Molecular and biochemical pharmacology of the histamine H₄ receptor

Abstract

The elucidation of the human genome has had a major impact on histamine receptor research. The identification of the human H₄ receptor by several groups has been instrumental for a new appreciation of the role of histamine in the modulation of immune function. In this review, we summarize the historical developments and the molecular and biochemical pharmacology of the H₄ receptor.

Introduction

Histamine is known since long to be an important mediator in different (patho)physiological conditions. In 1966, Ash and Schild suggested the presence of two distinct histamine receptor subtypes (Ash and Schild, 1966) based on the pharmacological effects of known histaminergic ligands on, for example, airway smooth muscle (H₁) and the heart, uterus and stomach (H₂). Sir James Black et al. at SK&F paved the way for the final acceptance of two distinct histamine receptor subtypes with the development of the first selective histamine H₂ antagonists metiamide and burimamide (Black et al., 1972). Moreover, the recognition that different actions of histamine could be effectively inhibited by selective receptor antagonist eventually resulted in the development of various blockbuster H₁ and H₂ antagonists.

In 1983 a new histamine H₃ receptor subtype was identified by Arrang et al. in the rat brain on the basis of classical pharmacology (Arrang et al., 1983). For a long time, this new histamine receptor subtype was not fully endorsed by the pharmaceutical industry because of the lack of molecular details of the receptor protein. While the gene for the histamine H₁ (Yamashita et al., 1991) and H₂ receptor (Gantz et al., 1991) were both cloned in 1991, for many years no definite proof of the molecular architecture of the H₃ receptor was available. Finally, in 1999 a team of J&J researchers led by Tim Lovenberg cloned the complementary DNA (cDNA) of an α₂ adrenergic-like EST sequence that, after characterization, turned out to encode for the human histamine H₃ receptor (Lovenberg et al., 1999). This cloning of the H₃ receptor gene resulted in a rapid increase in knowledge of its molecular pharmacology and biochemistry (e.g. expression, signal transduction, receptor isoforms) (Lovenberg et al., 1999; Drutel et al., 2001; Hancock, 2006; Bongers et al., 2007). Combined with the development of recombinant cell systems, these developments led to a huge interest from pharmaceutical companies (Leurs et al., 2000). Many new selective ligands have since then been reported in (patent) literature (Celanire et al., 2005; Wijtmans et al., 2007), and the H₃ receptor is currently seen as a new target for the treatment of a variety of central nervous system disorders (e.g. attention deficit hyperactivity disorder, Alzheimer's disease and schizophrenia) (Leurs et al., 1995; Esbenshade et al., 2008).

The cloning of the H₃ receptor gene also marked the entry of the histamine H₄ receptor into the G protein-coupled receptor (GPCR) family of histamine receptors. Using the DNA sequence of the histamine H₃ receptor, several research groups independently identified a previously unexplored GPCR sequence in the human genome as a new histamine H₄ receptor (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). The new receptor showed a distinct pharmacology (see below) is highly expressed in immune cells (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001) and is now regarded as an interesting target for, for example, inflammatory disorders and pruritis (de Esch et al., 2005; Thurmond et al., 2008). The first identification of the H₄ receptor led to a further increase in the interest in histamine receptor pharmacology and a substantial rise in H₄ receptor-related papers (112 in the period 2001–2009) and patents (36 in the period 2002–2008) in the last few years. In the present review we will highlight the molecular biology and pharmacology of the latest member of the histamine receptor family.

Cloning of the histamine H₄ receptor gene

As reported above, the H₄ receptor cDNA was finally identified in the human genome database on the basis of its overall homology (37%) to the H₃ receptor sequence (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). The homology of the H₄ receptor to the H₁ and H₂ receptor is only ∼19%, explaining why this subtype was only found after the long awaited identification of the H₃ receptor gene in 1999 (Lovenberg et al., 1999). The size of the open reading frame of the H₄ receptor cDNA is 1173 bp, encoding for a 390 amino acid protein (Figure 1), belonging to the large family of GPCRs. Besides a high sequence homology, the H₄ receptor also shares its gene structure with the H₃ receptor (Coge et al., 2001a; Coge et al., 2001b). The human H₄ receptor gene is present in a single copy on chromosome 18q11.2. The gene spans 16.98 kb (position 20 294 591 to 20 311 567) and, like the H₃ receptor (Drutel et al., 2001), is interrupted by two large introns (7867 bp and >17 500 bp). The introns divide the coding regions into three parts, encoding amino acid number 1–65, 66–119 and 120–390. Such an intron–exon distribution has led for the H₃ receptor to the generation of a large number of alternatively spliced GPCR variants, sometimes with clearly altered functionalities (Drutel et al., 2001; Leurs et al., 2005). However, for the H₄ receptor at present only two non-signalling, non-7TM H₄ receptor isoforms have been identified (van Rijn et al., 2008) (Figure 1), designated as H₄(67) and H₄(302). The polypeptide of H₄(67) is prematurely terminated at residue 67, while H₄(302) lacks residues 68–155 (Figure 1, vide infra) (van Rijn et al., 2008).

Figure 1.

Snake plot of the human histamine H₄ receptor. The full-length receptor consists of 390 amino acids, which form seven transmembrane helices, three extracellular loops and three intracellular loops, with an extracellular N-terminal and an intracellular C-terminal peptide; the dotted line represents the putative disulfide bridge that links the cysteines in the third transmembrane and second extracellular loop. Residues Asp^3.32 and Glu^5.46 that play important roles in histamine binding are marked with bold border. The H₄R₆₇ isoform only contains the first 67 N-terminal residues (marked in white), while the H₄R₃₀₂ lacks the residues marked in grey. The conserved residues in the family A of G protein-coupled receptors are depicted in black circles, while the putative glycosylation sites (Asn⁵ and Asn⁹) are indicated with arrows. A potential palmitoylation site (Cys³⁷⁴) at the C-terminal tail is suggested to be close to the membrane following membrane insertion of a putative attached palmitic acid.

The original H₄ receptor amino acid sequence as reported by Oda et al. (2000) deviates at three positions from the sequences reported by four other research groups (Nakamura et al., 2000; Liu et al., 2001a; Morse et al., 2001; Zhu et al., 2001), that is, Val instead of Ala at position 138, Arg instead of His at position 206 and Arg instead of Gln at position 253. The Val¹³⁸Ala and Arg²⁰⁶His polymorphisms have been confirmed by single nucleotide polymorphism (SNP) analysis involving samples from White, Chinese, Japanese, Afro-American and sub-Saharan African people (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=59340). The occurrence of Ala¹³⁸ or His²⁰⁶ alleles is higher than that of Val¹³⁸ or Arg²⁰⁶ (91.1–100% vs. 0–9.2%). Two other SNPs have been found as well in the coding region, resulting in Cys²⁸⁴Ser mutation and a frame shift at residue L³⁷⁹. Besides those, 37 SNPs are found in intron 1, 45 in intron 2 and 21 in 3′-untranslated region (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=59340). So far no functional comparisons of the various SNP variants or linkage studies have been reported.

As with the other histamine receptor family members, the H₄ receptor protein possesses all consensus motifs identified for the Class A rhodopsin-like GPCRs, that is, N1.50³³, D2.50⁶¹, R3.50¹¹², W4.50¹⁴⁰, P5.50¹⁸⁶, P6.50³¹⁸ and P7.50³⁵⁵[using the standardized GPCR nomenclature according to Ballesteros–Weinstein (Ballesteros and Weinstein, 1995) (Figure 1)]. With respect to histamine binding, a conserved aspartate residue in the third transmembrane (TM) aminergic GPCRs helix (D3.32, Figure 1) is also present in this newest member of the aminergic GPCR family. As for the other aminergic GPCRs, D3.32 is believed to interact with the protonated amine group of the agonist histamine (Shin et al., 2002). Moreover, in both H₃ and H₄ receptors a second negatively charged amino acid, E5.46, is present in the fifth TM helix (Figure 1). This amino acid has been shown to play an essential role in the binding of histamine as determined by site-directed mutagenesis analysis and computational analysis (Shin et al., 2002; Uveges et al., 2002). As is illustrated in Figure 2, E5.46 is a key anchor for the imidazole ring of histamine. The binding pocket for the imidazole ring is formed by E5.46, T5.42 and W6.48 (Jongejan et al., 2008). The latter residue adopts the ‘active’ rotamer conformation (Jongejan et al., 2005) by forming a hydrogen bond to the aforementioned E5.46 (box, Figure 2).

Figure 2.

Schematic representation of a homology model of the human histamine H₄ receptor. The figure shows the seven transmembrane domains and the position of the ligand binding site. In the box details of the histamine binding site, as determined using ab initio calculations is shown (reproduced with permission from Jongejan et al., 2008).

The H₄R has two potential glycosylation sites (Asn⁵ and Asn⁹) (Figure 1, vide infra), whereas Cys³⁷⁴ might serve as a potential site for palmitoylation. The H₄R also contains cysteine residues that potentially form a disulphide bridge, connecting the third TM with the second extracellular loop (EL2) (Figure 1). These cysteine residues are conserved among the GPCRs, and the disulphide bridge has been evidenced in the crystal structure of bovine rhodopsin and adrenergic β₁ and β₂ receptors (Palczewski et al., 2000; Cherezov et al., 2007; Warne et al., 2008). The disulfide bridge will bring some structural constraint to the extracellular loop two, which may be important in view of the suggested involvement of the EL2 in the binding of histamine (Lim et al., 2008).

Species differences

Following the identification of the human H₄R, the cDNA sequence of mouse, rat, guinea pig, pig, dog and monkey H₄ receptors have been reported and functionally expressed (Liu et al., 2001b; Oda et al., 2002; 2005). The monkey H₄ receptor (Macaca fascicularis) shows a high sequence homology (92%) with the human orthologue (Oda et al., 2005). The H₄ receptors of the other species are only moderately homologous to the human H₄R, with sequence homology between 67% and 72% (Liu et al., 2001b; Oda et al., 2002; 2005; Jiang et al., 2008). Forty (partial) amino acid sequences of H₄R orthologues from 34 other species have been extracted from various genomic ENSEMBL databases (http://www.ensembl.org/Homo_sapiens/Gene/Compara_Ortholog?db=core;g=ENSG00000134489;r=18:20294591-20313918;t=ENST00000256906), among others horse (Equus caballus), cow (Bos taurus), cat (Felis catus), rhesus monkey (Macaca mullata), chimpanzee (Pan troglodytes) and dolphin (Tursiops truncates) H₄ receptor sequences are known (Figure 3). The H₄R sequences from cynomogus (Macaca fascicularis) and pig (Sus scrofa), which are not listed as orthologues in the ENSEMBL database are also included in the phylogenetic tree in Figure 3. Because some of the sequences are still incomplete, changes in the phylogenetic tree are to be expected. Fish (zebra fish, fugu, tetraodon, medaka and stickleback) as well as frog (Xenopus tropicalis) have each two orthologues. However, these orthologues, together with the sequences from chicken (Gallus gallus) and opossum, show only 28–42% homology to the human H₄ receptor, while all the others show a substantial higher homology to the human H₄ receptor (>50%). In fact, these orthologues are more similar to the human H₃R. Consequently, in the phylogenetic tree these H₃/H₄ orthologues cluster in a separate group (Figure 3). Within the extracted (partial) GPCR sequences, the typical aminergic GPCR features mentioned earlier (e.g. Asp^3.32 in TM3 and Glu^5.46 in TM5) can often be found. Detailed analysis of most of these species variants is for the moment however lacking, but may provide useful tools to dissect receptor–ligand binding. Previously, Lim et al. (2008), for example, showed that a sequence difference in the extracellular loop EL2 is responsible for the lower affinity of histamine for rat and mouse H₄ receptors. Comparing the binding profiles of rat, mouse, guinea-pig, porcine, monkey and dog H₄ receptors, one can identify for ligands like clozapine and clobenpropit (see also paragraph on H₄ agonist) also substantial differences in binding affinity between the various species (Liu et al., 2001b;Oda et al., 2002; 2005). Differences in pharmacological activities of H₄ receptor ligands between the different species might hamper preclinical development of future H₄ receptor drugs.

Figure 3.

Phylogenetic tree of H₄ receptor orthologues. The sequences are obtained from http://www.ensembl.org and the phylogram was created with ClustalW.

Signal transduction of the H₄ receptor

The H₄ receptor is coupled to pertussis toxin (PTX)-sensitive Gα_i/o proteins, thereby inhibiting forskolin-induced cAMP and eventually the modulation of transcription of genes regulated by cAMP-responsive elements in cell lines recombinantly expressing the H₄ receptor (Oda et al., 2000; Liu et al., 2001a; Zhu et al., 2001). In transfected HEK-293 cells H₄ receptor stimulation also leads to a strong increase in mitogen-activated protein kinase phosphorylation via a PTX-sensitive pathway (Morse et al., 2001). Furthermore, the H₄ receptor also signals to calcium via either PTX-sensitive Gα_i/o proteins (Hofstra et al., 2003) or the promiscious Gα₁₆ protein (Oda et al., 2000; Morse et al., 2001), which is selectively expressed in immune cells (Wilkie et al., 1991). In recombinant cell lines the H₄ receptor constitutively inhibits forskolin-induced cAMP-responsive element-mediated reporter gene expression and activates [³⁵S]GTPγS binding (Liu et al., 2001a; Morse et al., 2001; Lim et al., 2005). The agonist-independent activity can be inhibited by H₄ inverse agonists, such as thioperamide.

Activation of endogenously expressed H₄ receptors in for example eosinophils and mast cells leads to PTX-sensitive calcium mobilization (Buckland et al., 2003; Hofstra et al., 2003), via the activation of phospholipase C (Hofstra et al., 2003). The increased cytosolic calcium concentration is intimately linked to actin polymerization, cell shape change and eventually to the migration of mast cells, eosinophils and monocyte dendritic cells (Buckland et al., 2003; Hofstra et al., 2003; Ling et al., 2004; Gutzmer et al., 2005; Barnard et al., 2008).

Looking back in immunology literature with this new knowledge, early examples of H₄ receptor-mediated signalling by histamine can be identified. Already in the 1975 Clark et al. observed histamine-induced eosinophil chemotaxis that could not be blocked by the known H₁ and H₂ receptor antagonists (Clark et al., 1975). Furthermore, in 1994 Raible et al. identified in eosinophils a PTX-sensitive cytosolic calcium increase induced by histamine, which could be inhibited by the presumed selective H₃ receptor antagonist thioperamide. The potent H₃ receptor reference agonist R-α-methylhistamine also induced a calcium increase, but at much lower potency than that of histamine itself. Based on these observations, the presence of a new histamine receptor subtype was suggested (Raible et al., 1994). Alas, this suggestion was largely ignored by the histamine research community and has only been fully exploited more than a decade after the discovery.

Anatomical Topography of the H₄ receptor

The tissue distribution of the H₄ receptor has been profiled at the mRNA level in a wide range of human (using reverse transcription polymerase chain reaction) and mouse (using in situ hybridization) tissues (Zhu et al., 2001). In human tissues, H₄ receptor expression was most abundant in bone marrow, peripheral blood, spleen, thymus, small intestine, colon, heart and lung (Nakamura et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). Discrepancies have occurred in both human and rodent tissue based on these mRNA studies, which highlights the necessity for selective immunological anti-H₄ receptor probes.

Anti-H₄ receptor antibodies have been crucial tools to study the H₄ receptor (van Rijn et al., 2006; 2008; Dijkstra et al., 2007; Baumer et al., 2008; Dijkstra et al., 2008; Morini et al., 2008). The majority of cells expressing H₄ receptors are indeed hematopoietic in lineage, including neutrophils, mast cells, eosinophils, basophils, dendritic cells, monocytes and T cells (e.g. van Rijn et al., 2006; Dijkstra et al., 2007; Baumer et al., 2008; Dijkstra et al., 2008), but importantly, it has recently been shown that the H₄ receptor is not exclusively expressed on hematopoietic cells. The H₄ receptor has been identified in subsets of endocrine cells in the gastrointestinal tract (distinct from H₃R containing cells) (Morini et al., 2008) and has been shown also to be expressed on dermal fibroblasts (Ikawa et al., 2008). Immunohistochemical studies have shown that H₄ receptors are present on nerves from the human nasal mucosa (Nakaya et al., 2004) and in the enteric nervous system on the myenteric plexus of the rodent fundus (H₃R absent) (PL Chazot, D Grandi, FC Shenton, G Morini, unpublished). Indeed, importantly, as with the other histamine receptor subtypes, the H₄ receptor is expressed in the central nervous system (Connelly, et al., 2009). In agreement, the H₄ receptor was detected on subpopulations of small and medium diameter cells within the lumbar sensory dorsal root ganglia and laminae I/II of the dorsal horn of the lumbar spinal cord (Strakhova et al., 2009; PL Chazot, NL Lethbridge, unpublished). These latter results indicate that the H₄ receptor is present on synaptic terminals of primary sensory afferent neurons. Therefore, the H₄ receptor is likely to subserve distinct roles in many parts of the body, and therefore may represent a target for an expanded array of therapeutic arenas, including neuropathic pain.

H₄ receptor biochemistry

Immunological approaches have demonstrated that the H₄ receptor comprises robust dimeric structures (van Rijn et al., 2006; 2008). It is clear from immunoblots that higher molecular weight species (consistent with dimers and/or higher oligomers) are present in both recombinant and native tissue (Figure 4). Immunoblotting identified three major protein species in human spleen lysate. Interestingly a low molecular weight species was clearly visible in the spleen lysate at 31 kDa, likely to be the receptor monomer. In contrast, monomeric species were not evident in either lymphocytes (polyhaemagglutinnin blasts) or in brain membranes (van Rijn et al., 2006; Connelly et al., 2009). The observed sizes detected in immunoblotting studies are not always completely consistent with H₄R dimeric structures, with sizes ranging between 60 and 80 kD in HEK-293 H₄R-transfected cells or in other native tissue preparations (Figure 4, van Rijn et al., 2006; Connelly et al., 2009). Several possibilities could account for these observations, including tissue-specific complements of H₄R isoforms and differential tissue-specific post-translational modifications (e.g. palmitoylation, glycosylation).

Figure 4.

Anti-hH_4(374–390) receptor antibody reacts with human H₄ receptor protein in spleen tissue lysate and sections. (A) Immunoblot of human spleen lysate (10 µg). Anti-H_4(374–390) antibodies were used at 2 µg·mL⁻¹, incubated overnight at 4°C and developed as described in van Rijn et al. (2006). In addition to the putative monomer at M_w 31 kDa, two higher molecular weight species were detected at 59 and 66 kDa. (B) Immunostaining of human spleen slice (bar = 50 µm) using anti-H_4(374–390) at 1 µg·mL⁻¹ using an immunohistochemical protocol described in Chazot et al. (2001).

Anti-H₄ receptor antibodies were also used to investigate the expression and potential role of the newly described human H₄ receptor isoforms: H₄(302) and H₄(67) (van Rijn et al., 2008). Because mRNAs coding for all three isoforms are found in several white blood cell types, hetero-oligomerization may occur in native tissue. The two shorter isoforms are both non-functional as regards ligand binding and signalling. However they may play a regulatory role as they appear to reduce number of histamine binding to the H₄(390) receptor by 55% [H₄(302)] and 30% ]H₄(67)] in heterologous expression studies. Based on surface biotinylation experiments, the three were expressed at the cell surface, although the shorter versions with greatly reduced efficiency. Furthermore co-expression of the H₄(390) with either H₄(302) or H₄(67) dose-dependently reduced surface expression of the full length receptor (van Rijn et al., 2008). These results provide further evidence to support the role of splice isoforms as dominant negative regulatory elements, which maybe a common theme in GPCR-regulatory pathways (Bakker et al., 2006).

The majority of secreted and cell surface proteins that transit to the cell surface through the endoplasmic reticulum are N-glycosylated (95% of GPCRs) (Lanctot et al., 2005). In order for a GPCR to elicit intracellular signalling appropriately, the correct quantity of properly folded functional receptors must be available in the plasma membrane. It is equally essential that signalling can be terminated at the appropriate time and the receptors either recycled or permanently degraded. The regulatory mechanisms controlling export trafficking of GPCRs, including glycosylation, have recently been reviewed (Duvernay et al., 2005). The role of post-translational changes to receptors is complex and an area where there is growing interest. Several studies have shown that glycosylation is involved in GPCR transport from the endoplasmic reticulum through the Golgi to the cell surface (reviewed in Duvernay et al., 2005). Both the H₃ and H₄ receptors are indeed N-glycosylated (van Rijn et al., 2006; Shenton and Chazot, 2006). Interestingly, receptor dimerization in vitro does not appear to be dependent on N-glycosylation. However, in the case of endogenous H₄ receptor expressed in human polyhaemagglutinnin blasts, chemical deglycosylation appears to destabilize the preformed dimeric species to individual monomers (van Rijn et al., 2006). The pharmacological influence of N-glycosylation of the H₄ receptor is yet to be explored.

Pharmacological tools to study the H₄ receptor

Already with the early description of histamine responsiveness of eosinophils in 1994, a clearly different pharmacology was described in comparison with the known receptor subtypes (Raible et al., 1994). The subsequent cloning of the H₄ receptor cDNA and the analysis of the expressed protein also resulted in the rapid recognition of a completely distinct H₄ receptor pharmacology (Hough, 2001). Since then, major progress has been made in the discovery of selective H₄ receptor agonists and antagonists.

The early pharmacological and (patho)physiological characterization of the H₄ receptor was performed using agonists that were originally developed for the homologous H₃R receptor. Typically, these initial H₃ and H₄ receptor dual activity reference compounds all contain an imidazole heterocycle. The most important examples are the H₃ and H₄ receptor agonist, immepip (2) and the H₃ antagonist and H₄ agonist clobenpropit (3) (Lim et al., 2005) (Figure 5).

Figure 5.

First and second generation H₄ receptor agonists with different selectivity profiles.

The first ligand that was optimized for selective H₄ receptor activation is OUP-16 (4) (Hashimoto et al., 2003) (Figure 5). This full agonist has a 40-fold selectivity for the H₄ receptor over the H₃ receptor. More recently, the potent H₄ receptor agonist 4-methylhistamine (5) was described after evaluation of a large number of histaminergic compounds (Lim et al., 2005). Although originally developed in the H₂ receptor research programme, 4-methylhistamine has a high affinity for the H₄ receptor, while displaying at least a 100-fold selectivity over the other histamine receptor subtypes, including the H₂ receptor. The first identified non-imidazole H₄ receptor agonist was clozapine (6) (Oda et al., 2000). This anti-psychotic agent has affinity for a large number of GPCRs, but almost exclusively acts as antagonist. The dibenzodiazepine structure was optimized for H₄ receptor affinity, leading to the dual activity H₁ receptor antagonist and H₄ receptor agonist VUF6884 (7) (Smits et al., 2006). Although promiscuous GPCR binders, this class of ligands provides detailed insight of the H₄ receptor agonist binding site (Smits et al., 2006; Jongejan et al., 2008). Another non-imidazole H₄ receptor agonist is dimaprit (8) and its analog VUF8430 (9) (Lim et al., 2005; Lim et al., 2006). The latter is a full agonist with high affinity for the H₄ receptor and a 30-fold selectivity over the H₃ receptor. In combination with 4-methylhistamine, VUF8430 forms a new useful pair of tools to study H₄ receptor pharmacology (see Lim et al., 2009).

As is true for the first H₄ receptor agonists, the first antagonists that were used to characterize the H₄ receptor were imidazole-containing, dual activity H₃ and H₄ receptor ligands. Of particular use has been the H₃ and H₄ receptor inverse agonist thioperamide (10) (Figure 6; Buckland et al., 2003; Hofstra et al., 2003; Bell et al., 2004; Takeshita et al., 2004; Damaj et al., 2007). The H₄ receptor research field has been greatly helped by the early discovery and disclosure of the selective non-imidazole neutral antagonist JNJ7777120 (11). The compound resulted from a high-throughput screening campaign and subsequent hit optimization efforts at Johnson and Johnson Pharmaceuticals (Carruthers et al., 2002). JNJ7777120 is equipotently active at human, mouse and rat H₄Rs (Jablonowski et al., 2003; Thurmond et al., 2004). This H₄R reference antagonist is more than a thousand fold selective over other histamine receptor subtypes and was reported to have no cross-reactivity in a panel of 50 other receptors, enzymes, transporters and ion-channels. JNJ7777120 has reasonable oral bioavailability (22%), but its use in disease models is hampered by a short in vivo half-life of about 0.8 h (Venable et al., 2005). Efforts to obtain compounds with improved properties by scaffold hopping led to VUF6002 (12) (Figure 6; Terzioglu et al., 2004; Venable et al., 2005). Although this benzimidazole derivative has an improved liver microsomes stability, the half-life remains limited (t_1/2= 1.0 h).

Figure 6.

First and second generation H₄ receptor antagonists. Except for thioperamide (10) the antagonists display at least a 100-fold selectivity for the histamine H₄ receptor over the histamine H₃ receptor.

Another series of H₄R compounds that was recently claimed in a patent by Johnson and Johnson contain methylpiperazine substituted 2-quinoxalinones (Edwards and Venable, 2005), including compound 13. At VU University Amsterdam, the construction of a H₄R pharmacophore model and rational design approaches resulted in the potent quinazoline compound 14 (Leurs et al., submitted). This compound was shown to have anti-inflammatory properties in vivo in the rat.

Bayer Healthcare AG has recently patented aminopyrimidines (e.g. 15) as H₄ receptor antagonists (Sato et al., 2005a,b). Since this disclosure, several other companies have claimed other pyrimidines as H₄ receptor ligands, including Abbott Laboratories who developed the rotationally restricted analogue A-943931 (16). This compound has been thoroughly characterized and displays a more than 640-fold selectivity over the H₃ receptor, a 190-fold selectivity over α₁ adrenergic receptors and a 470-fold selectivity over 5-HT_1d receptors. A-943931 is reported to have excellent antagonistic activity both in vitro and in vivo across multiple species, while displaying excellent metabolic stability and good oral bioavailability (90%). This compound is a good anti-inflammatory agent in mice and also displays good efficacy in rat pain models (Cowart et al., 2008). Further exploration of the aminopyrimidine theme resulted in the recently described H₄ receptor antagonist A-987306 (17), a potent and selective compound with an excellent pharmacokinetic profile, including a half-life of 3.7 h after p.o. dosing (Liu et al., 2008). A-987306 (17) has anti-inflammatory activity in a peritonitis model and is especially potent in a pain assay in rats, as was shown by the blockage of carrageenan induced thermal hyperalgesia.

In order to study the pharmacology of the H₄ receptor, radioligand binding studies are instrumental. As with the H₃ receptor, the H₄ receptor binds the endogenous agonist histamine with high, nanomolar affinity (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). Consequently, [³H]histamine has been effectively used as radioligand since the discovery of the H₄ receptor (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). In some studies also [³H]N^α-methylhistamine has been used as radiotracer (Liu et al., 2001a), but this has not become very popular. In 2004 J&J described the use of [³H]JNJ7777120 as antagonist radioligand for the H₄ receptor (Thurmond et al., 2004). This new radioligand binds differently than histamine in the H₄R ligand binding pocket (Jongejan et al., 2008), allowing the analysis of, for example, distinct H₄ receptor mutants that have lost high affinity [³H]histamine binding (Jongejan et al., 2008).

Final remarks

With the cloning of the gene of the histamine H₄ receptor in 1999/2000 the field of histamine research has been fueled with new excitement. Several laboratories have already identified a set of useful tools (agonists, antagonists, anti-H₄ receptor antibodies), allowing the field to address the function of the H₄ receptor in both the periphery and the brain. Moreover, in the last few years a large increase in knowledge on the biochemical pharmacology of the H₄ receptor has been obtained. In view of the first preclinical in vivo data for H₄ receptor ligands, this new histamine receptor family member is expected to become an important target in the area of inflammatory disorders and neuropathic pain. Moreover, also pruritis is associated with H₄ receptor function as itching can be induced by H₄ agonists such as clobenpropit and 4-methylhistamine and blocked by H₄ antagonists such as JNJ7777120. It might nevertheless be an interesting challenge to develop H₄ receptor drugs, as so far relatively high levels of H₄ receptor antagonists are required to inhibit in vivo inflammatory effects despite the high potency and selectivity of the tool compounds. This may reflect limitations of the tool compounds with respect to for example pharmacokinetic properties, as well as for example high local histamine concentrations at the H₄ receptor.

Glossary

Abbreviations:

cDNA

complementary DNA

EL2

second extracellular loop

GPCR

G protein-coupled receptor

PTX

pertussis toxin

SNP

single nucleotide polymorphism

TM

transmembrane domain

Conflict of interest

None.