A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H₃ Receptor

Abstract

The histamine H 3 receptor (H 3 R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H 3 R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H 3 R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H 3 R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H 3 R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H 3 R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

Structural and Molecular Insight into Piperazine and Piperidine Derivatives as Histamine H3 and Sigma-1 Receptor Antagonists with Promising Antinociceptive Properties

“…As it is well-known that σ 1 R antagonism enhances opioid analgesia, ,, we tested the effects of compounds 5 and 11 on antinociception induced by the opioid agonist loperamide. It is worth mentioning that loperamide is known to lack affinity for σ 1 R; S1RA was used as a σ 1 R reference antagonist.…”

“…As it is well-known that σ 1 R antagonism enhances opioid analgesia, ,, we tested the effects of compounds 5 and 11 on antinociception induced by the opioid agonist loperamide. It is worth mentioning that loperamide is known to lack affinity for σ 1 R; S1RA was used as a σ 1 R reference antagonist. Both S1RA and 5 were administered intraplantarally (ipl) at a dose of 100 μg, while 11 (due to solubility problems) was tested at a dose of 50 μg.…”

“…Compounds (as oxalate salts) were tested in H 3 R in vitro binding studies, using methods described previously. , Ligands were tested at 5 to 11 appropriate concentrations in a [ 3 H] N α -methylhistamine ( K D = 3.08 nM) radioligand depletion assay to determine the affinity at human recombinant histamine H 3 R stably expressed in HEK293 cells. Radioligand binding experiments at the H 1 R, H 2 R, and H 4 R were performed as previously described in Rosier et al and Bartole et al with HEK293T-SP-FLAG-hH x R ( x = 1, 2, or 4) expressing the respective hHR. The following radioligands and concentrations were used: [ 3 H]­mepyramine (hH 1 R, K d = 5.1 nM, c = 5 nM; Novandi Chemistry AB, Södertälje, Sweden), [ 3 H]­UR-DE257 (hH 2 R, K d = 66.9 nM, c = 50 nM), [ 3 H]­UR-PI294 (hH 4 R, K d = 3.6 nM, c = 4 nM).…”

“…Radioligand binding experiments at the H 1 R, H 2 R, and H 4 R were performed as previously described in Rosier et al 70 73 . Data represent mean values ± CI from three independent experiments, each performed in triplicate.…”

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors

“…Detailed structure–function knowledge is typically necessary to identify ligand positions that can be harnessed for linker/fluorophore functionalization. This is particularly challenging for small ligands but has been successfully accomplished for adenosine, − histamine, − and ß-adrenergic , receptors. Melatonin receptors present a particularly difficult case in this respect.…”