Binding Characteristics of Cetirizine and Levocetirizine to Human H1 Histamine Receptors: Contribution of Lys

Competition experiments with [H]mepyramine showed that cetirizine and its enantiomers, levocetirizine and (S)-cetirizine, bound with high affinity and stereoselectivity to human H1 histamine receptors (Ki values of 6, 3, and 100 nM, respectively). Cetirizine and levocetirizine were 600-fold more selective for H1 receptors compared with a panel of receptors and channels. Binding results indicated that the interaction between cetirizine, its enantiomers, and histamine is compatible with a competitive behavior, in contrast with the noncompetitive profile of cetirizine and levocetirizine observed in isolated organs. Binding kinetics provided a suitable explanation for this observation, because levocetirizine dissociated from H1 receptors with a half-time of 142 min; that of (S)-cetirizine was only 6 min, implying that the former could act as a pseudo-irreversible antagonist in functional studies. The carboxylic function of levocetirizine seemed responsible for its long dissociation time. Indeed, hydroxyl or methyl ester analogs dissociated more rapidly from H1 receptors, with half-times of 31 min and 7 min, respectively. The importance of the carboxylic function of levocetirizine for the interaction with the H1 receptor was further supported by the results from the mutation of Lys to Ala. This mutation decreased the dissociation half-time of levocetirizine from 142 to 13 min and reduced its affinity from 3 to 12 nM, whereas the affinity and dissociation kinetics of hydroxyl and methyl ester analogs were hardly affected. The mutation of Thr reduced the binding stereoselectivity by selectively enhancing the affinity of the distomer. The bioamine histamine produces a variety of physiological and pathophysiological effects through binding and activation of histamine receptors belonging to the superfamily of seven transmembrane G-protein-coupled receptors (Hill et al., 1997). Today, four human histamine receptor subtypes have been cloned: H1 (Moguilevsky et al., 1994), H2 (Gantz et al., 1991), H3 (Lovenberg et al., 1999), and, more recently, H4 (Oda et al., 2000). H1 receptors induce smooth muscle contraction and increase vascular permeability and H1 antagonists constitute a medication of choice to alleviate the symptoms of allergies. Cetirizine and levocetirizine are second-generation antihistamines. As opposed to first generation drugs, exemplified by hydroxyzine, chlorpheniramine, diphenhydramine, or ketotifen, second-generation drugs are nonsedating or less sedating, probably because of an improved H1 binding selectivity and reduced brain penetration (Timmerman, 1999). Structure-activity relationships and site-directed mutagenesis experiments performed with the guinea pig H1 receptor have provided data that have led to model pharmacophores of H1 antagonists (ter Laak et al., 1995; Wieland et al., 1999). Since the cloning of the H1 receptor, several studies have been published on mutant receptors designed to better identify the binding pocket and the amino acids residues involved in the binding of histamine and histamine antagonists (Fig. 1). This is of particular interest today in light of recent findings showing that most histamine H1 antagonists exhibit inverse agonist properties (Bakker et al., 2000). Asp, located in the third transmembrane domain of the human receptor, is crucial for the affinity of histamine and histamine antagonists (Ohta et al., 1994); this amino acid is a hallmark of GABBREVIATIONS: [H]RX821002, 1,4-[6,7(n)-H]benzodiazoxan-2-methoxy-2-yl)-2-imidazoline hydrochloride; [H]mepyramine, pyridinyl-5[H]pyrilamine; SCH23390 R( )-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SPA, scintillation proximity assay; -MEM, -Modified Eagle’s minimal essential medium; CHO, Chinese hamster ovary; [H]NMS, l-N-methyl-[H]scopolamine methyl chloride; [H]N -methylhistamine, N-[methyl-H]methylhistamine; [H]8-OH-DPAT, [propyl-2,3-ring-1,2,3-H]8-hydroxy-dipropylaminotetralin; [H]CGP12177, [5,7-H]( )CGP-12177; [H]spiperone, [benzene ring-H]spiperone; [H]DPCPX, 8-[dipropyl-2,3-H(N)]cyclopenthyl-1,3-dipropylxanthine; [H]ketanserin, [ethylene-H]ketanserin hydrochloride; [H]tiotidine, [methyl-H]tiotidine; [H]prazosin, [7-methoxy-H]prazosin. 0026-895X/02/6102-391–399$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 2 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1317/961623 Mol Pharmacol 61:391–399, 2002 Printed in U.S.A. 391 at A PE T Jornals on D ecem er 9, 2017 m oharm .aspeurnals.org D ow nladed from protein-coupled receptors, whose natural ligands are bioamines, and is responsible for forming an ionic bond with the protonated nitrogen of the neurotransmitter (Hibert et al., 1991). Asn (Leurs et al., 1994; Ohta et al., 1994; Moguilevsky et al., 1995) and Lys (Leurs et al., 1995) are also involved in histamine binding to the H1 receptor in human and guinea pig, respectively. By analogy with the histamine H2 receptor, Thr 194 was expected to participate in the binding of histamine to human H1 receptors, but the mutation of this residue into Ala led to a receptor that kept its ability to bind histamine and histamine antagonists with affinities very similar to that of the wild-type receptor (Leurs et al., 1994; Ohta et al., 1994). However, we have shown that the mutation of Thr to Ala decreased the stereoselectivity of the enantiomers of cetirizine by increasing the affinity of the distomer (Moguilevsky et al., 1995). Finally, Lys, the guinea pig equivalent of human Lys, was reported to interact with the carboxylic acid moiety of two second-generation antagonists, acrivastine and cetirizine (Wieland et al., 1999). In this report, we further explore the binding characteristics of cetirizine and its enantiomers to the wild-type human H1 receptor in comparison with receptors bearing mutations at key amino acids (Lys and Thr) known to be involved in ligand binding. Close structural analogs of cetirizine were also included in this study to explore the role of the carboxyl group in binding to the H1 receptor, under both equilibrium and nonequilibrium conditions. Materials and Methods