KRAS is often found mutated in lethal cancers and should be an important target for anticancer drug development. However, no effective inhibitor has been reported so far, prompting the scientific community to describe the RAS proteins as nearly "undruggable". Recent approaches developed to modulate KRAS protein expression comprises the targeting of G-quadruplex (G4) structures formed within the nuclease hypersensitive element of KRAS promoter region, by designing small and specific ligands to stabilize the tertiary fold and reduce gene expression. In this work, we report in vitro and in silico studies of novel acridine orange (AO) derivatives (C3-C8), developed as G4 stabilizing agents. The results show that the ligands bind with high affinity and stabilize KRAS22-RT G4 with modest specificity over duplex DNA. The most promising ligand C8 stabilizes the structure by ≈ 40 °C. Molecular docking using NMR-derived distance restraints reveal atomic details about the ligand structural features in the interaction with KRAS22-RT G4. In vitro studies with HeLa cells show that the ligands are cytotoxic with IC50 values between 0.9 μM and 5.7 μM. Moreover, the ligands tend to localize in the nucleus as shown by confocal fluorescence microscopy. Overall, these results show that the reported AO ligands display favourable properties as G4 ligands and this study provides structural detail for the development of lead KRAS G4 ligands.

Centro de Ciências e Tecnologias Nucleares Instituto Superior Técnico Universidade de Lisboa Estrada Nacional 10 2695 066 Bobadela LRS Portugal

CICS UBI Centro de Investigação em Ciências da Saúde Universidade da Beira Interior Av Infante D Henrique 6200 506 Covilhã Portugal

Institute of Biophysics AS CR v v i Kralovopolska 135 612 65 Brno Czech Republic

Univ Bordeaux ARNA Laboratory INSERM U1212 CNRS UMR 5320 IECB F 33600 Pessac France

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