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 1 H/31 P NMR spectroscopy. Binding induces chemical shift changes in GNH (~0.3ppm), T2/T7/A3 base protons and sequence specific shifts in 31 P resonances. Absence of large downfield shifts in 31 P 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.