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CELLULAR AND MOLECULAR

Evaluation of Histamine H₁-, H₂-, and H₃-Receptor Ligands at the Human Histamine H₄ Receptor: Identification of 4-Methylhistamine as the First Potent and Selective H₄ Receptor Agonist

Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (H.D.L., R.M.v.R., R.A.B., R.L.); and Johnson & Johnson Pharmaceutical Research & Development, L.L.C., San Diego, California (P.L., R.L.T.)

Received for publication April 13, 2005
Accepted June 1, 2005.

	Abstract

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The histamine H₄ receptor (H₄R) is involved in the chemotaxis of leukocytes and mast cells to sites of inflammation and is suggested to be a potential drug target for asthma and allergy. So far, selective H₄R agonists have not been identified. In the present study, we therefore evaluated the human H₄R (hH₄R) for its interaction with various known histaminergic ligands. Almost all of the tested H₁R and H₂R antagonists, including several important therapeutics, displaced less than 30% of specific [³H]histamine binding to the hH₄R at concentrations up to 10 µM. Most of the tested H₂R agonists and imidazole-based H₃R ligands show micromolar-to-nanomolar range hH₄R affinity, and these ligands exert different intrinsic hH₄R activities, ranging from full agonists to inverse agonists. Interestingly, we identified 4-methylhistamine as a high-affinity H₄R ligand (K_i = 50 nM) that has a >100-fold selectivity for the hH₄R over the other histamine receptor subtypes. Moreover, 4-methylhistamine potently activated the hH₄R (pEC₅₀ = 7.4 ± 0.1; = 1), and this response was competitively antagonized by the selective H₄R antagonist JNJ 7777120 [1-[(5-chloro-1H-indol-2-yl)-carbonyl]-4-methylpiperazine] (pA₂ = 7.8). The identification of 4-methylhistamine as a potent H₄R agonist is of major importance for future studies to unravel the physiological roles of the H₄R.

The presence of the hH₄R on leukocytes and mast cells suggests that this new histamine receptor plays an important role in the modulation of the immune system. This hypothesis is supported by the fact that IL-10 and IL-13 modulate hH₄R expression (Morse et al., 2001) and that binding sites for cytokine-regulated transcription factors, such as interferon-stimulated response element, interferon regulatory factor-1, nuclear factor-B, and nuclear factor-IL6, are present upstream of the hH₄R gene (Cogé et al., 2002). Physiological roles of the hH₄R include the control of IL-16 release by human CD8⁺ T cells (Gantner et al., 2002), chemotactic responses and cytoskeletal changes of human eosinophils (O'Reilly et al., 2002; Buckland et al., 2003; Ling et al., 2004), chemotaxis and intracellular calcium mobilization in mast cells (Hofstra et al., 2003), and control of leukotriene B₄ production by mast cells that subsequently leads to neutrophil recruitment into peritoneum (Takeshita et al., 2003; Thurmond et al., 2004). These studies suggest that the hH₄R is a potential drug target for immune system-related diseases.

Until recently, potent and selective H₄R ligands were not available. In the early studies, the H₃R antagonist thioperamide was identified as an equally effective H₄R antagonist (Oda et al., 2000; Hough, 2001). High-throughput screening and subsequent medicinal chemistry efforts recently identified the indolylpiperazine JNJ 7777120 (Jablonowski et al., 2003; Thurmond et al., 2004) and the related benzimidazole analog VUF 6002 (Terzioglu et al., 2004) as selective and potent H₄R antagonists. Studies directed toward selective H₄R agonists have so far been less successful. Burimamide, clozapine, and clobenpropit are all known to act as H₄R agonists, and clozapine and clobenpropit have been proven useful for initial pharmacological studies (Gantner et al., 2002; Buckland et al., 2003; Bell et al., 2004; Ling et al., 2004). Currently, the most selective H₄R agonist is the imifuramine analog OUP-16, which displays a 15-fold selectivity for the H₄R compared with its binding affinity for the H₃R (Hashimoto et al., 2003). However, the lack of selectivity of currently known agonists for the H₄R limits their use as H₄R agonists.

In our search for selective H₄R agonists, many known histaminergic ligands of different structural classes, including several important therapeutics, were evaluated for their interaction with the hH₄R. Our studies resulted in the identification of 4-methylhistamine, a presumed moderately active and selective H₂R agonist (Durant et al., 1975), as a high-affinity H₄R agonist with a more than 100-fold selectivity over the H₁R, H₂R, and H₃R.

	Materials and Methods

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Materials. Aminopotentidine, amthamine dihydrobromide, amselamine dihydrobromide, burimamide oxalate, and burimamide analogs (Vollinga et al., 1995); clobenpropit dihydrochloride, dimaprit dihydrobromide, histaprodifen dimaleate, homohistamine dihydrobromide, imbutamine dihydrobromide, imetit dihydrobromide, impentamine dihydrobromide, immepip dihydrobromide, immethridine dihydrobromide, iodophenpropit dihydrobromide, JNJ 7777120, 2-(3-bromophenyl)histamine dihydrobromide, methimmepip dihydrobromide, 2-pyridylethylamine (PEA) dihydrochloride, 2-(2-thiazolyl)ethylamine (TEA) dihydrochloride, thioperamide fumarate, and VUF 8328 were synthesized at the Department of Medicinal Chemistry (Vrije Universiteit Amsterdam, Amsterdam, The Netherlands). Famotidine, ketotifen fumarate, and 8R-lisuride were purchased from MP Biomedicals (Irvine, CA). Amoxapine, d-chlorpheniramine maleate, clozapine, cimetidine, N-desmethyl clozapine, diphenhydramine hydrochloride, doxepin hydrochloride, forskolin, histamine dihydrochloride, imipramine hydrochloride, loxapine, mepyramine (pyrilamine maleate), (R)--methylhistamine dihydrochloride, (S)--methylhistamine dihydrochloride, N-methylhistamine dihydrochloride, N-oxide clozapine, octoclothepin, pertussis toxin, polyethyleneimine, ranitidine hydrochloride, tripelennamine hydrochloride, and triprolidine hydrochloride were purchased from Sigma/RBI (Natick, MA). 2-Nitrophenol--D-pyranoside and G418 (geneticin) were from Duchefa (The Netherlands); promethazine was from VUMC Pharmacy Amsterdam (Amsterdam, The Netherlands); fexofenadine was from Ultrafine Chemicals (Manchester, UK); tiotidine was from Imperial Chemical Industries PLC (London, UK); and [³H]N-methylhistamine (85 Ci/mmol), [³H]histamine (12.4 Ci/mmol), and [³H]mepyramine (23 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Boston, MA). [¹²⁵I]Iodoaminopotentidine and [¹²⁵I]iodophenpropit were labeled at the Department Nuclear Medicine and PET Research (Vrije Universiteit Medical Centre, Amsterdam, The Netherlands) as described previously (Jansen et al., 1992), whereas [³H]JNJ 7777120 (84 Ci/mmol) was synthesized at Johnson & Johnson Pharmaceutical Research and Development, L.L.C. (La Jolla, CA) (Thurmond et al., 2004). Gifts of astemizole (Janssen Pharmaceuticals, Antwerp, Belgium); cyproheptadine hydrochloride (MSD, Haarlem, The Netherlands); cetirizine hydrochloride and hydroxyzine dihydrochloride (UCB Pharma, Brussels, Belgium); ebastine (Almirall Prodesfarma, Barcelona, Spain); loratidine (Schering Plough, Kenilworth, NJ); mianserin hydrochloride and ORG3770 (Organon NV, Oss, The Netherlands); mifentidine (Instituto De Angeli, Milan, Italy); mizolastine (Synthélabo Recherche, Bagneux, France); proxyfan dihydrochloride and iodoproxyfan dihydrochloride (Dr. J. A. M. Christiaans; Kovalainen et al., 1999); S(+)- and R(-)-sopromidine (Institute of Pharmacy, Free University Berlin, Berlin, Germany); and R-(+)- and S-(-)-terfenadine carboxylate (Sepracor, Marlborough, MA), 2-methylhistamine dihydrochloride, 4-methylhistamine dihydrochloride, and impromidine dihydrochloride (GlaxoSmithKline, Welwyn Garden City, Hertfordshire, UK) are greatly acknowledged.

Cell Culture. SK-N-MC cell lines, which stably express either the human H₃R (SK-N-MC/hH₃) or H₄R (SK-N-MC/hH₄) as well as a cAMP-responsive element (CRE)-driven -galactosidase reporter gene SK-N-MC/hH₃ or SK-N-MC/hH₄ cells (Lovenberg et al., 1999; Liu et al., 2001a), were cultured in Eagle's minimum essential medium supplemented with 5% fetal calf serum, 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 600 µg/ml G418 at 37°C in 5% CO₂ and 95% humidity.

Radioligand Binding Assays. The SK-N-MC/hH₃ cell homogenates were incubated for 40 min at 25°C with approximately 1 nM [³H]N-methylhistamine in 25 mM KPO₄ buffer and 140 mM NaCl (pH 7.4 at 25°C), with or without competing ligands, whereas the SK-N-MC/hH₄ cell homogenates were incubated 1 h at 37°C in 10 nM [³H]histamine and 50 mM Tris-HCl (pH 7.4 at 37°C), with or without competing ligands. Bound radioligands were collected on 0.3% polyethyleneimine-pretreated Whatman GF/C [and washed three times with 3 ml of ice-cold washing buffer (4°C) containing 25 mM Tris-HCl and 140 mM NaCl (pH 7.4 at 4°C) for the hH₃R and 50 mM Tris-HCl (pH 7.4 at 4°C) for the hH₄R]. Binding analysis of 10 nM [³H]JNJ 7777120 and 0.1 nM [¹²⁵I]iodophenpropit to the hH₄R was performed with the same conditions as described for [³H]histamine. In saturation binding analysis, the nonspecific binding of [³H]histamine or [³H]JNJ 7777120 was determined with 1 µM clobenpropit. The binding analysis of [³H]mepyramine and [¹²⁵I]iodoaminopotentidine binding to human H₁R and human H₂R, respectively, was performed according to Bakker et al. (2004). The binding data were analyzed with Prism 4.0 (GraphPad Software Inc., San Diego, CA), and data are presented as mean ± S.E.M. Mouse and rat H₄R radioligand binding assays were performed according to Liu et al. (2001b).

Colometric Cyclic AMP Assay. A reporter CRE--galactosidase reporter gene assay was used to determine (inverse) agonistic or antagonistic activity of either the hH₃R or hH₄R. Approximately 4 million cells/96-well plate of SK-N-MC/hH₃ and SK-N-MC/hH₄ cells were exposed for 6 h to histaminergic ligands in serum-free Eagle's minimum essential medium containing 1 µM forskolin. Thereafter, the medium was discarded, the cells were lysed in 100 µl of assay buffer (100 mM sodium phosphate buffer at pH 8.0, 4 mM 2-nitrophenol--D-pyranoside, 0.5% Triton X-100, 2 mM MgSO₄, 0.1 mM MnCl₂, and 40 mM -mercaptoethanol), incubated overnight at room temperature, and the -galactosidase activity was determined at 420 nm with a PowerwaveX340 plate reader (Bio-Tek Instruments Inc., Winooski, VT). The OD₄₂₀ might differ between experiments due to intra-assay variability; therefore, intrinsic activity of agonists was determined relatively to activity of histamine.

View larger version (26K): [in this window] [in a new window]   Fig. 1. The cell line SK-N-MC/hH4 stably expresses functional hH4R and CRE-control -galactosidase. The homogenate of SK-N-MC/hH4 cells shows a saturable binding for the H4R agonist [3H]histamine (A) and also for the H4R antagonist [3H]JNJ 7777120 (B). The binding of [3H]histamine to the hH4R is inhibited by H3/4R ligands (C). The full agonist histamine and partial agonist clobenpropit hH4R inhibit the 1 µM forskolin-induced CRE activation, as measured by a -galactosidase reporter gene, whereas the inverse agonist thioperamide dose-dependently blocks the hH4R constitutive activity (D). Data shown are from representative experiments, each performed in triplicate.

	Results

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Pharmacological Characterization of the hH₄R Expressed in SK-N-MC Cells. Stable transfection of the hH₄R cDNA in SK-N-MC cells resulted in the expression of functional hH₄R proteins. The hH₄R could be labeled with both agonist and antagonist radioligands. The H₄R agonist [³H]histamine shows saturable binding to the expressed H₄R with a minimal amount of nonspecific binding (Fig. 1A). Analysis of the [³H]histamine saturation binding yielded a K_d value of 11 ± 1.0 nM (n = 6) and a B_max value of 1.8 ± 0.4 pmol/mg protein. Recently, JNJ 7777120 was described as a selective H₄R antagonist (Jablonowski et al., 2003). In our hands, the nonimidazole JNJ 7777120 shows a 300-fold selectivity for the hH₄R(pK_i = 7.8 ± 0.1 against [³H]histamine) over the hH₃R (pK_i = 5.3 ± 0.1 against [³H]N-methylhistamine), allowing the use of [³H]JNJ 7777120 to label the H₄R (Thurmond et al., 2004). The H₄R antagonist [³H]JNJ 7777120 exhibits a somewhat higher level of nonspecific binding to hH₄R expressing SK-N-MC cells, but it also binds saturably and shows an equipotent affinity (K_d = 11 ± 3.6 nM; n = 3) and results in a B_max value of 1.7 ± 0.4 pmol/mg protein (Fig. 1B). The binding of either 10 nM [³H]histamine (Fig. 1C) or 10 nM [³H]JNJ 7777120 (data not shown) to the hH₄R is fully displaced by histamine (pK_i = 7.8 ± 0.1), the H_3/4R antagonist thioperamide (pK_i = 6.9 ± 0.1) and the H₄R agonist/H₃R antagonist clobenpropit (pK_i = 8.1 ± 0.1), in a good agreement with the results reported previously (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001).

The SK-N-MC/hH₄ cells used in this study coexpress a CRE-controlled -galactosidase reporter gene and can therefore also be used for a functional analysis of H₄R ligands. Stimulation of the hH₄R with histamine resulted in the inhibition (58 ± 3%; n = 16) of the forskolin-stimulated (1 µM) cAMP-mediated reporter gene transcription with a pEC₅₀ value of 7.7 ± 0.1 (n = 16) (Fig. 1D). Treatment of SK-N-MC/hH₄ cells with the G_i/o protein inhibitor pertussis toxin (100 ng/ml for 16 h) completely inhibited histamine induced responses, confirming the coupling of the H₄R to G_i/o proteins (Oda et al., 2000; Liu et al., 2001a; Zhu et al., 2001). In our hands, histamine exerted the maximally observed level of inhibition in this assay and is therefore referred to as a full agonist (intrinsic activity = 1). As reported previously (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001), clobenpropit acts as a potent partial hH₄R agonist with a pEC₅₀ value of 7.7 ± 0.1 (n = 3) and an intrinsic activity of 0.8 (Fig. 1D).

Treatment of SK-N-MC/hH₄ cells with pertussis toxin (100 ng/ml for 16 h) resulted in an increase of 1 µM forskolin-stimulated CRE activity by 130 ± 3%, suggesting that the hH₄R shows a detectable level of constitutive activity in the SK-N-MC/hH₄ cells. In line with previous observations on inverse agonism by thioperamide (Liu et al., 2001a; Morse et al., 2001), 1 µM forskolin-stimulated CRE activity was increased by thioperamide with a pEC₅₀ value of 7.0 ± 0.1 (Fig. 1D). The inhibition of the constitutive activity of the hH₄Rby thioperamide was of the same magnitude as observed after treatment with pertussis toxin and thioperamide is referred to as a full inverse hH₄R agonist (intrinsic activity = -1).

In SK-N-MC/hH₄ cells the cAMP-driven -galactosidase reporter-gene transcription also can be activated by endogenously expressed G_s protein-coupled adrenergic receptors (Bahouth et al., 2001). The ₂ adrenergic receptor agonist feneterol induced -galactosidase activity to a similar extent to that of forskolin, with a pEC₅₀ value of 6.9 ± 0.1 (n = 6). H₄R activation by histamine inhibited the 100 nM feneterol-induced -galactosidase activity for 39 ± 3% with a pEC₅₀ value of 7.4 ± 0.1 (n = 7). However, inverse agonistic activity of thioperamide, at the hH₄R, could not be easily demonstrated with a feneterol-based assay system (data not shown). The evaluation of the functional activity of all the various histaminergic ligands was therefore performed using forskolin (1 µM) stimulated SK-N-MC/hH₄ cells.

All compounds were preliminarily tested as displacers of [³H]histamine binding to the hH₄R expressed in SK-N-MC/hH₄ cells at a concentration of 10 µM. Compounds inhibiting the specific binding of 10 nM [³H]histamine to the hH₄R by 30% are expected to have a K_i > 10 µM based on the Cheng and Prusoff equation (Cheng and Prusoff, 1973): K_i = IC₅₀/(1 + [radioligand]/K_d) and were excluded for further testing. Active compounds (displacement 30%) were tested more extensively in both [³H]histamine displacement studies and the CRE--galactosidase-based functional H₄R assay.

Most H₁R Ligands Are Devoid of H₄R Activity. Histamine potently displaces [³H]histamine from the hH₄R with a pK_i value of 7.8 ± 0.1 (Table 1), whereas H₁R agonists with substituents at the 2-position of the imidazole ring show significantly lower affinities. Substitution of the imidazole ring with either a small methyl or large 3-bromophenyl substituent is not tolerated and causes an almost 100-fold drop of affinity. Bulkier substituents at the 2-position (1,1-diphenylpropyl in histaprodifen) even result in a total loss of affinity for the hH₄R (Table 1). Agonists, lacking the imidazole ring, such as TEA, PEA, or 8R-lisuride (Bakker et al., 2004), are also not active at the hH₄R (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Activity of H1R and H2R ligands at the hH4R The ligands were tested as described under Materials and Methods. Data shown are mean ± standard error of mean of at least three independent experiments, each performed in triplicate. pKi values were determined with [3H]histamine displacement assay; pEC50 values show the inhibition of 1 µM forskolin-induced CRE--galactosidase activity in SK-N-MC/hH4 cells.

After an initial report that the H₄R can be labeled with [³H]mepyramine (Nguyen et al., 2001), a large number of H₁R antagonists (Table 1), including many clinically relevant drugs, were evaluated for their hH₄R affinity as well. Almost all tested H₁R antagonists, including mepyramine, showed pK_i values <5 (Table 1) and did not show functional activity at 1 and 10 µM at the hH₄R (data not shown). Although structurally similar to some tricyclic H₁R antagonists devoid of H₄R affinity, clozapine binds with moderate potency to the hH₄R (pK_i = 6.7 ± 0.1) and exerts full agonistic activity at the hH₄R with a pEC₅₀ value of 6.8 ± 0.1 (n = 5). N-Desmethyl clozapine, a clozapine metabolite, showed a slightly decreased affinity (pK_i = 6.5 ± 0.1), whereas N-oxide clozapine, another clozapine metabolite, is totally devoid of hH4R affinity. Furthermore, we evaluated clozapine analogs of therapeutic importance as well. Loxapine and amoxapine showed >10-fold lower affinity (pK_i 5.4 ± 0.1 and 5.3 ± 0.1, respectively), whereas octoclothepin did not show binding for the hH₄R at all.

Some H₂R Ligands Act as hH₄R Agonists. Within the series of known H₂R agonists that we have tested, only some ligands retain H₄R activity. Replacement of the imidazole ring of histamine in the selective H₂R agonists amthamine and amselamine (Leurs et al., 1994) results in a total loss of hH₄R activity at concentrations up to 10 µM. Dimaprit, a H₂R agonist/H₃R antagonist lacking an imidazole group, binds the H₄R with moderate affinity, showing a pK_i value of 6.5 ± 0.1, and exerts partial H₄R agonistic activity (Table 1). Impromidine, which was reported to bind to both H₂R and H₃R, also binds potently to the hH₄R with a pK_i value of 7.6 ± 0.1 and acts as a partial H₄R agonist ( = 0.5). Both the R- and S-enantiomers of the related sopromidine bind, respectively, >10 and >100 times less potently. In fact, the first reported H₂R selective agonist 4-methylhistamine (Durant et al., 1975) is the only known H₂R agonist that also acts as full agonist at the H₄R (Table 1). 4-Methylhistamine binds two times less potently than histamine to the hH₄R, exhibiting a pK_i value of 7.3 ± 0.1 (n = 3).

Most tested H₂R antagonists, including cimetidine, mifentidine, aminopotentidine, ranitidine, famotidine, and tiotidine, only displaced <30% of 10 nM [³H]histamine binding to the hH₄R. Only the H_2/3R ligand burimamide shows a high affinity for the hH₄R pK_i = 7.4 ± 0.1 (Table 1). Moreover, burimamide acted as a potent, albeit partial H₄R agonist (pEC₅₀ = 7.7 ± 0.1; = 0.7). Previously, we reported on various burimamide analogs as H₃R antagonists (Vollinga et al., 1995). In our search for H₄R selective ligands, various burimamide analogs were therefore investigated for their H₄R activity. In this series of compounds, the presence of an isopropyl (VUF 4683 and VUF 4616) or cyclohexyl (VUF 4617) moiety adjacent to the thiourea group improved the affinity for the hH₄R (Table 2). Interestingly, this series of closely related compounds exerts partial agonistic, neutral antagonistic, and inverse agonistic activities at the hH₄R (Table 2; Fig. 2A). Substitution on the thiourea with aromatic substituents, like a benzyl group in VUF 4686, results in a reduced H₄R agonistic activity. A total loss of agonistic H₄R activity, but not affinity (pK_i = 7.6 ± 0.1) is surprisingly observed for VUF 4614. As can be seen in Fig. 2B, VUF 4614 was able to competitively block the hH₄R agonistic responses of histamine, resulting in a pA₂ value of 6.8. Finally, within this series we identified VUF 4742 as an hH₄R inverse agonist (Fig. 2A). This burimamide analog bound with moderate affinity to the H₄R(pK_i = 6.9 ± 0.1; n = 4) and acted as a full inverse agonist with a pEC₅₀ value of 7.2 ± 0.1 (n = 5), in accordance with its binding affinity.

View this table: [in this window] [in a new window]   TABLE 2 Activity of burimamide analogs at the hH4R The ligands were tested as described under Materials and Methods. Data shown are mean ± standard error of mean of at least three independent experiments, each performed in triplicate. pKi values were determined with [3H]histamine displacement assay; pEC50 values show the inhibition of 1 µM forskolin-induced CRE--galactosidase activity in SK-N-MC/hH4 cells.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Burimamide analogs exert different intrinsic activities at the hH4R. A, burimamide acts as a H4R partial agonist, which dose-dependently inhibits 1 µM forskolin-induced CRE activation in SK-N-MC/hH4 cells. Various burimamide analogs act as H4R agonists (VUF 4683, burimamide, and VUF 4686), a neutral H4R antagonist (VUF 4614), or an inverse H4R agonist (VUF 4742). B, neutral H4R antagonist VUF 4614 competitively antagonizes the histamine response at the hH4R, resulting in a rightward shift of histamine dose-response curve. Data shown are from representative experiments, each performed in triplicate.

Evaluation of H₃R Ligands at the hH₄R. The H₄R shares its highest sequence homology with the H₃R, and it is therefore not surprising that in the initial studies some H₃R ligands were identified as H₄R ligands as well. We therefore characterized in this study a large set of known H₃R ligands for their interaction with the H₄R. The histamine analogs N-methylhistamine, (R)--methylhistamine, and (S)--methylhistamine show an almost 2 order of magnitude lower affinity for the hH₄R than for the hH₃R. However, the hH₄R retains some level of stereoselectivity for (R)-- (pK_i = 6.6 ± 0.1) and (S)--methylhistamine (pK_i = 5.4 ± 0.1) (Table 3). Increasing the spacer length between imidazole and amine group from two carbon atoms (histamine, pK_i = 7.8 ± 0.1) to three carbon atoms (homohistamine, pK_i = 7.5 ± 0.1) slightly decreases the affinity for the hH₄R, whereas four carbon atoms (imbutamine, pK_i = 8.0 ± 0.1) results in a slightly higher hH₄R affinity. A further increase of the spacer length proved to be detrimental for hH₄R affinity. The highly potent H₃R agonist impentamine shows only moderate affinity at the H₄R(pK_i = 6.6 ± 0.1) (Table 3). Interestingly, besides the affinity, impentamine also looses intrinsic activity for the hH₄R ( = 0). Previously identified H₃R agonists, including immepip, imetit, and VUF 8328 (an imetit analog) (Wieland et al., 2001), also potently bind the hH₄R with pK_i values of 7.7 ± 0.1, 8.2 ± 0.1, and 8.0 ± 0.1, respectively. At the hH₄R these ligands also act as agonists, but exert somewhat lower intrinsic activity ( values of 0.9, 0.9, and 0.6, respectively) (Table 3). As reported previously (Kitbunnadaj et al., 2004), the recently identified H₃R agonist immethridine (pK_i = 9.1 ± 0.1) binds much less potently to the hH₄R (pK_i = 6.6 ± 0.1) and is also not able to fully activate the H₄R (Table 3). In agreement with our findings with N-methylhistamine, the methylated immepip analog methimepip shows a large selectivity for the hH₃R (pK_i = 9.0 ± 0.1) over the hH₄R (pK_i = 5.7 ± 0.1), as reported previously (Kitbunnadaj et al., 2005). Also various H₃R antagonists bind to the hH₄R (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Zhu et al., 2001). In our hands the isothiourea-based H₃R antagonists clobenpropit and iodophenpropit both potently bind to the hH₄R (Table 3). Yet, both ligands have very distinguishable intrinsic activities at the H₄R. Clobenpropit, which acts as inverse agonist at the hH₃R (Wieland et al., 2001) behaves as a potent partial agonist at the hH₄R (Table 3; Fig. 3, A and B). In contrast, iodophenpropit, which also acts as an inverse agonist at the hH₃R (Wieland et al., 2001), behaves as a neutral antagonist at the hH₄R with a pK_i value of 7.9 ± 0.1 (n = 6) (Table 3; Fig. 3, A and B). As expected, iodophenpropit competitively antagonized the action of histamine at the hH₄R, yielding a linear Schild-plot and a pA₂ value of 8.0 (Fig. 3, C and D), in accordance with its binding affinity. Besides clobenpropit, also the known H₃R ligands proxyfan and the related iodoproxyfan show reasonable hH₄R affinity with pK_i values of 7.3 ± 0.1 and 7.9 ± 0.1, respectively. As observed for their action at the hH₃R, both compounds act as partial agonist at the hH₄R (Table 3).

View this table: [in this window] [in a new window]   TABLE 3 Activity of H4R ligands at the hH3R and hH4R The ligands were tested as described under Materials and Methods. Data shown are mean ± standard error of mean of at least three independent experiments, each performed in triplicate. pKi values for hH3R were determined with [3H]histamine displacement assay; pKi values for hH4R were determined with [3H]N-methylhistamine displacement assay. pEC50 values show the inhibition of 1 µM forskolin-induced CRE--galactosidase activity in SK-N-MC/hH3 or SK-N-MC/hH4 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Effects of H3R ligands at the H4R. A, effects of histamine, thioperamide, clobenpropit, and iodophenpropit at the hH3R expressed in SK-N-MC cells, as determined by modulation of the forskolin-induced CRE-mediated -galactosidase activity. B, histamine and clobenpropit dose-dependently inhibit forskolin-induced responses activity in SK-N-MC/hH4 cells, whereas thioperamide acts as inverse agonist at the hH4R. Iodophenpropit does not change forskolin-induced responses activity, acting as a neutral antagonist. C, iodophenpropit (IPP) antagonizes the effects of histamine at the hH4R, resulting in a rightward shift of histamine dose-response curve. D, Schild plot analysis of iodophenpropit-mediated antagonism responses. Data shown are from representative experiments, each performed in triplicate.

Evaluation of the Potential Use of [¹²⁵I]Iodophenpropit as H₄R Radioligand. As reported in the previous section and in other studies, the hH₄R can be labeled with either [³H]histamine or the H₄R antagonist [³H]JNJ 7777120 (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001; Thurmond et al., 2004). Previously, we described [¹²⁵I]iodophenproprit as a suitable high affinity H₃R radioligand (Jansen et al., 1992). Considering, the relatively high affinity of iodophenpropit at the hH₄R and its high sensitivity, we investigated the potential of this radioligand to label the H₄R. Due to the hH₄R affinity of [¹²⁵I]iodophenpropit, saturation binding experiments were not feasible. We therefore used homologous [¹²⁵I]iodophenpropit displacement analysis to determine a K_d value of 34.4 ± 4.1 nM for [¹²⁵I]iodophenpropit. The B_max value obtained using [¹²⁵I]iodophenpropit-binding displacement experiments (3.8 ± 0.4 pmol/mg protein) is approximately 2 times higher than those obtained with either [³H]histamine (1.8 ± 0.4 pmol/mg protein) and [³H]JNJ 7777120 (1.7 ± 0.4 pmol/mg protein). [¹²⁵I]Iodophenpropit binding to membranes of SK-N-MC/hH₄ cells was competitively displaced by a variety of H_3/4R ligands (Fig. 4), despite a high level of nonspecific binding of approximately 60% as determined with 10 µM imetit. However, iodophenpropit and the related clobenpropit also displaced the nonspecific binding, resulting in a multiple site binding profile. The pK_i values of compounds for the hH₄R obtained using [¹²⁵I]iodophenpropit displacement studies are consistent with their corresponding values obtained using displacement of either [³H]histamine or [³H]JNJ 7777120 binding to the hH₄R; only the pK_i value of thioperamide obtained using [¹²⁵I]iodophenpropit displacement studies seems to deviate somewhat form the pK_i values obtained using either [³H]histamine or [³H]JNJ 7777120 displacement studies (Fig. 4; Table 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Displacement of [125I]iodophenpropit binding to the hH4R by different concentrations of H3/4 ligands. The nonspecific activity was determined with 1 µM imetit. The data, shown as percentage of specific binding, fit according to a one-site ligand-receptor model. Data shown are from representative experiments, each performed in triplicate.

4-Methylhistamine as a Selective H₄R Agonist. After our initial observation of the relative high affinity of 4-methylhistamine for the hH₄R, this histamine analog was evaluated in more detail. 4-Methylhistamine not only has high affinity for the hH₄R(pK_i = 7.3 ± 0.1; n = 3) but also exhibits considerable selectivity for the hH₄R over the other three human histamine receptors (Fig. 5A). The human histamine H₁, H₂, and H₃ receptors were tested for their interaction with 4-methylhistamine, using respectively 1 nM [³H]mepyramine (K_d = 1.6 nM), 0.5 nM [¹²⁵I]iodoaminopotentidine (K_d = 0.5 nM) and 1 nM [³H]N-methylhistamine (K_d = 2.9 nM) binding to homogenates of transfected cells. As can be seen in Fig. 5A, 4-methylhistamine shows highest affinity for the hH₄R and binds considerably less potently to the other histamine receptors, resulting in a >100-fold and >100,000-fold selectivity over the H₃R and H₂R, and H₁R, respectively. 4-Methylhistamine not only binds to the hH₄R but also has a high affinity, albeit reduced compared with the hH₄R, for the mouse and rat H₄R with K_i values of 73 and 55 nM, respectively (Fig. 5B). Moreover, 4-methylhistamine exerts full agonistic activity at the hH₄R (Fig. 5C), resulting in a pEC₅₀ value of 7.4 ± 0.1 ( = 1; n = 5). In contrast, 4-methylhistamine exhibits only moderate affinity for the hH₂R (pK_i = 5.1 ± 0.1; n = 3) and hH₃R (pK_i = 5.2 ± 0.1; n = 4), and partial agonistic hH₃R activity (Fig. 5D). The hH₄R agonistic effects of 4-methylhistamine can be antagonized by the selective H₄R antagonist JNJ 7777120 (Fig. 5E). Schild-plot analysis of the JNJ 7777120 antagonism of the 4-methylhistamine-induced hH₄R-mediated inhibition of forskolin-induced -galactosidase activity yields a pA₂ value of 7.8 (data not shown), which is in agreement with the hH₄R affinity of JNJ 7777120 (Table 3) (Jablonowski et al., 2003; Thurmond et al., 2004). At the mouse and rat H₄R, 4-methylhistamine also acts as a full H₄ agonist, although with reduced pEC₅₀ values of 5.8 ± 0.1 and 5.6 ± 0.1, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 5. 4-Methylhistamine is a potent and selective H4R full agonist. A, 4-methylhistamine displacement of the binding of 1 nM [3H]mepyramine, 0.5 nM [125I]iodoaminopotentidine, 1 nM [3H]N-methylhistamine, and 10 nM [3H]histamine to the human H1, H2, H3, and H4 receptor, respectively. The chemical structure of 4-methylhistamine is presented as an insert. B, 4-methylhistamine displacement of [3H]histamine binding to mouse, rat, and human H4Rs. C and D, functional effects of 4-methylhistamine at the human H3R (D) and the human H4R (C) compared with the effects of histamine. E, 4-methylhistamine exerts a full hH4R agonistic activity that is competitively antagonized by JNJ 7777120. Data shown are from representative experiments, each performed in triplicate.

Previously, the H₄R has been shown to be involved in the regulation of eosinophil and mast cell function (O'Reilly et al., 2002; Buckland et al., 2003; Hofstra et al., 2003; Takeshita et al., 2003; Ling et al., 2004; Thurmond et al., 2004). Indeed, 4-methylhistamine also acted as H₄R agonist at human eosinophils and induced a rapid change in eosinophil cell shape, as measured by the gated autofluorescence forward scatter assay (Ling et al., 2004). The H₄R agonist 4-methylhistamine acted as a full agonist and dose-dependently induced a change in forward scatter (Fig. 6A) with an EC₅₀ value of 0.36 ± 0.09 µM, which was 3-fold less active compared with histamine. The effects of 4-methylhistamine on human eosinophils were not inhibited by H₁R antagonist mepyramine or H₂R antagonist ranitidine, but they could be antagonized by the H₄R antagonist JNJ 7777120 (Fig. 6B). H₃R antagonists were not included in this assay because Ling et al. (2004) have previously demonstrated that the H₃R antagonist JNJ 637940 did not affect histamine-induced eosinophil shape change and that the H₃R was not expressed in eosinophils. Finally, 4-methylhistamine was tested as an H₄R agonist at mouse BMMCs as described previously (Hofstra et al., 2003). Like histamine, 4-methylhistamine dose-dependently induced migration of murine BMMCs with an EC₅₀ value of 12 µM (Fig. 6C), again a somewhat lower potency compared with histamine (Hofstra et al., 2003). Furthermore, we found that the effect of 4-methylhistamine on murine BMMCs was completely inhibited by the selective H₄R antagonist JNJ 7777120 in a dose-dependent manner (Fig. 6D).

View larger version (23K): [in this window] [in a new window]   Fig. 6. Physiological effects of 4-methylhistamine on hematopoietic cells in vitro. A, effects of 4-methylhistamine on human eosinophil shape change. B, effect of 4-methylhistamine on human eosinophil shape change inhibited by the H4R antagonist JNJ 7777120, but not by H1R or H2R antagonist (mepyramine and ranitidine, respectively). C, 4-methylhistamine induction of migration of murine BMMCs. D, 4-methylhistamine-induced murine BMMC migration completely inhibited by the H4R-selective antagonist JNJ 7777120 in a dose-dependent manner. Data shown are from representative experiments, each performed either in triplicate (A and C) or in duplicate (B and D).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
With the addition of the H₄R to the histamine receptor family (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001), this potential new drug target has created a lot of excitement in the field. The predominant expression of the histamine H₄R on hematopoietic cells (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Zhu et al., 2001) and the H₄R effects on, e.g., eosinophil and mast cell functions (Gantner et al., 2002; O'Reilly et al., 2002; Buckland et al., 2003; Hofstra et al., 2003; Takeshita et al., 2003; Ling et al., 2004; Thurmond et al., 2004) imply that this new histamine receptor subtype may play a role in various allergic and inflammatory conditions. So far, the search for selective H₄R ligands has resulted in the discovery of potent neutral hH₄R antagonists as JNJ 7777120 (Jablonowski et al., 2003; Thurmond et al., 2004) and VUF 6002 (Terzioglu et al., 2004), whereas potent and selective H₄R agonists or inverse agonists have so far not been described. In search for new hH₄R ligands, we therefore screened a library of known histaminergic ligands, using SK-N-MC cells stably expressing the hH₄R. In this cell line the hH₄R binds [³H]histamine and [³H]JNJ 7777120 with high affinity (Fig. 1, A and B) and functionally inhibits forskolin-induced CRE-mediated responses through pertussis toxin-sensitive G_i/o proteins (Fig. 1D). In these cells the hH₄R also exhibits constitutive activity, which is blocked by pertussis toxin or the nonselective inverse agonist thioperamide (Fig. 1D).

Considering the H₄R shares its highest sequence similarity with the H₃R, it is not surprising that the H₄R is targeted by various imidazole containing H₃R ligands (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Zhu et al., 2001). The standard H₃R inverse agonist thioperamide (Arrang et al., 1983) also acts as an inverse agonist at the hH₄R. Moreover, in the present study we confirm that the presumed H₃R agonists immepip, imetit, (R)--methylhistamine, and imbutamine also act as potent hH₄R agonists. Furthermore, the H₄R is activated by the H₂R/H₃R antagonist burimamide, the H₃R antagonists clobenpropit, and the H₃R agonist iodoproxyfan, indicating that for hH₄R agonism considerable structural diversity (piperidine, isothiourea, thiourea, and ether) in the side chain of imidazole ring is allowed, including aromatic substitutions as indicated by the hH₄R agonism displayed by clobenpropit. However, our detailed analysis of various H₃R ligands indicates that hH₄R efficacy can be modulated by differential hydrophobic substitution on the side chain. In the burimamide series we observed that differential substitution on the thiourea group gives rise to H₄R (partial) agonists, a neutral antagonist (VUF 4614; pK_i = 7.6) and a full inverse agonist (VUF 4742; pK_i = 6.9). In addition, in the clobenpropit series, we observe that a slight change on the isothiourea substituent results in a modulation of H₄R efficacy. The clobenpropit analog iodophenpropit (a phenylethyl substituent instead of a benzyl group) retains high H₄R affinity (pK_i = 7.9), but it has lost agonistic activity completely. In this study, we identified iodophenpropit as a high-affinity, neutral antagonist for the H₄R. In view of the 15 nM affinity of iodophenpropit for the hH₄R, we evaluated [¹²⁵I]iodophenpropit as a potential new H₄R radioligand. The hH₄R can be labeled to the same extent with both the agonist [³H]histamine and the neutral antagonist [³H]JNJ 7777120. Surprisingly, the B_max value determined with [¹²⁵I]iodophenpropit was twice as much as that determined with either [³H]histamine or [³H]JNJ 7777120, suggesting that the radioligands might bind to different hH₄R subpopulations, similarly to recent findings on the binding of two H₁R radioligands to the H₁R (Booth et al., 2002). Yet, the potential existence of different H₄R subpopulations needs further investigation. The binding of the three radioligands to membranes of SK-N-MC/hH₄ cells was displaced by a variety of H_3/4R ligands, and the pK_i values obtained from these displacement studies show a high correlation. Despite being shown as a potential hH₄R radioligand, [¹²⁵I]iodophenpropit has to be used with caution, as in our hands a high level of nonspecific binding limits its use.

From our screening of many H₁R ligands, only the tricyclic clozapine shows reasonable H₄R affinity, as reported previously (Oda et al., 2000; Liu et al., 2001a; Zhu et al., 2001). Despite their structural similarity to clozapine, other tested H₁R antagonists do not show any appreciable affinity for the hH₄R. We can therefore not confirm that mepyramine binds to the hH₄R (Nguyen et al., 2001), either studied by displacement of [³H]histamine binding to the hH₄R, by saturation [³H]mepyramine binding assays (data not shown), or by functional H₄R assays. Clinically used H₁R antagonists, such as cetirizine, ebastine, fexofenadine, and loratidine, demonstrate significant in vitro anti-inflammatory activity, which are not related to their H₁R activity (Gelfand et al., 2004). The data from our study do not support the involvement of the hH₄R in the anti-inflammatory effects of these H₁R antagonists.

An important finding of this study is the discovery of 4-methylhistamine as a potent and selective hH₄R agonist in both recombinant and endogenously expressing H₄R systems. Whereas this compound is originally described as a relatively selective H₂R agonist (Durant et al., 1975), our present data show that this histamine analog exhibits more than 100-fold selectivity over the recombinant H₁R, H₂R, and H₃Rs. 4-Methylhistamine not only acts as a full agonist at the recombinant hH₄R but also induces migration of mouse bone marrow derived mast cells and a shape change of human eosinophils. Both processes have recently been shown to be induced by histamine via interaction of the H₄R (Hofstra et al., 2003; Ling et al., 2004). The relative potencies of histamine and 4-methylhistamine on human eosinophils are similar to those observed in recombinant systems. Similar to the observations with histamine (Hofstra et al., 2003; Ling et al., 2004), the potency of 4-methylhistamine on mouse mast cells is somewhat lower than in recombinant systems. Although the mouse H₄R shows a lower affinity for both histamine and 4-methylhistamine compared with the hH₄R, the low potency at BMMCs seems not merely an issue of species difference, but also it might be related to a low H₄R expression level. In fact, the H₄R in BMMCs is present at a very low density because it cannot be detected by radioligand binding studies (R. L. Thurmond, unpublished observations). Moreover, the cellular environment might dictate the potency of an agonist, such as composition of G proteins and accessories proteins in the cells (Kenakin, 2004).

In conclusion, from a large screening of many known histamine receptor ligands we have identified a variety of compounds with interesting H₄R activities. The major significance of these findings is the reevaluation of numerous histaminergic ligands at the new histamine receptor subtypes. Based upon our data, many imidazol-containing H₃R ligands, including various H₃R reference compounds, show potent H₄R activities and should be treated with caution. More recently developed H₃R agonists, such as immethridine (Kitbunnadaj et al., 2004) or methimmepip (Kitbunnadaj et al., 2005), or nonimidazole H₃R antagonists, such as JNJ 6379490 (Ling et al., 2004) or A-349821 (Esbenshade et al., 2004), hardly act at the H₄R and will therefore provide good tools to selectively target the H₃R. In the series of tested H₃R ligands, we have identified iodophenpropit as potent neutral H₄R antagonist and the burimamide analog VUF 4742 as the second identified H₄R inverse agonist. From the screening of H₂R ligands, we have identified 4-methylhistamine as the first high-affinity H₄R agonist (K_i = 50 nM) that has a >100-fold selectivity for the hH₄R over the other histamine receptor subtypes. The identification of 4-methylhistamine as a potent H₄R agonist will be of major importance for future studies to unravel the physiological roles of the H₄R.

	Acknowledgements

 
We acknowledge the technical assistance on Wen Jiang, Steven Nguyen, and Pragnya Desai.

	Footnotes

 
R.L. is a recipient of a PIONIER award of the Technology Foundation (Stichting voor de Technische Wetenschapppen) of the Netherlands Foundation of Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.087965.

ABBREVIATIONS: H_xR, histamine H_x receptor; hH₄R, human histamine H₄ receptor; IL, interleukin; JNJ 7777120, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine; VUF 6002, 1-[(5-chloro-1H-benzimidazol-2-yl)carbonyl]-4-methylpiperazine; VUF 4742, N-(4-chlorobenzyl)-N'-[5-(4(5)-imidazolyl)pentyl]thiourea; OUP-16, 2-cyano-1-methyl-1–3-{(2R,5R)-5-[1H-imidazol-4(5)-yl]tetrahydrofuran-2-yl}methylguanidine; PEA, 2-pyridylethylamine; TEA, 2-(2-thiazolyl)ethylamine; VUF 8328, S-[3-(4(5)-imidazolyl)propyl]isothiourea; ORG3770, 1,2,3,4,10,14b-hexahydro-2-methylpyrazino[2,1-a]pyrido[2,3-c][2]benzazepine; CRE, cAMP response element; BMMC, bone marrow mast cell; JNJ 637940, 7-methyl-2-[4-(3-piperidin-1-yl-propoxy)-phenyl]-imidazo[1,2-a]pyridine; A-349821, 4'-{3-[(R,R)2,5-dimethyl-pyrrolidin-1-yl]-propoxy)-biphenyl-4-yl}-morpholin-4-yl-methanone; VUF 4683, 1-[4-(imidazol-4-yl)-butyl)-3-isopropyl-thiourea; VUF 4616, 1-[5-(imidazol-4-yl)-pentyl]-3-isopropyl-thiourea; VUF 4617, 1-cyclohexyl-3-[5-(imidazol-4-yl)-pentyl]-thiourea.

¹ These authors contributed equally.

Address correspondence to: Dr. R. Leurs, Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: r.leurs{at}few.vu.nl

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