Una sonda de fotoafinidad selectiva que permite evaluar la expresión de los receptores cannabinoides CB2 y la participación de los ligandos en las células humanas
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Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
Published on: August 4, 2015
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Marjolein Soethoudt, Sara C Stolze, Matthias V Westphal1
1Laboratorium für Organische Chemie , Eidgenössische Technische Hochschule Zürich , Vladimir-Prelog-Weg 3 , Zürich 8093 , Switzerland.
Journal of the American Chemical Society
|February 9, 2018
Resumen
Los investigadores desarrollaron LEI121, una nueva sonda fotorreactiva para el receptor cannabinoide tipo 2 (CB2R). Esta herramienta permite estudios de compromiso objetivo y visualización de CB2R en células inmunes humanas, ayudando al descubrimiento de fármacos.
Área de la Ciencia:
- Farmacología
- Biología Química
- Inmunología
Sus antecedentes:
- Los receptores acoplados a proteínas G (GPCR) son objetivos farmacológicos cruciales.
- El desarrollo de herramientas químicas para estudiar los GPCR, como el receptor cannabinoide tipo 2 (CB2R), es esencial para comprender su papel en la enfermedad.
- CB2R es un objetivo prometedor para el tratamiento de enfermedades inflamatorias y lesiones tisulares.
Objetivo del estudio:
- Desarrollar una sonda química nueva, selectiva y fotorreactiva para CB2R.
- Para demostrar la utilidad de la sonda en la evaluación de la participación del objetivo y la visualización de la expresión del receptor endógeno.
- Para validar la aplicación de la sonda en las células inmunes humanas para la investigación traslacional.
Principales métodos:
- Síntesis y caracterización de LEI121, una sonda fotorreactiva bifuncional con un mango de alquina.
- Etiquetado de fotoafinidad de CB2R utilizando el LEI121.
- Introducción de los grupos de reporteros a través de la química del clic.
- Análisis de citometría de flujo de la expresión y el compromiso endógenos de CB2R en HL-60 y células inmunes humanas primarias.
Principales resultados:
- LEI121 es la primera sonda bifuncional selectiva CB2R que se une covalentemente a CB2R después de la fotoactivación.
- El mango de alquina en el LEI121 permite una fácil introducción de moléculas reporteras.
- LEI121 permitió con éxito los estudios de compromiso objetivo y la visualización de CB2R endógeno en células inmunes humanas.
- La sonda demostró eficacia en el monitoreo de la expresión y el compromiso de GPCR.
Conclusiones:
- Las sondas fotorreactivas diseñadas estratégicamente, como LEI121, son herramientas valiosas para estudiar los GPCR.
- La química de fotoclic en tándem con LEI121 permite el monitoreo de la expresión y el compromiso endógenos de GPCR en células humanas.
- Este enfoque es muy prometedor para el desarrollo de biomarcadores en el descubrimiento de fármacos traslacionales para enfermedades relacionadas con CB2R.
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