A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3Receptor

Article abstract of DOI:10.1021/acs.jmedchem.1c01089

The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

Full text of DOI:10.1021/acs.jmedchem.1c01089

pubs.acs.org/jmc
Article
A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET
Binding Studies and Single-Molecule Microscopy at the Histamine
H3 Receptor
Niklas Rosier, Lukas Grätz,* Hannes Schihada, Jan Möller, Ali Isb̧ ilir, Laura J. Humphrys, Martin Nagl,
Ulla Seibel, Martin J. Lohse, and Steffen Pockes*
Cite This: https://doi.org/10.1021/acs.jmedchem.1c01089
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The histamine H3 receptor (H3R) is considered an
attractive drug target for various neurological diseases. We here
report the synthesis of UR-NR266, a novel fluorescent H3R ligand.
Broad pharmacological characterization revealed UR-NR266 as a
sub-nanomolar compound at the H3R with an exceptional
selectivity profile within the histamine receptor family. The
presented neutral antagonist showed fast association to its target
and complete dissociation in kinetic binding studies. Detailed
characterization of standard H3R ligands in NanoBRET competi-
tion binding using UR-NR266 highlights its value as a versatile
pharmacological tool to analyze future H3R ligands. The low
nonspecific binding observed in all experiments could also be
verified in TIRF and confocal microscopy. This fluorescent probe
allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical
analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a
selective compound.
information on ligand−receptor interactions.14−16 Among
INTRODUCTION
■
many imaging technologies, total internal reflection fluores-
The histamine H3 receptor (H3R) is a seven-transmembrane
domain receptor and belongs to the superfamily of G-protein
cence (TIRF) microscopy represents the most advanced
method for single-molecule imaging of individual proteins
coupled receptors (GPCRs).1 The H3R represents one of four
within (or close to) the plasma membrane.17,18 Although
members of the histamine receptor family and mediates its G-
several fluorescent H3R ligands have already been re-
protein-dependent signaling mainly via Gi-proteins.2,3 Due to
ported,19−22 none of them has been shown to be suitable for
the fact that the H3R is almost exclusively expressed in the
brain,1,4−6 it is an interesting target for numerous diseases of
single-molecule studies. We therefore aimed to develop a tool
the central nervous system, such as Parkinson’s,7 Hunting-
suitable for single-molecule imaging and as a tracer probe for
ton’s,8,9 and Alzheimer’s diseases,10 as well as tic disorders.11 In
binding studies of new putative drug candidates for the H3R in
a recently developed NanoBRET binding assay that can be
2016, the inverse agonist pitolisant entered the market as the
employed in high throughput analyses.23
first (and until now only) drug targeting the H3R for the
treatment of narcolepsy.12 In addition, pitolisant has shown
RESULTS AND DISCUSSION
promising results in initial studies for the treatment of Prader−
Willi Syndrome.13 Accordingly, the H3R is a highly interesting
target for CNS-related diseases and urgently needs to be
studied in more detail at the molecular level.
Fluorescently labeled ligands and related methods have
gained increasing importance in modern GPCR research. The
methods to be mentioned here include resonance energy
transfer (RET) assays, fluorescence anisotropy, and fluores-
cence correlation spectroscopy. Over the last decade, time-
■
Design Rationale. Fluorescent receptor ligands can be
considered as a combination of three distinct parts: the parent
ligand, often referred to as a pharmacophore, a linker region,
Received: June 17, 2021
̈
resolved Forster/bioluminescence resonance energy transfer
(TR-FRET/BRET) techniques have become increasingly
important in the study of ligand binding at GPCRs to provide
https://doi.org/10.1021/acs.jmedchem.1c01089
© XXXX American Chemical Society
J. Med. Chem. XXXX, XXX, XXX−XXX
A

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 1. Synthesis of the Pharmacophoric H3R Scaffold 5 (A) and the Linking PEG Structure 9 (B)
a
Reagents and conditions: (a) 1-Br-3-Cl-propane, K2CO3, MeCN, reflux, 18 h, 89%; (b) piperidine, Na2CO3, KI, MeCN, reflux, 20 h, 76%; (c)
ethyl isonipecotate, NaBH(OAc)3, CHCl3, rt, overnight, 95%; (d) 2 N HCl, THF, rt, overnight, 92%; (e) methane sulfonyl chloride, NEt3, DCM,
rt, overnight, 78%; (f) NaN3, EtOH/DMF (4:1), reflux, overnight, 90%; (g) PPh3, 1 N HCl/THF/EtOAc (5:1:5), rt, overnight, 77%.
a
Scheme 2. Synthesis of the 5-TAMRA-Labeled Fluorescent H3R Ligand 12
a
Reagents and conditions: (a) 1. 5, EDC·HCl, HOBt, DIPEA, DCM/DMF (1:1), rt, 30 min; 2. 9, 100 °C, MW, 30 min, 65%; (b) 1. 10, PPh3,
THF, 4 h, 45 °C; 2. H2O, 2 h, 45 °C, 60%; (c) 1. 11, 5-TAMRA NHS ester, NEt3, DMF, rt, 2.5 h; 2. 10% aq. TFA, rt, 15 min, 82%.
and a fluorescent dye. Each of them has to be chosen
depending on the intended use of the final fluorescent ligand.24
The H3 receptor antagonist JNJ-5207852, one of the most
affine H3 receptor ligands reported so far, has been used as the
parent ligand. Besides its high H3R affinity, it shows an
excellent selectivity not only within the histamine receptor
family but also with respect to 50 other different targets.25
These properties combined with synthetic accessibility and
good water solubility make it an excellent pharmacophore for a
fluorescent ligand. Structure activity relationships showed a
high tolerance of the ligand toward structural changes at the
para position of the benzylic piperidine moiety.26 Thus, this
position of the molecule has been chosen to attach the linker
via an amide group.
In terms of the linker moiety, a flexible polyethyleneglycol-
based (PEG-based) structure with a length of 13 atoms was
used, which we assumed would be sufficient to reach outside
the binding pocket, thus reducing the probability of a negative
impact of the linker on ligand binding. PEG-based linkers are
often used in fluorescent ligands, as they are chemically stable,
show higher water solubility than alkylic structures, and are less
susceptible to interact with cell membranes.27
The choice of the fluorescent dye must be taken carefully for
any fluorescent ligand and depends mainly on the intended
application of the ligand. With the aim of a versatile fluorescent
ligand for use in the NanoBRET binding assay and
fluorescence microscopy, the 5-TAMRA fluorescent dye was
chosen. The suitability of this dye for NanoBRET binding has
been shown for different receptors,15,16 as it led to a better
signal to noise ratio and less adhesion to the plastic vessel than,
e.g., BODIPY labeled ligands at the H2 receptor.28
Furthermore, 5-TAMRA was reported as a fluorescent dye
for single molecule imaging in TIRF microscopy,29,30 and due
to its hydrophilicity, it is less prone to interact with cell
membranes than BODIPY,31 resulting in a lower nonspecific
binding in fluorescence microscopy techniques.
Chemistry. The synthesis of the fluorescent ligand’s
pharmacophore 5 was carried out, following publications by
Apodaca et al. and Wingen et al. with minor modifications,
obtaining excellent yields (Scheme 1A).25,26 Starting from 4-
hydroxybenzaldehyde (1), 2 was obtained in a nucleophilic
B
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. Representative isotherms from saturation binding experiments with 12 at the NLuc-hH3R (A, NanoBRET) and at the wild-type hH3R (B,
flow cytometry), both stably expressed in HEK293 cells.
Table 1. Comparison of Thermodynamic and Kinetic Binding Constants of 12 at the Wild-Type hH3R and the NLuc-hH3R
flow cytometry
NanoBRET binding
a
b
c
d
c
e
f
g
compound
pKd (sat)
pKd (sat)
9.80 0.07
kobs (min−1
)
τass (min)
koff (min−1
)
τdiss (min)
kon (min−1 nM−1
)
pKd (kin)
9.66 0.15
12
9.71 0.04
0.51 0.02
1.97 0.14
0.16 0.01
6.45 0.62
0.71 0.04
a
Data represent mean values SEM from three independent experiments each performed in duplicate. Flow cytometric measurements performed
b
with HEK293-SP-FLAG-hH3R (wild-type hH3R) cells. Data represent mean values SEM from three independent experiments each performed
in triplicate. NanoBRET binding experiments performed with live HEK293 cells stably expressing the NLuc-hH3R. Data represent mean values
SEM from four independent experiments. Association time constant: τass = 1/kobs. Data represent mean values CI (95%) from four independent
experiments. Dissociation time constant: τdiss = 1/koff. Data represent mean values CI (95%) from four independent experiments. Association
rate constant: kon = (kobs − koff)/c(12). Indicated errors were calculated according to the Gaussian law of error propagation. Kd (kin) = koff/kon;
pKd (kin) = −log Kd (kin). Indicated errors were calculated according to the Gaussian law of error propagation.
c
d
e
f
g
substitution reaction with 1-bromo-3-chloropropane. The
same reaction type using piperidine resulted in aldehyde 3. A
reductive amination with ethyl isonipecotate, using sodium
triacetoxyborohydride as a reductive agent in chloroform,
yielded 4. Acid catalyzed hydrolysis of 4 afforded the free acid
5 as a dihydrochloride.26
The synthesis of the linker (Scheme 1B), which is necessary
for the connection of the pharmacophore with the fluorophore,
started from the commercially available tetraethylene glycol 6.
In the first step, mesylate groups were introduced (7) as good
leaving groups, followed by subsequent treatment with sodium
azide to afford 8.32,33 A selective “Staudinger”-type reduction
of a single azide group in a biphasic water/ethyl acetate
mixture afforded the final linker 9 with 77% yield.34
Subsequently, 5 was coupled with 9 using EDC/HOBt as
coupling reagents under microwave irradiation to afford
product 10,26 which was reduced to the primary amine 11
by a “Staudinger” reaction (Scheme 2). In the final step, 11
was coupled with the 5-TAMRA NHS ester in DMF to afford
fluorescent ligand UR-NR266 (12, Scheme 2).28 After
preparative HPLC purification, 12 was obtained with high
purity (99%) and yield (82%). The fluorescent ligand was
further examined for its chemical stability in aqueous solution
and showed no decomposition within an incubation period of
6 months (cf. Figure S1, Supporting Information (SI)).
Fluorescence properties of 12 (in PBS containing 1% bovine
serum albumin (BSA)) were ascertained by recording its
excitation and emission spectra shown in Figure S2 (SI) and
measuring the quantum yield (Φ = 35.09% (in PBS), Φ =
33.31% (in PBS + 1% BSA); cf. Table S1 in the SI). The 5-
TAMRA-labeled ligand exhibited an excitation maximum at
555 nm and an emission maximum at 585 nm.
the compound was tested for suitability in the NanoBRET
binding assay at the NLuc-hH3R stably expressed in HEK293
cells. BRET saturation binding experiments provided a binding
constant for 12 in the sub-nanomolar range (pKd = 9.80
0.07, cf. Figure 1A; Table 1) and very low nonspecific binding.
In order to investigate a potential influence of the receptor
modification on ligand binding, 12 was additionally examined
in flow cytometry using wild-type hH3R cells (HEK293-SP-
FLAG-hH3R) (cf. Figure 1B).
The receptor affinity of 12 to wild-type H3Rs (pKd = 9.71
0.04, Table 1) was in the same range as for the NLuc-hH3Rs
showing no significant difference (p < 0.05, two-tailed t-test).
This experimental setup further confirmed the minimal
nonspecific binding of this ligand observed in the BRET
assay. To further characterize the fluorescent tracer, radio-
ligand competition binding experiments were performed at the
H1‑4R, providing information on receptor subtype selectivity
within the histamine receptor family. The ligand exhibited
receptor affinity to the H3R in a sub-nanomolar range with a
pKi of 9.52 0.08 and an outstanding selectivity profile being
at least 100,000-fold selective toward the human H1R, H2R and
H4R (cf. Figure 2; Table 2). Precursor compounds 4, 5, and 11
and reference compound JNJ-5207852 (JNJ) displayed H3R
affinities in the same range (Table 2). While pKi data of 4 are
in very good agreement with literature data (9.56 vs 9.6426),
we saw a difference for compound 5 (8.80 vs 7.8926).
Being aware of the fact that the fluorescent ligand’s
pharmacological mode of action is crucial, e.g., since an
agonist might distort apparent affinities of competitive ligands
due to internalization processes and induction of ternary
complex formation (ligand/H3R/G-protein), we wanted to
gain insights into the functional behavior of 12. Therefore, we
employed a recently developed BRET-based Gi2 biosensor
detecting G protein activation as a decrease in BRET between
NLuc-tagged Gαi2 and cpVenus-tagged Gγ2.37 We first
determined the potency of the selective H3R agonist imetit
using the Gi2 biosensor and HEK293A cells overexpressing the
Pharmacological Characterization. The first step in the
pharmacological characterization of the fluorescent ligand 12,
structurally derived from the H3R antagonist JNJ-5207852
(JNJ)25 (Figure S4, SI), was the investigation of the binding
behavior in different assays. To achieve one of our main goals,
C
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
enantiomers RAMH and SAMH, a phenomenon that has been
described several times at the hH3R (Table 3).40,44−46
Fluorescence Microscopy. To visualize the binding and
dissociation kinetics of the fluorescent ligand, we used confocal
microscopy imaging. In this experiment, time-lapse images of
HEK293-SP-FLAG-hH3R cells were acquired (Figure 6).
Compared to the kinetic NanoBRET experiments, we used
10 times higher concentrations of 12 to facilitate visualization
with a confocal microscope. After the addition of 12 (c = 5
nM), the fluorescence signal on the cell surface increased and
reached a plateau within 4 min (Figure 7A). The fluorescent
ligand’s dissociation was initiated by the addition of 2.5 μM
clobenpropit (500-fold excess). The addition of clobenpropit
(after 220 s) decreased the fluorescence signal, indicating the
dissociation of 12 (Figure 7B). We observed a τ value of 0.48
0.07 min for the association and 3.50 0.46 min for the
dissociation of the fluorescent ligand (Table 4). These
experiments demonstrated again that the binding of the
fluorescent ligand to the H3R occurs very rapidly and that the
bound ligand can be displaced completely, similar to the results
observed in the BRET binding assay. Due to the use of
different concentrations (see above), a difference in kobs values
was to be expected (cf. Tables 1 and 4). However, the kon and
Figure 2. Displacement curves from radioligand competition binding
experiments performed with compound 12 and the respective
radioligands (cf. Table 2 footnotes).
wild-type H3R (Figure S3, SI).37 As expected, 12 acted as a
neutral antagonist (Figure 3A) revealing a pKb value of 9.04
0.03 in competition experiments with the selective H3R agonist
imetit (Figure 3B). However, before using 12 as a molecular
tool to characterize other H3R ligands, the kinetic behavior of
the fluorescent ligand should be determined first. The
NanoBRET setup is ideally suited for this purpose due to
the possibility to perform real-time kinetic measurements. 500
pM 12 was used to measure ligand association at the H3R
(Figure 4). Ligand binding to the receptor saturated after
about 15 min (τass = 1.97 0.14 min), and the ligand fully
k
off values, which would be expected to be the same, differ by a
factor of nearly 2 (cf. Tables 1 and 4). This could be a result of
the receptor’s modification with the NanoLuc, which may
influence the binding kinetics. Consequently, the kinetic pKd
values from the NanoBRET (pKd = 9.66 0.15) and confocal
experiments (pKd = 9.10 0.06) differ but are still in good
agreement, so both assays can be considered complementary
(cf. Tables 1 and 4). Additionally, we tested the photo-
bleaching properties of 12 by immobilizing the fluorescent
ligand in a 0.5% agarose film in 1 μM final concentration. In
these experiments, the ligand shows negligible photobleaching
(4%, N = 4) after 20 min, which is unlikely to affect the
observed association and dissociation kinetics of 12. The
biphasic curve describing the photobleaching properties is
shown in Figure S28 in the SI.
dissociated with a tau (τ) value of 6.45
0.62 min upon
addition of an excess of clobenpropit (500-fold, c = 250 nM,
Figure 4; Table 1). All kinetic parameters describing the
binding of 12 in the BRET binding assay are presented in
Table 1.
The complete reversibility of receptor binding makes the
fluorescent ligand a suitable tool for use in competition binding
studies, in our case, performed with a selection of standard
H3R agonists and antagonists (see structures in Figure S4, SI).
We selected histamine (his),38 imetit (imet),39 (R)-(−)-α-
methylhistamine (RAMH),40 and (S)-(+)-α-methylhistamine
(SAMH)40 as agonists and clobenpropit (clob),41 Z27743747
(Z-cmpd),42 thioperamide (thio),40 pitolisant (pito),43 and
JNJ-5207852 (JNJ)25 as the inverse agonists/antagonists. For
all ligands, total displacement could be determined with Hill
slopes around 1 (Figure 5). Overall, our data are in good
agreement with data from the literature (cf. Table 3), apart
from RAMH and SAMH showing larger deviations. However,
it is remarkable that the assay can distinguish between the two
Due to the low nonspecific binding and the high affinity of
our compound, we wondered about its suitability for total
internal reflection fluorescence (TIRF) single-molecule mi-
croscopy as shown previously for a few other fluorescent
GPCR ligands.49−51 Indeed, its favorable properties were
confirmed in microscopy experiments and allowed the
acquisition of single-molecule TIRF movies in the presence
Table 2. Binding and Functional Data of 12 on Human Histamine Receptor Subtypes
a
b
radioligand competition binding
H3R selectivity
Ki (H1,2,4R)/Ki (H3R)
hH1,2,4
>100,000
Gi2 activation
pKi
pKb
c
d
e
f
g
compound
hH1R
N
hH2R
N
hH3R
N
hH4R
N
R
hH3R
N
12
4
5
11
JNJ
<4
3
<4
3
9.52 0.08
9.56 0.12
8.80 0.11
9.40 0.10
9.68 0.01
3
3
3
3
3
<4
3
9.04 0.03
n.d.
n.d.
n.d.
n.d.
3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a
Competition binding assay at HEK293-SP-FLAG-hH1R, HEK293-SP-FLAG-hH2R, HEK293-SP-FLAG-hH3R, or HEK293-SP-FLAG-hH4R cells.
b
c
Competition binding experiment at HEK293A cells stably expressing the Gi2 BRET sensor with the wild-type hH3R. Displacement of 5 nM
[3H]mepyramine (Kd = 4.5 nM). Displacement of 20 nM [3H]UR-DE25735 (Kd = 66.9 nM). Displacement of 2 nM [3H]UR-PI29436 (Kd = 5
d
e
f
g
nM). Displacement of 15 nM [3H]histamine (Kd = 15.88 nM). Inhibition of imetit-induced (c = 1 nM, EC50 = 0.85 nM) Gi2 activation. Data
shown are mean values SEM of N independent experiments, each performed in triplicate. Data were analyzed by nonlinear regression and were
best fitted to sigmoidal concentration−response curves. Displacement curves are presented in Figures 2 and 3.
D
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 3. Concentration−response curves (CRCs) for Gi activation of 12 in the absence (A) and presence (B) of 1 nM imetit at HEK293A cells
transiently expressing the Gi2 BRET sensor along with the wild-type hH3R.
Figure 4. BRET-based specific binding kinetics of the fluorescent ligand 12 at the NLuc-hH3R, stably expressed in HEK293 cells. (A) Graph shows
association of 12 (c = 500 pM) to the receptor and dissociation of 12 induced by addition of clobenpropit (c = 250 nM, 500-fold excess) from a
representative experiment. (B) Scatter dot plot displaying the quantification of tau (τ) values for association/dissociation of the fluorescent ligand.
Data were analyzed from four independent experiments and are represented as mean SD.
Table 3. Binding Data (pKi Values) of Standard H3R
Ligands Determined at the Human H3R in the NanoBRET
a
Binding Assay
NanoBRET UR-NR266
(12)
references
compound
pKi
N
pKi
his
imet
6.23 0.08
7.90 0.11
7.20 0.02
5.79 0.11
9.30 0.08
7.71 0.01
7.28 0.18
9.04 0.12
10.22 0.02
4
3
3
3
3
3
3
3
3
6.3;47 6.5;23 7.6;48 8.045
8.3;47 8.845
RAMH
SAMH
clob
Z-cmpd
thio
8.4;44 8.2;45 8.346
6.4;47 7.6;44 7.2;45 7.346
9.6;47 9.5;23 8.645
7.4;42 7.346
7.3;47 7.4;23 7.345
8.6;47 8.643
9.225
Figure 5. Displacement curves from BRET competition binding
experiments of the fluorescent ligand 12 (c = 500 pM) and reported
H3 receptor ligands at HEK293 cells, stably expressing the NLuc-
hH3R. “Vehicle” denotes the condition where the cells were not
incubated with 12. Abbreviations used: histamine (his), imetit (imet),
(R)-(−)-α-methylhistamine (RAMH), (S)-(+)-α-methylhistamine
(SAMH), clobenpropit (clob), Z27743747 (Z-cmpd), thioperamide
(thio), pitolisant (pito), and JNJ-5207852 (JNJ).
pito
JNJ
a
Data represent mean values
SEM from N independent
experiments, each performed in triplicate. NanoBRET experiments
were performed at live HEK293 cells stably expressing the NLuc-
hH3R as described in the Experimental Section. The standard H3R
ligands used are depicted in Figure S4 in the Supporting Information.
Abbreviations used: histamine (his), imetit (imet), (R)-(−)-α-
methylhistamine (RAMH), (S)-(+)-α-methylhistamine (SAMH),
clobenpropit (clob), Z27743747 (Z-cmpd), thioperamide (thio),
pitolisant (pito), and JNJ-5207852 (JNJ).
of 3 nM 12 in the imaging buffer with negligible background
fluorescence (Figure 8A). We further tested the labeling
efficiency under these conditions by using a H3R control
construct, which was C-terminally tagged with the photostable
fluorescent protein mNeonGreen. Dual color acquisition
revealed colocalization, a labeling efficiency of 96.1
2.1%,
and a nonspecific binding of 2.5 0.8% under these conditions
(Figure 8B). Single-particle tracking of the acquired TIRF
movies and subsequent diffusion analysis of the receptor tracks
(Figure 8C,D) showed similar receptor dynamics for the H3R
Off-Target Studies. Once selectivity within the histamine
receptor family was established, we sought to identify any
binding of 12 to other GPCR groups. We performed an off-
target screen using 14 different GPCRs. Therefore, we used the
BRET-based binding assay with transient NLuc-receptor
expression for our off-target screen. Unfortunately, the capacity
(diffusion coefficient of 0.085
0.006 μm2/s) as shown
previously for other class A GPCRs.50,51
E
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
hM4R was unsurprising as the binding sites of these receptors
share features with the hH3R, and several characteristics of
known H3R ligands have been proven to be useful in
developing muscarinic antagonists.56,57 It should be noted
that BRET signals from binding experiments to different
NLuc-GPCR constructs can only be compared with caution
given the different N-terminal lengths and localizations of
orthosteric binding pockets. Therefore, the low BRET signals
observed at hM2R and hM4R (only ∼10% of the BRET signal
detected with NLuc-hH3R) do not provide a quantitative value
but rather an estimate for the binding of 12 to the receptors.
Nevertheless, we believe that the low BRET signals for the
muscarinic receptors using a maximal concentration of 200 nM
do not indicate any off-target issues, as 12 has sub-nanomolar
affinity for the target hH3R.
CONCLUSIONS
■
In UR-NR266 (12), we described a sub-nanomolar affinity
fluorescent ligand at the H3R with an outstanding selectivity
profile within the histamine receptor family. The TAMRA-
labeled neutral antagonist was broadly characterized analyti-
cally and pharmacologically and showed excellent kinetic and
fluorescence properties for further investigations. These results
ensure excellent suitability for the recently described Nano-
BRET assay. The successful use as a fluorescent probe in the
NanoBRET binding assay highlights the versatility of 12 and
its suitability as a powerful tool in the urgently needed research
for new drug candidates at the H3R. A comprehensive off-
target screening at 14 different receptors reveals 12 as a
selective compound.
Due to its high affinity and exceptional receptor selectivity
combined with high brightness and the proven low nonspecific
binding, 12 shows remarkable results in fluorescence
microscopy and is the first fluorescent ligand to enable single
molecule imaging of the H3R. Furthermore, it allows the
presence of saturating concentrations in the imaging buffer
during single-molecule image acquisition. This accounts for the
obvious advantage of continuous labeling of receptors in the
course of the experiment and compensates for potential
fluorophore photobleaching. Thus, 12 is a versatile tool
suitable for binding and also imaging studies at the H3R. To
the best of our knowledge, this is unique within the panel of
available H3R fluorescent ligands lifting the study of H3R-
Figure 6. Time-lapse confocal microscopy images of the fluorescent
ligand 12: imaging of the HEK293-SP-FLAG-hH3R cells treated with
a 5 nM fluorescent ligand displayed a time-dependent increase of the
fluorescence signal on the cell surface, indicating ligand binding to the
H3R. Competing 12 with the unlabeled H3R antagonist clobenpropit
decreased the fluorescence signal in a time-dependent manner
(addition of clobenpropit after 220 s).
of the unpublished NLuc-receptor constructs to bind their
endogenous ligands could not be thoroughly controlled given
the lack of validated fluorescent ligands. Instead, it was only
presumed based on sufficient surface expression of these
constructs and based on the experience that N-terminal
insertion of the NLuc usually does not affect the binding ability
of class A GPCRs.15,52−55 Receptor cell surface expression was
confirmed by an ELISA using a NanoLuc antibody (Cat. No.
MAB100261, Bio-Techne, Minneapolis, MN, USA). Using 200
nM of 12, we observed little off-target effects with only the
muscarinic hM2R and hM4R displaying mild but significant
response (13.2
4.5 and 6.7
5.3% of hH3R response,
respectively; Figure 9). Some ligand binding at the hM2R and
Figure 7. Association and dissociation kinetics of the fluorescent ligand: (A) representative graph displaying the quantification of the fluorescence
signal obtained from experiments as in Figure 1. The fluorescence signal (in arbitrary units) was plotted as a function of time, and association/
dissociation kinetics were calculated by fitting an exponential decay function on the signal (from baseline to saturation, and from saturation to
decay to the baseline). Addition of the competitor clobenpropit after 220 s. (B) Scatter dot plot displaying the quantification of tau (τ) values for
association/dissociation of the fluorescent ligand. Five cells from three independent experiments were analyzed. Data are represented as mean
SD.
F
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 4. Kinetic Binding Constants of 12 at the Wild-Type hH3R in Confocal Microscopy
confocal microscopy
a
b
a
c
d
e
compound
kobs (min−1
)
τass (min)
koff (min−1
)
τdiss (min)
kon (min−1 nM−1
)
pKd (kin)
12
2.13 0.14
0.48 0.07
0.29 0.02
3.50 0.46
0.37 0.03
9.10 0.06
a
Five cells from three independent experiments were analyzed. Data represent mean values SEM. Confocal microscopy measurements performed
at HEK293-SP-FLAG-hH3R (wild-type hH3R) cells. Five cells from three independent experiments were analyzed. Data represent mean values
CI (95%). Association time constant: τass = 1/kobs. Five cells from three independent experiments were analyzed. Data represent mean values CI
b
c
d
(95%). Dissociation time constant: τdiss = 1/koff. Association rate constant: kon = (kobs − koff)/c(12). Indicated errors were calculated according to
e
the Gaussian law of error propagation. Kd (kin) = koff/kon; pKd (kin) = −log Kd (kin). Indicated errors were calculated according to the Gaussian
law of error propagation.
Figure 8. (A) Top panel: wild-type H3 receptors labeled with 3 nM UR-NR266 (12). Bottom panel: single particle tracking of individual wild-type
H3 receptors and classification of diffusion classes (magenta = directed diffusion, blue = confined diffusion, cyan = Brownian motion, and brown =
immobile). Shown images are representative of four independent experimental days. (B) Labeling efficiency measured by colocalization of the two
colors in their corresponding detection channels (GFP/Cy3) as shown in the representative image. Labeling efficiencies are for the unlabeled
control 2.5 1.8 and 96.1 5.2% at 3 nM 12, respectively. Data are mean SD and originate from three independent experiments. Each data
point refers to one cell. (C) Diffusion analysis based on single-particle tracking gives a diffusion coefficient of 0.085 0.006 μm2/s. The diffusion
type classification (D) is composed by 6.9 0.8% immobile, 13.5 0.7% confined, 73.3 1.3% Brownian motion, and 6.3 0.3% directed tracks
undergoing the corresponding type of diffusion. Data are mean SD and originate from three independent experiments. Each data point refers to
one cell.
Germany), or TCI Europe (Zwijndrecht, Belgium)) and were used
as received. All solvents were of analytical grade. The syntheses of
compounds 2−4 and 7−9 have already been described in the
literature.25,26,33,58,59 The fluorescent dye 5-TAMRA NHS ester was
purchased from Lumiprobe (Hannover, Germany). Deuterated
solvents for nuclear magnetic resonance (1H NMR and 13C NMR)
spectra were purchased from Deutero GmbH (Kastellaun, Germany).
All reactions carried out with dry solvents were accomplished in dry
flasks under a nitrogen or argon atmosphere. For the preparation of
buffers, HPLC eluents, and stock solutions, Millipore water was used.
Column chromatography was accomplished using Merck silica gel
Geduran 60 (0.063−0.200 mm) or Merck silica gel 60 (0.040−0.063
mm) (flash column chromatography). The reactions were monitored
by thin layer chromatography (TLC) on Merck silica gel 60 F254
aluminum sheets, and spots were visualized under UV light at 254 nm
by potassium permanganate or ninhydrin staining. Microwave assisted
reactions were performed on an Initiator 2.0 synthesizer (Biotage,
Uppsala, Sweden). Lyophilization was done with a Christ alpha 2−4
LD equipped with a Vacuubrand RZ 6 rotary vane vacuum pump.
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were
dependent disease-related functions onto another level.
Especially, the suitability in TIRF and confocal microscopy
makes 12 a valuable tool to further investigate the role of the
H3R in the central nervous system, as the H3R was reported to
form heteroreceptor complexes in combination with other
GPCRs and ion channels.7,9,10 This could be of great interest
for research on neurodegenerative diseases, e.g., Alzheimer’s
disease, as these receptor complexes have arisen as promising
targets to prevent neuronal cell death.10
EXPERIMENTAL SECTION
■
Chemistry. Commercially available chemicals (4-hydroxybenzal-
dehyde, 1-bromo-3-chloropropane, piperidine, sodium triacetoxybor-
ohydride, ethyl isonipecotate, tetraethylene glycol, methanesulfonyl
chloride, EDC, HOBt) and all other chemicals and solvents were
purchased from standard commercial suppliers (Merck (Darmstadt,
Germany), Sigma-Aldrich (Munich, Germany), Acros Organics (Geel,
Belgium), Alfa Aesar (Karlsruhe, Germany), abcr (Karlsruhe,
G
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
analyzed compound in % relative to the total peak area (UV detection
at 220 nm). The HPLC purity (see Figure S1, SI) of the final
compound 12 was 99%. The tested compound 12 has been screened
for PAINS and aggregation by publicly available filters (http://zinc15.
docking.org/patterns/home, http://advisor.docking.org).60,61 The
compound has not been previously reported as PAINS or an
aggregator. None of the data showed abnormalities, e.g., high Hill
slopes, what could be a hint for PAINS.61
Synthesis and Analytical Data. 4-(3-Chloropropoxy)-
benzaldehyde (2).25 A suspension of 4-hydroxybenzaldehyde (5.00
g, 40.94 mmol, 1 eq), 1-bromo-3-chloropropane (8.06 mL, 81.88
mmol, 2 eq), and K2CO3 (16.85 g, 121.92 mmol, 3 eq) in MeCN was
heated to reflux for 18 h. After cooling to room temperature, the solid
was filtered off and the filtrate was dried in vacuo. The resulting
residue was dissolved in EtOAc and washed with water and brine and
dried over Na2SO4, and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography
(PE/EtOAc 9:1). 2 (7.26 g, 89%) was obtained as a yellow oil. Rf =
1
0.40 (PE/EtOAc 8:1). H NMR (300 MHz, CDCl3) δ 9.89 (s, 1H),
Figure 9. Off-target affinity in BRET binding experiments of the
fluorescent ligand 12 (c = 200 nM) in HEK293T cells transiently
expressing NLuc-receptor constructs. Data were normalized to the
hH3R (100%) and empty vector control (0%). Transfection with an
empty pcDNA vector backbone is expressed as “pcDNA”. Receptor
abbreviations are as follows: H3, histamine H3; β1, β1 adrenoceptor;
CB1/2, cannabinoid receptor type 1/2; A3, adenosine A3; mSMO,
7.95−7.75 (m, 2H), 7.09−6.91 (m, 2H), 4.21 (t, J = 5.9 Hz, 2H),
3.76 (t, J = 6.2 Hz, 2H), 2.36−2.18 (m, 2H). 13C NMR (75 MHz,
CDCl3) δ 190.8, 163.7, 132.0, 130.1, 114.8, 64.6, 41.2, 32.0. HRMS
(ESI-MS): m/z [M + H+] calculated for C10H12ClO2 : 199.0520,
+
found 199.0516; C10H11ClO2 (198.65).
4-(3-(Piperidin-1-yl)propoxy)benzaldehyde (3).25 2 (7.22 g, 36.34
mmol, 1 eq), piperidine (5.38 mL, 54.51 mmol, 1.5 eq), Na2CO3
(5.78 g, 54.51 mmol, 1.5 eq), and KI (0.30 g, 1.82 mmol, 5 mol %)
were heated to reflux in MeCN for 20 h. The solvent was removed
under reduced pressure, and the residue was dissolved in DCM. The
organic phase was washed with water and brine and dried over
Na2SO4, and the solvent was removed under reduced pressure.
Column chromatography (DCM/MeOH 9/1 + 0.1% NEt3) afforded
3 (6.79 g, 76%) as a yellow oil. Rf = 0.48 (DCM/MeOH/7 N NH3 in
mouse smoothened; D1/2long/3, dopamine D1/2long/3; M1/2/3/4/5
,
muscarinic acetylcholine M1/2/3/4/5; AT1, angiotensin II type 1. All
receptor sequences are human, save the smoothened receptor
originating from the mouse. Lines represent mean
SD of four
experiments, with each experimental condition performed in
duplicate. Statistical significance was assessed by one-way ANOVA
followed by Fisher’s LSD post-hoc test against “pcDNA”; *: p < 0.05;
****: p < 0.0001.
1
MeOH 95:4:1). H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 7.88−
7.74 (m, 2H), 7.06−6.92 (m, 2H), 4.09 (t, J = 6.4 Hz, 2H), 2.51−
2.31 (m, 6H), 2.06−1.94 (m, 2H), 1.63−1.54 (m, 4H), 1.50−1.38
(m, 2H). 13C NMR (101 MHz, CDCl3) δ 190.8, 164.2, 132.0, 129.8,
114.8, 66.9, 55.7, 54.7, 26.7, 26.0, 24.4. HRMS (ESI-MS): m/z [M +
recorded on a Bruker (Karlsruhe, Germany) Avance 300 (1H: 300
MHz, 13C: 75 MHz), 400 (1H: 400 MHz, 13C: 101 MHz), or 600
(1H: 600 MHz) spectrometer using perdeuterated solvents. The
chemical shift δ is given in parts per million (ppm). Multiplicities
were specified with the following abbreviations: s (singlet), d
(doublet), t (triplet), q (quartet), quin (quintet), m (multiplet),
and br (broad signal) as well as combinations thereof. 13C NMR-
peaks were determined by DEPT 135 and DEPT 90 (distortionless
enhancement by polarization transfer). NMR spectra were processed
with MestReNova 11.0 (Mestrelab Research, Compostela, Spain).
High-resolution mass spectrometry (HRMS) was performed on an
Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS system (Agilent
Technologies, Santa Clara, CA) using an ESI source. Preparative
HPLC was performed with a system from Waters (Milford,
Massachusetts, USA) consisting of a 2524 binary gradient module,
a 2489 detector, a prep inject injector, and a fraction collector III. A
YMC-Triart C18 (150 × 20 mm, 5 μm; YMC Co. Ltd., Kyoto, Japan)
served as the stationary phase. As the mobile phase, 0.1% TFA in
Millipore water and acetonitrile (MeCN) were used. The temperature
was 25 °C, the flow rate was 20 mL/min, and UV detection was
performed at 220 nm. Analytical HPLC experiments were performed
on a 1100 HPLC system from Agilent Technologies equipped with an
Instant Pilot controller, a G1312A Bin Pump, a G1329A ALS
autosampler, a G1379A vacuum degasser, a G1316A column
compartment, and a G1315B DAD detector. The column was a
Phenomenex Kinetex XB-C18 column (250 × 4.6 mm, 5 μm)
(Phenomenex, Aschaffenburg, Germany), tempered at 30 °C. As the
mobile phase, mixtures of MeCN and aqueous TFA were used (linear
gradient: MeCN/TFA (0.1%) (v/v) 0 min: 10:90, 25−35 min: 95:5,
36−45 min: 10:90; flow rate = 1.00 mL/min, t0 = 3.21 min). Capacity
factors were calculated according to k = (tR − t0)/t0. Detection was
performed at 220 nm. Furthermore, filtration of the stock solutions
with PTFE filters (25 mm, 0.2 μm, Phenomenex Ltd., Aschaffenburg,
Germany) was carried out before testing. Compound purities
determined by HPLC were calculated as the peak area of the
+
H+] calculated for C15H22NO2 : 248.1645, found 248.1673;
C15H21NO2 (247.34).
Ethyl 1-(4-(3-(Piperidin-1-yl)propoxy)benzyl)piperidine-4-car-
boxylate (4).26 Sodium triacetoxyborohydride (5.35 g, 25.23 mmol,
1.3 eq) was added to a solution of 3 (4.80 g, 19.41 mmol, 1 eq) and
ethyl isonipecotate (3.29 mL, 21.35 mmol, 1.1 eq) in CHCl3 at room
temperature, and the reaction was stirred for 20 h. A saturated
solution of sodium bicarbonate was added, and the organic phase was
separated. The aqueous phase was extracted with DCM, the
combined organic phases were washed with brine and dried over
Na2SO4, and the solvent was removed under reduced pressure.
Column chromatography (DCM/MeOH 95/5 + 0.1% NH3) afforded
4 (7.18 g, 95%) as a yellow oil. Rf = 0.28 (DCM/MeOH/7 N NH3 in
MeOH 95:4:1). 1H NMR (300 MHz, CDCl3) δ 7.22−7.16 (m, 2H),
6.87−6.78 (m, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.99 (t, J = 6.4 Hz, 2H),
3.41 (s, 2H), 2.83 (d, J = 11.6 Hz, 2H), 2.53−2.36 (m, 6H), 2.32−
2.18 (m, 1H), 2.05−1.90 (m, 4H), 1.91−1.68 (m, 4H), 1.65−1.54
(m, 4H), 1.50−1.39 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ 175.3, 158.1, 130.3, 114.2, 66.5, 62.7, 60.3, 56.1, 54.6,
52.8, 41.3, 28.3, 26.8, 25.9, 24.4, 14.2. HRMS (ESI-MS): m/z [M +
+
H+] calculated for C23H37N2O3 : 389.2799, found 389.2800;
C23H36N2O3 (388.55).
1-(4-(3-(Piperidin-1-yl)propoxy)benzyl)piperidine-4-carboxylic
Acid Dihydrochloride (5).26 2 N aqueous HCl was added to a
solution of 4 (2.06 g, 5.30 mmol, 1 eq) in THF and stirred at room
temperature overnight. The solvent was evaporated, and the product
was dried in vacuo. A sticky brown oil was obtained (5, 2.10 g, 92%).
1H NMR (300 MHz, CD3OD) δ 7.55−7.47 (m, 2H), 7.07−6.98 (m,
2H), 4.27 (s, 2H), 4.14 (t, J = 5.8 Hz, 2H), 3.64−3.44 (m, 4H),
3.14−2.92 (m, 4H), 2.72−2.57 (m, 1H), 2.34−1.77 (m, 12H). 13C
NMR (101 MHz, CD3OD) δ 173.6, 159.9, 132.8, 121.2, 114.8, 64.9,
59.7, 54.4, 53.1, 51.0, 38.2, 25.3, 23.8, 22.9, 21.3. HRMS (ESI-MS):
H
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
m/z [M + H+] calculated for C21H33N2O3 : 361.2486, found
361.2490; C21H32N2O3 × 2 HCl (433.42).
was obtained for 11 (79 mg, 60%). Rf = 0.22 (DCM/MeOH/7 N
NH3 in MeOH 90:9:1). H NMR (400 MHz, DMSO-d6) δ 10.05−
1
((Oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) Dimetha-
nesulfonate (7).58 Triethylamine (43.06 mL, 308.92 mmol, 6 eq) was
added at 0 °C to a solution of tetraethylene glycol (6, 10.00 g, 51.49
mmol, 1 eq) and methanesulfonyl chloride (17.69 g, 154.46 mmol, 3
eq) in DCM, and the reaction was stirred at room temperature for 20
h. Water was added, and the organic phase was separated, washed
with brine, and dried over Na2SO4, and the solvent was removed
under reduced pressure. Column chromatography (DCM/MeOH
98/2) afforded 7 (14.03 g, 78%) as a slightly yellow oil. Rf = 0.70
(DCM/MeOH 95:5). 1H NMR (300 MHz, CDCl3) δ 4.42−4.29 (m,
4H), 3.80−3.70 (m, 4H), 3.69−3.57 (m, 8H), 3.06 (s, 6H). 13C NMR
(75 MHz, CDCl3) δ 70.6, 70.5, 69.3, 69.0, 37.7. HRMS (ESI-MS):
9.65 (m, 2H), 8.04 (t, J = 5.4 Hz, 1H), 7.90 (br s, 3H), 7.45−7.37 (m,
2H), 7.06−6.93 (m, 2H), 4.27−4.26 (m, 2H), 4.06 (t, J = 5.9 Hz,
2H), 3.66−3.43 (m, 12H), 3.42−3.28 (m, 4H), 3.26−3.13 (m, 4H),
3.02−2.75 (m, 6H), 2.42−2.29 (m, 1H), 2.20−2.05 (m, 2H), 1.92−
1.54 (m, 9H), 1.46−1.28 (m, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 173.3, 159.5, 159.0, 158.7, 133.3, 122.2, 118.5, 115.6, 115.1, 70.2,
70.1, 70.1, 70.0, 69.4, 67.1, 65.5, 59.0, 53.8, 52.6, 51.0, 39.0, 38.9,
26.2, 23.9, 23.0, 21.7. HRMS (ESI-MS): m/z [M + H+] calculated for
+
C29H51N4O5 : 535.3854, found 535.3855; C29H50N4O5 × 3 TFA
(876.81).
2-(6-(Dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-
((1-oxo-1-(1-(4-(3-(piperidin-1-yl)propoxy)benzyl)piperidin-4-yl)-
5,8,11-trioxa-2-azatridecan-13-yl)carbamoyl)benzoate Dihydrotri-
fluoroacetate (12). 11 (7.89 mg, 9 μmol, 1.5 eq) was dissolved in
DMF (30 μL). NEt3 (6.68 mg, 66 μmol, 11 eq) and 5-
carboxytetramethylrhodamine succinimidyl ester (5-TAMRA NHS
ester) (3.17 mg, 6 μmol, 1 eq) in DMF (60 μL) were added, and the
reaction was shaken for 2.5 h in the dark at room temperature. The
reaction was quenched with 10% aqueous TFA (20 μL), and the
crude product was purified by preparative HPLC. A pink solid was
obtained for 12 (5.7 mg, 4.9 μmol, 82%). RP-HPLC: 99%, (tR = 9.84
+
m/z [M + H+] calculated for C10H23O9S2 : 351.0778, found
351.0783; C10H22O9S2 (350.40).
1-Azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane (8).33
7
(5.00 g, 14.27 g, 1 eq) and sodium azide (3.71 g, 57.08 mmol, 4
eq) were dissolved in EtOH/DMF (4/1) and heated to reflux for 20
h. The solvent was removed under reduced pressure, the residue was
dissolved in Et2O, the organic phase was washed subsequently with
water and saturated ammonium chloride solution and dried over
Na2SO4, and the solvent was removed under reduced pressure. The
product was dried in vacuo. A colorless oil was obtained for 8 (3.14 g,
1
min, k = 2.07). H NMR (600 MHz, DMSO-d6) δ 9.84−9.57 (m,
1
1H), 9.48 (br s, 1H), 8.95 (t, J = 5.5 Hz, 1H), 8.65 (br s, 1H), 8.28
(d, J = 7.9 Hz, 1H), 8.01 (t, J = 5.7 Hz, 1H), 7.54 (br s, 1H), 7.45−
7.38 (m, 2H), 7.10−6.97 (m, 4H), 6.94 (br s, 2H), 6.56 (br s, 2H),
4.20 (s, 2H), 4.05 (t, J = 6.0 Hz, 2H), 3.64−3.43 (m, 18H), 3.29−
3.04 (m, 16H), 2.94−2.82 (m, 4H), 2.39−2.32 (m, 1H), 2.18−2.08
(m, 2H), 2.04−1.60 (m, 9H), 1.45−1.31 (m, 1H). HRMS (ESI-MS):
90%). H NMR (300 MHz, CDCl3) δ 3.71−3.62 (m, 12H), 3.43−
3.33 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 70.7, 70.1, 50.7. HRMS
+
(ESI-MS): m/z [M + H+] calculated for C8H17N6O3 : 245.1357,
found 245.1359; C8H16N6O3 (244.26).
2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethan-1-amine (9).59
8
(2.00 g, 8.19 mmol, 1 eq) was dissolved in a mixture of 1 N aqueous
HCl, THF, and EtOAc (5/1/5 v/v/v). Triphenylphosphine (2.14 g,
8.19 mmol, 1 eq) in EtOAc was added slowly at room temperature,
and the reaction was stirred overnight. The reaction was basified with
aqueous NaOH solution, the solvent was removed under reduced
pressure, and the crude product was purified by column
chromatography (DCM/MeOH 95/5 + 0.1% NH3). A slightly yellow
oil was obtained for 9 (1.37 g, 77%). Rf = 0.32 (DCM/MeOH/7 N
+
m/z [M + H+] calculated for C54H71N6O9 : 947.5277, found
947.5293; C54H70N6O9 × 2 TFA (1175.23).
Fluorescence Properties. Excitation and emission spectra of 12
were recorded in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4, pH 7.4) containing 1% BSA (Sigma-
Aldrich, Munich, Germany) using a Cary Eclipse spectrofluorometer
(Varian Inc., Mulgrave, Victoria, Australia) at 22 °C, in acryl cuvettes
1
(10 × 10 mm, Sarstedt, Numbrecht, Germany). The slit adjustments
̈
NH3 in MeOH 95:4:1). H NMR (300 MHz, CDCl3) δ 3.70−3.59
(m, 12H), 3.40 (m, 2H), 3.04 (t, J = 5.0 Hz, 2H). 13C NMR (75
MHz, CDCl3) δ 70.5, 70.4, 70.1, 69.9, 69.4, 50.7, 40.4. HRMS (ESI-
(excitation/emission) were 5/10 nm for excitation spectra and 10/5
nm for emission spectra. Net spectra were calculated by subtracting
the respective vehicle reference spectrum, and corrected emission
spectra were calculated by multiplying the net emission spectra with
the respective lamp correction spectrum. The quantum yield of 12
was determined according to previously described procedures23,62
with minor modifications using a Cary Eclipse spectrofluorometer
(Varian Inc., Mulgrave, Victoria, Australia) at 22 °C, using acryl
+
MS): m/z [M + H+] calculated for C8H19N4O3 : 219.1452, found
219.1453; C8H18N4O3 (218.26).
N-(2-(2-Azidoethoxy)ethyl)-1-(4-(3-(piperidin-1-yl)propoxy)-
benzyl)piperidine-4-carboxamide (10). 5 (100 mg, 0.23 mmol, 1
eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (48 mg, 0.25
mmol, 1.1 eq), 1-hydroxybenzotriazole (34 mg, 0.25 mmol, 1.1 eq),
and DIPEA (119 mg, 0.92 mmol, 4 eq) were dissolved in DMF/DCM
(1/1) in a microwave vial and stirred at room temperature for 30 min.
9 (50 mg, 0.23 mmol, 1 eq) was added, and the reaction was stirred
30 min at 100 °C in a microwave reactor (sealed vial). The solvent
was removed under reduced pressure, and the crude product was
purified by column chromatography (DCM/MeOH 95/5 + 0.1%
NH3). A brown oil was obtained for 10 (85 mg, 65%). Rf = 0.36
cuvettes (10 × 10 mm, Sarstedt, Numbrecht, Germany) and cresyl
̈
violet perchlorate (Biomol GmbH − Life Science Shop, Hamburg,
Germany) as a red fluorescent standard. Absorption spectra were
recorded by UV/Vis spectroscopy (350−850 nm, scan rate: 300 nm/
min, slits: fixed 2 nm) at a concentration of 2 μM for cresyl violet (in
EtOH, λabs,max = 575 nm) and 12 (in PBS buffer and PBS + 1% BSA,
λabs,max = 550 nm). The quantum yields were calculated for three
different slit adjustments (exc./em.): 5/5, 10/5, and 10/10 nm. The
means of the quantum yields, absorption and emission maxima, and
absorbance are presented in Table S1 in the Supporting Information.
BRET-Based Saturation/Real-Time Kinetics/Competition
Binding at the NLuc-H3R. BRET-based saturation/real-time
kinetics/competition binding at the NLuc-H3R were performed as
previously described by Bartole et al. with minor modifications.23 For
the determination of nonspecific binding, clobenpropit (500-fold
excess) was used instead of thioperamide. Dissociation was initiated
by addition of 250 nM clobenpropit (500-fold excess) instead of
thioperamide. For competition binding experiments, 12 was used in a
concentration of 500 pM. Histamine dihydrochloride (his) was from
TCI Chemicals (Tokyo, Japan). Imetit dihydrobromide (imet), (R)-
(−)-α-methylhistamine dihydrobromide (RAMH), (S)-(+)-α-meth-
ylhistamine dihydrobromide (SAMH), clobenpropit dihydrobromide
(clob), and thioperamide maleate (thio) were from Tocris Bioscience
(Ellisville, MO, USA). Z27743747 (Z-cmpd) was from Enamine Ltd.
1
(DCM/MeOH/7 N NH3 in MeOH 90:9:1). H NMR (300 MHz,
CDCl3) δ 7.20−7.12 (m, 2H), 6.84−6.75 (m, 2H), 6.08 (t, J = 5.2
Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 3.66−3.55 (m, 10H), 3.53−3.46
(m, 2H), 3.37 (m, 6H), 2.87 (m, 2H), 2.50−2.30 (m, 6H), 2.13−1.84
(m, 5H), 1.73 (m, 4H), 1.56 (m, 4H), 1.47−1.33 (m, 2H). 13C NMR
(101 MHz, CDCl3) δ 175.1, 158.1, 130.2, 130.2, 114.1, 70.7, 70.6,
70.6, 70.2, 70.1, 69.9, 66.5, 62.6, 56.0, 54.6, 53.0, 50.7, 43.4, 39.0,
28.9, 26.8, 25.9, 24.4. HRMS (ESI-MS): m/z [M + H+] calculated for
+
C29H49N6O5 : 561.3759, found 561.3767; C29H48N6O5 (560.74).
N-(2-(2-Aminoethoxy)ethyl)-1-(4-(3-(piperidin-1-yl)propoxy)-
benzyl)piperidine-4-carboxamide (11). Triphenylphosphine (60 mg,
0.23 mmol, 1.5 eq) was added to a solution of 10 (85 mg, 0.15 mmol,
1 eq) in THF, and the reaction was stirred for 4 h at 45 °C. Water was
added, and the reaction was stirred for another 2 h at the same
temperature. THF was removed under reduced pressure, and
trifluoroacetic acid (200 μL) was added to the aqueous solution.
The crude product was purified by preparative HPLC. A colorless oil
I
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(Kyiv, Ukraine). Pitolisant hydrochloride (pito) was kindly provided
by Prof. Dr. Katarzyna Kiec-Kononowicz (Jagiellonian University,
Krakow). Significant differences between pKd values were assessed
using a two-tailed t-test (p < 0.05).
(Gain 4000) monochromator settings, respectively, and an integration
time of 0.3 s. Raw BRET ratios were defined as acceptor emission/
donor emission. The three BRET ratios prior to ligand/vehicle
addition were averaged and defined as BRETbasal. To quantify ligand-
induced BRET changes, ΔBRET was calculated for each well and
time point as a percent over basal ([(BRETstim − BRETbasal)/
BRETbasal] × 100). Subsequently, the average ΔBRET of vehicle-
treated control wells was subtracted. Ligand concentration response
curves were generated based on vehicle-corrected ΔBRET measured
as a mean of three subsequent reads, 23 min after ligand addition.
Live Cell Confocal Microscopy. HEK293 cells stably transfected
with a vector plasmid for H3R expression were maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) (PAN Biotech,
Aidenbach, Germany) supplemented with 10% Fetal Bovine Serum
(FBS) (PAN , Aidenbach, Germany Biotech), 1% L-Glutamine (PAN
Biotech, Aidenbach, Germany), penicillin (100 U/mL), and
streptomycin (100 μg/mL) at 37 °C and 5% CO2. Cells were
passaged every 2−3 days.
For imaging, 300,000 HEK293-H3R cells were seeded on poly-D-
lysine coated coverslips in 6-well plates. 24 h later, coverslips were
transferred to an Attofluor cell chamber (Thermo Fisher Scientific,
Nidderau, Germany) and supplemented with Fluorobrite DMEM
medium (Thermo Fisher, Nidderau, Germany), and then the
chamber was placed on a microscope stage.
Imaging was performed using a Leica SP8 laser scanning confocal
microscope equipped with a 40×/1.25 NA oil immersion objective, a
white light laser (WLL), and photon counting hybrid (HyD)
detectors. For excitation, the WLL was set to 552 nm at 10%
power. Fluorescence was detected within an emission window of
557−764 nm. Image size was set to 512 × 512 pixels, and the
scanning speed was 700 Hz. After adjusting the focus, time-lapse cell
imaging was performed with a 1 s interval between each image. After
imaging for 10 s of baseline, the fluorescent ligand was added by using
a micropipette at a final concentration of 5 nM. After imaging for 4−5
min, clobenpropit was added at a final concentration of 2.5 μM,
maintaining the 5 nM fluorescent ligand concentration, and imaging
was completed after 20 min.
Time-lapse confocal images were processed using the ImageJ
software. Contrast was adjusted for each file to facilitate the
visualization of the fluorescence signal. Total fluorescence (in
arbitrary units) was plotted as a function of time using the Origin
2018 software. Association and dissociation kinetics were calculated
using the “ExpDec” function in the software. Each kinetic data was
pooled and plotted as a scatter dot-plot using the GraphPad 7
software.
Experiments to demonstrate the photobleaching properties were
performed according to the method described above. The fluorescent
ligand was immobilized in a 0.5% agarose film in 1 μM final
concentration of the fluorescent ligand UR-NR266 (12) and imaged
over a period of 20 min.
TIRF-Imaging. For single-molecule experiments, CHO-cells
(ATTC/LGC Standards, Wesel, Germany) were seeded 24 h before
transfection. Transfection was done 4−6 h before TIRF-imaging using
Lipofectamine 2000 (Thermo Fisher Scientific, Nidderau, Germany).
For each single well of a 6-well cell culture plate (Brand, Wertheim,
Germany), 2 μg of the desired DNA was diluted in 500 μL of Opti-
MEM (Thermo Fisher Scientific, Nidderau, Germany) and mixed
with another dilution containing 6 μL of Lipofectamine 2000
transfection reagent in 500 μL of Opti-MEM. After incubation at
25 °C for 20 min, this mixture (total = 1 mL) was added to a single
well of cell culture plate, containing 1 mL of DMEM-F12 medium
(Thermo Fisher Scientific, Nidderau, Germany). After 4−6 h,
expression levels were sufficient for single-molecule experiments and
the medium was exchanged with a new DMEM-F12 medium.
All TIRF-imaging experiments were performed with transiently
transfected CHO cells as described above. To measure the labeling
efficiency of UR-NR-266 (12), a C-terminally mNeonGreen tagged
H3R was expressed (H3-mNG). Cells were labeled with 10 nM of 12
for 15 min. After labeling, cells were washed once with PBS and taken
for imaging to an Attofluor Cell Chamber (Invitrogen, Carlsbad,
Flow Cytometry. Flow cytometric saturation binding experiments
at the H3 receptor were performed with a FACSCanto II flow
cytometer (Becton Dickinson, Heidelberg, Germany) equipped with
an argon laser (488 nm) and a red diode laser (640 and 635 nm).
Fluorescence signals were recorded using the following instrument
settings: excitation: 488 nm, emission: 585 21 nm (PE channel). All
samples were prepared and incubated in 1.5 mL cups (Eppendorf,
Hamburg, Germany). Cells were seeded 5 to 6 days prior to the
experiment in cell culture flasks. On the day of the experiment, the
cells were treated with trypsin/EDTA (0.05%/0.02%) (Sigma-
Aldrich, Munich, Germany), detached, and suspended in the cell
culture medium followed by centrifugation. The cells were
resuspended in Leibovitz’s L-15 medium (Fisher Scientific, Nidderau,
Germany) with 1% BSA (in the following, referred to as L-15
medium). The cell density was adjusted to 100,000 cells/mL. For the
total binding experiments, 2.5 μL of a solution of the fluorescent
ligand (100-fold concentrated to final concentration) in the L-15
medium and 2.5 μL of L-15 medium were added to 245 μL of the cell
suspension. For the nonspecific binding experiments, 2.5 μL of
solution of the fluorescent ligand (100-fold concentrated to final
concentration) and 2.5 μL of a 1 mM solution of clobenpropit in the
L-15 medium were added to 245 μL of the cell suspension. NR266
(12) was used at final concentrations of 0.02−10 nM. All samples
were incubated at 22 °C in the dark under shaking for 1 h. All
experiments were performed in duplicate.
Radioligand Competition Binding. Radioligand competition
binding experiments were performed as previously described by
Pockes et al. with minor modifications.48 All experiments were carried
out on whole HEK cells instead of Sf9 membranes. Generation of the
stable HEK293-SP-FLAG-hH1R and HEK293-SP-FLAG-hH2R cell
lines was conducted as described for the HEK293-SP-FLAG-hH3R
and HEK293-SP-FLAG-hH4R.23 Ligand dilutions of 12 were
prepared 10-fold concentrated in L-15 with 1% BSA, and 10 μL/
well was transferred to a flat-bottom polypropylene 96-well microtiter
plate (Greiner Bio-One, Frickenhausen, Germany), as well as 10 μL/
well of the respective radioligand. The cells were adjusted to a density
of 1.25 × 106 cells/mL, and 80 μL of the cell suspension was added to
each well (total volume of 100 μL). All data were analyzed using
GraphPad Prism8 software (San Diego, CA, USA). The normalized
competition binding curves were then fitted with a four-parameter
logistic fit yielding pIC50-values. These were transformed into pKi-
values using the Cheng−Prusoff equation.63
BRET Measurements of Ligand-Induced Gi2 Activation. The
Gi2 BRET sensor was generated as previously described.37 For the
BRET measurements, HEK293A cells were transiently transfected in a
suspension with wild-type H3R and Gi2 BRET sensor (1:1 plasmid
ratio) using Lipofectamine 2000 (Thermo Fisher Scientific, Nidderau,
Germany) (2 μL transfection reagent/μg total plasmid) and seeded
onto poly-D-lysine-precoated, white 96-well plates (Greiner Bio-One,
Frickenhausen, Germany) (30,000 cells/well). 48 h after transfection,
cells were washed with HBSS (Gibco/Life Technologies, Carlsbad,
USA) and incubated with a 1/1000 stock solution of furimazine
(Promega, Mannheim, Germany). 5 min later, baseline BRET was
recorded in three consecutive reads (4 min), 10-fold serial ligand
dilutions or vehicle control was added, and the ligand-induced BRET
ratio was recorded in 17 consecutive reads (36 min). For competition
experiments, cells grown in 96-well plates were washed 48 h after
transfection as described above and incubated with a 1/1000 stock
solution of furimazine along with the indicated concentrations of
compound 12 or ultrapure water (solvent control). After recording
the basal BRET ratio, all wells pre-incubated with 12 were stimulated
with 1 nM imetit. Wells pre-incubated with ultrapure water were
treated with HBSS (vehicle control for imetit treatment). All
experiments were conducted using a CLARIOstar plate reader
(BMG Labtech, Ortenberg, Germany) recording NLuc and cpVenus
emission intensities with 450/80 nm (Gain 3600) and 530/30 nm
J
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
USA) in sterile filtered PBS (Sigma-Aldrich, Munich, Germany)
containing 3 nM 12. For single-molecule imaging, a TIRF illuminated
Nikon Eclipse Ti2 microscope (Nikon, Tokyo, Japan) equipped with
a 100× 1.49NA automated correction collar objective and 405, 488,
561, and 647 nm laser diodes coupled via an automated N-Storm
module and four iXon Ultra 897 EMCCD cameras (Andor, Tokyo,
Japan) was used. The objective and sample were kept at 20 °C during
imaging. The automated objective collar was on, exposure times were
set to 40 ms, and the perfect focus system (Nikon, Tokyo, Japan) was
activated. Emission of TAMRA was recorded using a Cy3 Filter
(Chroma, Vermont, USA), and mNeonGreen was recorded using a
GFP Filter (Chroma, Vermont, USA).
Image analysis of the obtained TIRF-movies was done by first
cropping the image to the desired size, region, and frame-number
using Fiji.64 Afterward, movies were loaded in the Matlab environ-
ment (MathWorks) using u-Track,65 and requested parameters were
adjusted according to the abovementioned imaging conditions. Spot-
Detection, Tracking, and Motion-Analysis modules were then applied
and analyzed as previously described, as well as the assignment of
diffusion classes.51,66
Single-molecule imaging (AVI)
Single-particle tracking (AVI)
AUTHOR INFORMATION
Corresponding Authors
■
Lukas Grätz − Institute of Pharmacy, University of
Regensburg, Regensburg 93053, Germany;
Email: lukas.graetz@ur.de
Steffen Pockes − Institute of Pharmacy, University of
Regensburg, Regensburg 93053, Germany; Department of
Neurology, University of Minnesota, Minneapolis, Minnesota
55455, United States; Department of Medicinal Chemistry,
Institute for Therapeutics Discovery and Development,
University of Minnesota, Minneapolis, Minnesota 55414,
United States; orcid.org/0000-0002-2211-9868;
Email: steffen.pockes@ur.de
Authors
Off-Target Screening Using the NanoBRET Binding Assay.
The NLuc construct of the mouse smoothened receptor53 was kindly
provided by Prof. Dr. Gunnar Schulte (Karolinska Institutet), the
adenosine A3 receptor15 by Prof. Dr. Stephen J. Hill (University of
Nottingham), the β1 adrenoceptor by Dr. Ulrike Zabel (University of
Niklas Rosier − Institute of Pharmacy, University of
Regensburg, Regensburg 93053, Germany
Hannes Schihada − Section of Receptor Biology & Signaling,
Dept. of Physiology & Pharmacology, Karolinska Institutet,
Stockholm 171 77, Sweden; orcid.org/0000-0002-1889-
1636
̈
Jan Möller − Max Delbruck Center for Molecular Medicine,
Berlin 13125, Germany; Institute of Pharmacology and
Toxicology and Rudolf Virchow Center, University of
Wu
̈
rzburg), the muscarinic acetylcholine M2 receptor54 by Dr. Lukas
̈
Gratz (University of Regensburg), and the dopamine D1 receptor by
̈
Denise Monnich (University of Regensburg). All other constructs
̈
were made for these purposes by Dr. Lukas Gratz and Dr. Hannes
Schihada. For the BRET-based off-target screening, HEK293T cells
were transiently transfected in a suspension with 1 μg of cDNA per
mL of cell suspension (300,000 cells/mL). The receptors were the
histamine H3, β1 adrenoceptor, cannabinoid receptor types 1 and 2,
adenosine A3, mouse smoothened, dopamine D1/2long/3, muscarinic
acetylcholine M1/2/3/4/5, and angiotensin II type 1 receptor and were
prepared by replacing the receptor sequence of the NLuc-A3
construct. After 5 min of incubation, the mixtures were combined
and incubated at room temperature for a further 10 min. HEK293T
cells were suspended at 300,000 cells/mL in DMEM (10% FCS), and
1 mL of cell suspension was added to each receptor/PEI mix. Seeding
consisted of 100 μL of cDNA/cell/PEI mixture per well on a poly-D-
lysine (mol wt 70,000−150,000, Sigma-Aldrich, Taufkirchen,
Germany) coated white 96 well plate (cellGrade, Brand, Wertheim,
Germany). Cells were used 36 h after transfection. Media was
aspirated and washed with HBSS/0.5% BSA, before being replaced
with 90 μL of HBSS/0.5% BSA plus furimazine (1:1000 dilution) and
incubated for 5 min at 37 °C. Bioluminescence and fluorescence were
read on the Tecan GENios Pro, using the same parameters as the
other BRET experiments at 37 °C. The baseline was read for 5 cycles,
and the response post ligand addition was read for another 10 min.
Buffer control and 200 nM 12 were used in duplicate for each
receptor.
Wu
Ali Isb
rzburg, Wurzburg 97070, Germany
̈ ̈
̧
ilir − Max Delbruck Center for Molecular Medicine,
̈
Berlin 13125, Germany; Institute of Pharmacology and
Toxicology and Rudolf Virchow Center, University of
Wurzburg, Wurzburg 97070, Germany
̈ ̈
Laura J. Humphrys − Institute of Pharmacy, University of
Regensburg, Regensburg 93053, Germany
Martin Nagl − Institute of Pharmacy, University of
Regensburg, Regensburg 93053, Germany
Ulla Seibel − Institute of Pharmacy, University of Regensburg,
Regensburg 93053, Germany
̈
Martin J. Lohse − Max Delbruck Center for Molecular
Medicine, Berlin 13125, Germany; Institute of Pharmacology
and Toxicology and Rudolf Virchow Center, University of
Wurzburg, Wurzburg 97070, Germany; ISAR Bioscience
̈ ̈
Institute, Planegg 82152, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c01089
Author Contributions
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01089.
■
N.R. contributed to the following: chemistry, structural
analysis, FACS experiments, fluorescence properties, radio-
ligand binding studies, and manuscript writing. L.G. con-
tributed to the following: conception, project administration,
NanoBRET studies, and manuscript writing. H.S. contributed
to the following: the Gi2-BRET sensor and studies and
manuscript writing. J.M. contributed to the following: TIRF
microscopy and manuscript writing. A.I. contributed to the
following: confocal microscopy and manuscript writing. L.J.H.
contributed to the following: off-target screening, live cell
ELISA, and manuscript writing. M.N. contributed to the
following: chemistry, radioligand binding studies, and manu-
script writing. U.S. contributed to the following: cell culture
and manuscript writing. M.J.L. contributed to the following:
providing infrastructure and manuscript writing. S.P. con-
sı
Chemical stability (Figure S1), fluorescence properties
(Figure S2, Table S1), imetit-induced Gi2 activation
(Figure S3), structures of competitive histamine
receptor ligands (Figure S4), kinetic binding data from
confocal microscopy (Figure S5), supplementary data
from TIRF microscopy (Figure S6), synthesis of 1-[4-
(3-piperidin-1-ylpropoxy)benzyl]piperidine (JNJ-
5207852) (Scheme S1), NMR spectra (Figures S7−
S26), live cell ELISA for surface expression (Figure
S27), photobleaching (Figure S28) (PDF)
Molecular Formula Strings (CSV)
K
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
tributed to the following: conception, project administration,
FACS experiments, and manuscript writing. The manuscript
was written through contributions of all authors. All authors
have given approval to the final version of the manuscript.
Kb in M; pKi, negative logarithm of Ki in M; ppm, parts per
million; PTFE, polytetrafluoroethylene; RAMH, (R)-(−)-α-
methylhistamine; RP-HPLC, reversed-phase HPLC; Q-TOF,
quadrupole time of flight; RET, resonance energy transfer; sat,
saturation; SAMH, (S)-(+)-α-methylhistamine; SD, standard
deviation; s, second; SEM, standard error of the mean; t0, dead
time; 5-TAMRA NHS-ester, 5-carboxytetramethylrhodamine
succinimidyl ester; thio, thioperamide; TIRF, total internal
reflection fluorescence; tR, retention time; TR-FRET, time-
Funding
This work was supported by the Deutsche Forschungsgemein-
schaft (DFG, German Research Foundation − 427840891) to
H.S. Financial support by the Deutsche Forschungsgemein-
schaft (DFG, Research Training Group GRK1910) is gratefully
acknowledged (L.G., L.J.H.). Financial support by the graduate
school “Receptor Dynamics” of the Elite Network of Bavaria
(ENB) is gratefully acknowledged (N.R., H.S., J.M., A.I., M.N.,
S.P.). Financial support by the EU training network Oncornet
and the SFB 1423 is gratefully acknowledged (A.I., M.J.L.).
̈
resolved Forster resonance energy transfer; τass, association
time constant; τdiss, dissociation time constant; WLL, white
light laser; Z-cmpd, Z27743747
REFERENCES
■
(1) Arrang, J.-M.; Garbarg, M.; Schwartz, J.-C. Auto-Inhibition of
Brain Histamine Release Mediated by a Novel Class (H3) of
Histamine Receptor. Nature 1983, 302, 832−837.
(2) Clark, E. A.; Hill, S. J. Sensitivity of Histamine H3 Receptor
Agonist-Stimulated [35S] GTPγ [S] Binding to Pertussis Toxin. Eur. J.
Pharmacol. 1996, 296, 223−225.
Notes
The authors declare no competing financial interest.
Ligand and plasmids are available from the corresponding
authors upon reasonable request.
(3) Takeshita, Y.; Watanabe, T.; Sakata, T.; Munakata, M.; Ishibashi,
H.; Akaike, N. Histamine Modulates High-Voltage-Activated Calcium
Channels in Neurons Dissociated from the Rat Tuberomammillary
Nucleus. Neuroscience 1998, 87, 797−805.
(4) Arrang, J.-M.; Devaux, B.; Chodkiewicz, J.-P.; Schwartz, J.-C. H3-
Receptors Control Histamine Release in Human Brain. J. Neurochem.
1988, 51, 105−108.
(5) Lovenberg, T. W.; Roland, B. L.; Wilson, S. J.; Jiang, X.; Pyati, J.;
Huvar, A.; Jackson, M. R.; Erlander, M. G. Cloning and Functional
Expression of the Human Histamine H3 Receptor. Mol. Pharmacol.
1999, 55, 1101−1107.
(6) Rapanelli, M. The Magnificent Two: Histamine and the H3
Receptor as Key Modulators of Striatal Circuitry. Prog. Neuro-
Psychopharmacol. Biol. Psychiatry 2017, 73, 36−40.
(7) Ferrada, C.; Ferré, S.; Casadó, V.; Cortés, A.; Justinova, Z.;
Barnes, C.; Canela, E. I.; Goldberg, S. R.; Leurs, R.; Lluis, C.; Franco,
R. Interactions between Histamine H3 and Dopamine D2 Receptors
and the Implications for Striatal Function. Neuropharmacology 2008,
55, 190−197.
(8) Goodchild, R. E.; Court, J. A.; Hobson, I.; Piggott, M. A.; Perry,
R. H.; Ince, P.; Jaros, E.; Perry, E. K. Distribution of Histamine H3
-Receptor Binding in the Normal Human Basal Ganglia: Comparison
with Huntington’s and Parkinson’s Disease Cases: Histamine H3-
Receptors in the Human Basal Ganglia. Eur. J. Neurosci. 1999, 11,
449−456.
(9) Moreno-Delgado, D.; Puigdellívol, M.; Moreno, E.; Rodríguez-
Ruiz, M.; Botta, J.; Gasperini, P.; Chiarlone, A.; Howell, L. A.;
Scarselli, M.; Casadó, V.; Cortés, A.; Ferré, S.; Guzmán, M.; Lluís, C.;
Alberch, J.; Canela, E. I.; Ginés, S.; McCormick, P. J. Modulation of
Dopamine D1 Receptors via Histamine H3 Receptors Is a Novel
Therapeutic Target for Huntington’s Disease. eLife 2020, 9,
No. e51093.
(10) Rodríguez-Ruiz, M.; Moreno, E.; Moreno-Delgado, D.;
Navarro, G.; Mallol, J.; Cortés, A.; Lluís, C.; Canela, E. I.; Casadó,
V.; McCormick, P. J.; Franco, R. Heteroreceptor Complexes Formed
by Dopamine D1, Histamine H3, and N-Methyl-D-Aspartate
Glutamate Receptors as Targets to Prevent Neuronal Death in
Alzheimer’s Disease. Mol. Neurobiol. 2017, 54, 4537−4550.
(11) Rapanelli, M.; Frick, L.; Pogorelov, V.; Ohtsu, H.; Bito, H.;
Pittenger, C. Histamine H3R Receptor Activation in the Dorsal
Striatum Triggers Stereotypies in a Mouse Model of Tic Disorders.
Transl. Psychiatry 2017, 7, e1013−e1013.
(12) Syed, Y. Y. Pitolisant: First Global Approval. Drugs 2016, 76,
1313−1318.
(13) Pullen, L. C.; Picone, M.; Tan, L.; Johnston, C.; Stark, H.
Cognitive Improvements in Children with Prader-Willi Syndrome
Following Pitolisant TreatmentPatient Reports. J. Pediatr. Pharma-
col. Ther. 2019, 24, 166−171.
ACKNOWLEDGMENTS
■
We thank Prof. Dr. Sigurd Elz and Prof. Dr. Gunnar Schulte for
providing infrastructure and Dr. Ulrike Zabel for support in
cloning the NLuc tagged β1 adrenoceptor. We thank Denise
Mönnich for support in cloning the NLuc tagged D1 receptor.
We thank Prof. Dr. Gunnar Schulte (mouse smoothened
receptor) and Prof. Dr. Stephen J. Hill (adenosine A3 receptor)
for providing the respective NLuc receptor constructs.
ABBREVIATIONS
■
aq, aqueous; a.u., arbitrary units; AU, absorption units; Bq,
becquerel; Bmax, total number of receptors in a sample; br,
broad; BRET, bioluminescence resonance energy transfer;
BSA, bovine serum albumin; CDCl3, deuterated chloroform;
CHO, Chinese hamster ovary cells; CI, confidence interval;
clob, clobenpropit; cpm, counts per minute; CRC, concen-
tration response curve; Cy3, cyanine dye 3; DAD, diode array
detector; dd, doublet of doublets; DIPEA, diisopropylethyl-
amine; DMEM, Dulbecco’s modified Eagle medium; DMSO-
d6, deuterated DMSO; dt, doublet of triplets; EDC·HCl, 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;
eq, equivalent; EtOAc, ethyl acetate; EtOH, ethanol; FCS,
fetal bovine serum; G418, geneticin; Gαi2, α-subunit of the Gi2
protein that mediates inhibition of adenyl cyclase; Gβ1γ2, G
protein β1- and γ2-subunit; HBSS, Hank’s balanced salt
solution; HOBt, 1-hydroxybenzotriazole; H2O, water;
HEK293, human embryonic kidney 293 cells; HEK293T,
human embryonic kidney 293 T cells; HEPES, 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; His, hista-
mine; HxRs, histamine receptor subtype x; imet, imetit; k,
retention (or capacity) factor (HPLC); Kb, dissociation
constant obtained from functional assays; Kd, dissociation
constant obtained from a saturation binding experiment; Ki,
dissociation constant obtained from a competition binding
experiment; kin, kinetic; kobs, observed association rate
constant; koff, dissociation rate constant; kon, association rate
constant; MeCN, acetonitrile; CD3OD, deuterated methanol;
MeOH, methanol; MW, microwave; N, normality; Nano-
BRET, NanoLuc luciferase-based bioluminescence resonance
energy transfer; NEt3, triethylamine; NLuc, NanoLuc frag-
ment; PE, petroleum ether; pEC50, negative logarithm of the
half-maximum activity concentration in M; pH, potential or
power of hydrogen; pito, pitolisant; pKb, negative logarithm of
L
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(14) Emami-Nemini, A.; Roux, T.; Leblay, M.; Bourrier, E.;
Lamarque, L.; Trinquet, E.; Lohse, M. J. Time-Resolved Fluorescence
Ligand Binding for G Protein−Coupled Receptors. Nat. Protoc. 2013,
8, 1307−1320.
(15) Stoddart, L. A.; Johnstone, E. K. M.; Wheal, A. J.; Goulding, J.;
Robers, M. B.; Machleidt, T.; Wood, K. V.; Hill, S. J.; Pfleger, K. D. G.
Application of BRET to Monitor Ligand Binding to GPCRs. Nat.
Methods 2015, 12, 661−663.
(16) Stoddart, L. A.; Kilpatrick, L. E.; Hill, S. J. NanoBRET
Approaches to Study Ligand Binding to GPCRs and RTKs. Trends
Pharmacol. Sci. 2018, 39, 136−147.
(17) Axelrod, D. Cell-Substrate Contacts Illuminated by Total
Internal Reflection Fluorescence. J. Cell Biol. 1981, 89, 141−145.
(18) Sako, Y.; Minoghchi, S.; Yanagida, T. Single-Molecule Imaging
of EGFR Signalling on the Surface of Living Cells. Nat. Cell Biol.
2000, 2, 168−172.
(19) Kuder, K.; Kiec-Kononowicz, K. Fluorescent GPCR Ligands as
New Tools in Pharmacology. Curr. Med. Chem. 2008, 15, 2132−2143.
(20) Kuder, K. J.; Kottke, T.; Stark, H.; Ligneau, X.; Camelin, J.-C.;
for Human Nonlysosomal Glucosylceramidase. J. Am. Chem. Soc.
2017, 139, 14192−14197.
(34) Iyer, S. S.; Anderson, A. S.; Reed, S.; Swanson, B.; Schmidt, J.
G. Synthesis of Orthogonal End Functionalized Oligoethylene Glycols
of Defined Lengths. Tetrahedron Lett. 2004, 45, 4285−4288.
(35) Baumeister, P.; Erdmann, D.; Biselli, S.; Kagermeier, N.; Elz, S.;
Bernhardt, G.; Buschauer, A. [3H]UR-DE257: Development of a
Tritium-Labeled Squaramide-Type Selective Histamine H2 Receptor
Antagonist. ChemMedChem 2015, 10, 83−93.
(36) Igel, P.; Schnell, D.; Bernhardt, G.; Seifert, R.; Buschauer, A.
1
Tritium-Labeled N -[3-(1 H -Imidazol-4-Yl)Propyl]- N2-Propio-
nylguanidine ([3H]UR-PI294), a High-Affinity Histamine H3 and H4
Receptor Radioligand. ChemMedChem 2009, 4, 225−231.
(37) Schihada, H.; Shekhani, R.; Schulte, G. Quantitative assessment
of constitutive G protein-coupled receptor activity with BRET-based
G
protein biosensors. bioRxiv 2021, DOI: 10.1101/
2021.02.05.429900.
(38) Windaus, A.; Vogt, W. Synthesis of Imidazolylethylamine. Ber.
Dtsch. Chem. Ges. 1907, 40, 3685−3691.
(39) Garbarg, M.; Arrang, J. M.; Rouleau, A.; Ligneau, X.; Tuong, M.
D.; Schwartz, J. C.; Ganellin, C. S-[2-(4-Imidazolyl) Ethyl]
Isothiourea, a Highly Specific and Potent Histamine H3 Receptor
Agonist. J. Pharmacol. Exp. Ther. 1992, 263, 304−310.
(40) Arrang, J.-M.; Garbarg, M.; Lancelo, J.-C.; Lecomte, J.-M.;
Pollard, H.; Robba, M.; Schunack, W.; Schwartz, J.-C. Highly Potent
and Selective Ligands for Histamine H3-Receptors. Nature 1987, 327,
117−123.
(41) Van der Goot, H.; Schepers, M. J. P.; Sterk, G. J.; Timmerman,
H. Isothiourea Analogues of Histamine as Potent Agonists or
Antagonists of the Histamine H3-Receptor. Eur. J. Med. Chem.
1992, 27, 511−517.
(42) Schaller, D.; Hagenow, S.; Stark, H.; Wolber, G. Ligand-Guided
Homology Modeling Drives Identification of Novel Histamine H3
Receptor Ligands. PLoS One 2019, 14, No. e0218820.
(43) Ligneau, X.; Perrin, D.; Landais, L.; Camelin, J.-C.; Calmels, T.;
Berrebi-Bertrand, I.; Lecomte, J.-M.; Parmentier, R.; Anaclet, C.; Lin,
J.-S.; Bertaina-Anglade, V.; la Rochelle, C. D.; d’Aniello, F.; Rouleau,
A.; Gbahou, F.; Arrang, J. M.; Ganellin, C. R.; Stark, H.; Schunack,
W.; Schwartz, J.-C. BF2.649 [1-{3-[3-(4-Chlorophenyl) propoxy]-
propyl}piperidine, Hydrochloride], a Nonimidazole Inverse Agonist/
Antagonist at the Human Histamine H3 Receptor: Preclinical
Pharmacology. J. Pharmacol. Exp. Ther. 2007, 320, 365−375.
(44) Wulff, B. S.; Hastrup, S.; Rimvall, K. Characteristics of
Recombinantly Expressed Rat and Human Histamine H3 Receptors.
Eur. J. Pharmacol. 2002, 453, 33−41.
(45) Lim, H. D.; van Rijn, R. M.; Ling, P.; Bakker, R. A.; Thurmond,
R. L.; Leurs, R. Evaluation of Histamine H1-, H2-, and H3-Receptor
Ligands at the Human Histamine H4 Receptor: Identification of 4-
Methylhistamine as the First Potent and Selective H4 Receptor
Agonist. J. Pharmacol. Exp. Ther. 2005, 314, 1310−1321.
(46) Schihada, H.; Ma, X.; Zabel, U.; Vischer, H. F.; Schulte, G.;
Leurs, R.; Pockes, S.; Lohse, M. J. Development of a Conformational
Histamine H3 Receptor Biosensor for the Synchronous Screening of
Agonists and Inverse Agonists. ACS Sens. 2020, 5, 1734−1742.
(47) Mocking, T. A. M.; Verweij, E. W. E.; Vischer, H. F.; Leurs, R.
Homogeneous, Real-Time NanoBRET Binding Assays for the
Histamine H3 and H4 Receptors on Living Cells. Mol. Pharmacol.
2018, 94, 1371−1381.
́
Seifert, R.; Kiec-Kononowicz, K. Search for Novel, High Affinity
Histamine H3 Receptor Ligands with Fluorescent Properties.
Inflammation Res. 2010, 59, 247−248.
(21) Tomasch, M.; Schwed, J. S.; Paulke, A.; Stark, H. BodilisantA
Novel Fluorescent, Highly Affine Histamine H3 Receptor Ligand.
ACS Med. Chem. Lett. 2013, 4, 269−273.
(22) Kuder, K.; Kiec-Kononowicz, K. Fluorescent GPCR Ligands as
New Tools in Pharmacology-Update, Years 2008- Early 2014. Curr.
Med. Chem. 2014, 21, 3962−3975.
(23) Bartole, E.; Grätz, L.; Littmann, T.; Wifling, D.; Seibel, U.;
Buschauer, A.; Bernhardt, G. UR-DEBa242: A Py-5-Labeled
Fluorescent Multipurpose Probe for Investigations on the Histamine
H3 and H4 Receptors. J. Med. Chem. 2020, 5297.
(24) Drakopoulos, A.; Decker, M. Development and Biological
Applications of Fluorescent Opioid Ligands. ChemPlusChem 2020, 85,
1354−1364.
(25) Apodaca, R.; Dvorak, C. A.; Xiao, W.; Barbier, A. J.; Boggs, J.
D.; Wilson, S. J.; Lovenberg, T. W.; Carruthers, N. I. A New Class of
Diamine-Based Human Histamine H3 Receptor Antagonists: 4-
(Aminoalkoxy)Benzylamines. J. Med. Chem. 2003, 46, 3938−3944.
̌
́
(26) Wingen, K.; Schwed, J. S.; Isensee, K.; Weizel, L.; Zivkovic, A.;
Odazic, D.; Stark, H. Benzylpiperidine Variations on Histamine H3
Receptor Ligands for Improved Drug-Likeness. Bioorg. Med. Chem.
Lett. 2014, 24, 2236−2239.
(27) Kshirsagar, T.; Nakano, A. H.; Law, P.-Y.; Elde, R.; Portoghese,
P. S. NTI4F: A Non-Peptide Fluorescent Probe Selective for
Functional Delta Opioid Receptors. Neurosci. Lett. 1998, 249, 83−86.
(28) Grätz, L.; Tropmann, K.; Bresinsky, M.; Muller, C.; Bernhardt,
̈
G.; Pockes, S. NanoBRET Binding Assay for Histamine H2 Receptor
Ligands Using Live Recombinant HEK293T Cells. Sci. Rep. 2020, 10,
13288.
(29) Ru
Genotyping by TIRF Microscopy. J. fluoresc. 2008, 18, 1021−1026.
(30) Laasfeld, T.; Ehrminger, R.; Tahk, M.-J.; Veiksina, S.; Kolvart,
̈
ttinger, S.; Lamarre, B.; Knight, A. E. Single Molecule
̃
K. R.; Min, M.; Kopanchuk, S.; Rinken, A. Budded Baculoviruses as a
Receptor Display System to Quantify Ligand Binding with TIRF
Microscopy. Nanoscale 2021, 13, 2436−2447.
(31) Qiu, B.; Simon, M. C. BODIPY 493/503 Staining of Neutral
Lipid Droplets for Microscopy and Quantification by Flow
Cytometry. Bio-Protoc. 2016, 6, No. e1912.
(48) Pockes, S.; Wifling, D.; Keller, M.; Buschauer, A.; Elz, S. Highly
Potent, Stable, and Selective Dimeric Hetarylpropylguanidine-Type
Histamine H2 Receptor Agonists. ACS Omega 2018, 3, 2865−2882.
(49) Gentzsch, C.; Seier, K.; Drakopoulos, A.; Jobin, M.-L.;
(32) Newman, M. S.; Barbee, T. G.; Blakesley, C. N.; Zia-ud-Din;
Gromelski, S., Jr.; Khanna, V. K.; Lee, L.-F.; Radhakrishnan, J.; Robey,
R. L. High-Dilution Cyclization of Polyoxapentacosanodinitriles. J.
Org. Chem. 1975, 40, 2863−2870.
Lanoiselée, Y.; Koszegi, Z.; Maurel, D.; Sounier, R.; Hubner, H.;
̈
(33) Lahav, D.; Liu, B.; van den Berg, R. J. B. H. N.; van den
Nieuwendijk, A. M. C. H.; Wennekes, T.; Ghisaidoobe, A. T.; Breen,
I.; Ferraz, M. J.; Kuo, C.-L.; Wu, L.; Geurink, P. P.; Ovaa, H.; van der
Marel, G. A.; van der Stelt, M.; Boot, R. G.; Davies, G. J.; Aerts, J. M.
F. G.; Overkleeft, H. S. A Fluorescence Polarization Activity-Based
Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors
Gmeiner, P.; Granier, S.; Calebiro, D.; Decker, M. Selective and
Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow
Single-Molecule Imaging of Μ-Opioid Receptor Dimerization. Am.
Ethnol. 2020, 132, 6014−6020.
(50) Sungkaworn, T.; Jobin, M.-L.; Burnecki, K.; Weron, A.; Lohse,
M. J.; Calebiro, D. Single-Molecule Imaging Reveals Receptor−G
M
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Protein Interactions at Cell Surface Hot Spots. Nature 2017, 550,
543−547.
(51) Möller, J.; Isbilir, A.; Sungkaworn, T.; Osberg, B.; Karathanasis,
C.; Sunkara, V.; Grushevskyi, E. O.; Bock, A.; Annibale, P.;
disruption of dynamic CXCR4 dimerization by subpocket-specific
inverse agonists. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 29144−
29154.
Heilemann, M.; Schutte, C.; Lohse, M. J. Single-Molecule Analysis
̈
Reveals Agonist-Specific Dimer Formation of μ-Opioid Receptors.
Nat. Chem. Biol. 2020, 16, 946−954.
(52) Soave, M.; Stoddart, L. A.; Brown, A.; Woolard, J.; Hill, S. J.
Use of a New Proximity Assay (NanoBRET) to Investigate the
Ligand-Binding Characteristics of Three Fluorescent Ligands to the
Human β1 -Adrenoceptor Expressed in HEK-293 Cells. Pharmacol.
Res. Perspect. 2016, 4, No. e00250.
(53) Kozielewicz, P.; Bowin, C.-F.; Turku, A.; Schulte, G. A
NanoBRET-Based Binding Assay for Smoothened Allows Real-Time
Analysis of Ligand Binding and Distinction of Two Binding Sites for
BODIPY-Cyclopamine. Mol. Pharmacol. 2020, 97, 23−34.
(54) Grätz, L.; Laasfeld, T.; Allikalt, A.; Gruber, C. G.; Pegoli, A.;
Tahk, M.-J.; Tsernant, M.-L.; Keller, M.; Rinken, A. BRET-and
Fluorescence Anisotropy-Based Assays for Real-Time Monitoring of
Ligand Binding to M2 Muscarinic Acetylcholine Receptors. Biochim.
Biophys. Acta Mol. Cell Res. 2021, 1868, 118930.
(55) Allikalt, A.; Purkayastha, N.; Flad, K.; Schmidt, M. F.; Tabor,
̈
A.; Gmeiner, P.; Hubner, H.; Weikert, D. Fluorescent Ligands for
Dopamine D2/D3 Receptors. Sci. Rep. 2020, 10, 21842.
(56) Pándy-Szekeres, G.; Munk, C.; Tsonkov, T. M.; Mordalski, S.;
Harpsøe, K.; Hauser, A. S.; Bojarski, A. J.; Gloriam, D. E. GPCRdb in
2018: Adding GPCR Structure Models and Ligands. Nucleic Acids Res.
2018, 46, D440−D446.
́
(57) Staszewski, M.; Nelic, D.; Jonczyk, J.; Dubiel, M.; Frank, A.;
́
Stark, H.; Bajda, M.; Jakubik, J.; Walczynski, K. Guanidine
Derivatives: How Simple Structural Modification of Histamine H3R
Antagonists Has Led to the Discovery of Potent Muscarinic M2R/
M4R Antagonists. ACS Chem. Neurosci. 2021, 12, 2503−2519.
(58) Perepichka, I. F.; Besbes, M.; Levillain, E.; Sallé, M.; Roncali, J.
Hydrophilic Oligo(Oxyethylene)-Derivatized Poly(3,4-Ethylenediox-
ythiophenes): Cation-Responsive Optoelectroelectrochemical Proper-
ties and Solid-State Chromism. Chem. Mater. 2002, 14, 449−457.
(59) Goswami, L. N.; Houston, Z. H.; Sarma, S. J.; Jalisatgi, S. S.;
Hawthorne, M. F. Efficient Synthesis of Diverse Heterobifunctional-
ized Clickable Oligo (Ethylene Glycol) Linkers: Potential Applica-
tions in Bioconjugation and Targeted Drug Delivery. Org. Biomol.
Chem. 2013, 11, 1116−1126.
(60) Baell, J. B.; Holloway, G. A. New Substructure Filters for
Removal of Pan Assay Interference Compounds (PAINS) from
Screening Libraries and for Their Exclusion in Bioassays. J. Med.
Chem. 2010, 53, 2719−2740.
(61) Aldrich, C.; Bertozzi, C.; Georg, G. I.; Kiessling, L.; Lindsley,
C.; Liotta, D.; Merz, K. M.; Schepartz, A.; Wang, S. The Ecstasy and
Agony of Assay Interference Compounds. ACS Cent. Sci. 2017, 3,
143−147.
(62) Keller, M.; Erdmann, D.; Pop, N.; Pluym, N.; Teng, S.;
Bernhardt, G.; Buschauer, A. Red-Fluorescent Argininamide-Type
NPY Y1 Receptor Antagonists as Pharmacological Tools. Bioorg. Med.
Chem. 2011, 19, 2859−2878.
(63) Cheng, Y.-C.; Prusoff, W. H. Relationship between the
Inhibition Constant (Ki) and the Concentration of the Inhibitor
Which Causes 50 per Cent Inhibition (I50) of an Enzymatic Reaction.
Biochem. Pharmacol. 1973, 22, 3099−3108.
(64) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.;
Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.;
Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.;
Tomancak, P.; Cardona, A. Fiji: An Open-Source Platform for
Biological-Image Analysis. Nat. Methods 2012, 9, 676−682.
(65) Jaqaman, K.; Loerke, D.; Mettlen, M.; Kuwata, H.; Grinstein,
S.; Schmid, S. L.; Danuser, G. Robust Single-Particle Tracking in Live-
Cell Time-Lapse Sequences. Nat. Methods 2008, 5, 695−702.
(66) Isb̧ ilir, A.; Möller, J.; Arimont, M.; Bobkov, V.; Perpiná-Viciano,
̃
C.; Hoffmann, C.; Inoue, A.; Heukers, R.; de Graaf, C.; Smit, M. J.;
Annibale, P.; Lohse, M. J. Advanced fluorescence microscopy reveals
N
https://doi.org/10.1021/acs.jmedchem.1c01089
J. Med. Chem. XXXX, XXX, XXX−XXX