RESUMEN
The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.
Asunto(s)
G-Cuádruplex , MicroARNs , Humanos , Ligandos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Carcinogénesis , NucleolinaRESUMEN
The development of novel biomarkers for early-stage diagnosis of prostate cancer (PCa) has attracted the attention of researchers in the last decade. Nucleolin (NCL) has emerged as a possible biomarker of PCa due to its high expression levels in the surface of PCa cells and affinity towards parallel G4s since it contains four RNA-binding domains (RBDs). Herein, we developed a novel strategy based on a microfluidic platform for the detection of NCL in biological samples, such as human plasma. The RNA G4 (rG4) sequence found in human precursor microRNA 92b (pre-miR-92b) was used as a molecular recognition probe since it forms a single dominant parallel rG4 conformation in the presence of 0.1 mM K+ as confirmed by NMR spectroscopy. The additional stability of the rG4 structure was provided by the acridine orange derivative ligand C8, which stabilizes the pre-miR-92b rG4 structure, as denoted by an increase in more than 30 °C of its melting temperature. FRET-melting assay revealed a remarkable synergistic effect of NCL RBD1,2 and C8 on the stabilization of the pre-miR-92b rG4. The binding of pre-miR-92b to NCL RBD1,2 was determined by in silico studies, which revealed a binding pocket formed by a 12-residue linker between RBD1 and RBD2. Both, pre-miR-92b rG4 and pre-miR-92b rG4/C8 complex demonstrated high affinity towards NCL RBD1,2, as proved by fluorimetric titrations (KD range between 10-12 and 10-9 M). The stability and nuclease resistance of pre-miR-92b rG4 and pre-miR-92b rG4/C8 complex were evaluated as molecular recognition probes to capture and detect NCL. Finally, the microfluidic platform detects NCL in complex biological samples, such as human plasma. Overall, this work demonstrates the usefulness of the microfluidic platform based on the pre-miR-92b to detect NCL and the possibility to be used as a valuable biomedical tool in PCa diagnosis.
Asunto(s)
G-Cuádruplex , MicroARNs/química , MicroARNs/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Humanos , Técnicas Analíticas Microfluídicas/métodos , Simulación del Acoplamiento Molecular/métodos , Unión Proteica/fisiología , Estructura Secundaria de Proteína , NucleolinaRESUMEN
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.
Asunto(s)
Naranja de Acridina/química , Naranja de Acridina/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , G-Cuádruplex/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Naranja de Acridina/metabolismo , Transporte Biológico , Proliferación Celular/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Células HeLa , Humanos , Espacio Intracelular/metabolismo , LigandosRESUMEN
Current methods for sentinel lymph node (SLN) mapping involve the use of radioactivity detection with technetium-99m sulfur colloid and/or visually guided identification using a blue dye. To overcome the kinetic variations of two individual imaging agents through the lymphatic system, we report herein on two multifunctional macromolecules, 5a and 6a, that contain a radionuclide ((99m)Tc or (68)Ga) and a near-infrared (NIR) reporter for pre- and/or intraoperative SLN mapping by nuclear and NIR optical imaging techniques. Both bimodal probes are dextran-based polymers (10 kDa) functionalized with pyrazole-diamine (Pz) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating units for labeling with fac-[(99m)Tc(CO)3](+) or (68)Ga(III), respectively, mannose units for receptor targeting, and NIR fluorophore units for optical imaging. The probes allowed a clear visualization of the popliteal node by single-photon emission computed tomography (SPECT/CT) or positron emission tomography (PET/CT), as well as real-time optically guided excision. Biodistribution studies confirmed that both macromolecules present a significant accumulation in the popliteal node (5a: 3.87 ± 0.63% IA/organ; 6a: 1.04 ± 0.26% IA/organ), with minimal spread to other organs. The multifunctional nanoplatforms display a popliteal extraction efficiency >90%, highlighting their potential to be further explored as dual imaging agents.