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plemented with 20 l of scintillation cocktail (Rotiscint Eco plus,
Roth, Karlsruhe, Germany) and incubated in the dark under shaking
(200 rpm). The counting was performed with a Micro Beta2 1450
scintillation counter. The data obtained by the [35S]GTP␥S bind-
ing assay were analysed by nonlinear regression and best-fitted
to sigmoidal concentration-response curves. All data are means of
at least three independent experiments SEM, each performed in
triplicate (unless otherwise indicated by a footnote in the respec-
tive table). For data analysis the software Prism 5.01 (GraphPad
each compound, at least twenty concentration response-curves
were measured.
For analysis of the data, the Schild equation (r − 1) = [B]/KB was
used [51]. Therein, r represents the concentration ratio (concentra-
tion of histamine, required to elicit a given response in the presence
of an antagonist divided by the concentration of histamine required
to produce exactly the same response in the absence of an antag-
the equilibrium dissociation constant for the antagonist B with the
receptor. The affinities of all analyzed antagonists, except 7b, were
expressed as pA2 values, determined according to the method of
Arunlakshana and Schild [52], using at least three different antago-
nist concentrations in a range of at least 1 or 2 orders of magnitude.
The slope s and the pA2 value were calculated by fitting the data
points by linear regression using GraphPad Prism 5.01 (GraphPad
Software Inc., San Diego, CA, USA). For compound 7b, which showed
a depression already at antagonist concentrations ≥ 3.16 nM, the
Schild equation was used to estimate KB for single antagonist con-
centrations, where a depression was not observed. The statistical
error was denoted as SEM.
2.8. GTPase assay
The GTPase assay at the hH1R was performed, as described pre-
viously [28,34]. Briefly, Sf9 membranes, coexpressing the hH1R and
RGS4 were thawed and sedimented by centrifugation (13000g at
4 ◦C, 10 min). The resulting membrane pellet was resuspended in
10 mM Tris-HCl (pH 7.4). The reaction mixture in each vessel con-
tained Sf9 cell membranes, coexpressing hH1R and RGS4 (10–20 g
protein/cup), 1.0 mM MgCl2, 100 M adenylyl imidodiphosphate,
100 M ATP, 100 nM GTP, 100 M EDTA, 5 mM creatine phosphate,
40 g creatine kinase, 0.2% (w/v) BSA in 50 mM Tris-HCl (pH 7.4),
and the investigated ligand at various concentrations (concentra-
tion range from 1 nM to 1 mM, as appropriate). All reaction mixtures
(80 l) were incubated at 25 ◦C for 3 min. Afterwards, 20 l of [␥-
32P]GTP (0.1 Ci/cup) were added into each tube. After incubation
at 25 ◦C for 20 min, the reactions were terminated by addition of
900 ml of slurry (5% (w/v) activated charcoal and 50 mM NaH2PO4
(pH 2.0)). Thereafter, the mixtures were centrifuged at 15000g,
room temperature, for 15 min. 600 l of the supernatant fluid were
collected, and 32Pi was determined by Cherenkov counting. Because
the analyzed compounds had been identified as neutral antago-
nists, the GTPase assay was performed in the antagonist mode in
the presence of 200 nM histamine. All data are means of at least
three independent experiments SEM, each performed in tripli-
cate (unless otherwise indicated by a footnote in the respective
table). For data analysis the software Prism 5.01 (GraphPad Soft-
ware Inc., San Diego, CA, USA) was used.
2.10. Molecular modelling
Models of the hH1R and the hH4R in its inactive state were gen-
erated by homology modelling using the crystal structure of the
hH1R with the antagonist doxepine bound (3RZE) [53], as tem-
plate, as described previously using the software SYBYL 7.0 (Tripos
Inc) [17,19]. Briefly, the cocrystallized lysozyme was deleted and
the N-terminus and the E2-loop were completed, using a pre-
viously described protocol [54]. Due to its length, the I3-loops
of both receptors were not modelled. However, to close the gap
between the intracellular parts of TM V and TM VI, the amino
acids of the I3-loop (hH1R: Leu231 to Val404, hH4R: Gly215 to His292
)
were replaced by eight alanines. Internal water molecules were
included into both models, as described [54]. The compounds 7e,
(R)-16e and (S)-16e were manually docked into the binding pocket
of the hH1R and the hH4R using SYBYL 7.0 (Tripos Inc.). The result-
ing ligand-receptor-complexes were minimized and embedded
in a POPC lipid bilayer. For the subsequent molecular dynamic
(MD) studies, the software package GROMACS 4.0.2 (http://www.
gromacs.org) was used. The force field parameters for the lig-
according to Gasteiger-Hückel using SYBYL 7.0. The force field
sodium and chloride ions were included into the simulation boxes
to achieve electroneutrality using the commands genbox and genion
of the software package GROMACS 4.0.2. All simulation boxes con-
tained a number of 115 POPC molecules and about 11000 to 12000
water molecules. The ffG53a6 force field [55] was used for the
hH1R and the hH4R. After minimization of the simulation boxes, the
MD simulations (5 ns equilibration phase, at least 10 ns productive
phase) were performed with GROMACS 4.0.2. Each simulation was
performed four times, using different seed values.
2.9. Histamine H1 receptor assay on the isolated guinea-pig ileum
the gpH1R. For this purpose, assays on the isolated guinea-pig ileum
with cumulative concentration effect curves of histamine in the
presence of an antagonist were performed, as described in detail
previously [48]. Briefly, guinea-pigs of either sex (250 g–500 g)
were stunned by a blow on the neck and exsanguinated by cut-
ting the carotid artery. After removing the ileum, it was cut
into segments of 1.5 cm–2.5 cm length. The ileum segments were
mounted isotonically in 20 ml organ baths, containing aqueous
Tyrode’s solution (NaCl 137 mM, KCl 2.7 mM, CaCl2 1.8 mM, MgCl2
1.0 mM, NaH2PO4 0.4 mM, NaHCO3 11.9 mM, glucose 5.0 mM) [49]
and were connected to a transducer (Type TIT 1100, FMI GmbH,
Seeheim, preload 5 mN), was warmed to a constant temperature
of 37 ◦C and gassed with 95% O2 and 5% CO2. The contraction
of the ileum segments was measured by means of an ampli-
fier (Transducer coupler 4711, FMI GmbH, Seeheim, Germany)
and recorded by a 6-channel-x/t-writer (Kompensograph C 1015,
Siemens, Germany). To block cholinergic muscarinic receptors,
atropine was added to achieve a final concentration of 0.05 M [50].
After 30 min of equilibration, the tissues were prestimulated three
times with histamine (first 1 M, then 10 M) and washed out
for 8 min, followed by a recovery period of 10 min. Subsequently,
cumulative concentration-response curves were registered for his-
tamine in the concentration range of 10 nM to 30 M. After adding
the antagonist and an incubation period of 15 min, cumulative
3. Results and discussion
3.1. Synthesis
All loxapine derived compounds 7a–7i, 8a–f, 9a–c were
prepared according to a synthetic pathway already described