Bodilisant - A novel fluorescent, highly affine histamine H3 receptor ligand

Article abstract of DOI:10.1021/ml300383n

A piperidine-based lead structure for the human histamine H₃ receptor (hH₃R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH₃R ligand with affinity in the nanomolar concentration range (K_i hH₃R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH₃R preference. Bodilisant was used for visualization of hH₃R in hH₃R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH₃R.

Full text of DOI:10.1021/ml300383n

Letter
pubs.acs.org/acsmedchemlett
BodilisantA Novel Fluorescent, Highly Affine Histamine H3
Receptor Ligand
Miriam Tomasch,† J. Stephan Schwed,† Alexander Paulke,‡ and Holger Stark*,†
†Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany
‡Goethe University, Kennedyallee 104, 60596 Frankfurt am Main, Germany
S
* Supporting Information
ABSTRACT: A piperidine-based lead structure for the human
histamine H3 receptor (hH3R) was coupled with the BODIPY
fluorophore and resulted in a strong green fluorescent
(quantum yield, 0.92) hH3R ligand with affinity in the
nanomolar concentration range (Ki hH3R = 6.51
3.31
nM), named Bodilisant. Screening for affinities at histamine
and dopamine receptor subtypes showed high hH3R
preference. Bodilisant was used for visualization of hH3R in
hH3R overexpressing HEK-293 cells with fluorescence
confocal laser scanning microscopy. In addition, in native
human brain tissues, Bodilisant showed clear and displaceable
images of labeled hH3R.
KEYWORDS: histamine, H3 receptor, nonimidazole derivative, GPCR, BODIPY, HEK-293 cells, tissue labeling,
fluorescence confocal laser scanning microscopy, displacement, pharmacological tool
human tissue10 based on earlier results (Mirisant-405, Mirisant-
he human histamine H3 receptor is one of the four human
Thistamine receptor subtypes (hH
470, and Benz-Mirisant-405).11−13 Because these early ligands
were not usable under all conditions, we wanted to design
fluorescent hH3R ligands with more properties in common in
one molecule, that is, higher hH3R affinity, versatile fluorescent
wavelength, optimized fluorescence intensity, and thus a better
signal-to-noise ratio. The decision has been made for 4,4-
difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye as a
fluorophore due to its high extinction coefficient, its sharp
fluorescence emission peaks with high quantum yield,14 its
insensitivity to pH and solvent polarity, and its greater chemical
and photochemical stability. Therefore, BODIPY is often used
as a sensor for analytical detection of transmitters, like metal
ions, reducing agents, nitrogen-monoxide,15 or hydroperox-
ides,16 or as labeling reagent for peptides.17 Recently, there are
efforts to design BODIPY-labeled GPCR ligands, such as
purinergic receptor ligands,18−21 β2-adrenoceptor agonists,22
M1 muscarinic receptor ligands,23 and dopamine D1 and D2
receptor ligands.24
1−4R). It is a membrane-
bound class A family G-protein-coupled receptor (GPCR) (Gi/o
coupled) mainly expressed in the central nervous system
(CNS) acting as an auto- as well as a heteroreceptor.1 The
human histamine H3 receptor (hH3R) modulates the release of
several neuronal neurotransmitters.2,3 Because of different
central effects of several hH3R antagonists in preclinical and
clinical trials, we have seen a need for labeled hH3R ligands to
be taken as diagnostic tools to further investigate neurological
disorders in the CNS based on receptor distribution,
occupation, and regulation. In histochemistry, fluorescent-
labeled antibodies are of common use.4 For the manufacturing
process of antibodies, mostly animal experiments are
required.5,6 These antibodies have to be labeled in a follow-
up work step with a second labeled antibody for immuno-
fluorescence; merely a few primary antibodies are directly
labeled.7 Most antibodies are sensitive to temperature and have
to be stored in freezers. The main disadvantage as compared to
small fluorescent GPCR ligands is their undisplacement
properties without target destruction. Small fluorescent
GPCR ligands can be displaced by other ligands and
consequently enable the development of fluorescence displace-
ment assays. There are many efforts to design fluorescent
GPCR ligands on the strength of their prominence as the
largest and most versatile group of cell surface receptors,
therefore responsible for various pharmacological functions.8,9
Recent research in our working group resulted in chalcone-
based fluorescent hH3R ligands able to label hH3R in cells and
Different histamine receptor ligands were linked with
BODIPY derivatives. In 2012, the human histamine H1
receptor (hH1R) ligand mepyramine-BODIPY 630−650 was
published.25 Aminopotentidine was fluorescence-tagged but
with moderate H2R affinity,26 and a red fluorescent
clobenpropit-based H3R ligand is available: H3/H4-633-AN
Received: November 12, 2012
Accepted: December 12, 2012
Published: December 12, 2012
© 2012 American Chemical Society
269
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Bodilisant
a
Reagents and conditions: (i) SOCl2, 0 °C → 60 °C, toluene, 3 h. (ii) Methyl 4-hydroxybenzoate, K2CO3, KI, acetone, reflux, 72 h. (iii) LiOH, THF;
H2O, 60 °C, 1 h, precipitation with HCl. (iv) SOCl2, 70 °C, 5 h. (v) 2,4-Dimethyl-1H-pyrrol, CH2Cl2, RT → 40 °C, 1 h. (vi) NEt3, BF3 × Et2O,
toluene, 80 °C, 15 min.
(Abcam, Cambridge, MA). This purchasable compound
possesses moderate affinities at H3R and does not act
specifically selective toward the other histamine receptors.
In brief, the initializing precursor 3-(piperidin-1-yl)propan-1-
ol hydrochloride (I) was synthesized by alkylation of piperidine
with 3-chloropropan-1-ol as described in the literature27 and
chlorinated.28 The intermediate II was used for alkylation of
methyl 4-hydroxybenzoate by Williamson ether reaction.29
Methyl ether III28 was cleaved in basic milieu. Resulting acid
IV28 was chlorinated with thionyl chloride to the appropriate
acid chloride V, which was reacted via condensation with 2,4-
dimethyl-1H-pyrrole to VI. Final ring closure to Bodilisant was
achieved with triethyl amine and boron trifluoride diethyl
etherate.16 Bodilisant was purified via column chromatography
(Scheme 1). For detailed synthesis procedures and analytical
data, see the Supporting Information.
For the design of our novel BODIPY-labeled hH3R ligands,
we chose ciproxifan and related nonimidazoles as lead
structures. The imidazole moiety of ciproxifan was replaced
by the piperidine group (see Pitolisant). Nonimidazole
ciproxifan analogues have been claimed to possess a highly
reduced interaction with the cytochrome P450 system. The
basic piperidine element was labeled with a propyl group
(spacer A). An ether as a polar group links the phenyl group
(spacer B). Previously, we have coupled spacer B with
cyanoisoindole13 as a fluorophore. In Mirisant-405, the phenyl
group of spacer B was extended by a tetralone group.10 Via
aldol condensation, the tetralone group was converted to the
fluorescent hH3R ligand, which is part of the fluorophore. In
Bodilisant, the phenyl group of spacer B is part of the
fluorophore moiety. The BODIPY group represents the
lipophilic residue as an element in the pharmacophore (cf.
Figure 1).
The affinity of the newly designed Bodilisant at the hH3R in a
displacement assay32 is seven times higher than that of the
reference compound, ciproxifan (hH3R Ki = 46
Bodilisant is 10 times more potent at the hH3R (hH3R Ki =
4 nM).37
Figure 1. Design strategy for the novel fluorescent human histamine
H3 receptor ligand.
6.51
3.31 nM) than recently described fluorescent
chalcones,10 indicating the BODIPY-core as a tolerated
lipophilic residue in the binding pocket.
receptors are about one log unit and at the human dopamine
D1 and D5 receptors about two log units lower than that at
hH3R (hD1R Ki = 12513 9937 nM; hD2R Ki = 1971 216
nM; hD3R Ki = 1492 537 nM; and hD5R Ki = 5210 1739
nM). Bodilisant has clearly only marginal affinity at human
dopamine receptor subtypes investigated as compared to
histamine receptor subtypes, especially to hH3R (Supporting
Information).
Fluorescence absorption and emission measurements were
carried out in buffer (12.5 mM MgCl2, 100 mM NaCl, and 75
mM Tris/HCl, pH 7.4) at a concentration of 10 mM to imitate
fluorescence microscopy conditions. An absorption maximum
For selectivity validation, Bodilisant has been screened on
affinities at related hH1R and human histamine H4 receptor
(hH4R).33−35 Bodilisant possesses the highest affinity at the
hH3R with selectivity ratios higher than 250 and 1100 for hH1R
and hH4R, respectively (hH1R Ki = 1662 88 nM; hH4R = Ki
7476 3853 nM) (Supporting Information).
Affinities at human dopamine receptor subtypes for
Bodilisant have been determined due to colocalization of
these aminergic GPCRs in different brain tissues.3,36,38
Affinities of Bodilisant at the human dopamine D2 and D3
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ACS Medicinal Chemistry Letters
Letter
at 468 nm has been found in aqueous media. Two emission
maxima could be detected: 494 and 563 nm (Figure 2). The
green fluorescent BODIPY could be detected. Consequently,
Bodilisant was not internalized. HEK-293 cells that do not
overexpress hH3R were used as a control. On the basis of
Bodilisant's illuminating power, fluorescence microscopy
conditions could be adjusted that negligible autofluorescence
occurs. In contrast to antibodies, small fluorescent GPCR
ligands are displaceable. To confirm this thesis, displacement
experiments with 10 μM pitolisant were successfully carried
out. Marginal remaining signal could be detected.
To confirm localization of hH3R on hH3R overexpressing
HEK-293 cells, an anti-hH3R rabbit monoclonal IgG1 antibody
and a red fluorescent Texas Red dye conjugated goat antirabbit
IgG (H+L) secondary antibody were used for immunostaining
(Supporting Information).
Figure 2. Fluorescence emission spectrum of Bodilisant.
After successful staining of hH3R on hH3R overexpressing
HEK-293 cells, we tested Bodilisant on human brain tissue. As
reported from Martinz-Mir et al., hH3R are expressed in the
cerebral cortex, nucleus caudatus, and globus pallidus.2 Nuclei
of synapses were stained with DAPI. After staining with 1 μM
Bodilisant, hH3R next to synapses nuclei and distant hetero
hH3R were labeled (Supporting Information). These signals
were deleted after treatment with 500 μM pitolisant or 1 mM
ciproxifan. Also, Bodilisant shows high displaceable properties
in brain tissue with structurally different unlabeled hH3R
ligands. In paraffin tissue of globus pallidus, hH3R were
detectable and structures of histaminergic nerve tracts (Figure
4). These findings confirm Bodilisant as a labeling tool in
stored tissues.
Stokes shift between the absorption and the first emission
maximum is very low (26 nm). To avoid additionally recording
the excitation energy, the second emission maximum was only
used for fluorescence microscopy.
Measurement of Bodilisant's quantum yield resulted in a
value of 0.92 for which it is similar to sodium fluorescein
(quantum yield, 0.91).30,31 Consequentially, Bodilisant's
illuminating power is as high as sodium fluorescein, a standard
for strong fluorescence substances. An advantage over sodium
fluorescein is the stability of luminescence. Fluorescein
compounds are easily quenched, which has led to their
application in MELC technology (multiepitope ligand cartog-
raphy).39 Bleaching experiments for 1 h with Bodilisant were
not successful (results not shown).
To minimize unspecific binding, cells were incubated with
1−3% BSA solution for 30 min. Bodilisant was incubated in a
concentration range between 1 and 10 nM. The best signal-to-
noise ratio was achieved with a concentration of 8 nM.
Bodilisant labeled hH3R with strong green fluorescence signals
in the outer cell membrane of hH3R overexpressing human
embryonergic kidney cell line (HEK-293 cells), where these
membrane-bound GPCRs are localized (Figure 3 and
Supporting Information). No overlay of blue fluorescent 4′,6-
diamidino-2-phenylindole (DAPI), staining cell nuclei, and
Figure 4. Staining of hH3R with Bodilisant on human globus pallidus
slices. (A) Detail of a Bodilisant-labeled globus pallidus slice. Blue
areas mark Bodilisant-labeled hH3R and histaminergic nerves. (B)
Zoom of panel A. Structures of the globus pallidus and hH3R are
visible. White arrows show some histaminergic nerves in the lower part
of picture.
In our work, we achieved the optimization of physicochem-
ical and pharmacological properties via introduction of a
BODIPY group as compared to our previously reported
chalcone-based fluorescent hH3R ligands.10 The novel BODIPY
dye is a high-affinity hH3R ligand with excellent fluorescence
properties. Bodilisant is a strong illuminating green fluorescent
compound with high affinity and good selectivity ratios. The
investigation on hH3R overexpressing HEK-293 cells and in
hH3R rich human brain tissue demonstrated Bodilisant's
application as an useful pharmacological tool for receptor
imaging. Bodilisant can be used in low concentrations (1−10
nM) for the detection of hH3R in hH3R overexpressing cells
and in a concentration of approximately 1 μM in hH3R-rich
tissue. Bodilisant is applicable in aqueous neutral buffer.
Figure 3. Labeling of hH3R with Bodilisant on hH3R-HEK-293 cells in
the outer cell membrane. Several hH3R-HEK-293 cells were visualized.
In all cells, Bodilisant enriches in the outer cell membrane, where
hH3R are mainly expressed.
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Letter
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ASSOCIATED CONTENT
* Supporting Information
Synthesis and analytical data of compounds V, VI, and
Bodilisant and pharmacological and imaging procedures with
additional material. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
*Tel: 049(0)69-798-29302. Fax: 049(0)69-798-29258. E-mail:
h.stark@pharmchem.uni-frankfurt.de.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Funding
This work was kindly supported by the Hesse LOEWE
Schwerpunkte NeFF, OSF, and AFA as well as by the FP7 EU
COST Actions BM0806, CM1103, and CM1007.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We greatly thank Prof. J.-C. Schwartz (Bioprojet, Saint-
■
Greg
́
oire, France) for providing HEK-293 cells stably
expressing the recombinant hH3R. The CHO-K1 cells stably
expressing the hH1R were generously gifted by Prof. H.
Timmermann and Prof. R. Leurs (Amsterdam, The Nether-
lands). We gratefully thank Prof. R. Seifert (Hannover,
Germany) for providing Sf9 cells and baculovirus stock
solutions encoding for the hH4R and G-protein Gαi2 and
Gβ1γ2 subunits. Cell lines expressing dopamine D3 and D2
receptors were provided courtesy of Dr. P. Sokoloff (INSERM,
Paris, France) and Dr. J. Shine (The Garvan Institute of
Medical Research, Sydney, Australia). Access to cell lines
expressing dopamine D1 and D5 receptors was kindly enabled
by Prof. J. Lehmann (Jena, Germany). We are greatly thankful
to Christine Elbert for excellent human brain tissue
preparations.
Wilhelms-Universitat Bonn: Bonn, Germany, 2002.
̈
(19) Briddon, S. J.; Middleton, R. J.; Yates, A. S.; George, M. W.;
Kellamb, B.; Hill, S. J. Application of fluorescence correlation
spectroscopy to the measurement of agonist binding to a G-protein
coupled receptor at the single cell level. Faraday Discuss. 2004, 126,
197−207.
ABBREVIATIONS
■
(20) Briddon, S. J.; Middleton, R. J.; Cordeaux, Y.; Flavin, F. M.;
Weinstein, J. A.; George, M. W.; Kellam, B.; Hill, S. J. Quantitative
analysis of the formation and diffusion of A1-adenosine receptor−
antagonist complexes in single living cells. Proc. Natl. Acad. Sci. U.S.A.
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BODIPY-630/650 labelled fluorescent adenosine receptor agonists.
Med. Chem. Comm. 2012, 3, 333−338.
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visualisation of BODIPY-TMR-CGP: A long-acting fluorescent β2-
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hH1R, human histamine H1 receptor; hH3R, human histamine
H3 receptor; hH4R, human histamine H4 receptor; GPCR, G-
protein-coupled receptor; CNS, central nervous system;
BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; HEK-293
cell, human embryonergic kidney cell line; DAPI, 4′,6-
diamidino-2-phenylindole
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