Journal of the American Chemical Society
Article
diaryl-hydrazine, and deprotection of TBS ether (38% yield
over 4 steps). Azo-HU308-2 and -3 were prepared via cross-
coupling with corresponding potassium trifluoroborate re-
agents45,46 8 and 9 under conditions reported by Molander
and co-workers (Pd(OAc)2, PCy3, Cs2CO3, THF/water).47
After TBS ether deprotection with Bu4NF, azo-HU308-2 and
-3 were obtained in excellent yields of 89 and 94% over 2 steps,
respectively.
We proceeded to characterize the photophysical properties
of azo-HU308s. In their resting state, they reside in the
thermodynamically favored trans-configuration. Illumination at
365 nm (UV-A) leads to a photostationary state (PSS)
favoring the cis-isomer. Irradiation at 455 nm (blue light) leads
to a new PSS with trans as the dominant isomer (Figures 2, S1,
Figure S6B, bottom), demonstrating the suitability of this
fluorescent probe for specific visualization of CB2.
Classically, CB2 receptors are known to couple to Gαi/o
proteins, which inhibit adenylyl cyclase activity.16,51,52 More
recent studies suggest that CB2 can also couple to other G
proteins and effector pathways.18,53 CB2 shows pronounced
biased signaling on activation by different ligands, and the
effects of CB2 activation on intracellular Ca2+ concentration
([Ca2+]i) have only been described on few occa-
sions.22−24,54−56 To the best of our knowledge, CB2-mediated
modulation of [Ca2+]i has not been reported for HU308
derivatives; therefore, we were intrigued to study Ca2+
signaling in living cells using HU308 and its photoswitchable
derivatives. To assess the effect of HU308 in modulating CB2-
mediated Ca2+ release, AtT-20(CB2) cells were loaded with
Ca2+-sensitive Fluo-4-AM dye and subjected to real-time
fluorescent Ca2+ imaging (Figure 3C).57 Ionomycin (10 μM),
a Ca2+ ionophore that permeates cellular membranes, was
added for normalization purposes at the end of the experiment.
Upon the addition of HU308 (20 μM), a large increase in
Fluo-4 fluorescence intensity was observed, indicating that
CB2 activation causes an increase in [Ca2+]i (Figure 3C,D
black, Figure S7A). Irradiation at 375 nm (UV-A) did not
affect [Ca2+]i before or after HU308 addition, confirming that
light alone does not affect [Ca2+]i in AtT-20(CB2) cells and
that the effect of HU308 on [Ca2+]i is not photosensitive.
Lower concentrations of HU308 induced Ca2+ transients to a
lesser degree (Figure S7B), and we determined an EC50 of
∼6.4 μM (Figure S7C). As the binding affinity for HU308 is
typically reported in the low nM range,31 this could indicate
that this phenomenon is induced specifically at high CB2
receptor occupancies.54
In control experiments, vehicle addition sometimes
produced a small Ca2+ transient, which was not affected by
irradiation (Figure S8A,B). Additionally, the effect of HU308
was substantially diminished in AtT-20-wild-type cells (Figure
3D, green), confirming the involvement of CB2 in initiating
the Ca2+ response. The remaining small Ca2+ transient is likely
an artifact by the mechanical force resulting from drug addition
and was not blocked by the presence of CB2 inverse agonist
AM63058 (20 μM) (Figure S8C).
Next, we investigated the effect of several CB2 antagonists/
inverse agonists on the action of HU308 in AtT-20(CB2) cells
(Figure 3E). In comparison to HU308 addition alone (20 μM,
black), SR14452859 (20 μM, blue) did not have a profound
effect, while AM630 (20 μM, green) dramatically reduced the
Ca2+ transient. Interestingly, the addition of HU308 in the
presence of JTE90760 (40 μM, red) resulted in a delayed and
persistent Ca2+ transient. These results confirm the involve-
ment of CB2 in driving this cellular response and highlight the
intricacies of biased signaling mediated by a chemically diverse
set of CB2 inverse agonists.
Figure 2. Photoisomerization of azo-HU308s. (A) azo-HU308-3 is
isomerized between its cis- and trans-configurations with UV-A and
blue irradiation, respectively. (B) UV−vis spectra of azo-HU308-3
(50 μM in DMSO) in its dark-adapted (black), UV-A-adapted (gray),
and blue light-adapted (blue) photostationary states.
and S2). Trans- and cis-isomer ratios at their photostationary
states were quantified by HPLC analysis with UV detection at
the isosbestic wavelengths. At 365 nm, azo-HU308-1-3 attain
maximum cis-content of 80, 71, and 49%, respectively.
Subsequently, at 455 nm, a new PSS is reached, with trans-
content of 83, 86, and 87% (Figures S3 and S4). Additionally,
azo-HU308s were moderately bistable, with cis-azo-HU308-1-
3 relaxing to their trans-isomers with thermal half-lives (t1/2 at
rt and pH = 7) of 2.1, 6.5, and 1.6 h in H2O, respectively
(Figure S5).48
Optical Control of Intracellular Ca2+ via CB2. A
transformed mouse pituitary tumor AtT-20 cell line which
stably expresses CB2 receptors (AtT-20(CB2) cells) reported
by Mackie and co-workers was selected to explore the activity
of the HU308-based photoswitches.49 To confirm CB2
expression and membrane localization in this cell line, we
utilized our recently reported HU308 derivative tagged with
Alexa488 (Figure S6A).33 This nonpermeable fluorescent
probe binds specifically to CB2 receptors, and it allows real-
time receptor visualization in living cells using fluorescence
microscopy. For comparison, both AtT-20(CB2) and AtT-20-
wild-type cells lacking CB249,50 were incubated in parallel with
fluoroprobe (150 nM) and nuclear stain Hoechst33342 (20
μM). In AtT-20(CB2) cells, Alexa488-fluorescence was
observed on the outer plasma membrane of most cells (Figure
3A, white arrows; Figure S6B, top), confirming that CB2 is
expressed on the cell surface. We did not observe plasma
membrane fluorescence in AtT-20-wild-type cells (Figure 3B;
Cytosolic Ca2+ is known to originate from both extracellular
matrix as well as various intracellular reservoirs, including
endo/sarcoplasmic reticulum.24 We thus sought to determine
the mechanism by which HU308 and CB2 modulate [Ca2+]i
and to identify the source of the observed Ca2+ ions. First, Ca2+
in the extracellular imaging buffer was sequestered by addition
of the chelator ethylene glycol-bis(2-aminoethyl ether)-
N,N,N′,N’-tetraacetic acid (EGTA, 0.1 mM), which prevents
Ca2+ entry into the cell. Under these conditions, HU308
addition caused a rise in [Ca2+]i of approximately the same
magnitude; however, the effect subsided more rapidly when
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J. Am. Chem. Soc. 2021, 143, 736−743