Descripción del proyecto
Un protocolo de ensayo de unión de ligandos sienta las bases de nuevas opciones de tratamiento
La acidosis del tejido encefálico es una consecuencia del aumento de la tensión de dióxido de carbono o de una acumulación de los ácidos producidos por el metabolismo. Normalmente, la acidificación local del tejido encefálico sirve como señal neuronal, transducida a través de los canales iónicos que detectan el ácido (ASIC1a, por sus siglas en inglés). La acidosis local se ha relacionado con afecciones como el dolor crónico, el ictus isquémico y trastornos psiquiátricos. Recientemente, se ha señalado la posible función terapéutica de los ASIC1a, pero en la actualidad no hay fármacos específicos disponibles para los canales en condiciones patológicas. El proyecto hsPCF-FRET, financiado con fondos europeos, pretende caracterizar la unión del neuropéptido dinorfina «grande» a los ASIC1a en tiempo real e identificar los sitios de unión y las interacciones. Los resultados contribuirán al futuro diseño de inhibidores de ASIC1a con el potencial para tratar el dolor crónico y la isquemia.
Objetivo
Under physiological conditions, localized acidification of brain tissue serves as neuronal signal that get synaptically transduced via acid-sensing ion channels (ASIC1a). Local acidosis has, however, also been linked to some of the most prevalent neurological disorders such as chronic pain, ischemic stroke and psychiatric diseases. ASIC1a has thus emerged as drug target with great potential, but no drugs are currently available that specifically target the channels under pathological conditions. A few known neuropeptides modulate ASIC1a and could thus serve as scaffolds for a new generation of ASIC1a-selective drugs to, for example, treat pain without the typical downsides of opioids. Advances have, however, been hampered by the limited understanding of detailed protein-peptide interactions. Thus, the aim of the proposed project is to directly characterize the binding of the neuropeptide Big Dynorphin to ASIC1a in real time. Here, I will use a unique in-house developed high-sensitivity fluorescence patch-clamp electrophysiology setup and establish a protocol for a FRET-based ligand-binding assay. Together with site-directed mutagenesis, this approach will be able to identify state-dependent binding sites and key interactions, and allow direct analysis of binding affinity and kinetics under pathological conditions; all in intact membranes and with unprecedented (microsecond) temporal resolution. This information will aid future design of ASIC inhibitors with the potential to treat chronic pain and ischemia. The technology developed for this work will also enable ligand-binding studies of other membrane proteins in living cells and with high temporal resolution and will thus be of great potential value for a broad field. The project will expand my existing electrophysiology skills and add highly versatile expertise in fluorescent measurements. I thus anticipate my project to have significant personal and scientific impact beyond the scope of this proposal.
Ámbito científico
Programa(s)
Régimen de financiación
MSCA-IF-EF-ST - Standard EFCoordinador
1165 Kobenhavn
Dinamarca