Effect of H4R antagonist N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamides and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole on histamine and 4-methylhistamine-induced mast cell response

Article abstract of DOI:10.1080/10799893.2016.1247863

Context: The histamine plays a decisive role in acute and chronic inflammatory responses and is regulated through its four types of distinct receptors designated from H1 to H4. Recently histamine 4 receptor (H4R) antagonists have been reported to possess various pharmacological effects against various allergic diseases. Objective: To investigate the inhibitory effect of N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamide (Compound A) and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole (Compound L) on H4R-mediated calcium mobilization, cytokine IL-13 production, ERK1/2, Akt and NF-κB activation in human mastocytoma cells-1 (HMC-1). Materials and methods: Compounds A and L were synthesized chemically and their inhibitory effect on intracellular calcium release was analyzed by Fluo-4 calcium assay, cytokine measurement through ELISA and activation of signaling molecules by western blot. Results: Pre-treatment with compounds A and L significantly reduced the H4R-mediated intracellular calcium release. Histamine and 4-methylhistamine (4-MH) induced Th2 cytokine IL-13 production in HMC-1 cells, was inhibited by compound A (77.61%, 74.25% at 1 μM concentration) and compound L (79.63%, 81.70% at 1 μM concentration). Furthermore, histamine induced the phosphorylation of ERK1/2, Akt and NF-κB was suppressed by compounds A and L at varying levels, ERK1/2 (88%, 86%), Akt (88%, 89%) and NF-κB (89%, 87%) in HMC-1 cells. Discussion and conclusions: Taken together these data demonstrate that compound A and compound L may block H4R-mediated downstream signaling events.

Full text of DOI:10.1080/10799893.2016.1247863

Journal of Receptors and Signal Transduction
ISSN: 1079-9893 (Print) 1532-4281 (Online) Journal homepage: http://www.tandfonline.com/loi/irst20
Effect of H4R antagonist N-(2-aminoethyl)-5-
chloro-1H-indol-2-carboxamides and 5-chloro-2-
(piperazin-1-ylmethyl)-1H-benzimidazole on
histamine and 4-methylhistamine-induced mast
cell response
Gomathi Nagarajan, Vairamani Mariappanadar, Muthu Tamizh, Ilango
Kaliappan & Berla Thangam Elden
To cite this article: Gomathi Nagarajan, Vairamani Mariappanadar, Muthu Tamizh, Ilango
Kaliappan & Berla Thangam Elden (2016): Effect of H4R antagonist N-(2-aminoethyl)-5-
chloro-1H-indol-2-carboxamides and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole on
histamine and 4-methylhistamine-induced mast cell response, Journal of Receptors and Signal
Transduction, DOI: 10.1080/10799893.2016.1247863
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Date: 07 November 2016, At: 02:07

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION, 2016
http://dx.doi.org/10.1080/10799893.2016.1247863
RESEARCH ARTICLE
Effect of H4R antagonist N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamides and
5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole on histamine and
4-methylhistamine-induced mast cell response
Gomathi Nagarajana, Vairamani Mariappanadara, Muthu Tamizhb, Ilango Kaliappanc and Berla Thangam Eldena
b
aDepartment of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India; Interdisciplinary Institute of
c
Indian System of Medicine (IIISM), SRM University, Kattankulathur, Tamil Nadu, India; Department of Pharmaceutical Chemistry,
SRM College of Pharmacy, SRM University, Kattankulathur, Tamil Nadu, India
ABSTRACT
ARTICLE HISTORY
Received 25 July 2016
Revised 30 August 2016
Accepted 8 September 2016
Published online 3 November
2016
Context: The histamine plays a decisive role in acute and chronic inflammatory responses and is regu-
lated through its four types of distinct receptors designated from H1 to H4. Recently histamine 4 recep-
tor (H4R) antagonists have been reported to possess various pharmacological effects against various
allergic diseases.
Objective: To investigate the inhibitory effect of N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamide
(Compound A) and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole (Compound L) on H4R-mediated
calcium mobilization, cytokine IL-13 production, ERK1/2, Akt and NF-jB activation in human mastocy-
toma cells-1 (HMC-1).
KEYWORDS
H4R antagonist; mast cells;
histamine; 4-methylhist-
amine; Compound A;
Compound L
Materials and methods: Compounds A and L were synthesized chemically and their inhibitory effect
on intracellular calcium release was analyzed by Fluo-4 calcium assay, cytokine measurement through
ELISA and activation of signaling molecules by western blot.
Results: Pre-treatment with compounds A and L significantly reduced the H4R-mediated intracellular
calcium release. Histamine and 4-methylhistamine (4-MH) induced Th2 cytokine IL-13 production in
HMC-1 cells, was inhibited by compound A (77.61%, 74.25% at 1 lM concentration) and compound L
(79.63%, 81.70% at 1 lM concentration). Furthermore, histamine induced the phosphorylation of ERK1/
2, Akt and NF-jB was suppressed by compounds A and L at varying levels, ERK1/2 (88%, 86%), Akt
(88%, 89%) and NF-jB (89%, 87%) in HMC-1 cells.
Discussion and conclusions: Taken together these data demonstrate that compound A and com-
pound L may block H4R-mediated downstream signaling events.
Introduction
human central nervous system (4). Compared with H1R
and H2R, the lately discovered H4R has more selective
expression pattern and found mainly in the cells of hem-
atopoietic origin, in particular mast cells, basophils, eosino-
phils and T-cells (3,5–8). H1R antagonists also referred as
antihistamines have long been used to treat allergies, offer-
ing symptomatic relief in atopic nasal, conjunctival and
skin disease. However, H1R antagonists are not optimally
effective in asthma (9), where histamine is particularly
involved in immune cell chemotaxis and pro-inflammatory
responses. Recent reports indicate that, H4R is involved in
the control of immune cell trafficking, pro-inflammatory
mediator’s release, increased expression of adhesion mole-
cules and rearrangement of the actin cytoskeleton leading
to immune cell migration from the bloodstream into the
sites of inflammation (10,11). Recently, we also reported
that in human mast cells H4R activation caused the pro-
duction of various inflammatory mediators including, vari-
ous cytokines, chemokines which are associated with
allergic asthma and other inflammatory diseases (12).
Histamine has long been known to be the mediator that
orchestrates inflammatory and allergic responses acting
mainly through histamine receptors. During inflammation,
histamine released from preformed stores in mast cells and
basophils, acts on vascular smooth muscle cells and endothe-
lial cells, leading to increased vasodilation and vascular per-
meability (1). Histamine contributes to the progression of
allergic inflammatory responses by enhancing the secretion
of proinflammatory cytokines like IL-1a, IL-1b, IL-6, as well as
chemokines like RANTES and IL-8 both in several cell types
and local tissues (2).
The pleiotropic effects of histamine are mediated by four
subtypes of histamine receptors namely H1R, H2R, H3R and
histamine 4 receptor (H4R) and belong to G-protein coupled
receptor family (3). The most characteristic roles for H1R acti-
vation are smooth muscle contraction and increase in vascu-
lar permeability. Many of its functions contribute to allergic
responses. The H2R functions as a key modulator for gastric
acid secretion and H3R is predominantly expressed in the
CONTACT Dr E. Berla Thangam Elden
berlathagam.e@ktr.srmuniv.ac.in
Department of Biotechnology, School of Bioengineering, SRM University,
Kattankulathur 603203, Tamil Nadu, India
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
G. NAGARAJAN ET AL.
H4R plays a major role in immunomodulatory functions, assay kit from Invitrogen, IL-13 ELISA kit from R&D Systems
therefore, it is now considered as a new drug target for the (Minneapolis, MN). H4R siRNA, siRNA transfection medium,
treatment of various inflammatory diseases including allergy
and asthma (5). Experiments using the dual H3R/H4R antag-
onist thioperamide have shown the pathophysiological sig-
nificance of H4R in inflammatory conditions, such as allergic
asthma and other allergic disorders (6). Recent reports indi-
cate that the H4R antagonist 1-[(5-chloro-1H-indol-2-yl)car-
bonyl]-4-methylpiperazine (JNJ7777120) was able to inhibit
histamine induced chemotaxis and Ca2þ influx in mouse
bone marrow derived mast cells, as well as tracheal mast cell
migration from the connective tissue toward the epithelium
(4,13,14). Furthermore, JNJ7777120 significantly blocked neu-
trophil infiltration in a mast cell-dependent mouse zymosan
induced peritonitis model (4). In addition, only a few H4R
antagonists were reported, and for instance A-940894,
INCB38579 and VUF6007 were reported to have anti-inflam-
matory and anti-pruritic effects (4,15–18). Thus, only a limited
number of selective H4R antagonists have been discovered
so far.
Though JNJ7777120 is considered to be the standard
antagonist for H4R (16,19), the recent data show that
JNJ7777120 activates b-arrestin in Gai/o protein independent
manner and exhibit partial agonist effect which indicates
that it has dual activity and also it has a relatively short
plasma half-life and limited bioavailability (4,5). Therefore, fur-
ther investigations on the development of novel lead com-
pounds to explore their therapeutic potential in treating
allergic diseases are required. In our previous study, we have
generated selective histamine receptor H4R antagonists using
bioinformatics tools (20). Here in this study, we chemically
synthesized (Scheme 1) and tested whether these com-
pounds can inhibit H4R-mediated downstream signaling
events in human mast cells (human mastocytoma cells-1
(HMC-1)).
transfection reagent and 4-methylhistamine (4-MH) (H4R
agonist), phospho-Akt antibody, phospho-ERK1/2 (Thr 177/
Tyr160) and phospho-NF-jB antibody were purchased from
Santa Cruz Biotechnology (Dallas, TX). Total ERK 1/2 antibody
and total Akt antibody were purchased from Cell Signaling
(Burlington, NC). Super Signal West Pico chemiluminescent
substrate was purchased from Thermo Scientific and all the
other basic chemicals were from Sigma (St. Louis, MO).
IMDM, FBS, glutamine, penicillin and streptomycin were pur-
chased from Gibco, Invitrogen, Carlsbad, CA.
Methods
Synthesis of title compounds
Synthesis of N-(2-aminoethyl)-5-chloro-1H-indol-2-carbox-
amide (compound A) 5-Chloroindol-2-carboxylic acid
(100 mg) dissolved in 10.0 ml of benzene was refluxed with
SOCl2 (1 ml) for 5 h at 100 ꢀC. The excess of SOCl2 was
removed by distillation. The acid chloride was added to
ethylenediamine (2.0 ml) and stirred overnight at room tem-
perature. The precipitated solid was removed by centrifuga-
tion and the supernatant solvent was removed. The
precipitate was dissolved in ethylacetate (10.0 ml), washed
with water (2 ꢁ 5 ml) and the organic layer was evaporated
to get a solid, after drying over anhydrous sodium sulfate.
Yield: 40.0 mg. The purity of the compound was checked by
thin layer chromatography (methanol 100%) and the struc-
ture of the compound was confirmed by NMR spectroscopy,
d values (assignment); 6.8, 7.2, 7.3, 7.6 (aromatic ring hydro-
gen); 3.5 (N–CH2); 2.98 (CH2–N). The molecular weight was
confirmed by electrospray ionization mass spectrometry; m/z
238 [M þ 1]þ(Figure 1(A,B)) (21,22).
Synthesis of 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimi-
dazole (compound L)
Materials and methods
Step 1: Synthesis of N-carboxymethyl piperazine The solution
of chloroacetic acid (1.98 g) and sodium hydroxide (0.8 g) in
water is added drop wise to the stirred solution of piperazine
(8.0 g) and sodium hydroxide (2.0 g) in water. After 12 h the
Materials
JNJ7777120, histamine, 5-chloroindol-2-carboxylic acid, 4-
chloro-1,2-phenylene diamine and chloroacetic acid were
purchased from Sigma–Aldrich (St. Louis, MO), Fluo-4 calcium separated solid was filtered and the filtrate was cooled in an
Scheme 1. Structure of Compound A and Compound L.

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
3
Figure 1. Characterization of compound A and compound L. (A) The electrospray ionization mass spectrometry of compound A with peak 238. (B) The NMR spec-
trum for compound A. (C) The electrospray ionization mass spectrometry of compound L with peak 251. (D) The NMR spectrum for compound L.

4
G. NAGARAJAN ET AL.
Figure 1. Continued.
ice bath. The solution was made alkaline with 10% sodium
hydroxide solution and extracted with ethylacetate (20 ml) to
remove excess of piperazine. Further, it was acidified and
extracted with ethylacetate (20 ml) to remove excess of
chloroacetic acid. The filtrate was then treated with excess of
methanol. The precipitate formed was centrifuged and the
supernatant was decanted. The precipitate was dried and a
white solid was obtained. Yield: 8.0 g, the molecular weight
of the synthesized compound was confirmed by electrospray
ionization mass spectrometry; m/z: 145 [M þ 1]þ.
Cytotoxicity assay
Cell toxicity of H4R antagonists compound A and L was eval-
uated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay on HMC-1 cells. Briefly, the cells were
seeded on to 96-well plates at a density of 0.1 ꢁ 105 cells/
well in IMDM medium supplemented with 1% BSA. Different
concentrations in the range 1 nM–100 lM of compounds A
and L were added to the cells and were incubated for 24 h.
MTT reagent was added and incubated for 4 h. The farma-
zone crystals formed were dissolved in DMSO and the
absorbance was taken at 570 nm using ELISA reader (Biotek
Synergy HT multimode reader).
Step 2: Synthesis of compound L 4-Chloro-1,2-phenylene
diamine (1.0 g) was taken in 50 ml of xylene and reflexed
with N-carboxymethyl piperazine (0.8 g) for 6 h. Xylene was
decanted and the residue was dissolved in ethyl acetate and
the compound was purified by column chromatography over
silica gel. Yield: 70 mg. The purity of the compound was
checked by thin layer chromatography (methanol:ethylace-
tate 1:1) and the structure of the compound was confirmed
by NMR spectroscopy, d values (assignment): 7.3, 7.5, 7.6
(aromatic ring hydrogens); 3.9 (C–CH2–N); 3.2 (N–CH2); 2.8
(CH2–NH). The molecular weight of the synthesized com-
pound was confirmed by electrospray ionization mass spec-
trometry; m/z 251 [M þ 1]þ(Figure 1(C,D)).
Small interfering RNA (siRNA) mediated gene silencing
of histamine H4R in HMC-1 cells
The HMC-1 cells (50,000 cells/well) were plated in six-well
plates. For histamine H4R gene silencing, a human siRNA tar-
geting H4 receptor (Santa Cruz Biotechnology, Dallas, TX)
was employed. The siRNA transfection was performed accord-
ing to the manufacturer’s instruction. In brief, cells were incu-
bated with transfection mixture containing 100 nmol/L of
siRNA for 5–6 h and then incubated with 1 ml of serum-
containing media further for 24 h.
Culture of HMC-1 cell line
Measurement of intracellular calcium levels
HMC-1 cells were kindly provided by Dr. Joseph H Butterfield,
Mayo Clinic (Rochester, MN), and cultured in IMDM supple-
mented with 10% FBS, glutamine (2 mM), penicillin (100 IU/
ml) and streptomycin (100 ng/ml).
Intracellular calcium levels were measured in HMC-1 cells
using Fluo-4 calcium assay kit and performed according to
the manufacturer’s instructions. In brief, HMC-1 cells

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
5
and siRNA mediated H4R transfected cells were washed
and suspended in assay buffer and plated in black flat-
bottom 96-well plates. Mast cells (2 ꢁ 105) were pre-treated
with JNJ7777120 (10 lM), compound A and compound L
(10 lM, 1 lM) for 10 min, labeled with Fluo-4 dye and incu-
bated for 1 h at 37 ꢀC. The plates were then read at excitation
494 nm and emission 516 nm. The basal reading for 100 s was
taken, then the cells were stimulated with histamine (10 lM)
or 4-MH (10 lM) and change in fluorescence was measured
for a total of 600 s. (Biotek Synergy HT multimode reader).
Estimation of IL-13 by ELISA
The HMC-1 cells (1 ꢁ 106 cells/ml per well in a basal medium)
were washed and preincubated with JNJ7777120 (10 lM),
compound A, compound L (10 lM, 30 min) and then stimu-
lated with 4-MH or histamine (10 lM). Supernatants were col-
lected and stored at ꢂ80 ꢀC until further use. Concentration of
IL-13 in supernatant was estimated using sandwich ELISA kit
according to the manufacturer’s instructions. The reactions
were measured at 450 nm (Biotek Synergy HT multimode
reader).
Figure 2. Effect of compounds A and L on cytotoxicity in HMC-1 cells. The data
show the % cell viability of HMC-1 cells after the treatment of cells with differ-
ent concentrations (100 lM–1 nM) of compounds A and L. Values are expressed
as mean SD of three experiments done in triplicates. ns was considered as
statistically non-significant.
100 lM. The compounds were not found to be toxic to the
cells at different concentration used (Figure 2).
Effect of compounds A and L on histamine and 4-MH
induced calcium mobilization
Western blotting
The HMC-1 cells (1 ꢁ 106 cells/ml) were lysed in radioimmu-
noprecipitation assay (RIPA) buffer. The proteins concentra-
tion was quantified by Bradford method and was separated
on sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). The blots were probed with rabbit anti-human
H4R antibody, anti-phospho-ERK1/2, anti-phospho-Akt anti-
body and anti-phospho-NF-jB p65 antibody. Respective blots
were stripped and reprobed with b-actin, antibodies followed
by anti-mouse-IgG-HRP antibodies (Santa Cruz). The bands
were visualized by SuperSignal West Pico chemiluminescent
substrate (Thermo scientific) and the image was captured
using multi-imaging system (Cell Biosciences). Images from
multiple gels (at least three determinations) were quantitated
using the fluorchem Q software.
A rise in intracellular calcium ion concentration triggers cellu-
lar activation, which in turn leads to the release of inflamma-
tory mediators from mast cells. Histamine a natural agonist
for H4R and 4-MH, a synthetic agonist which has high affinity
and binds preferentially to H4R were used as agonist in the
study. We examined the effect of compounds A and L on his-
tamine and 4-MH induced intracellular calcium release in
HMC-1 cells. We found that histamine and 4-MH (10 lM)
induce the intracellular calcium release, whereas JNJ7777120
(10 lM), compounds A and L (10 lM, 1 lM) inhibited the
intracellular calcium release in HMC-1 cells. Interestingly,
compound L was found to be more effective than compound
A at 10 lM concentrations in inhibiting the intracellular cal-
cium release in response to histamine and 4-MH (Figure
3(A,B)). The H4R siRNA gene silencing decreased the 4-MH-
induced intracellular calcium release. In addition, no signifi-
cant effect was observed on JNJ7777120, compounds A and
Statistical analysis
The difference in estimated parameters between the groups
was analyzed using one-way ANOVA with Bonferroni’s test.
Data are expressed as mean SD. All the parameters were
analyzed at 99% confidence intervals and p values of <.01
were considered to be statistically significant except for den-
drogram analysis where the parameters were analyzed at
95% confidence intervals and p values of; <.05 were consid-
ered to be statistically significant. Statistical analysis of the
data was performed using GraphPad Prism version 6.00, ver-
sion 7.00, San Diego, CA.
L
pre-treatment on H4R-mediated intracellular calcium
release. These data indicate that compounds A and L can
inhibit the H4R activation in HMC-1 cells.
Effect of compounds A and L on H4R mediated IL-13
release in HMC-1 cells
Since IL-13 is thought to be a critical mediator of allergic
asthma, we examined the effect of compounds A and L on
H4R mediated IL-13 release by using histamine and 4-MH in
HMC-1 cells. As shown in Figure 4, histamine (10 lM) was
able to induce IL-13 secretion up to 1576 pg/ml. Pre-treat-
ment with the compounds A and L at 1 lM concentration
decreased the histamine (10 lM) induced IL-13 cytokine
release up to 352.84 pg/ml (77.61%) and 321.03 pg/ml
Results
Cytotoxic effect of compound A and compound L on
HMC-1 cells
The compounds A and L were checked for its cytotoxic effect
on HMC-1 cells at different concentrations from 1 nM to (79.63%). 4-MH (10 lM) induced IL-13 (1494 pg/ml) was

6
G. NAGARAJAN ET AL.
Figure 3. Effect of compounds A and L on histamine and 4-MH induced intracellular calcium release in HMC-1 cells. Data showing the inhibitory effect of com-
pounds A and L (pretreated for 10 min) on intracellular calcium release at the concentration of 10 lM and 1 lM against (A) histamine (10 lM) and (B) 4-MH (10 lM)
with H4R standard antagonist JNJ7777120 (10 lM), (C) Effect of compounds A and L on 4-MH induced intracellular calcium release in H4R siRNA transfected cells.
Data shown are representative of three similar experiments.
inhibited by compounds A and L to the level of 384.65 pg/ml
(74.25%) and 273.31 pg/ml (81.70%), respectively.
Discussion
In the present study, we chemically synthesized the H4R
antagonist, compound A and compound L that were gen-
erated using bioinformatics structure-based virtual screen-
ing strategies (20) and its inhibitory effect on
H4R-mediated mast cell functions were studied using HMC-
1 cells. Our compounds A and L were able to inhibit the
effect of histamine and 4-MH induced calcium mobilization
and Th2 cytokine IL-13 release. We have also shown that
the compounds were able to inhibit the activation of
ERK1/2, Akt and NF-jB when stimulated with histamine in
HMC-1 cells.
Effect of compounds A and L on ERK 1/2, Akt and NF-jB
signalling molecules in HMC-1 cells
Furthermore, the effect of compounds A and L on signal-
ing molecules such as NF-jB, ERk1/2 and Akt in hista-
mine-induced mast cell activation was checked. We found
that histamine (10 lM) induced phosphorylation of ERK1/2,
Akt and NF-jB p65 in HMC-1 cells. Compound A (10 lM,
1 lM, 100 nM) was able to inhibit histamine-induced ERK1/
2, Akt and NF-jB phosphorylation at (88%), (88%) and
(89%), respectively. Compound L (10 lM, 1 lM, 100 nM)
The discovery of H4R led to diligent efforts on the develop-
was also able to inhibit phosphorylation of ERK1/2, ment of antagonists targeting H4R based on its involvement
Akt and NF-jB at (86%), (89%), (87%) in HMC-1 cells
in inflammatory and immunomodulatory functions (23–30). So
(Figure 5).
far several antagonists such as JNJ7777120, A-940894,

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
7
Figure 4. Effect of compounds A and L on histamine and 4-MH induced IL-13 release in HMC-1 cells. The figure shows the level of IL-13 production after 24 h
stimulation with histamine and 4-MH (10 lM) and inhibitory effect of JNJ7777120 (10 lM), compounds A and L (pre-treated for 10 min at the concentration of
10 lM, 1 lM and 100 nM) against histamine (10 lM) (A, B) and 4-MH (10 lM) (C, D) and JNJ7777120 (10 lM). Values are expressed as mean SD of three experi-
ꢃꢃ
ments done in triplicates. ##p < .001 significantly different from control. p < .001 and
ꢃꢃꢃ
p < .0001 significantly different from histamine or 4-MH.
A-943931, A-987306, INCB38579, VUF6007, VUF11489, were with gastric acid secretion. H3R is involved in neurotransmis-
sion and is not expressed in HMC-1 cells. The stimulation of
H4R reduces forskolin-induced cAMP formation activation of
MAPK and enhanced Caþþ mobilization and chemotaxis,
without affecting degranulation, which enables the selective
recruitment of effector cells and enhancement of allergic
responses (53).
Accumulated evidence suggests that selective H4R antag-
onists have shown to inhibit histamine-induced chemotaxis
and calcium responses in eosinophil and bone marrow-
derived mast cells (8,15,17). Histamine-mediated calcium
release was inhibited by A-940894 dose dependently in
mouse bone marrow-derived mast cells (15). The previous
report by Shin et al. (18) has shown that INCB38579 a small
molecule antagonist inhibited H4R-mediated calcium mobil-
ization in HEK-293 cells stably co-expressing the human,
mouse or rat histamine H4R. JNJ7777120 and thioperamide
were also shown to inhibit the calcium response in bone
marrow-derived mast cells when stimulated with histamine
(4,30). In the present study, compounds A, L and JNJ7777120
tested for their effect against H4R and has shown distinct
effect on different cell types and various animal models of
allergy and inflammation (4,15–18,23,32–49,55). PF-3893787
(NCT00856687), UR-63325 (NCT01260753), KD1157 (54),
JNJ39758979 (NCT01679951, NCT00941707, NCT01862224,
NCT01823016 and NCT02295865) are in various stage of clin-
ical trials. JNJ39758979 has shown preclinical and safety in vol-
unteers (50,51).
Among the antagonists, an indolecarboxamide-containing
JNJ7777120 was considered as the first standard selective
H4R antagonist (54). JNJ7777120 is used in various studies by
different groups. Previous studies have reported that H3R
antagonist such as thioperamide, clobenpropite and H3R
agonist such as imetit and R-a-methylhistamine bind to the
H4R with different affinity from that of the H3R and further it
has shown to inhibit or activate the H4R (55).
Recent reports suggest that mast cells express H1R, H2R
and H4R but not H3R except for brain mast cells. The Gaq/11
coupled H1R and Ga/io coupled H4R play a major role in
allergic inflammation. The Gas-coupled H2R is associated have shown to inhibit intracellular calcium release in HMC-1

8
G. NAGARAJAN ET AL.
Figure 5. Effect of compounds A and L on histamine-induced downstream signaling in HMC-1 cells. HMC-1 cells (1 ꢁ 106) were pretreated with compounds A and
L for 10 min and stimulated with histamine (10 lM). Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2, anti-phospho-Akt
and anti-phospho-NFjB antibody. Respective blots were stripped and reprobed with total or b-actin antibody followed by anti-mouse IgG-HRP antibodies.
Immunoreactive bands were visualized by SuperSignal West Pico chemiluminescent substrate. Blots shown here is representative of three similar experiments. Bar
graph represents summary of ERK1/2, Akt and NFjB phosphorylation. Bands were quantified by densitometry and expressed as percentage. Values are analyzed
using one-way ANOVA with Bonferroni’s test. Data were expressed as mean SD of three experiments. #p < .05 was considered significantly different from control.
ꢃ
p < .05 was considered significantly different from histamine.
cells. Since H4R expression is silenced by H4R siRNA, there is JNJ7777120 has anti-puritic and anti-inflammatory function in
very less or no binding site available for the compounds. a mouse model (18). H4R antagonist treatment has shown
Therefore, there is no significant effect observed with com- significantly to reduce the level of IL-13 in bronco alveolar
pounds A and L on 4-MH induced intracellular calcium lavage fluid and tissue of mice model of allergic asthma (9).
release in H4R siRNA transfected HMC-1 cells. Whereas, com- Therefore, in the present study we compared the potencies
pounds A and L showed inhibition in H4R-mediated intracel- of compound A and compound L in the inhibition of IL-13
lular calcium release in HMC-1 cells. This shows that release. Compound A and compound L were found to inhibit
compounds A and L may be specific to H4R and doesn’t IL-13 release to basal level against 4-MH and histamine.
show any effect through other receptors (H1, H2 and H3) at
the used concentrations.
NF-jB is a central transcription factor which plays a major
role in regulating the immune response to allergy, inflamma-
Selective antagonism or gene knockout of H4R has been tion and infection. Previously it was thought that H1R acti-
demonstrated to reduce allergic airway inflammation in a vates NF-jB in cos-7 cells, later it was found that H4R was
mouse model (30,52). The H4R antagonist JNJ7777120 and also involved in the activation of NF-jB (56,57). Furthermore,
VUF6007 have shown anti-inflammatory effect in rat model previous reports have shown that histamine-induced phos-
(31). Furthermore, it has shown that H4R antagonist phorylation of ERK, MEK and Akt in mast cells, whereas

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
9
11.
12.
13.
14.
Zampeli E, Tiligada E. The role of histamine H4 receptor in
JNJ7777120 inhibited histamine induced NF-jB, Akt and ERK
phosphorylation in mast cells and has also shown to inhibit
the phosphorylation of NF-jB in a rat model of inflamed
knee tissue (58–60). Similarly, in the present study our com-
pounds A and L were also able to inhibit histamine induced
ERK1/2, Akt and NF-jB phosphorylation in HMC-1 cells.
Taken together, our data demonstrate that compound A
and compound L were shown to inhibit the H4R mediated
calcium mobilization, IL-13 cytokine release and able to
inhibit the downstream signaling molecules such as ERK1/2,
Akt and NF-jB.
immune and inflammatory disorders. Br
J
Pharmacol
2009;157:24–33.
Angel Jemima E, Prema A, Berla Thangam E. Functional character-
isation of histamine H4 receptor on human mast cells. Mol
Immunol 2014;62:19–28.
Seifert R, Strasser A, Schneider EH, et al. Molecular and cellular
analysis of human histamine receptor subtypes. Trends Pharmacol
Sci 2013;34:33–58.
Geng S, Gao YD, Yang J, et al. Potential role of store-operated
Ca2þ entry in Th2 response induced by histamine in human mono-
cyte-derived dendritic cells. Int
J Immunopharmacol 2012;12:
358–67.
15.
Strakhova MI, Cuff CA, Manelli AM, et al. in vitro and in vivo char-
acterization of A-940894: a potent histamine h4 receptor antagon-
ist with anti-inflammatory properties. Br J Pharmacol 2009;157:
44–54.
Conclusions
16.
17.
Jablonowski JA, Grice CA, Chai W, et al. The first potent and
selective non-imidazole human histamine H4 receptor antagonists.
J Med Chem 2003;46:3957–60.
Reher TM, Neumann D, Buschauer A, Seifer R. Incomplete activa-
tion of human eosinophils via the histamine H4-receptor; evidence
for ligand-specific receptor conformations. Biochem Pharmacol
2012;84:192–203.
Shin N, Covington M, Bian D, et al. INCB38579, a novel potent his-
tamine H4 receptor small molecule antagonist with anti-inflamma-
tory and anti-pruritic functions. Eur J Pharmacol 2012;675:47–56.
Venable JD, Cai H, Chai W, et al. Preparation and biological evalu-
ation of indole, benzimidazole, and thienopyrrole piperazine car-
boxamides: potent human histamine H4 antagonists. J Med Chem
2005;48:8289–98.
Therefore, the present study suggests that compounds A and
L can inhibit H4R-mediated allergic and inflammatory reac-
tion and can also provide new therapeutic agents for various
allergic and inflammatory diseases. Further, the effect of
these compounds on allergic asthma mice model is under
progress.
18.
19.
Acknowledgements
We are grateful to Dr. Joseph H. Butterfield (Mayo Clinic, Rochester, MN)
for HMC-1 cells. This work was supported by SRM University, Chennai,
Tamil Nadu, India.
20.
21.
Annan N, Silverman RB. New analogues of N-(2-aminoethyl)-4-
chlorobenzamide (RO 16-6491). Some of the most potent mono-
amine oxidase-B inactivators. J Med Chem 1993;36:3968–70.
Engle M, Frohner W, Stroba A, Biondi RM. Preparation of 4-hetro-
cycyl-3-arylbutanoic acid 3,4-di-aryl butanoic acid 4-hetrocyclyl-3-
aryl-2-pentonoic acid, and 3,5-diaryl-2-pentenoic acid derivatives
as allosteric protein kinase modulators. Universitaet Des
Sarnalandes, Application. WO201004311 A1; Apr-22, 2010.
Christopher F, Berla Thangam E, Xavier M. A bioinformatics search
for selective histamine H4 receptor antagonists through structure-
based virtual screening strategies. Chem Biol Drug Des
2012;79:749–59.
Disclosure statement
The authors declare no conflict of interest.
References
22.
23.
24.
25.
1.
Panettieri RA Jr. Airway smooth muscle: immunomodulatory cells
that modulate airway remodelling? Respir Physiol Neurobiol
2003;137:277–93.
2.
3.
Umetsu D, McIntire J, Akbari O, et al. Asthma: an epidemic of dys-
regulated immunity. Nat Immunol 2002;3:715–20.
Zhang M, Thurmond RL, Dunford PJ. The histamine H4 receptor: a
novel modulator of inflammatory and immune disorders.
Pharmacol Ther 2007;113:594–606.
Mowbray CE, Bell SA, Clarke NP, et al. Challenges of drug discov-
ery in novel target space. The discovery and evaluation of PF-
3893787: a novel histamine H4 receptor antagonist. Bioorg Med
Chem Lett 2011;21:6596–602.
Terzioglu Van Rijn RM, Bakker RA, et al. Synthesis and structure-
activity relationships of indole and benzimidazole piperazine as
histamine H4 receptor antagonists. Bioorg Med Chem Lett
2004;14:5251–6.
4.
5.
Thurmond RL, Desai PJ, Dunford PJ, et al. A potent and selective
histamine H4 receptor antagonist with anti-inflammatory proper-
ties. J Pharmacol Exp Ther 2004;309:404–13.
Seifert R, Schneider EH, Dove S, et al. Pardoxical Stimulatory effect
of the standard Histamine H4 receptor antagonist JNJ7777120: the
H4 receptor joins the club of 7 transmembrane domine receptors
exhibiting functional selectivity. J Mol Pharmacol 2011;79:631–8.
Oda T, Morikawa N, Saito Y, et al. Molecular cloning and character-
ization of novel type of histamine receptor preferentially
expressed in leukocytes. J Biol Chem 2000;275:36781–6.
Liu C, Ma X, Jiang X, et al. Cloning and pharmacological character-
ization of a fourth histamine receptor (H4) expressed in bone mar-
row. Mol Pharmacol 2001;59:420–6.
ꢀ
ꢀ
ꢀ
Kiss R, Noszal B, Racz A, et al. Binding mode analysis and enrich-
ment studies on homology models of the human histamine H4
receptor. Eur J Med Chem 2008;43:1059–70.
26.
27.
Lane CAL, Hay D, Mowbray CE, et al. Synthesis of novel histamine
H4 receptor antagonists. Bioorg Med Chem Lett 2012;22:1156–9.
Audaloussi M, Lim HD, Van der Meer T, et al. A novel series of his-
tamine H4 receptor antagonists based on the pyrido[3,2-d]pyrimi-
dine scaffold: comparison of hERG binding and target residence
time with PF-3893787. Bioorg Med Chem Lett 2013;23:2663–70.
Więcek M, Kottke T, Ligneau X, et al. N-Alkenyl and cycloalkylcar-
bamates as dual acting histamine H3 and H4 receptor ligands.
Bioorg Med Chem 2011;19:2850–8.
Smits RA, Lim HD, Van der Meer T, et al. Ligand based design of
novel histamine H4 receptor antagonists; fragment optimization
and analysis of binding kinetics. Bioorg Med Chem Lett
2012;22:461–7.
6.
7.
28.
29.
8.
Ling P, Ngo K, Nguyen S, et al. Histamine H4 receptor mediates
eosinophil chemotaxis with cell shape change and adhesion mol-
ecule upregulation. Br J Pharmacol 2004;142:161–71.
9.
Cowden JM, Riley JP, Ying Ma J, et al. Histamine H4 receptor
antagonism diminishes existing airway inflammation and dysfunc-
tion via modulation of Th2 cytokine. Respir Res 2010;11:86.
Schroeder JT, Macglashan DW Jr, Lichtenstein LM. Human baso-
phils: mediator release and cytokine production. Adv Immunol
2001;77:93–122.
10.
30.
Hofstra CL, Desai PJ, Thurmond RL, Fung-Leung W-P. Histamine H4
receptor mediates chemotaxis and calcium mobilization of mast
cells. J Pharmacol Exp Ther 2003;305:1212–21.

10
G. NAGARAJAN ET AL.
31.
Coruzzi G, Adami M, Guaita E, et al. Antiinflammatory and antino-
ciceptive effects of the selective histamine H4-receptor antagonists
JNJ7777120 and VUF6002 in a rat model of carrageenan-induced
acute inflammation. Eur J Pharmacol 2007;563:240–4.
Dunford PJ, O’Donnell N, Riley JP, et al. The histamine H4 receptor
mediates allergic airway inflammation by regulating the activation
of CD4þ T cells. J Immunol 2006;176:7062–70.
Cowden JM, Yu F, Banie H, et al. The histamine H4 receptor medi-
ates inflammation and Th17 responses in preclinical models of
arthritis. Ann Rheum Dis 2014;73:600–68.
Cowden JM, Yu F, Challapalli M, et al. Antagonism of the hista-
mine 4 receptor reduces LPS-induced TNF production in vivo.
Inflamm Res 2013;62:599–607.
against Carrageenan-induced hyperalgesia. J Med Chem 2008;
51:7094–8.
47.
Cowart MD, Altenbach RJ, Liu H, et al. Rotationally constrained
2,4-diamino-5,6-disubstituted pyrimidines: a new class of histamine
H4 receptor antagonists with improved druglikeness and in vivo
efficacy in pain and inflammation models. J Med Chem 2008;51:
6547–57.
Savall BM, Chavez F, Tays K, et al. Discovery and SAR of 6-alkyl-
2,4-diaminopyrimidines as histamine H4 receptor antagonists.
J Med Chem 2014;57:2429–39.
32.
33.
34.
35.
48.
49.
Ballerini C, Aldinucci A, Luccarini I, et al. Antagonism of histamine
H4 receptor exacerbates clinical and pathological signs of experi-
mental autoimmune encephalomyelitis. Br
J Pharmacol 2013;
Cowden JM, Zhang M, Dunford PJ, Thurmond RL. The hista-
mine H4 receptor mediates inflammation and pruritus in Th2-
170:67–77.
50.
Murata Y, Song M, Kikuchi H, et al. Phase 2a, randomized, double-
blind, placebo-controlled, multicentre, parallel-group study of a
H4R-antagonist (JNJ-39758979) in Japanese adults with moderate
atopic dermatitis. J Dermatol 2015;42:129–39.
Thurmond RL. The histamine H4 receptor: from orphan to the
clinic. Front Pharmacol 2015;6:1–11.
dependent
dermal
inflammation.
J.
Investig
Dermatol
2010;130:1023–33.
36.
37.
Beerman SG, Glade S, Jonigk D, et al. Opposite effects of mepr-
amine on JNJ7777120- induced amelioration of experimentally
induced asthma in mice in sensitization and provocation. PLoS
One 2012;7:e30285.
Somma T, Cinci L, Formicola G, et al. A selective antagonist of his-
tamine H4 receptors prevents antigen induced airway inflamma-
tion and bronchoconstriction in guinea pigs: involvement of
lipocortin-I. Br J Pharmacol 2013;170:200–13.
Thurmond RL, Chen B, Dunford PJ, et al. Clinical and preclinical
characterization of the histamine H4 receptor antagonist JNJ-
39758979. J Pharmacol Exp Ther 2014;667:383–8.
Thurmond RL, Kazerouni K, Chaplan SR, Greenspan AJ, Peripheral
neuronal mechanism of itch: histamine and itch. Itch:
mechanisms and treatment. Boca Raton, FL: CRC Press 2014;Chap.
10:143–92.
Rossbach K, Wendorff S, Sander K, et al. Histamine H4 receptor
antagonism reduces hapten-induced scratching behaviour but not
inflammation. Exp Dermatol 2009;18:57–63.
Seike M, Furuya K, Omura M, et al. Histamine H4 receptor antag-
onist ameliorates chronic allergic contact dermatitis induced by
repeated challenge. Allergy 2010;65:319–26.
Beaumer W, Stahl J, Sander K, et al. Lack of preventing effect of
systemically and topically administered histamine H1 or H4 recep-
tor antagonists in a dog model of acute atopic dermatitis. Exp
Dermatol 2011;20:577–81.
Suwa E, Yamaura K, Oda M, et al. Histamine H4 receptor antagon-
ist reduces dermal inflammation and pruritus in a hapten-induced
experimental model. Eur J Pharmacol 2011;667:383–8.
Matsushita A, Seike M, Okawa H, et al. Advantages of histamine
H4 receptor antagonist usage with H1 receptor antagonist for the
treatment of murine allergic contact dermatitis. Exp Dermatol
2012;21:714–15.
51.
52.
Hartwig C, Munder A, Glage S, et al. The histamine H4 receptor
(H4R) regulates eosinophilic inflammation in ovalbumin-induced
experimental allergic asthma in mice. Eur
J Immunol 2015;
45:1129–40.
53.
Mirzahosseini A, Dalmadi B, Csutora p. Histamine receptor H4 reg-
ulates mast cell degranulation and IgE induced FceRI upregulation
in murine bone marrow-derived mast cells. Cell Immunol
2013;283:38–44.
Engelhardt H, de Esch IJP, Kuhn D, et al. Detailed structure-activity
relationship of indolecarboxamides as H4 receptor ligands. Eur J
Med Chem 2012;54:660–8.
Lim HD, van Rijn RM, Ling P, et al. Evaluation of histamine H1-,
H2-, and H3-receptor ligands at the human histamine H4
receptor: identification of 4-methylhistamine as the first potent
38.
39.
54.
55.
40.
41.
42.
and selective H4 receptor agonist.
J Pharmacol Exp Ther
2005;314:1310–21.
56.
Remko AB, Stefan BJ, Schoonus, et al. Histamine H(1)-receptor acti-
vation of nuclear factor-kappa B: roles for G beta gamma- and G
alpha(q/11)-subunits in constitutive and agonist-mediated signal-
ling. Mol Pharmacol 2001;60:1133–42.
Gutzmer R, Mommert S, Gschwandtner M, et al. The histamine H4
receptor is functionally expressed on T(H)2 cells. J Allergy Clin
Immunol 2009;123:619–25.
Desai P, Thurmond RL. Histamine H4 receptor activation enhances
LPS-induced IL-6 production in mast cells via ERK and PI3K activa-
tion. Eur J Immunol 2011;41:1764–73.
Ahmad SF, Mushtaq AA, Khairy MA, et al. Regulation of TNF-
a and NFj-B activation through the JAK/STAT signalling path-
way downstream of histamine 4 receptor in a rat model of
LPS-induced joint inflammation. Immunobiology 2015;220:
889–98.
57.
58.
59.
43.
44.
45.
46.
Ohsawa Y, Hirasawa N. The antagonism of histamine H1 and H4
receptors ameliorates chronic allergic dermatitis via anti-pruritic
and anti-inflammatory effect in NC/Nga mice. Allergy 2012;67:
1014–22.
Liu H, Altenbach RJ, Carr TL, et al. Cis-4-(piperazine-1-yl)-
5,6,7a,8,9,10,11,11a-octahydrobenzofuro[2,3-h] quinazoline-2-amine
(A-987306), a new H4r antagonist that blocks pain responses
60.
Angel Jemima E, Prema A, Berla Thangam E. H4R activation
utilizes distinct signalling pathways for the production of
RANTES and IL-13 in human mast cells. J Recept Sig Transd
2016. [Epub ahead of print]. doi: 10.1080/10799893.2016.
1203938.