NMR based structure reveals groove binding of mitoxantrone to two sites of [d-(TTAGGGT)]4 having human telomeric DNA sequence leading to thermal stabilization of G-quadruplex.

Interaction of mitoxantrone (MTX) with G-quadruplex, leading to inhibition of telomerase enzyme and anticancer action, is not understood. Titrations of MTX with [d-(TTAGGGT)]4, comprising human telomere single repeat sequence, have been monitored by fluorescence and 1H/31P NMR spectroscopy. Binding induces chemical shift changes in GNH (~0.3ppm), T2/T7/A3 base protons and sequence specific shifts in 31P resonances. Absence of large downfield shifts in 31P signals and presence of all sequential NOEs show that classical intercalation of drug between base quartets does not occur. The upfield shift (~0.53ppm) in 2/3H, 1/4OH and minor shift in 6/7H aromatic protons of MTX in complex rule out end stacking as a possible mode of interaction. The 26 short inter molecular contacts of 1/4OH, 11NH, 6/7H and 12CH2 protons of MTX with T1, T2, G6, G7 protons in the structure of complex obtained by restrained Molecular Dynamics simulations show binding at grooves accompanied by 4 hydrogen bonds and partial stacking of MTX with base pairs. Interaction causes thermal stabilization, ΔTm=35°C, which may be attributed to telomerase inhibition. The present study is the first report on solution structure of mitoxantrone-[d-(TTAGGGT)]4 complex and is significant for structure based designing of anthraquinone derivatives as future drugs.

Molecular Recognition of Parallel DNA Quadruplex d(TTAGGGT)4 by Mitoxantrone: Binding with 1:2 Stoichiometry Leading to Thermal Stabilization and Telomerase Inhibition.

The interaction of the anthraquinone derivative mitoxantrone, a semisynthetic anti-cancer drug with two non-planar side chains, with heptamer G-quadruplex d(TTAGGGT)4 , which contains the human telomere DNA sequence, was evaluated by differential scanning calorimetry, fluorescence Job plotting, absorption, and NMR and CD spectroscopy. Binding led to thermal stabilization of DNA (ΔTm =13-20 °C). The spectra revealed that two mitoxantrone molecules bind externally at two sites of the DNA quadruplex as monomers, by partial insertion of the chromophore and side-chain interaction at the grooves. The inhibition of telomerase (IC50 =2 µM), as determined by a TRAP assay, can be attributed to thermal stabilization of the DNA quadruplex because of the interactions with mitoxantrone. The studies revealed highly specific molecular recognition between a ligand and a parallel-stranded G-quadruplex; this might serve as a platform for the rational design of new drugs.

NMR-based structure of anticancer drug mitoxantrone stacked with terminal base pair of DNA hexamer sequence d-(ATCGAT)2.

Mitoxantrone is a promising antitumor drug having considerably reduced cardiotoxicity as compared to anthracyclines. Its binding to deoxyhexanucleotides sequence d-(ATCGAT)2 has been studied by proton and phosphorous-31 nuclear magnetic resonance spectroscopy. The stoichiometry reveals that 1:1 and 2:1 mitoxantrone-d(ATCGAT)2 complexes are formed in solution. Significant upfield shifts in 6H/7H, 2H/3H, 11NH, and 12NH protons (∼.5 ppm) of mitoxantrone and T6NH imino protons (∼.3 ppm) are observed. The phosphorous resonances do not shift significantly indicating that the base pairs do not open at any nucleotide step along the sequence of hexamer. Several inter-molecular Nuclear Overhauser Enhancement connectivities between mitoxantrone and hexanucleotide protons indicate that mitoxantrone chromophore stacks with terminal A1-T6 base pair and side chains involving 12CH2, 12NH, and 14OH protons are in close proximity of A1, T2, A5, and T6 bases. Absorption and emission spectra show red shift in wavelength maxima, which is characteristic of stacking interaction. At higher mitoxantrone to nucleic acid ratios, electrostatic interactions are dominant. The 2:1 drug/DNA stoichiometric structure obtained by restrained Molecular Dynamics simulations shows considerable distortions in backbone torsional angles and helicoidal parameters although structural fluctuations in 25 ps analysis of trajectory are found to be negligible. Mitoxantrone binds as a monomer at either or both ends of hexamer externally with side chains interacting specifically with DNA. The findings are relevant to the understanding of pharmacological action of drug.

On the affinity and specificity of quercetin for DNA.

The potential of quercetin (QUE), being a member of the whole family of structurally different flavonoids, to serve as an anti-tumor agent has been recognized, but not fully understood. The interactions between DNA and a series of the flavonoids have so far been mainly investigated using a variety of experimental techniques. Herein, the specificity of QUE for DNA is explored using sophisticated density functional theory (DFT) methods employed to generate the optimized structure of QUE in complex with adenine (A), guanine (G), thymine (T) and cytosine (C), respectively. As far as a preference of QUE is concerned, structural and energetic as well as NMR chemical shift arguments clearly indicate a highest for G and a lowest for C. This observation is further substantiated by analyzing the binding modes of QUE docked in a quadruplex receptor structure of DNA and in a duplex receptor structure of DNA. Among all possible single point mutations of the DNA quadruplex and duplex residues, several critical ones causing a conspicuous stabilizing effect on the original complexes of QUE with the DNA receptors are identified. Consequently, several fundamental standpoints shedding new light on the molecular mechanisms underlying the interactions between QUE and DNA are discussed.