The structure-based traceless specific fluorescence labeling of the smoothened receptor

Article abstract of DOI:10.1039/c9ob00654k

The smoothened receptor (SMO) mediates the hedgehog (Hh) signaling pathway and plays a vital role in embryonic development and tumorigenesis. The visualization of SMO has the potential to provide new insights into its enigmatic mechanisms and associated disease pathogenesis. Based on recent progress in structural studies of SMO, we have designed and characterized a group of affinity probes to facilitate the turn-on fluorescence labeling of SMO at the ?-amine of K395. These chemical probes were derived from a potent SMO antagonist skeleton by the conjugation of a small non-fluorescent unit, O-nitrobenzoxadiazole (O-NBD). In this context, optimal probes were developed to be capable of efficiently and selectively lighting up SMO regardless of whether it is in micelles or in native membranes. More importantly, the resulting labeled SMO only bears a very small fluorophore and allows for the recovery of the unoccupied pocket by dissociation of the residual ligand module. These advantages should allow the probe to serve as a potential tool for monitoring SMO trafficking, understanding Hh activation mechanisms, and even the diagnosis of tumorigenesis in the future.

Full text of DOI:10.1039/c9ob00654k

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Biomol. Chem., 2019, DOI: 10.1039/C9OB00654K.
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DOI: 10.1039/C9OB00654K
Structure-Based Traceless Specific Fluorescence Labeling of
Smoothened Receptor†
Dongxiang Xue1,2,3,4, Lintao Ye , Jun Zheng , Yiran Wu , Xianjun Zhang , Yueming Xu , Tao Li ,
1
4
1
1
1
1
1
,4
1,4
4
1,4
1*
1*
Raymond C. Stevens , Fei Xu , Min Zhuang , Suwen Zhao , Fei Zhao , and Houchao Tao
1iHuman Institute, ShanghaiTech University, Ren Building, 393 Middle Huaxia Rd, Pudong New
District, Shanghai 201210, China. Email: zhaofei@shanghaitech.edu.cn and
taohch@shanghaitech.edu.cn
2Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Building 3, Room 426, Shanghai, 201203, China
3University of Chinese Academy of Sciences, No. 19A, Yuquan Road, Beijing 100049, China
4School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8obxxxxxd
1

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ABSTRACT: Smoothened receptor (SMO) mediates Hedgehog (Hh) signaling pathway and plays
a vital role in embryonic development and tumorigenesis. Visualization of SMO bears the potential
to prompt new insights into its enigmatic mechanisms and associated disease pathogenesis. Based
on recent progress in structural studies of SMO, we designed and characterized a group of affinity
probes facilitating turn-on fluorescence labeling SMO at the ε-amine of K395. These chemical
probes were derived from a potent SMO antagonist skeleton by conjugation of a small non-
fluorescent unit O-nitrobenzoxadiazole (O-NBD). In this context, the optimal probes were evolved
to be capable of efficiently and selectively lighting up SMO regardless of whether it is in micelles
or in native membranes. More importantly, the resulting labeled SMO only bears a very small
fluorophore and allows for recovery of unoccupied pocket by dissociation of the residual ligand
module. These advantages should promise the probe to serve as a potential tool for monitoring
SMO trafficking, understanding Hh activation mechanisms, and even diagnosis of tumorigenesis
in the future.
2

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Introduction
G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins that
mediate transmembrane transduction of extracellular signals, including endogenous chemical
1
ligands and exogenous stimuli. Smoothened receptor (SMO) is a Frizzled (class F) GPCR that
regulates the physiologically important Hedgehog (Hh) signaling pathway.2-3 This pathway
exhibits high-level activity in embryonic development of tissues and organs while maintaining
only low-level activity in adult tissues.4-5 Dysregulation of the pathway, usually constitutive
activation by oncogenetic mutation, results in several human cancers, including basal cell
carcinoma and medulloblastoma.6-8 Thus, the SMO has been an intensively pursued target for anti-
tumor agent developments.9-12 Since 2012, two drugs, vismodegib (GDC-0449) and Sonidegib
(
LDE225), have been marketed.13-14 Research on SMO has witnessed tremendous progress in
1
5
structural studies. To date several high resolution structures have emerged of 7-transmebrane
domain (TMD) 16-17 and extracellular domain (ECD)18-19 SMO as well as the very recent two-
domain structures.20-21 Nevertheless, many elements of SMO-related biology are not yet fully
5
answered , highlighting the urgency for development of new research tools and alternative
biophysical methodologies.
Fluorescence imaging has revolutionized to be a powerful approach to investigate biological
2
2
processes virtually under the microscope. Fluorescence microscopy of particular protein is highly
dependent on precision introduction of fluorophores that minimally perturb the receptor function.
Generally, protein labeling has been achieved through various well-developed methods.23-24
Genetically encoded fluorescence tag is one of most prevailing and straightforward strategy to
yield fluorescence labeling proteins. However, self-fluorescence fusion proteins and other
chemoenzymatic tag strategy series, are based on the introduction of a relatively large fragment
3

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that are likely associated with unpredictable interference.25-26 Besides, antibody- or nanobody-
based technology suffers from unreliable accessibility of high-quality sources. Though the
incorporation of unnatural amino acid (uaa) exemplifies the size advantage showing great potency
2
7
in GPCR research, it requires expert manipulation and engineered expression systems. In contrast,
small molecule-based strategies can offer several advantages of which the most important are low-
cost and the ability to readily label endogenous proteins without engineering.28-29
Fluorescent ligands, composed of ligand and fluorophore, have often been used in the imaging of
protein-ligand interaction and ligand evaluation.28, 30 To date, the most promising fluorescent probe
for SMO is CP-bodipy, which is derived from the naturally occurring SMO ligand cyclopamine
3
1
32
(
CP). CP-bodipy has been widely used in the discovery of drug-like agonists and antagonists.
However, CP was recently found to act as a dual-role ligand since it can bind to two domains as
1
9
either an agonist or antagonist ; a characteristic that had confused our understanding of SMO
biology. Here, in contrast to generally applied chemical probes, we turned to a ligand-directed
grafting strategy to generate unoccupied fluorescent SMO, in which a fluorescent group could be
specifically transferred to the receptor33 whose function will be recovered. Presumably, this
labeling strategy with less interference would allow investigations of more complex SMO biology.
4

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Results & discussion
Design of SMO ligand-based O-NBD conjugated probes. We employed a ligand-directed
grafting strategy to specific labeling of SMO. Generally, the affinity labeling probe comprises two
fragments, one for the directing ligand that can recognize SMO precisely and the other for the
transferred labeling group (Figure 1A). With careful investigation of the TMD ligand binding
modes in X-ray crystallography (Figures S1 and S2), we hypothesized that residue K395 could be
a potential modification site. The modification of this seemingly unsettled residue would
presumably have less interference on TMD ligand binding. Importantly, K395, which locates in
the outer edge of the binding pocket, also exhibits close spatial proximity to the TMD ligands.21
Particularly, the distance was found to be within 4 Å between the nucleophilic ε-amine of K395
and the closest point in the corresponding co-crystallized ligand, including 4-fluorine in
LY2940680 (Figure 1B), methyl in cyclopamine, hydroxyl group in AntaXV, pyridine nitrogen in
SAG-1.5, and the nitro group in the TC114 (Figure S2).
To accommodate the amine chemistry of the selected lysine, we drew on the successful experience
of O-NBD fragment to achieve fluorescence transmission.34 As expected, O-NBD acted as a
versatile ‘turn-on’ florescent widget for the S Ar reaction with ε-amine of lysine under
N
physiological conditions. Applied here is the common skeleton of LY2940680 and TC114 as the
starting anchor fragment to prompt the specific transferring reaction of O-NBD. In our previous
efforts in the digestion of the modular contribution of LY2940680 and in the development of
optimal co-crystallized ligand TC114, we found that the substituents in the head benzoic fragment
are highly tolerated. Initially, a trifluoromethyl group was removed in the initial design so as to
simplify the synthesis (Figure 1C).21, 35
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Evolution of probes for rapid labeling of SMO. With the chosen ligand and labeling group, we
set out to measure the linker. To this end, a range of linker lengths of one to five atoms was
considered in the possible reaction orientation between a swinging lysine and the reactive O-NBD
group. The linker was pre-installed in the benzoic moiety that could be integrated in a convergent
synthetic route. Our synthesis commenced by the coupling of the secondary amine with hydroxyl-
tethered benzoic acids, followed by the O-arylation with F-NBD in the presence of triethylamine
and 4-dimethylaminopyridine at room temperature to give corresponding O-NBD conjugated
probes (Scheme S1 and S2).
Labeling with designed probes was performed on detergent-solubilized SMO and monitored by
the fluorescence increase (λ = 488 nm; λ = 530 nm). The reaction was set in the physiological
ex
em
buffer (25 mM HEPES, pH 7.4, 25 °C) with 1:1 ratio of labeling compounds and protein (2.5 μM
each). To determine the corresponding labeling rate, we performed both in situ fluorescence
determinations using a microplate fluorescence reader and fluorescent sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). Under these conditions, we observed that probe
3
with a 3-atom linker reacted fastest, closely followed by probe 2 with 2-atom linker (Figures 2C,
2
D and S3). According to our previous work, a phenyl ether analog would be more potent than
3
5
benzyl ether. Furthermore, phenol generated from probe 1 after the S Ar reaction, would be a
N
seemingly much better leaving group than alcohols from other probes even though the reaction of
probe 1 still runs much slower, probably due to the steric hindrance of the crowded di-aryl ether
that blocks the nucleophilic attack from K395. Probes 4 and 5 with longer linkers dramatically
slowed the reaction, which correlated with the greater separation of the desired reaction partners.
Overall, we observed a clear relationship between the linker lengths and the labeling efficiencies.
6

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To further accelerate the labeling process, we modified the directing anchor fragment of the probe.
Probe 6 was built by re-introducing the trifluomethyl group to the benzoyl head of probe 2.
Meanwhile, given the important role of bioisosteres in drug evolution, we designed probes 7-10
3
6
by introducing nitrogen in the benzoic and phthalazine moiety (Scheme S1-S3). The efficiency
of probes 6-10 were evaluated as above (Figures 2C, 2E and S4). Contrary to the expectation that
the addition of a trifluoromethyl group to the benzoyl would dramatically enhance the antagonism
of SMO, an even lower labeling rate was observed for probe 6. This contradiction reflected the
inconsistency of ligand efficacy and labeling efficiency. Similarly, we observed that nitrogen
introduction to the probe generally improved the labeling reaction, though their corresponding
precursors showed deteriorated potency in luciferase reporter assay (Figure S5). As shown,
introducing a single N atom to 3-position of the benzoyl (7) or to 4-position of the phthalazine (9)
notably increased the labeling rate compared to probe 2. A further replacement of phenyl in probe
9
7
by 3-pyridyl, generated probe 10 with a slight improvement. In contrast, replacing 3-pyridyl in
by a pyrazinyl group (8) largely decreased the labeling rate.
Confirmation of specificity of SMO labeling by probe 9. Most GPCRs are known for their low
expression and susceptibility to an unnatural membrane environment. A fusion partner is usually
required to improve both expression and stability. Here we applied the construct that has
successfully yielded high resolution crystal structure of two-domain SMO, co-bound with
2
1
TC114. In this construct, a flavodoxin (FLA, MW 16 KD) fusion protein added 4 extra lysines
in the intracellular loop 3 (ICL3) so that there are 19 lysines in total, making it even more
challenging to specifically target K395 while keeping others unlabeled.
To determine the labeling specificity, we designed two K395 mutant constructs (K395A and
K395R). All the SMO constructs, together with adenosine A2A receptor (another GPCR that does
7

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not specifically bind to SMO ligands such as LY2940680), were treated with probe 9. The only
bright band in the fluorescent SDS-PAGE corresponded to the original SMO, while all others were
blank, indicating the specific labeling on K395 (Figures 3A, 3B, S6 and S7). Not surprisingly, pre-
treating SMO with 10 equivalents of TC114 significantly obstructed the labeling process of probe
9
(Figures 3C and S8). Moreover, digestion-MS/MS analysis also confirmed that the NBD
modified site was indeed K395 (Figure 3D). In addition, probes 2, 6, 7, 8, 10 underwent selective
labeling on K395 with different efficiencies as well (Figure S9).
Specifically labeling SMO on membrane using probe 9 and 10. To test the feasibility of probe
9
and 10 to light up SMO in native membrane environments, an on-membrane labeling experiment
was then carried out. The membrane with over-expressed SMO was treated with 0.1, 1, and 10 μM
of probes, followed by an SDS-PAGE analysis (Figure 4). The fluorescence results showed that
both probe 9 and 10 could selectively label SMO at the concentration of 1 μM and no other protein
bands were clearly observed even at higher concentration. However, we also observed the side
reaction between the probe and the membrane lipid with primary amine. This might be ascribed to
the high hydrophobicity (ClogP ~ 5) and the linear shape (similar to cholesterol) of the probe which
makes it highly detained in the lipid part. To this end, the two probes might not be applicable to
target SMO in live cell. The on-membrane-labeled SMO protein was later purified and analyzed
by HPLC with size exclusive columns and UV/fluorescence detectors (Figures S10 and S11),
providing another operation to obtain the purified fluorescence labeling SMO protein.
8

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Conclusion
In summary, we have developed a series of turn-on probes for site selective labeling on SMO.
Guided by rich crystal structures of SMO, the probe was designed based on the interaction between
the target residue and ligand. The labeling was enhanced by carefully regulating properties of the
linker and the anchor ligand. The strategy has generated labeling probes with high-selectivity and
convenient wash-free determination. This method worked well not only with purified SMO protein
but also for on-membrane labeling. As fluorescence group transferring reagents, the ligand
moieties of these probes were able to dissociate or be replaced by other SMO ligands, providing
the opportunity for further treatment of the SMO receptor. Taking advantage of a small
fluorescence group, these probes can serve as useful tools for mechanistic study of SMO
conformational change in vitro. The strategy exhibits promising prospects for various types of
1
9
probes labeling on SMO receptor, such as F probes for NMR study, radioactive isotopes or biotin
tag for immunology study. However, probes in this context seemed to be not applicable to live-
cell imaging at present owing to undesired reactions with biological membrane components along
with modest stability in aqueous buffer (Figure S11), further optimization is still on the way.
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Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
The authors (FX, SZ and HT) are thankful to Shanghai Municipal Government, ShanghaiTech
University for startup financial support, and National Key R&D Program of China
(2018YFA0507000) for support. This work was also partially funded by the National Natural
Science Foundation of China (No. 21672147 to HT and 21708026 to FZ). We thank J. Liu, Q. Shi
and L. Yang for assistance in the membrane preparation, protein purification and NMR data
collection respectively at the Core Facility of iHuman Institute. We thank Y. Zhao for the generous
donation of NIH-3T3 cell line for the assay. We thank Z.-J. Liu, D. Wu and D. Liu for their
insightful discussion and assistance in manuscript preparation.
NIH3T3 cell line for the assay was generously provided by Dr. Zhao Yun from Shanghai Institute
of Biological Sciences, Chinese Academy of Sciences. HEK293 cells for the expression of
engineered protein was purchased from ATCC. Both of the cell lines were maintained at the core
facility of iHuman Institute, ShanghaiTech University.
1
0

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Figure. 1. Rational design and synthesis of SMO ligand-derived labeling probes. (A) General
scheme for the SMO labeling using ligand directed residue modification. The directing ligand of
SMO is indicated as blue rectangle. Labeling and labeled groups are indicated as grey and green
stars, respectively, that indicated off and on states. (B) K395 in the ECL2 of SMO is within about
3
.4 Å of the 4-fluorine of LY2940680 from the crystal structure (PDB: 4JKV). (C) Schematic
illustration of SMO antagonist derived probes.
1
1

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Figure 2. Probe evolution in linker and anchor unit. (A) Chemical structures of probes 1-5 with
linker length from one to five atoms. (B) Chemical structures of probes 6-10 showing modification
in ligand skeleton. (C) Normalized fluorescence (λ = 488 nm; λ = 530 nm) intensity of SDS-
ex
em
PAGE of the FLA-SMO construct treated with probes 1-10. (D) SDS gel of the FLA-SMO
construct treated with probes 1-5. The gel was photographed under a fluorescence detector and
CBB staining. (E) SDS-PAGE of a FLA-SMO construct treated with probes 6-10 as well as probe
2
.
1
2

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Figure 3. Selective labeling on K395 of SMO. (A) SDS gel of the FLA-SMO construct
SMO_K395), the K395A mutant (SMO_K395A), the K395R mutant (SMO_K395R), and A2A
treated with probe 9. The gel was photographed under a fluorescence detector (λ = 488 nm; λ
(
ex
em
=
530 nm). (B) Normalized fluorescence intensity of SDS-PAGE of FLA-SMO constructs and
A2A treated with probe 9. (C) SDS-PAGE of FLA-SMO construct (SMO_K395) treated with
probe 9 and/or TC114. The gel was photographed under a fluorescence detector or CBB staining.
(D) Digestive MS analysis.
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Figure 4. Probe 9 and 10 selectively labeled SMO on membrane. SDS-PAGE of membrane
with over-expressed SMO after being treated with 100 nM, 1 μM and 10 μM probe 9 or probe 10
at 4 °C. The SDS gel was visualized by CBB staining (A) or a fluorescence detector with the
excitation wavelength at 488 nm and the emission wavelength at 530 nm (B).
1
4

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Organic & Biomolecular Chemistry
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DOI: 10.1039/C9OB00654K
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