Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells

Article abstract of DOI:10.1021/jacs.7b11281

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB₂R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB₂R-selective bifunctional probe that covalently captures CB₂R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB₂R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Full text of DOI:10.1021/jacs.7b11281

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Article
A selective photoaffinity probe enables assessment of cannabinoid
CB2 receptor expression and ligand engagement in human cells
Marjolein Soethoudt, Sara C. Stolze, Matthias V. Westphal, Luuk van Stralen, Andrea
Martella, Eva J. van Rooden, Wolfgang Guba, Zoltan V. Varga, Hui Deng, Sander I. van
Kasteren, Uwe Grether, Adriaan P. IJzerman, Pal Pacher, Erick M. Carreira, Herman
S. Overkleeft, Andreea Ioan-Facsinay, Laura H. Heitman, and Mario van der Stelt
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b11281 • Publication Date (Web): 08 Feb 2018
Downloaded from http://pubs.acs.org on February 8, 2018
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A selective photoaffinity probe enables assessment of cannabinoid CB2
receptor expression and ligand engagement in human cells
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Marjolein Soethoudt,1,2 Sara C. Stolze,3,# Matthias V. Westphal,4 Luuk van Stralen,5 Andrea Martella,1 Eva J.
van Rooden,1 Wolfgang Guba,6 Zoltan V. Varga,7 Hui Deng,1,$ Sander I. van Kasteren,3 Uwe Grether,6 Adriaan
P. IJzerman,2 Pal Pacher,7 Erick M. Carreira,4 Herman S. Overkleeft,3 Andreea IoanꢀFacsinay,5 Laura H.
Heitman,2 Mario van der Stelt1*
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1Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands
2Research division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC,
Leiden, The Netherlands
3BioꢀOrganic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands
4Laboratorium für Organische Chemie, Eidgenössische Technische Hochschule Zürich, VladimirꢀPrelogꢀWeg 3, 8093 Zürich, Switzerland
5Department of Rheumatology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands
6Roche Innovation Center Basel, F. HoffmannꢀLa Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland
7Laboratory of Cardiovascular Physiology and Tissue Injury, National Institute on Alcohol Abuse and Alcoholism, National Institutes of
Health, 5625 Fishers Lane, Rockville, MD 20852, United States of America
#Current address: Max Planck Institute for Plant Breeding research, CarlꢀvonꢀLinnéꢀWeg 10, 50829, Köln, Germany
$West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, China
*corresponding author: Mario van der Stelt, m.van.der.stelt@chem.leidenuniv.nl
Keywords: GPCRs, CB2 receptor, target engagement, photoꢀaffinity labeling, bioꢀorthogonal chemistry
Abstract
Chemical tools and methods that report on G proteinꢀcoupled receptor (GPCR) expression levels and receptor
occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive
probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and
inflammatory diseases. LEI121 is the first CB2Rꢀselective bifunctional probe that covalently captures CB2R
upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups.
LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HLꢀ60 as well
as primary human immune cells using flow cytometry. Our findings show that strategically functionalized
probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoꢀ
click chemistry and hold promise as biomarkers in translational drug discovery.
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Introduction
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G proteinꢀcoupled receptors (GPCR) comprise a 700ꢀmembered family of sevenꢀtransmembrane domain
proteins expressed at the cell membrane.1,2 They convey extracellular signals from different types of stimuli,
such as light, (peptide) hormones and neurotransmitters, to intracellular second messenger systems, thereby
allowing cells to respond to their environment. GPCRs are involved in the regulation of many physiological
processes, including vision, behavior, mood, energy balance, immunity and inflammation.3 GPCRs are also an
important class of drug targets and offer great potential for the discovery of new therapeutics for a variety of
diseases.1 The type 2 cannabinoid receptor (CB2R),4 subject of the hereꢀpresented study, is a promising GPCR
for the treatment of tissue injury and inflammatory diseases.5,6
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The CB2R plays an important role in cell migration and immunosuppression. It shares extensive
sequence homology with the type 1 cannabinoid receptor (CB1R, 44% overall homology, and 68% homology in
the ligandꢀbinding domain),4 which is highly expressed in the central nervous system.7 Both CB1R and CB2R are
activated by ꢁ9ꢀtetrahydrocannabinol (ꢁ 9ꢀTHC, Figure S1), the main psychoactive constituent of marijuana.8
CB2R is predominantly found in cells of the immune system and is upregulated during various
pathophysiological conditions.5,9 Selective activation of the CB2R may confer therapeutic benefits without
inducing adverse side effects associated with CB1R modulation.10 This spurred drug discovery efforts by
academic groups and the pharmaceutical industry resulting in the identification of highly selective CB2R
agonists, such as LEI101, HU308 and HU910 (for structures see Figure S1).11ꢀ14 These CB2R agonists show
robust efficacy in various animal models of chronic and inflammatory pain, diabetic neuroꢀ and nephropathy,
liver cirrhosis, and ischemicꢀreperfusion injury.11ꢀ14
The successful development of new drugs strongly depends on our understanding of their underlying
molecular and cellular mechanism of action.15,16 An important step that drives the drug discovery processes is the
determination of the cellular expression profile of the target protein in humans.17 This provides a challenge for
the study of GPCRs, because they are usually expressed at very low levels in native cells and tissues.18 In
addition, GPCRs are known for their inducible nature, which includes the adaptation from inactive to active
conformations and internalization and desensitization upon durable activation. These factors may lead to variable
surface expression of the GPCRs. Antibodies can be used to detect surface expression of GPCRs, but specific
antibodies against CB2R are currently lacking, which hampers the detection of CB2R by standard biochemical
methods.18ꢀ20
Another important aspect in drug development is to verify that the drug candidate fully engages with its
intended target in vivo.15 Information on target engagement at a certain concentration will help to select the best
compound as a drug candidate and may guide the dose selection by providing information on full target
engagement, while minimizing the risk for untoward offꢀtarget interactions by preventing overexposure.
Currently, there are no biomarkers for target occupancy of CB2R available, complicating the translation of
preclinical data and dose selection of CB2R agonists in the clinic.12,21
Chemical probes are highly useful tools to map ligandꢀprotein interactions in living systems.17 Positron
emission tomography (PET) tracers, for instance, are widely applied to determine receptor occupancy of drug
candidates in patients. However, the disadvantages of such probes are that they require a facility for
radiolabeling and have limited cellular resolution.
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Fluorescent and biotinylated probes are also used to study GPCR function,22ꢀ30 but the size of the reporter group
may interfere with receptor affinity and/or selectivity,22,23,25,26 may increase nonspecific binding23,26,27 and lead to
a decrease in metabolic stability.22 Another drawback is that such probes rely on nonꢀcovalent interactions with
the receptor, which can be easily disrupted by various experimental conditions. The latter can be avoided by
using photoaffinity probes that employ a lightꢀresponsive element to covalently crosslink the compound with its
target protein upon irradiation.31,32ꢀ34 Photoreactive probes have been previously used to map GPCR binding sites
and ligandꢀreceptor interactions with or without the use of radioactive isotopes for detection,34ꢀ36 and to capture
the receptor in heterologous overexpression systems,37 but not for GPCR target engagement in endogenous
expressing systems. To circumvent the problems associated with large reporter groups or radioactive
isotopes,35,36 photoaffinity probes, containing a strategic ligation handle (e.g. alkyne or azide), to introduce a
fluorescent or affinity tag (e.g. biotin) after crosslinking to a protein, have emerged as powerful tools to visualize
small moleculeꢀprotein interactions in living systems.15,38ꢀ40 This strategy is known as twoꢀstep photoaffinityꢀ
based protein profiling (pAfBPP).31
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Inspired by these established and emerging concepts, we describe herein a twoꢀstep pAfBPP strategy to
visualize endogenous CB2R expression and target engagement in primary human cells. To the best of our
knowledge, this is the first report of a twoꢀstep photoaffinity probe being used for such studies on a GPCR.
Photoaffinity probe LEI121 contains a photoactivatable diazirine group, enabling CB2R crosslinking upon UVꢀ
irradiation and an alkyne moiety for bioꢀorthogonal conjugation to various reporter groups. LEI121 enabled
target engagement studies and visualization of CB2R on HLꢀ60 cells and primary human immune cells. Our
results show that strategically functionalized photoreactive probes can monitor endogenous GPCR expression
and ligand engagement. Such probes hold promise as biomarkers for target engagement studies in translational
drug discovery.
Results
Design and synthesis of LEI121
The ideal bioꢀorthogonal photoaffinity probes are normally composed of three distinct features:31 1) a
recognition element that binds to the intended target in a potent and selective manner, 2) a photoactivatable
group, which upon irradiation, forms a reactive intermediate capable of covalently binding the target protein, but
is otherwise stable in the absence of the external activating signal, and 3) a ligation handle that can be used to
couple the probe to different reporter tags using bioꢀorthogonal chemistry. The latter two functionalities should
be small and properly positioned to minimally affect the probe’s binding affinity for the target or the selectivity
of the probe. In addition, the probe should have favorable physicoꢀchemical properties, displaying aqueous
solubility with a minimum of nonꢀspecific interactions. In view of the stringent set of criteria, we choose 3ꢀ
cyclopropylꢀ1ꢀ(4ꢀ(6ꢀ((1,1ꢀdioxidothiomorpholino)methyl)ꢀ5ꢀfluoropyridinꢀ2ꢀyl)benzyl)imidaꢀzoleidineꢀ2,4ꢀ
dione (LEI101, (1), Figure 1a) as our starting point to develop a new photoaffinity probe for the CB2R. LEI101
is a highly selective CB2R agonist with favorable physicoꢀchemical properties, such as low molecular weight
(MW 473 Da), low lipophilicity (cLogP 1.0) and high solubility (89 mg/L).11 LEI101 has oral efficacy in a
neuropathic pain model and in a clinically relevant murine model of nephropathy in a CB2Rꢀdependent
manner.11
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To identify appropriate positions in the scaffold of LEI101 for the introduction of the two key elements, a
photoreactive group and ligation handle, we conducted a careful analysis involving the available structureꢀ
activity relationship (SAR) data previously reported for LEI101.41 Furthermore, we performed a docking study
of LEI101 with a homology model of the human CB2R (hCB2R), based on a recently published crystal structure
of the hCB1R in complex with the antagonist AM6538 (Figure 1b).42 We surmised that opening the hydantoin
moiety would enable the introduction of exitꢀvectors incorporating functional groups to introduce a
photoreactive group and a ligation handle. We selected trifluoromethylꢀdiazirineꢀbenzoyl as a relatively small
photoreactive group and a propargyl substituent as ligation handle. Combining these functional groups with the
biaryl scaffold of LEI101 led to the design of the CB2R selective photoaffinity probe LEI121 (2) (Figure 1a).
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Figure 1. Design, synthesis and two-step photo-affinity labeling of LEI121. A) LEI121 was designed by replacing the hydantoin group of
LEI101 by a central amide that carries both the photoꢀreactive diazirine moiety (blue), and the ligation handle (red). B) LEI101 was docked
into a CB2R homology model based on a recently published CB1R crystal structure.42 C) Synthesis of LEI121, reagents and conditions: a)
NaBH4, DCM:MeOH (2:1), rt, 99%; b) (4ꢀFormylphenyl)boronic acid, Pd(PPh3)4, K2CO3, toluene:EtOH (4:1), 80ºC, 89%; c) DiPEA, MsꢀCl,
DCM, 0ºC, quant.; d) Thiomorpholine 1,1ꢀdioxide, K2CO3, ACN, 60ºC, 95%; e) Nꢀmethylmorpholine, HOBt, EDC, DCM, quant.; f) HCl,
dioxane, 60%; g) K2CO3, NaBH(OAc)3, MeOH:DCM (1:1), 28%; h) Diazirine 13, EDC, HOBt, DCM, 14%. D) Twoꢀstep photoꢀaffinity
labeling. After incubation of proteome or whole cells with the probe, UVꢀirradiation causes the formation of a carbene, which is able to
insert itself into a CꢀH, OꢀH or NꢀH bond of the targeted protein. The resulting proteinꢀprobe complex is tagged using a copperꢀcatalyzed
azideꢀalkyne cycloaddition (CuAAC) with the following conditions: NaAsc, CuSO4, THPTA and tag (14), (15) or (16) to enable SDSꢀ
PAGE, mass spectrometry, or flow cytometry analysis, respectively.
The synthesis of LEI121 commenced with reduction of commercially available aldehyde 3, followed by Suzuki
coupling with 4ꢀformylboronic acid to afford intermediate 5, which was converted into compound 7 via
mesylation and nucleophilic substitution. Building block 11 was synthesized in two steps from Bocꢀprotected
glycine 8 following literature procedures.43
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Probe precursor 12 was obtained via reductive amination of aldehyde 7 using building block 11. Peptide
coupling of amine 12 with commercially available diazirine 13 furnished LEI121 (Figure 1c). Figure 1d shows
the twoꢀstep photoꢀaffinity labeling workflow with LEI121: after covalent CB2R adduct formation by insertion
of a reactive carbene, generated upon photoactivation of LEI121 (irradiation at 350 nm), ligation of CY5ꢀN3
(14), biotinꢀN3 (15) or AlexaFluorꢀ647ꢀN3 (16) is effected by Cu(I)ꢀcatalyzed azideꢀalkyne cycloaddition
(CuAAC, “click”ꢀreaction).44,45 The conjugated constructs enable subsequent visualization by inꢀgel
fluorescence imaging after electrophoresis on polyacrylamide (SDSꢀPAGE), mass spectrometry, and FACS
analysis, respectively.
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Molecular pharmacology of LEI121 on human CB2 and CB1 receptors
We first determined the affinity of LEI121 (without UVꢀirradiation) for the hCB2R in a radioligand binding
competition assay employing [3H]ꢀCP55940 (a tritiated high affinity ligand for CB2R) using membranes derived
from recombinant hCB2Rꢀoverexpressing Chinese hamster ovary (CHO) cells (CB2RꢀCHO cells), as described
previously.12 LEI121 displaced [3H]ꢀCP55940 in a concentrationꢀdependent manner (pKi = 7.2 ± 0.4), similarly
to LEI101 (pKi = 7.5 ± 0.1) (Table S1).11 Importantly, LEI121 did not show any affinity (pKi < 5) for hCB1R in
a similar competition assay (Table S1).12 Next, we tested whether UVꢀirradiation would result in covalent
binding of LEI121 to CB2R. Indeed, specific binding of [3H]ꢀCP55940 to hCB2R membranes was significantly
reduced in samples pretreated with LEI121 and crossꢀlinked by UVꢀirradiation (λ = 350 nm, 5 minutes), using a
CaproBoxTM 46
, a device used for controlled irradiation of biological samples with simultaneous cooling at 4°C,
to counteract the heat induced by the irradiation. Specific binding of [3H]ꢀCP55940 remained unchanged when
the irradiation step was omitted or the nonꢀphotoreactive ligand ꢁ9ꢀTHC was used (Figure 2a). These findings
indicate that LEI121 undergoes covalent and irreversible crosslinking to the CB2R binding site upon UVꢀ
irradiation.
Figure 2. Molecular pharmacology of LEI101 vs. LEI121. A) Reduced [3H]CP55940 binding to CB2R in LEI121ꢀtreated membranes from
CB2Rꢀoverexpressing CHO cells was observed after UVꢀirradiation and washout, but not in THCꢀ or nonꢀirradiated LEI121ꢀtreated
membranes. Data presented are the mean ± SEM of three (two in case of THC) independent experiments performed in duplicate. Statistics
performed is a twoꢀtailed tꢀtest (****pꢀvalue < 0.0001). BꢀC) βꢀArrestin recruitment and G protein activation were measured as described
previously,12,47 showing inverse agonistic activity of LEI121 (■) (βꢀarrestin recruitment pEC50 (Emax): 7.3 ± 0.3 (ꢀ12 ± 4); G protein activation
pEC50 (Emax): 6.6 ± 0.2 (ꢀ50 ± 7)), which is in contrast to the agonism activity of LEI101 (●)11 (βꢀarrestin recruitment pEC50 (Emax): 7.0 ± 0.3
(41 ± 6); G protein activation pEC50 (Emax): 6.6 ± 0.2 (65 ± 8). Efficacy (Emax) is normalized to the effect of 10 µM CP55940. Data are
presented as the mean ± SEM of three independent experiments performed in duplicate, except for βꢀArrestin recruitment of LEI121 (4
experiments in duplicate).
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Since CB2R ligands can modulate different intracellular signal transduction pathways, we determined the
potency of LEI121 in two functional assays by measuring βꢀarrestin recruitment and G protein activation.12,47
As previously reported, LEI101 behaved as partial agonist in both assays (Figure 2b,c and Table S1).11 LEI121
did not activate either of the two pathways, but was able to reduce the constitutive activity of the receptor,
thereby classifying as an inverse agonist (See Figure 2b,c and Table S1). Although the exact reason for the
switch in functional activity between LEI101 and LEI121 is currently unknown, it is well known that structural
changes within a chemical series may result in a reversal of functional activity (Figure S2).48
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Visualization of CB2 receptors by LEI121 using affinityꢀbased protein profiling
LEI121 was tested for its ability to visualize CB2R by twoꢀstep pAfBPP. To this end, membrane preparations of
hCB2Rꢀoverexpressing CHO cells were incubated with LEI121. Crossꢀlinking was again effected by UVꢀ
irradiation (λ = 350 nm). Next, the membranes were subjected to copper (I)ꢀcatalyzed click reaction conditions,
utilizing Cy5ꢀN3 (14) as the fluorescent azide to analyze the probeꢀprotein complex by SDSꢀPAGE and inꢀgel
fluorescence imaging. In this manner two major bands with an apparent molecular weight of ~47 and ~41 kDa
(Figure 3a) were visualized, and these were absent in membranes from wild type CHO cells treated in the same
manner. Heatꢀinduced denaturation prior to probe incubation also resulted in a loss of fluorescent bands,
indicating that the recognition is dependent on an intact threeꢀdimensional protein conformation. The bands were
also absent in nonꢀUV treated samples, demonstrating that the probe does not covalently interact with CB2Rs in
the absence of irradiation. Furthermore, omission of the clickꢀmixture showed that labeling was dependent on
copper (I)ꢀcatalyzed azide alkyne click ligation (Figure 3a). CB2R has a glycosylation site on its Nꢀterminus,49
and a glycosylated form of CB2R (~46 kDa) has previously been reported.50 Therefore we wondered whether the
two fluorescent bands could reflect different glycosylation forms. We treated the membranes with a glycosidase
(PNGaseF) to remove Nꢀlinked glycans. This resulted in decreased fluorescence of the 47 kDa band, while the
fluorescence intensity of the 41 kDa band, which corresponds to the molecular weight of the CB2RꢀprobeꢀCy5
adduct, increased (Figure 3b). Of note, longer incubation times or higher amounts of PNGaseF resulted in a loss
of signal, which might reflect protein precipitation due to loss of protein solubility. Inꢀgel digestion and mass
spectrometry analysis identified CB2R peptides in both bands (Table S2). Taken together, these results could
suggest that the highꢀMW labeled bands represent glycosylated forms of the receptor, although other postꢀ
translational modifications may also be present on the CB2R. Next, we validated that the band intensity was
dependent on probe and protein concentration (Figure S3). With the total amount of CB2R (Bmax) for the
membrane preparations of this cell line set at around ~15 pmol/mg protein (as determined by radioligand
saturation studies), we calculated the detection limit of the probe to be around 9 fmol of CB2R protein. Of note,
labeling of CB2R, albeit less intense, could also be obtained with two other photoaffinity probes, behaving as
CB2R agonists: LEI120, a close analog of LEI121, and RO7239315, which has a completely different chemotype
that is based on the structure of HU308 (Supplementary Figures 4 and 5, respectively).14 Interestingly, LEI120
and three analogues of RO7239315 showed less labeling (Supplementary Figures 4 and 5), respectively. This
indicated that a subtle change in positioning of the diazirine of the probes in the binding site plays an important
role in the labeling efficiency. It is hypothesized that the switch in functional behavior of LEI121 and LEI120 is
associated with the basic tertiary amine in LEI120, whereas the corresponding nitrogen in LEI121 is a tertiary
amide without hydrogenꢀbonding acceptor or donor properties.
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The CB2R binding cavity is not favorable for charged interactions around the position of the nitrogen. Therefore,
LEI121 has more favorable interactions than LEI120 with the receptor, which might explain its higher labeling
efficiency.
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Figure 3: Validation of CB2R labeling by LEI121 in CB2R-overexpressing CHO cells. A) LEI121 labels displaceable bands ( = CB2R)
specifically in CB2Rꢀoverexpressing membranes, not in wildtype CHO membranes, and only in presence of all necessary components (active
protein, probe, UVꢀirradiation, ligation components). B) Addition of PNGase F shows a decrease of higher MW glycosylated CB2R bands,
and an increase in fullꢀlength, probeꢀbound CB2R signal (~41 kDa). The gel shown is a representative result of three independent
experiments. C) Isolation of CB2R was achieved in live CB2Rꢀoverexpressing cells by ligation with biotinꢀN3 (15), followed by avidin
enrichment, trypsinization and proteomics. CB2R peptides identified from 3 independent experiments are highlighted in magenta. D)
Quantification of doseꢀdependent displacement of LEI121ꢀlabeled bands in SDSꢀPAGE experiments by different cannabinoid ligands.
Results shown are the mean ± SEM of 2 (CP55940 and 2ꢀAG) or 3 (SR144528 and HU910) independent experiments. The inlay shown is a
representative gel of concentrationꢀdependent displacement of LEI121 labeling by 2ꢀAG (see also Figure S8).
To unequivocally prove that CB2R is captured by LEI121, we incubated live human CB2Rꢀoverexpressing CHO
cells with LEI121. Subsequent UVꢀirradiation and ligation with biotinꢀN3 (15) for affinity enrichment on avidin
agarose beads enabled receptor identification by mass spectrometryꢀbased proteomics. In three independent
experiments, we identified CB2Rꢀspecific peptides that belonged to the extraꢀ and intracellular regions of the
receptor (Figure 3c, Table S3). The identified CB2R peptides were absent in samples from wild type CHO cells
and nonꢀirradiated samples. CHOꢀCB2R cells pretreated with CP55940, revealed a 78 ± 6% inhibition of CB2R
target engagement by LEI121 (Figure S6a,b).
Taken together, these results show that LEI121 is able to capture the hCB2R using tandem photoꢀclick
chemistry. Of note, five other proteins were identified as potential offꢀtargets of LEI121, i.e. multidrug resistance
protein 1, protein disulfide isomerase, mitochondrial carnitine/acylcarnitine carrier protein, Glutathione Sꢀ
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transferase Mu 6 and 26S proteasome nonꢀATPase regulatory subunit 3 (Figure S7). CP55940 only reduced the
abundance of CB2R (Figure S6c), which indicates that the displaceable signal by CP55940 is CB2Rꢀspecific.
Next, we explored whether the probe could be used in a competition format to test target engagement of
various ligands representing distinct chemical classes and exhibiting different functional activities. Highly
selective CB2R agonists (LEI101, HU910 and HU308), CB2R inverse agonists (SR144528 and AM630), CBR
agonist CP55940 and the endocannabinoid 2ꢀarachidonoylglycerol (2ꢀAG) (for structures see Figure S1) all
prevented labeling of both bands by LEI121 (Figure 3a,d). Concentrationꢀdependent displacement was observed
for SR144528, HU910, 2ꢀAG and CP55940 (Figure S8). CP55940, the most potent CB2R ligand in the series,
showed the strongest reduction in CB2R labeling by LEI121 (Figure 3a; Figure S8). Of note, LEI121 was not
able to label CB1R in a similar experiment using membranes of CB1Rꢀoverexpressing CHO cells (Figure S9).
Collectively, the results showed that LEI121 is able to capture and visualize the human CB2R and its ligand
engagement in heterologous overexpression systems.
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Visualization and target engagement of endogenous CB2R in human cells
To test the ability of LEI121 to visualize endogenous CB2R expression in live human cells, we used the human
promyelocytic leukemia cell line HLꢀ60, a fastꢀgrowing cell line widely used to study endogenously expressed
CB2R.9,51,52 Since CB2R expression in these cells was too low to reveal a specific CB2R band using gelꢀbased
fluorescence imaging, we turned to fluorescenceꢀactivated cell sorting (FACS), a specialized form of flow
cytometry. AlexaFluorꢀ647ꢀN3 (16) was preferred over the Cy5 dye (14) due to the lower background
fluorescence in FACS analysis. Incubation of HLꢀ60 cells with LEI121 followed by tandem photoꢀclick
chemistry resulted in a population of cells that demonstrated increased fluorescence (Figure 4). Preꢀincubation
with CP55940 or SR144528 showed significant reductions in mean fluorescence intensity, thereby indicating
that CB2R was successfully engaged by these ligands (Figure 4), as CB2R is the only shared protein target of
these molecules (Figure S6).
Figure 4. Visualization of endogenous CB2R expression in live HL-60 cells by LEI121. A) Representative dot plot of the selected HLꢀ60
cell population for each individual experiment. BꢀC) Representative histograms showing fluorescence intensity differences between the
untreated sample (AF647ꢀfluorophore only), the LEI121ꢀtreated sample and the sample with CP55940 (CP, B) or SR144528 (SR, C). D)
Pretreatment with CP55940 (10 µM) and SR144528 (20 µM) induced ~50% and ~25% displacement, respectively, of the labeling induced
by LEI121. Statistics performed was a twoꢀtailed tꢀtest and the results shown are the mean ± SEM of the backgroundꢀcorrected, normalized
mean fluorescence intensity (MFI) values of three independent experiments performed in duplicate (**pꢀvalue < 0.01, **** pꢀvalue <
0.0001).
Finally, we assessed the ability of LEI121 to visualize endogenous CB2R expression in primary human cells. To
this end, peripheral blood mononuclear cells (PBMCs) from four healthy donors were isolated. PBMCs contain
different immune cell subtypes that can be distinguished based on the expression of specific cell surface markers
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using a panel of fluorescently labeled antibodies. For example, antibodies against Cluster of Differentiation 3
(CD3) detect the total Tꢀcell population, whereas Tꢀhelper cells and cytotoxic Tꢀcells are recognized by
antibodies against CD4 and CD8, respectively. B lymphocytes are targeted by antibodies against CD19 and
monocytes by antibodies against CD14.53 To assess which specific cell population expresses CB2R, we
optimized PBMC labeling conditions by LEI121 in the presence of a panel of fluorescently labeled antibodies.
Incubation of PBMCs with LEI121 followed by tandem photoꢀclick chemistry led to increased mean
fluorescence intensity (MFI) in each cell population (Figure 5b, middle panels). To determine the amount of
specific CB2R labeling, we performed the same experiment with a preꢀincubation using an excess of CP55940,
which resulted in decreased MFI in most cell populations (Figure 5b, middle panels). Quantification of this
effect revealed a significant reduction in LEI121 labeling by CP55940 in CD19+ B cells, CD14+ monocytes and
CD3+ T cells (Figure 5b, right panels). The highest specific fluorescent labeling was found in CD19+ Bꢀcells,
followed by CD14+ monocytes, whereas the signal was minimal in Tꢀcells (Figure 5c, bar graph). These results
indicate that CB2R is expressed in only a selection of human immune cells. Using quantitative polymerase chain
reaction (qPCR) as an orthogonal technique, we determined CB2R mRNA levels in the immune cell populations
studied, and found that CB2R mRNA levels were also highest in Bꢀcells, followed by monocytes and Tꢀcells
(Figure 5c, Northern blot).
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Figure 5. Visualization of endogenous CB2R expression in PBMCs by LEI121. A) Representative dot plot of the selected PBMC
population (debris, doublets and dead cells excluded). B) From left to right: representative dot plots of selected positive populations,
representative histograms of these populations to show fluorescence intensity differences, and scatter plots showing % labeling by LEI121 ꢀ
/+ CP55940 of four donors, in triplicate (n=12), normalized to the average maximum LEI121 signal per donor. The line represents the mean.
Statistics was performed using a twoꢀtailed tꢀtest (*pꢀvalue < 0.05, **** pꢀvalue < 0.0001, ns = not significant) on the mean backgroundꢀ
corrected, normalized MFI values of LEI121 ꢀ/+ CP55940 (N=4). Significant displacement with CP55940 was observed in CD3+ T cells,
CD14+ monocytes and CD19+ B cells. C) The specific fluorescent signal of LEI121 indicates the level of CB2R expression in these cell
types. The highest signal was found in CD19+ B cells, which is also the case for the level of CB2R mRNA expression measured for these cell
types. β2 Microglobulin mRNA expression per cell type was determined as control. Bar graph shows the mean ± SEM of backgroundꢀ
corrected specific fluorescence of LEI121 (details for calculations in B and C: see Table S3 and Data analysis).
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Recently, drug discovery research has focused on the development of selective CB2R agonists for the treatment
of tissue injury and inflammatory diseases that avoid inducing CB1Rꢀmediated psychoactive side effects. CB2R
knockout mice show enhanced pathology in various inflammatory disease models, including heart, liver or
kidney injury and inflammatory pain, thereby supporting the notion that CB2R plays an essential role in these
conditions. Despite compelling proofꢀofꢀconcept data obtained in preclinical pain models, two CB2R agonists
lacked efficacy in phase 2 clinical trials.12,21 The reasons for this nonꢀtranslatability are yet unclear. To validate
the hypothesis that the CB2R is a suitable therapeutic target for e.g. inflammatory pain, evidence is required that
the receptor is expressed by relevant cell types in pain signaling pathways and, importantly, that investigational
drugs fully engage with CB2R in clinical trials. The absence of specific antibodies to detect CB2R protein at the
site of inflammation in patients and the lack of biomarkers to study their engagement is currently hampering the
clinical development of CB2R agonists. To address these challenges, new chemical tools are necessary to
determine cellular CB2R expression and to show receptor occupancy.
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The work we have described allowed visualization of CB2R expression and target engagement in human
cells by photoaffinityꢀbased protein profiling and fluorescenceꢀactivated cell sorting (FACS). The probe LEI121
was designed and synthesized to possess a diazirine as photoreactive group to capture CB2R and an alkyne as
ligation handle to enable visualization or isolation of the protein by conjugation to fluorophores or biotin,
respectively. The study demonstrates that LEI121 is a potent CB2R inverse agonist and selective over the closely
related CB1R. Using tandem photoꢀclick chemistry, we noted that photoaffinity labeling of CB2Rꢀoverexpressing
CHO cell membranes resulted in visualization of two major species, corresponding to different glycosylated
forms of the receptor. The labeling was dependent on UVꢀirradiation and copperꢀcatalyzed conjugation of a
fluorophore and could be prevented by preꢀincubation with various, structurally diverse CB2R ligands. In
addition, we have shown that LEI121 labels endogenous CB2R on HLꢀ60 cells and primary human immune
cells, which could be prevented by various, structurally diverse CB2R ligands.
The development of LEI121 as a twoꢀstep photoaffinity probe provides new opportunities to study
CB2R biology. For example, LEI121 may serve as an alternative to the highly unselective CB2R antibodies.
Using LEI121, we have shown that Bꢀcells exhibited the highest CB2R levels, followed by monocytes, while
minimal receptor levels were found in Tꢀcells, which is in line with the CB2R mRNA levels as determined in this
study and as previously reported.9 We envision that LEI121 may be used to profile CB2R expression levels in
PBMCs of patients suffering from inflammatory pain and/or other inflammatory diseases, such as rheumatoid
arthritis, multiple sclerosis and Crohn’s disease. Next, isolation of CB2R using the biotinꢀreporter and affinity
enrichment from primary cells and tissues may facilitate the identification of potential protein interaction
partners of the receptor. Additionally, the photoreactive probe may help stabilizing CB2R to facilitate
crystallization studies of the protein.
Activityꢀbased protein profiling (ABPP) using chemical probes with electrophilic warheads has been
successfully applied to assess target engagement in living systems.15,38,39,54 ABPP is, however, limited to protein
families such as serine hydrolases, kinases and proteases, which possess nucleophilic amino acids in their
binding site that undergo covalent bond formation with the probe. The ABPP strategy has not been applied to the
study of GPCRs due to a lack of mechanismꢀbased rationale for probe design.
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To the best of our knowledge, the present study represents the first successful application of twoꢀstep
photoaffinityꢀbased protein profiling to monitor endogenous GPCR expression and occupancy by small
molecules in living human cells. Projecting forward it is likely that strategically functionalized photoreactive
probes can be used in preclinical animal models as well as in clinical settings to guide dose selection, thereby
advancing translational drug discovery.
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Experimental Section
Materials and general remarks
All materials used are specified in the supporting information, as well as the synthetic procedures of LEI121,
LEI120 and RO7239315. Molecules shown are drawn using Chemdraw, graphs and statistics were performed
with Graphpad Prism 7, BioꢀRad Imagelab was used for gel analysis and quantification, online TOPO software
was used to generate the snake plot in Figure 3, FlowJo V10.1 (Miltenyi Biosciences) was used to analyze
FACS data. In case of PBMC experiments, DIVA software from BD Biosciences was used for compensation of
the fluorescence.
Molecular modeling of LEI101 and LEI121
The Xꢀray structure of CB1R with the stabilizing antagonist AM653842 (PDB entry 5TGZ) was used to build a
CB2R homology model for the docking of LEI101, as described in the supporting information.
Molecular pharmacology of LEI121
CB2R affinity, G protein activation and βꢀarrestin recruitment were measured as reported previously.12
Radioligand binding on LEI121 pre-treated membranes from CB2R-overexpressing CHO cells
[3H]ꢀCP55940 specific binding was determined using membranes of CB2Rꢀoverexpressing CHO cells, preꢀ
treated with LEI121 (+/ꢀ UV) and THC in a washout experiment similar to previously reported procedures.32
Specifications are provided in the supporting information.
Two-step covalent SDS-PAGE visualization of LEI121 labeling of CB2R
CB2RꢀCHO and WT CHO cell membrane aliquots were pretreated with competitor or vehicle, followed by
incubation with LEI121. The samples were diluted, irradiated using the CaproboxTM (λ=350 nm). Control
samples were denatured before UVꢀtreatment and NoꢀUV controls were kept in the dark using aluminum foil
until the ligation reaction was performed with Cy5ꢀN3 (14) for 1 hr at rt. Samples without click mix received
MilliQ with the same % of DMSO. Finally, samples were denatured in 4x Laemmli sample buffer, and resolved
by SDSꢀPAGE electrophoresis followed by inꢀgel fluorescence scanning using a BioꢀRad Chemidoc at the Cy5
channel. Inꢀgel digestion was performed after coomassie staining according to published procedures.55
Specifications are provided in the supporting information.
Two-step photo-affinity enrichment and mass spectrometry-based proteomics of CB2R
Cells were grown as specified in the supporting information. Live wild type or CB2Rꢀoverexpressing CHO
cells were pretreated with DMSO or CP55940 and incubated with LEI121 (total volume: 3 mL).
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The incubation solution was removed and replaced by 1.5 mL fresh buffer, then the plates were immediately
irradiated (except the No UV control) for 5 min with CaproboxTM (λ=350 nm), and the cells were harvested. The
cells were lysed and the membrane and cytosol fractions were separated. Ligation reaction was performed with
biotinꢀN3 (15). Protein was precipitated and washed to remove all click mix components, then denatured,
reduced and alkylated. The protein was added to avidin beads and incubated. The avidin beads were washed 4x,
then digested overnight with trypsin containing buffer. Samples were quenched with formic acid (FA) and beads
were removed using a biospin column. Peptides were added to C18 stagetips, washed, and then eluted. Peptides
were concentrated using an Eppendorf SpeedVac and dissolved in LC/MS solution. Samples were measured
using a NanoACQUITY UPLC System coupled to a SYNAPT G2ꢀSi high definition mass spectrometer (Waters)
and analyzed as specified in the supporting information, which contains also all other specifications of the
pulldown procedure.
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Two-step photo-affinity visualization of endogenous CB2R by LEI121 using FACS
All specifications of FACS experiments in HLꢀ60 cells and freshly isolated human PBMCs are provided in the
supporting information. Briefly, after extensive washing steps, the cells were counted and resuspended at 1 x
106 cells/mL and 499 µL cell suspension was added per sample. Pretreatment with competitor or DMSO was
followed by treatment with LEI121 or DMSO. Unbound molecules were removed by centrifugation and the cells
were resuspended in PBS (HLꢀ60 cells), or antibody/isotype mixture or 1% BSA/PBS (PBMCs), followed by a
short incubation time in case of the antibody stain. Then, the cell suspensions were irradiated with CaproboxTM
(350 nm), then transferred to a Vꢀbottom 96ꢀwell plate and PBS was removed by centrifugation. Cells were
fixed, washed, and blocked for 20 min with 1% BSA/PBS. The cells were pelleted and resuspended in ligation
mixture containing AF647ꢀN3 (16). The cells were pelleted, washed, resuspended in 1% BSA/PBS and analyzed
with a Guava easyCyte HT (HLꢀ60 cells) or a LSRII (PBMCs). Compensation of fluorescence was done using
stained and unstained compensation beads using DIVA software (BD Biosciences) and further analysis was
performed using FlowJo v10.1 (Miltenyi Biosciences).
Gene expression analysis of CB2R mRNA in human PBMC populations
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, collected from multiple healthy
human donors. Different cell types were separated by positive magnetic selection, and RNA was isolated and
amplified by PCR. Amplicons were resolved on 2% agarose gels and analyzed using a G:Box gel documentation
system (Syngene, Frederick, MD, USA). Specifications are provided in the
supporting information.
Supporting information available
Experimental procedures, supporting figures, supporting tables and compound characterization. Mass
spectrometry proteomics data have been deposited to the proteomeXchange Consortium via the PRIDE partner
repository with the dataset identifier PXD008460.56
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Acknowledgments
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M.S., L.H.H. and M.v.d.S. were supported by an ECHOꢀSTIP grant from the Netherlands Organization for
Scientific Research (NWO, grant: 711.014.009). The Dutch Ministry of Education (Gravity Program ‘The
Institute of Chemical Immunology’) is acknowledged for financial support. The authors thank Caprotec for
kindly providing the CaproboxTM. Hans van den Elst and Fons Lefeber are acknowledged for technical support.
Dr. Richard van den Berg is acknowledged for critical feedback on the supplementary information of the
manuscript. Elisabeth Zirwes, Anja Osterwald and Christoph Ullmer are acknowledged for the in vitro
characterization of compounds 27, 28, 41 and RO7239315.
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Author Contributions
M.S. has designed and synthesized probes LEI121 and LEI120, developed, optimized and performed the
biological assays and wrote the manuscript. S.C.S aided in the development of the gelꢀbased imaging and
pulldown protocol. M.V.W. designed, synthesized and chemically characterized probes RO7239315, 27, 28 and
41 under supervision of U.G. and E.M.C., L.v.S. aided in the development of the FACS experiments in human
PBMCs under supervision of A.IꢀF. A.M. performed the radioligand experiments on LEI121ꢀpretreated CB2Rꢀ
overexpressing CHO membranes. E.J.v.R. measured and analyzed proteomics samples. W.G. created the CB2R
homology model and performed docking studies. Z.V.V. measured mRNA expression in human PBMCs. H.D.
synthesized the Cy5ꢀN3 used in this study. M.V.W, S.v.K, U.G., A.P.IJ, E.M.C, P.P., A.IꢀF and H.S.O.
contributed to the experimental design and writing of the manuscript.. L.H.H. and M.v.d.S conceived, and
contributed to the experimental design, supervised the project and wrote the manuscript.
Conflict of interest
The authors declare no conflict of interest
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