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

Abstract

The pharmacodynamics of drug-candidates is often optimized by metrics that describe target binding (Kd or Ki value) or target modulation (IC50). However, these metrics are determined at equilibrium conditions, and consequently information regarding the onset and offset of target engagement and modulation is lost. Drug-target residence time is a measure for the lifetime of the drug-target complex, which has recently been receiving considerable interest, as target residence time is shown to have prognostic value for the in vivo efficacy of several drugs. In this study, we have investigated the relation between the increased residence time of antihistamines at the histamine H1 receptor (H1R) and the duration of effective target-inhibition by these antagonists. Hela cells, endogenously expressing low levels of the H1R, were incubated with a series of antihistamines and dissociation was initiated by washing away the unbound antihistamines. Using a calcium-sensitive fluorescent dye and a label free, dynamic mass redistribution based assay, functional recovery of the H1R responsiveness was measured by stimulating the cells with histamine over time, and the recovery was quantified as the receptor recovery time. Using these assays, we determined that the receptor recovery time for a set of antihistamines differed more than 40-fold and was highly correlated to their H1R residence times, as determined with competitive radioligand binding experiments to the H1R in a cell homogenate. Thus, the receptor recovery time is proposed as a cell-based and physiologically relevant metric for the lead optimization of G protein-coupled receptor antagonists, like the H1R antagonists. Both, label-free or real-time, classical signaling assays allow an efficient and physiologically relevant determination of kinetic properties of drug molecules.

Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes

“…Previous research on GPCRs synthesized without G-proteins indicated that the GPCRs had low affinity for some antagonists and agonists because they were not in their active state ( Fitzsimons et al, 2004 ). However, homogenates of HEK293T cells transiently expressing the HRH1 demonstrated that the affinity for pyrilamine was comparable to that described in previous studies ( Bosma et al, 2017 ) because this homogenate included G-proteins. To achieve a synthesis of active state GPCRs, which have the same binding affinity for all ligands, a future study of the co-synthesis of HRH1 and G-proteins is suggested.…”

A structure–kinetic relationship study using matched molecular pair analysis

“…The kinetic dataset KIND (KINetic Dataset) contains a total of 3812 structures and their kinetic data triplets (k on , k off , K D ). It has been compiled from 21 publications 16,19,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] and the K4DD database (for details see the ESI, † the dataset is provided in KIND.xlsx). For the literature search, only papers containing numerical values for all three parameters investigated (K D , k on and k off ) were selected.…”

Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor

“…A chemically diverse set of unlabeled H 1 R ligands (structures are depicted in Supplementary Fig. 2), including reference molecules with known differences in their H 1 R binding kinetics, was selected for characterization of their H 1 R binding kinetics using either [ 3 H]mepyramine or [ 3 H]levocetirizine 13,23,31 . To guide the design of competitive association experiments, binding affinities (K i ) of the unlabeled ligands were first determined by equilibrium competition binding.…”

“… a pK d,kin = k off /k on. b Values were reported before except for (S)fexofenadine, (R)fexofenadine and (S)cetirizine 23 . c RT = residence time = 1/k off. …”

“…Other employed, commercially available ligands were: triprolidine hydrochloride monohydrate (Tocris Bioscience, Bristol, United Kingdom), doxepin hydrochloride (Tocris Bioscience, E/Z mixture with a ~85:15 ratio), Olopatadine hydrochloride (BOC Sciences, Shirley, NY, USA), acrivastine (BOC Sciences), levocetirizine dihydrochloride (Biotrend, Cologne, Germany), S-cetirizine dihydrochloride (TLC PharmaChem, Mississauga, Canada), Mepyramine maleate (Research Biochemicals International, Natick, MA, USA), R-fexofenadine (Sepracor Inc., Marlborough, MA, USA), S-fexofenadine (Sepracor Inc.), desloratadine (HaiHang Industry, Jinan City, China), Terfenadine (MP biomedicals, Santa Ana, CA, USA). VUF14454, VUF14544, VUF14506, VUF14493 and mianserin were synthesized at the Vrije Universiteit Amsterdam and were fully characterized with respect to purity and identity 22,23 . [ 3 H]levocetirizine (25.9 Ci/mmol) was synthesized at AstraZeneca and was fully characterized with respect to purity and identity.…”