BRET-based β-arrestin2 recruitment to the histamine H 1 receptor for investigating antihistamine binding kinetics

The Target Residence Time of Antihistamines Determines Their Antagonism of the G Protein-Coupled Histamine H1 Receptor

“…Pharmacological tool compounds were acquired from the following commercial sources: probenecid, olopatadine hydrochloride and acrivastine from Sigma–Aldrich (St. Louis, MO, United States); Histamine hydrochloride from TCI chemicals (Portland, OR, United States); mepyramine maleic acid from Research Biochemicals International (Natick, MA, United States); levocetirizine dihydrochloride from Biotrend (Köln, Germany); doxepin hydrochloride and triprolidine hydrochloride from Tocris Bioscience (Bristol, United Kingdom); desloratadine from HaiHang Industry, Co., Ltd (Jinan City, China). HeLa cells and HEK293T cells were from an in-house eukaryotic cell biobank as described in previous publications ( Bakker et al, 2004 ; Bosma et al, 2016 ). Compounds VUF14454, VUF14493, VUF14506, and VUF14544 were synthesized at the Vrije Universiteit Amsterdam and were fully characterized with respect to purity and identity.…”

“…HEK293T cells and HeLa cells were cultured in a humidified atmosphere at 37°C with 5% CO 2 in DMEM and DMEM/F12 medium respectively which was supplemented with 10% FBS and 1x penicillin/streptomycin. Cell pellets of HEK293T cells transiently expressing the HA-hH 1 R were derived as described before ( Bosma et al, 2016 ). Cell pellets of HeLa cells were derived by flushing cells from a sub confluent 10 cm 2 dish.…”

“…Characterization of [ 3 H]mepyramine and unlabeled ligands using radioligand binding experiments was described extensively before with minor changes ( Bosma et al, 2016 ). In short, cell pellets were reconstituted in binding buffer [50 mM Na 2 HPO 4 /KH 2 PO 4 pH 7.4] and homogenized.…”

“…Kinetic binding rate constants of [ 3 H]mepyramine were determined to be 0.22 min -1 ( k off ) and 1.1 × 10 8 M -1 min -1 ( k on ) ( Bosma et al, 2016 ). To determine the kinetic binding rate constants of unlabeled ligands (0.5–3 μg) a HEK293T cell homogenate transiently expressing the H 1 R was co-incubated with a single concentration [ 3 H]mepyramine 1.5–12 nM for various incubation times (0–81 min) at 25°C in the presence and absence of three concentrations unlabeled ligand.…”

“…Additionally, compounds with a long residence time show prolonged drug-target occupancies beyond the point at which pharmacologically active drug concentrations are present in the blood ( Ramsey et al, 2011 ; Bradshaw et al, 2015 ). A long residence time (>1 h) was, e.g., observed for several clinically used antihistamines that bind to the histamine H 1 receptor (H 1 R), an important drug target for the treatment of, e.g., allergic rhinitis ( Anthes et al, 2002 ; Gillard et al, 2002 ; Slack et al, 2011b ; Bosma et al, 2016 ). In analogy with insurmountable antagonism observed in neuronal signaling, it has been described that histamine levels after allergen challenge are only transiently increased, implying that an insurmountable mode of antagonism could effectively block high concentrations of histamine ( Petersen et al, 1996 ).…”