Specific Stabilization of c-MYC and c-KIT G-Quadruplex DNA Structures by Indolylmethyleneindanone Scaffolds.

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.

Dihydrochelerythrine and its derivatives: Synthesis and their application as potential G-quadruplex DNA stabilizing agents.

A convenient route was envisaged toward the synthesis of dihydrochelerythrine (DHCHL), 4 by intramolecular Suzuki coupling of 2-bromo-N-(2-bromobenzyl)-naphthalen-1-amine derivative 5 via in situ generated arylborane. This compound was converted to (±)-6-acetonyldihydrochelerythrine (ADC), 3 which was then resolved by chiral prep-HPLC. Efficiency of DHCHL for the stabilization of promoter quadruplex DNA structures and a comparison study with the parent natural alkaloid chelerythrine (CHL), 1 was performed. A thorough investigation was carried out to assess the quadruplex binding affinity by using various biophysical and biochemical studies and the binding mode was explained by using molecular modeling and dynamics studies. Results clearly indicate that DHCHL is a strong G-quadruplex stabilizer with affinity similar to that of the parent alkaloid CHL. Compounds ADC and DHCHL were also screened against different human cancer cell lines. Among the cancer cells, (±)-ADC and its enantiomers showed varied (15-48%) inhibition against human colorectal cell line HCT116 and breast cancer cell line MDA-MB-231 albeit low enantio-specificity in the inhibitory effect; whereas DHCHL showed 30% inhibition against A431 cell line only, suggesting the compounds are indeed cancer tissue specific.

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

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.

Targeting promoter G-quadruplex DNAs by indenopyrimidine-based ligands.

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.