Activity-based probe for specific photoaffinity labeling γ-aminobutyric acid B (GABAB) receptors on living cells: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/jm800140f

A trimodular activity-based probe was designed, synthesized, characterized, and applied to photoaffinity label the GABA_B receptors transiently expressed in Chinese hamster ovary (CHO) cells. The probe exhibits specific binding activity at the ligand-binding pocket of GB1 subunits and high specificity of photoaffinity labeling, which makes the probe valuable for studying the localization and function of GABA_B receptors on living cells.

Full text of DOI:10.1021/jm800140f

J. Med. Chem. 2008, 51, 3057–3060
3057
Letters
Activity-Based Probe for Specific
Photoaffinity Labeling γ-Aminobutyric Acid
B (GABAB) Receptors on Living Cells:
Design, Synthesis, and Biological Evaluation
Xin Li,‡,§ Jian-Hua Cao,†,§ Ying Li,†,§ Philippe Rondard,#
Yang Zhang,† Ping Yi,† Jian-Feng Liu,*,† and Fa-Jun Nan*,‡
Key Laboratory of Molecular Biophysics of Ministry of Education,
School of Life Science and Technology, Huazhong UniVersity of
Science and Technology, 1037 Luo Yu Road, Wuhan, Hubei, China,
National Center for Drug Screening, State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Shanghai Institutes
of Biological Sciences, Chinese Academy of Sciences, 189 Guo Shou
Jing Road, Shanghai 201203, China, and Institute of Functional
Genomics, CNRS UMR5203, INSERM U661, UniVersity of
Montpellier 1 and 2, Montpellier, F-34094, France
Figure 1. Structures of probe 1 and known GB1 antagonist 11
(CGP64213).
an IRS located in its C-terminal tail.12 In contrast, the GB2
subunit can activate G-proteins upon assembly of the het-
erodimer with GB1 at the cell surface but cannot directly bind
GABA.13 Interestingly, the GB2 subunit is also required for
high affinity of GABA to the GB1 subunit.14 Moreover, GB2
facilitates GB1 targeting to the cell surface by masking its IRS
when forming heterodimers. However, the mechanisms that
regulate GABAB receptors oligomerization at the plasma
membrane remain largely unknown.
The development of small molecular fluorescent probes
specifically targeting unmodified or native GABAB receptors
represents a major challenge. Such molecules will be powerful
tools to probe the localization and function of GABAB receptors
in living cells, as previously described for other proteins.15
Activity-based protein profiling (ABPP) is a chemical strategy
that utilizes activity-based probes (ABPs) to profile the func-
tional state of proteins in complex proteomes.16 To date, ABPs
have been developed for many enzyme classes and produced
global portraits of enzyme activity that depict specific physi-
ological and pathological processes.17 Nonetheless, the approach
has not been applied to study the location of important receptor
proteins in living cells.
In this paper, we report the design, synthesis, and biological
application of an activity-based fluorescent probe for photoaf-
finity labeling GABAB receptors on living cells. The probe,
which is termed probe 1, is designed based on 11, a GB1
selective high-affinity antagonist.18 The structure template of
probe 1 consists of the main structure moiety in 11 for binding
GABAB receptors, a photolabile diazirine group that effectively
generates a covalent, irreversible linkage between the probe and
the GABAB receptors after UV irradiation and a fluorescent
reporter tag for visualizing the localization of the labeled
proteins, as outlined in Figure 1. The photoinduced cross-linking
of probe 1 to the receptor is designed to dramatically increase
labeling efficacy. The trifluoromethylaryldiazirine moiety was
incorporated as the photochemically reactive functional group
because of the advantage of its excellent chemical stability prior
to photolysis.19 We selected 4, 4-difluoro-4-bora-3a,4a-diaza-
s-indacene (BODIPY) as the fluorophore because of its many
attractive spectral characteristics and its hydrophobic nature.20
The synthesis of probe 1 is outlined in Scheme 1. The key
intermediates 3-5 (Figure 2) were prepared individually via
procedures similar to those reported previously with some
ReceiVed February 19, 2008
Abstract: A trimodular activity-based probe was designed, synthesized,
characterized, and applied to photoaffinity label the GABAB receptors
transiently expressed in Chinese hamster ovary (CHO) cells. The probe
exhibits specific binding activity at the ligand-binding pocket of GB1
subunits and high specificity of photoaffinity labeling, which makes
the probe valuable for studying the localization and function of GABAB
receptors on living cells.
γ-Aminobutyric acid (GABA) is the main inhibitory neu-
rotransmitter in the mammalian central nervous system and
exerts its effects through two ligand-gated channels, GABAA
and GABAC receptors, and the metabotropic GABAB receptors.
GABAB receptors are widely expressed in the brain or spinal
cord during development and are located in both pre- and
postsynaptic compartments.1 They mediate slow synaptic inhibi-
tion and are involved in numerous types of nociception,
cognitive impairment, epilepsy, spasticity, and drug addiction.2
GABAB receptors belong to the family 3 of G-protein-coupled
receptors (GPCRs). Structurally, GABAB receptors possess an
extracellular domain so-called Venus flytrap module that binds
GABA and a heptahelical domain responsible for the recognition
and activation of heterotrimeric G-proteins. Functional GABAB
receptors were reported to repress Ca2+ influx3 and trigger K+
channels opening4,5 via coupling to Gi/o-type protein.6,7 GABAB
receptors can also modulate the level of cyclic adenosine
monophospate (cAMP)8 and the phosphorylation of ERK1/2 and
CREB.a,9 GABAB receptors form heterodimers composed of two
subunits: GB1 and GB2.10 The GB1 subunit has been shown
to bind GABA but is unable to activate G-proteins.11 It also
fails to reach the cell surface when expressed alone because of
* To whom correspondence should be addressed. Phone/Fax: 86-21-
50800954. E-mail: fjnan@mail.shcnc.ac.cn (for F.-J.N.) or jfliu@
mail.hust.edu.cn (for J.-F.L.).
‡
Chinese Academy of Sciences.
Authors contributed equally to this work.
Huazhong University of Science and Technology.
University of Montpellier 1 and 2.
§
†
#
a Abbreviations: ERK, extracellular signal-regulated kinase; cAMP, cyclic
adenosine monophosphate; CREB, cAMP-responsive element binding
protein; IRS, intracellular retention signal.
10.1021/jm800140f CCC: $40.75
2008 American Chemical Society
Published on Web 05/01/2008

3058 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Scheme 1. Synthesis of Probe 1a
Letters
Figure 3. Probe 1 inhibits GABAB receptor activation. HEK293 cells
cotransfected with the cDNA of wild-type GB1 and GB2 subunits, and
the chimera Gqi9, were incubated with the indicated concentrations of
probe 1 for 15 min before stimulation with 10 µM of GABA for 30
min. IP formation data are expressed as the percentage of the response.
IC50 value of probe 1 is 1.03 µM. Inner panel, 10 µM probe 2 is not
able to inhibit the IP production induced by 10 µM GABA, compared
to 10 µM probe 1. Values are the mean of triplicates from one
representative of three independent experiments.
a Reagents and conditions: (a) tert-butyl 2-(2-(2-bromoethoxy)ethoxy)-
ethylcarbamate, K2CO3, (n-C4H9)4NI, DMF, 60 °C, 24 h, 70%; (b) (i) HCl/
EtOAc, room temp, 1 h, (ii) chloroacetyl chloride, NaHCO3, 1,4-dioxane,
room temp, 24 h, 65%, two steps; (c) (i) NaN3, NaI, acetone, 60 °C, 12 h,
(ii) (n-C4H9)4NMnO4, pyridine, room temp, 12 h, 86%, two steps; (d)
N-hydroxysuccinimide, EDC·HCl, CH2Cl2, room temp, 12 h, 83%; (e) 3,
i-Pr2NEt, MeOH, room temp, 12 h, 89%; (f) 4, CuSO4, vitamin C, MeOH/
H2O, room temp, 1 h, 82%.
Figure 4. Probe 1 specifically labels GB1 subunit expressed on the
surface of living cells. CHO cells transiently transfected with the
indicated constructs were treated with probe 1 followed by UV
irradiation and then observed in TIRF microscopy (OLYMPUS IX-
70, oil immersion, ×100 objective) using filter sets for probe 1. Upper
panels are bright field images, while the lower panels represent
fluorescent images under probe 1 channel. Scale bar represents 10 µm.
Figure 2. Structures of the three elements comprising probe 1.
Scheme 2. Synthesis of Probe 2a
receptors and represents an activity-based fluorescent probe. The
selectivity of the probe 1 on other GABA receptors was not
determined.
Probe 1 was further evaluated for its capacity to label the
GABAB receptors in living cells. CHO cells transiently express-
ing GB1-ASA14 (a GB1 mutant able to reach the cell surface
alone) were incubated with probe 1 for 15 min. After photolysis
under UV for 15 min at room temperature, the cells were washed
with phosphate buffered saline (PBS) twice and observed by
total internal reflection fluorescent (TIRF) microscopy to acquire
detailed information about protein expressed on the cell sur-
face.25 As shown in Figure 4, probe 1 specifically labels GB1
expressed on the cell surface. Similar results were obtained on
CHO cells transiently cotransfected with GB1-wt and GB2-wt.
However, CHO cells transfected only with wild-type GB1 that
remains intracellular, or GB2 with no affinity for 11, could not
be labeled with probe 1. All these results were in agreement
with the previously reported structural properties11–13 of the
GABAB receptor and illustrate the specificity of the labeling
experiment. To show that probe 1 specifically labels the GB1
subunits expressed on the cell surface, we conducted double
labeling experiments using fluorescence confocal microscopy.
The DsRed markers26 combined with green fluorescent probe
1 markers for double labeling enable us to optically distinguish
and quantitatively analyze the specificity of labeling in living
cells. CHO cells transiently cotransfected with DsRed2-tagged
GB1-wt and GB2-wt, DsRed2-tagged GB1-ASA, or DsRed2-
tagged GB1-wt were labeled with probe 1 as previously
a Reagents and conditions: (a) (i) NaBH4, EtOH, room temp, 1 h; (ii)
HCl/EtOAc, room temp, 1 h; (iii) N-hydroxysuccinimidyl-4-(4,4-di-
fluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl)butyric acid,
i-Pr2NEt, room temp, 12 h, 82.1%, three steps.
modifications.21–23 The azide group was attached to the phe-
nyldiazirine 5 via a poly(ethylene glycol) (PEG) linker, and the
benzaldehyde was oxidized to the corresponding benzoic acid,
which was subsequently activated with N-hydroxysuccinimide
to give compound 9. Coupling 9 with the bioactive ligand 3
gave 10, which was subsequently reacted with the fluorophore
4 to give probe 1 employing the copper-catalyzed [3 + 2]
azide-alkyne cycloaddition.24
As a control compound, probe 2 (Figure 1), which has no
bioactive ligand toward GABAB receptors but only contains the
photoaffinity and fluorophore groups, was also synthesized by
reduction and deprotection of 6, followed by coupling with the
activated ester of the fluorophore (Scheme 2).
Prior to the labeling studies, the probes were first evaluated
for their bioactivity as GABAB receptor antagonists. As shown
in Figure 3, probe 1 significantly inhibited GABAB receptors
activation induced by GABA with an IC50 of 1.03 µM, while
probe 2 had no such inhibitory effect (Figure 3). These results
confirmed that probe 1 retains substantial affinity to GABAB

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3059
In summary, an activity-based trimodular probe was designed,
synthesized, and applied to the specific labeling of GABAB
receptors transiently expressed in living cells. Pharmacological
studies show that the probe conserved reasonable antagonist
potency for GABAB receptors. Subsequent photoaffinity labeling
and different fluorescent microscopy experiments also show that
the probe labels the functional GABAB receptors on the cell
surface with high specificity in an activity-based manner. Several
lines of evidence support this view. First, only GB1 with its
IRS mutated (GB1-ASA) or the heterodimeric functional
GABAB could be labeled but not GB2 expressed alone. Second,
the labeling could be significantly completed by pretreatment
with the bioactive moiety of the probe. Finally, probe 2, which
has no affinity to GABAB receptors, failed to label the GB1-
ASA transiently expressed on the CHO cells surface. Moreover,
the approach has the advantage of forming a permanent covalent
bond between the probe and the receptors through photolysis,
thus allowing direct study of the dynamics of these receptors
in living cells together with its biochemical analysis. Although
many fluorescent photoaffinity probes have been reported,27 it
is important to note that our experiments were performed on
living cells. The functional multiplicity and the specificity of
the labeling of the probe suggest useful applications for this
method.
Figure 5. Probe 1 does not label intracellular GABAB receptors in
living cells. CHO cells were transiently cotransfected with the indicated
constructs. Images were detected by LSCM (OLYMPUS, FV-1000,
×60 objective). The colocalization of the DsRed2-tagged receptor with
probe 1 results in the apparition of a third color in the overlay image:
yellow for red and green merge. Probe 1 channel was detected as
previously, whereas DeRed-tagged constructs were detected after
excitation (ex) at 550 nm and emission (em) at 585 nm. Scale bar
represents 10 µm.
Acknowledgment. This work was supported by National
Basic Research Program of China (Grant 2007CB914200),
Chinese Academy of Sciences (Grant KSCX1-YW-R-12, to F.-
J.N), National Natural Science Foundation of China (Grants
30725049 and 30623008 to F.-J.N. and Grants 30530820 and
30470368 to J.-F.L.), and 863 Program of China (Grant
2006AA02Z326 to J.-F.L.). We also thank Dr. Jean-Philippe
Pin for providing us the GB1-ASA construct and Dr. Eric
Chevet for helpful discussions.
Supporting Information Available: Synthesis and chromato-
graphic data of 6-10 and probes 1 and 2, biological methods, and
HPLC conditions and traces for probes 1 and 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 6. The specificity of labeling depends on the active moiety.
Pretreatment of CHO cells expressing GB1-ASA with 11 significantly
eliminates the fluorescent intensity compared with no pretreatment.
Probe 2 treatment produces no labeling. Results were obtained by TIRF
microscopy.
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