A furanyl acryl conjugated coumarin as an efficient inhibitor and a highly selective off-on fluorescent probe for covalent labelling of thioredoxin reductase

Article abstract of DOI:10.1039/c4cc02119c

A simple and novel furanyl acryl conjugated coumarin fluorophore was designed as an effective thioredoxin reductase (TrxR)-responding fluorescent probe by an activity-guided approach. Basically, the α,β-unsaturated ketone moiety in the probe structure could quench the fluorescence of the coumarin, but upon the covalent modification of TrxR, a significant fluorescence could be generated, which has been confirmed to be obviously selective over Trx, GSH, Cys and DTT. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02119c

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A furanyl acryl conjugated coumarin as an
efficient inhibitor and a highly selective off–on
fluorescent probe for covalent labelling of
thioredoxin reductase†
Cite this: Chem. Commun., 2014,
50, 6987
Received 21st March 2014,
Accepted 8th May 2014
Lei Huang,a Yu Chen,a Baoxia Liang,a Bengang Xing,b Gesi Wen,a Shuni Wang,a
DOI: 10.1039/c4cc02119c
Xin Yue,a Cuige Zhu,a Jun Dua and Xianzhang Bu*a
www.rsc.org/chemcomm
A simple and novel furanyl acryl conjugated coumarin fluorophore protein labelling to benefit the fluorescent imaging and analysis are
was designed as an effective thioredoxin reductase (TrxR)-responding highly desirable. Unfortunately, to the best of our knowledge, such
fluorescent probe by an activity-guided approach. Basically, the a,b- unique fluorescent probes with functions for covalent modification
unsaturated ketone moiety in the probe structure could quench the of TrxR have not been exploited.
fluorescence of the coumarin, but upon the covalent modification of
TrxR contains a conserved C-terminal active site (–Gly–Cys–Sec–
TrxR, a significant fluorescence could be generated, which has been Gly), where Cys497 and Sec498 are essential for the reducing
confirmed to be obviously selective over Trx, GSH, Cys and DTT.
activity.6 In recent years, a lot of compounds with Michael acceptor
groups have been proved to be inhibitors toward TrxR, mostly
Thioredoxin reductase (TrxR EC 1.8.1.9) is a ubiquitous flavo- attributed to their activity with the nucleophilic selenol or sulfhydryl
enzyme present in all living cells from archaea to humans. It groups.7 Curcumin is a dietary yellow spice and pigment isolated
belongs to a well-known class of homodimeric pyridine nucleo- from the rhizome of Curcuma longa with a broad range of bioacti-
tide disulfide oxidoreductases.1 TrxR together with thioredoxin vities. Previously, Holmgren et al. first reported that the activity of
(Trx) and NADPH composes the thioredoxin system which efficiently TrxR could be inhibited by curcumin, and the irreversible inhibition
reduces protein disulfide bonds and serves as an important regulator was probably caused by first alkylation of Sec498 present in the
of the intracellular redox status.2
catalytically active site of TrxR, followed by alkylation of the Cys497
Currently, TrxR has been found to be over expressed in a with another curcumin.8 In our previous study, we synthesized a
number of human tumors and it is believed to play an important series of symmetrical and asymmetrical curcumin analogs (2a for
role in tumor proliferation and drug resistance.3 Therefore, it is of example, Scheme 1) and found that the furan moiety containing
considerable significance to develop a specific probe to detect TrxR analogs exhibit much higher inhibitory effects against TrxR than
in the biological system. So far, several fluorescent probes toward the other curcumin analogs, and the furanyl–acryl moiety may serve as a
Trx system have been reported. For example, TRFS-Green and Mito- possible pharmacophore during the interaction with TrxR.9
Naph, whose fluorescence off–on change is induced by disulfide
To search for a specific covalent probe for fluorescence imaging
cleavage, were used to visualize TrxR and Trx activity, respectively.4 of TrxR, a coumarin fluorophore conjugated with a substituted
However, although these noncovalent labelling probes could attach aromatic ring through an a,b-unsaturated ketone unit was proposed
to the protein active site, the released fluorophore accumulates only first. We hypothesize that the incorporation of the aromatic ring
around the protein binding domain, therefore nonspecific dissocia- could elicit intramolecular charge transfer (ICT)10 from the electron-
tion could be easily induced under highly diluted conditions, which rich diethylamino group at the 7-position of the coumarin to the
may potentially limit their further applications in biological studies.5
Unlike the less stable noncovalent labelling TrxR probes, the
covalent labelling probes which can specifically react with TrxR,
and meanwhile, greatly minimize the diffusion of the probe after
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006,
P. R. China. E-mail: phsbxzh@mail.sysu.edu.cn
b Division of Chemistry and Biological Chemistry, School of Physical &
Mathematical Sciences, Nanyang Technological University, Singapore, 637371,
Singapore. E-mail: bengang@ntu.edu.sg
† Electronic supplementary information (ESI) available: Experimental details and
additional spectra. See DOI: 10.1039/c4cc02119c
Scheme 1 Design of a thioredoxin reductase-responding fluorescent probe.
Chem. Commun., 2014, 50, 6987--6990 | 6987
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electron-poor aromatic ring through the extended conjugation
system, and the fluorescence of coumarin was therefore quenched.
However, in the presence of TrxR, the extended conjugate of the
a,b-unsaturated ketone could be selectively disrupted and the ICT
process was effectively eliminated,11 thus, resulting in an obvious
turn-on fluorescent response.
Numerous thiol-specific probes which contain a Michael
acceptor have been developed on the basis of the Michael addition
reaction.12 In current work, however, a simple conjugation of the
fluorophore with an a,b-unsaturated ketone is insufficient to
achieve the high selectivity, mainly attributed to the reactivities of
general thiol containing compounds with the a,b-unsaturated Fig. 1 SDS-PAGE analysis of TrxR labelling. (a) Coomassie blue staning
and (b) the fluorescence image with excitation l = 365 nm. Lane 1, 3, 5:
Mw marker; lane 2: TrxR; lane 4: TrxR + TR-green; lane 6: IAM inhibited
TrxR + TR-green.
ketone moiety. Though the –SeH in the binding site of TrxR is
more active than the –SH,13 the abundant cellular thiol bearing
small molecules such as GSH may also contribute to the non-
specific binding, and thus potentially decrease the signal to noise
ratio (S/N) of the protein detection. In order to further improve the
selectivity, a highly efficient activity-guided approach was employed,
in which TrxR inhibitory assay was adopted to screen the specific
probes. Based on the high inhibitory activity toward TrxR, the
affinity of such inhibitor based probes toward TrxR could be pre-
dicted, and thus allowed the selective identification over other thiol
containing small molecules. As proof-of-concept, Scheme 1 and data
in the ESI† indicate the rational design and synthesis of a series of
proposed coumarin derivatives from available starting materials.
Upon their structural identification by 1H NMR, 13C NMR, and
HRMS spectroscopy, their activities toward TrxR inhibition and
specific protein labelling were further investigated.
Typically, the TrxR inhibitory activities of all compounds were
determined by the 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) assay.
The IC50 (half maximal inhibitory concentration) values are sum-
marized in Table S1 (ESI†). The results show that TR-green bearing
a 5-methylfuran moiety exerted a much higher inhibitory activity
(IC50: 0.52 mM) than those of the other compounds, which is
consistent with our results reported previously.9
Encouraged by the potent inhibition properties of TR-green, we
next investigated the possibility of the fluorescent turn-on behaviour
of TR-green by the Michael addition reaction. As shown in the ESI†
(Scheme S2 and Fig. S1), obvious fluorescence could be induced by
the Michael addition between TR-green and N-acetylcysteamine
(NAC) after 24 h of incubation. Though significant fluorescence
enhancement needs a long time and the catalysis of an alkali, these
results confirmed the turn-on effect of the probe.
shift of the absorption maximum from 470 nm to 440 nm was
observed (Fig. 2a), indicating the formation of the conjugated
structure after the reaction. Furthermore, a new emission peak
at 500 nm appeared and a dramatic fluorescence enhancement
was also observed (Fig. 2b). Furthermore, the linear correlation
between the fluorescence intensity and the TrxR concentration
(Fig. 2b) indicate that TrxR can be detected down to nanomolar
concentrations, which is comparable to the fluorescent probes
for TrxR sensing reported recently.4
The selectivity of TR-green toward TrxR over other biological
species was then investigated. The fluorescence change in TR-green
in response to TrxR was measured in the presence of different amino
acids, essential metal ions, and hydrogen peroxide. As depicted in
Fig. S4 (ESI†), the reactivity of TR-green is highly selective for TrxR.
To obtain insights into its possible origin, we also carried out kinetic
analyses. The time course of the fluorescence intensity of TR-green in
the presence of TrxR, Trx (the substrate of TrxR), BSA (a general
protein with a free sulfhydryl), GSH, Cys and DTT in PBS buffer is
displayed in Fig. 3a. TrxR was distinguished from other species by a
rapid increase of the fluorescence intensity, and the initial reaction
rate (DF/Dt) in the presence of TrxR (1.51) was obviously faster than
that of Trx (2.51 Â 10À1), BSA (1.20 Â 10À1), GSH (4.10 Â 10À2), Cys
(3.23 Â 10À2) and DTT (4.90 Â 10À2), although the concentrations of
GSH, Cys and DTT are much higher (1000 times) than TrxR (Fig. 3b,
Fig. S3, ESI†). The reaction rate constants were calculated from the
Moreover, the binding affinity of TR-green toward TrxR was also
investigated. Basically, purified TrxR was incubated with TR-green
for two hours. Subsequent SDS-PAGE analysis demonstrated that a
fluorescent band appeared in the gel, and the molecular weight
(58 kDa and 116 kDa) was in accordance with that of the TrxR and
TrxR dimer, respectively. In contrast, pre-treatment of TrxR with
iodoacetamide, a potent reagent to block the functions of TrxR,14
resulted in much weaker fluorescence. These SDS-PAGE results
clearly indicated that the specific covalent conjugation of TR-green
to the TrxR structure and the modification of TR-green by TrxR
(Fig. 1) would significantly turn on the fluorescence.
Fig. 2 Photophysical changes of TR-green during its reaction with TrxR.
(a) UV/vis absorption and (b) fluorescence changes of TR-green (1.0 mM)
with increasing concentrations of TrxR (0–1.5 mM) and the linear correlation.
All spectra were acquired 1 h after addition of TrxR at room temperature in
The optical properties of TR-green were also tested in 100 mM
PBS (pH = 7.4). Upon addition of TrxR, an obvious 30 nm blue PBS buffer (pH 7.4), with excitation l = 440 nm.
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Fig. 4 Confocal fluorescence images of A549/CDDP cells. The cells with
(A–C) or without (D–F) pre-incubation with NEM (50 mM) followed by
treatment with 2.5 mM TR-green for 20 min. Then the cellular TrxR were
stained with the TrxR1 (B-2) mouse antibody and Dylight 549-Goat
Anti-Mouse IgG (red). Images for TR-green (A, D) and IgG (red) (B, E) were
obtained using excitation at 405 and 547 nm, respectively. C, F are the
merged images from A/B and D/E respectively.
Fig. 3 (a) Time course fluorescence response of TR-green (1.0 mM) towards
TrxR (1.0 mM), Trx (1.0 mM), BSA (1.0 mM), GSH (1 mM), Cys (1 mM), and DTT
(1 mM). (b) The bars represent the initial slope (100 s) of the fluorescence
variation at 500 nm as a function of time (DF/Dt).
reactions between TR-green and different concentrations of TrxR,
Trx, GSH, Cys and DTT. As summarized in Table 1, the reaction
kinetics with TrxR was approximately 4748 times higher than with
GSH. Interestingly, Trx, which is regarded as a di-thiol molecule and
being much more active than DTT in redox reactions, shows a
reaction rate constant lower than that of TrxR by almost 40 times.
Accordingly, we demonstrated that TR-green exhibited no apparent
inhibitory activity toward Trx (Fig. S5, ESI†). These results further
evidenced the selectivity of TR-green toward TrxR.
To verify if the fluorescence enhancement was induced by the
Michael addition between TR-green and TrxR, TrxR was pre-treated
with iodoacetamide (IAM) to alkylate the free –SeH and –SH groups
first, and the kinetic curves were recorded. Fig. S2 (ESI†) shows that
pre-treatment with IAM could greatly decrease the fluorescence
intensity. Meanwhile, BSA could also turn on the fluorescence to a
much lower extent, and be decreased by the IAM treatment.
In order to further demonstrate that TR-green can be effective
under living cell conditions, the confocal imaging studies were con-
ducted. Basically, the cisplatin-resistant A549 (A549/CDDP) tumor
cells were chosen in this typical study mainly due to the elevated TrxR
level in this cell line.15 The cellular TrxR was stained with the TrxR1-
(B-2) mouse antibody and Dylight 549-Goat Anti-Mouse IgG (red).
Upon incubation with TR-green (2.5 mM) for 20 minutes, strong
fluorescence could be observed inside the cells (Fig. 4B). In contrast,
when the cells were pre-incubated with the –SeH/–SH alkylating agent
NEM (N-ethylmaleimide)16 for 30 minutes followed by treatment with
TR-green, the fluorescence intensity decreased obviously (Fig. 4E).
These results suggest that this probe works well in living cells.
In conclusion, we presented a simple and new covalent
fluorescent labelling probe for thioredoxin reductase based
on a Michael-type reaction strategy. With a high inhibitory
activity toward TrxR, the probe shows a fast response. By the
SDS-PAGE analysis, we found that TR-green covalently modified
TrxR at its C-terminal active site and emitted significant fluores-
cence. The in vitro kinetic analysis showed that the reaction rate
constant for TrxR is much higher than those for other tested
biological species and displayed good selectivity. The in vivo perfor-
mance of TR-green for TrxR was evidenced by confocal microscopy.
A TrxR inhibitor markedly decreased the intracellular fluorescence,
which further validated the covalent turn-on effect of TR-green.
Considering that TrxR has been evidenced to be overexpressed and
constitutively active in cancer cells, we expect that this probe could
be employed in further biological applications.
We acknowledge the Natural Science Foundation of China
(No. 30973619 and 81172931) and the National High-tech R&D
863 Program (No. 2008AA02Z304) for support for this work.
Notes and references
´
1 E. S. J. Arner and A. Holmgren, Eur. J. Biochem., 2000, 267, 6102–6109.
2 (a) P. Y. Gasdaska, M. M. Berggren, M. J. Berry and G. Powis, FEBS
Lett., 1999, 442, 105–111; (b) J. Lu, Cancer Res., 2006, 66, 4410–4418.
3 (a) K. Becker, C. Herold Mende, J. J. Park, G. Lowe and R. H. Schirmer,
J. Med. Chem., 2001, 44, 2784–2792; (b) M. Ueno, J. Biol. Chem., 1999,
274, 35809–35815.
4 (a) L. Zhang, D. Duan, Y. Liu, C. Ge, X. Cui, J. Sun and J. Fang, J. Am.
Chem. Soc., 2013, 136, 226–233; (b) M. H. Lee, J. H. Han, J. H. Lee, H. G.
Choi, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012, 134, 17314–17319.
5 S. Mizukami, Y. Hori and K. Kikuchi, Acc. Chem. Res., 2014, 47, 247–256.
6 (a) T. Sandalova, L. Zhong, Y. Lindqvist, A. Holmgren and G. Schneider,
Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 9533–9538; (b) L. Zhong, E. S. J.
Arner and A. Holmgren, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5854–5859.
7 (a) E. H. Chew, A. A. Nagle, Y. Zhang, S. Scarmagnani, P. Palaniappan,
T. D. Bradshaw, A. Holmgren and A. D. Westwell, Free Radicals Biol.
Med., 2010, 48, 98–111; (b) J. Fang and A. Holmgren, J. Am. Chem. Soc.,
2006, 128, 1879–1885.
Table 1 Rate constants for the reaction of TR-green with TrxR, Trx, GSH,
Cys and DTT
8 J. Fang, J. Biol. Chem., 2005, 280, 25284–25290.
Entry
ka
k/kGSH
9 (a) X. Qiu, Z. Liu, W. Y. Shao, X. Liu, D. P. Jing, Y. J. Yu, L. K. An,
S. L. Huang, X. Z. Bu, Z. S. Huang and L. Q. Gu, Bioorg. Med. Chem.,
2008, 16, 8035–8041; (b) B. Zhou, J. Huang, Y. Zuo, B. Li, Q. Guo,
B. Cui, W. Shao, J. Du and X. Bu, Eur. J. Pharmacol., 2013, 707, 130–139.
10 S. Ji, J. Yang, Q. Yang, S. Liu, M. Chen and J. Zhao, J. Org. Chem.,
2009, 74, 4855–4865.
TrxR
Trx
GSH
Cys
(1.32 Æ 0.22) Â 106
(3.26 Æ 0.32) Â 104
(2.78 Æ 0.33) Â 102
(3.38 Æ 0.38) Â 101
(4.29 Æ 0.40) Â 102
4748.02
117.27
1.00
0.12
1.54
DTT
11 (a) H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee, J. L. Sessler,
T. W. Kim, C. Kang and J. S. Kim, Biomaterials, 2012, 33, 945–953;
a
The rate constants are expressed as (M s)À1
.
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(b) H. S. Jung, K. C. Ko, G. H. Kim, A. R. Lee, Y. C. Na, 13 (a) C. Jacob, G. I. Giles, N. M. Giles and H. Sies, Angew. Chem., Int. Ed.,
C. Kang, J. Y. Lee and J. S. Kim, Org. Lett., 2011, 13, 1498–1501;
(c) X. Zhou, X. Jin, G. Sun, D. Li and X. Wu, Chem. Commun., 2012,
48, 8793.
2003, 42, 4742–4758; (b) H. Maeda, K. Katayama, H. Matsuno and
T. Uno, Angew. Chem., Int. Ed., 2006, 45, 1810–1813.
14 Y. H. Jan, D. E. Heck, J. P. Gray, H. Zheng, R. P. Casillas, D. L. Laskin
and J. D. Laskin, Chem. Res. Toxicol., 2010, 23, 1045–1053.
12 (a) H. S. Jung, T. Pradhan, J. H. Han, K. J. Heo, J. H. Lee, C. Kang and
J. S. Kim, Biomaterials, 2012, 33, 8495–8502; (b) G. J. Kim, K. Lee, 15 S. E. Eriksson, S. Prast Nielsen, E. Flaberg, L. Szekely and E. S. J.
´
H. Kwon and H. J. Kim, Org. Lett., 2011, 13, 2799–2801; (c) W. Lin, Arner, Free Radicals Biol. Med., 2009, 47, 1661–1671.
L. Yuan, Z. Cao, Y. Feng and L. Long, Chem. – Eur. J., 2009, 15, 16 J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem.
5096–5103.
Soc., 2010, 132, 1216–1217.
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