Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERß, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.

Studies of ternary systems of sulfadiazine with Beta-cyclodextrin and aminoacids / Estudios de sistemas ternarios de sulfadiazina con Beta-ciclodextrina y aminoácidos

Introduction: Cyclodextrins (CD), are known to form inclusion complexes with a variety of guest molecules both in solution and in the solid state. This can lead to the alteration of properties of guest molecules. Unfortunately, the complexation efficiency of CD is rather low, and can be enhanced by formation of ternary complexes using aminoacids (AA). Sulfadiazine (SDZ) is an antibiotic with extremely low water solubility which limits its therapeutic applications and bioavailability. Objetives: The aim of this work was to increase the aqueous solubility of SDZ by preparing ternary complexes of this drug with β-cyclodextrin (βCD) and an AA as a third auxiliary substance. Materials y Methods: Complex formation was studied by phase solubility analysis (PSA), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TG) and scanning electron microscopy (SEM). Results: The apparent stability constants (KC) of the multicomponent complexes were calculated from the solubility diagrams. By the analysis of the NMR spectra, it could be said that the shifts of some protons evidenced the important role of the AA in the formation of multicomponent complexes. Among the AA, Arginine (ARG) proved to have better solubilizing properties for SDZ, reaching an improvement up to 70 times. The use of DSC, TG and SEM suggested the formation of new solid phases between SDZ:βCD:AA. Conclusions: As a result of this research, it was determined that ternary products were more effective in improving drug solubility than the corresponding SDZ:βCD binary system

Introducción: Las ciclodextrinas (CD), son capaces de formar complejos de inclusión con una variedad de moléculas huésped, tanto en solución y en estado sólido, pudiendo producir la modificación de las propiedades de las moléculas huésped. Desafortunadamente, la eficiencia de la formación de complejos con CD es baja, lo cual se puede mejorar mediante la formación de complejos ternarios utilizando aminoácidos (AA). Sulfadiazina (SDZ) es un antibiótico con muy baja solubilidad en agua que limita su biodisponibilidad y sus aplicaciones terapéuticas. Objetivos: El objetivo de este trabajo fue aumentar la solubilidad acuosa de SDZ mediante la preparación de complejos ternarios con β-ciclodextrina (βCD) y un AA como una tercera sustancia. Materiales y Métodos: La formación de complejos se estudió por análisis de solubilidad de fases (PSA), resonancia magnética nuclear (RMN), calorimetría diferencial de barrido (DSC), análisis termogravimétrico (TG) y microscopía electrónica de barrido (SEM). Resultados: Las constantes de estabilidad aparente (KC) de los complejos multi-componentes se calcularon a partir de los diagramas de solubilidad. Por el análisis de los espectros de RMN se constató, por los corrimientos de algunos protones, el papel importante de los AA en la formación de complejos ternarios. Entre los AA, arginina (ARG) demostró tener mejores propiedades de solubilización para SDZ, alcanzando una mejora de hasta 70 veces. El uso de DSC, TG y SEM sugirió la formación de nuevas fases sólidas entre SDZ:βCD:AA