Astemizole-based turn-on fluorescent probes for imaging hERG potassium channel

Article abstract of DOI:10.1039/c8md00562a

Based on the scaffold of astemizole, three novel turn-on fluorescent probes (N1-N3) for human ether-a-go-go-related gene (hERG) potassium channel were developed herein. These probes have reasonable fluorescence properties, acceptable cell toxicity, and potent inhibitory activity, all of which contribute to cell imaging at the nanomolar level. Overall, these probes have the potential for setting up a screening system for hERG channels.

Full text of DOI:10.1039/c8md00562a

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DOI: 10.1039/C8MD00562A
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RESEARCH ARTICALE
Astemizole-based turn-on fluorescent probes for imaging hERG
potassium channel
Xiaomeng Zhanga, Tingting Liu a, Beilei Wanga, Yuqi Gaoa, Pan Liua, Minyong Lia, Lupei Dua , *
Received 00th January 20xx,
Accepted 00th January 20xx
Based on the scaffold of astemizole, three novel turn-on fluorescent probes (N1-N3) for Human Ether-a-go-go-Related
Gene (hERG) potassium channel were developed herein. These probes have reasonable fluorescent properties, acceptable
cell toxicity, and potent inhibitory activity, all of which contribute to cell imaging at the nanomolar level. Overall, these
probes have the potential for setting up a screening system for hERG channels.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
target for tumor research.
The Human Ether-a-go-go-Related Gene encodes hERG
At the present time, small-molecule fluorescent probes have
potassium channel (Kv11.1), key effector of cardiac been diffusely used in pharmacology and biology for the detection
a
repolarization.1 hERG channel is a voltage-dependent channel of proteins, nucleic acids, and other vital biological molecules,
known for its role in repolarizing the cardiac action potential. The owing to their unique properties, such as high sensitivity, simplicity,
alteration of hERG by mutation2 or pharmacological inhibition3 and convenient operation. When they label targets, they can
produces Long QT syndrome and the lethal cardiac arrhythmia provide real-time information and trace the dynamic process on cell
torsade de pointes. In recent years, more and more drugs have even animal level with high spatial-temporal resolution.10-12
serious adverse cardiac reactions due to their blockade on the hERG
Now, it has been well studied that when the ligands bind with
channel. Most of them were withdrawn from the market, such as
hERG channel, a hydrophobic interaction between the ligands and
Cissapride, Tefenadine, Astemisole and Grepafloxacin.4 Currently, all
the residue Phe 656 or Tyr 652 of hERG channel plays a critical role
drugs should be measured the affinity with hERG channel to
evaluate their cardiotoxicity which is required by FDA. Recently, the
MacKinnon group5 discovered that hERG channel is open with the
voltage sensors when it is in depolarized conformation and hERG
channel’s idiosyncratic sensitivity to many drugs may be associated
with the very small volume of central cavity, which is embraced by
four deep hydrophobic pockets. However, there is still no simple
and effective method to high-throughput screening drugs with
hERG channel.
for high-affinity binding.1, 13, 14 So we decide to use the molecular
mechanism to design environment-sensitive probes. In fact, probes
with this off-on mechanism have been utilized to detect structural
characterization of the ligand-binding domain, protein−protein
interaction, and protein conformation dynamics.15-17 Generally,
three parts are included in
a typical fluorescent probe: a
pharmacophore, a fluorophore, and a linker. Astemizole, the most
potent inhibitor of the hERG channel, was chosen as
pharmacophore. In addition, some fluorophores with good
Recently, many studies have shown that compared with the fluorescent properties could be used in small-molecule fluorescent
corresponding normal cells, the expression of hERG channel is up- probes, such as naphthalimide18 and dansyl derivative.19 In order to
regulated in some tumor cell lines6-8such as neuroblastoma, breast keep the inhibitory activity of the probe, the major interaction sites
cancer, and colon cancer cells to facilitate cell proliferation, of Astemizole’s bonding were retained. Herein, the naphthalimide
invasion, and tumor angiogenesis.9 However, there is no and dansyl fluorophore were conjugated to the recognition moiety
explanation for the up-regulation of hERG channel and the role of using an aliphatic spacer. Soon afterwards, several small-molecule
hERG channel in these cancers. For the purpose of better analysis fluorescent probes (N1−N3) for the hERG channel were synthesized
and molecular imaging of the hERG channel, there is a stringent (Scheme 1).
demand for a simple, cost-effective, and safe method to label hERG
channels. It may help explain the role of hERG channel in these
N
N
N
N
cancers so as to make hERG channel become a biomarker and new
O
O
N
O
S
O
O
N
N
HN
O
O
O
F
F
F
F
N
N
N
N
a. Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE),
School of Pharmacy, Shandong University, Jinan, Shandong 250012, China. Email:
dulupei@sdu.edu.cn
† The authors declare no competing interests.
‡ Electronic Supplementary Information (ESI) available. See DOI:
10.1039/x0xx00000x
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
N
N1
N2
N3
Fluorophore
Hydrophobic interaction
Tyr652 or Phe 656
Pharmacophore
Linker
Scheme 1 Design strategy of the fluorescent probes
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Soon afterwards, the inhibitory activitiesDoOfI:th10e.s10e3p9r/oCb8eMsDa0g0a5i6n2sAt
the hERG channel were measured by radio-ligand binding assays
using hERG transfected HEK293 cells. 20-22The results showed that
probe N1 displayed best inhibitory effects against the hERG
channel, and the calculated IC50 and Ki values are 0.053 and 0.030
μM, respectively, which are slightly lower than astemizole (0.011
and 0.006 μM, respectively). Probes N2 and N3 also disclosed
potent inhibitory activity against the hERG channel, with IC50 values
of 0.183 and 0.186 μM, respectively, although lower than that of
probe N1.
Results and discussion
Chemistry
Subsequently, the fluorescent derivatives of astemizole were
synthesized through a convenient reaction as shown in Scheme 2.
Compound 3 and a (b, c) in the presence of K2CO3 in acetonitrile
yielded the fluorescent compound N1 (N2, N3) through
a
substitution reaction. Further details about the synthesis of the
fluorophores and recognition moiety can be found in the
Supporting Information.
Table 2 Inhibitory activity of the synthesized probes against the
F
hERG potassium
Kib (μM)
0.030
0.103
0.104
0.006
N
compd
N1
IC50 (μM)
0.053
NH
N
O
N1
N2
n=3
n=4
N
N
N2
0.183
n
i
O
N3
0.186
Astemizole
0.011
n
Br
a
See Supporting Information. bThe inhibition constant (Ki) was
O
N
O
calculated from each IC50 value using the Cheng−Prusoff equation.
F
N
a
b
n=3
n=4
N
N
Cytotoxicity assay
NH
O
S
O
The cytotoxicity of these probes was evaluated by CCK-8 assays
using hERG transfected HEK293. The results demonstrated that the
IC50 of probes N1−N3 were 3.55±0.28, 2.43±0.12, and 7.03±0.14μM
in hERG -HEK293 cells.
HN
S
O
N•HBr
O
O
3
N
c
Table 3 Cytotoxicity results for the synthesized probes
ii
compd
N1
IC50 (μM)
3.55±0.28
2.43±0.12
7.03±0.14
17.37±1.07
F
N
N
NH
N2
N
N3
N
O
S
N3
NH
O
Astemizole
Scheme 2 Synthetic routes of the fluorescent probes. (i) K2CO3,
acetonitrile, 80 °C; (ii) K2CO3, acetonitrile, 80 °C.
Fluorescent image assay
As mentioned above, the pharmacophore is environment-
sensitive so that the probe was proposed to have a turn-on
mechanism for hERG channel. To test this hypothesis, a series of
concentrations of hERG transfected HEK293 cell membrane was
incubated with the probe N1 (5 μM). As good as anticipated, with
increasing amount of cell membrane, fluorescence intensity was
gradually enhanced (Fig. 1). When incubated with 0.8 mg/mL cell
membrane, the fluorescence intensity was 12-fold higher than the
blank group.
Spectroscopic properties of the probes
The spectroscopic properties of the probes were measured in 10
μM solution of the corresponding probe (N1, N2, N3) in PBS
(pH=7.4). The results indicated that all probes possessed good
fluorescent properties. Compared to probe N3, the maximum
emission of the probes N1 (N2) is red-shifted reaching 535 (545)
nm, but the fluorescence quantum yields are relatively low (Table
1).
Table 1 Photophysical Properties of Synthesized Probes
Subsequently, the selectivity of fluorescence intensity for hERG
potassium channel was also examined. In the assay, taking account
of the forming of nonspecific binding with small molecules, trypsin
and bovine serum albumin (BSA) were selected as the control
group. Probe N1 (5 μM) was incubated with trypsin, BSA or hERG
transfected HEK293 cell membrane at the same concentration (1
mg/mL). As shown in Fig. 2, there is a suitable increase for trypsin
and BSA, which manifested that probe N1 may form some
compd
N1
λmax
440
445
330
λex
λem
535
545
495
Φ (%)
440
430
335
5
5
N2
N3
15
Binding affinity of probes
Med. Chem.Commun
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ResVeiaewrcAhrticAlerOtinclilnee
DOI: 10.1039/C8MD00562A
nonspecific binding with trypsin and BSA. Additionally, when probe N3 for hERG channels in living cells was conducted on hERG
N1 (5 μM) was incubated with cell membrane (1 mg/mL) and transfected HEK293 cells. The microscopic imaging results indicated
Astemizole (a potent hERG channel inhibitor, 25 μM), the that these probes exhibit rapid responses and strong fluorescence
fluorescence intensity was decreased compared with the group that to hERG - HEK293 cells (Fig. 3). Astemizole, a potent inhibitor of the
was only incubated with cell membrane. However, the decrease hERG channels, was chosen to incubate the cells with each probe.
degree of fluorescence intensity was not complete, which may be When the cells were coincubated with Astemizole and probe, the
caused by unavoidable nonspecific binding between probe N1 and microscopic imaging results showed that inhibition of hERG by
other components in the cell membrane, especially the astemizole resulted in a decrease of fluorescence intensity, which
hydrophobic components.
confirmed that the hERG channel can be well selectively labeled by
the probe. Particularly, because of their turn-on mechanism, there
was not needed a complex washing procedure, which ensured a
convenient imaging process.23, 24 Additionally, we also investigated
cell autofluorescence and the effect of astemizole on cell
autofluorescence (see Supporting Information). The microscopic
imaging results showed that the autofluorescence of the cells is
negligible in both the presence and absence of astemizole (Fig. S9)
so that it would not interfere with the imaging of cells using probes
N1-N3. In general, the results showed that these probes all possess
superior selectivity for hERG channels and could be used in the
detection of hERG potassium channel.
Fig. 1 Fluorescent emission spectra of 5 μM probe N1 incubated
with different concentrations of hERG transfected HEK293
membrane (0.8, 0.6, 0.4, 0.2, 0.1, and 0 mg/mL) for 20−30 min in
the assay buffer (50 mM Tris-HCl, 1 mM MgCl2, 10 mM KCl) at room
temperature (λex = 440 nm).
Fig. 3 Fluorescence microscopy imaging of hERG transfected
HEK293 cells incubated with 0.3 μM probe N1 (A1, bright field; A2,
GFP channel), 0.3 μM probe N2 (B1, bright field; B2, GFP channel),
and 0.5 μM probe N3 (C1, bright field; C2, DAPI channel),
respectively. The imaging of inhibition of the hERG channels was
accomplished by incubating astemizole (3 μM; 3 μM; 5 μM ) with
probe N1 (0.3 μM; A3, bright field; A4, GFP channel), N2 (0.3 μM;
B3, bright field; B4, GFP channel), and N3 (0.5 μM; C3, bright field;
C4, DAPI channel). All cells were incubated with each probe at 37 °C
for 10 min and washed immediately. The background was adjusted
by ImageJ software. Imaging was performed using a Zeiss Axio
Observer A1 microscope with a 63× objective lens.
Fig. 2 (A)Fluorescent emission spectra of 5 μM probe N1 which is
respectively incubated with 1 mg/mL trypsin, 1 mg/mL BSA, 1
mg/mL hERG transfected HEK293 membrane, and 1 mg/mL cell
membrane combined with hERG channel inhibitor Astemizole (10
μM) for 20−30 min in the assay buffer (50 mM Tris-HCl, 1 mM
MgCl2, 10 mM KCl) at room temperature (λex = 440 nm). (B) The
corresponding fluorescent intensity changes (normalized based on
the last point that is seen as 1) at 535 nm (λex = 440 nm).
Conclusions
In conclusion, we developed three high-affinity environment-
sensitive probe, with commendable fluorescent properties, which
can be utilized to the localization and visualization of hERG channel.
These probes have been successfully utilized to label the hERG
channels in hERG transfected HEK293 (hERG- HEK293) cells at the
On account of their good fluorescent properties, acceptable cell
toxicity, and potent inhibitory activity, probes N1-N3 can be used
for hERG channel imaging in living cell in order to expand the
application of our probes. Thus, microscopic imaging of probes N1-
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DOI: 10.1039/C8MD00562A
20. G. J. Diaz, K. Daniell, S. T. Leitza, R. L. Martin, Z. Su, J. S.
McDermott, B. F. Cox and G. A. Gintant, J Pharmacol Toxicol
Methods, 2004, 50, 187-199.
nanomolar level. Because of the environment-sensitive switch in
the structure, it is not needed a washing procedure, so this method
is convenient compared with other imaging techniques, such as
fluorescent protein-based approaches and immuno-fluorescence.
Meanwhile, the synthesis of these probes is convenient and
21. X. P. Huang, T. Mangano, S. Hufeisen, V. Setola and B. L. Roth,
Assay Drug Dev Technol, 2010, 8, 727-742.
22. S. A. Titus, D. Beacham, S. A. Shahane, N. Southall, M. Xia, R.
Huang, E. Hooten, Y. Zhao, L. Shou, C. P. Austin and W. Zheng,
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affordable. When compared with our previously published
astemizole-based fluorescent probes,
23, 24
these probes have
better turn-on effect and higher fluorescent intensity. They also
have good fluorescent properties, acceptable cell toxicity, and
potent inhibitory activity, which make the probes favourable for cell
imaging. Therefore, these probes are anticipated to be applied in
the study of hERG channels. However, there is still a lot of work to
be done, such as setting a screening system for hERG channels.
Acknowledgements
The present project was supported by grants from the Shandong
Natural Science Foundation (No. ZR2017MH101) and the Key
Research and Development Project of Shandong Province (No.
2017CXGC1401).
Conflicts of Interest
The authors declare no conflict of interest.
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Astemizole-based turn-on fluorescent probes for imaging hERG potassium
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DOI: 10.1039/C8MD00562A
channel
Xiaomeng Zhanga, Tingting Liu a, Beilei Wanga, Yuqi Gaoa, Pan Liua, Minyong Lia, Lupei Dua , *
N
O
N
O
F
N
N
N
H
N
Hydrophobic interaction
Tyr652 or Phe 656