Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 983.36 of the human histamine H4 receptor

Article abstract of DOI:10.1111/bph.12113

Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H₄ receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182^5.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98^3.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98^3.36 by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H₄ receptor research. Experimental Approach We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH₄ receptor radioligand binding, G protein activation and β-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. Key Results VUF14480 was shown to be a partial agonist of hH₄ receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH₄ receptor with submicromolar affinity. Serine substitution of C98^3.36 prevented this covalent interaction. Conclusion and Implications VUF14480 is thought to bind covalently to the hH₄ receptor-C98^3.36 residue and partially induce hH₄ receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH₄ receptor. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013. 170.issue-1 2013 The Authors. British Journal of Pharmacology

Full text of DOI:10.1111/bph.12113

DOI:10.1111/bph.12113
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Themed Issue: Histamine Pharmacology Update
Correspondence
R Leurs, Division of Medicinal
Chemistry, Faculty of Sciences,
Amsterdam Institute for
RESEARCH PAPER
Molecules, Medicines and
Systems (AIMMS), VU University
Amsterdam, De Boelelaan 1083,
1081 HV Amsterdam, The
Netherlands. E-mail: r.leurs@vu.nl
----------------------------------------------------------------
Design and pharmacological
characterization of
*These authors contributed
equally to this work.
VUF14480, a covalent partial
agonist that interacts with
cysteine 983.36 of the human
histamine H4 receptor
----------------------------------------------------------------
Keywords
covalent binder; histamine H4
receptor; GPCR; pyrimidine
----------------------------------------------------------------
Received
31 October 2012
Revised
20 December 2012
Accepted
23 December 2012
S Nijmeijer1*, H Engelhardt1,2*, S Schultes1,2, A C van de Stolpe1,
V Lusink1, C de Graaf1, M Wijtmans1, E E J Haaksma2, I J P de Esch1,
K Stachurski2, H F Vischer1 and R Leurs1
1Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules,
Medicines and Systems (AIMMS), VU University Amsterdam, Amsterdam, The Netherlands, and
2Department of Medicinal Chemistry, Boehringer Ingelheim RCV GmbH & Co KG, Vienna,
Austria
BACKGROUND AND PURPOSE
The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the
2-amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this
receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue
C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning
as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide
a valuable tool for H4 receptor research.
EXPERIMENTAL APPROACH
We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand
binding, G protein activation and b-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder
was assessed both chemically and pharmacologically.
KEY RESULTS
VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and b-arrestin2 recruitment.
VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C983.36 prevented this
covalent interaction.
CONCLUSION AND IMPLICATIONS
VUF14480 is thought to bind covalently to the hH4 receptor-C983.36 residue and partially induce hH4 receptor-mediated G
protein activation and b-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode
of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further
investigate the structure of the active hH4 receptor.
LINKED ARTICLES
This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit
http://dx.doi.org/10.1111/bph.2013.170.issue-1
© 2013 The Authors
British Journal of Pharmacology © 2013 The British Pharmacological Society
British Journal of Pharmacology (2013) 170 89–100 89

S Nijmeijer et al.
BJP
Abbreviations
PEI, polyethylenimine; TM, transmembrane; VUF14480, 4-(4-methylpiperazin-1-yl)-6-phenyl-2-vinylpyrimidine;
VUF14481, 2-methyl-4-(4-methylpiperazin-1-yl)-6-phenylpyrimidine
2,4 diaminopyrimidines, quinoxalines and quinazolines as H4
receptor antagonists (Smits et al., 2008; 2010; Sander et al.,
Introduction
The human (h) histamine H4 receptor belongs to the GPCR
subfamily of four histamine receptors and is considered to be
an important receptor in the regulation of inflammatory
processes (Nijmeijer et al., 2012a). Therapeutic intervention
of this receptor is thought to be beneficial in disorders such
as allergic asthma, pruritus and rheumatoid arthritis (Leurs
et al., 2011). Since the identification of this histamine recep-
tor in 2000, many carboxamides and pyrimidines have been
found to be H4 receptor antagonists (Engelhardt et al., 2009;
Smits et al., 2009; Figure 1A). The indolecarboxamide JNJ
7777120 was one of the first compounds found to be a
selective H4 receptor antagonist as a result of a high through-
put screening campaign followed by lead optimization
(Jablonowski et al., 2003) (Figure 1A). Originally it was
thought that JNJ 7777120 was only an antagonist, as it inhib-
its histamine-induced Gai protein signalling, but, recently,
from its effects on H4 receptor-mediated b-arrestin2 signalling
it has also been shown to be a partial agonist (Rosethorne
and Charlton, 2011; Nijmeijer et al., 2012b). More rational
approaches, such as scaffold-optimization and -hopping or
fragment-based drug design, have led to the development of
2009).
Computer-aided drug discovery has provided valuable
insight into the shape and properties of GPCR ligand-binding
pockets. Three-dimensional models based on the rhodopsin
(Palczewski et al., 2000) or b2-adrenoceptor X-ray structures
(Cherezov et al., 2007), in combination with site-directed
mutagenesis and structure activity relationship data, were
used to identify interaction points in the hH4 receptor that
are crucial for ligand binding (Jongejan et al., 2008; Lim et al.,
2010; Istyastono et al., 2011a,b; Wijtmans et al., 2011;
Schultes et al., 2013b). In 2011, the more closely related
doxepin-bound histamine hH1 receptor crystal structure was
elucidated (Shimamura et al., 2011). On the basis of this
crystal structure-based homology model of the hH1 receptor,
we recently investigated the binding mode of indolecarboxa-
mides and 2-aminopyrimidines in the binding pocket of the
hH4 receptor, (Figure 1A, B) (Schultes et al., 2013a,b). The
proposed binding mode of 2-aminopyrimidines involves a
strong interaction between the 2-amino substituent and
glutamic acid residue E1825.46 in transmembrane (TM) helix 5
of the hH4 receptor (the superscript residue numbers are
A
B
Cl
N
O
Cl
N
5.39
N
L
45.49
N
N
N
E
N
H
HN
O
OH
Compound 10 (pKi 8.4)
3.33
JNJ 7777120 (pKi 8.3)
Y
6.51
N
Y
N
N
N
NH2
5.46
VUF10460 (pKi 8.3)
E
N
N
3.32
D
N
N
3.36
C
N
N
N
N
VUF14480 (pKi 6.3)
VUF14481 (pKi 6.4)
Figure 1
Rational design of a covalent hH4 receptor ligand. (A) Chemical structures of hH4 receptor antagonists. JNJ 7777120, Compound 10 from Janssen
Pharmaceutica (Chavez et al., 2008), 2-amino pyrimidine VUF10460, VUF14480 and VUF14481. (B) Binding mode of VUF10460 in the previously
validated hH4 receptor homology model (Schultes et al., 2013b) based on the hH1 receptor crystal structure template (Shimamura et al., 2011).
The amino group at position 2 of 2-aminopyrimidines interacts with E1825.46 (Schultes et al., 2013b). In addition, substituents at this position are
also in close proximity to C983.36 as indicated by the green dotted line (distance ring carbon to thiol group of C983.36 is 4.2 Å). The backbone of
TM helices 5, 6 and 7 (right to left) are presented as yellow helices, TM3 is presented as a yellow ribbon in the front. Important binding residues
are depicted as ball-and-stick models with grey carbon atoms. Oxygen, nitrogen sulfur and polar hydrogen atoms are coloured red, blue, yellow
and light blue respectively. H-bonds between VUF10460 and hH4 receptors are depicted by black dotted lines.
90 British Journal of Pharmacology (2013) 170 89–100

Characterization of a covalent hH4 receptor partial agonist
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according to the Ballesteros Weinstein numbering; Ballesteros
and Weinstein, 1995). Residues in extracellular loop 2 have
been numbered 45.x, in which 45 refers to the position
between helix 4 and 5 and x refers to a number that indicates
its position relative to the conserved cysteine residue 45.50
(De Graaf et al., 2008). Moreover, the model predicts that
substituents at position 2 of the pyrimidine are in close
proximity (<2 Å) to the cysteine residue C983.36 in TM3
(Figure 1B). In addition, substituents of considerable size can
be present at this position without loss of affinity (Figure 1A,
compound 10). Interestingly, the nucleophilic thiol side
chain of cysteines can form an irreversible bond with
unsaturated carbonyl groups (i.e. Michael acceptor; Ekici
et al., 2004; Tsou et al., 2005; Klutchko et al., 2006; Pan et al.,
2007; Garuti et al., 2011). Hence, we hypothesized that the
introduction of an ethenyl at position 2 of the pyrimidine
ring might result in a compound that can covalently interact
with C983.36. Covalent binders are considered useful as tool
compounds, for example, when studying protein structure.
This would be particularly useful in the current era, where
numerous GPCR crystal structures are being elucidated, as the
need for a ligand that has a long residence time might be
beneficial for co-crystallization with its receptor (Rosenbaum
et al., 2011). Moreover, a covalent interaction in the binding
pocket can unequivocally confirm a proposed ligand-binding
model.
consisted of 25 cycles of 95°C-30 s, 60°C-30 s, 72°C-60 s. The
two PCR products were isolated from agarose gel. The PCR
products from both mutagenesis reactions were fused in a
self-primed PCR and subsequently amplified using flanking
primers PCR programme: 25 cycles of 95°C-30 s, 55°C-30 s,
72°C-90 s. Fused PCR products were isolated from agarose gel,
digested with KpnI and XbaI and inserted into the expression
vector pcDEF3. The sequences of the C983.36S DNA obtained
were verified.
Construction of hH4 receptor-Rluc8 was as described pre-
viously (Nijmeijer et al., 2010). The b-arrestin2-mVenus con-
struct was created using the same procedure. DNA plasmid
encoding the mVenus protein was a kind gift of Dr J. A.
Javitch (Columbia University, New York, NY, USA) (Guo et al.,
2008).
Cell culture, transfection and
membrane preparation
HEK293T cells were cultured in DMEM supplemented with
10% FBS, 50 IU·mL-1 penicillin, and 50 mg·mL-1 streptomycin
at 37°C and 5% CO2. Two million cells were seeded per 10 cm
dish one day prior to transfection. Approximately 4E6 cells
were transfected with 5 mg of cDNA using the polyethylen-
imine (PEI) method. Briefly, for binding experiments, 2.5 mg
hH4R(-C983.36S) cDNA was supplemented with empty pcDEF3
plasmid to produce a total of 5 mg cDNA; for BRET-based
b-arrestin2 recruitment, 1 mg hH4 receptor(-C983.36S)-Rluc8
and 4 mg b-arrestin2-mVenus were mixed with 20 mg of
25 kDa linear PEI in 500 mL of 150 mM NaCl. This transfec-
tion mixture was incubated at 22°C for 10–30 min. Mean-
while, the medium in the 10 cm dish was replaced with 6 mL
of fresh culture medium and transfection mix was then added
dropwise to the cells. Two days after transfection, transfected
cells were washed once with PBS and subsequently scraped
from their culture dish in 1 mL of PBS. Crude membrane
pellets were collected by centrifugation at ~2000¥ g for
10 min at 4°C and stored at -20°C until further use.
In this study, we describe the rational design of the pyri-
midine derivate VUF14480 (Figure 1A) that covalently binds
and partially activates the hH4 receptor. The pyrimidine com-
pound contains a Michael acceptor that is in close proximity
to C983.36, which was identified as an anchor point for a
covalent interaction.
Methods
Materials
Cell culture media were bought from PAA (Pasching, Austria),
[3H]-histamine (10.6–15.3 Ci·mmol-1) and [35S]-GTPgS (1250
Ci·mmol-1) were purchased from Perkin Elmer (Groningen,
The Netherlands). Chemicals and reagents were obtained
from commercial suppliers and were used without further
purification. L-glutathione reduced was bought from Sigma-
Aldrich (Saint Louis, MO, USA) and L-cysteine ethyl ester
hydrochloride was purchased from Janssen Chimica (Beerse,
Belgium). The synthesis of VUF14480 and VUF14481 is
described in the Supporting Information.
S-alkylation of glutathione or cysteine ethyl
ester by VUF14480 or VUF14481
VUF14480 and VUF14481 were mixed with glutathione or
cysteine ethyl ester in a 1:1 molar ratio and incubated over-
night at 22°C. The incubated mixtures were subsequently
separated and analysed by LCMS.
[3H]-histamine displacement binding
Crude membrane pellet was dissolved in 50 mM Tris-HCl
(pH 7.4 at 22°C) and incubated with increasing concentra-
tions (10 pM–100 mM) of the indicated compounds and
[3H]-histamine (~10 nM) for 1.5 h with crude membrane sus-
pensions. The reaction was terminated by filtration through a
PEI (0.5%) soaked GF/C plate (Perkin Elmer), followed by
three washes with ice-cold 50 mM Tris-HCl (pH 7.4 at 4°C).
Microscint O scintillation fluid was added and radioactivity
was counted in a Wallac Microbeta counter (Perkin Elmer).
Site-directed mutagenesis of C983.36 and
construction of hH4 receptor-Rluc8 and
b-arrestin2-mVenus
The C983.36S was constructed via PCR-mediated mutagenesis.
Wild-type (WT)-hH4 receptors (pcDEF3) were amplified in the
presence of 0.1 mM dNTPs and 10 U Pfu polymerase and
10 pM forward plasmid primer (5′-CATTCTCAAgCCTCAgA
CAgTgg-3′) in combination with 10 pM reverse mutagenesis
primer (5′-CAgATgCTgTAgACAACAgATAg-3′), and 0.5 mM
forward mutagenesis primer (5′-CTATCTgTTgTCTACAgCAT
CTg-3′) in combination with 0.5 mM reverse plasmid primer
(5-gAgCTCTAgCATTTAggTgACAC-3′). The PCR programme
Membrane preparation for
[35S]-GTPgS binding
Two days after transfection, cells were washed with 2 mL PBS
and harvested in 2.5 mL membrane buffer (15 mM Tris,
British Journal of Pharmacology (2013) 170 89–100 91

S Nijmeijer et al.
BJP
1 mM EGTA, 0.3 mM EDTA and 2 mM MgCl2; pH 7.4 at 4°C).
The cell homogenate was sonicated for 20 s and centrifuged
for 30 min, 2000¥ g at 4°C. The supernatant was aspirated
and the membrane pellet was resuspended in 250 mL Tris-
sucrose (20 mM Tris and 250 mM sucrose, pH 7.4 at 4°C) per
dish. Membranes were stored at -80°C until further use.
group was removed and rebuilt using the MOE version
2011.10 (MOE, 2011). The models were subjected to energy
minimization using the MMFF94x force field with fixed posi-
tion of the backbone atoms of the protein.
Results
[35S]-GTPgS-binding assay
Membranes (10 mg per well) were incubated in GTPgS assay
buffer (50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4
at 22°C, supplemented with 0.2 mg·mL-1 saponin, 3 mM GDP
and 0.5 nM [35S]-GTPgS) with increasing amount (1 nM–
10 mM) of the indicated compounds for 1 h at 22°C. [35S]-
GTPgS binding was terminated by rapid filtration through
unifilter GF/B plates (Perkin Elmer). Filter plates were washed
three times with ice-cold washing buffer (50 mM Tris-HCl
and 5 mM MgCl2, pH 7.4 at 4°C). Microscint O scintillation
fluid (Perkin Elmer) was added and the radioactivity was
counted in a Wallac Microbeta counter.
Synthesis VUF14480 and VUF14481
VUF14480 was designed and synthesized based on the
previously suggested binding mode of 2-aminopyrimidines
(Figure 1B) and the hypothesis that an ethenyl substituent at
position 2 of the pyrimidine scaffold can act as a Michael
acceptor (Liu et al., 1999; Karagiorgou et al., 2005). Due to
the electron-deficient heteroaromatic pyrimidine ring, the
ethane residue is polarized so that the nucleophilic thiol
side chain of cysteine can react at the b-carbon of the
alkene. Furthermore, based on the binding model of
2-aminopyrimidines (Figure 1B), the thiol from C983.36
displays
a conformation similar to the proposed 2-
BRET-based b-arrestin2 recruitment
ethenylpyrimidine, which is favourable for the Michael-type
addition reaction (Tsou et al., 2005). As a control, we synthe-
sized VUF14481 with a methyl substituent at the 2-position
and a similar proposed binding mode, but without the option
to function as a Michael acceptor (Figure 1A, Supporting
Information).
One day post-transfection, cells were seeded in a poly-L-
lysine-coated, white bottom, 96 well plate. Next day,
the medium was aspirated and replaced with PBS.
Coelenterazine-h (5 mM final concentration) and indicated
ligands (10 mM final) were added to the cells and incubated
for 20 min at 37°C. Subsequently, BRET (em. 535 nm) and
Rluc8 (em. 460 nm) were measured on a Victor3 (Perkin
Elmer). BRET ratios (BRET divided by Rluc8 emission signal)
were corrected for BRET ratios measured in cells that only
express hH4 receptor (C983.36S)-Rluc8.
Covalent binding of VUF14480 and
VUF14481 to glutathione or cysteine
ethyl ester
VUF14480 and VUF14481 were first tested for their ability to
react with either glutathione or the cysteine ethyl ester. To
this end, both compounds were mixed with either glutath-
ione or cysteine ethyl ester in a 1:1 molar ratio and incubated
overnight at 22°C. The incubated mixtures were subsequently
separated and analysed by LCMS (Figure 2). Incubation of
VUF14480 with glutathione resulted in a reaction product
(588 g·mol-1), which corresponded to the sum of the masses
of VUF14480 (281 g·mol-1) and glutathione (307 g·mol-1). In
addition, we observed a peak with mass 295 g·mol-1 that
could be explained by a reaction product that is double pro-
tonated (i.e. the m/z ratio consequently resulted in m+2/2)..
The reaction between VUF14480 and the cysteine ethyl ester
yielded an expected mass peak of 430 g·mol-1 as well as
Pre-incubation experiments prior to
heterologous displacement binding
Crude membrane fractions were pre-incubated for 1 h at 22°C
on a roller bench with either 50 mM Tris-HCl or increasing
amounts (100 nM–10 mM) of VUF14480, VUF14481 or JNJ
7777120. Next, treated membrane fractions were washed
three times with 1 mL 50 mM Tris-HCl (pH 7.4). Washing
includes incubation for 10 min at 22°C on a roller bench and
every 2 min a 30 s vortexing step. Finally, membranes were
dissolved in 50 mM Tris-HCl. Aliquots, 50 mL, of the pre-
treated membranes were subsequently used in the previously
described [3H]-histamine binding studies.
216 g·mol-1 that corresponded to
a double protonated
Data analysis and statistical procedures
product. Moreover, we observed a small peak of 281 g·mol-1,
which equals the mass of compound VUF14480. In contrast,
the control compound VUF14481 did not react with either
glutathione or the cysteine ethyl ester in a covalent manner
and only the mass of the initial starting compounds was
detected (Figure 2, Table 1).
All data were analysed with GraphPad Prism 5 software
(GraphPad Software Inc., La Jolla, CA, USA). Homologous and
heterologous displacement-binding data were fitted to a one-
site competition model. Functional concentration response
curves were fitted to
a three-parameter concentration
response model. Statistical analyses were performed using
Student’s unpaired t-test (****P < 0.0001; ***P < 0.001; **P <
0.01; *P < 0.05).
Determination of the affinity of compounds
VUF14480 and VUF14481 for the
Construction of three-dimensional binding
mode models for VUF14481 and VUF14480
in the hH4 receptor-binding pocket
hH4 receptor
To determine their binding affinity for the hH4 receptor, both
compounds were tested in a heterologous [3H]-histamine dis-
placement experiment (n = 3). VUF14480 and VUF14481 had
comparable binding affinities for the hH4 receptor in the low
The binding mode of 2-aminopyrimidines was used as an
initial binding model (Schultes et al., 2013b). The 2-amino
92 British Journal of Pharmacology (2013) 170 89–100

Characterization of a covalent hH4 receptor partial agonist
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N
SH
O
GSH adduct
DMSO
O
O
O
N
H
N
+
HO
N
OH
N
N
H
NH2
N
N
O
Glutathione + VUF14480
N
N
S
100
O
O
H
N
HO
N
H
OH
NH2
O
m/z
N
SH
GSH
VUF14481
O
O
O
N
H
N
+
HO
N
OH
N
N
H
NH2
O
Glutathione + VUF14481
N
100
N
N
N
m/z
100
SH
O
O
O
H
N
HO
N
H
OH
NH2
O
m/z
Figure 2
LCMS analysis of glutathione addition to VUF14480 and VUF14481. VUF14480 (top panel) or VUF14481 (bottom panel) were mixed with
glutathione in a 1:1 molar ratio and incubated overnight at 22°C. The incubated mixtures were subsequently separated and analysed by LCMS.
(Top panel) Reaction product (588 g·mol-1; m+1) was formed that corresponded to the mass of VUF14480 (280 g·mol-1) and glutathione
(307 g·mol-1). Peak with mass 295 g·mol-1 (m+2/2) corresponded to a double protonated product. (Bottom panel) VUF14481 (269 g·mol-1; m+1)
and glutathione (307 g·mol-1; GSSG m+2/2) could only be detected separately and no product was formed.
British Journal of Pharmacology (2013) 170 89–100 93

S Nijmeijer et al.
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Table 1
LCMS analysis of glutathione or cysteine ethyl ester addition reaction to VUF14480 and VUF14481
Compound
Retention time (min)
Observed mass (g·mol-1
)
Expected mass (g·mol-1
)
VUF14480
2.57–2.68
1.56–1.77
281
269
281
VUF14481
269
Glutathione
307
Cysteine ethyl ester
VUF14480 + glutathione
VUF14481 + glutathione
VUF14480 + cysteine ethyl ester
VUF14481 + cysteine ethyl ester
149
1.98–2.12
588/295
269/307
430/216/281
269
588
1.51–1.72/1.11–1.24
2.24–2.40
269/307
430
1.53–1.76
269/149
VUF14480 and VUF14481 were incubated in a 1:1 molar ratio to glutathione or cysteine ethyl ester. Reaction products were analysed by
LCMS.
A
B
125
100
75
50
25
0
125
Histamine
VUF14480
VUF14481
****
100
75
50
25
0
****
****
-9
-8
-7
-6
-5
HA
VUF
14480
VUF
14481
Log [compound] (M)
Figure 3
Functional characterization of VUF14480 and VUF14481. (A) GTPgS-binding assay. Membranes (10 mg per well) from cells expressing hH4
receptors were incubated with increasing amounts (1 nM–10 mM) of histamine, VUF14480 or VUF14481 for 1 h at 22°C. (B) BRET-based
b-arrestin2 recruitment assay. Cells expressing the hH4 receptor-Rluc8 and b-arrestin2-mVenus were incubated with 10 mM histamine (HA),
VUF14480 or VUF14481 in absence (open columns) and presence (hatched columns) of thioperamide (10 mM) for 20 min at 37°C. Results shown
are from at least three pooled experiments performed in triplicate. Data were normalized to the histamine response (100%) and fitted to a
three-parameter response model. Error bars indicate SEM values.
micromolar range (pKi = 6.3 Ϯ 0.1 and pKi = 6.5 Ϯ 0.1,
respectively; Table 2, Figure 1A). Importantly, it should be
noted that the pKi value of the possible covalent binder
VUF14480 is not a true pKi value due to the absence of
equilibrium binding between both competing ligands and
receptor.
receptor agonist (Emax = 49 Ϯ 5%) with a potency (pEC50 =
6.3 Ϯ 0.2) that corresponded to its affinity (pKi = 6.4 Ϯ
0.1; Figure 3A, Table 2). Moreover, both VUF14481 and
VUF14480 induced b-arrestin2 recruitment to the hH4
receptor, as evaluated in a BRET-based assay (n = 3) that
could be inhibited by pre-incubation with the H3/H4
receptor antagonist thioperamide (Figure 3B). Interestingly,
b-arrestin2 recruitment upon VUF14480 stimulation could
not be completely blocked by thioperamide; this might be
due to a possible covalent, non-displaceable interaction of
VUF14480 with the hH4 receptor.
Functional characterization of VUF14480
and VUF14481
Functional characterization of these compounds in a GTPgS-
binding assay on hH4 receptor-expressing membranes (n = 3)
showed that VUF14480 is a partial agonist (Emax = 60 Ϯ 8%) in
comparison to histamine (Emax = 100 Ϯ 4%, pEC50 = 7.5 Ϯ 0.2).
The potency of compound VUF14480 (pEC50 = 6.0 Ϯ 0.2) was
comparable to its affinity (pKi = 6.3 Ϯ 0.1; Figure 3A, Table 2).
In addition, VUF14481 was also identified as a partial hH4
Investigating the covalent interaction of
compound VUF14480 with hH4 receptors
Identification of both compounds as partial hH4 receptor
agonists prompted us to investigate the irreversible charac-
94 British Journal of Pharmacology (2013) 170 89–100

Characterization of a covalent hH4 receptor partial agonist
BJP
teristics of compound VUF14480 by determining its ability to
irreversibly block [3H]-histamine hH4 receptor-binding sites.
To this end, crude membrane fractions of HEK293T cells,
expressing the hH4 receptor, were pre-incubated for 1 h with
increasing concentrations (100 nM–10 mM) of VUF14480,
VUF14481 or the selective H4 receptor ligand JNJ 7777120.
Pretreated membrane fractions were then washed very exten-
sively to ensure dissociation of compounds that were not
covalently bound. Pretreatment with VUF14480 resulted
in a concentration-dependent decrease (5–50%) in specific
[3H]-histamine binding. However, the affinity of histamine
for the hH4 receptor was not affected by VUF14480 pretreat-
ment (Figure 4A). In contrast, membrane fractions pretreated
with VUF14481 (Figure 4B) or JNJ 7777120 (Figure 4C)
showed no decrease in specific [3H]-histamine binding, ensur-
ing the efficiency of the washing procedure, even for JNJ
7777120, which has a long residence time.
(pEC50 = 6.2 Ϯ 0.2) and VUF14480 (pEC50 = 6.2 Ϯ 0.3) to induce
G protein activity were comparable to their respective affini-
ties (pKi = 6.8 Ϯ 0.2 and pKi = 5.8 Ϯ 0.1) for this mutant.
Although the possible covalent interaction between C983.36
and VUF14480 did not appear to contribute to binding
VUF14480
A
125
N
N
100
N
N
75
50
25
0
0 µM
0.1 µM
1 µM
C983.36 as an anchor point for the
covalent bond
10 µM
Although the above-mentioned binding experiment indicates
VUF14480 has a covalent interaction of with hH4 receptors, it
does not confirm the hypothesized interaction with the
C983.36 residue. We therefore mutated the C983.36 residue into
a serine residue. [3H]-histamine bound with a ~4-fold lower
-11 -10 -9 -8 -7 -6 -5 -4
Log [histamine] (M)
affinity to HEK293T membranes expressing hH4-C983.36
S
VUF14481
B
receptor as compared to hH4-WT receptors (i.e. Kd = 27 Ϯ 6 nM
and Kd = 7 Ϯ 1 nM, respectively) as measured in equilibrium
saturation-binding experiments (n = 3, data not shown).
125
100
75
50
25
0
N
N
Protein expression levels of hH4-C983.36
S receptors and
N
N
hH4-WT receptors were comparable, with Bmax values of 1.3 Ϯ
0.2 and 1.3 Ϯ 0.1 pmol·mg-1 protein respectively. Affinities
of VUF14481 and VUF14480 for the hH4-C983.36S receptor
mutant were determined in a heterologous [3H]-histamine
displacement binding experiment. VUF14481 had an affinity
(pKi 6.8 Ϯ 0.2) for hH4-C983.36S receptors, which was not
significantly higher (P > 0.05) than that observed for the
hH4-WT receptors (pKi = 6.4 Ϯ 0.1). In contrast, VUF14480 had
an affinity (pKi 5.8 Ϯ 0.1) for hH4-C983.36S receptors, which
was slightly lower, but not significantly (P > 0.05) different
from its affinity for hH4-WT receptors (pKi 6.3 Ϯ 0.1). Efficacy
(Emax) values for VUF14481 did not differ significantly (P >
0.05) for hH4-C983.36S receptors (46 Ϯ 7%) and hH4-WT recep-
tors (49 Ϯ 5%; Figures 3A and 5A). In contrast, a significant
(P < 0.01) decrease in Emax was observed for the effects of
VUF14480 on hH4-C983.36S receptors (32 Ϯ 5%) compared to
hH4-WT receptors (59 Ϯ 8%). Potency values of VUF14481
0 µM
0.1 µM
1 µM
10 µM
-11 -10 -9 -8 -7 -6 -5 -4
Log [histamine] (M)
JNJ 7777120
C
Cl
125
100
75
50
25
0
N
N
᭤
N
H
O
Figure 4
[3H]-histamine binding to hH4 receptor-expressing HEK293T cell
membranes pre-incubated with compounds VUF14481, VUF14480
or JNJ7777120. Crude membrane fractions of hH4 receptor-
transfected cells were pre-incubated with increasing concentrations
of VUF14480, VUF14481 or JNJ7777120, at 22°C for 1 h. Pretreated
membranes fractions were washed at least three times very exten-
sively with 50 mM Tris-HCl before further incubation at 22°C for 1 h
with ~10 nM [3H]-histamine and increasing amounts (10 pM–
10 mM) of non-labelled histamine. Curves are fitted to a one-site
competition-binding model. Data shown are pooled data from three
experiments performed in triplicate, error bars indicate SEM values.
0 µM
0.1 µM
1 µM
10 µM
-11 -10 -9 -8 -7 -6 -5 -4
Log [histamine] (M)
British Journal of Pharmacology (2013) 170 89–100 95

S Nijmeijer et al.
BJP
A
B
125
100
75
50
25
0
125
Histamine
VUF14480
VUF14481
****
100
75
50
25
0
*
-9
-8
-7
-6
-5
HA
VUF
14480
VUF
14481
Log [compound] (M)
Figure 5
Functional characterization of VUF14480 and VUF14481 on hH4-C983.36S receptor. (A) GTPgS-binding assay. Membranes (10 mg per well) from
cells expressing hH4 receptor-C983.36S were incubated with increasing amounts (1 nM–10 mM) of histamine, VUF14480 or VUF14481 for 1 h at
22°C. (B) BRET-based b-arrestin2 recruitment assay. Cells expressing the hH4 receptor-C983.36S-Rluc8 and b-arrestin2-mVenus were incubated with
10 mM histamine (HA), VUF14480 or VUF14481 in the absence (open columns) and presence (hatched columns) of thioperamide (10 mM) for
20 min at 37°C. Results shown are from at least three pooled experiments performed in triplicate. Data were normalized to the histamine response
(100%) and fitted to a three-parameter response model. Error bars indicate SEM values.
affinity to the receptor and consequently potency of the
compound, this bond seemed to stabilize an active hH4 recep-
tor conformation. Histamine showed a decreased potency
(pEC50 = 6.4 Ϯ 0.2) compared to its affinity (pKi = 7.2 Ϯ 0.1) for
the hH4-C983.36S receptor (Figure 5A). In contrast, only a
minor decrease in potency (pEC50 = 7.5 Ϯ 0.2) was observed for
histamine at the hH4-WT receptor (pKi = 8.1 Ϯ 0.1). A BRET-
based b-arrestin2 recruitment assay (n = 3) revealed that
histamine, VUF14481 and VUF14480 all induced b-arrestin2
recruitment via the hH4-C983.36S receptor (Figure 5B), which
was less pronounced for VUF14480 and VUF14481 than that
observed via the hH4-WT receptor. Interestingly, complete
inhibition of VUF14480-induced b-arrestin2 recruitment via
hH4-C983.36S receptors by the H3/H4 receptor antagonist thi-
operamide (Figure 3B) but not that induced via the hH4-WT
receptor (Figure 5B), confirmed the hypothesized covalent
tion 2 of the pyrimidine scaffold to C983.36 resulted in the
formation of a covalent bond between VUF14480 and C983.36
(Figure 7B).
Discussion and conclusion
The interaction between a ligand and GPCRs is in general
reversible. However, covalent ligand–GPCR interactions are
also observed. For example, retinal binds covalently to rho-
dopsin via a protonated Schiff base linkage between its alde-
hyde group and the amino group of K2967.43 (Palczewski et al.,
2000), whereas b-funaltrexamine binds covalently to the rat m
opioid receptor (Chen et al., 1995). The use of these com-
pounds as valuable tools for structural research and for iden-
tification of accessible amino acids in ligand-binding pockets
is widely acknowledged (Javitch et al., 1999; Buck et al., 2005;
Potashman and Duggan, 2009; Chapman et al., 2010;
Manglik et al., 2012). In this study, we use the covalent inter-
action to confirm a hypothesized ligand position in the
binding pocket.
A detailed binding mode of 2-aminopyrimidines in the
hH4 receptor-binding pocket has recently been postulated,
suggesting a strong interaction between the amino group
at position 2 of the pyrimidine and hH4 receptor-E1825.46
(Schultes et al., 2013b). Based on this binding mode, we
anticipated that substituents at position 2 of the pyrimidine
scaffold are in close proximity to hH4 receptor-C983.36. We
therefore proposed that the nucleophilic thiol group of this
cysteine is able to form a covalent interaction with an ethenyl
group at position 2 of the pyrimidine scaffold of VUF14480,
which functions as a Michael acceptor. Indeed, from the
results of the LCMS analysis, VUF14480 is able to form a
covalent adduct with glutathione or cysteine ethyl ester. In
accord with this, the related structural control VUF14481 did
not react with glutathione or the cysteine ethyl ester.
interaction between VUF14480 and C983.36
.
Next, the hH4-C983.36S receptor mutant was tested in a
similar pre-incubation binding experiment as conducted
for the hH4-WT receptor to test if VUF14480 was able to
decrease total [3H]-histamine binding. Pre-incubation of
membrane fractions expressing hH4-C983.36S receptors with
either VUF14480 (Figure 6A) or VUF14481 (Figure 6B) fol-
lowed by extensive washing to remove non-covalently bound
ligands, did not decrease the specific [3H]-histamine binding.
Binding models for VUF14481 and
VUF14480 in the hH4 receptor-binding pocket
Binding models for VUF14481 and VUF14480 were
constructed based on our previously published 2-
aminopyrimidine-binding position in hH4 receptors (Schultes
et al., 2013b) and in combination with the newly identified
covalent interaction of VUF14480 with C983.36. Whereas
VUF14481 binds to the hH4R in a non-covalent manner
(Figure 7A), the close proximity of the ethenyl group at posi-
96 British Journal of Pharmacology (2013) 170 89–100

Characterization of a covalent hH4 receptor partial agonist
BJP
VUF14480
A
125
N
N
100
N
N
75
50
25
0
0 µM
0.1 µM
1 µM
10 µM
-11 -10 -9 -8 -7 -6 -5 -4
Log [histamine] (M)
VUF14481
B
125
N
N
100
75
50
25
0
N
N
0 µM
0.1 µM
1 µM
10 µM
-11 -10 -9 -8 -7 -6 -5 -4
Log [histamine] (M)
Figure 6
[3H]-histamine binding to hH4-C983.36S receptor expressing HEK293T
cell membranes pre-incubated with VUF14480 or VUF14481. Crude
membrane fractions of hH4-C983.36S receptor transfected cells were
pre-incubated with increasing concentrations of VUF14480 or
VUF14481 at 22°C for 1 h. Pretreated membranes were subsequently
washed extensively and at least three times with 50 mM Tris-HCl
before further incubation at 22°C for 1 h with ~10 nM [3H]-histamine
and increasing amounts (10 pM–10 mM) of non-labelled histamine.
Curves are fitted to a one-site competition-binding model. Data
shown are pooled data from three experiments performed in tripli-
cate, error bars indicate SEM values.
Both VUF14480 and VUF14481 were found to bind to the
hH4 receptor with submicromolar affinities, which is a 100-
fold less than that found previously for 2-aminopyrimidines
(Schultes et al., 2013b). This affinity decrease can be
explained by the loss of the interaction between a protonated
2-amino group and hH4 receptor-E5.46. In fact, a similar reduc-
tion (10- to 100-fold) in affinity for 2-aminopyrimidines was
observed when E1825.46 was mutated into a non-charged
glutamine residue (Schultes et al., 2013b).
In contrast to previously described 2-aminopyrimidines
antagonists (Engelhardt et al., 2009), VUF14481 and
VUF14480 partially induced hH4 receptor-mediated G protein
activation. The efficacy of VUF14481 and VUF14480 was not
British Journal of Pharmacology (2013) 170 89–100 97

S Nijmeijer et al.
BJP
A
B
Figure 7
Proposed binding modes of VUF14481 (A, green) and VUF14480 (B, magenta) in previously validated hH4 receptor homology model (Schultes
et al., 2013b). Rendering and colour coding is the same as in Figure 1.
only limited to G protein signalling but also extended to the
recruitment of b-arrestin2 to hH4 receptors. It was very inter-
esting to observe that only a small replacement of the
2-amino group by a methyl or ethenyl group changed these
pyrimidines from being an antagonist (data not shown) into
a partial agonist. Interestingly, S1113.36 was previously identi-
fied as an important link between histamine binding and
conformational changes in helices 6 and 7 of the histamine
H1 receptor (Jongejan et al., 2005). Based on in silico-guided
mutagenesis studies, S1113.36 was proposed to act as a rotamer
toggle switch that, upon agonist binding, initiates the acti-
vation of the receptor through N1987.45 (Jongejan et al.,
2005).
adenosine receptor antagonist FSCPX has been shown to
decrease the binding sites in its receptor in a concentration-
dependent manner (Lorenzen et al., 2002).
Based on our 2-aminopyrimidine binding mode of the
hH4 receptor, we hypothesized that VUF14480 covalently
interacts with C983.36, located in the third transmembrane
helix (TM3) of the hH4 receptor-binding site. However, from
the pre-incubation experiments, we could not exclude a pos-
sible role of other putative surface-accessible cysteine residues
in the hH4 receptor. In the hH4 receptor, three cysteine resi-
dues are located in the TM helices (i.e. C873.25, C983.36 and
C3156.47) and one is located in the extracellular loop (EL)2
(C16445.50). C873.25 is thought to form a disulphide bridge with
the cysteine residue in EL2, which leaves C3156.47 and C983.36
as the two possible interaction sites. Interestingly, C3156.47
has been demonstrated to be the site for the covalent bond
between human cannabinoid receptor CB2 and the ligand
AM841 (Mercier et al., 2010; Szymanski et al., 2011).
However, this residue is located outside the proposed binding
pocket of the hH4 receptor and might only be accessible to
ligands that can enter the GPCR via the lipid bilayer, as has
been proposed for AM841 (Hurst et al., 2010). Serine substi-
tution of the hH4 receptor-C983.36 prevented the decrease in
[3H]-histamine-binding sites induced by pre-incubation of
VUF14480 that was observed with WT hH4 receptors, con-
firming the role of this residue in the formation of the cova-
lent bond. It is noted that in the dopamine D2 receptor,
C1183.36 was identified as a solvent accessible residue pointing
into the binding cavity by using methanethiosulfonate rea-
gents (Javitch et al., 1994). Moreover, C3.36 in a2A-, a2B- and
Functional experiments are the preferred method for
identifying covalent antagonists and even weak partial
agonists.
A covalent antagonist will not only shift a
concentration-response curve (depending on receptor
reserve) to the right, but more importantly will (eventually)
decrease the maximal agonist-induced response by reducing
the number of available receptors (Kenakin et al., 2006). Due
to the agonist effects of VUF14480, we tested the covalent
character of this compound by showing that it had the ability
to permanently decrease the number of available histamine-
binding sites. Binding experiments are generally not the pre-
ferred method for proving covalent binding of a compound
to a GPCR, as it is difficult to distinguish irreversible from
pseudo-irreversible interactions (Kenakin et al., 2006). To
make sure we were not looking at pseudo-irreversible intere-
actions caused by slow dissociation rates, we also tested the
effects of another hH4 receptor ligand, JNJ 7777120, in our
experiments. JNJ 7777120 has been reported to have a rela-
tively slow dissociation rate compared to other hH4 receptor
compounds tested (Smits et al., 2012). Extensive washing of
membranes that had been pre-incubated with JNJ 7777120
resulted in a full recovery of available histamine-binding
sites. This proves that extensive washing will result in full
dissociation of a compound with a relatively long hH4
receptor residence time. Interestingly, VUF14480, but not
VUF14481, concentration-dependently reduced specific [3H]-
histamine binding, indicating that VUF14480 irreversibly
occupies the hH4 receptor-binding pocket. Similarly, the A1
a
2C-adrenoceptors was shown to covalently interact with the
reactive aziridinium derivative of phenoxybenzamine (Frang
et al., 2001). Collectively, these data provide substantial evi-
dence that this conserved cysteine residue, present in 24 out
of 42 bioamine receptors and 30 out of 369 non-olfactory
human GPCRs (Surgand et al., 2006), is accessible for ligands
in these amine GPCR families.
In conclusion, the rational design of a covalent hH4 recep-
tor ligand has successfully led to the synthesis and pharma-
cological identification of VUF14480. This compound is
thought to bind covalently to the hH4 receptor-C983.36 residue
98 British Journal of Pharmacology (2013) 170 89–100

Characterization of a covalent hH4 receptor partial agonist
BJP
Frang H, Cockcroft V, Karskela T, Scheinin M, Marjamaki A (2001).
Phenoxybenzamine binding reveals the helical orientation of the
third transmembrane domain of adrenergic receptors. J Biol Chem
276: 31279–31284.
and partially induce hH4 receptor-mediated
G protein
activation and b-arrestin2 recruitment. Moreover, our obser-
vations confirm the previously proposed binding mode of
2-aminopyrimidines (Schultes et al., 2013b). The possible
covalent partial hH4 receptor agonist VUF14480 will be a
useful tool to stabilize the receptor into an active confirma-
tion and further investigate the structure of the active hH4
receptor, as recently shown for the agonist FAUC50 that cova-
lently binds to b2-adrenoceptor H2.64C and facilitates GPCR
crystallization (Rosenbaum et al., 2011).
Garuti L, Roberti M, Bottegoni G (2011). Irreversible protein kinase
inhibitors. Curr Med Chem 18: 2981–2994.
de Graaf C, Foata N, Engkvist O, Rognan D (2008). Molecular
modeling of the second extracellular loop of G protein-coupled
receptors and its implication on structure-based virtual screening.
Proteins 71: 599–620.
Guo W, Urizar E, Kralikova M, Mobarec JC, Shi L, Filizola M et al.
(2008). Dopamine D2 receptors form higher order oligomers at
physiological expression levels. EMBO J 27: 2293–2304.
Acknowledgements
Hurst DP, Grossfield A, Lynch DL, Feller S, Romo TD, Gawrisch K
et al. (2010). A lipid pathway for ligand binding is necessary for a
cannabinoid G protein-coupled receptor. J Biol Chem 285:
17954–17964.
Herman Lim and Kamar Azijli are acknowledged for making
the hH4-C983.36S receptor mutant. Hans Custers is acknowl-
edged for his help with the LCMS experiments. S. N., H. V., C.
dG. and R. L. participate in the European COST Action
BM0806. C. dG. is sponsored by The Netherlands Organiza-
tion for Scientific Research NWO: VENI Grant 700.59.408.
Istyastono EP, de Graaf C, de Esch IJ, Leurs R (2011a). Molecular
determinants of selective agonist and antagonist binding to the
histamine H receptor. Curr Top Med Chem 11: 661–679.
Istyastono EP, Nijmeijer S, Lim HD, van de Stolpe A, Roumen L,
Kooistra AJ et al. (2011b). Molecular determinants of ligand binding
modes in the histamine H(4) receptor: linking ligand-based
three-dimensional quantitative structure-activity relationship
(3D-QSAR) models to in silico guided receptor mutagenesis studies.
J Med Chem 54: 8136–8147.
Conflict of interest
The authors state no conflict of interest.
Jablonowski JA, Grice CA, Chai W, Dvorak CA, Venable JD,
Kwok AK et al. (2003). The first potent and selective non-imidazole
human histamine H4 receptor antagonists. J Med Chem 46:
3957–3960.
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Additional Supporting Information may be found in the
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Table S1 Chromatography methods.
100 British Journal of Pharmacology (2013) 170 89–100