The formation of G-quadruplex structures can regulate telomerase activity and the expression of oncogenes at the transcriptional and translational levels. Therefore, stabilization of G-quadruplex DNA structures by small molecules has been recognized as a promising strategy for anticancer drug therapy. One of the major challenges in this field is to impart stabilizing molecules with selectivity toward quadruplex structures over duplex DNAs, and to maintain specificity toward a particular quadruplex topology. Herein we report the synthesis and binding interactions of indenopyrimidine derivatives, endowed with drug-like properties, with oncogenic promoters of c-myc and c-kit, telomeric and duplex DNAs. The results show specific stabilization of promoter over telomeric quadruplexes and duplex DNAs. Molecular modeling studies support the experimental observations by unraveling the dual binding mode of ligands by exploiting the top and bottom quartets of a G-quadruplex structure. This study underscores the potential of the indenopyrimidine scaffold, which can be used to achieve specific G-quadruplex-mediated anticancer activity.

Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (c-MYC, c-KIT1, and c-KIT2), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (InEt2 and InPr2) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the c-MYC and c-KIT quadruplex DNAs (ΔT1/2 up to 24 °C) over telomeric and duplex DNAs (ΔT1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (Ka ∼ 10(5) to 10(6) M(-1)). Low IC50 (∼2 μM) values were emerged for these ligands from a Taq DNA polymerase stop assay with the c-MYC quadruplex forming template, whereas the telomeric DNA template showed IC50 values >120 μM. Molecular modeling and dynamics studies demonstrated the 5'- and 3'-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications.

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 (ΔTm = 10-15 °C) as compared to antiparallel promoter quadruplex (h-RAS1), telomeric quadruplex and duplex DNAs (ΔTm = 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.

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DNA can fold into non-canonical structures such as Gquadruplexes (G4 s) in addition to adopting the double helical structure. Considering the relationship between stabilization of G4 structure and anticancer effects, development of G4 interactive compounds has been of significant interest. Past years have witnessed the discovery of scaffolds targeting G4 structures based on planar, multi-aromatic ring compounds.With an aim to engineer drug-like properties, we designed and synthesized pyridopyrimidinone based selective G4 DNA stabilizing agents 1-3, and further they were evaluated for G4 DNA recognition properties. CD melting studies revealed the preferential stabilization of parallel topology of promoter c-MYC and c-KIT G4 DNAs by the ligands, especially 2 and 3, over the different topologies of telomeric G4 DNA. UV melting experiments suggested that no significant stabilization was observed for duplex DNA. Further, the results from ITC experiments substantiated the preferential stabilization of parallel topology of c-MYC G4 DNA over telomeric and duplex DNA by the ligands 2. These data showed that ligand 2 has moderate binding affinity to the c-MYC G4 DNA and is~49-fold and~25fold selective over the telomeric G4 DNA and the duplex DNA respectively. The molecular modeling and dynamics studies of the ligand 2 in complex with c-MYC and c-KIT1 G4 DNAs showed that this ligand stacks on the 5'-quartet of c-MYC and 3'-quartet of c-KIT1 G4 DNA structures.