Fluorescent H2Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications

Article abstract of DOI:10.1021/acsmedchemlett.0c00033

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH2R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH2R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pKi: 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pKi: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pKd values comparable to the pKi values. The fluorescent probes 21, 23, and 25 could be used to localize H2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

Full text of DOI:10.1021/acsmedchemlett.0c00033

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Letter
2
Fluorescent H Receptor Squaramide-Type Antagonists:
Synthesis, Characterization and Applications
Sabrina Biselli, Inês Alencastre, Katharina Tropmann, Daniela Erdmann, Mengya
Chen, Timo Littmann, André F. Maia, Maria Gomez-Lazaro, Miho Tanaka, Takeaki
Ozawa, Max Keller, Meriem Lamghari, Armin Buschauer, and Günther Bernhardt
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00033 • Publication Date (Web): 20 Jul 2020
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ACS Medicinal Chemistry Letters
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Fluorescent H Receptor Squaramide-Type Antagonists: Synthesis,
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Characterization and Applications
,
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Sabrina Biselli,* Inês Alencastre, Katharina Tropmann, Daniela Erdmann, Mengya Chen,
‡
,╪
§,
§,♦
†
†
Timo Littmann, André F. Maia, ° Maria Gomez-Lazaro, Miho Tanaka, Takeaki Ozawa, Max
‡
§,♦
‡,
,‡
∥
Keller, Meriem Lamghari, Armin Buschauer and Günther Bernhardt*
‡
Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitätsstrasse 31, D-93053
Regensburg, Germany
§
i3S - Instituto de Investigação e Inovação em Saúde da Universidade do Porto, Porto, Portugal
♦
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal
°
†
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Bunkyo-ku, Hongo, Tokyo 113-0033, Japan
ABSTRACT: Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding
studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH R, a series of squaramides
2
labeled with pyridinium or cyanine fluorophores (19-27), was synthesized and characterized. The highest hH R affinities in
2
radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK : 7.71-7.76) and
i
cyanine labeled antagonists 23 and 25 (pK : 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies
i
(saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry and high content
imaging. Saturation binding experiments revealed pK values comparable to the pK values. The fluorescent probes 21, 23 and 25
d
i
could be used to localize H receptors in HEK cells and to determine the binding affinities of unlabeled compounds.
2
KEYWORDS: histamine H receptor, squaramides, fluorescence labeling, flow cytometry, high content imaging, confocal microscopy
2
The histamine H receptor (H R), an aminergic GPCR, is one
NBD-analogs resulted in fluorescent ligands with high affinity
23,24
2
2
of the histamine receptor subtypes (H R) which mediate the
for the H R (5, 6: pA or pK > 7.0) (Figure 1).
However, 5
1
-4
2
2
i
action of the biogenic amine histamine (1). Activation of H R
and 6 were inapplicable for cell-based methods e.g. flow
2
1,2
results e.g. in gastric acid secretion, and positive inotropic
cytometry due to the high cellular autofluorescence at the
3
23,24
and chronotropic effects . In humans, the H R is located on
emission wavelength, giving low signal-to-noise ratios.
In
2
2
4,5
parietal cells in the stomach, in the brain, on neutrophils
and eosinophils as well as on smooth muscle cells . However,
order to expand the range of applications, well characterized
6
7
fluorescent H R ligands labeled with red-emitting
2
the (patho)-physiological role of the H R, especially in the
brain, is still far from being understood.
During the past few decades, fluorescence labeled GPCR
ligands have gained increasing importance as molecular tools
for the investigation of ligand-receptor-interactions as they
represent a complement or even an attractive alternative to
fluorophores (emission wavelength >600 nm) are required.
First attempts to the development of such ligands were already
made by labeling piperidinomethylphenoxypropylamino
26
2
2
5
pharmacophores with the cyanine dye S0436 (7 and 8
2
6
(Note to the paper authored by Petrache et al. , the ligands in
that paper were prepared and pharmacologically characterized
in our laboratories. The authors sketchily (compounds were
radioligands with respect to waste disposal, safety protocols
8-11
26
and costs.
Various fluorescent ligands for aminergic
“chemical synthesized” ) published very preliminary data
12-14
GPCRs have been reported, for example for muscarinic
,
without our knowledge and consent in a case of severe scien-
15,16
17,18
19-21
25
adrenergic , histamine H1
and H3
receptors. There
tific misconduct)), BODIPY 650/665 (9 ) or BODIPY
2
7
are different fluorophore core structures available for
630/650 (10 ) (Figure 1). AB118175, an aminopotentidine
derivative labeled with BODIPY 630/650 (chemical structure
not disclosed, alleged structure of 10 was deducted by the
2
2
8
labeling , e.g. BODIPY, rhodamine, dansyl/NBD or cyanine.
Most of the fluorescent ligands reported for the H R are emit-
2
2
3,24
27
ting at wavelengths below 550 nm.
ed fluorescent H R ligands
The majority of report-
consist of
provided molecular formula (C H BF N O S) ), is the only
4
8
56
2
9
4
a
commercially available fluorescent ligand for H R, but the
2
2
piperidinomethylphenoxypropylamino pharmacophore, de-
rived either from roxatidine (2), iodoaminopotentidine (3) or
BMY 2536 (4), which were linked to the fluorophore by an
alkyl chain (Figure 1). Labeling with relatively small
chromophores such as the N-methylanthranilic acid amide or
exact chemical structure is elusive and the high costs com-
promise its appropriateness as a H R fluorescent ligand. Addi-
2
tionally, AB118175 and the other red-emitting H R fluores-
2
cent ligands lack a comprehensive pharmacological character-
ization (only functional data available, no subtype selectivity)
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and the suitability for binding studies by fluorescence-based
techniques (e.g. confocal microscopy, flow cytometry) was
The synthesis of the amine precursors 16-18 and BMY 2536
29,31
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(4) according to previously published procedures,
is de-
2
5-27
only partially reported.
Furthermore, ligands 8 and 9 were
scribed in the supporting information. The Py-5 labeled fluo-
2
6
reported to have only moderate H R activities (pK : 6.89 and
rescent ligands 19-21 were synthesized by direct coupling of
2
b
2
5
32
6
.59 ).
Py-5 (12, chameleon label ) with the respective amine precur-
sor 16-18 under basic conditions (Scheme 1). The reaction
progressed rapidly accompanied by an immediate change in
color from blue to red. The cyanine labeled fluorescent ligands
22-27 were derived from the amine precursors 16-17 by amide
coupling using succinimidyl esters of the respective fluores-
cent dyes (13b-15b).
Here, we describe the synthesis and characterization of high
affinity fluorescent H R antagonists with improved optical and
2
physicochemical properties to gain access to a wide range of
potential applications, in particular to confocal microscopy
and to high throughput/content imaging.
0
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Replacing the propionic acid amide of the high affinity
The fluorescence quantum yields of representative compounds
(20, 21, 23, 25, 27) were determined in PBS at pH 7.4 and
PBS containing 1% (w/v) of BSA (Table S1, Figure S61,
Supporting Information). For all investigated compounds,
fluorescence quantum yields, determined in PBS containing
1% BSA, were higher compared to the quantum yields deter-
mined in PBS devoid of protein. Fluorescence is strongly
dependent on the environment of the fluorophore; this phe-
nomenon can be explained by intermolecular hydrophobic and
electrostatic interactions of the fluorescent ligand with pro-
3
28
radioligand [ H]UR-DE257 (11) (K value: 31 nM) , a
d
2
9
squaramide derived from BMY 2536 (4) , by red-emitting
fluorophores was the starting point for the development of
high affinity fluorescent hH R ligands. As the physicochemi-
2
cal properties of the fluorescent labels can considerably effect
3
0
e.g. non-specific binding or internalization, we chose two
different types of red-emitting fluorophores with various elec-
trical charge: the positively charged pyrilium dye (Py-5, 12)
and differently charged cyanine dyes (positive: S0223 (13a),
neutral: S0436 (14a) or negative: S0386 (15a), free acids)
33
teins. Additionally, ligands are flexible in solution and be-
(Scheme 1).
come more rigid upon binding, which generally leads to an
3
3
increase in quantum yield. Therefore, fluorescent ligands are
not suitable for the determination of absolute values (e.g.
number of specific binding sites Bmax), but are valuable tools
for the determination of dissociation constants (pK , and pK ),
d
i
which are accessible by measuring relative fluorescence inten-
sities, by analogy with the determination of count rates
(
cpm/cps) in competition radioligand binding. The Py-5 la-
beled compounds 20 and 21 showed an excitation maximum at
81 nm and an emission maximum at 646 nm in the presence
4
of BSA. The cyanine labeled compounds 23 and 25 showed an
excitation maximum at 663-667 nm and an emission maxi-
mum at 672-676 nm in the presence of BSA. Therefore, the
Py-5 labeled compounds 19-21 can be excited with an argon
laser at 488 nm and the cyanine labeled compounds 22-27 by a
red laser at 635 nm.
The fluorescent ligands 19-27, BMY2536 (4) and the amine
precursors 16-18 were investigated in equilibrium competition
binding experiments on membrane preparations from Sf9
insect cells expressing the hH R-G
fusion protein, co-
2
sαS
expressing hH R and RGS4, or co-expressing either the hH R
1
3
or the hH R and G and Gβ1γ2 proteins (Table 1; Table S2,
4
αi2
3
4
Figures S62 and S63, Supporting Information). Radioligand
competition binding experiments revealed that most of the
fluorescent labels were tolerated with no or only a slight de-
crease in affinity. Exceptions were the cyanine labeled ligands
2
6 and 27, in which the introduction of the S0387 fluorophore
with a negative charge resulted in a decrease in hH R affinity
2
(
pK : 26, 5.69; 27, 5.88) compared to the corresponding
i
amine-precursors (pK : 16, 6.52; 17, 7.87). The Py-5 labeled
i
ligands 19-21 showed, irrespective of linker length, high hH R
2
affinities (pK : 7.71-7.76) in the same range as the parent
i
compound BMY 25368 (4) (pK : 7.80). In case of 19, the
i
hH R affinity even increased with labeling (pK of amine
2
i
precursor 16: 6.52, pK of 19: 7.75). In the cyanine dye series,
i
ligand 23, precursor 17 (n=6, Table 1) labeled with
fluorophore S0223 (positive charge), and ligand 25, precursor
17 (n=6, Table 1) labeled with fluorophore S0436 (no charge),
showed the highest hH R affinity (pK : 23, 7.67; 25, 7.11).
Figure 1. Chemical structures of histamine (1), reported H R
2
standard antagonists (2-4), fluorescent H R ligands (5-10) and the
2
2
i
3
5,23-29
radioligand [ H]UR-DE257 (11).
Interestingly, labeling with Py-5, S0223 and S0436 led to an
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Scheme 1. Chemical structures of Py-5 (12), the free acids S0223 (13a), S0436 (14a), S0387 (15a) and the succinimidyl esters
S2197 (13b), S0S0536 (14b), S0586 (15b), as well as the synthesis of the fluorescent ligands 19-27.
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9
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0
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0
Reagents and conditions: i) DMF, TEA or DIPEA, rt, 90-120 min, 24-32%; ii) DMF, DIPEA, rt, 45-90 min, 18-44%.
increase in hH R and hH R receptor affinity up to two orders
characterized in a GTPγS binding assay on membrane prepara-
1
3
of magnitude compared to the corresponding amine precursor
Table S2, Figure S62, Supporting Information). The
pyridinium labeled ligands 19-21 and the cyanine labeled
tions of Sf9 insect cells expressing the hH R-G fusion pro-
2
sαS
3
3
(
tein (Table 1; Figure S64, Supporting Information). The
investigated compounds proved antagonists with the calculat-
ed pK values being in a good agreement with the pK values
ligands 23 and 25 showed a slight preference for the hH R
2
b
i
over the hH R and hH R. In case of the cyanine labeled ligand
from radioligand competition binding.
1
3
2
4, precursor 16 (n=4) labeled with fluorophore S0436 (no
Furthermore, representative fluorescent ligands (20, 21, 23 and
charge), the preference changed in favor of the hH R. Interest-
26) were investigated for hH R agonism in a β-arrestin2 re-
3
2
ingly, cyanine ligand 22, precursor 16 (n=4) labeled with
cruitment assay on HEK293T-β-Arr2-hH R cells (Figure S65,
2
fluorophore S0223 (positive charge), showed a high hH R
Supporting Information). None of the investigated ligands
showed any β-arrestin2 recruitment, indicating that no β-
arrestin2 mediated internalization of the receptor-ligand-
complex took place.
1
affinity (pK of 7.88) with a 20-fold selectivity for the hH R
i
1
over the hH R. The compounds 19-21, 23 and 25, which were
2
investigated for hH R affinity, showed a 70 to 150-fold selec-
4
tivity for the hH R over hH R (Table S2, Figure S63, Support-
The hH R binding of the fluorescent ligands 21, 23 and 25 was
2
4
2
ing Information).
The amine precursors 16-18, BMY 25368 (4) and representa-
tive fluorescent ligands (20, 21, 23 and 25) were functionally
also investigated by confocal microscopy (Figure 2). After 20
min of incubation, all investigated ligands were still localized
at the cell membrane of HEK293T-hH R-qs5 cells.
2
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Table 1. H R affinities and antagonistic activities as well as HR selectivity profiles.
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
compd.
n
Fluorophore
hH R
HR selectivity
profile
2
pKib
d
c
d
d
e
ratios of Ki
pK flow cytom.
pK high content imaging
pK (pEC )
b 50
H R / H R / H R /
1
2
3
automated cell imaging flow
f
imaging
cytometry
H R
4
His (1)
-
-
6.53 ± 0.04
n.a.
n.a.
n.a.
n.a.
(5.80 ± 0.06)
7.03 ± 0.02
5.76 ± 0.22
- / 1 / 0.5 / 0.1
- / 1 / 1300 / -
33 / 1 / 36 / 33
g
4
-
-
7.80 ± 0.01
6.52 ± 0.04
7.87 ± 0.02
7.86 ± 0.02
7.75 ± 0.02
7.71 ± 0.04
7.76 ± 0.01
6.57 ± 0.02
7.67 ± 0.07
6.49 ± 0.04
7.11 ± 0.01
5.69 ± 0.08
5.88 ± 0.09
n.a.
n.a.
n.a.
n.a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
4
6
7
4
6
7
4
6
4
6
4
6
-
n.a.
1
1
7
8
-
n.a.
n.a.
n.a.
6.73 ± 0.08 740 / 1 / 620 / 740
-
n.a.
n.a.
n.a.
7.06 ±0.13
n.d.
380 / 1 / 550 / 720
6 / 1 / 20 / 70
5 / 1 / 4 / 120
4 / 1 / 6 / 160
1 / 20 / 8 / 180
2 / 1 / 3 / 50
2 / 2 / 1 / -
19
Py-5
Py-5
Py-5
S0223
S0223
S0436
S0436
S0387
S0387
7.55 ± 0.02
7.84 ± 0.07
7.73 ± 0.09
6.54 ± 0.05
7.89 ± 0.07
6.25 ± 0.21
7.32 ± 0.02
n.d.
7.13 ± 0.01
n.d.
7.06 ± 0.03
n.d.
2
2
0
1
7.21 ± 0.04
7.85 ± 0.10
n.d.
7.05 ± 0.04
6.92 ± 0.06
7.82 ± 0.10
6.77 ± 0.09
7.79 ± 0.13
n.d.
7.19 ± 0.03
n.d.
22
2
2
3
4
n.d.
7.73 ± 0.04
n.d.
n.d.
25
n.d.
6.49 ± 0.03
n.d.
6 / 1 / 3 / 130
- / 1 / 2 / -
2
2
6
7
n.d.
n.d.
6.35 ± 0.04
n.d.
n.d.
n.d.
a
b
3
Length of the linker given as the number of carbon atoms. Determined by radioligand competition binding with [ H]UR-DE257 (K =
d
1
2.2 nM, c= 20 nM) at membrane preparations of Sf9 insect cells expressing the hH R-G fusion protein; mean values ± SEM from three
2 sαS
c
independent experiments (each performed in triplicate). Determined by flow cytometric saturation binding at HEK293T-hH R-qs5 cells;
2
d
mean values ± SEM from three independent experiments (each performed in duplicate). Determined by high-content imaging saturation
e
binding at HEK293T-hH R-qs5 cells; mean values ± SEM from 2-3 experiments (each performed in duplicate). Determined by GTPγS
2
binding assay at membrane preparations of Sf9 insect cells expressing the hH R-G fusion protein; pK values of neutral antagonists were
2
sαS
b
determined in the antagonist mode versus histamine (c= 1 µM); mean values ± SEM from 2-3 independent experiments (each performed in
f
g
triplicate). Calculated from the K values obtained by conversion of the respective pK value (Table S2, Supporting Information). Data
i
i
2
8
c,d
was previously reported as K value by Baumeister et al. and the raw data was re-analyzed to give the pK values. Non-specific binding
i
i
was determined in the presence of famotidine (300-fold excess). The incubation period was 60 min (b, d, e, f) or 90 min (c). n.d.: not de-
termined. n.a.: not applicable.
The ligands 21, comprising a positively charged pyridinium
moiety, and 25, comprising an electroneutral cyanine moiety,
showed low non-specific binding at a concentration of 100 nM
in the presence of famotidine (300-fold excess), whereas 23,
comprising a positively charged cyanine moiety, showed high-
er non-specific binding (Figure 2).
25), had a positive effect regarding non-specific binding
(~10%).
The association and dissociation kinetics of 21, 23 and 25
were determined on HEK293T-hH R-qs5 cells at 37 °C using
flow cytometry (Figure S68, Table S3, Supporting Infor-
2
mation). The fluorescent ligands showed a fast (k (21):
on
-
1
-1
Fluorescent ligands 19-25, which showed moderate to high
hH R affinity (pK >6.0) were used for flow cytometric bind-
0.0043 min nM ) or moderate (k (23, 25): 0.0009-0.002
on
-1 -1
min nM ) association rate and incomplete dissociation. A
similar dissociation behavior was reported for the closely
2
i
ing studies at HEK293T-hH R-qs5 cells (Table 1; Figure S66-
2
3
28
S68, Supporting Information). All investigated ligands afford-
related radioligand [ H]UR-DE257. A possible explanation
for the (pseudo-)irreversible binding of these ligands is a slow
dissociation from the receptor or binding to two different
receptor states: a fast reversible and a tight-binding state (for
ed pK values which were in good agreement with the pK
d
i
values. Within the pyridinium labeled ligands 19-21, non-
specific binding was low (<10% of total binding around the
28
K ). The cyanine labeled ligands 22 and 23 with a positive
d
more detailed information, see Supporting Information). The
charge of the fluorophore showed slightly higher non-specific
binding (~20%). The introduction of a sulfonic acid group into
the cyanine moiety resulting in an uncharged fluorophore (24,
estimated pKd(kin) values of 21, 23 and 25, were consistent with
the pK values determined in saturation binding experiments,
d
suggesting that binding followed at least in part the law of.
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Figure 2. Visualization of binding of the fluorescent ligands 21, 23 and 25 (all 100 nM) to the membrane of HEK293T-hH R-qs5 cells
2
determined by confocal microscopy after 20 min of incubation at rt. Non-specific binding was determined in the presence of famotidine
(300-fold excess). Images were acquired with a Zeiss Axiovert 200M microscope equipped with the LSM 510 Laser scanner. A 63x/1.40
oil immersion objective was used.
mass action. The (pseudo-)irreversibility of binding might be
suboptimal for e.g. competition binding experiments, but
might be advantageous in microscopy or even in in vivo (im-
aging) experiments The fluorescent ligands 19, 21-25 and 27
were also analyzed by automated cell imaging, enabling
residual fluorescent ligand was preferentially located at the
cell membrane, and there was only very low fluorescence in
the cytoplasm.
Results from saturation binding studies with 19 and 21 on
suspended HEK293T-hH R-qs5 cells using an imaging flow
2
measurement of hH R binding on live and adherent
cytometer were in good agreement with the results from auto-
mated cell imaging (Table 1; Figure S73-74, Supporting In-
formation). The applicability of fluorescent ligands 21 and 25
for the determination of binding affinities of unlabeled ligands
was demonstrated by flow cytometric and high content imag-
ing competition binding assays (a more detailed description is
provided in the Supporting information, Figure S75, Table
S4).
For none of the investigated fluorescent ligands was a negative
influence on cell viability during the incubation periods of the
applied assays observed (flow cytometry and high content
imaging (Figures S76-S78, Supporting Information).
2
HEK293T-hH R-qs5 cells. Figure 3 shows representative
2
images after incubation of the cells with the fluorescent lig-
ands 21 (250 nM), 23 (75 nM), 25 (75 nM) and 27 (500 nM)
at room temperature for 60 min, followed by a washing step.
All fluorescent ligands were localized at the cell membrane.
The pK values determined by saturation binding by automat-
d
ed cell imaging were generally in good agreement with those
obtained by flow cytometry (Table 1; Figure S69-S70, Sup-
porting Information).
The background fluorescence (binding to plastics) was low in
case of fluorescent ligands with no or with negative charge of
the fluorophore (24, 25, 27: <15% of total binding around the
Taken together, we showed that the presented fluorescent
ligands are useful molecular tools for non-radioactive binding
studies using different techniques such as confocal microsco-
py, (imaging) flow cytometry and automated cell imaging.
The cyanine dye labeled ligands proved to be useful candi-
dates for in vivo imaging as a high tissue permeability requires
excitation and emission wavelengths in the far-red / near-
K ), but relatively high, if the ligand contained a fluorophore
d
with positive charge (21: 25%; 22, 23: 30-40%). An exception
was compound 19, which was also labeled with a positively
charge Py-5, but showed a lower background fluorescence
than 21 (~12%).
Investigation of the association and dissociation kinetics of the
cyanine labeled ligand 25 with automated cell imaging re-
3
5
infrared (>650 nm) and the pyridinium labeled ligands 19-21
-
1
-1
vealed a much faster association (k : 0.0098 min nM ) com-
might be suitable molecular tools (BRET acceptors) for Nano-
on
3
6
pared to flow cytometry, but also an incomplete dissociation
BRET assays .
(
Figure S71-S72, Table S3, Supporting Information). The
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Figure 3. Visualization of binding of the fluorescent ligands 21 (250 nM), 23 (75 nM), 25 (75 nM) and 27 (500 nM) to the membrane of
HEK293T-hH R-qs5 cells (red) determined by automated cell imaging after 60 min of incubation at rt. Hoechst 33342 was used as a nu-
2
clear stain (blue). Fluorescence was mainly associated with the plasma membrane. Non-specific binding was determined in the presence of
famotidine (300-fold excess). Images were acquired with a IN Cell Analyzer 2000.
19, 21-25 and 27 at HEK293T-hH R-qs5 cells studied by auto-
mated cell imaging (including Figures S69-S72); Saturation bind-
ASSOCIATED CONTENT
Supporting Information
2
ing of 19 and 21 at HEK293T-hH R-qs5 cells studied by imaging
2
The Supporting Information is available free of charge on the
ACS Publications website.
flow cytometry (including Figures S73 and S74); Competition
binding studies with 21 and 25 (including Figure S75 and Table
S4); Rough estimate of cell viability (including Figures S76-S78).
Synthesis of the intermediates 30-36, BMY 25368 (4) and the
amine precursors 16-18 (including Schemes S1 and S2); Experi-
Molecular formula strings (XLSX)
1
13
mental section (including Figures S1-S9); H and C-NMR spec-
tra of 30-36, 4, 16-27 (including Figures S10-S37); HPLC chro-
matograms of 16-27 and BMY 2536 (4) (including Figures S38-
S40); HRMS spectra of compounds 30-36, 4, 16-27 (including
Figures S41-S60), Determination of quantum yields (including
Figure S61 and Table S1); Radioligand competition binding
studies and GTPγS binding assay (including Figures S62-S64 and
Table S2); Investigation of β-arrestin2 recruitment by a split
AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: sabrina.biselli@ur.de (S.B.),
guenther.bernhardt@ur.de (G.B.)
Tel.: (+49)941 9432925 (S.B.), (+49)941 9434822 (G.B.)
Fax: (+49)941 9434820 (S.B., G.B.)
luciferase assay at HEK-hH R cells (including Figure S65); Bind-
2
Present Addresses
ing studies of 19-25 at HEK293T-hH R-qs5 cells studied by flow
2
●Agrolab Labor GmbH, 84079 Bruckberg, Germany
cytometry (including Figures S66-S68 and Table S3); Binding of
6
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25
⁑
Zentrale Arzneimittelüberwachung Bayern, Germany
with [ I]iodoaminopotentidine. J. Neurochem. 1992, 59, 290-
1
2
3
4
5
6
7
8
9
|Sanofi Investment Co., Ltd., Chaoyang District, Beijing, China
AbbVie Deutschland GmbH & Co. KG, 67061 Ludwigshafen
am Rhein, Germany
299.
5.
╪
Ruat, M.; Traiffort, E.; Bouthenet, M. L.; Schwartz, J.
C.; Hirschfeld, J.; Buschauer, A.; Schunack, W. Reversible and
Irreversible labeling and autoradiographic localization of the
cerebral histamine H -receptor using [I-125] iodinated probes. P.
Natl. Acad. Sci. USA 1990, 87, 1658-1662.
6.
Evidence for ligand-specific conformations of the histamine H2-
receptor in human eosinophils and neutrophils. Biochem.
Pharmacol. 2012, 84, 1174-1185.
Author Contributions
2
S.B., D.E., K.T. and M.C. performed the synthesis. S.B., I.A.,
T.L., A.F.M. and M.G-L. performed functional and binding stud-
Reher, T. M.; Brunskole, I.; Neumann, D.; Seifert, R.
ies. M.T. cloned the vector hH R-ELucC/ELucN-β-arrestin2
2
under supervision of T.O.. G.B. and A.B. initiated and planned
the project. G.B., M.K., M.L. and A.B. supervised the research.
S.B. and G.B. wrote the manuscript. All authors have given ap-
proval to the final version of the manuscript.
7.
Mitznegg, P.; Schubert, E.; Fuchs, W. Relations
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between the effects of histamine, pheniramin and metiamide on
spontaneous motility and the formation of cyclic AMP in the
isolated rat uterus. Naunyn Schmiedeberg´s Arch. Pharmacol.
1975, 287, 321-327.
Funding
This work was funded by the Graduate Training Programs
GRK760 (D.E., G.B. and A.B.) and GRK1910 (S.B., K.T., M.C.,
T.L., M.K. and A.B.) of the Deutsche Forschungsgemeinschaft
8.
Iliopoulos-Tsoutsouvas, C.; Kulkarni, R. N.;
Makriyannis, A.; Nikas, S. P. Fluorescent probes for G-protein-
coupled receptor drug discovery. Expert Opin. Drug Discov.
(DFG) and the Elite Network of Bavaria (T.L. and A.B.).
2
9
018, 13, 933-947.
Sridharan, R.; Zuber, J.; Connelly, S. M.; Mathew, E.;
.
Notes
Dumont, M. E. Fluorescent approaches for understanding
interactions of ligands with G protein coupled receptors. Biochim.
Biophys. Acta 2014, 1838, 15-33.
The authors declare no competing financial interest.
∥
Deceased, July 18, 2017.
1
0.
Rinken, A.; Lavogina, D.; Kopanchuk, S. Assays with
detection of fluorescence anisotropy: Challenges and possibilities
for characterizing ligand binding to GPCRs. Trends Pharmacol.
Sci. 2018, 39, 187-199.
ACKNOWLEDGMENT
The authors thank Paul Baumeister, Xueke She and Edith Bartole
for helpful advices and Maria Beer-Krön, Elvira Schreiber, Dita
Fritsch and Sieglinde Dechant for excellent technical assistance.
We also thank Johannes Felixberger and Johannes Mosandl for
1
1.
Ciruela, F.; Jacobson, K. A.; Fernandez-Duenas, V.
Portraying G protein-coupled receptors with fluorescent ligands.
ACS Chem. Biol. 2014, 9, 1918-1928.
1
2.
Daval, S. B.; Valant, C.; Bonnet, D.; Kellenberger, E.;
preparation of the HEK293T-hH R-qs5 and HEK293T-β-Arr2-
2
Hibert, M.; Galzi, J. L.; Ilien, B. Fluorescent derivatives of AC-42
to probe bitopic orthosteric/allosteric binding mechanisms on
muscarinic M receptors. J. Med. Chem. 2012, 55, 2125-2143.
hH R cells. The authors acknowledge the support of the project
2
P2020-PTDC/BIM-MED/4041/2014 and of the i3S Scientific
Platforms BioSciences Screening and Bioimaging, members of
the PPBI (PPBI-POCI-01-0145-FEDER-022122).
1
1
3.
Tahtaoui, C.; Parrot, I.; Klotz, P.; Guillier, F.; Galzi, J.
L.; Hibert, M.; Ilien, B. Fluorescent pirenzepine derivatives as
potential bitopic ligands of the human M muscarinic receptor. J.
Med. Chem. 2004, 47, 4300-4315.
14.
Sreckovic, S.; Watson, J. Design and synthesis of a fluorescent
muscarinic antagonist. Bioorg. Med. Chem. Lett. 2008, 18, 825-
827.
1
ABBREVIATIONS
BSA, bovine serum albumin; BRET, bioluminescence energy
transfer; cpm/cps, counts per minute/counts per second; DIPEA,
diisopropylethylamine; GPCR, G-protein coupled receptor;
GTPγS, guanosine 5´-thiotriphosphate; HEK293T, human embry-
onic kidney 293T; NBD, 4-nitrobenzo-2-oxa-1,3-diazole; PBS,
Jones, L. H.; Randall, A.; Napier, C.; Trevethick, M.;
15.
Baker, J. G.; Adams, L. A.; Salchow, K.; Mistry, S. N.;
phosphate buffered saline; pK , negative logarithm of the dissoci-
Middleton, R. J.; Hill, S. J.; Kellam, B. Synthesis and
characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-
s-indacene-labeled fluorescent ligands for human beta-
adrenoceptors. J. Med. Chem. 2011, 54, 6874-6887.
b
ation constant obtained from functional assays; pK , negative
d
logarithm of the dissociation constant obtained from saturation
binding experiments; pK , negative logarithm of the dissociation
i
constant obtained from competition binding experiments; Sf9,
Spodoptera frugiperda cells, TEA, triethylamine; TFA,
trifluoroacetic acid.
16.
Sugawara, T.; Hirasawa, A.; Hashimoto, K.; Tsujimoto,
G. Differences in the subcellular localization of alpha1-
adrenoceptor subtypes can affect the subtype selectivity of drugs
in a study with the fluorescent ligand BODIPY FL-prazosin. Life.
Sci. 2002, 70, 2113-2124.
REFERENCES
1
7.
Li, L.; Kracht, J.; Peng, S.; Bernhardt, G.; Buschauer,
1.
Black, J. W.; Duncan, W. A.; Durant, C. J.; Ganellin, C.
A. Synthesis and pharmacological activity of fluorescent
histamine H receptor antagonists related to mepyramine. Bioorg.
Med. Chem. 2003, 13, 1245-1248.
R.; Parsons, E. M. Definition and antagonism of histamine H2-
receptors. Nature 1972, 236, 385-390.
1
2
.
Domschke, W.; Domschke, S.; Classen, M.; Demling,
1
8.
Rose, R. H.; Briddon, S. J.; Hill, S. J. A novel
L. Histamine and cyclic 3',5'-AMP in gastric acid secretion.
Nature 1973, 241, 454-455.
fluorescent histamine H1 receptor antagonist demonstrates the
advantage of using fluorescence correlation spectroscopy to study
the binding of lipophilic ligands. Br. J. Pharmacol. 2012, 165,
3
.
Reinhardt, D.; Schmidt, U.; Brodde, O. E.; Schumann,
H. J. H - and H -receptor mediated responses to histamine on
1
2
1
1
789-1800.
9. Isensee, K.; Amon, M.; Garlapati, A.; Ligneau, X.;
contractility and cyclic AMP of atrial and papillary muscles from
guinea-pig hearts. Agents Actions 1977, 7, 1-12.
Camelin, J. C.; Capet, M.; Schwartz, J. C.; Stark, H. Fluorinated
non-imidazole histamine H3 receptor antagonists. Bioorg. Med.
Chem. Lett. 2009, 19, 2172-2175.
4
.
Traiffort, E.; Pollard, H.; Moreau, J.; Ruat, M.;
Schwartz, J. C.; Martinez-Mir, M. I.; Palacios, J. M.
Pharmacological characterization and autoradiographic
localization of histamine H receptors in human brain identified
20.
Amon, M.; Ligneau, X.; Schwartz, J. C.; Stark, H.
2
Fluorescent non-imidazole histamine H3 receptor ligands with
7
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ACS Medicinal Chemistry Letters
Page 8 of 8
nanomolar affinities. Bioorg. Med. Chem. Lett. 2006, 16, 1938-
1940.
1.
histamine H receptor antagonist. ChemMedChem 2015, 10, 83-
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
93.
29.
2
Amon, M.; Ligneau, X.; Camelin, J. C.; Berrebi-
Algieri,
A.
A.;
Crenshaw,
and a
R.
R.
1,2-
Bertrand, I.; Schwartz, J. C.; Stark, H. Highly potent
Diaminocyclobutene-3,4-diones
pharmaceutical
fluorescence-tagged nonimidazole histamine H receptor ligands.
composition containing them. FR 2505835, 1982. Chem. Abstr.
99:22320.
3
ChemMedChem 2007, 2, 708-716.
2
2.
Probes. John Wiley & Sons, Inc: Hoboken, 2015.
23. Li, L.; Kracht, J.; Peng, S.; Bernhardt, G.; Elz, S.;
Sabnis, R. W. Handbook of Fluorescent Dyes and
30.
Keller, M.; Mahuroof, S.; Hong Yee, V.; Carpenter, J.;
Schindler, L.; Littmann, T.; Pegoli, A.; Hübner, H.; Bernhardt, G.;
Gmeiner, P.; Holliday, N. D. Fluorescence labeling of
neurotensin(8–13) via arginine residues gives molecular tools
with high receptor affinity. ACS Med. Chem. Lett. published
Buschauer, A. Synthesis and pharmacological activity of
fluorescent histamine H2 receptor antagonists related to
potentidine. Bioorg. Med. Chem. Lett. 2003, 13, 1717-1720.
online
10.1021/acsmedchemlett.1029b00462.
31. Brown, T. H.; Young, R. C. Dioxocyclobutene
compounds. EP 105702, 1986. Chem. Abstr. 101:130290
32. Wetzl, B. K.; Yarmoluk, S. M.; Craig, D. B.; Wolfbeis,
November
2019,
DOI:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4.
Malan, S. F.; van Marle, A.; Menge, W. M.; Zuliani, V.;
Hoffman, M.; Timmerman, H.; Leurs, R. Fluorescent ligands for
the histamine H2 receptor: synthesis and preliminary
characterization. Bioorg. Med. Chem. 2004, 12, 6495-6503.
25.
Xie, S. X.; Petrache, G.; Schneider, E.; Ye, Q. Z.;
O. S. Chameleon labels for staining and quantifying proteins.
Angew. Chem. Int. Ed. Eng.l 2004, 43, 5400-5402.
Bernhardt, G.; Seifert, R.; Buschauer, A. Synthesis and
pharmacological characterization of novel fluorescent histamine
33.
Dunn, S. M. J.; Fluorescence measurements of receptor-
H -receptor ligands derived from aminopotentidine. Bioorg. Med.
ligand interactions. In handbook of neurochemistry and molucular
2
rd
Chem. Lett. 2006, 16, 3886-3890.
neurobiology, 3 ed.;Lajtha, A; Baker, G.; Dunn, S.; Holt, A.,
2
6.
Petrache, G.; Pavelescu, M. D. The pharmacological
Eds.; Springer Science + Buisness Media, LLC.: New York,
2007; 133-148.
activity of some new fluorescent small molecule histamine H2
receptor (H R) ligands, derived from aminopotentidine and
34.
Kelley, M. T.; Bürckstümmer, T.; Wenzel-Seifert, K.;
2
squaramide, in the GTPase assay. Rev. Med. Chir. Soc. Med. Nat.
Iasi. 2010, 114, 255-259.
Dove, S.; Buschauer, A.; Seifert, R. Distinct interaction of human
and guinea pig histamine H -receptor with guanidine-type
2
2
7.
Hill, S. J.; Kellam, B.; Briddon, S. J. Fluorescence-
agonists. Mol. Pharmacol. 2001, 60, 1210-1225.
based high content screening of compounds for functional
response or pharmacological properties. 2006032926, 2006.
Chem. Abstr. 144:343538.
35.
Chen, C.; Hua, Y.; Hu, Y.; Fang, Y.; Ji, S.; Yang, Z.;
Ou, C.; Kong, D.; Ding, D. Far-red/near-infrared fluorescence
light-up probes for specific in vitro and in vivo imaging of a
tumour-related protein. Sci. Rep. 2016, 6, 23190.
28.
Baumeister, P.; Erdmann, D.; Biselli, S.; Kagermeier,
3
N.; Elz, S.; Bernhardt, G.; Buschauer, A. [ H]UR-DE257:
Development of a tritium-labeled squaramide-type selective
36.
Dale, N. C.; Johnstone, E. K. M.; White, C. W.; Pfleger,
K. D. G. NanoBRET: The bright future of proximity-based
assays. Front. Bioeng. Biotechnol. 2019, 7, 56.
For Table of Contents Use Only
Title: Fluorescent H Receptor Squaramide-Type Antagonists: Synthesis, Characterization and Applications
2
Authors: Sabrina Biselli, Inês Alencastre, Katharina Tropmann, Daniela Erdmann, Mengya Chen, Timo Littmann, André F. Maia,
Maria Gomez-Lazaro, Miho Tanaka, Takeaki Ozawa, Max Keller, Meriem Lamghari, Armin Buschauer and Günther Bernhardt
Confocal microscopy (HEK cells)
100 nM 25
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