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.

通常在致死性癌症中发现KRAS发生突变，并且应该成为抗癌药物开发的重要目标。但是，到目前为止，尚无有效抑制剂的报道，这促使科学界将RAS蛋白描述为几乎“不可吸收的”。开发来调节KRAS蛋白表达的最新方法包括通过设计小的和特异性的配体来稳定三倍折叠并减少基因表达，从而靶向在KRAS启动子区域的核酸酶超敏元件内形成的G-四链体（G4）结构。在这项工作中，我们报告在体外和在计算机芯片上新颖的吖啶橙的研究（AO）衍生物（C ₃ -C ₈），用作G4稳定剂。结果表明，这些配体以高亲和力结合并稳定了KRAS22-RT G4，其相对于双链DNA具有适度的特异性。最有前途的配体C _8可在≈40°C的温度下稳定结构。使用NMR衍生的距离限制的分子对接揭示了与KRAS22-RT G4相互作用时有关配体结构特征的原子细节。HeLa细胞的体外研究表明，配体具有细胞毒性，IC50值在0.9μM至5.7μM之间。此外，如共聚焦荧光显微镜所示，配体倾向于位于核内。总体而言，这些结果表明，所报道的AO​​配体表现出与G4配体相同的良好性能，并且该研究为铅KRAS G4配体的开发提供了结构细节。