Fluorescein Derivatives as Bifunctional Molecules for the Simultaneous Inhibiting and Labeling of FTO Protein

Article abstract of DOI:10.1021/jacs.5b06690

The FTO protein is unequivocally reported to play a critical role in human obesity and in the regulation of cellular levels of m⁶A modification, which makes FTO a significant and worthy subject of study. Here, we identified that fluorescein der

Full text of DOI:10.1021/jacs.5b06690

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Communication
Fluorescein Derivatives as Bifunctional Molecules for
the Simultaneous Inhibiting and Labeling of FTO Protein.
Tianlu Wang, Tingting Hong, Yue Huang, Haomiao Su, Fan Wu, Yi Chen, Lai Wei, Wei Huang, Xiaolan Hua,
Yu Xia, Jinglei Xu, Jianhua Gan, Bifeng Yuan, Yuqi Feng, Xiaolian Zhang, Cai-Guang Yang, and Xiang Zhou
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b06690 • Publication Date (Web): 12 Oct 2015
Downloaded from http://pubs.acs.org on October 12, 2015
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Fluorescein Derivatives as Bifunctional Molecules for the Simul-
taneous Inhibiting and Labeling of FTO Protein
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Tianlu Wang‡ †, Tingting Hong‡ †, Yue Huang‡ §, Haomiao Su†, Fan Wu†, Yi Chen†, Lai Wei†, Wei
Huang†, Xiaoluan Hua†, Yu Xia†, Jinglei Xu†, Jianhua Gan⊥, Bifeng Yuan†, Yuqi Feng†, Xiaolian
Zhang#, Cai-Guang Yang*,§ & Xiang Zhou*,
†
†College of Chemistry and Molecular Sciences, Institute of Advanced Studies, Wuhan University, Wuhan, Hubei, 430072, P. R. China
§State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China
⊥School of Life Sciences, Fudan University, Shanghai, China
#School of Medicine, Wuhan University
Supporting Information Placeholder
Inspired by the previous work12c, we tried to screen potent FTO
ABSTRACT: The FTO protein is unequivocally reported to play
inhibitors from many fluorescent molecules having structures
similar to MA. To our delight, we surprisingly found that fluoresꢀ
cein and some of its derivatives could be utilized as bifunctional
molecules for selectively inhibiting FTO demethylation and diꢀ
rectly labeling FTO protein at the same time.
Recently, activityꢀbased protein profiling (ABPP) has been
widely implemented to monitor enzyme activity directly in native
biological systems through designing activeꢀsiteꢀdirected covalent
probes to specifically perturb target enzymes13. A few photoreacꢀ
tive groups are employed in ABPP to covalently label interacting
proteins, such as aryl azides, diazirines and benzophenones14.
Here, through introducing a diazirine group to fluorescein derivaꢀ
tives, we can further provide a platform for the downstream bioꢀ
chemical analyses of FTO, such as in situ FTO labeling within the
native cellular environment and FTO enrichment for subsequent
proteomeꢀwide identification.
a critical role in human obesity and in the regulation of cellular
levels of m6A modification, which makes FTO a significant and
worthy subject of study. Here, we identified that fluorescein deꢀ
rivatives can selectively inhibit FTO demethylation. And the
mechanisms behind these activities were elucidated after we deꢀ
termined the Xꢀray crystal structures of FTO/ fluorescein and
FTO/ 5ꢀaminofluorescein. Furthermore, these inhibitors can also
be applied to the direct labeling and enrichment of FTO protein
combined with photoaffinity labeling assay.
As a prevalent postꢀtranscriptional modification in mRNA for
higher eukaryotes 1, m6A (N6ꢀmethyladenosine) was found to be
enriched primarily near stop codons and in 3’UTRs 2. The recent
discoveries of a m6A methyltransferase tricomplex (METTL3–
METTL14–WTAP) 3 and two demethylases (FTO and ALKBH5)
revealed new insights into the dynamic regulation of m6A
methylation on gene expression. Together with the m6Aꢀbinding
We initially designed and synthesized eleven fluorescein derivaꢀ
tives, which were named as FL1ꢀ11. The preliminary inhibition
evaluation of these compounds was performed via a LCꢀMS assay
(Figure S1) and the robust restriction endonuclease digestion
assay (Figure S2a and S2c). Our results revealed that 100 ꢁM of
compounds FL1ꢀ8 could completely abolish the demethylation of
FTO in vitro, while FL9ꢀ11 displayed poor or no inhibitory activiꢀ
ty under the same condition. Moreover, these fluorescein anaꢀ
logues were shown to be inactive against ALKBH5 demethylation
even at 100 ꢁM (Figure S2b), which demonstrated that FL1ꢀ8
displayed high selectivity towards FTO over ALKBH5 in vitro.
To further investigate the structureꢀactivity relationships of theꢀ
se inhibitors, we precisely validated their FTO inhibitory activities
through IC50 values measurement using a LCꢀMS based assay
(Figure 1b and S3). The results revealed that all eight fluorescein
analogues showed good inhibitory activities towards FTO, with
IC50 values between 1.0 and 7.0 ꢁM. In addition, the amide derivaꢀ
tives of 5ꢀaminofluorescein presented equal or even higher inhibiꢀ
tory activities compared with 5ꢀaminofluorescein, which offers a
new perspective for further structural optimization.
4,
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protein (YTHDF2 and YTHDF1) , researchers have found that
perturbation of the activities of these enzymes may result in the
disturbance of the translation status and RNA metabolism, which
further highlights the fundamental physiological roles of m6A
methylation in biological processes and diseases 7.
Initially, the fat mass and obesityꢀassociated (FTO) protein was
known to be involved in human obesity and energy homeostasis 8.
As a member of the AlkB dioxygenase family, FTO was also identiꢀ
fied to be the first RNA demethylase that catalyses the demethylaꢀ
tion of m6A in an Fe (II)ꢀ and 2ꢀoxoglutarateꢀdependent manner 5,
9, 10. Later, m6A demethylation was also found to be linked to adiꢀ
pogenesis through FTOꢀdependent m6A demethylation 11. Reꢀ
cently, some studies aiming at exploiting small molecules as funcꢀ
tional probes for studying FTO have been reported 12. These small
molecules exhibit good inhibitory activities on m6A demethylation
in vitro and in vivo. Among them meclofenamic acid (MA) was
identified as a highly selective inhibitor of FTO12c and dihydroxyꢀ
furan sulfonamides were demonstrated with anticonvulsant activiꢀ
ty other than inhibiting FTO demethylation12e. However, to our
knowledge, no compounds equipped with the ability to simultaꢀ
neously selectively inhibit and label FTO have been developed to
date.
Among these inhibitors, FL6 and FL8 were chosen to modulate
the level of m6Ain the mRNA of living cells because of their better
cell permeability. Firstly, a MTT assay was performed to find that >
95% of the cells remained viable after treatment with 20ꢀ150 ꢁM
FL6 and FL8 (Figure S4). Using DMSOꢀtreated HeLa
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ID code 4ZS3 (FTO/ FL1) and 4ZS2 (FTO/ FL4). In the strucꢀ
tural complexes, we observed that the esters were hydrolyzed to
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carboxyl acids
(Figure 2a and 2d), which form hydrogenꢀ
bonding with Ser 229 (Figure 2b and 2e). The phenyl ring is surꢀ
rounded by the side chains of adjacent residues Val83, Ile85,
Leu90, Pro93, and Leu109, providing extensive hydrophobic inꢀ
teractions to stabilize the inhibitor binding (Figure 2b and 2e and
S9a). This hydrophobic nucleotide recognition lid (NRL) motif
contributes to the highly selective inhibition of FTO over
ALKBH5 by fluorescein (Figure S2b) 18. In addition, it is applied
to explain why compounds FL9ꢀ11 failed to inhibit FTO (Figure
S1), The bulky substituent on either the 5ꢀ or 6ꢀposition of the
phenyl ring would severely hinder the binding to FTO (Figure 2
and Figure S9). Overall, quite similar to MA for FTOꢀbinding
(Figure S8a), fluorescein inhibitors partially occupy the nucleoꢀ
tide binding site (Figure S8b), and do not disturb the cofactor 2ꢀ
OG or metal ion binding to FTO (Figure S8c).Therefore, these
fluorescein inhibitors are not acting as an adenine mimetic, or
stacking with active site residues which are involved in nucleotide
recognition. It is worthy to note that the 5ꢀamino group in FL1
forms an extra hydrogenꢀbonding with the backbone carbonyl of
Leu 91 (Figure 2b). Generally, the additional interaction would
contribute to an enhanced binding. However, this hydrophilic
amino substituent is close to the hydrophobic NRL motif (Figure
S9b and S9c), which appears to be less favorable for the FTO/ FL1
interaction, explaining why FL1 displays weaker inhibitory activity
than FL4 (Figure 1b).
Furthermore, these two structures clearly reveal that that the 4ꢀ
position in the phenyl ring of fluorescein is fully exposed to the
solvent. Therefore, we anticipate that a proper substituent on this
site would minimally disturb the binding to FTO. Thus, we synꢀ
thesized 4ꢀaminofluorescein (FL12) and found a slight, but not
statistically significant, decrease in its IC50 value (4.37 ꢁM) comꢀ
pared with that of 5ꢀaminofluorescein (FL1) (Figure S18 and
Table S6). In summary, this investigation of the structureꢀactivity
relationships showed good agreement between the observed activꢀ
ity of the inhibitors and the structural feature of FTO/ FL interacꢀ
tions.
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Figure 1. Fluorescein derivatives as potent inhibitors against FTO
demethylation. a) Scheme of demethylation of m6A to A by FTO
demethylase, which can be selectively inhibited by fluorescein
derivatives. b) IC50 values of FL1ꢀ8 against FTO demethylation.
c) The cellular levels of m6A in mRNA were increased when the
HeLa cells were treated with 50 ꢁM FL6 or FL8. *P < 0.01. Error
bar, mean ± SEM for n = 3 experiments.
cells as control, we could observe a significant increase in the level
of m6A modification in mRNA for the cells that were treated with
50 ꢁM FL6 or FL8. These results revealed that these two fluoresꢀ
cein derivatives could efficiently inhibit FTO demethylation inꢀ
side live cells (Figure 1c).
Next, in order to investigate the inhibition mechanism of these
13
fluorescein analogs, differential scanning fluorometry (DSF)
was firstly carried out (Table S1). We observed that the presence
of 80ꢀfold compounds FL1ꢀ8 could significantly increase the meltꢀ
ing temperature (Tm) of the FTO protein, which revealed that
these inhibitors could directly bind to FTO in vitro. In contrast,
FL9ꢀ11 minimally altered the Tm values of the FTO protein,
which is consistent with their poor inhibitory activities against
FTO in vitro.
Due to the fluorescence properties of these inhibitors 19, they
were further designed for the labeling of FTO. Considering that
the photoaffinity labeling was successfully demonstrated by the
capture of the binding proteins in a distanceꢀdependent manner,
particularly under native cellular conditions 20, we introduced
diazirine into FL2 as a photoreactive group for the purpose of
FTO labeling and enrichment (Figure 3a).
In addition, we also performed aggregation tests in order to rule
out the possibility of these fluorescent analogues inhibiting FTO
protein through aggregateꢀbased mechanism16. Although we obꢀ
served a slight decrease in IC50 values of FL2 and FL4 against
FTO demethylation after preincubation, the results of both dyꢀ
namic light scattering (DLS) and detergentꢀbased assay confirmed
that these fluorescein derivatives didn’t act as promiscuous inhibiꢀ
tors (Figure S5 and Table S2, S3, S4). Then by increasing the
concentrations of ferrous ion or 2ꢀoxoglutarate (2ꢀOG), we found
that even the presence of 2 mM ferrous ion or 2ꢀOG had little
impact on the inhibitory activity of fluorescein, which thus exꢀ
cludes the possibility of fluorescein acting as a ferrous ion chelator
or mimicking 2ꢀOG to inhibit FTO demethylation (Figure S6).
In order to gain insight into the inhibitory mode of action from
the atomic level, we determined the Xꢀray crystal structures of
FTO/ fluoresceins complexes. The structures were solved by moꢀ
lecular replacement and refined to a resolution of 2.15 Å for
FTO/ FL4 and 2.45 Å for FTO/ FL1, respectively (Figure S7, and
Table S5). The atomic coordinates and structure factors have
been deposited in the RCSB Protein Data Bank under accession
The inhibitory activity of FL2ꢀDZ was first measured, and
minor change in its IC50 value (4.49 ꢁM) was observed compared
with FL2 due to the small size of a diazirine substitution (Figure
S10). Nonspecific crosslinking between BSA and FL2ꢀDZ can be
remarkably suppressed with increasing salt concentrations and
completely disappeared under 0.4 M PBS and 0.5 M NaCl (Figure
S11 and S12). The selective photoaffinity labeling of FTO by
FL2ꢀDZ was further confirmed by comparison with other AlkB
enzymes. A mixture of FTO and ALKBH5 was treated with FL2ꢀ
DZ under the conditions described above, followed by UV irradiaꢀ
tion (λ = 365 nm, 20 min) and SDSꢀPAGE analysis. As shown in
Figure 3b, no labeling band corresponding to ALKBH5 was obꢀ
served. The same results were also obtained with two other human
AlkB demethylases, ALKBH2 and ALKBH3 21, 22 (Figure 3b). This
extraordinary selectivity of FL2ꢀDZ for FTO demonstrates its
prospective application in the identification of FTO in living cells.
Because of its excellent cell permeability and biochemical activiꢀ
ties, the FL2ꢀDZ probe was added to the medium of HeLa cells to
demonstrate its bioimaging capacity for the labeling of endogenꢀ
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Figure 2. Structural complexes of inhibitors bound
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to the FTO protein. a) The 2FoꢀFc map contoured
to 1.0 sigma confirms that FL1 indeed binds to
FTO. The FTO protein is depicted as a cartoon and
coloured in green, the inhibitor FL1 is depicted in
stick and coloured in cyan, and the map is coloured
in orange. b) Interaction networks between the
FTO protein and FL1 in the active site pocket. The
environmental residues involved in the interactions
are shown as sticks. Dark dotted lines indicate the
hydrogen bonds. c) Surface view showing the vaꢀ
cancy to hold inhibitor FL1. d) The m│Fo│ꢀ
D│Fc│ map was calculated with the PHENIX
program suite after removing the inhibitor from the
complex model and subsequent simulated annealꢀ
ing. The OMIT map density contoured to 3.0 sigma
is coloured in magenta. e) Close view of the interꢀ
action between FTO and FL4 in the binding pockꢀ
et. f) Surface potential view of the pocket in FTO
for binding to FL4.
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protein in living cells and FTO enrichment. We anticipate that
this potential useful tool can facilitate the study of the roles of
FTO in m6Aꢀmediated epigenetic processes. Finally, it is worthy
to note that when fluorescein and some of its derivatives are apꢀ
plied to imaging studies to investigate some cellular biochemistry,
their potent ability to inhibit FTO demethylation should also be
taken into account.
ous FTO demethylase in situ. After being covalently crossꢀlinked
to its targeted proteins by UV irradiation, we observed strong
green fluorescence throughout the nucleus, which was well coꢀ
localized with the red signals obtained from the immunofluoresꢀ
cence (IF) results using antiꢀFTO (Figure 3c). We then transfectꢀ
ed the HeLa cells with FTO siRNA to silence the fto gene (Figure
S13a), and a significant decrease in the green signals was obꢀ
served, which is consistent with the red signals corresponding to
the IF results (Figure 3c). As expected, HeLa cells with stable
overexpression of wideꢀtype FTO showed a notable increase in
both green fluorescence and red signals (Figure S13b and S15).
The tracking of FTO using the FL2ꢀDZ probe was further demonꢀ
strated in living cells that were exposed to oxidative stress, heat
shock or knockdown of METTL3 (Figure S14 and S16).
Lastly, by using a small terminal alkyne as the reporter, we furꢀ
ther applied this specific FL1ꢀDZꢀalkyne probe to the identificaꢀ
tion of cellular FTO by pullꢀdown and western blotting through
the click chemistry with biotinꢀN3 (Figure 4a). The newly deꢀ
signed FL1ꢀDZꢀalkyne probe was firstly detected for its biological
activities through IC50 measurements (Figure 4b). Next, the selecꢀ
tivity and sensitivity of the probe were further verified through the
enrichment of lowꢀabundance FTO in the diluted HeLa cell lyꢀ
sates (Figure S17b). Then, the FL1ꢀDZꢀalkyne probe was successꢀ
fully applied to the identification of the cellular FTO in cell lysates.
For comparison, we performed the labeling of both endogenous
FTO in HeLa cells and recombinant FTO overexpressed in stable
cell lines. The captured proteins were enriched by strepavidinꢀ
agarose beads and separated by SDSꢀPAGE. The inꢀgel fluoresꢀ
cence scanning showed a clear labeled protein band for both cell
lysates that matches the expected molecular weight of FTO (Figꢀ
ure 4c, the upper gel), which was further identified through westꢀ
ern blotting (Figure 4c, the bottom gel).
In summary, we have explored fluorescein and its derivatives as
bifunctional molecules for selectively inhibiting FTO demethylaꢀ
tion and specifically photoaffinity labeling of FTO. We also proꢀ
vide molecular insights to elucidate the structureꢀactivity relationꢀ
ships of these fluorescent inhibitors through crystallography and
pave the way for the structural optimization of FTO demethylaꢀ
tion inhibitors. Besides, this strategy realized the downstream
biochemical analysis of FTO, such as visualization of the FTO
Figure 3. Selective labeling of FTO using probe FL2ꢀDZ. a)
Scheme of the photoaffinity labeling of FTO demethylase by FL2ꢀ
DZ under UV irradiation. b) Selective labeling of FTO over other
human AlkB enzymes. FTO and other AlkB enzymes (ALKBH2,
ALKBH3, and ALKBH5) were treated with FL2ꢀDZ, followed by
UV irradiation (λ = 365 nm, 20 min). c) In situ labeling of the
FTO in HeLa cells or siRNA FTO HeLa cells with the FL2ꢀDZ
probe (20 ꢁM).
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Figure 4. Affinity labeling and enrichment of FTO in cell lysates
using FL1ꢀDZꢀalkynyl probe. a) Scheme of the pullꢀdown of FTO
by using FL1ꢀDZꢀalkynyl. b) The inhibition curves of FL1ꢀDZꢀ
alkynyl against FTO demethylation. Error bar, mean ± SEM for n
= 3 experiments. c) Inꢀgel fluorescence scanning of the proteins
enriched by FL1ꢀDZꢀalkynyl from HeLa cells and stable cell lines
that overexpress FTO; the protein was further verified through
western blotting using antiꢀFTO (bottom).
Supporting Information
Experiment methods, synthesis and characterization data, IC50
measurement, Thermal shifts of FTO protein, Photoaffinity labelꢀ
ing, Western blot quantification of FTO.
AUTHOR INFORMATION
Corresponding Author
*xzhou@whu.edu.cn
*yangcg@simm.ac.cn
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank the National Basic Research Program of China (973 Program)
(2012CB720600, 2012CB720603, and 2012CB720604 to X.Z.;
2015CB910603 to C.ꢀG.Y.), the National Science Foundation of China
(21432008, 91413109, and 91213302 to X.Z.; 21372237 and 91313303 to
C.ꢀG.Y.), and the National Grand Program on Key Infectious Disease
(2012ZX10003002ꢀ014 to X.Z.). We also thank all beamline staff for data
collection support at the BL19U of the Shanghai Synchrotron Radiation
Facility and Yuntao Zhu and Professor Xing Chen (Peking University) for
offering us linker L2.
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Falnes, P. O.; Krokan, H. E.; Klungland, A. EMBO. J. 2006, 25, 2189.
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