates obtained mixing the ruthenium complex and the phospholipid carrier at 50/50 molar ratio is realized by combining a variety of techniques, including dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR), electron paramagnetic resonance (EPR), and zeta potential measurements. The in vitro bioactivity profile of the Ru-loaded nanoparticles is investigated on human and non-human cancer cell lines, showing IC₅₀ values in the low μM range against MCF-7 and WiDr cells, that is, proving to be 10–20-fold more active than AziRu, a previously synthesized NAMI-A analog, used for control. Fluorescence microscopy studies demonstrate that the amphiphilic Ru-complex/DOTAP formulations, added with rhodamine-B, are efficiently and rapidly incorporated in human MCF-7 breast adenocarcinoma cells. The intracellular fate of the amphiphilic Ru-complexes was investigated in the same in vitro model by means of an ad hoc designed fluorescently tagged analog, which exhibited a marked tendency to accumulate within or in proximity of the nuclei

Fluorescence Enhancement upon G‑Quadruplex Folding: Synthesis, Structure, and Biophysical Characterization of a Dansyl/Cyclodextrin-Tagged Thrombin Binding Aptamer

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3′-end and a β-cyclodextrin residue at the 5′-end, has been efficiently synthesized exploiting Cu­(I)-catalyzed azide–alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K⁺ and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3′-end, into the apolar cavity of the β-cyclodextrin at the 5′-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity