Targeting Oncogene Promoters and Ribosomal RNA Biogenesis by G-Quadruplex Binding Ligands Translate to Anticancer Activity.

G-Quadruplex (GQ) nucleic acids are promising therapeutic targets in anticancer research due to their structural robustness, polymorphism, and gene-regulatory functions. Here, we presented the structure-activity relationship of carbazole-based monocyanine ligands using region-specific functionalization with benzothiazole (TCA and TCZ), lepidine (LCA and LCZ), and quinaldine (QCA and QCZ) acceptor moieties and evaluated their binding profiles with different oncogenic GQs. Their differential turn-on fluorescence emission upon GQ binding confirmed the GQ-to-duplex selectivity of all carbazole ligands, while the isothermal titration calorimetry results showed selective interactions of TCZ and TCA to and GQs, respectively. The aldehyde group in TCA favors stacking interactions with the tetrad of GQ, whereas TCZ provides selective groove interactions with GQ. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) showed that these molecules interfere with the recruitment of specific transcription factors at and promoters and stabilize the promoter GQ structures to inhibit their constitutive transcription in cancer cells. Their intrinsic turn-on fluorescence response with longer lifetimes upon GQ binding allowed real-time visualization of GQ structures at subcellular compartments. Confocal microscopy revealed the uptake of these ligands in the nucleoli, resulting in nucleolar stress. ChIP studies further confirmed the inhibition of Nucleolin occupancy at multiple GQ-enriched regions of ribosomal DNA (rDNA) promoters, which arrested rRNA biogenesis. Therefore, carbazole ligands act as the "double-edged swords" to arrest c-MYC and BCL-2 overexpression as well as rRNA biogenesis, triggering synergistic inhibition of multiple oncogenic pathways and apoptosis in cancer cells.