Experimental and theoretical characterization of the strong effects on DNA stability caused by half-sandwich Ru(II) and Ir(III) bearing thiabendazole complexes.

The synthesis and characterization of two half-sandwich complexes of Ru(II) and Ir(III) with thiabendazole as ancillary ligand and their DNA binding ability were investigated using experimental and computational methods. 1H NMR and acid-base studies have shown that aquo-complexes are the reactive species. Kinetic studies show that both complexes bind covalently to DNA through the metal site and non covalently through the ancillary ligand. Thermal stability studies, viscosity, circular dichroism measurements and quantum chemical calculations have shown that the covalent binding causes breaking of the H-bonding between base pairs, bringing about DNA denaturation and compaction. Additionally, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations shed light into the binding features of the Ru(II) and Ir(III) complexes and their respective enantiomers toward double-helical DNA, highlighting the important role played by the NˆN ancillary ligand once the complexes are covalently linked to DNA. Moreover, metal quantification in the nucleus of SW480 colon adenocarcinoma cells were carried out by inductively coupled plasma-mass spectrometry (ICP-MS), both complexes are more internalized than cisplatin after 4 h of exposition. However, in spite of the dramatic changes in the helicity of the DNA secondary structure induced by these complexes and their nuclear localization, antiproliferative studies have revealed that both, Ru(II) and Ir(III) complexes, cannot be considered cytotoxic. This unexpected behavior can be justified by the fast formation of aquo-complexes, which may react with components of the cell culture medium or the cytoplasm compartment in such a way that they may become deactivated before reaching DNA.

Fishing for G-Quadruplexes in Solution with a Perylene Diimide Derivative Labeled with Biotins.

A new fluorescent, non-cytotoxic perylene diimide derivative with two biotins at the peri position, PDI2B, has been synthesized. This molecule is able to interact selectively with G-quadruplexes with scarce or no affinity towards single- or double-stranded DNA. These features have made it possible to design a simple, effective, safe, cheap, and selective method for fishing G-quadruplex structures in solution by use of PDI2B and streptavidin coated magnetic beads. The new cyclic method reported leads to the recovery of more than 80 % of G-quadruplex structures from solution, even in the presence of an excess of single-stranded or duplex DNA as competitors. Moreover, PDI2B is a G4 ligand that can display higher thermal stabilization and greater affinity for 2- over 3-tetrad quadruplexes, which constitutes a novel type of behavior.

Kinetic evidence for interaction of TMPyP4 with two different G-quadruplex conformations of human telomeric DNA.

BACKGROUND: Stabilization of G-quadruplex helices by small ligands has attracted growing attention because they inhibit the activity of the enzyme telomerase, which is overexpressed in >80% cancer cells. TMPyP4, one of the most studied G-quadruplex ligands, is used as a model to show that the ligands can exhibit different binding features with different conformations of a human telomeric specific sequence. METHODS: UV-Vis, FRET melting Assay, Isothermal Titration Calorimetry, Time-resolved Fluorescence lifetime, T-Jump and Molecular Dynamics. RESULTS: TMPyP4 yields two different complexes with two Tel22 telomeric conformations in the presence of Na+ or K+. T-Jump kinetic experiments show that the rates of formation and dissociation of these complexes in the ms time scale differ by one order of magnitude. MD simulations reveal that, in K+ buffer, "hybrid 1" conformation yields kinetic constants on interaction with TMPyP4 one order lower than "hybrid 2". The binding involves π-π stacking with external loop bases. CONCLUSIONS: For the first time we show that for a particular buffer TMPyP4 interacts in a kinetically different way with the two Tel22 conformations even if the complexes formed are thermodynamically indistinguishable. GENERAL SIGNIFICANCE: G-quadruplexes, endowed with technological applications and potential impact on regulation mechanisms, define a new research field. The possibility of building different conformations from same sequence is a complex issue that confers G-quadruplexes very interesting features. The obtaining of reliable kinetic data constitutes an efficient tool to determine reaction mechanisms between conformations and small molecules.

Role of Seroalbumin in the Cytotoxicity of cis-Dichloro Pt(II) Complexes with (N

Given the potent anticancer properties of cis-diamminedichloroplatinum(II) and knowing its mode of action, we synthesized four new cis-[PtCl2(N^N)] organoplatinum complexes, two with N-substituted pbi ligands (pbiR = 1-R-2-(2-pyridyl)benzimidazole) (namely, 1 and 2) and two more with 4,4'-disubstituted bpy ligands (bpy = 2,2'-bipyridine) (namely, 3 and 4). We explored their cytotoxicity and ability to bind to deoxyguanosine monophosphate (dGMP), DNA, and albumin models. By 1H NMR and UV-vis spectroscopies, circular dichroism, agarose gel electrophoresis, differential scanning calorimetry measurements, and density functional theory calculations, we verified that only 3 can form aquacomplex species after dimethyl sulfoxide solvation; surprisingly, 1, 2, and 3 can bind covalently to DNA, whereas 4 can form a noncovalent complex. Interestingly, only complexes 1 and 4 exhibit good cytotoxicity against human ovarian carcinoma (HeLa) cell line, whereas 2 and 3 are inactive. Although lung carcinoma (A549) cells are more resistant to the four platinum complexes than HeLa cells, when the protein concentration in the extracellular media is lower, the cytotoxicity becomes substantially enhanced. By native electrophoresis of bovine seroalbumin (BSA) and inductively coupled plasma mass spectrometry uptake studies we bear out, on one hand, that 2 and 3 can interact strongly with BSA and its cellular uptake is negligible and, on the other hand, that 1 and 4 can interact with BSA only weakly, its cellular uptake being higher by several orders. These results point up the important role of the protein binding features on their biological activity and cellular uptake of cis-"PtCl2" derivatives. Our results are valuable in the future rational design of new platinum complexes with improved biological properties, as they expose the importance not only of their DNA binding abilities but also of additional factors such as protein binding.

Combined spectroscopic and theoretical analysis of the binding of a water-soluble perylene diimide to DNA/RNA polynucleotides and G-quadruplexes.

We present here a combined spectroscopic and theoretical analysis of the binding of N,N'-bis(2-(1-piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide dichloride (PZPERY) to different biosubstrates. Absorbance titrations and circular dichroism experiments, melting studies and isothermal calorimetry (ITC) titrations reveal a picture where the binding to natural double-stranded DNA is very different from that to double and triple-stranded RNAs (poly(A)∙poly(U) and poly(U)∙poly(A)⁎poly(U)). As confirmed also by the structural and energetic details clarified by density functional theory (DFT) calculations, intercalation occurs for DNA, with a process driven by the combination of aggregates disruption and monomers intercalation. Oppositely, for RNAs, no intercalation but groove binding with the formation of supramolecular aggregates is observed. Among all the tested biosubstrates, the affinity of PZPERY towards DNA G-quadruplexes (G4) is the greatest one with a preference for human telomeric G4s. Focusing on hybrid G4 forms, either sitting-atop ("tetrad-parallel") or lateral ("groove-parallel") binding modes were considered in the discussion of the experimental results and molecular dynamics (MD) simulations. Both turned out to be possible concurrently, in agreement also with the experimental binding stoichiometries higher than 2:1.