Evaluation of Histamine H1-, H2-, and H3-Receptor Ligands atthe Human Histamine H4 Receptor: Identification of 4-Methylhistamine as the First Potent and Selective H4Receptor Agonist

Herman D. Lim,1 Richard M. van Rijn,1 Ping Ling, Remko A. Bakker, Robin L. Thurmond,and Rob LeursLeiden/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 April 13, 2005; accepted June 1, 2005

ABSTRACTThe histamine H4 receptor (H4R) is involved in the chemotaxisof leukocytes and mast cells to sites of inflammation and issuggested to be a potential drug target for asthma and allergy.So far, selective H4R agonists have not been identified. In thepresent 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, includingseveral important therapeutics, displaced less than 30% ofspecific [3H]histamine binding to the hH4R at concentrations upto 10 M. Most of the tested H2R agonists and imidazole-based H3R ligands show micromolar-to-nanomolar range hH4Raffinity, and these ligands exert different intrinsic hH4R activi-

ties, ranging from full agonists to inverse agonists. Interestingly,we identified 4-methylhistamine as a high-affinity H4R ligand(Ki 50 nM) that has a 100-fold selectivity for the hH4R overthe other histamine receptor subtypes. Moreover, 4-methylhis-tamine potently activated the hH4R (pEC50 7.4 0.1; 1),and this response was competitively antagonized by the selec-tive H4R antagonist JNJ 7777120 [1-[(5-chloro-1H-indol-2-yl)-carbonyl]-4-methylpiperazine] (pA2 7.8). The identification of4-methylhistamine as a potent H4R agonist is of major impor-tance for future studies to unravel the physiological roles of theH4R.

Histamine exerts many (patho-)physiological effectsthrough its interaction with four histamine receptor subtypesthat all belong to the family of G protein-coupled receptors(Hough, 2001). The histamine H1 receptor (H1R) and H2receptor (H2R) were pharmacologically identified long beforetheir cDNAs were cloned (Gantz et al., 1991; Yamashita et

al., 1991), and they have been successful blockbuster targetsfor more than two decades. The cDNA encoding the hista-mine H3R was cloned more recently (Lovenberg et al., 1999),and bioinformatic analysis of human genome databases re-sulted in identification of the gene encoding the human H4R(hH4R) based on its sequence homology to the H3R gene(37%) (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001;Nguyen et al., 2001; Zhu et al., 2001). Although the hH3R ismainly present in the nervous system, the hH4R is distrib-uted mainly in hematopoietic cells (Oda et al., 2000; Liu etal., 2001a; Morse et al., 2001; Zhu et al., 2001). The H4Rshows a different pharmacological profile compared with theclosely related H3R, although many H3R ligands also interact

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

1 These authors contributed equally.Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.105.087965.

ABBREVIATIONS: HxR, histamine Hx 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-13-{(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; VUF4617, 1-cyclohexyl-3-[5-(imidazol-4-yl)-pentyl]-thiourea.

0022-3565/05/3143-13101321$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 3Copyright 2005 by The American Society for Pharmacology and Experimental Therapeutics 87965/3047246JPET 314:13101321, 2005 Printed in U.S.A.

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with the H4R. Like the H3R, the H4R couples to pertussistoxin-sensitive Gi/o proteins and thereby inhibits forskolin-induced cAMP production (Oda et al., 2000; Liu et al., 2001a;Zhu et al., 2001). In addition, the H4R also activates mitogen-activated protein kinase (Morse et al., 2001) and mobilizescalcium in eosinophils and mast cells (Buckland et al., 2003;Hofstra et al., 2003).The presence of the hH4R on leukocytes and mast cells

suggests that this new histamine receptor plays an impor-tant role in the modulation of the immune system. Thishypothesis is supported by the fact that IL-10 and IL-13modulate hH4R expression (Morse et al., 2001) and thatbinding sites for cytokine-regulated transcription factors,such as interferon-stimulated response element, interferonregulatory factor-1, nuclear factor-B, and nuclear factor-IL6, are present upstream of the hH4R gene (Coge et al.,2002). Physiological roles of the hH4R include the control ofIL-16 release by human CD8 T cells (Gantner et al., 2002),chemotactic responses and cytoskeletal changes of humaneosinophils (OReilly et al., 2002; Buckland et al., 2003; Linget al., 2004), chemotaxis and intracellular calcium mobiliza-tion in mast cells (Hofstra et al., 2003), and control of leuko-triene B4 production by mast cells that subsequently leads toneutrophil recruitment into peritoneum (Takeshita et al.,2003; Thurmond et al., 2004). These studies suggest that thehH4R is a potential drug target for immune system-relateddiseases.Until recently, potent and selective H4R ligands were not

available. In the early studies, the H3R antagonist thioper-amide was identified as an equally effective H4R antagonist(Oda et al., 2000; Hough, 2001). High-throughput screeningand subsequent medicinal chemistry efforts recently identi-fied the indolylpiperazine JNJ 7777120 (Jablonowski et al.,2003; Thurmond et al., 2004) and the related benzimidazoleanalog VUF 6002 (Terzioglu et al., 2004) as selective andpotent H4R antagonists. Studies directed toward selectiveH4R agonists have so far been less successful. Burimamide,clozapine, and clobenpropit are all known to act as H4Ragonists, and clozapine and clobenpropit have been provenuseful 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 imi-furamine analog OUP-16, which displays a 15-fold selectivityfor the H4R compared with its binding affinity for the H3R(Hashimoto et al., 2003). However, the lack of selectivity ofcurrently known agonists for the H4R limits their use as H4Ragonists.In our search for selective H4R agonists, many known

histaminergic ligands of different structural classes, includ-ing several important therapeutics, were evaluated for theirinteraction with the hH4R. Our studies resulted in the iden-tification of 4-methylhistamine, a presumed moderately ac-tive and selective H2R agonist (Durant et al., 1975), as ahigh-affinity H4R agonist with a more than 100-fold selectiv-ity over the H1R, H2R, and H3R.

Materials and MethodsMaterials. Aminopotentidine, amthamine dihydrobromide, am-

selamine dihydrobromide, burimamide oxalate, and burimamide an-alogs (Vollinga et al., 1995); clobenpropit dihydrochloride, dimapritdihydrobromide, histaprodifen dimaleate, homohistamine dihydro-

bromide, imbutamine dihydrobromide, imetit dihydrobromide, im-pentamine dihydrobromide, immepip dihydrobromide, immethridinedihydrobromide, iodophenpropit dihydrobromide, JNJ 7777120, 2-(3-bromophenyl)histamine dihydrobromide, methimmepip dihydrobro-mide, 2-pyridylethylamine (PEA) dihydrochloride, 2-(2-thiazolyl)eth-ylamine (TEA) dihydrochloride, thioperamide fumarate, and VUF8328 were synthesized at the Department of Medicinal Chemistry(Vrije Universiteit Amsterdam, Amsterdam, The Netherlands). Fa-motidine, ketotifen fumarate, and 8R-lisuride were purchased fromMP Biomedicals (Irvine, CA). Amoxapine, d-chlorpheniramine mal-eate, clozapine, cimetidine, N-desmethyl clozapine, diphenhydra-mine hydrochloride, doxepin hydrochloride, forskolin, histamine di-hydrochloride, imipramine hydrochloride, loxapine, mepyramine(pyrilamine maleate), (R)--methylhistamine dihydrochloride, (S)--methylhistamine dihydrochloride, N-methylhistamine dihydrochlo-ride, N-oxide clozapine, octoclothepin, pertussis toxin, polyethylenei-mine, ranitidine hydrochloride, tripelennamine hydrochloride, andtriprolidine hydrochloride were purchased from Sigma/RBI (Natick,MA). 2-Nitrophenol--D-pyranoside and G418 (geneticin) were fromDuchefa (The Netherlands); promethazine was from VUMC Phar-macy Amsterdam (Amsterdam, The Netherlands); fexofenadine wasfrom Ultrafine Chemicals (Manchester, UK); tiotidine was from Im-perial Chemical Industries PLC (London, UK); and [3H]N-methyl-histamine (85 Ci/mmol), [3H]histamine (12.4 Ci/mmol), and[3H]mepyramine (23 Ci/mmol) were from PerkinElmer Life and An-alytical Sciences (Boston, MA). [125I]Iodoaminopotentidine and[125I]iodophenpropit were labeled at the Department Nuclear Medi-cine and PET Research (Vrije Universiteit Medical Centre, Amster-dam, 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. (LaJolla, CA) (Thurmond et al., 2004). Gifts of astemizole (JanssenPharmaceuticals, Antwerp, Belgium); cyproheptadine hydrochloride(MSD, Haarlem, The Netherlands); cetirizine hydrochloride and hy-droxyzine dihydrochloride (UCB Pharma, Brussels, Belgium); ebas-tine (Almirall Prodesfarma, Barcelona, Spain); loratidine (ScheringPlough, Kenilworth, NJ); mianserin hydrochloride and ORG3770(Organon NV, Oss, The Netherlands); mifentidine (Instituto De An-geli, Milan, Italy); mizolastine (Synthelabo 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-methyl-histamine dihydrochloride, and impromidine dihydrochloride(GlaxoSmithKline, Welwyn Garden City, Hertfordshire, UK) aregreatly 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 acAMP-responsive element (CRE)-driven -galactosidase reportergene SK-N-MC/hH3 or SK-N-MC/hH4 cells (Lovenberg et al., 1999;Liu et al., 2001a), were cultured in Eagles minimum essential me-dium supplemented with 5% fetal calf serum, 0.1 mg/ml streptomy-cin, 100 U/ml penicillin, and 600 g/ml G418 at 37C in 5% CO2 and95% humidity.Radioligand Binding Assays. The SK-N-MC/hH3 cell homoge-

nates were incubated for 40 min at 25C with approximately 1 nM[3H]N-methylhistamine in 25 mM KPO4 buffer and 140 mM NaCl(pH 7.4 at 25C), with or without competing ligands, whereas theSK-N-MC/hH4 cell homogenates were incubated 1 h at 37C in 10 nM[3H]histamine and 50 mM Tris-HCl (pH 7.4 at 37C), with or withoutcompeting ligands. Bound radioligands were collected on 0.3% poly-ethyleneimine-pretreated Whatman GF/C [and washed three timeswith 3 ml of ice-cold washing buffer (4C) containing 25 mM Tris-HCland 140 mM NaCl (pH 7.4 at 4C) for the hH3R and 50 mM Tris-HCl(pH 7.4 at 4C) for the hH4R]. Binding analysis of 10 nM [

3H]JNJ7777120 and 0.1 nM [125I]iodophenpropit to the hH4R was performedwith the same conditions as described for [3H]histamine. In satura-

4-Methylhistamine Is a Potent and Selective Histamine H4R Agonist 1311

tion binding analysis, the nonspecific binding of [3H]histamine or[3H]JNJ 7777120 was determined with 1 M clobenpropit. The bind-ing analysis of [3H]mepyramine and [125I]iodoaminopotentidinebinding to human H1R and human H2R, respectively, was performedaccording to Bakker et al. (2004). The binding data were analyzedwith Prism 4.0 (GraphPad Software Inc., San Diego, CA), and dataare presented as mean S.E.M. Mouse and rat H4R radioligandbinding 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 orantagonistic activity of either the hH3R or hH4R. Approximately 4million cells/96-well plate of SK-N-MC/hH3 and SK-N-MC/hH4 cellswere exposed for 6 h to histaminergic ligands in serum-free Eaglesminimum essential medium containing 1 M forskolin. Thereafter,the medium was discarded, the cells were lysed in 100 l of assaybuffer (100 mM sodium phosphate buffer at pH 8.0, 4 mM 2-nitro-phenol--D-pyranoside, 0.5% Triton X-100, 2 mM MgSO4, 0.1 mMMnCl2, and 40 mM -mercaptoethanol), incubated overnight at roomtemperature, and the -galactosidase activity was determined at 420nm with a PowerwaveX340 plate reader (Bio-Tek Instruments Inc.,Winooski, VT). The OD420 might differ between experiments due tointra-assay variability; therefore, intrinsic activity of agonists wasdetermined relatively to activity of histamine.

Primary Cell Experiments. Cell culture of BALB/c mice-derivedbone marrow mast cells (BMMCs) and in vitro BMMC chemotaxisassay was performed as described previously (Hofstra et al., 2003).Purification of human polymorphonuclear leukocytes and the humaneosinophil shape change assay were performed as described previ-ously (Ling et al., 2004). The mouse-derived BMMCs were obtainedfollowing approved protocols that follow National Institutes ofHealth/International Animal Care and Use Guidelines.

ResultsPharmacological Characterization of the hH4R Ex-

pressed in SK-N-MC Cells. Stable transfection of the hH4RcDNA in SK-N-MC cells resulted in the expression of func-tional hH4R proteins. The hH4R could be labeled with bothagonist and antagonist radioligands. The H4R agonist[3H]histamine shows saturable binding to the expressed H4Rwith a minimal amount of nonspecific binding (Fig. 1A).Analysis of the [3H]histamine saturation binding yielded aKd value of 11 1.0 nM (n 6) and a Bmax value of 1.8 0.4pmol/mg protein. Recently, JNJ 7777120 was described as aselective H4R antagonist (Jablonowski et al., 2003). In our

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 showsa 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 M forskolin-induced CREactivation, as measured by a -galactosidase reporter gene, whereas the inverse agonist thioperamide dose-dependently blocks the hH4R constitutiveactivity (D). Data shown are from representative experiments, each performed in triplicate.

1312 Lim et al.

hands, the nonimidazole JNJ 7777120 shows a 300-fold se-lectivity for the hH4R (pKi 7.8 0.1 against [

3H]histamine)over the hH3R (pKi 5.3 0.1 against [

3H]N-methylhista-mine), allowing the use of [3H]JNJ 7777120 to label the H4R(Thurmond et al., 2004). The H4R antagonist [

3H]JNJ7777120 exhibits a somewhat higher level of nonspecificbinding to hH4R expressing SK-N-MC cells, but it also bindssaturably and shows an equipotent affinity (Kd 11 3.6nM; n 3) and results in a Bmax value of 1.7 0.4 pmol/mgprotein (Fig. 1B). The binding of either 10 nM [3H]histamine(Fig. 1C) or 10 nM [3H]JNJ 7777120 (data not shown) to thehH4R is fully displaced by histamine (pKi 7.8 0.1), theH3/4R antagonist thioperamide (pKi 6.9 0.1) and the H4Ragonist/H3R antagonist clobenpropit (pKi 8.1 0.1), in agood agreement with the results reported previously (Oda etal., 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 there-fore also be used for a functional analysis of H4R ligands.Stimulation of the hH4R with histamine resulted in the in-hibition (58 3%; n 16) of the forskolin-stimulated (1 M)cAMP-mediated reporter gene transcription with a pEC50value of 7.7 0.1 (n 16) (Fig. 1D). Treatment of SK-N-MC/hH4 cells with the Gi/o protein inhibitor pertussis toxin (100ng/ml for 16 h) completely inhibited histamine induced re-sponses, confirming the coupling of the H4R to Gi/o proteins(Oda et al., 2000; Liu et al., 2001a; Zhu et al., 2001). In ourhands, histamine exerted the maximally observed level ofinhibition in this assay and is therefore referred to as a fullagonist (intrinsic activity 1). As reported previously (Odaet al., 2000; Liu et al., 2001a; Morse et al., 2001), clobenpropitacts as a potent partial hH4R agonist with a pEC50 value of7.7 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 M forskolin-stimulated CRE activity by 130 3%, suggesting that thehH4R shows a detectable level of constitutive activity in theSK-N-MC/hH4 cells. In line with previous observations oninverse agonism by thioperamide (Liu et al., 2001a; Morse etal., 2001), 1 M forskolin-stimulated CRE activity was in-creased by thioperamide with a pEC50 value of 7.0 0.1 (Fig.1D). The inhibition of the constitutive activity of the hH4R bythioperamide was of the same magnitude as observed aftertreatment with pertussis toxin and thioperamide is referredto 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 endog-enously expressed Gs protein-coupled adrenergic recep-tors (Bahouth et al., 2001). The 2 adrenergic receptor ago-nist feneterol induced -galactosidase activity to a similarextent to that of forskolin, with a pEC50 value of 6.9 0.1(n 6). H4R activation by histamine inhibited the 100 nMfeneterol-induced -galactosidase activity for 39 3% with apEC50 value of 7.4 0.1 (n 7). However, inverse agonisticactivity of thioperamide, at the hH4R, could not be easilydemonstrated with a feneterol-based assay system (data notshown). The evaluation of the functional activity of all thevarious histaminergic ligands was therefore performed usingforskolin (1 M) 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 M. Compounds inhibiting

the specific binding of 10 nM [3H]histamine to the hH4R by30% are expected to have a Ki 10 M based on the Chengand Prusoff equation (Cheng and Prusoff, 1973): Ki IC50/(1 [radioligand]/Kd) and were excluded for further testing.Active compounds (displacement 30%) were tested moreextensively in both [3H]histamine displacement studies andthe CRE--galactosidase-based functional H4R assay.Most H1R Ligands Are Devoid of H4R Activity. Hista-

mine potently displaces [3H]histamine from the hH4R with apKi value of 7.8 0.1 (Table 1), whereas H1R agonists withsubstituents at the 2-position of the imidazole ring showsignificantly lower affinities. Substitution of the imidazolering with either a small methyl or large 3-bromophenyl sub-stituent is not tolerated and causes an almost 100-fold dropof affinity. Bulkier substituents at the 2-position (1,1-diphe-nylpropyl in histaprodifen) even result in a total loss ofaffinity for the hH4R (Table 1). Agonists, lacking the imida-zole ring, such as TEA, PEA, or 8R-lisuride (Bakker et al.,2004), are also not active at the hH4R (Table 1).After an initial report that the H4R can be labeled with

[3H]mepyramine (Nguyen et al., 2001), a large number ofH1R antagonists (Table 1), including many clinically relevantdrugs, were evaluated for their hH4R affinity as well. Almostall tested H1R antagonists, including mepyramine, showedpKi values 5 (Table 1) and did not show functional activityat 1 and 10 M at the hH4R (data not shown). Althoughstructurally similar to some tricyclic H1R antagonists devoidof H4R affinity, clozapine binds with moderate potency to thehH4R (pKi 6.7 0.1) and exerts full agonistic activity atthe hH4R with a pEC50 value of 6.8 0.1 (n 5). N-Desmethyl clozapine, a clozapine metabolite, showed aslightly decreased affinity (pKi 6.5 0.1), whereas N-oxideclozapine, another clozapine metabolite, is totally devoid ofhH4R affinity. Furthermore, we evaluated clozapine analogsof therapeutic importance as well. Loxapine and amoxapineshowed 10-fold lower affinity (pKi 5.4 0.1 and 5.3 0.1,respectively), whereas octoclothepin did not show binding forthe hH4R at all.Some H2R Ligands Act as hH4R Agonists. Within the

series of known H2R agonists that we have tested, only someligands retain H4R activity. Replacement of the imidazolering of histamine in the selective H2R agonists amthamineand amselamine (Leurs et al., 1994) results in a total loss ofhH4R activity at concentrations up to 10 M. Dimaprit, aH2R agonist/H3R antagonist lacking an imidazole group,binds the H4R with moderate affinity, showing a pKi value of6.5 0.1, and exerts partial H4R agonistic activity (Table 1).Impromidine, which was reported to bind to both H2R andH3R, also binds potently to the hH4R with a pKi value of7.6 0.1 and acts as a partial H4R agonist ( 0.5). Both theR- and S-enantiomers of the related sopromidine bind, re-spectively, 10 and 100 times less potently. In fact, thefirst reported H2R selective agonist 4-methylhistamine (Du-rant et al., 1975) is the only known H2R agonist that also actsas full agonist at the H4R (Table 1). 4-Methylhistamine bindstwo times less potently than histamine to the hH4R, exhib-iting a pKi value of 7.3 0.1 (n 3).Most tested H2R antagonists, including cimetidine, mifen-

tidine, aminopotentidine, ranitidine, famotidine, and tioti-dine, only displaced30% of 10 nM [3H]histamine binding tothe hH4R. Only the H2/3R ligand burimamide shows a highaffinity for the hH4R pKi 7.4 0.1 (Table 1). Moreover,

4-Methylhistamine Is a Potent and Selective Histamine H4R Agonist 1313

burimamide acted as a potent, albeit partial H4R agonist(pEC50 7.7 0.1; 0.7). Previously, we reported onvarious burimamide analogs as H3R antagonists (Vollinga etal., 1995). In our search for H4R selective ligands, variousburimamide analogs were therefore investigated for theirH4R activity. In this series of compounds, the presence of anisopropyl (VUF 4683 and VUF 4616) or cyclohexyl (VUF4617) moiety adjacent to the thiourea group improved theaffinity for the hH4R (Table 2). Interestingly, this series ofclosely related compounds exerts partial agonistic, neutralantagonistic, and inverse agonistic activities at the hH4R(Table 2; Fig. 2A). Substitution on the thiourea with aromaticsubstituents, like a benzyl group in VUF 4686, results in areduced H4R agonistic activity. A total loss of agonistic H4Ractivity, but not affinity (pKi 7.6 0.1) is surprisinglyobserved for VUF 4614. As can be seen in Fig. 2B, VUF 4614was able to competitively block the hH4R agonistic responses

of histamine, resulting in a pA2 value of 6.8. Finally, withinthis series we identified VUF 4742 as an hH4R inverse ago-nist (Fig. 2A). This burimamide analog bound with moderateaffinity to the H4R (pKi 6.9 0.1; n 4) and acted as a fullinverse agonist with a pEC50 value of 7.2 0.1 (n 5), inaccordance with its binding affinity.Evaluation of H3R Ligands at the hH4R. The H4R

shares its highest sequence homology with the H3R, and it istherefore not surprising that in the initial studies some H3Rligands were identified as H4R ligands as well. We thereforecharacterized in this study a large set of known H3R ligandsfor their interaction with the H4R. The histamine analogsN-methylhistamine, (R)--methylhistamine, and (S)--methylhistamine show an almost 2 order of magnitude loweraffinity for the hH4R than for the hH3R. However, the hH4Rretains some level of stereoselectivity for (R)-- (pKi 6.6 0.1) and (S)--methylhistamine (pKi 5.4 0.1) (Table 3).

TABLE 1Activity of H1R and H2R ligands at the hH4RThe ligands were tested as described under Materials and Methods. Data shown are mean standard error of mean of at least three independent experiments, eachperformed in triplicate. pKi values were determined with

3Hhistamine displacement assay; pEC50 values show the inhibition of 1 M forskolin-induced CRE--galactosidaseactivity in SK-N-MC/hH4 cells.

pKi at hH1R H1R Activity pKi pEC50

H1R liganda

Histamine 4.2 0.1b Agonist 7.8 0.1 7.7 0.1 12-Methylhistamine Agonist 6.1 0.1 5.9 0.1 0.82-(3-Bromophenyl)histamine Agonist 6.0 0.1 5.9 0.2 0.6Histaprodifen 5.7 0.1d Agonist 5 N.D. N.D.TEA 4d Agonist 5 N.D. N.D.PEA 3.8 0.1d Agonist 5 N.D. N.D.8R-Lisuride 7.9 0.1c Agonist 5.3 0.1 Azatidine Antagonist 5 N.D. N.D.Cetirizine 8.0 0.1e Antagonist 5 N.D. N.D.Clozapine 9.4 0.1f Antagonist 6.7 0.1 6.8 0.1 1Amoxapine 7.4 0.1f Antagonist 5.3 0.1 6.7 0.1 0.5Loxapine 8.3 0.1f Antagonist 5.4 0.1 6.2 0.1 0.8Octoclothepin 8.6 0.1f Antagonist 5 N.D. N.D.Ebastine 8.0 0.1b Antagonist 5 N.D. N.D.Fexofenadine 8.0 0.3e Antagonist 5 N.D. N.D.Hydroxyzine 8.7 0.1e Antagonist 5 N.D. N.D.Loratidine 6.8 0.1b Antagonist 5 N.D. N.D.Mizolastine 9.1 0.1b Antagonist 5 N.D. N.D.Mepyramine 8.7 0.1b Antagonist 5 N.D. N.D.R-()-Terfenadine 7.0 0.1b Antagonist 5 N.D. N.D.

pKi at hH2R H2R Activity pKi pEC50

H2R ligandHistamine 4.3 0.1g Agonist 7.8 0.1 7.7 0.1 14-Methylhistamine 5.1 0.1 Agonist 7.3 0.1 7.4 0.1 1Amthamine 5.2 0.1g Agonist 5.3 0.1 0Amselamine 5.0 0.1g Agonist 5.6 0.1 0Dimaprit 4.6 0.1g Agonist 6.5 0.1 5.8 0.2 0.8Impromidine 6.3 0.1g Agonist 7.6 0.1 7.6 0.1 0.5S-()-Sopromidine Agonist 5.5 0.1 0R-()-Sopromidine Agonist 6.1 0.1 0Aminopotentidine 7.8 0.1g Antagonist 5 N.D. N.D.Burimamide 5.4 0.2g Antagonist 7.4 0.1 7.7 0.1 0.7Cimetidine 6.2 0.2g Antagonist 5 N.D. N.D.Famotidine 7.8 0.1g Antagonist 5 N.D. N.D.Mifentidine Antagonist 5 N.D. N.D.Ranitidine 7.1 0.1g Antagonist 5 N.D. N.D.Tiotidine 7.8 0.2g Antagonist 5 N.D. N.D.

, intrinsic activity (1 designated for full agonistic, 0 for neutral antagonist, and 1 for full inverse agonistic activity); N.D., not determined; , due to non-H4R-mediatedeffects of 8R-lisuride, the pEC50 value was not determined.

a H1R antagonists astemizole, chlorpheniramine, cyproheptadine, desipramine, dexchlorpheniramine, diphenhydramine, doxepine, imipramine, ketotifen, mianserine,octoclothepin, ORG3770, promethazine, S-()-terfenadine, tripelennamine, and triprolidine also have pKi values 5.

b pKi values determined with 3Hmepyramine displacement assay (Bakker et al., 2001).

c pKi value determined with 3Hmepyramine displacement assay (Bakker et al., 2004).

d pKi values determined with 3Hmepyramine displacement assay (Govoni et al., 2003).

e pKi values determined with 3Hmepyramine displacement assay (Gillard et al., 2002).

f Potencies as inverse H1R agonists determined with receptor selection and amplification technology assay (Bakker, 2003).g pKi values determined with

125Iiodoaminopotentidine displacement assay (Leurs et al., 1994).

1314 Lim et al.

Increasing the spacer length between imidazole and aminegroup from two carbon atoms (histamine, pKi 7.8 0.1) tothree carbon atoms (homohistamine, pKi 7.5 0.1) slightlydecreases the affinity for the hH4R, whereas four carbonatoms (imbutamine, pKi 8.0 0.1) results in a slightlyhigher hH4R affinity. A further increase of the spacer lengthproved to be detrimental for hH4R affinity. The highly potentH3R agonist impentamine shows only moderate affinity atthe H4R (pKi 6.6 0.1) (Table 3). Interestingly, besides theaffinity, impentamine also looses intrinsic activity for thehH4R ( 0). Previously identified H3R agonists, includingimmepip, imetit, and VUF 8328 (an imetit analog) (Wielandet al., 2001), also potently bind the hH4R with pKi values of7.7 0.1, 8.2 0.1, and 8.0 0.1, respectively. At the hH4Rthese 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 (pKi 9.1 0.1) binds much less potently to the hH4R (pKi 6.6 0.1) and is also not able to fully activate the H4R (Table 3). Inagreement with our findings with N-methylhistamine, themethylated immepip analog methimepip shows a large selec-tivity for the hH3R (pKi 9.0 0.1) over the hH4R (pKi 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 cloben-propit and iodophenpropit both potently bind to the hH4R(Table 3). Yet, both ligands have very distinguishable intrin-sic activities at the H4R. Clobenpropit, which acts as inverse

TABLE 2Activity of burimamide analogs at the hH4RThe ligands were tested as described under Materials and Methods. Data shown are mean standard error of mean of at least three independent experiments, eachperformed in triplicate. pKi values were determined with

3Hhistamine displacement assay; pEC50 values show the inhibition of 1 M forskolin-induced CRE--galactosidaseactivity in SK-N-MC/hH4 cells.

Ligand n R pKi at hH2R pKi pEC50

Burimamide 4 Methyl 5.4 0.2a 7.4 0.1 6.8 0.1 0.7VUF 4681 4 Ethyl 7.6 0.1 7.0 0.1 0.8VUF 4682 4 n-Propyl 8.0 0.1 7.2 0.1 0.8VUF 4683 4 Isopropyl 8.1 0.1 7.7 0.1 0.8VUF 4686 4 Benzyl 7.3 0.1 7.1 0.3 0.3VUF 4687 4 Phenylethyl 7.2 0.1 5.9 0.3 0.3VUF 4614 5 Ethyl 5.0 0.1b 7.6 0.1 0VUF 4616 5 Isopropyl 5.0 0.1b 7.9 0.1 c N.D.VUF 4617 5 Cyclohexyl 5.4 0.1b 7.9 0.1 c N.D.VUF 4742 5 4-Chlorobenzyl 5.8 0.2b 6.9 0.1 7.2 0.1 1

, intrinsic activity (1 designated for full agonistic, 0 for neutral antagonist, and 1 for full inverse agonistic activity); N.D., not determined.a pKi values determined with

125Iiodoaminopotentidine displacement assay (Leurs et al., 1994).b pKi values determined with

125Iiodoaminopotentidine displacement assay (Vollinga et al., 1995).c Compounds that exerted a biphasic response: a slight decrease of forskolin stimulated -galactosidase activity at concentrations up to 1 M and an increase at

concentrations 10 M. The latter effect is not mediated by the hH4R, because it cannot be blocked by H4R antagonist JNJ 7777120.

Fig. 2. Burimamide analogs exert different intrinsic activities at the hH4R. A, burimamide acts as a H4R partial agonist, which dose-dependentlyinhibits 1 M forskolin-induced CRE activation in SK-N-MC/hH4 cells. Various burimamide analogs act as H4R agonists (VUF 4683, burimamide, andVUF 4686), a neutral H4R antagonist (VUF 4614), or an inverse H4R agonist (VUF 4742). B, neutral H4R antagonist VUF 4614 competitivelyantagonizes the histamine response at the hH4R, resulting in a rightward shift of histamine dose-response curve. Data shown are from representativeexperiments, each performed in triplicate.

4-Methylhistamine Is a Potent and Selective Histamine H4R Agonist 1315

agonist at the hH3R (Wieland et al., 2001) behaves as apotent partial agonist at the hH4R (Table 3; Fig. 3, A and B).In contrast, iodophenpropit, which also acts as an inverseagonist at the hH3R (Wieland et al., 2001), behaves as aneutral antagonist at the hH4R with a pKi value of 7.9 0.1(n 6) (Table 3; Fig. 3, A and B). As expected, iodophenpropitcompetitively antagonized the action of histamine at thehH4R, 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 proxyfanand the related iodoproxyfan show reasonable hH4R affinitywith pKi values of 7.3 0.1 and 7.9 0.1, respectively. Asobserved for their action at the hH3R, both compounds act aspartial agonist at the hH4R (Table 3).Evaluation of the Potential Use of [125I]Iodophen-

propit as H4R Radioligand. As reported in the previoussection and in other studies, the hH4R can be labeled witheither [3H]histamine or the H4R antagonist [

3H]JNJ 7777120(Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyenet al., 2001; Zhu et al., 2001; Thurmond et al., 2004). Previ-ously, we described [125I]iodophenproprit as a suitable highaffinity H3R radioligand (Jansen et al., 1992). Considering,the relatively high affinity of iodophenpropit at the hH4R andits high sensitivity, we investigated the potential of thisradioligand to label the H4R. Due to the hH4R affinity of[125I]iodophenpropit, saturation binding experiments werenot feasible. We therefore used homologous [125I]iodophen-propit displacement analysis to determine a Kd value of34.4 4.1 nM for [125I]iodophenpropit. The Bmax value ob-tained using [125I]iodophenpropit-binding displacement ex-periments (3.8 0.4 pmol/mg protein) is approximately 2times higher than those obtained with either [3H]histamine(1.8 0.4 pmol/mg protein) and [3H]JNJ 7777120 (1.7 0.4pmol/mg protein). [125I]Iodophenpropit binding to mem-branes of SK-N-MC/hH4 cells was competitively displaced bya variety of H3/4R ligands (Fig. 4), despite a high level ofnonspecific binding of approximately 60% as determined

with 10 M imetit. However, iodophenpropit and the relatedclobenpropit also displaced the nonspecific binding, resultingin a multiple site binding profile. The pKi values of com-pounds for the hH4R obtained using [

125I]iodophenpropit dis-placement studies are consistent with their correspondingvalues obtained using displacement of either [3H]histamineor [3H]JNJ 7777120 binding to the hH4R; only the pKi valueof thioperamide obtained using [125I]iodophenpropit dis-placement studies seems to deviate somewhat form the pKivalues obtained using either [3H]histamine or [3H]JNJ7777120 displacement studies (Fig. 4; Table 4).4-Methylhistamine as a Selective H4R Agonist. After

our initial observation of the relative high affinity of 4-meth-ylhistamine for the hH4R, this histamine analog was evalu-ated in more detail. 4-Methylhistamine not only has highaffinity for the hH4R (pKi 7.3 0.1; n 3) but also exhibitsconsiderable selectivity for the hH4R over the other threehuman histamine receptors (Fig. 5A). The human histamineH1, H2, and H3 receptors were tested for their interactionwith 4-methylhistamine, using respectively 1 nM [3H]me-pyramine (Kd 1.6 nM), 0.5 nM [

125I]iodoaminopotentidine(Kd 0.5 nM) and 1 nM [

3H]N-methylhistamine (Kd 2.9nM) binding to homogenates of transfected cells. As can beseen in Fig. 5A, 4-methylhistamine shows highest affinity forthe hH4R and binds considerably less potently to the otherhistamine 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 ahigh affinity, albeit reduced compared with the hH4R, for themouse and rat H4R with Ki values of 73 and 55 nM, respec-tively (Fig. 5B). Moreover, 4-methylhistamine exerts full ag-onistic activity at the hH4R (Fig. 5C), resulting in a pEC50value of 7.4 0.1 ( 1; n 5). In contrast, 4-methylhista-mine exhibits only moderate affinity for the hH2R (pKi 5.1 0.1; n 3) and hH3R (pKi 5.2 0.1; n 4), andpartial agonistic hH3R activity (Fig. 5D). The hH4R agonisticeffects of 4-methylhistamine can be antagonized by the se-

TABLE 3Activity of H4R ligands at the hH3R and hH4RThe ligands were tested as described under Materials and Methods. Data shown are mean standard error of mean of at least three independent experiments, eachperformed in triplicate. pKi values for hH3R were determined with

3Hhistamine displacement assay; pKi values for hH4R were determined with 3HN-methylhistamine

displacement assay. pEC50 values show the inhibition of 1 M forskolin-induced CRE--galactosidase activity in SK-N-MC/hH3 or SK-N-MC/hH4 cells.

LigandhH3R hH4R

pKi pEC50 pKi pEC50

Histamine 8.0 0.1 8.3 0.1 1 7.8 0.1 7.7 0.1 1N-Methylhistamine 8.4 0.1 9.4 0.1 1 6.5 0.1 6.1 0.1 1(R)--Methylhistamine 8.2 0.1 9.5 0.1 1 6.6 0.1 6.2 0.1 1(S)--Methylhistamine 7.2 0.1 8.0 0.1 1 5.4 0.1 4.9 0.1 1Homohistamine 7.3 0.1 7.4 0.2 0.9 7.5 0.1 6.7 0.1 0.8Imbutamine 8.4 0.1 9.2 0.1 1 8.0 0.1 7.5 0.1 0.8Impentamine 8.3 0.1 8.4 0.1 0.9 6.6 0.1 0Immepip 9.3 0.1 10.4 0.1 1 7.7 0.1 7.8 0.1 0.9Methimmepip 9.0 0.1 9.5 0.2 0.8 5.7 0.1 5.3 0.2 0.5Immethridine 9.1 0.1 9.8 0.1 0.9 6.6 0.1 6.0 0.1 0.5Imetit 8.8 0.1 9.9 0.1 1 8.2 0.1 7.9 0.1 0.9VUF 8328 8.5 0.1 9.3 0.2 1 8.0 0.1 7.9 0.1 0.6Clobenpropit 8.6 0.1 9.4 0.1 1 8.1 0.1 7.7 0.1 0.8Iodophenpropit 8.2 0.1 8.5 0.1 1 7.9 0.1 0Impromidine 6.8 0.1 6.5 0.3 0.5 7.6 0.1 7.6 0.1 0.5Burimamide 7.9 0.1 7.0 0.1 0.5 7.4 0.1 7.7 0.1 0.7Thioperamide 7.3 0.1 7.5 0.1 1 6.9 0.1 7.0 0.1 1Proxyfan 7.9 0.1 8.5 0.1 1 7.3 0.1 7.2 0.1 0.5Iodoproxyfan 9.2 0.1 10.3 0.1 1 7.9 0.1 7.9 0.1 0.7JNJ 7777120 5.3 0.1 6.0 0.1 0.7 7.8 0.1 0

, intrinsic activity (1 designated for full agonistic, 0 for neutral antagonist, and 1 for full inverse agonistic activity).

1316 Lim et al.

lective H4R antagonist JNJ 7777120 (Fig. 5E). Schild-plotanalysis of the JNJ 7777120 antagonism of the 4-methylhis-tamine-induced hH4R-mediated inhibition of forskolin-in-

duced -galactosidase activity yields a pA2 value of 7.8 (datanot shown), which is in agreement with the hH4R affinity ofJNJ 7777120 (Table 3) (Jablonowski et al., 2003; Thurmondet al., 2004). At the mouse and rat H4R, 4-methylhistaminealso acts as a full H4 agonist, although with reduced pEC50values of 5.8 0.1 and 5.6 0.1, respectively.Previously, the H4R has been shown to be involved in the

regulation of eosinophil and mast cell function (OReilly etal., 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 agonistat human eosinophils and induced a rapid change in eosino-phil cell shape, as measured by the gated autofluorescenceforward scatter assay (Ling et al., 2004). The H4R agonist4-methylhistamine acted as a full agonist and dose-depen-dently induced a change in forward scatter (Fig. 6A) with anEC50 value of 0.36 0.09 M, which was 3-fold less activecompared with histamine. The effects of 4-methylhistamineon human eosinophils were not inhibited by H1R antagonistmepyramine or H2R antagonist ranitidine, but they could beantagonized by the H4R antagonist JNJ 7777120 (Fig. 6B).H3R antagonists were not included in this assay because

Fig. 3. Effects of H3R ligands at the H4R. A, effects of histamine, thioperamide, clobenpropit, and iodophenpropit at the hH3R expressed in SK-N-MCcells, as determined by modulation of the forskolin-induced CRE-mediated -galactosidase activity. B, histamine and clobenpropit dose-dependentlyinhibit forskolin-induced responses activity in SK-N-MC/hH4 cells, whereas thioperamide acts as inverse agonist at the hH4R. Iodophenpropit does notchange 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 shownare from representative experiments, each performed in triplicate.

Fig. 4. Displacement of [125I]iodophenpropit binding to the hH4R bydifferent concentrations of H3/4 ligands. The nonspecific activity wasdetermined with 1 M imetit. The data, shown as percentage of specificbinding, fit according to a one-site ligand-receptor model. Data shown arefrom representative experiments, each performed in triplicate.

4-Methylhistamine Is a Potent and Selective Histamine H4R Agonist 1317

Ling et al. (2004) have previously demonstrated that the H3Rantagonist JNJ 637940 did not affect histamine-induced eo-sinophil shape change and that the H3R was not expressed ineosinophils. Finally, 4-methylhistamine was tested as anH4R agonist at mouse BMMCs as described previously (Hof-stra et al., 2003). Like histamine, 4-methylhistamine dose-dependently induced migration of murine BMMCs with anEC50 value of 12 M (Fig. 6C), again a somewhat lowerpotency compared with histamine (Hofstra et al., 2003). Fur-thermore, we found that the effect of 4-methylhistamineon murine BMMCs was completely inhibited by the selectiveH4R antagonist JNJ 7777120 in a dose-dependent manner(Fig. 6D).

Discussion

With the addition of the H4R to the histamine receptorfamily (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001;Nguyen et al., 2001; Zhu et al., 2001), this potential new drugtarget has created a lot of excitement in the field. The pre-dominant expression of the histamine H4R on hematopoieticcells (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001;Zhu et al., 2001) and the H4R effects on, e.g., eosinophil andmast cell functions (Gantner et al., 2002; OReilly 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 thisnew histamine receptor subtype may play a role in various

TABLE 4hH4R affinity for selected H4R ligands as determined with displacement of the binding of

3Hhistamine, 3HJNJ 7777120, or 125IiodophenpropitData shown are mean standard error of mean of at least three independent experiments, each performed in triplicate.

CompoundpKi

3HHistamine 3HJNJ 7777120 125IIodophenpropit

Histamine 7.8 0.1 7.7 0.1 7.6 0.24-Methylhistamine 7.3 0.1 7.6 0.1 7.4 0.1Immepip 7.7 0.1 7.9 0.1 7.7 0.1Clozapine 6.7 0.1 6.4 0.1 6.5 0.2Clobenpropit 8.1 0.1 7.9 0.1 7.8 0.1Iodophenpropit 7.9 0.1 7.7 0.1 7.7 0.1Thioperamide 6.9 0.1 7.1 0.1 7.4 0.1JNJ 7777120 7.8 0.1 7.8 0.2 7.8 0.1

Kd (nM) 20.9 1.6 11.1 3.6 34.4 4.1a

Bmax (pmol/mg protein) 1.8 0.4 1.7 0.4 3.8 0.4a Determined by homologous displacement analysis, using the equation Kd IC50 radioligand.

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. Thechemical structure of 4-methylhistamine is presented as an insert. B, 4-methylhistamine displacement of [3H]histamine binding to mouse, rat, andhuman 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 representativeexperiments, each performed in triplicate.

1318 Lim et al.

allergic and inflammatory conditions. So far, the search forselective H4R ligands has resulted in the discovery of potentneutral hH4R antagonists as JNJ 7777120 (Jablonowski etal., 2003; Thurmond et al., 2004) and VUF 6002 (Terzioglu etal., 2004), whereas potent and selective H4R agonists orinverse agonists have so far not been described. In search fornew hH4R ligands, we therefore screened a library of knownhistaminergic ligands, using SK-N-MC cells stably express-ing the hH4R. In this cell line the hH4R binds [

3H]histamineand [3H]JNJ 7777120 with high affinity (Fig. 1, A and B) andfunctionally inhibits forskolin-induced CRE-mediated re-sponses through pertussis toxin-sensitive Gi/o proteins (Fig.1D). In these cells the hH4R also exhibits constitutive activ-ity, which is blocked by pertussis toxin or the nonselectiveinverse agonist thioperamide (Fig. 1D).Considering the H4R shares its highest sequence similarity

with the H3R, it is not surprising that the H4R is targeted byvarious imidazole containing H3R ligands (Oda et al., 2000;Liu et al., 2001a; Morse et al., 2001; Zhu et al., 2001). Thestandard 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 imbu-tamine also act as potent hH4R agonists. Furthermore, theH4R is activated by the H2R/H3R antagonist burimamide, theH3R antagonists clobenpropit, and the H3R agonist iodo-proxyfan, indicating that for hH4R agonism considerablestructural diversity (piperidine, isothiourea, thiourea, andether) in the side chain of imidazole ring is allowed, includingaromatic substitutions as indicated by the hH4R agonismdisplayed by clobenpropit. However, our detailed analysis ofvarious H3R ligands indicates that hH4R efficacy can bemodulated by differential hydrophobic substitution on theside chain. In the burimamide series we observed that dif-ferential substitution on the thiourea group gives rise to H4R(partial) agonists, a neutral antagonist (VUF 4614; pKi 7.6)and a full inverse agonist (VUF 4742; pKi 6.9). In addition,in the clobenpropit series, we observe that a slight change onthe isothiourea substituent results in a modulation of H4Refficacy. The clobenpropit analog iodophenpropit (a phenyl-ethyl substituent instead of a benzyl group) retains high H4Raffinity (pKi 7.9), but it has lost agonistic activity com-pletely. In this study, we identified iodophenpropit as a high-affinity, neutral antagonist for the H4R. In view of the 15

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 murineBMMCmigration completely inhibited by the H4R-selective antagonist JNJ 7777120 in a dose-dependent manner. Data shown are from representativeexperiments, each performed either in triplicate (A and C) or in duplicate (B and D).

4-Methylhistamine Is a Potent and Selective Histamine H4R Agonist 1319

nM affinity of iodophenpropit for the hH4R, we evaluated[125I]iodophenpropit as a potential new H4R radioligand. ThehH4R can be labeled to the same extent with both the agonist[3H]histamine and the neutral antagonist [3H]JNJ 7777120.Surprisingly, the Bmax value determined with [

125I]iodophen-propit was twice as much as that determined with either[3H]histamine or [3H]JNJ 7777120, suggesting that the ra-dioligands might bind to different hH4R subpopulations, sim-ilarly to recent findings on the binding of two H1R radioli-gands to the H1R (Booth et al., 2002). Yet, the potentialexistence of different H4R subpopulations needs further in-vestigation. The binding of the three radioligands to mem-branes of SK-N-MC/hH4 cells was displaced by a variety ofH3/4R ligands, and the pKi values obtained from these dis-placement studies show a high correlation. Despite beingshown as a potential hH4R radioligand, [

125I]iodophenpropithas to be used with caution, as in our hands a high level ofnonspecific binding limits its use.From our screening of many H1R ligands, only the tricyclic

clozapine shows reasonable H4R affinity, as reported previ-ously (Oda et al., 2000; Liu et al., 2001a; Zhu et al., 2001).Despite their structural similarity to clozapine, other testedH1R antagonists do not show any appreciable affinity for thehH4R. We can therefore not confirm that mepyramine bindsto the hH4R (Nguyen et al., 2001), either studied by displace-ment of [3H]histamine binding to the hH4R, by saturation[3H]mepyramine binding assays (data not shown), or by func-tional H4R assays. Clinically used H1R antagonists, such ascetirizine, ebastine, fexofenadine, and loratidine, demon-strate significant in vitro anti-inflammatory activity, whichare not related to their H1R activity (Gelfand et al., 2004).The data from our study do not support the involvement ofthe hH4R in the anti-inflammatory effects of these H1R an-tagonists.An important finding of this study is the discovery of

4-methylhistamine as a potent and selective hH4R agonist inboth recombinant and endogenously expressing H4R sys-tems. Whereas this compound is originally described as arelatively selective H2R agonist (Durant et al., 1975), ourpresent data show that this histamine analog exhibits morethan 100-fold selectivity over the recombinant H1R, H2R, andH3Rs. 4-Methylhistamine not only acts as a full agonist atthe recombinant hH4R but also induces migration of mousebone marrow derived mast cells and a shape change of hu-man eosinophils. Both processes have recently been shown tobe induced by histamine via interaction of the H4R (Hofstraet al., 2003; Ling et al., 2004). The relative potencies ofhistamine and 4-methylhistamine on human eosinophils aresimilar to those observed in recombinant systems. Similar tothe observations with histamine (Hofstra et al., 2003; Ling etal., 2004), the potency of 4-methylhistamine on mouse mastcells is somewhat lower than in recombinant systems. Al-though the mouse H4R shows a lower affinity for both hista-mine and 4-methylhistamine compared with the hH4R, thelow potency at BMMCs seems not merely an issue of speciesdifference, but also it might be related to a low H4R expres-sion level. In fact, the H4R in BMMCs is present at a very lowdensity because it cannot be detected by radioligand bindingstudies (R. L. Thurmond, unpublished observations). More-over, the cellular environment might dictate the potency ofan agonist, such as composition of G proteins and accessoriesproteins in the cells (Kenakin, 2004).

In conclusion, from a large screening of many known his-tamine receptor ligands we have identified a variety of com-pounds with interesting H4R activities. The major signifi-cance of these findings is the reevaluation of numeroushistaminergic ligands at the new histamine receptor sub-types. Based upon our data, many imidazol-containing H3Rligands, including various H3R reference compounds, showpotent H4R activities and should be treated with caution.More recently developed H3R agonists, such as immethridine(Kitbunnadaj et al., 2004) or methimmepip (Kitbunnadaj etal., 2005), or nonimidazole H3R antagonists, such as JNJ6379490 (Ling et al., 2004) or A-349821 (Esbenshade et al.,2004), hardly act at the H4R and will therefore provide goodtools to selectively target the H3R. In the series of tested H3Rligands, we have identified iodophenpropit as potent neutralH4R antagonist and the burimamide analog VUF 4742 as thesecond identified H4R inverse agonist. From the screening ofH2R ligands, we have identified 4-methylhistamine as thefirst high-affinity H4R agonist (Ki 50 nM) that has a100-fold selectivity for the hH4R over the other histaminereceptor subtypes. The identification of 4-methylhistamineas a potent H4R agonist will be of major importance for futurestudies to unravel the physiological roles of the H4R.

Acknowledgments

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

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Address correspondence to: Dr. R. Leurs, Leiden/Amsterdam Center forDrug Research, Department of Medicinal Chemistry, Vrije Universiteit Am-sterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail:r.leurs@few.vu.nl

4-Methylhistamine Is a Potent and Selective Histamine H4R Agonist 1321