Bisindolylmaleimide Ligands Stabilize c-MYC G-Quadruplex DNA Structure and Downregulate Gene Expression.

G-Quadruplex (G4) structures play a pivotal role in diverse biological functions, including essential processes, such as telomere maintenance and gene regulation. G4 structures formed in functional regions of genomes are actively pursued toward therapeutics and are targeted by small-molecule ligands that alter their structure and/or stability. Herein, we report the synthesis of bisindolylmaleimide-based (BIM) ligands, which preferentially stabilize parallel G4 structures of c-MYC and c-KIT oncogenes over the telomeric h-RAS1 G4 and duplex DNAs. The preferential stabilization of parallel G4s with BIM ligands is further validated by the DNA polymerase stop assay, where stop products were only observed for templates containing the c-MYC G4 sequence. Nuclear magnetic resonance (NMR) titration studies indicate that the lead ligand BIM-Pr1 forms a 2:1 complex with c-MYC G4 DNA with a KD of 38 ± 5 µM. The BIM ligand stacks at the 5' and 3' quartets, with molecular modeling and dynamics studies supporting the proposed binding mode. The ligand is cytotoxic to HeLa cells and downregulates c-MYC gene expression. Collectively, the results present bisindolylmaleimide scaffolds as novel and powerful G4 targeting agents.

Stabilization and fluorescence light-up of G-quadruplex nucleic acids using indolyl-quinolinium based probes.

G-Quadruplexes (G4s) are four-stranded motifs formed by G-rich nucleic acid sequences. These structures harbor significant biological importance as they are involved in telomere maintenance, transcription, and translation. Owing to their dynamic and polymorphic nature, G4 structures relevant for therapeutic applications need to be stabilized by small-molecule ligands. Some of these ligands turn on fluorescence upon binding to G4 structures, which provides a powerful detection platform for G4 structures. Herein, we report the synthesis of fluorescent ligands based on the indolyl-quinolinium moiety to specifically stabilize G4 structures and sense DNA. CD titration and melting experiments have shown that the lead ligand induces the formation of parallel G4 with preferential stabilization of the c-MYC and c-KIT1 promoter G4s over the telomeric, h-RAS1 G4, and duplex DNA. Fluorimetric titration data revealed fluorescence enhancement when these ligands interact with G4 DNA structures. The fluorescence lifetime experiment of the ligand with different DNAs revealed three excited state lifetimes (ns), which indicates more than one binding site. MD studies showed that the ligand exhibits 3 : 1 stoichiometry of binding with c-MYC G4 DNA and revealed the unique structural features, which impart selectivity toward parallel topology. The ligand was found to have low cytotoxicity and exhibited preferential staining of DNA over RNA. Collectively, the results presented here offer avenues to harness indolyl-quinolinium scaffolds for sensing and selective stabilization of G4 structures.

Estrone-Based Derivatives Stabilize the c-MYC and c-KIT G-Quadruplex DNA Structures.

G-rich sequences are present across the genome and can fold to form dynamic secondary structures, namely, G-quadruplexes (G4). These structures play a pivotal role in regulating numerous biological processes including replication, transcription, and translation. Therefore, targeting these structures using molecular scaffolds is an attractive approach to modulating their functions. Herein, we report the synthesis of three estrone-based derivatives (Est-1, Est-2, and Est-3) with a nonplanar core and a cationic alkyl side chain as G4 stabilizers. CD melting and polymerase stop assay results indicate that these ligands preferentially stabilize parallel c-MYC and c-KIT1 G4s over the other G4s and duplex DNAs. The ligand Est-3 shows cytotoxicity against cancer cell lines and effectively downregulates the c-KIT gene in HepG2 cell lines. Molecular modeling and dynamics studies showed that the ligand prefers stacking over the 5'-quartet of c-MYC G4 using the aromatic ring of the ligand. Overall, the findings of this study demonstrate that even G4 ligands can accommodate nonplanar scaffolds, which opens up new avenues for ligand design.