Chemical probes for the recognition of cannabinoid receptors in native systems

Article abstract of DOI:10.1002/anie.201200467

Receptors made visible: The described biotin-tagged small-molecule probes with excellent affinities for the CB₁ and CB₂ cannabinoid receptors (CB₁R and CB₂R) enable direct visualization of these receptors in nat

Full text of DOI:10.1002/anie.201200467

Angewandte
Chemie
DOI: 10.1002/anie.201200467
Small-Molecule Probes
Chemical Probes for the Recognition of Cannabinoid Receptors in
Native Systems**
Lidia Martꢀn-Couce, Mar Martꢀn-Fontecha, ꢁscar Palomares, Leyre Mestre, Arnau Cordomꢀ,
Miriam Hernangomez, Sara Palma, Leonardo Pardo, Carmen Guaza, Marꢀa L. Lꢂpez-
Rodrꢀguez,* and Silvia Ortega-Gutiꢃrrez*
Dedicated to Professor Miguel ꢄngel Miranda on the occasion of his 60th birthday
The endogenous cannabinoid system (ECS) regulates a broad
number of physiological processes, and perturbations in its
normal functioning are linked to many disorders.[1] In this
respect, the development of high-sensitivity and high-
throughput analytical tools that afforded a broader view of
the ECS would be highly valuable. As such, small-molecule
fluorescent probes could provide dynamic information con-
cerning the direct spatial and temporal expression levels of
cannabinoid receptors as has been recently reported for some
specific classes of enzymes.[2] Accordingly, the development of
small-molecule probes that are able to recognize cannabinoid
receptors is an area of current interest, because they could
complement and even overcome some of the drawbacks of
the available antibodies.[3] Recent attempts toward this goal
have been mainly focused on the CB2 receptor (CB2R = can-
nabinoid receptor type 2). However, these probes show
moderate affinities [Ki = (260–387) nm], and their application
in native systems is limited.[4] Similarly, endocannabinoid-
based probes display modest affinities for CB1 and CB2
receptors [(84.7–450) nm],[5] a fact that could limit their use
in complex systems. Therefore, we focused our efforts on
the synthetic high-affinity cannabinoid ligands HU210
[Ki(CB1R) = 0.061 nm, Ki(CB2R) = 0.52 nm],[6] and HU308
[Ki(CB1R) > 10000 nm, Ki (CB2R) = 22.7 nm][7] (Scheme 1).
Among the different tags, biotin was selected owing to its
versatility for detection by a variety of readily available
(strept)avidin conjugates. A closer look to the structure of
these ligands revealed that the most straightforward possibil-
ity was to attach the tag at the free hydroxy groups
(Scheme 1).
[*] Dr. L. Martꢀn-Couce, Prof. Dr. M. Martꢀn-Fontecha,
Prof. Dr. M. L. Lꢁpez-Rodrꢀguez, Dr. S. Ortega-Gutiꢂrrez
Department of Organic Chemistry
Universidad Complutense de Madrid
28040 Madrid (Spain)
E-mail: mluzlr@quim.ucm.es
siortega@quim.ucm.es
Scheme 1. Structures of the synthetic cannabinoid ligands HU210 and
HU308 and of synthesized probes 1–3.
Dr. ꢃ. Palomares, S. Palma
Department of Biochemistry and Molecular Biology
Universidad Complutense de Madrid
28040 Madrid (Spain)
Ligand HU210 was prepared as previously described.[8]
For the introduction of the tag, we made some attempts to
selectively acylate the allylic hydroxy group using either
Mitsunobu conditions[9] or the HfCl4·2THF catalyst.[10] How-
ever, none of them gave good yields in our hands and we
carried out the reaction between ligand HU210 and N-
(+)-biotinyl-6-aminohexanoic acid in the presence of 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylami-
nopyridine. Under these conditions, formation of bisacylated
product was not observed, starting material was partly
recovered, and compounds 1 and 2 were obtained after
separation by column chromatography with 25% and 14%
yields, respectively. Ligand HU308 was obtained as previously
described[7] from commercially available a-pinene. Esterifi-
cation of ligand HU308 with N-(+)-biotinyl-6-aminohexanoic
acid gave derivative 3 in 55% yield. Compounds 1–3 were
Dr. L. Mestre, Dr. M. Hernangomez, Dr. C. Guaza
Cajal Institute, CSIC
Dr. Arce 37, 28002 Madrid (Spain)
Dr. A. Cordomꢀ, Prof. Dr. L. Pardo
Universitat Autꢄnoma de Barcelona
08193 Bellaterra, Barcelona (Spain)
[**] This work was supported by grants from the Spanish Ministerio de
Economꢀa y Competitividad (MINECO, SAF2010-22198, SAF2010-
17501, and SAF2008-04053, and predoctoral fellowship to L.M.-C.)
and Comunidad de Madrid (S2010/BMD-2353). The authors thank
the Spanish Society of Medicinal Chemistry (SEQT) and Glaxo-
SmithKline for the young researcher award to L.M.-C. S.O.-G. and
O.P. are Ramon y Cajal Scholars funded by MINECO and the
European Social Fund.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201200467.
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tested for their affinities to CB1 and CB2 receptors, showing Ki
values in the nanomolar range (Table 1). It is especially
relevant that probes 1 and 2, derived from ligand HU210,
keep a high affinity for both cannabinoid receptors and that
probe 3 keeps the selectivity feature of its parent compound,
HU308, with high affinity for the CB2 receptor whilst being
inactive at the CB1 receptor.
Table 1: Binding affinities of synthesized probes 1–3 to the CB1 and CB2
receptors.
Compound
Ki(CB1R) [nm][a]
Ki(CB2R) [nm][a]
1
2
3
2.4Æ0.4
11Æ2
1.6Æ0.4
3Æ1
>5000
44Æ4
[a] Ki values are given as the meanÆstandard error from 2–3 inde-
pendent experiments performed in triplicate.
To propose structural hypotheses for the binding of these
compounds, we constructed three-dimensional models of the
CB1 and CB2 receptors using the crystal structure of the
homologous sphingosine 1-phosphate receptor 1 (S1P1)[11] as
a template (the Supporting Information and Figure S1).
Docking of ligands HU210 and HU308 into the receptor
models (the Supporting Information and Figure S2) places
the hydroxy groups pointing toward the extracellular environ-
ment. However, the entrance of the ligand into the binding
pocket has been proposed very recently to be from the lipid
bilayer, in particular through an opening between trans-
membrane helices (TMs) 1 and 7 (Figure 1A).[11] Thus, in
ligand HU210, the hydroxy group attached to the phenyl ring
is more distant to this opening than its allylic hydroxy group.
As a consequence, probes 1 (Figure 1A,B) and 3 (Figure 1C)
expand their biotin chains through this channel more
favorably than probe 2 (not shown). Importantly, attachment
of the biotin chain to ligands HU210 or HU308 decreases the
binding affinity in a more pronounced manner in CB1R than
CB2R (Table 1). According to the Ballesteros–Weinstein
numbering scheme the side chains at positions 1.32 (Q in
CB1R and K in CB2R), 1.35 (I in CB1R and V in CB2R), 7.34
(V in CB1R and A in CB2R), and 7.36 (A in CB1R and A in
CB2R) define this channel between TMs 1 and 7. Thus, we
hypothesize that this effect is due to the presence of amino
acid residue K1.32 in the CB2 receptor, which stabilizes the
carbonyl group of the biotin chain in a more significant
manner than residue Q1.32 of the CB1 receptor.
Compounds 1–3 were used for in vitro labeling of
cannabinoid receptors in transiently transfected hippocampal
neuronal (HT22) cells (data not shown). In these experiments,
probe 1 performed better than 2, either because of the higher
exposure of the biotin moiety as suggested by the computa-
tional model or because of the slightly higher affinity for
CB1R (Table 1). Therefore, we selected probes 1 and 3 for
their use in native systems, and neurons and microglia were
chosen as relevant systems for CB1 and CB2 receptors,
respectively. Probe 1 is able to label neurons in primary
culture (Figure 2A,B). Next, we checked the specificity of the
labeling by parallel experiments in the presence of an excess
Figure 1. Molecular dynamics (MD) simulations of the ligand–receptor
complexes (the Supporting Information). Molecular surface of the
S1P1-based homology model of the human CB1R, showing the
entrance/exit of the ligand to/from the binding pocket. The structures
of probe 1 (in green, panels A and B) bound to CB1R and probe 3 (in
orange, panel C) bound to CB2R are shown, computed during the MD
trajectories. Helices color code: TM1 white, TM2 yellow, TM3 red,
TM4 gray, TM5 green, TM6 blue, and TM7 brown.
of ligand HU210, which abolished labeling (Figure 2C). As
expected, probe 3, with no affinity for CB1R, did not show any
significant fluorescence in neurons (Figure 2D). Notably, this
labeling is fully compatible with the simultaneous use of
primary antibodies in colabeling studies (Figure 3). Probe
1 shows the predominant location of CB1 receptor in the
neuronal soma (Figure 3A), whereas the anti-MAP2 anti-
body labels the entire neuron (Figure 3B), including axon and
dendrites, as expected from this marker. Labeling of CB1
receptor by probe 1 (Figure 3D) is further confirmed by
double staining with a commercial antibody against this
receptor (Figure 3E,F).
To further explore the potential of the probes in a different
native cell system, we assessed their performance in micro-
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Figure 2. Labeling of CB1R in neurons in primary culture with probe 1.
A) Neurons were incubated with 1 (0.5 mm). Excess of the probe was
then removed, and the bound probe was detected with streptavidin-
Alexa Fluor 488 (green). B) Colabeling of probe 1 and anti-MAP2
antibody (MAP2=microtubule-associated protein 2) visualized with
a secondary antibody conjugated to Alexa Fluor 594 (red). C) To
assess specificity, cells were labeled with 1 (0.5 mm) in the presence of
an excess of ligand HU210 (100 mm). D) Neurons incubated with the
CB2R-selective probe 3. All samples were imaged under the same
conditions by using a Zeiss fluorescence inverted microscope. Nuclei
are shown in blue. Bars: 25 mm.
Figure 4. Labeling of CB2R in rat microglial cells in primary culture
with 0.5 mm of probes 1 (A) and 3 (B). Excess of the probe was then
removed, and the bound probe was detected with streptavidin-Alexa
Fluor 488 (green). C) To assess specificity, cells were labeled under the
same conditions with the probe but in the presence of an excess of
ligand HU210 (100 mm). D) Immunostaining of microglial cells with
anti-Iba1 antibody (red, Iba1=ionized calcium-binding adaptor mole-
cule 1). All samples were imaged under the same conditions by using
a Zeiss fluorescence inverted microscope. Nuclei are shown in blue.
Bars: 25 mm.
Importantly, in microglial cells, labeling with probe 3
clearly reflects the changes in CB2R expression between
a resting state (Figure 5, panels A–C) and an activated (pro-
inflammatory) phenotype induced by lipopolysaccharide
(LPS) exposure (Figure 5, panels D–F).
Considering the relevance of the ECS in the regulation of
the immune system we studied the potential of these probes in
flow cytometry. The application of this methology would
facilitate the profiling of cannabinoid receptors at the single-
Figure 3. A–C) Double labeling of neurons with probe 1 (green, A) and
with an anti-MAP2 antibody (red, B). Panel C shows merged image.
D–F) Double labeling of neurons with probe 1 (green, D) and with an
anti-CB1R antibody (red, E). Panel F shows merged image. Images
were obtained by using a Leica fluorescence confocal microscope. In
D–F nuclei are shown in blue. Bars: 100 mm (A–C) and 5 mm (D–F).
glial cells, also known as the macrophages of the brain. These
cells express CB2R, which is believed to play a fundamental
role in their immune-related functions.[12] In agreement with
the binding profile of the probes, both probes 1 and 3 allowed
visualization of CB2R in microglia (Figure 4A,B) but not in
the presence of an excess of ligand HU210 (Figure 4C).
Again, the intensity of the fluorescence signal is comparable
with the one obtained with an antibody against the microglial
marker Iba-1 (Figure 4D).
Figure 5. Labeling of CB2R with probe 3 in rat microglial cells in
resting (A–C) and activated (D–F) state after pro-inflammatory stim-
ulus (LPS exposure). Microglia in primary culture were labeled with
anti-Iba1 (in red, A and D) and with probe 3 (in green, B and E).
Merged images are shown in panels C and F. Samples were imaged
under the same conditions by using a Leica fluorescence confocal
microscope. Nuclei are shown in blue. Bar (A–F): 5 mm.
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cell level and could contribute to shed light on the function of
the ECS in the immune system, a field of utmost current
interest.[13] Probes were tested in the monocytic cell line THP-
1, which has been described to have a functional endocanna-
binoid system.[14] Cells were incubated with probe 1 (1 mm)
and then stained with streptavidin-Alexa Fluor 488 and
analyzed by flow cytometry (Figure 6). Although the expres-
sion of CB1 and CB2 receptors is moderate in unstimulated
THP-1 cells as assessed by western blot (data not shown), our
experiments cleary demonstrate the suitability of probe 1 to
visualize cannabinoid receptors in this cell line by flow
cytometry.
Keywords: bioorganic chemistry · cannabinoid ligands ·
chemical probes · membrane proteins · protein models
.
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Figure 6. Flow cytometry analysis of THP-1 cells using probe 1.
Representative dot plots of THP-1 cells labeled with A) vehicle or
B) probe 1, followed by addition of streptavidin-Alexa Fluor 488.
Forward scatter (FSC) indicates the size of the gated cells. C) Histo-
grams display THP-1 cells stained with vehicle (gray) or probe
1 (black).
In conclusion, the probes described herein are, to our
knowledge, the first small-molecule tools suitable for visual-
ization of CB1 and CB2 receptors in native cell systems of
physiological relevance with at least comparable potency to
antibodies. Taking into account the practical limitations that
these antibodies have, these probes increase the arsenal of
chemical tools for the study of cannabinoid receptors and may
help to dissect the complex roles of the ECS. Further
extension of the versatility of this approach is currently
ongoing.
Received: January 17, 2012
Revised: May 14, 2012
Published online: && &&, &&&&
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Communications
Small-Molecule Probes
L. Martꢁn-Couce, M. Martꢁn-Fontecha,
ꢂ. Palomares, L. Mestre, A. Cordomꢁ,
M. Hernangomez, S. Palma, L. Pardo,
C. Guaza, M. L. Lꢃpez-Rodrꢁguez,*
S. Ortega-Gutiꢄrrez*
&&&& — &&&&
Receptors made visible: The described
biotin-tagged small-molecule probes with
excellent affinities for the CB1 and CB2
cannabinoid receptors (CB1R and CB2R)
enable direct visualization of these
receptors in native cellular systems,
including neurons (see picture), micro-
Chemical Probes for the Recognition of
Cannabinoid Receptors in Native
Systems
glia, and immune cells. This method
could overcome some of the limitations
of current methodologies and may help to
dissect the complexity of the endogenous
cannabinoid system.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
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These are not the final page numbers!