Structure‐Informed Design of Distinct Parallel G‐Quadruplex Stabilizers for KRAS ‐Driven Cancer Therapy

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.