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Current Topics in Medicinal Chemistry, 2011, 11, 661-679 66

1568-0266/11 $58.00+.00 2011 Bentham Science Publishers Ltd.

Molecular Determinants of Selective Agonist and Antagonist Binding tothe Histamine H4Receptor

Enade P. Istyastono, Chris de Graaf, Iwan J.P. de Esch and Rob Leurs*

Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Department of Pharmaco-chemistry, Faculty of Exact Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The

Netherlands

Abstract:The deorphanization of the histamine H4 receptor (H4R) has led to a significant number of scientific publica-

tions and patent applications. Whereas some histamine H1, H2and H3 receptor ligands were found to have significant af-

finity for H4R, several agonists and antagonists with high affinity for H4R and selectivity over the other histamine recep-

tors were successfully designed and synthesized. Moreover, site-directed mutation studies on H4R have been performed

and reveal detailed information on receptor-ligand interactions. This review will focus on the most important H 4R ligand

scaffolds and their structure-activity relationships and selectivity over other histamine receptors and specific H4R func-

tional activity. Experimental data are used to construct and validate high resolution three-dimensional receptor-ligand

models and, vice versa, in silicomodels are used to design and rationalize experimental studies to probe receptor-ligand

interactions.

Keywords: Histamine H4, structure-activity relationships, site-directed mutations, receptor-ligand interactions.

1. INTRODUCTION

Histamine (1) (Fig. 1) is an endogenous amine that actsas an important mediator in different (patho)physiologicalconditions. Until the end of 2000, histamine was thought toact via three receptors, i.e.histamine H1 receptor (H1R), his-tamine H2 receptor (H2R), and histamine H3 receptor (H3R).These receptors all belong to the G-protein-coupled receptors(GPCR) family. The H1R and H2R have served as successfultargets for the treatment of allergic conditions and gastriculcers, respectively [1]. The H3R was identified in the ratbrain in 1983 using classical pharmacological approaches

[2]. Although early indications hinted at putative clinicalapplications for H3R ligands, e.g. ADHD, obesity, and cogni-tive disorder, it took more than 15 years for the H 3R to at-tract serious interest from the pharmaceutical industry [3, 4].The long awaited cloning of human H3R (hH3R) gene in1999 rapidly enabled modern drug discovery approachesincluding high-throughput screening (HTS) and several H3Rligands are currently being investigated in clinical trials [4].The human H4R (hH4R) was subsequently identified in 2000,based on its sequence homology to the hH3R gene (31% atthe protein level and 54% within the transmembrane domain)[5-12]. In the beginning, this new histamine receptor wasreported to be expressed mainly in bone marrow, peripheralblood, spleen, thymus, small intestine, colon, heart and lung[5-7, 11, 12]. Recent findings show that the H4R is also ex-pressed in the above mentioned tissues in the CNS [13, 14].The identification of the hH4R has led to the identification ofthe complementary DNA (cDNA) sequence of mouse, rat,

Address correspondence to this author at the Leiden/Amsterdam Center forDrug Research (LACDR), Division of Medicinal Chemistry, Department of

Pharmacochemistry, Faculty of Exact Sciences, VU University Amsterdam,De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands;Tel: +31205987579; Fax: +31 20 5987610; E-mail: [email protected]

guinea pig, pig, dog and monkey H4receptors [15-17]. Thedifferences in the binding of reference H4R ligands in thedifferent H4R species orthologs were just recently publishedand provide important information for a better understandingin animal models of H4R-related diseases [18, 19].

Accompanying the discovery of H4R, several weak topotent H4R ligands were found by testing a set of knownH1R, H2R, and H3R ligands at the hH4R receptor (Fig. 1) [610, 17, 20]. (R)--Methylhistamine (2), N-methylhistamine(3), immepip (4), impromidine (5), dimaprit (6), imetit (7)clobenpropit (8), clozapine (9) were recognized as hH4R

(partial) agonists, while thioperamide (10) was found as ahH4R partial inverse agonist [6, 7, 10]. Subsequently, severamedicinal chemistry programs led to the identification oother new hH4R ligands [21-24]. Among these ligands areOUP-16 (11) and JNJ 7777120 (12) (Fig. 2), which serve aan agonist and an antagonist at the hH4R, respectively [2225]. At this point, the difficulty of designing selective andpotent ligands for the hH4R due to the high sequence similarity with the hH3R was acknowledged [1]. Following theidentification of several potent and selective H4R ligandsmore extensive testing of the H1R, H2R, H3R ligands at thehH4R revealed that 4-methylhistamine (13) (Fig. 2) is a potent and selective hH4R agonist (> 100 fold compare to othehistamine receptors) and this compound has been very usefufor further characterization of H4R pharmacology [20]. TheH4R was shown to play important roles in important diseaselike asthma, allergic rhinitis and pruritis [26-32]. Consequently, the number of biology related publications on H4Rhas steadily increased over time (Fig. 3). (Fig. 3) shows thathe initial increase in the number of chemistry related scientific publications on H4R in 2003 was not continued andeven dropped to zero in 2007, as result of a significant in-crease in patent applications on H4R in that year [33]. A sub-stantial number of chemistry-related scientific publications

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662 Current Topics in Medicinal Chemistry, 2011, Vol. 11,No. 6 Istyastono et a

Fig. (1). H1R, H2R and H3R ligands tested at the hH4R. The pKiand functional activity () data were obtained from Lim, et al.(2005) [20].

Fig. (2). Several potent and selective hH4R ligands discovered in the earlier stage of the quest of the hH 4R ligands discovery. The affinity data

were obtained from Hashimoto, et al.(2003), Jablonowski, et al.(2003), and Lim, et al.(2005) [20-22].

has, however, been disclosed in the past two years (Fig. 3)[33].

Extensive reviews of H4R ligands [28, 33] and H4R

pharmacology [34] have recently appeared. A concise over-view and discussion of experimental data on the molecularfeatures of H4R-ligand interactions is however lacking. Thecurrent article reviews the most important H4R ligand scaf-folds and their structure-activity relationships (SAR), as wellas site-directed mutagenesis (SDM) studies probing the H4Rligand binding pocket. Experimental data are integrated intothree-dimensional H4R-ligand models in order to obtain in-sights into H4R-ligand interactions at an atomic level. Sec-tion 2 gives an overview of different H4R ligand chemo-types. (Figs. 4-15) summarize the SAR of these chemotypes,while biological activity data of H4R ligands are presented in

Table 1. Section 3 discusses H4R species differences inligand binding (Table 2) and the use of SDM studies (Table3) to probe the H4R binding pocket (Fig. 16). Finally, insection 4, SAR and SDM information are combined to constructhree-dimensional H4R models to give more detailed insightinto the molecular determinants of H4R ligand binding (Fig

18).

2. STRUCTURE-ACTIVITY RELATIONSHIPS OFH4R LIGANDS

The first generation of the H4R ligands were discoveredby examining ligands of other histamine receptor subtype[6, 7, 10, 20]. It turned out that most of the high affinity imidazole-containing hH3R ligands are also potent H4R ligand[1, 35-42]. In order to obtain H4R ligands with higher selec

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Molecular Determinants of Selective H4R Agonist and Antagonist Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 6 663

Fig. (3). Number of H4R-related publications. Methodology: A literature search using histamine h4 as keywords in PubMed database

(www.pubmed.com) was performed to count of H4R-related publications (status: January 2010).

Fig. (4). Structure-activity relationship of histamine derivatives as agonists at the hH 4R. As reference in this and the subsequent figures ihistamine (1) (pKi= 7.8 0.1) [20]. The compound will be considered as equal to histamine (~) if the pKiis between 7.3 to 8.3 (7.8 0.5)

while more potent compounds () are compounds with pKimore than 8.3, and vice versa, less potent compounds (

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Table 1. Biological Activity of Some hH4R Ligands at the hH4R, hH3R, hH2R, and hH1R

Biological Activity

H4R H3R H2R H1RCmpd

pKi pEC50 pKi pEC50 pKi pEC50 pKi pEC50 Ref

1 7.8 7.7 1.0 8.0 8.3 1.0 4.3 AGO 4.2 AGO [20]

2 6.6 6.2 1.0 8.2 9.5 1.0 [20]

3 6.5 6.1 1.0 8.4 9.4 1.0 [20]

4 7.7 7.8 0.9 9.3 10.4 1.0 [20]

5 7.6 7.6 0.5 6.8 6.5 -0.5 [20]

6 6.5 5.8 0.8 4.6 AGO [20]

7 8.2 7.9 0.9 8.8 9.9 1.0 [20]

8 8.1 7.7 0.8 8.6 9.4 -1.0 [20]

9 6.7 6.8 1.0 9.4 ANT [20, 47]

10 6.9 7.0 -0.1 7.3 7.5 -1.0 [20]

11 6.9 7.1 1.0 5.7 5.5 0.8 [22]

12 7.8 ANT 5.3 6.0 -0.7 < 5.0 < 5.0 [20, 21]

13 7.3 7.4 1.0 5.2 pAGO 5.1 AGO < 5.0 [20]

14 7.5 6.7 0.8 7.3 7.4 0.9 [20]

15 8.0 7.5 0.8 8.4 9.2 1.0 [20]

16 6.6 < 5.0 8.3 8.4 0.9 [20, 41]

17 6.1 < 5.0 8.0 7.5 0.6 [20, 41]

18 5.6 6.5 6.1 0.9 [38]

19 6.2 7.6 7.5 1.1 [38]

20 5.4 4.9 1.0 7.2 8.0 1.0 [20]

21 6.4 7.7 7.9 0.6 [38]

22 5.7 [38]

23 5.7 5.3 0.5 9.0 9.5 0.8 [20, 38]

24 6.6 6.0 0.5 9.1 9.8 0.9 [20, 38]

25 8.3 1.0 8.4 0.3 6.1 0.0 4.9 0.2 [37]

26 8.5 0.9 8.8 0.4 6.4 0.0 5.5 0.3 [37]

27 8.6 1.0 8.8 0.4 6.9 0.0 5.7 0.3 [37]

28 8.6 0.9 8.9 0.3 7.0 0.0 5.6 0.4 [37]

29 7.5 0.9 -0.3 0.1 0.1 [36]

30 8.0 7.9 0.6 8.5 9.3 1.0 [20]

31 7.6 0.0 8.1 [35]

32 7.8 0.5 8.1 [35]

33 8.0 0.2 8.1 [35]

34 7.6 0.4 8.3 [35]

35 8.0 1.0 8.5 [35]

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(Table 1) contd

Biological Activity

H4R H3R H2R H1RCmpd

pKi pEC50 pKi pEC50 pKi pEC50 pKi pEC50 Ref

36 7.6 0.5 8.2 [35]

37 7.9 0.9 8.1 [35]

38 7.2 0.5 7.7 [35]

39 7.3 0.4 8.0 [35]

40 7.2 0.4 7.7 [35]

41 8.8 1.0 8.2 [35]

42 5.1 [48]

43 7.5 7.3 1.0 6.5 1.0 pAGO [48]

44 7.4 [47]

45 7.6 7.7 1.0 5.0 5.0 8.1 8.2 -1.0 [47]

46 7.4 [21]

47 7.7 [21]

48 7.1 ANT 5.4 6.0 -1.0 [32, 56]

49 8.2 ANT 6.0

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(Table 2) contd.

pKiat H4R Species VariantCompound

Human Monkey Pig Dog Mouse Rat Guinea pig

Clobenpropit (8) 7.5 7.5 6.6 6.5 7.3 7.3 8.2

JNJ 7777120 (12) 8.3 7.5 6.3 7.1 8.4 8.4 6.0

VUF 6002 (48) 7.5 6.7 5.1 6.2 6.9 7.3 5.8

Thioperamide (10) 7.1 7.1 7.0 6.4 7.6 7.5 7.1

62 7.8 7.2 5.9 8.1 7.8 6.9

63a 7.3 7.4 5.3 6.7 6.5 6.3

63b 7.5 7.6 5.1 8.1 7.7 7.3

63c 7.3 6.7 5.0 7.7 6.7 6.5

Fig. (5). Structure-activity relationship of guanidines at the hH4R. The designations refer to those in Fig. 4.

Fig. (6). Structure-activity relationship of isothioureas at the hH4R. The designations refer to those in Fig. 4.

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Fig. (7). Structure-activity relationship of guanidine-isothioureas at the hH4R. The designations refer to those in Fig. 4.

Fig. (8). Structure-activity relationship of dibenzodiazepines at the hH4R. The designations refer to those in Fig. 4.

Fig. (9). Structure-activity relationship of indolylpiperazine derivatives at the hH4R. JNJ 7777120 (12), the first potent and selective hH4R

non imidazole containing compound belongs to this series. The Kiof JNJ 7777120 (12) is 41 nM (pKi~ 8.4) [19, 21]. The designations refe

to those in Fig. 4.

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Fig. (10). A-987306, a potent and selective hH4R antagonist be-

longs to aminopyrimidines series. For an extensive review on this

particular see references [28, 33].

2.1. Histamine Derivatives

Fig (4). presents the schematic SAR of histamine deriva-tives on hH4R. The hH4R affinity of the endogenous ligand,histamine (1) (pKi= 7.8) is comparable to its hH3R affinity(pKi = 8.0), but better than the affinity at hH 1R (pKi = 4.2)and hH2R (pKi= 4.3) [20]. Elongation of the spacer betweenthe imidazole ring and the basic amine from two carbon

atoms (1) to three (homohistamine (14)) and four (imbu-tamine (15)) carbon atoms (Fig. 4) does not affect hH4Raffinity (pKi= 7.5 and 8.0, respectively) [20, 41]. However,(Fig. 4) also shows that a further increase of the spacerlength to five carbon atoms (impentamine (16)) or six carbonatoms (17) is detrimental (pKi = 6.6 and 6.1, respectively)[20, 41]. It is also detrimental to modify the amine group ofcompound 14 to an hydroxyl group (18) or a methyl group(19) (pKi = 5.6 and 6.2, respectively) as can be seen from(Fig. 4) [38]. Methylating the ethylamine sidechain of hista-mine (1) is detrimental (Fig. 4), both at theN(compound 3)and the carbon position (R-enantiomer (2) and S-enantiomer (20)) [20]. These compounds (2, 3 and 20) allshow an hH4R pKi value of less than 7, with the (R)-enan-

tiomer (2) shows ~1 log unit better affinity than its (S)-enantiomer (20) [20]. Changing the basic amine group ofcompound 1 to piperidine moiety (21) (Fig. 4) results in asharp decrease in affinity (pKi= 6.4), but reducing the spacerbetween imidazole and piperidine from two carbon atoms toone carbon atom (immepip 4) regains the hH4R affinity (pKi= 7.7) (Fig. 4) [20, 41]. However, modification of compound4by moving the nitrogen atom of the piperidine ring from 4-in compound 4to 3- or 2- is detrimental. Further decreasingin the spacer length (22) reduces also the hH4R affinity (pKi= 5.7) (Fig. 2) [41]. Similar to methylation of the ethylaminemoiety of histamine (1), methylating the basic amine moietyof compound 4 (compound 23) decreases the hH4R affinity(pKi = 5.7) (Fig. 4) [20, 40, 41]. Replacing the basic

piperidine moiety with a pyridine ring (24) reduces the affin-ity for ~ 1 log unit, similar to the non-basic histamine (Fig.

4) [38]. It can be hypothesized that the basic amine moiety is

essential to obtain high affinity for hH4R. Besides modification of the basic amine, substitution of the imidazole moietywas also performed and led to the discovery of 4methylhistamine (13) (Fig. 2and 4) as a potent hH4R agonis(pKi = 7.3; pEC50 = 7.4; = 1.0) with a >100-fold and>100,000-fold selectivity over the hH3R and hH2R, andhH1R, respectively [20]. All ligands mentioned in this para-graph, except compound 13, are also high affinity ligands for

hH3R (pKis > 6.5) [20, 38, 40, 41]. The ligands with comparable hH4R affinity with compound 1all show (partial) agonistics behavior (values range from 0.5 to 1.0) at the hH4R[20].

2.2. Guanidines

In early 2003, the discovery of the first selective and po-tent hH4R agonists (OUP-16 (11)) (Fig. 2) was published[22]. The compound (at hH4R: pKi = 6.9; pEC50 = 7.1; =1.0), having ~40-fold selectivity over hH3R, was part of aseries of tetrahydrofuranylimidazoles [22]. The imidazolemoiety of histamine (1)was kept in these compounds, whilethe basic amine and the two carbon atoms spacer between

the imidazole ring and the amine function were exchangedwith guanidines and furans. Recently, related compoundswith higher hH4R affinity and improved selectivity werediscovered by exploration of the compound 11 scaffold(combination of an imidazole ring with a modified guanidine), i.e. N

G-acylated imidazolylpropylguanidines and

cyanoguanidines [36, 37]. In NG-acylated imidazolylpropyl

guanidines (Fig. 5), bulky aromatic heterocycles are tolerableas N

G-acylated guanidine substituent [36]. Neverthelesssmaller alkyl substituents (methyl (25), ethyl (26), propy(27) and isopropyl (28)) lead to improved hH4R activity [36]All presented N

G-acylated imidazolylpropylguanidines withcomparable hH4R affinity with histamine (1) or better, show(partial) agonistic behavior (values range from 0.4 to 1.0

[36]. Another series, similar to compound 11, the cyanoguanidines series, were explored by modifying the spacer between the imidazole ring and the guanidine moiety, and substitution of the guanidine (Fig. 5) [37]. The data (Fig. 5show that: (i) the optimal spacer length between the imidazole ring and the guanidine moiety is four carbon atoms; (ii)introducing sulfur in the spacer and a 4-methyl substitutionof the imidazole moiety is detrimental, and (iii) non-polasubstituents at the 3-guanidine position are acceptable, with2-phenylthioethyl (29) as the substituent that give the bespharmacological profile in the cyanoguanidine series [37]. 2Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-(2-phenylthioethyl)-guanidine (29) is the best hH4R ligand in the series in termof potency (pEC50= 7.47), functional activity (= 0.93) and

selectivity over other histamine receptors (>10-fold and>100-fold selectivity over the hH3R, and hH2R and hH1Rrespectively [37]).

Fig. (11). Piperazine moiety and its isosteres, which are well tolerated in the aminopyrimidines series.

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Fig. (12). Structure-activity relationship of quinoxaline derivatives at the hH4R. The designations refer to those in Fig. 4.

Fig. (13). Structure-activity relationship of quinazoline derivatives at the hH4R. The designations refer to those in Fig. 4.

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2.3. Isothioureas

Isosteric replacement of guanidines can be performedwith isothioureas. Imetit (7), clobenpropit (8) and thiopera-mide (10) are isothiourea-containing hH4R ligands whichwere previously known as potent ligands for hH3R [6, 7, 10].(Fig. 6) shows that increasing the spacer length between theimidazole ring and the isothiourea group from two imetit (7)

to three carbon atoms (VUF 8328 30) is tolerated (pKi= 8.0)[20]. Additional benzyl substitution to compound 30 resultsin compound 31 with slightly lower hH4R affinity (pKi =7.6) [35]. However, substitution with 4-chlorobenzyl(clobenpropit (8)), 3-chlorobenzyl (32) or 2-chlorobenzyl(33) is well tolerated [35]. Interestingly, compounds 8, 32and 33differ in their functional activity (s = 0.8, 0.5, and0.2, respectively) [35]. Other 4-benzylsubstituted derivatives(4-fluorobenzyl (34), 4-iodobenzyl (35), 4-methylbenzyl(36) and 4-methoxybenzyl (37)) have comparable affinity tohistamine (1) and imetit (7) [35]. Increasing the spacerlength between the isothiourea and the phenyl moiety to two(38), three (39), or four (40) carbon atoms is detrimental forthe hH4R affinity [35]. Compounds so far presented in this

paragraph are also hH3R ligands with better affinity at hH3Rthan hH4R [35]. However, the opposite is true for compound

41 (a derivative of compound 30 with 3,4-dichlorobenzylsubstitution) (Fig. 6). Compound 41shows remarkably highaffinity at hH4R (pKi = 8.8) with ~4-fold selectivity overhH3R [35]. The compounds presented in this paragraph varyfrom neutral antagonist (= 0.0) (compound 31) to full ago-nist (= 1.0) (compound 41) [35].

2.4. Guanidine-Isothioureas

The previous paragraphs (section 2.1-2.3) present thedesign of potent and selective hH4R ligands by modificationof the basic amine from histamine (1) while maintaining theimidazole moiety. Some selective H4R ligands (compound

11, 13, and 29) could be discovered (Figs. 2and 5), but thehH4R affinity of these ligands is similar to the affinity ofhistamine (1). Most imidazole-containing hH4R ligands withhigher hH4R affinity show also high affinity at hH3R. Thishas stimulated the discovery of non-imidazole hH4R ligands,which were expected to be more selective over the other his-tamine receptors, especially the hH3R [28, 33, 43]. One ap-proach to escape from imidazole-containing compounds is toexplore derivatives of non-imidazole containing compounds(dimaprit (6) and clozapine (9), which were discovered earlyafter the discovery of the receptor [6, 7, 10, 20, 48]. (Fig. 7).shows that changing the N,N-dimethyl moiety of compound6to a guanidine moiety (42) decreases the hH4R affinity, but

decreasing the spacer length between the isothiourea andguanidine from three (42) to two carbon atoms (VUF 8430(43)) increases the hH4R affinity considerably (pKi = 7.5)[48]. Changing the isothiourea of VUF 8430 (43) to a secondguanidine group is not favorable for hH4R affinity (Fig. 7)[48]. VUF 8430 (43) shows full agonistic activity at hH4Rand ~30-fold selectivity over hH3R [49].

2.5. Dibenzodiazepines

Following the discovery of the hH4R, the tricyclic diben-zodiazepine clozapine (9; Fig. 1) was identified as non-imidazole hH4R agonist [6, 7, 10, 20, 48]. Altering the tri-

cyclic framework (-NH- to -O-) (44) increases the affinityconsiderably (pKi = 7.4) (Fig. 8) [47]. Alterations of thepiperazine side chain is detrimental, while various aromaticsubstituents are acceptable (Fig. 8). This has led to the discovery of VUF 6884 (45) (pKi= 7.6) [47]. VUF 6884 (45shows full agonistic activity at hH4R and ~300-fold selectivity over hH3R and hH2R, but has high hH1R affinity(pKi=8.2) and displays inverse agonistic activity at hH1R (=

-1) [47].

2.6. Indolylpiperazine Analogs

The first selective non-imidazole hH4R ligand was JNJ7777120 (12), reported in 2003 [25]. Whereas imidazolecontaining compounds can achieve selectivity of >100-foldover hH3R, JNJ 7777120 (12) combines an hH4R affinity inthe nanomolar range (pKi= 8.4) with a selectivity of >1000fold over other histamine receptors (Fig. 2) [25]. MoreoverJNJ 77777120 has become a reference hH4R antagonist withanti-inflammatory effects in a variety of animal models [125, 28, 32, 44, 50-55]. The discovery of JNJ 7777120 (12was initiated by a hightroughput screening at Johnson &

Johnson, which led to the discovery of an indolecarboxamide, indolylpiperazine (46) (pKi= 7.4) [21]. Methylation of the most basic amine (47) increases the hH4R affinity (pKi= 7.8 nM), but substitution of the most basic aminewith more bulky alkyl group (Fig. 9) is detrimental foligand affinity [21]. Attempts to substitute the methylpiperazine moiety to other moieties containing basic aminewith similar distance from the carbonyl group to the aminegroup were detrimental for affinity, except the replacemenof the methylpiperazine moiety with a methylpiperidinemoiety [56]. Substitution of the carbonyl group to methyleneor oxime (Fig. 9) diminishes ligand binding [19, 21], busubstitution of the indole moiety to its isosteres (Fig. 9), i.e.benzoimidazole (VUF 6002 48) or thienopyrroles, results in

affinities at the hH4R close to that of histamine (1) and JNJ7777120 (12) [28, 56, 57]. Decorating the indole moiety ofcompound 46 with a halogen atom, an amine group, a hy-droxyl group or a methyl group at the position 4-, 5-, 6- or 7is tolerated (Fig. 9)[21].

2.7. Aminopyrimidines

Similar to the discovery of indolylpiperazine (46), ahightroughput screening at Abbott Laboratories identified 2aminopyrimidines as a potential scaffold to be developedfurther [29, 30, 46], An extensive review on hH4R antagonists which covers this particular scaffold from both scientific publications and patent documents was recently pub

lished and will not be repeated here [28, 33]. A recent repre-sentative of this series, A-987306 (49) shown in (Fig. 10) ia potent hH4R antagonist with selectivity over other histamine receptors (pKis at hH4R, hH3R, hH2R and hH1R are8.2, 6.0,

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2.8. Quinoxalines and Quinazolines

After the construction of a pharmacophore model basedon JNJ 7777120 (12) and VUF 6884 (47), a quinoxalinefragment was discovered as a new potential H4R ligand scaf-fold [44]. (Fig. 12) shows that modification of the indolylmoiety to quinoxaline and keeping the N-methylpiperazineof JNJ 7777120 (12) resulted in compound 50with a hH4RpKiof 6.0 [44]. As shown in (Fig. 12), the introduction of abenzyl group at R3 leads to compound 51with good hH4Raffinity (pKi= 7.4) [44]. Additional chlorine substitutions atR6and R7(Fig. 12) to mimic the chlorine substituent in JNJ7777120 (12)and VUF 6884 (47) are favorable and result incompound 52 (Fig. 12) with the highest hH4R affinity in thisseries (pKi = 8.3) [44]. Compound 51 has selectivity overhH2R and hH3R and some affinity at hH1R (pKi= 6.1), whilecompound 52 shows even better selectivity over all otherhistamine receptors [44]. Compounds 51and 52have in vivoanti-inflammatory activity [44]. Another potential lead com-pound was found using a scaffold hopping approach [44].Modifying the quinoxaline fragment in compound 50 re-sulted in the quinazoline 53, which binds to hH4R in the mi-cromolar range [44, 45]. (Fig. 13) shows that introduction ofhydrophobic groups at the 4-position of compound 53 led tothe discovery of the benzyl moiety (compound 54) as thefavorable substituent (pKihH4R = 6.0) [45]. Similar to qui-noxalines, additional chlorine substitutions at R6 (55) arefavorable and increase the hH4R (pKihH4R = 6.6) [45]. Ben-zyl moiety alterations of compound 55 to its isosteres led tothe identification of a 2-thiophenyl substituent (compound56) which strongly increases hH4R affinity (pKihH4R = 8.1)[45]. Compounds containing a sulfonamide group at the 4-position were designed and further optimized leading tocompound 57(pKihH4R = 8.4), a potent hH4R inverse ago-nist [58].

2.9. 2-ArylbenzimidazolesCompared to previously described H4R ligand scaffolds,

2-arylbenzimidazoles (Fig. 14) are structurally distinct, con-taining a long linker between a basic amine moiety with thearomatic heterocycle [28, 59]. Arylbenzimidazole 58 (Fig.14) has a moderate hH4R affinity (pKi= 6.9) [28, 59]. (Fig.14) shows that rigidification of the linker between the phenylgroup with the N-methylpiperazine moiety does not increasehH4R affinity [28, 59]. Substitution of the phenyl moiety

with chlorine atoms (compound 60, pKi = 7.4; compound 61pKi = 7.7) increases hH4R affinity (Fig. 14) [28, 59]. Replacing the N-methylpiperazine moiety by a Nmethylhomopiperazine moiety (compound 61) results inspectacularly increased hH4R affinity (pKi= 9) [28, 59].

2.10. General Features of Highly Potent and SelectivehH4R Ligand

Table 1presents the biological activities of some hH4Rligands, including binding affinities and functional activities() at hH4R and other histamine receptors. From the variouspotent and selective hH4R ligands, some general pharmacophore features can be identified.

(Fig. 15) illustrates the features based on the alignment ohistamine (1), clozapine (9), and JNJ 7777120 (12) extractedfrom hH4R models in (Fig 17) [18]. H4R ligands comprise anessential basic amine group (alkylamine section 2.1; (Fig. 4)imidazole sections 2.1-2.3; (Figs. 4-6), guanidine section2.2 and 2.4; (Figs. 5, 7), isothiourea (sections 2.3-2.4; (Figs6-7), piperidine sections 2.1; (Fig. 4), piperazine section2.5-2.8; (Figs. 8-10, 12-13), pyrrolidinamine section 2.9

(Fig. 11), azetidinamine section 2.9; (Fig. 11), diazepanesection 2.5; (Fig. 8) which acts as H-bond donor (and inmost cases also as a cation) in protein-ligand interactionsindicated as HBD1 in (Fig. 15). This basic amine is connected to either a small polar group (imidazole (which alsocontains an aromatic (ARO) feature, sections 2.1-2.3; (Figs4-6), guanidine sections 2.2 and 2.4; (Figs. 5, 7), or isothiourea sections 2.3-2.4; (Figs. 6-7) or a nitrogen containingaromatic heterocycle (indole section 2.6; (Fig. 9)(dibenzo)diazepine section 2.5; (Fig. 8), aminopyrimidinesection 2.7; (Fig. 10), quinoxaline section 2.8; (Fig. 12) andquinazoline section 2.8; (Fig. 13), which is involved in aromatic interactions ARO in (Fig. 15) and acts as H-bond donor HBD2 in (Fig. 15). Additional lipophilic moieties fea

tures HYD1 and HYD2 in (Fig. 15) are linked to the nitrogencontaining aromatic heterocycle (ARO/HBD2) can furtheincrease hH4R affinity and selectivity of hH4R ligands (e.g4-methyl group attached to the imidazole ring of histamine(section 2.1), substitutions on the guanidine or isothioureamoiety (sections 2.2.-2.3), substitutions on the indole moietyof indolylpiperazines (section 2.6), substitutions on theaminopyrimidine moiety (section 2.7) and dibenzodiazepinemoiety (section 2.5)). The pharmacophore model in (Fig. 15is similar to the modified pharmacophore model for non

Fig. (14). Structure-activity relationship of arylbenzimidazole derivatives at the hH4R. The designations refer to those in Fig. 4.

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imidazole H3R antagonists recently proposed by Gemkow etal.(2009) [60]. The Gemkow model comprises a basic groupsimilar to feature HBD1 in (Fig. 15) and a lipo-philic/aromatic region similar to feature ARO in (Fig. 15)connected by a linker with an additional feature of a 2

ndbasic

group similar to feature HBD2 in (Fig. 15), a polar groupsimilar to feature HBD2 in (Fig. 15), or a 2nd lipophilic re-gion similar to feature HYD1 or HYD2 in (Fig. 15) [60].

This similarity between H4R and H3R ligand pharmacophoremodels underlines the challenge to design H4R ligands withselectivity over H3R (Table 1).

Fig. (15). General pharmacophore features of hH4R ligands. HBD1:

Hydrogen bond donor/cation; ARO: center of small polar

group/aromatic heterocycle; HBD2: Hydrogen bond donor attached

to ARO; HYD1and HYD2: lipophilic/aromatic moiety. HBD1 and

ARO are connected with a black thick line since these features are

essential. The lines connecting ARO to HYD1 or HYD2 are thinner

than the line connecting HBD1 to ARO, since HYD1 and HYD2

are optional. The lines connecting HBD1 to HYD1 and HYD2 are

presented as dashed lines because generally there is no direct link

from HBD1 to either HYD1 or HYD2.

Smaller polar compounds like histamine-analogues, gua-nidines, and isothioureas (Figs. 4-7) containing two polargroups (HBD1 and HBD2, and in the case of histamine alsoan aromatic feature (ARO)) generally act as agonists on H4R.Larger ligands (Figs. 8-10) containing an aromatic heterocy-cle (features ARO and HBD2) and 1 or 2 lipohilic moieties(Features HYD1 and HYD2) generally act as inverse ago-nists or neutral antagonists. Exceptions are dibenzodiazepi-nes (Fig. 8) which are H4R agonists but act as antagonists onH1R [28, 33, 47], and indole- and benzoimidazole-oximes(Fig. 16), analogues of antagonist JNJ 7777120, which act aspartial agonists at H4R [19]. Interestingly, modification of N-methylpiperazine (HBD1) to its isosteres (Fig. 11) decreases

the hH4R affinity of dibenzodiazepines (section 2.5), in-dolylpiperazine analogs (section 2.6), aminopyrimidines(section 2.7), quinoxalines and quinazolines (section 2.8),but not the affinity of aminopyrimidines (section 2.7). Thesize of feature HYD1 is limited (Fig. 15) in dibenzodiazepi-nes (Fig. 8) Polar groups can be added to HYD2, which canbe further increased in size in indolylpiperazines (Fig. 9) andquinazolines (13).

A quantitative structure-activity relationship (QSAR)model on a quinazolines series was recently generated toidentify the molecular determinants of H4R ligand binding

affinity [58], and QSAR models on a series of clobenpropianalogs have been constructed to explain H3R versus H4Rligand selectivity [35]. These QSAR studies show the importance of combined electronic and steric descriptors in thehH4R affinity [18, 58] and indicate that energy related stericdescriptors (stretch-bend conformational energy) are usefudescriptors to design H4R ligands with selectivity over H3R[35].

3. PROBING THE LIGAND BINDING POCKET

3.1. Species Differences

Various species orthologs of H4R were promptly clonedbased on their homology to the human H4R sequence, including those of mouse, rat, guinea pig, pig, monkey(Macaca fascilularis), and dog [15-18, 61]. The H4R specievariants share relatively low homology with the human H4R(65-71%), except for the monkey H4R that shares an overalamino acid homology of 91% [18]. These variations result insignificant differences in the affinity for the H4R endogenouagonist histamine (1) and some H4R antagonists (Table 2[15-19, 30, 61]. The species differences complicate preclini

cal development when studying animal models of diseases.

Species differences in ligand binding have been extensively and systematically investigated by expressing the human, monkey, pig, dog, guinea-pig, mouse and rat H4Rs inHEK 293T and SK-N-MC cells [18, 19]. The interactions ofthese H4R proteins with reference H4R ligands, includinghistamine (1), clobenpropit (8), clozapine (9), thioperaminde(10), JNJ 7777120 (12), 4-methylhistamine (13), VUF 8430(43), VUF 6002 (48), a benzimidazole-oxime derivative (62Fig. 16), and indole-oxime derivatives (compounds 63a-c(Fig. 16) have been evaluated (Table 2) [15-19, 61, 62].

Fig. (16). Structures of a benzoimidazole-oxime (62) and indole

oximes (63a-c), analogs of the H4R antagonist JNJ 7777120 (12

Fig 2and 9) that show H4R agonism [19].

Ligand binding affinities for different H4R specieorthologs are presented in Table 2[15-19, 61]. Histamine (1and 4-methylhistamine (13) bind with higher affinity to theH4R proteins of human, monkey, pig, and guinea pigthan tothe H4R receptors of dog, mouse, and rat (Table 2) [15-1861, 62]. Another selective H4R agonist, VUF 8430 (43) [48binds with lower aff inity is to pig and dog H4R [18]. Interestingly, the binding affinity of the large H4R agonist clozapine(9) is higher for monkey H4R but lower for other H4Rorthologs compared to human H4R [18]. The non-imidazoleantagonists JNJ 7777120 and VUF 6002 have significantly

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lower affinity for monkey, pig, dog, and guinea pig H 4Rorthologs than for human H4R [18, 19]. Thioperamide (10)on the other hand has equipotent affinity for the species vari-ants and consistently acts as an antagonist or inverse agonistat these species variants [18]. Ligand binding affinities fordifferent H4R species orthologs of other JNJ 7777120 (12)analogs (compounds 61, 62a-c) have been recently reported[19]. A similar trend in affinity for H4R orthologs is ob-

served, except for compound 63c, which displays lower af-finity for monkey H4R [19]. Notably, small modification ofJNJ 7777120 (12) by substitution of its carbonyl to oxime(compound 62b) changes the functional activity of thisligand from a neutral antagonist [21] or partial inverse ago-nist [63] to a partial agonist of human H4R [19].

3.2. Mutagenesis Studies

Several mutation studies have been employed to probeimportant residues involved in ligand binding to H4R [18, 62,64-66] (Table 3). The effect of some H4R mutations is foundto be ligand-dependent (Table 3). Residue D3.32, which isconserved in the histamine receptor (Fig. 17), is identified asan essential ionic interaction anchor for hH4R ligands (Table

3) [65, 66]. Mutation of this residue to Ala, Asp, or Asn isdetrimental for histamine (1) binding [66]. Also residue E5.46

is an important residue for the binding of histamine (1) andJNJ 7777120 [65, 66] (Table 3). The affinity of histamine (1)for the hH4R E

5.46A mutant can not be determined, while the

affinity of histamine (1) for the E5.46Q mutant can be meas-ured but is significantly decreased [65].The affinity of an-other small polar ligand, VUF 8430 (43) is also significantlydecreased in the E5.46Q mutant, while the affinity of largerligands like clozapine (9) and JNJ 7777120 (12) is not af-fected by this mutation [65].

Domain swapping of the protein sequences of humanH4R with mouse [62] and pig [18] H4Rs enabled the identifi-cation of TM helices 4 and 5 and extracellular loop 2 (EL2)as important regions determining H4R species differences.Individual residues at positions 4.57, 5.39, S5.43, and 45.55(in EL2) were subsequently identified by site-directedmutagenesis experiments as residues responsible for the ob-served species differences [18, 62]. The affinity of the hH4R

N4.57H mutant for VUF8430 (43), clozapine (9), and JNJ7777120 (12) for the hH4R N

4.57H mutant is significantly

descreased compared to wild-type hH4R (Table 3) [49], andexplains the low affininty of these ligands for dog and pighH4R (Table 2) [19, 49]. The decreased affinity for histamine(1) in the dog mimicking N4.57H mutant is however restoredin the pig mimicking N

4.57H/S

5.43L double mutant, explaining

the differences in binding between human, pig and dog H 4R

(Table 2).The affinity of histamine (1) and clozapine (9) is signifi

cantly decreased in the mouse mimicking human H4R F45.55V

mutant, suggesting the involvement of extracellular loop 2(ECL2) in stabilizing ligand binding (Table 3) [18]. The affinity for VUF 8430 (43) is only slightly affected in theF

45.55V mutant (Table 3), suggesting that also other residues

are responsible for differences in binding affinity of thisligand to human and mouse H4R (Table 2) [18]. The affinityof clozapine (9) is increased while the affinity of JNJ7777120 (12) is decreased in the monkey mimicking hH4RL

5.39V mutant [18], which is in line with the observed specie

differences (Table 2) [18]. While the previous mutation studies were performed to explain H4R species differences (Table 2), other mutations were designed to identify moleculardeterminants of histamine receptor subtype selectivity Table1, (Fig. 17). The H1R mimicking hH4R S

6.52F mutation

slighty decreases the affinity of histamine (1), and has a detrimental effect on histamine-induced activation of H4R [66]In section 4 the (ligand-specific) effects of the mutationdescribed in section 3 will be rationalized using threedimensional H4R receptor models.

4. THREE-DIMENSIONAL MODELS OF H4RLIGAND BINDING

4.1. GPCR Transmembrane Ligand Binding Pocket

Knowledge of the three-dimensional structure of GPCRcan provide important insights into receptor function andreceptor-ligand interactions, and can be used for the discovery of new ligands [67]. So far most structural GPCR modeling studies have been limited to either ab initiomodels [6869] or bovine rhodopsin (bRho)-based [70] homology mod-

Table 3. Affinity of hH4R Ligands for Different hH4R Mutantsa,b

hH4R MutantCompound

WT

(pKi) D3.32A D3.32E D3.32N N4.57H F45.55V L5.39V E5.46A E5.46Q S6.52FRef

Histamine (1) 7.7 N.D. N.D. N.D. ~/c

~ N.D.

[66]

[62, 65]

VUF 8430 (43) 7.6 ~ [18, 65]

Clozapine (9) 6.2 ~ [18, 62, 65]

JNJ 7777120 (12) 8.3 ~ [18, 65]

aThe affinity is considered as equal to wild type (~) if the affinity is between 0.5 log unit from the compound affinity to wild type. If the affinity more than 0.5 log unit compare to

the affinity to the wild type, it is considered that the mutation increases ( ) the affinity of the compound and vice versa, if the affinity less than 0.5 log unit, it is considered that themutation decreases () the affinity of the compound. N.B., no binding determinedbThe following are reported mutants that give less significant effects: N4.57H/S5.43L, M4.60V; S45.42A; F4.55L; F4.55L/S45.56K; I5.38V; T5.42A; S5.43A; S5.43L; T5.42A/S5.43A; N4.57A; N4.57Y;

S6.52A; S6.52F [18, 66].cThe difference in pKi value is equal, but the Kdvalue is significantly lower

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els. Recently, crystal structures of squid rhodopsin, the betaadrenergic receptors type 1 (ADRB1) and 2 (ADRB2), theA2A adenosine receptor (AA2AR), and the ligand-free opsin(Ops*) were solved [71-77]. The emergence of these GPCRcrystal structures, notably the inverse agonist and antagonistbound structures of homologous ADRB1 and ADRB2, offersnew opportunities for the construction of high resolutionhH4R models, that can explain and predict H4R-ligand bind-

ing [18, 65, 66, 78]. Most of the current GPCR crystal struc-tures are relatively homologous in their seven transmem-brane (TM) helical binding cavity, but structurally divergentin the intracellular as well as the extracellular loop regions ofthe receptors [71, 73]. Despite their structural inaccuraciesGPCR models have been efficiently used to find novelligands [67, 79]. Recent chemogenomical analyses have fur-thermore shown that the physicochemical properties of theTM cavities of GPCRs match their cognate ligands [80, 81].Despite the relatively low sequence identity between the TMdomains of rhodopsin-like (class A) GPCRs [82], there areseveral GPCR family-specific patterns and motifs [83] whichfacilitate their alignment [84]. The Ballesteros-Weinsteinnumbering scheme [85] is in fact based on the presence of

several highly conserved residues among class A GPCRs:N1.50 in TM 1 (1.30-1.59), D2.50 in TM2 (2.38-2.67), R3.50 inTM3 (3.22-3.54), W

4.50in TM4 (4.40-4.62), P

5.50in TM5

(5.35-5.60), P6.50 in TM6 (6.30-6.55), and P7.50 in TM7 (7.33-7.53). (Fig. 17) presents the sequences alignment of putativebinding residues in the helical transmembrane (TM) domain[80, 81] of all histamine receptors and a set of GPCRs forwhich ligand-bound crystal structures are available (humanadrenergic 2 (ADRB2), human adenosine 2A (AA2AR) andbovine rhodopsin (bRho)) [1, 71, 86]. Human H4R and H3Rshare high sequence homology in the putative ligand bindingpocket in the transmembrane (TM) helical domain ~85%sequence similarity [80, 81], see (Fig. 17). It is therefore notsurprising that H3R and H4R receptors share the same ligands

(Table 1). Residues shown to be involved in ligand bindingin histamine receptors based on site-directed mutagenesisexperiments (Table 2, paragraph 3) [18, 65, 66, 87-97] over-lap with ligand contact residues in the ADRB2 and bRhocrystal structures between TM helices 3-7 (Fig. 17) [71, 79,98], while the ligand binding pocket cavity in AA2AR is

located more towards the extracellular loops [79, 86]. Notably, D

3.32, a crucial residue in binding the positively charged

nitrogens in ligands of crystallized bioaminergic ADRB1and ADRB2 receptors [71, 77], is also essential for ligandbinding to histamine receptors [18, 65, 66, 87-93]. Furthermore, like in ADRB2 [71, 79], F6.52 is proposed to be involved in aromatic stacking interactions with ligands in H1R[88]. The resemblance in ligand binding pocket topology

[80, 81] makes ADRB1 and ADRB2 currently the most suitable crystal structure template for the construction of hista-mine receptor models. Despite the availability of suitableGPCR crystal structure templates, careful and critical receptor modeling strategies, guided and validated by experimen-tal data, are still needed to construct histamine receptor models which can be used for structure-based virtual screeningand ligand design [67, 79].

4.2. Experimentally Guided H4R Models

Receptor modeling studies have been performed to understand ligand interactions with H4R [18, 62, 64-66]. (Fig18) presents the binding poses of three representative H4Rligands as proposed by Lim and de Graaf, et al.(2010) [18]the full agonists histamine (1) (Fig. 18A) and clozapine (9(Fig. 18B), and the neutral antagonist JNJ 7777120 (12) (Fig18C). These binding modes are in line with the experimentadata presented in sections 2 and 3 and give insights intoatomic details of H4R-ligand interactions. While the orientation and interactions of histamine in the H4R binding pockecan be used as a receptor-ligand binding model of smalmolecule agonists (histamine derivatives (section 2.1), gua-nidines (section 2.2 and 2.4), and isothioureas (section 2.3)the binding modes of clozapine and JNJ 7777120 can beconsidered as templates for H4R interactions with guanidineisothioureas (section 2.4), dibenzodiazepines (section 2.5)indolylpiperazines (section 2.6), aminopyrimidines (section

2.7), quinoxalines (section 2.8.) and quinazolines (section2.8).

4.2.1. Histamine Binding Mode

Like in other bioaminergic receptors [81, 99] and as observed in the ligand-bound ADRB1 and ADRB2 crysta

Fig. (17). Sequences alignment of putative binding residues [80, 81] of human H 1R (HRH1_HUMAN; Accession Code: P35367), human

H2R (HRH2_HUMAN; Accession Code: P25021), human H3R (HRH3_HUMAN; Accession Code: Q9Y5N1), human H4R

(HRH4_HUMAN; Accession Code: Q9H3N8), together with three GPCRs for which crystal structures are available: human adrenergic 2receptor (ADRB2_HUMAN; Accession Code: P07550) [71], human adenosine 2A receptor (Uniprot code: AA2AR_HUMAN; Accession

Code: P29274) [86] and bovine rhodopsin (OPSD_BOVIN; Accession Code: P02699) [70]. Residues that are suggested to be involved in

ligand binding based on mutation studies in histamine receptors [18, 65, 66, 87-97] and ADRB2, AA2AR, and OPSD [79] are shaded grey,

while residues in contact with the ligand in the crystal structure [70, 71, 79, 86] are indicated with a black square border.aNumber of similar

residues (based on ClustalW2; http://www.ebi.ac.uk/Tools/clustalw2/index.html) divided by total putative binding residues;bNumber of

identical residues divided by total putative binding residues.

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structures [71, 77], the positively charged amine group ofhistamine corresponding to the HBD1 in (Fig. 15) forms atight H-bond with the negatively charged carboxylate groupof D3.32 in H4R [65, 66] in the pocket between TMs 2, 3,and 7 (subpocket i) (Fig. 18A). The imidazole group of his-tamine corresponding to HBD2 in (Fig. 15) forms an H-bondwith E

5.46 in the pocket between TMs 3, 4, 5, and 6 (sub-

pocket ii) (Fig. 18A). This binding mode is in line with ear-

lier SDM studies indicating the essential role of D3.32

andE

5.46in histamine binding in H4R (section 3.2, Table 2) [65,

66]. Whether the imidazole ring is positively charged or neu-tral or the carboxylate group of E

5.46is negatively charged or

neutral is under debate [65, 66]. While D3.32is conserved andshown to be involved in histamine binding in all histaminereceptors [18, 65, 66, 87-93], residues at positions 5.42, 5.43,and 5.46 of TM5 are not strictly conserved among histaminereceptors (Fig. 17) and suggested to participate to the selec-tive recognition of the histamine imidazole ring [65, 87].Negatively charged residues E5.46 (also present in H4R) andD

5.42are responsible for the selective binding of agonists in

H3R [66] and H2R [89], respectively. H1R lacks a negativelycharged residue in TM5 but utilizes an asparagines side

chain (N5.46

) for H-bonding the imidazole ring of histamine[92]. The imidazole ring pharmacophore feature ARO in(Fig. 15) stacks between the aromatic rings of Y3.33 (in thebioaminergic GPCR receptor cluster exclusively present inhistamine receptors and muscarinic receptors [81]) andY6.51 (Fig. 18A). The differences in affinity between hu-man, pig and dog H4R (Table 2) is explained by the in-volvement of residue 4.57 (Asn in human H4R, His in pigand dog H4R) in alternative H-bond networks determiningthe orientation of E5.46 [18]. An alternative binding pose ofhistamine is stabilized by the L

5.43in pig H4R, but not in dog

H4R, explaining the increased binding affinity for histaminein pig mimicking N

4.57H/S

5.43L double mutant of human H4R

over dog mimicking N4.57H single mutant (Table 2).

4.2.2. Binding Modes of Non-Imidazole Ligands (Clozap-ine and JNJ 7777120)

Like histamine (1) (Fig. 18A), clozapine (9) and JNJ7777120 (12) both form an H-bond between their positivelycharged piperidine nitrogen atom corresponding to thepharmacophore feature HBD1 in (Fig. 15) and the carboxy-late group of D3.32 (Fig. 18B-C). This binding mode is sup-ported by site-directed mutagenesis studies demonstratingthe essential role of this residue in JNJ 7777120 binding [65](Table 3). The full agonist clozapine (9) forces C3.36in its g-conformation (which has been associated with the activatedstate of H1R [90]) by placing its chlorinated aromatic ringpharmacophore feature HYD1 in (Fig. 15) in the hydropho-

bic subpocket ii (Fig. 18B). The neutral antagonist JNJ777120 (12) on the other hand stabilizes C3.36 in its inactive[90] trans conformation by accepting an H-bond with itspiperazine carbonyl oxygen (Fig. 18B). The non-chlorinatedaromatic ring of clozapine (9) pharmacophore feature HYD2in (Fig. 15) stacks between Y3.33and Y6.51, while the nitrogenatom in the tricyclic ring system pharmacophore featureHBD2 in (Fig. 15) donates an H-bond to E5.46 (Fig. 18B).Dibenzodiazepine structure-activity relationship analysisindeed indicates the steric restriction around the tricylic ni-trogen atom section 2.3, (Fig. 8). In addition, site-directedmutagenesis studies suggest that the clozapine binding mode

is not complementary with the orientation of E5.46in pig anddog mimicking N

4.57H mutant, explaining the large decrease

in clozapine (9) binding affinity at the N4.57H mutant (Table3) as well as at pig and dog H4Rs (Table 2). The proposedclozapine binding mode is also in line with recent modelingstudies identifying residues at position 3.36, 5.43, 5.46, and6.55 as important residues determining the selectivity profileof clozapine (9) and olanzapine for bioaminergic recepto

subtypes [100]. In H1R clozapine (9) acts as an antagonis(Table 1) by stabilizing the inactive C3.36in its inactive [90transconformation by accepting an H-bond with its sp2 diazepine nitrogen atom, and binds with higher affinity than toH4R (Table 1) by additional stacking interactions with F

6.5

(T6.55 in H4R). The orientation of the non-chlorinated aromatic ring of clozapine pharmacophore feature HYD2 in(Fig. 15) towards the extracellular regions of TM5 and TM6is furthermore supported by dibenzodiazepine structureactivity relationships section 2.3, (Fig. 8) showing the preference of 7- and 8-substitution over 2- and 3- substitution[47], as well as the positive effect of the monkey H 4R mimicking L5.39V mutant [18] (Tables 2-3). The sterically restricted valine residue forms a complementary cap with its

CG2 methyl group on top of the non-chlorinated aromaticring of clozapine (9). The leucine residue in wild-type human H4R on the other hand, is more flexible and can orientits iso-butyl side chain in a transconformation [101] pointing out of the binding pocket [18].

The antagonist JNJ 7777120 (12) donates an H-bondfrom its indole nitrogen pharmacophore feature HBD2 in(Fig. 15) to the carboxylate group of E5.46, as supported bystructure-activity relationships [21, 65] demonstrating theessential role of this H-bond donor functionality in indolylpiperazines section 2.4, (Fig. 9), and site-directedmutagenesis studies indicating the essential H-bond acceptofunctionality of E

5.46 [65] (Table 3). The chlorinated aro

matic ring of JNJ 7777120 (12) pharmacophore featureHYD2 in (Fig. 15) is placed between Y3.33and Y6.51and occupies a pocket between TMs 3, 5, 6, and the second ex-tracellular loop (Fig. 18C). Structure-activity relationships oindolylpiperazines section 2.4, (Fig. 9) show the preferencefor 4- and 5-substitution over 6- and 7-substitution, and thetolerance of polar groups at the 5-position of the aromaticring [21] dismissing a binding mode in which the chlorinatedaromatic ring binds in the highly hydrophobic pocket closeto W

6.48. The negative effect of the monkey H4R mimicking

L5.39V mutation [18] (Tables 2 and 3) further supports theproposed binding mode. In the mutant, the CG2 methygroup of the sterically restricted valine residue bumps intothe chlorine atom of JNJ 7777120 (12), while the more

flexible leucine residue [101] in the wild-type can avoid thisclash.

SAR studies on guanidine-isothioureas (Fig. 7), aminopyrimidines (Figs. 10,11), quinoxalines (Fig. 12) and quinazolines (Fig. 13) support the existence of two pockets whichcan accommodate hydrophobic ligand moieties [33]. Onepocket is located close to W6.48 between TM3, TM4, TM5and TM6 (accommodating pharmacophore feature HYD1(Fig. 15): the chlorinated ring of clozapine (9) (Fig. 18B)and another pocket is located towards the extracellular region between TM3, TM5, TM6, and the second (and possibly third) extracellular loop (accommodating pharmacophore

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feature HYD2 (Fig. 15): the non-chlorinated ring of clozap-ine (Fig. 18B) or chlorinated ring of JNJ 7777120 (Fig.18C). The size of the first pocket is limited (see SAR diben-zodiazepines (Fig. 8), while the second pocket can accom-modate polar and relatively large groups see SAR in-dolylpiperazines (Fig. 9) and quinazolines (13). Interest-ingly, mutation studies have indicated that the second ex-tracellular loop [62] and the top of TM5 [18] are important

determinants in H4R species selectivity. More systematicmutation studies are however needed to further probe thisregion of the binding pocket and to determine its role in his-tamine receptor subtype selectivity.

Fig. (18). Binding mode of histamine (1) (A), clozapine (9) (B) and

JNJ 7777120 (12) (C) in the hH4R binding pocket [18]. The ligands

are depicted as ball-and-sticks with magenta carbon atoms. The

backbone of TM helices 4, 5, 6, and 7 are represented by yellow

ribbons and part of TM3 is shown as ribbon (the top of the helix is

not shown for clarity). Important binding residues are depicted as

ball-and-sticks with grey carbon atoms.

5. CONCLUDING REMARKS

The discovery of the H4R has attracted pharmaceuticaindustries and academia to search for new and potent ligandfor this pharmaceutically important G protein coupled receptor. In the past few years, several agonists and antagonistswith high affinity for the H4R and selectivity over the othehistamine receptors were successfully designed and synthesized. A general H4R ligand pharmacophore can be derivedby comparison of the structure-activity relationships of privileged H4R ligand scaffolds, i.e. histamine derivatives, guanidines, isothioureas, guanidine-isothioureas, indolylpiperazine analogs, dibenzodiazepines, aminopyrimidinesquinoxalines and quinazolines. H4R species differences andsite-directed mutagenesis studies on the other hand, haverevealed some key residues that are involved in ligand bind-ing. Taken together, the experimentally identified H4R residues and ligand SAR data are used to construct and validatethree-dimensional H4R models which offer insight into theatomic details of H

4R-ligand interactions, and can be applied

to selective structure-based virtual screening and ligand design in the future.

ACKNOWLEDGEMENTS

This work was supported by the Top Institute Pharma[project number D1.105: the GPCR Forum], the NetherlandOrganization for Scientific Research [VENI Gran700.59.408], and COST Action BM0806.

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Received: March 02, 2010 Accepted: May 12, 2010

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