Novel G-quadruplex stabilizing agents: in-silico approach and dynamics

The stabilization of overhang G-rich repetitive DNA units at the 3-end of telomeres, which are well known to form functionally important G-quadruplex structures, is a current goal in designing novel anticancer drugs. In the present study, we have undertaken an in silico approach by molecular docking using a small molecule library to find potential G-quadruplex stabilizing agents. Two molecules, A, [N1-imino(2-pyridyl)methyl-3,4,5-trimethoxybenzene-1-carbohydrazide] and B, [(3-[4-({[3-({4-[(2cyanoethyl)(methyl)amino]benzylidene}amino)propyl]imino}methyl)(methyl) anilino]propanenitrile)], that had good docking scores have been investigated for interaction with G-quadruplexes in a Molecular Dynamics simulation study. Fluorescence spectroscopy of G-quadruplexes bound to the screened molecules A and B was used to experimentally validate the theoretical results. The binding of ligands A and B to G-quadruplexes resulted in blue shifts of 10-18nm, respectively, in the fluorescence emission spectra of the G-quadruplexes, demonstrating that both molecules bind to the G-face of the quadruplex. The same experiment was performed for the complexation of these small molecules with a G-rich DNA duplex, 5'-GCGCATGCTACGCG-3' 3'-CGCGTACGAGCGC-5'. Interestingly, no blue shift was observed in the fluorescence emission spectra of the DNA duplex in the presence of these small molecules. Thus, these findings indicated that these ligands very selectively bind to G-quadruplexes instead of the duplex DNA. In addition, a one-dimensional water ligand observed via a gradient spectroscopy Nuclear Magnetic Resonance (NMR) experiment showed that both molecules bound to the 23-mer G-quadruplex DNA. The molecular properties of the ligand-quadruplex complex have been analyzed with the help of the Adaptive Poisson-Boltzmann Solver, revealing that electrostatics govern the binding of the small molecules to G-quadruplexes. Both molecules were investigated in detail using solvation free energy calculations and Absorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) predictions, which provide insight into lead optimization for designing G-quadruplex stabilizing agents; therefore, these molecules have potential as new therapeutic agents.