Structural basis for asymmetric bis-intercalator targeting of DNA triplex junctions enabling dual inhibition of topoisomerase I and oncogene transcription.

Achieving multi-pathway suppression with minimal toxicity remains a major goal in anticancer drug design. Here, we present the structural and mechanistic basis for asymmetric bis-intercalators QA4 and QA5, which integrate weak (quinoxaline) and strong (acridine) chromophores through optimized alkyl linkers to achieve preference recognition of non-canonical DNA triplex junctions. High-resolution crystal structures reveal a spermine-stabilized triplex junction comprising minor-groove-mediated C:G-G triplets and sheared G-G interactions, distinct from classical Hoogsteen triplexes. QA compounds engage this topology via CpG step bis-intercalation, disrupting junction integrity and inducing localized DNA deformation. QA4, with a four-carbon linker, provides optimal spacing for dual chromophore engagement, producing pronounced helical distortion. Structure-function analyses indicate that acridine-mediated DNA distortion preferentially suppresses topoisomerase I, whereas quinoxaline-induced groove deformation is correlated with transcriptional repression of multiple oncogenes, including CEACAM6. In colorectal and lung cancer xenograft models, QA4 demonstrates potent antitumor activity with negligible hepatotoxicity. These findings define the structural principles underlying topology-driven DNA recognition and establish DNA triplex junctions as druggable targets for dual-function anticancer therapeutics.