Targeting Oncogene Promoters and Ribosomal RNA Biogenesis\nby G‑Quadruplex Binding Ligands Translate to Anticancer Activity

Abstract

G-Quadruplex (GQ)\nnucleic acids are promising therapeutic targets\nin anticancer research due to their structural robustness, polymorphism,\nand gene-regulatory functions. Here, we presented the structure–activity\nrelationship of carbazole-based monocyanine ligands using region-specific\nfunctionalization with benzothiazole (TCA and TCZ), lepidine (LCA\nand LCZ), and quinaldine (QCA and QCZ) acceptor moieties and evaluated\ntheir binding profiles with different oncogenic GQs. Their differential\nturn-on fluorescence emission upon GQ binding confirmed the GQ-to-duplex\nselectivity of all carbazole ligands, while the isothermal titration\ncalorimetry results showed selective interactions of TCZ and TCA to <i>c-MYC</i> and <i>BCL-2</i> GQs, respectively. The\naldehyde group in TCA favors stacking interactions with the tetrad\nof <i>BCL-2</i> GQ, whereas TCZ provides selective groove\ninteractions with <i>c-MYC</i> GQ. Dual-luciferase assay\nand chromatin immunoprecipitation (ChIP) showed that these molecules\ninterfere with the recruitment of specific transcription factors at <i>c-MYC</i> and <i>BCL-2</i> promoters and stabilize\nthe promoter GQ structures to inhibit their constitutive transcription\nin cancer cells. Their intrinsic turn-on fluorescence response with\nlonger lifetimes upon GQ binding allowed real-time visualization of\nGQ structures at subcellular compartments. Confocal microscopy revealed\nthe uptake of these ligands in the nucleoli, resulting in nucleolar\nstress. ChIP studies further confirmed the inhibition of Nucleolin\noccupancy at multiple GQ-enriched regions of ribosomal DNA (rDNA)\npromoters, which arrested rRNA biogenesis. Therefore, carbazole ligands\nact as the “double-edged swords” to arrest c-MYC and\nBCL-2 overexpression as well as rRNA biogenesis, triggering synergistic\ninhibition of multiple oncogenic pathways and apoptosis in cancer\ncells.