Journal
BIOCHIMIE
Volume 144, Issue -, Pages 144-152Publisher
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2017.11.004
Keywords
G-quadruplex; KRAS promoter; Acridine orange ligands; NMR spectroscopy; Fluorescent probes
Categories
Funding
- FCT -Foundation for Science and Technology [SFRH/BD/122953/2016]
- Acoes Integradas LusoFrancesas [TC-15/17]
- FCT project Projeto de Investigacao Exploratoria [IF/00959/2015]
- Fundo Social Europeu e Programa Operacional Potencial Humano [IF/00959/2015]
- FEDER funds through the POCI-COMPETE Operational Programme Competitiveness and Internationalisation in Axis I Strengthening research, technological development and innovation [POCI-01-0145-FEDER-007491]
- National Funds, FCT [UID/Multi/00709/2013]
- Conseil regional d'Aquitaine
- SYMBIT [CZ.02.1.01/0.0/0.0/15_ 003/0000477]
- ERDF [CZ.02.1.01/0.0/0.0/15_003/0000477]
- Ligue regionale contre le Cancer
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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 (C-3-C-8), 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 C-8 stabilizes the structure by approximate to 40 degrees 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 mu M and 5.7 mu M. 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. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
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