A NanoBRET-Based H(3)R Conformational Biosensor to Study Real-Time H(3) Receptor Pharmacology in Cell Membranes and Living Cells

Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H(3) receptor (H(3)R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H(3)R ligands. In the current study, we have further characterized this H(3)R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H(3)R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H(3)R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay.