Mutational analysis of the histamine H₁-receptor binding pocket of histaprodifens

Abstract

Histaprodifens constitute a new class of histamine H₁-receptor agonists. These ligands can be regarded as hybrid molecules, consisting of a histamine moiety linked at the two-position of the imidazole ring by a propyl chain to two phenyl rings, one of the characteristic features of several H₁-receptor antagonists. To delineate the binding site of various histaprodifen-like ligands, we generated mutant histamine H₁ receptors, in which various amino acids, involved in the binding of either histamine or H₁-receptor antagonists, were replaced by alanine. Wild-type and mutant H₁ receptors were transiently expressed in African green monkey kidney cells (COS-7) and evaluated for their interaction with histamine and various histaprodifens by [³H]mepyramine radioligand-binding studies and by nuclear factor κB (NF-κB) reporter-gene assays. Our data show that, within the histamine H₁-receptor binding pocket, histaprodifens interact with both agonist and antagonist binding sites, resulting in high affinity histamine H₁-receptor agonists.

Introduction

The histamine H₁ receptor plays an important role in allergic conditions like rhinitis, asthma, anaphylaxis and uriticaria (Hill et al., 1997). Consequently, histamine H₁-receptor antagonists constitute the medication of choice to alleviate symptoms of allergies. The rationalization of the molecular mechanism of action of these successful therapeutics have for many years been hampered by the lack of detailed knowledge of the histamine H₁ receptor. Using an expression cloning strategy, Yamashita et al. (1991) revealed that the bovine histamine H₁ receptor belongs to the large multigene family of G protein-coupled receptors. Using the bovine histamine H₁-receptor cDNA, the H₁-receptor genes from a variety of species, including man, have been cloned (for review, see Hill et al., 1997). The use of transfected cell lines recently showed that all therapeutically used histamine H₁-receptor antagonists are in fact inverse agonists, i.e. they stabilize the receptor in an inactive conformation Bakker et al., 2000, Bakker et al., 2001.

Moreover, following the cloning of the H₁ receptor, several site-directed mutagenesis studies have been conducted in order to identify the binding pocket of histamine and H₁-receptor antagonists. Asp¹⁰⁷ in transmembrane domain 3 is a conserved residue among all aminergic receptors and is thought to be responsible for an ionic interaction with the protonated nitrogen of biogenic amines (Shi and Javitch, 2002). In the human histamine H₁ receptor, Asp¹⁰⁷ is reported to be crucial for the binding of both histamine and histamine H₁-receptor antagonists Moguilevsky et al., 1998, Nonaka et al., 1998, Ohta et al., 1994. Mutation of a lysine in transmembrane domain 5 in the human (Lys¹⁹¹) and guinea pig (Lys²⁰⁰) histamine H₁ receptor to alanine leads to a decreased affinity for histamine Gillard et al., 2002, Leurs et al., 1995, Moguilevsky et al., 1998, Wieland et al., 1999. This lysine is suggested to interact with the proximal nitrogen (N^π) of the imidazole ring of histamine (Leurs et al., 1995). Asn¹⁹⁸ is proposed to interact with either the proximal Moguilevsky et al., 1995, Moguilevsky et al., 1998 or the distal (N^τ) Leurs et al., 1994, Ohta et al., 1994 nitrogen of the imidazole ring of histamine, whereas this residue is not involved in the interaction with antagonists. Involvement of the aromatic amino acids Phe⁴³³ and Phe⁴³⁶ in the guinea-pig histamine H₁ receptor (human: Phe⁴³² and Phe⁴³⁵, respectively) (transmembrane domain 6) in the binding of histamine and mepyramine has previously also been reported (Wieland et al., 1999). Both phenylalanines were identified as probable interaction points with the trans-aromatic ring of histamine H₁-receptor antagonists. Based on these observations, one can conclude that histamine and the H₁-receptor antagonists have distinct, but partially overlapping binding sites.

Recently, a new class of selective histamine H₁-receptor agonists, the histaprodifens, has been identified and pharmacologically characterized using guinea-pig ileum and rat aortic rings. Interestingly, histaprodifen (2-[2-diphenylpropyl)-1H-imidazol-4-yl]ethanamine), the prototype agonist of this family consists of a histamine moiety, representing the endogenous agonist ligand, linked by a propyl chain to two phenyl rings, that are a characteristic feature of the histamine H₁-receptor antagonist pharmacophore Ter Laak et al., 1995, Zhang et al., 1997. Fig. 1 depicts the structures of histamine, the H₁-receptor antagonist cetirizine and the ‘hybrid’ agonist histaprodifen. The aromatic rings confer high receptor affinity to these antagonists/inverse agonists. The combination of high affinity, via the diphenyl moiety, with the agonistic properties of histamine is hypothesized to be the rationale behind the reported potent agonism of the ‘hybrid’ histaprodifens (Elz et al., 2000b).

In this study, we have tested this last hypothesis and we have therefore characterized the interaction of histaprodifen and several of its analogues with the human H₁ receptor using [³H]mepyramine binding studies and a H₁-receptor-driven nuclear factor κB (NF-κB) reporter-gene assay. To delineate the histamine H₁-receptor binding sites of these ‘hybrid’ ligands we studied the interaction with Asp¹⁰⁷ (transmembrane domain 3), Lys¹⁹¹, Asn¹⁹⁸ (transmembrane domain 5), Phe⁴³⁵ (transmembrane domain 6) (agonist binding site) and Phe⁴³² (transmembrane domain 6) (antagonist binding site). These amino acids are conserved among all species.