Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
NMR study of interaction of mitoxantrone to G-quadruplex sequences leading to stabilization and inhibition of telomerase enzyme
Guanine rich single-stranded DNA occurring in human genome (e.g. telomere, promoters, oncogenes) have ability to assemble as G-quadruplex structures by folding. Ligands which induce and stabilize G-quadruplex structures cause telomerase inhibition and interfere with gene regulation. This has led to structure-based drug designing of molecules specific to G-quadruplex. Several ligands have been evaluated for telomerase inhibition but lack of structural data of G-quadruplex complexes has restricted the applications. Telomerase Repeat Amplification Protocol assay using Polymerase Chain Reaction to amplify newly synthesized telomeric repeats shows that Mitoxantrone, a semisynthetic anthraquinone derivative,
can inhibit telomerase activity with EC50 = 2 µM. Binding stoichiometry of mitoxantrone with tetramolecular parallel forms of G-quadruplex sequences, d-(TTGGGGT)4 and d-(TTAGGGT)4, which occur in Tetrahymena and human telomeric DNA, is found to be 4:1 and 2:1, respectively by the continuous variation analysis using fluorescent intensity measurements. Titration of DNA quadruplex sequences with mitoxantrone have been followed by proton and phosphorus-31nuclear magnetic resonance spectroscopy. The GNH protons shift upfield up to 0.24 ppm while aromatic ring protons of the mitoxantrone, 2/3H and 1/4OH, shift upfield by ∼0.46 ppm. The imino protons persist beyond 328 K in the presence of ligand indicating significant stabilization by ~25 K as compared to uncomplexed DNA sequence. 1/4OH ring protons are immobilized and make several intermolecular contacts with DNA protons. About 20-30 short inter molecular contacts observed in Nuclear Overhauser Enhancement spectroscopy (NOESY) experiments have been used to arrive at the conformation of ligand-DNA complex. The results of restrained Molecular Dynamics simulations show that two dimers, each formed by head to tail alignment of two molecules of mitoxantrone, bind outside DNA at the groove near T1pT2pG3 and G6pT7 sites in the complex with d-(TTGGGGT)4 sequence.
On the other hand only monomer binds at two opposite sites with G-quadruplex sequence d-(TTAGGGT)4 (picture on right side).
The structure is stabilized by partial stacking close to ends of helix and specific hydrogen bonds with O1' sugar of T2 base. The interactions of mitoxantrone with DNA are unique to each of these DNA sequences. These interactions may lead to thermal stabilization of G-quadruplexes which may be responsible for inhibition of telomerase enzyme.
Department of Biotechnology