Targeting oncogene promoter G-quadruplexes (G4s) is a compelling therapeutic strategy against human malignancies. However, clinical progress has been hindered by the lack of potent and structurally diverse G4-targeting ligands. Herein, using parallel KRAS proximal promoter G4 (KRAS-G4) as a model system, we screened an in-house natural product library and identified dehydroevodiamine (DEE) as a novel G4 stabilizer. Although DEE showed only modest anticancer activity, structural analyses determined its predominant binding mode to KRAS-G4. Leveraging these structural insights, we rationally designed and synthesized 15 DEE analogues. Among them, compound 7i emerged as the lead candidate, demonstrating a 9-fold higher binding affinity and up to 20-fold improvement in antiproliferative activity over DEE. We further determined the high-resolution NMR structure of the KRAS-G4-7i complex, uncovering a distinct dual-binding mode, featuring extensive π-π stacking interactions with outer G-tetrads and specific hydrogen bonding within groove regions. Functional analysis showed 7i effectively suppressed transcription of several G4-containing oncogenes, induced genome-wide G4 formation, and triggered DNA damage in colorectal cancer cells. Moreover, 7i significantly inhibited the growth of patient-derived colorectal tumor organoids. Overall, our findings establish a structural framework for rational design of parallel G4-targeting ligands and emphasize an alternative G4-based therapeutic strategy for KRAS-driven cancers.
Keywords: G‐quadruplex; KRAS; NMR structure; cancer therapy; dehydroevodiamine.
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