Bioluminescence Resonance Energy Transfer Based G Protein-Activation Assay to Probe Duration of Antagonism at the Histamine H3 Receptor

International Journal of Molecular Sciences
Tamara A M MockingHenry F Vischer

Abstract

Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the 'effective' target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβ1γ2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gαi-coupled histamine H3 receptor (H3R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess anta...Continue Reading

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Citations

Mar 19, 2020·International Journal of Molecular Sciences·Francisco Ciruela
Dec 15, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Gábor WágnerRob Leurs
Jan 16, 2021·International Journal of Molecular Sciences·Tausif AltamashKabir H Biswas

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Methods Mentioned

BETA
competition binding
BRET
biosensor

Software Mentioned

Prism
Graphpad Prism
GraphPad

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