Fluorescent light-up acridine orange derivatives bind and stabilize KRAS-22RT G-quadruplex

Area of Science:

• Medicinal Chemistry
• Molecular Biology
• Drug Discovery

Background:

• KRAS mutations are prevalent in lethal cancers, making KRAS a critical therapeutic target.
• RAS proteins are considered 'undruggable' due to the lack of effective inhibitors.
• Targeting KRAS G-quadruplex (G4) structures in the promoter region offers a novel strategy to reduce gene expression.

Purpose of the Study:

• To design and evaluate novel acridine orange (AO) derivatives as G4 stabilizing agents targeting the KRAS promoter.
• To investigate the in vitro and in silico binding affinity, specificity, and stabilizing effects of these ligands on the KRAS G4 structure.
• To assess the cytotoxicity and cellular localization of the developed AO derivatives.

Main Methods:

• Synthesis and characterization of novel acridine orange derivatives (C3-C8).
• In vitro G4 stabilization assays (thermal melting) and DNA binding studies.
• In silico molecular docking studies using NMR-derived distance restraints.
• In vitro cytotoxicity assays (IC50 values) and confocal fluorescence microscopy for cellular localization.

Main Results:

• The novel AO derivatives bind with high affinity and stabilize the KRAS22-RT G4 structure.
• Ligand C8 demonstrated significant stabilization of the G4 structure (≈40 °C).
• The ligands showed cytotoxicity in HeLa cells (IC50 values 0.9–5.7 μM) and localized in the nucleus.

Conclusions:

• The developed AO derivatives are effective G4 ligands with favorable properties for potential anticancer drug development.
• These ligands show promise for targeting KRAS G4 structures, offering a new avenue for modulating KRAS expression.
• The study provides crucial structural insights for the future development of lead KRAS G4 ligands.