Molecular recognition of parallel quadruplex [d-(TTGGGGT)]4 by mitoxantrone: binding with 1 : 4 stoichiometry leads to telomerase inhibition

Abstract

The interaction of mitoxantrone (MTX), an anticancer drug, with parallel stranded tetra-molecular G-quadruplex DNA [d-(TTGGGGT)]₄ has been studied using Surface Plasmon Resonance (SPR), thermal melting profiles, absorption, nuclear magnetic resonance and Circular Dichroism (CD) spectroscopy. Mitoxantrone binds to G-quadruplex DNA with the affinity constant K_A ∼ 3 × 10⁴ M⁻¹ and the binding stoichiometry of the mitoxantrone : DNA quadruplex complex is found to be 4 : 1. The DNA melting (T_m) experiments show that binding leads to an increase in T_m indicating the thermal stabilization of DNA. T_m increases with D/N, that is, the ratio of added mole equivalents of mitoxantrone (D) to DNA (N) and saturates at D/N = 4.0 yielding total ΔT_m = 26 °C. Proton and phosphorus-31 chemical shifts show that the base pairs of DNA do not open to allow intercalation of a mitoxantrone chromophore. The 2D NOESY spectra reveal that mitoxantrone binds as a stacked dimer having head to tail (anti-parallel) orientation at two opposite ends externally to the DNA quadruplex. CD spectra show the induced CD band of mitoxantrone having exciton splitting at 630–690 nm. The TRAP assay gives IC₅₀ ∼ 2 μM for inhibition of telomerase by mitoxantrone, which may be attributed to thermal stabilization of the DNA quadruplex. The present study suggests that a dimer formed by stacking of two mitoxantrone molecules might provide highly specific molecular recognition between ligands and a parallel stranded G-quadruplex and hence serve as a platform for rational design of new G-quadruplex groove binders.