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

Authors :
Tamara A. M. Mocking
Maurice C. M. L. Buzink
Rob Leurs
Henry F. Vischer
Source :
International Journal of Molecular Sciences, Vol 20, Iss 15, p 3724 (2019)
Publication Year :
2019
Publisher :
MDPI AG, 2019.

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 antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H3R responsiveness and re-equilibration of pre-bound antagonists between the tested H3R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix®) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H3R.

Details

Language :
English
ISSN :
14220067 and 20153724
Volume :
20
Issue :
15
Database :
Directory of Open Access Journals
Journal :
International Journal of Molecular Sciences
Publication Type :
Academic Journal
Accession number :
edsdoj.f53b481f374462a8a8f62afc675b1f1
Document Type :
article
Full Text :
https://doi.org/10.3390/ijms20153724