Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

Abstract

Receptor-binding affinity and ligand-receptor residence time are key parameters for the selection of drug candidates and are routinely determined using radioligand competition-binding assays. Recently, a novel bioluminescence resonance energy transfer (BRET) method utilizing a NanoLuc-fused receptor was introduced to detect fluorescent ligand binding. Moreover, this NanoBRET method gives the opportunity to follow fluorescent ligand binding on intact cells in real time, and therefore, results might better reflect in vivo conditions as compared with the routinely used cell homogenates or purified membrane fractions. In this study, a real-time NanoBRET-based binding assay was established and validated to detect binding of unlabeled ligands to the histamine H 3 receptor (H 3 R) and histamine H 4 receptor on intact cells. Obtained residence times of clinically tested H 3 R antagonists were reflected by their duration of H 3 R antagonism in a functional receptor recovery assay.

Covalent Inhibition of the Histamine H3 Receptor

“…We therefore believe that the identified covalent inverse agonist 44 could aid in stabilizing the H 3 R in the inactive conformation and as such function as a tool compound in H 3 R crystallization. The covalent binder 44 might also serve as a useful extension of the diverse H 3 R compound set for studying H 3 R signaling [39,44].…”

“…To further validate that 44 binds irreversibly to the H 3 R, recovery of [ 3 H]NAMH binding after washout of H 3 R-expressing cell homogenates preincubated with 10 µM 44 or 42 was compared to washout of three reference non-covalent H 3 R ligands (namely thioperamide, pitolisant, and histamine) with relatively long to short residence times [39]. Indeed, 10 µM 44 could significantly (p < 0.05, one-way ANOVA with Fisher's LSD test) inhibit recovery of [ 3 H]NAMH binding after washout, while washout of H 3 R expressing cell homogenates preincubated with 42, histamine, pitolisant, or thioperamide recovered [ 3 H]NAMH occupancy to vehicle levels ( Figure 5A).…”

“…Cell homogenates expressing the hH 3 R were harvested 48 h after transfection as reported previously [39].…”

“…Tert-butyl 4-(3-nitrophenyl)piperazine-1-carboxylate (39). A solution of bromide 38 (808 mg, 4.00 mmol) and NaOtBu (461 mg, 4.80 mmol) in dioxane (15 mL) was degassed with N 2 for 10 min.…”

NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing

“…Furthermore, the assay is truly homogenous with no wash or lysis step necessary, enabling kinetic measurements of ligand binding to be performed with relative ease which would be time-consuming and cumbersome in traditional radio-ligand binding studies. Indeed, due to its relative ease of use and adaptability, since the initial description of NanoBRET ligand binding, this method has been used in a variety of binding modes (saturation, kinetic or competition) to investigate fluorescent ligand binding at a number of GPCRs including: adenosine A1 and A3, as well as angiotensin II receptor type 1 17 ; β1 and β2-adrenoceptors 17,19 ; free fatty acid receptors 1 and 2 20,21 ; histamine H1, H3 and H4 receptors 22,23 ; relaxin family peptide receptor 3 24 ; and P2Y2 receptor 25 . It has also been used to study the receptor tyrosine kinase vascular endothelial growth factor 2 26 and its co-receptor neuropilin-1 27 .…”