Benzothiazole hydrazones of furylbenzamides preferentially stabilize c-MYC and c-KIT1 promoter G-quadruplex DNAs

Abstract

The stabilization of G-quadruplex DNA structures by using small molecule ligands having simple structural scaffolds has the potential to be harnessed for developing next generation anticancer agents. Because of the structural diversity of G-quadruplexes, it is challenging to design stabilizing ligands, which can specifically bind to a particular quadruplex topology. To address this, herein, we report the design and synthesis of three benzothiazole hydrazones of furylbenzamides having different side chains (ligands 1, 2 and 3), which show preferential stabilization of promoter quadruplex DNAs (c-MYC and c-KIT1) having parallel topologies over telomeric and duplex DNAs. The CD melting study revealed that all the ligands, in particular ligand 2, exhibit higher stabilization toward parallel promoter quadruplexes (ΔT_m = 10–15 °C) as compared to antiparallel promoter quadruplex (h-RAS1), telomeric quadruplex and duplex DNAs (ΔT_m = 0–3 °C). FID assay and fluorimetric titration results also reveal the preferential binding of ligands toward c-MYC and c-KIT1 promoter quadruplex DNAs over telomeric and duplex DNAs. Validating these results further, Taq DNA polymerase stop assay showed IC₅₀ ∼ 6.4 μM for ligand 2 with the c-MYC DNA template, whereas the same for the telomeric DNA template was found to be >200 μM. Molecular modeling and dynamics studies demonstrated a 1 : 1 binding stoichiometry in which stacking and electrostatic interactions play important roles in stabilizing the c-MYC G-quadruplex structure. Taken together, the results presented here provide new insights into the design of structurally simple scaffolds for the preferential stabilization of a particular G-quadruplex topology.