Targeting Parallel Topology of G-Quadruplex Structures by Indole- Fused Quindoline Scaffolds.

Preferential stabilization of G-quadruplex (G4) structures using small-molecule ligands has emerged as an effective approach to develop anticancer drugs. Herein, we report the synthesis of three indole-fused quindoline derivatives with varying lengths of side chains (InqEt1, InqEt2, and InqPr2) as selective ligands for promoter G4 structures. The ligands stabilize the parallel topology of c-MYC and c-KIT1 promoter G4 DNAs over telomeric and duplex DNAs, as evident from the circular dichroism melting and polymerase stop-assay experiments. The lead ligand, InqPr2, downregulates the gene expression of c-MYC and c-KIT in HeLa and HepG2 cells, respectively, leading to apoptotic cell death. Molecular modeling and dynamics studies support the 2:1 binding stoichiometry revealed from the Job plot analysis and show the ligand's structural features that enable the preferential binding to the parallel G4 structures over other topologies. Our studies show that indole-fused quindoline derivatives can be harnessed as new molecular scaffolds for selective targeting of parallel G4 topologies.

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.

Modulating the dynamics of NFκB and PI3K enhances the ensemble-level TNFR1 signaling mediated apoptotic response.

Cell-to-cell variability during TNFα stimulated Tumor Necrosis Factor Receptor 1 (TNFR1) signaling can lead to single-cell level pro-survival and apoptotic responses. This variability stems from the heterogeneity in signal flow through intracellular signaling entities that regulate the balance between these two phenotypes. Using systematic Boolean dynamic modeling of a TNFR1 signaling network, we demonstrate that the signal flow path variability can be modulated to enable cells favour apoptosis. We developed a computationally efficient approach "Boolean Modeling based Prediction of Steady-state probability of Phenotype Reachability (BM-ProSPR)" to accurately predict the network's ability to settle into different phenotypes. Model analysis juxtaposed with the experimental observations revealed that NFκB and PI3K transient responses guide the XIAP behaviour to coordinate the crucial dynamic cross-talk between the pro-survival and apoptotic arms at the single-cell level. Model predicted the experimental observations that ~31% apoptosis increase can be achieved by arresting Comp1 - IKK* activity which regulates the NFκB and PI3K dynamics. Arresting Comp1 - IKK* activity causes signal flow path re-wiring towards apoptosis without significantly compromising NFκB levels, which govern adequate cell survival. Priming an ensemble of cancerous cells with inhibitors targeting the specific interaction involving Comp1 and IKK* prior to TNFα exposure could enable driving them towards apoptosis.